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32 nd Congress of the ESP and XXXIII International Congress of the IAP

A Correction to this article was published on 04 February 2021

A Correction to this article was published on 01 February 2021

A Correction to this article was published on 16 January 2021

A Correction to this article was published on 18 December 2020

This article has been updated

Oral Free Paper Sessions

OFP-01 Joint Oral Free Paper Session: Uropathology /Nephropathology


Kidney biopsy codes for pathologists-mapping to SNOMED CT

A. Dendooven*, M.J. Helbert, H. Peetermans, S. Leh

*UZ Gent, Universiteit Antwerpen, Belgium

Background & objectives: The “Kidney Biopsy Codes (KBC)” project provides terms that allow any diagnosis and/or histomorphological pattern for a non-neoplastic kidney biopsy to be coded. We explored whether mapping to SNOMED CT is feasible, to enable aggregation and computerized exchange of data.

Methods: For KBC terms for which an unambiguous match with a pre-existing SNOMED CT concept was available, both ‘parent concept’ and the place in the SNOMED taxonomy were established. For remaining terms, we explored whether these could be defined by combining pre-existing (more simple) SNOMED CT concepts. This process ('post-coordination’) is well supported by SNOMED CT and allows extending its content.

Results: Of glomerular terms, 88/195 (45%) could be matched to SNOMED CT. %-matching was more successful for KBC terms designating disease concepts (56%) than patterns of injury (32%). For the majority of terms that could not be mapped, we found that these could indeed be defined as a compositional expression of pre-existing SNOMED CT concepts (post-coordination). We suggested concepts that are needed for this post-coordination.

Conclusion: SNOMED CT is considered the standard for documenting, encoding and exchanging medical data in/between health information systems. This proof-of-concept shows that mapping of KBC terms to SNOMED CT is feasible, in part directly, in part through post-coordination.


Molecular analysis of renal transplant biopsies comparing the Edmonton Molecular Microscope with the NanoString Human Organ Transplant Panel

J. Schmitz*, H. Stark, S. Bartels, D. Jonigk, P.F. Halloran, G. Einecke, J.H. Bräsen

*Institute of Pathology, Nephropathology Unit, Hannover Medical School, Germany

Background & objectives: Different molecular methods like microarrays or quantitative PCRs were used by several groups on renal transplant tissues. High-resolution determination of the inflammatory infiltrate by NanoString analysis (which was developed for formalin-fixed paraffin-embedded-derived RNA) should be a sufficient approach.

Methods: We used surveillance and indication biopsies from 63 patients whose time-matched second biopsy core had been frozen and analysed by microarray in the INTERCOM/INTERCOMEX study. After re-evaluation according to recent Banff consensus, RNA isolation was performed with Maxwell FFPE kits and led to sufficient RNA yields in 53 samples which were further processed for NanoString analysis (Human Organ Transplant panel).

Results: Morphologically, of the 53 samples analysed (samples from 2011/12 and 2015), twenty-five patients showed no signs of rejection, twelve had borderline rejection, four showed cellular rejection, seven had humoral rejection, and five presented with combined rejection. Preliminary analysis of gene expression by T-distributed Stochastic Neighbour Embedding (t-SNE), Random Forest and Principal Component Analysis (PCA) showed clear differences between samples with rejection (humoral and cellular) and without rejection. Rejection samples revealed high expression of chemokine ligands compared to rejection-free tissues. Borderline rejection shared a common pattern compared to samples without rejection. First results display good correlation with the former molecular diagnosis from the INTERCOM/INTERCOMEX study.

Conclusion: Molecular approach using the NanoString platform may supplement morphological diagnosis of renal grafts especially in unclear cases and thus enhance precision diagnostics with small tissue requirement. Morphological and molecular evaluation in the same biopsy core from FFPE tissue enables direct histological-molecular correlation. Additionally, this technology also improves our understanding of pathophysiology in renal and other transplants.

Funding by: Dr. Werner Jackstädt foundation


Arteriolar C4d: a potential prognostic marker in IgA nephropathy – a retrospective study in a Portuguese tertiary centre

P. Amoroso Canão*, B. Faria, Q. Cai, C. Henriques, A.C. Matos, F. Poppelaars, M. Gaya da Costa, M. R. Daha, R. Pestana Silva, M. Pestana, M. A. Seelen

*Centro Hospitalar Universitário de São João, Portugal

Background & objectives: IgA Nephropathy (IgAN) is the most common glomerulonephritis worldwide. C4d has been recognized as a marker associated with significantly reduced renal survival. We aimed to study the clinical significance of arteriolar immunoexpression of C4d in a cohort of IgAN patients.

Methods: We selected all kidney biopsies with the diagnosis of IgAN, between 2001 and 2017, and reviewed their clinical features; evaluated them according to the Oxford Classification of IgAN 2016 and assessed the presence of vascular lesions. We evaluated the arteriolar and glomerular immunoexpressions of C4d and their association with the baseline and follow-up clinico-histological data thought bivariate and regression analysis.

Results: Arteriolar immunoexpression of C4d was present in 21 (17%) biopsies and associated with mean arterial pressure (MAP), chronic microangiopathy and arterial intimal fibroelastosis. After adjusting to other significant predictors, such as baseline estimated glomerular filtration rate, MAP and the presence of crescents, this immunoexpression remained significantly associated (P values <0.001) with progressive kidney disease.

Conclusion: Arteriolar immunoexpression of C4d is a potential prognostic marker in IgAN. These findings raise the possibility of including immunohistochemistry for C4d in the evaluation of IgAN biopsies.


The impact of vitamin D3 (1,25-dihydroxyvitamin D3) and its role on to the epithelial to mesenchymal transition and the development of interstitial fibrosis in renal allografts

E.S. Ayva*, B.H. Ozdemir, E. Yılmaz Akcay, G. Ozdemir, A. Yucel Polat, M. Haberal

*Baskent University, School of Medicine, Department of Pathology, Turkey

Background & objectives: This study aimed to understand the role and the mechanism of action of vitamin D3 (1,25-dihydroxyvitamin D3) in the development of epithelial to mesenchymal transition (EMT) and interstitial fibrosis (IF) in renal transplant (Tx) patients.

Methods: Out of 161 cases, 70 treated with vitamin D3 (Group D) and 91 cases did not receive vitamin D3 (Group A). Cytoskeletal proteins F-actin, α-SMA and paxillin expression of tubules and the tubular TGF-β, TNF-α, and E-cadherin expression evaluated. Follow-up biopsies analysed for the development of IF during 18 and 24 months after Tx.

Results: Group D patients showed higher degrees of tubular E-cadherin expression than Group A (p<0.001). Tubular F-actin, α-SMA, paxillin, TGF-β and TNF-α found significantly low in Group D compared to Group A (p<0.001). A positive correlation found between the development of IF with the tubular F-actin, α-SMA, paxillin, TNF-α and TGF-β expression (p<0.001). The development of IF during in follow-up biopsies found lower in Group D compared to Group A (p<0.001)

Conclusion: The activation of EMT and the occurrence of IF found higher in Group A cases who had cytoskeleton reorganization (increased F-actin, α-SMA, paxillin expression), and E-cadherin downregulation. E-cadherin loss during EMT suggested to promoted by high TNF-α and TGF-β expression. Contrarily, Group D patients had a lower incidence of IF. Treatment with D3 abolishes the EMT promoted by TNF-α and TGF-β. Therefore, D3 therapy is beneficial in renal transplant patients with its antifibrotic property.


Rearrangement of the endothelial cell cytoskeleton in glomerular and peritubular capillaries following antibody-mediated rejection: its significance on the development of transplant glomerulopathy and interstitial fibrosis

E.S. Ayva*, B.H. Ozdemir, E. Yılmaz Akcay, G. Ozdemir, M. Haberal

*Baskent University, School of Medicine, Department of Pathology, Turkey

Background & objectives: This study aimed to understand the rearrangement of endothelial cell cytoskeleton (EC-CSK) both in glomeruli and peritubular capillaries (PTCs) in antibody-mediated rejection (AMR) and to investigate the influence of cytoskeleton rearrangement on the development of transplant glomerulopathy and interstitial fibrosis

Methods: Total 116 allograft biopsies which diagnosed with AMR were re-evaluated and the intensity of interstitial, glomerular and PTC inflammation were graded. The endothelial cell (EC) expression of F-actin, paxillin, vinculin, HIF-1α, VEGF, and eNOS were evaluated in glomeruli and PTCs. Follow-up biopsies analysed for the development of transplant glomerulopathy (TG) and interstitial fibrosis (IF).

Results: The expression of F-actin paxillin, vinculin, HIF-1α, VEGF and eNOS in EC increased with increasing degree of C4d expression, PTC destruction, interstitial inflammation, glomerulitis and PTC-itis in glomeruli and PTCs (p<0.001). The risk of the development of TG, IF, proteinuria, and graft loss found higher in recipients with a high degree of F-actin, paxillin, vinculin, HIF-1α, VEGF and eNOS expression (p<0.001).

Conclusion: We hypothesized that PTC destruction following AMR causes chronic hypoxia that induces HIF-1α, VEGF and eNOS expression that give rise to the reorganization of EC-CSK. Upon the reorganization of EC-CSK and the increased amount of endothelial VEGF together induce the formation of gaps between adjacent ECs, which in turn initiate EC permeability and influx of leukocytes and proteinuria. As a consequence, the increasing amount of inflammatory cells induces the early development of IF, TG, and consequently the premature graft loss.


Application of artificial intelligence (AI) in assessment of renal allograft biopsies: comparison with subjective Banff criteria for graft inflammation

M. Shamassi*, E.S. Ayva, N.K. Alham, I.S. Roberts

*Department of Anatomical Pathology, Monash Medical Centre, Monash Health, Melbourne, Australia

Background & objectives: The Banff classification is used routinely in reporting of transplant biopsies. The variable reproducibility of Banff criteria limits its clinical value. This study evaluated the use of AI to quantify graft inflammation and correlated inflammatory cell density with Banff scores.

Methods: Digital images from 119 allograft biopsies were scored by two renal pathologists according to Banff criteria. Images were then analysed using the Visiopharm Integrator System platform. An APP was designed to quantify inflammation within normal and fibrotic cortex, and an auxiliary APP to exclude glomeruli. The model was trained through one million iterations.

Results: The inter-observer agreement for i, ti and i-IFTA scores was moderate to good (kappa-scores 0.615, 0.566, 0.552 respectively). Banff scores correlated only weakly with the density of inflammatory cells assessed using AI. i-score: only for i0 vs i3 was there a significantly different inflammatory cell density. i-IFTA and ti scores: the density was significantly greater for Banff categories 1-3 than 0, but there were no significant differences between categories 1-3.

Conclusion: he density of inflammatory cells correlates weakly with Banff scores, likely reflecting the fact that a pathologist will only recognise cortex as inflamed when there is greater than a threshold level of inflammatory cell density in a particular area. It remains to be ascertained whether inflammatory cell density assessed by AI or subjective Banff scores correlate better with clinical outcome and response to treatment.


Carcinogen-induced tumour mouse model reveals dynamic molecular changes during basal bladder cancer progression

J. Fontugne*, J. Wong, N. Karboul, A. Rapinat, P. Maille, R. Leclere, A. Nicolas, D. Meseure, D. Gentien, S. Baulande, N. Servant, I. Bernard-Pierrot, M. Sibony, F. Radvanyi, Y. Allory

*Molecular Oncology team, UMR144, Institut Curie, Paris, France

Background & objectives: The basal subgroup of bladder cancer is often diagnosed at the muscle-invasive stage (MIBC). Identification of molecular changes during progression from non-muscle-invasive (NMIBC) to basal MIBC is challenging.

We used the N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) mouse model to study disease progression.

Methods: To study multiple stages of progression, 40 mice bladders were obtained at different time points following an oral BBN exposure of a maximum of 14 weeks. To characterize molecular changes occurring during progression from early stages to MIBC, we performed immunohistochemistry (IHC) - markers of basal (CK5, CK6, CK14) and luminal subtypes (FOXA1, CK20) - and RNA-seq for all stages.

Results: Morphological analysis identified a spectrum of lesions during BBN exposure: hyperplasia (n= 5), dysplasia (n=5), CIS (n=3), pTa (n=14), pT1 (n= 6), MIBC (n = 7). All tumours displayed a squamous or sarcomatoid component. IHC showed expression of basal markers within early stages of carcinogenesis, with a decrease in FOXA1 expression during progression. Molecular classifications using RNA-seq data highlighted an early and progressive switch to the complete basal phenotype. Further analysis of RNA-seq data identified clusters of genes with expression changes during progression. Assessment of the expression dynamics of each cluster of genes provided insights into specific biological processes involved in basal bladder cancer progression and invasion.

Conclusion: We used the BBN-induced cancer mouse model to longitudinally study carcinogenesis and progression of basal bladder cancer. Our study sheds light on the underlying biology of progression from NMIBC to basal MIBC and from squamous to the aggressive sarcomatoid variant.


TERT promoter mutation analysis is a valuable diagnostic adjunct to distinguish pseudosarcomatous spindle cell proliferations from sarcomatoid urothelial carcinoma of the urinary bladder

S. Bertz*, R. Stöhr, N.T. Gaisa, B. Wullich, A. Hartmann, A. Agaimy

*Institute of Pathology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany

Background & objectives: Bladder pseudosarcomatous myofibroblastic proliferations are diagnostically challenging due to occasional ALK-negativity, frequent cytokeratin expression, worrisome histology. Sarcomatoid urothelial carcinoma (sUC) may resemble inflammatory myofibroblastic tumour (IMT). TERT promoter mutations are frequent in urothelial cancers, but pseudosarcomatous lesions have not been investigated.

Methods: We performed comparative histomorphological and immunohistochemical analysis and TERT promoter mutation testing in 17 ALK-positive and -negative spindle cell lesions and 18 sUC. Screening for genetic rearrangement and/or FISH analysis was performed by RNA sequencing in the pseudosarcomatous myofibroblastic proliferations.

Results: 9/17 sUC showed at least focal IMT-like morphology. Atypical mitoses, presence of a differentiated urothelial component and presence of heterologous elements were the most reliable morphological features of sUC, if present. ALK (D5F3), p53, p63, Ki67-index, smActin and GATA3 were differentially expressed. Pancytokeratin staining was frequent in both groups, but the staining pattern differed significantly (diffuse in 17/17 pseudosarcomatous proliferations, focal in 6/15 sUC). TERT promoter mutations were found in 17/18 sUC but in none of 17 pseudosarcomatous myofibroblastic proliferations. RNA sequencing and FISH analysis revealed genetic rearrangements in 6/7 ALK positive and 1/10 ALK negative pseudosarcomatous myofibroblastic proliferations, including a novel FN1/RET gene fusion in an ALK-negative lesion

Conclusion: TERT promoter mutations and/or ALK expression/rearrangements in spindle cell lesions of the bladder are confirmatory for sUC or IMT in the majority of cases, respectively. In equivocal cases lacking TERT and ALK alterations, clinical history, classical histomorphological and immunohistochemical features should be assessed carefully. RNA sequencing is a sensitive method for assessment of gene rearrangement, including involvement of novel genes other than ALK.


Grading of intraductal carcinoma has minor impact on grade group assignment in prostate cancer biopsy and radical prostatectomy specimens

L. Rijstenberg*, E. Hollemans, T. Hansum, C. Kweldam, G. van Leenders

*Erasmus Medical Center, The The Netherlands

Background & objectives: Intraductal carcinoma (IDC) has an adverse outcome in prostate cancer patients but is not incorporated in tumour grading. Our objective was to determine the effect on global Grade group (GG) assignment in case IDC is included in grading.

Methods: A prostate cancer biopsy (n=1,031) and unrelated radical prostatectomy (n=835) cohort were reviewed for GG according to the 2014 ISUP guidelines, without IDC grade assignment. Presence and percentage of IDC were monitored. By accounting IDC as Gleason pattern 4 or 5 based on its morphology, the GG was recalculated.

Results: IDC was identified in 139/1,031 biopsies (13.5%). Assigning a Gleason pattern to IDC led to a change of GG in 17 cases (1.6%): 4/486 from GG1 to GG2, 9/375 GG2 to GG3, and 4/58 GG4 to GG5. In 213/835 (25.5%) prostatectomy specimens IDC was present. Grading of IDC based on its underlying pattern led to a GG change in 5 cases (0.6%): 1/307 from GG1 to GG2, 1/307 GG1 to GG3, 2/420 GG2 to GG3, and 1/50 GG4 to GG5.

Conclusion: We demonstrate that grading IDC led to a change in GG in <2% of prostate cancer biopsies and radical prostatectomies, indicating it has overall minimal impact on prostate cancer grade assignment.


A retrospective single-centre audit of block-taking in cystectomy specimens for malignancy

N. Herlihy*, A. Haider

*UCLH, United Kingdom

Background & objectives: There is no consensus on how many blocks should be submitted from cystectomy specimens for bladder tumours. Our department recommends ≤10 blocks. Our objective was to audit block-taking and assess how frequently the diagnosis could be reached with ≤10 blocks.

Methods: We performed a retrospective single-centre audit on cystectomy specimens for malignancy processed from January-December 2017. We performed a SNOMED search on Co-Path for all cystectomy cases for malignancy and collected data on patient demographics, presence of gross tumour, number of blocks taken from the bladder at initial cut-up and number of extra blocks (if any), and the histological diagnosis.

Results: 45 cases were suitable for inclusion. A median of 13 bladder blocks were taken per case (range, 4-28). Ten blocks or less from bladder were submitted in 10/45 cases (22.2%). More than 10 blocks were taken in a higher proportion of cases without gross tumour (16/19, 84.2%) compared to cases with gross tumour (19/26, 73.1%). Additional blocks were taken in 4/45 cases (8.8%). 2/10 specimens (20%) where less than ten blocks were taken initially required additional blocks. On review of the slides, the final histological diagnosis was achievable on the initial blocks in all four cases where additional blocks were taken. Estimated costs of oversampling for one year were £8,595.36.

Conclusion: The final histological diagnosis was achievable in all cystectomy cases for malignancy where ten blocks or less were taken from the bladder. In the majority of cases, more than ten blocks were taken, incurring significant potentially unnecessary costs. We suggest that no more than ten blocks should be taken from cystectomy specimens for malignancy, and that even fewer representative blocks can be taken if a gross lesion is identified.


Heterogeneity-analysis of molecular subtypes of muscle-invasive bladder cancer and their precursor lesions in multiregion mapped whole-organ bladders

V. Weyerer*, R. Stoehr, S. Bertz, S. Wach, H. Taubert, D. Sikic, B. Wullich, A. Hartmann, M. Eckstein

*Institute of Patholoy, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nuremberg, Germany

Background & objectives: Molecular subtypes of bladder cancer have been described which seem to have a predictive effect for chemotherapy regimens. Due to this clinical implication, the aim is to characterize in which extent precursor lesions and tumours exhibit a heterogeneity of subtypes.

Methods: 23 positions of three whole-organ mapped specimens were histomorphologically reviewed for classifying location of normal urothelium, precursor lesions and different areas of the tumour. Immunohistochemical analysis of luminal (CK20, GATA3, FOXA1) and basal markers (CD44, CK14 and CK5/6) was carried out in selected precursor lesions and tumours to map the distribution of luminal and basal subtypes across the different positions/lesions.

Results: Out of three analysed multiregion mapped bladder tumours, one showed a heterogenous luminal and double-negative molecular subtype in multiple tumour positions as well as a mixed morphology including conventional urothelial and neuroendocrine areas. Moreover, among the second analysed bladder tumour, all tumour positions showed a high expression of basal markers whereas the included Carcinoma in situ demonstrated a luminal subtype with almost total absence of basal markers. The third analysed mapped bladder tumour demonstrated a homogenous luminal molecular subtype in all precursor and tumorous lesions.

Conclusion: This first analysis of three multiregion mapped bladder tumours shows divergent results of subtype distribution: Heterogeneity of subtypes can be observed, but other tumours and associated precursor lesions show a homogenous distribution of subtypes. To further elucidate how often subtype heterogeneity occurs in whole multiregion mapped bladder tumours, data of further bladder specimens will be presented at the meeting.


Combining hypermethylated RASSF1A detection using droplet digital PCR with miR-371a-3p testing: an improved and highly sensitive panel of liquid biopsy biomarkers for testicular germ cell tumour patients

J. Lobo*, L. MJ van Zogchel, M. G Nuru, A. JM Gillis, E. van der Schoot, G. AM Tytgat, L. HJ Looijenga

*Portuguese Institute of Oncology - Porto, Department of Pathology, Portugal

Background & objectives: Classical serum tumour markers used routinely in the management of testicular germ cell tumour (TGCT) patients (AFP and HCG) are elevated in only 50-60% of the cases at time of diagnosis. miR-371a-3p is the most recent promising biomarker for TGCTs, but is not sufficiently informative for detecting mature teratoma, which is therapeutically relevant. We aimed to test the feasibility to use hypermethylated RASSF1A (RASSF1AM), detected in circulating cell-free DNA, combined with miR-371a-3p, as non-invasive diagnostic markers of TGCTs.

Methods: A total of 109 patient-derived serum samples and 29 sera from healthy young-adult males were included, along with representative cell lines and tumour tissue samples. We describe a novel droplet digital PCR (ddPCR) method for quantitatively assessing RASSF1AM in liquid biopsies.

Results: Promoter methylation of RASSF1A was detected in all TGCT tissues and cell lines by EPIC array, confirmed by ddPCR. Both miR-371a-3p (sensitivity=85.7%) and RASSF1AM (sensitivity=89.8%) outperformed the combination of AFP and HCG (sensitivity=65.5%) for TGCT diagnosis. RASSF1AM detected 88% of teratomas. In this representative cohort, 14 cases were negative for miR-371a-3p, all of which were detected by RASSF1AM, resulting in a combined sensitivity of 100%.

Conclusion: Cell-free detection of RASSF1AM combined with miR-371a-3p resulted in a 100% diagnostic sensitivity for TGCT patients. These results need to be validated in patient follow-up and detection of minimal residual disease.

Funding by: FCT/MCTES, project EpiMarkGermCell (PTDC/MECONC/ 29043/2017). JL is recipient of a fellowship from FCT - Fundação para a Ciência e Tecnologia—(SFRH/BD/132751/2017).


Inflammation burden in benign tissue is inversely associated with significant cancer burden in the prostate: lessons from the PROMIS study

V. Stavrinides*, L. Texeira Mendes, A. Freeman, N. Trahearn, A.E.S. Bosaily, L. Brown, L. Carmona Echeverria, R. Kaplan, C. Parker, S. Punwani, G. Attard, A. De Marzo, H. Whitaker, H. Ahmed, M. Emberton

*Division of Surgery and Interventional Science, University College London, United Kingdom

Background & objectives: Intraprostatic inflammation enables cancer and is an attractive therapeutic target, but its burden in biopsy-naive men is poorly defined. We analysed inflammation in 158 PROMIS participants who had MRI followed by 5-mm template mapping biopsies (TPM) regardless of MRI findings.

Methods: 235 men enrolled at University College London Hospital (2012-2015) and TPM outcomes were collected for 158 (77 pilots excluded). Cancer burden was recorded in 20 Barzell zones per prostate by an experienced uropathologist (Definition 1: Gleason≥4+3 and/or maximum length ≥6mm; Definition 2: 3+4 and/or 4-5mm; insignificant: 3+3, less than 3mm). Inflammation, PIN or ASAP were noted in cancer-free Barzell zones.

Results: There was a negative association between the proportion of benign Barzell zones reported as “heavily inflamed” (out of all cancer-free zones) and TPM cancer burden (1A). The fraction of non-cancerous, inflamed Barzell zones was significantly higher in men without/insignificant cancer, compared to those with Definition 1/2 disease (1B; Kruskal-Wallis, p<0.001; Tukey post hoc analysis; p<0.001). This difference persisted when men without/insignificant cancer were compared to those with significant disease of any definition (1C). The opposite trend was true for PIN/ASAP: there was a positive association between PIN content in benign Barzell zones and cancer burden (2A), although not statistically significant (2B, 2C: Kruskal-Wallis ANOVA, p=0.37; Wilcoxon test, p=0.11).

Conclusion: Heavy inflammation affects fewer benign areas in men with significant cancer compared to those without significant tumours in their prostate. We hypothesise that such inflammatory burden differences (1) enable the progression of significant cancer in the first place (2) are a consequence of tumour-related microenvironmental changes or (3) are driven by increased inflammation in the early stages of carcinogenesis. Whether these hypotheses are true requires further investigation.

Funding by: MRC Clinical Research Training Fellowship MR/S005897/1

OFP-02 Joint Oral Free Paper Session: Breast Pathology / Paediatric and Perinatal Pathology


Congenital vascular anomaly in alveolar capillary dysplasia with misalignment of pulmonary veins

L. Wang*, M. Alturkustani, Y. Lou, D. Li, J. Byers, L. Szymanski, D. Parham, W. Shi

*Children’s Hospital of Los Angeles, University of Southern California, USA

Background & objectives: Alveolar capillary dysplasia with “misalignment of pulmonary veins” (ACDMPV) is a rare, fatal abnormal pulmonary vascular developmental disease. The pathogenic mechanisms remain largely unknown. This study aims to characterize the microvascular alterations in the lungs of ACDMPV patients.

Methods: Lung histologic structures were analysed from 16 autopsies with ACDMPV. Lung endothelial cells, smooth muscle cells, and alveolar epithelial cells were examined by immunohistochemical and immunofluorescent stainings with antibodies recognizing different cellular markers for ACDMPV and age-matched controls. Statistical analysis was performed.

Results: The decreased capillary density or completely absence of capillary in the alveolar septa was observed while new microvascular formation with abnormal tortuosity in the thickened alveolar septa was present. Aberrant pulmonary veins were detected adjacent to small pulmonary arteries within the same bronchovascular bundle even though septal pulmonary veins were still seen in the ACDMPV lungs. Glut-1 protein, a marker for endothelial immaturity, was detected in the malpositioned and normalized pulmonary veins as well as abnormal septal neovasculature. In addition, hyperplasia of type 2 alveolar epithelial cells was observed.

Conclusion: The loss of vascular integrity and the destruction of the alveolar-capillary interface may drive abnormal growth and remodelling of the pulmonary microvasculature, which challenges the current concept of “misalignment of pulmonary veins”. Future studies are needed to understand the pathogenesis.


Clinical exome sequencing for the diagnosis of foetal skeletal dysplasias

A. Nadal*, M. Pauta, B. Campos, M. Segura, G. Arca, A. Borrell

*Pathology, Hospital Clínic, UB, IDIBAPS, Barcelona, Spain

Background & objectives: Skeletal dysplasias require genetics for definite diagnosis. Single gene sequencing (SGS), next generation sequencing (NGS) methods such as gene panel sequencing (GPS), clinical exome sequencing (CES, including all genes listed in OMIM) or whole exome sequencing are alternative approaches.

Methods: Nineteen foetuses from pregnancies interrupted because of skeletal dysplasia were genetically investigated either by SGS (eight) or NGS-based GPS (six) or CES (nine –three previously SGS and one GPS analysed-), and full autopsy including radiology.

Results: Pathogenic gene variants were found in 3/8 SGSs and in 10/12 NGS-based analysis. In six cases genetics confirmed the pathology diagnosis (thanatophoric dysplasia -twice-, spondylo-epiphyseal dysplasia (SEDC), osteogenesis imperfecta (OI), and Desbuquois syndrome), that could raise to seven if two negative SGS are included. Genetics provided previously unsuspected diagnosis in eight cases: de novo achondroplasia, OI, short-rib polydactyly type 3 (SRP3), anauxetic dysplasia, and LADD, Roberts, Steel, and Schwartz-Jampel syndromes. Pathology confirmed variants of unknown significance as actual pathogenic variants in seven cases (Steel, SEDC, Schwartz-Jampel, SRP3, Roberts, OI, and Desbuquois), all among CES-analysed cases.

Conclusion: CES identifies a wide range of skeletal dysplasias and previously unreported genetic variants carrying pathogenetic significance. CES methods would likely identify variants identified in SGS whereas the contrary is unlikely. Multidisciplinary approach results in the highest diagnostic yield and precision.

Funding by: Grant from Fondo de Investigaciones Sanitarias PI17/01153, Instituto de Salud Carlos III, and Fondo Europeo de Desarrollo Regional (FEDER), Ministerio de Economia y Competitividad, Spain


CD34 immunostain increases sensitivity and/or upgrades the diagnosis of foetal vascular malperfusion in placentas from ex-utero intrapartum treatment

J. Stanek*

*Cincinnati Children's Hospital, Division of Pathology, USA

Background & objectives: Previously we found an increase incidence of foetal vascular malperfusion (FVM) in placentas from EXIT (ex-utero intrapartum treatment) procedures. This retrospective analysis analyses the impact the recently introduced CD34 immunostain for the FVM diagnosis in placentas from EXIT procedures.

Methods: 105 placentas from EXIT procedures were studied. In 73 cases, the placental histological diagnosis of segmental FVM was made on H&E stained placental sections only (Group 1). In 32 cases, the CD34 component of a double E-cadherin/CD34 immunostain slides was also used to detect the segmental endothelial fragmentation (Group 2). 23 clinical and 47 independent placental phenotypes were statistically compared.

Results: The indications for EXIT procedures were: EXIT 48 to airway, 43 EXIT to ECMO and 14 EXIT to resection). Congenital diaphragmatic hernias were statistically significantly more common in Group 2 (34% vs 56%, p=0.03). There was no statistical significance between the groups in rates of segmental villous avascularity (29% vs 34%), but CD34 immunostain added and/or upgraded 12 more cases in Group 2, thus increasing the sensitivity of placental examination for FVM by 37%. There were no other statistically significantly differences in clinical and placental phenotypes, proving the otherwise comparability of the groups.

Conclusion: The use of CD34 immunostain increases the sensitivity of placental examination for FVM which thus may improve the neonatal management by revealing the increased likelihood of the potentially life-threatening neonatal complications.


A report of a very rare case of 47XYY disorder of sexual development presenting with an ambiguous genitalia and mixed gonadal dysgenesis with persistent Mullerian structures

K. Abrham*

*St. Paul's Hospital MMC, Ethiopia

Background & objectives: Introduction – A report of a very rare case of 47XYY DSD presenting with an ambiguous genitalia and mixed gonadal dysgenesis with persistent Mullerian structures.

Case presentation – An 11 month old infant presented with a chief complaint of ambiguous genitalia. Physical examination of the external genitalia revealed poorly developed bifid scrotum with bilaterally non palpable testes, phallus length of 4cm with distal hypospadia and chordae. Other systemic examination was unremarkable. Hormonal analysis showed Testosterone 2.15 ng/ml and Dihydrotestestrone <6 pg/ml.cont...

Methods: cont... . Serum electrolytes were within the normal limits. Abdomino-pelvic ultrasound reported non visualized testicles. Karyotype was determined from a cheek cell with a report of 47XYY. Surgical exploration done for DSD showed Uterus with cervix, Bilateral fallopian tubes with fimbrated ends and Left side ovary like structure. No right side ovary , testicular structure or cord was seen.

Results: Histopathology showed Fallopian Tube histology and cryptorchid testis

Conclusion - 47,XYY syndrome occurs in about 1 in 1,000 new-born boys. Most males with 47,XYY syndrome have normal production of the male sex hormone testosterone and normal sexual development. Although many males with this condition are taller than average, the chromosomal change sometimes causes no unusual physical features. Mixed gonadal dysgenesis (MGD) is one of the most frequent causes of male sexual ambiguity.

Conclusion: The most common karyotype is 45,X/46,XY mosaicism

In our case the association of 47XYY DSD with ambiguous genitalia and mixed gonadal dysgenesis with persistent Mullerian structures is a rare finding, and to our knowledge, very few are reported so far


Paediatric non-Hodgkin lymphomas: a 10-year study from a terciary centre in Coimbra

C. Faria*, M.J. Julião, J. Fraga, A. Lai, M.B. Pimentão, R. Almeida, J. Madeira, J. Gama, M.J. Brito, R. Pina

*Centro Hospitalar e Universitário de Coimbra, Portugal

Background & objectives: Non-Hodgkin lymphoma is a common malignancy in children and adolescents in Europe. Burkitt lymphoma remains the most frequent followed by lymphoblastic lymphoma and diffuse large B-cell lymphoma. The outcome has improved, and prognosis is in general good at paediatric age.

Methods: Retrospective study of children (age<18 years old) with histopathologically diagnosis of Non-Hodgkin lymphoma from 2009 to 2019. We enrolled 43 patients in total, predominantly male (72.1%), being the youngest case with 3 years old and the oldest one with 17 years old.

Results: Diffuse large B-cell lymphoma (DLBCL) represented 25.6% of cases, Burkitt lymphoma 23.3%, lymphoblastic T-cell lymphoma 16.3%, anaplastic large cell lymphoma 16.3%, lymphoblastic B-cell lymphoma 6.9%, follicular lymphoma 4.7%, primary cutaneous anaplastic lymphoma 2.3%, plasmablastic lymphoma 2.3% and hepatoesplenic T-cell lymphoma 2.3%. Three of DLBCL were post-transplant lymphoproliferative disorders (PTLDs).

Five patients died (11.6%), two were PTLDs after cardiac transplant, one PTLD after bone marrow transplant and the others had primary cutaneous anaplastic lymphoma (death by suicide, 3 years off treatment) and hepatoesplenic T-cell lymphoma with advanced disease at the time of the diagnosis, infection and progression of the disease. B symptoms were also a factor for worse prognosis.

Conclusion: Non-Hodgkin lymphoma remains an aggressive neoplasia; however effective treatment has improved the overall survival and 5-year disease free rates. In our series, we also conclude that specific types of lymphoma have a worse outcome, as well as association with other comorbidities.


Frequency distribution of breast cancer biomarkers from a large volume laboratory – a single institution experience

J. Carter, A. Plagge, K. Del Rosario, K. Geiersbach, T. Mounajjed, B. Chen*

*Mayo Clinic, USA

Background & objectives: Oestrogen receptor (ER), progesterone receptor (PR) and HER2 are established clinical biomarkers in breast carcinoma (BC). We evaluated trends in biomarker data (2007-2019), including newly implemented decile scoring in ER/PR, and compared internal cases (IC) and external cases (EC).

Methods: ER/PR (2011-2019) and HER2 (2007-2019) data on invasive breast carcinoma were analysed. Per ASCO/CAP guidelines, ER (clone SP1) and PR (clone 1E2) IHC were scored as <1%, 1-10%, and > 10% (or deciles of 10-100% from 2017). HER2 IHC/digital image analysis (Ventana Pathway HER2 (4B5) with Aperio software was scored 0 - 3+; 2+ cases were reflexed to FISH.

Results: Among 19,193 BC, (IC=6833, EC=12,360), year-to-year ER positive rates (>1%) ranged narrowly (79-81%) with PR ranging from 70-79%.By decile scoring (N=2300), ER was: score<1% (range 18-21%), score 1-10% (2.7-3.2%), score>90% (67-71%), others ≤3%. PR: score <1% (28-30%); score1-10% (7-10%); score>90% (27-31%), others ≤10%. Year-to-year HER2 IHC (total N=40,137) ranged: 0/1+ (67-80%); 2+(12-24%), and 3+(7.4-10.2%). The HER2 2+/FISH+ rate fluctuated between 13% and 28%. IC and EC results were similar.

Conclusion: Over the past decade, ER/PR IHC positive rates remained stable. The bimodal distribution of ER decile scores supports previous studies that most breast cancers are either ER negative or diffusely positive whereas PR scores vary more widely. Variations in the HER2 IHC2+ rate reflected the equivocal nature of this category, but interestingly also coincided with changes in the ASCO/CAP HER2 FISH guidelines. Frequency distributions for ER, PR and HER2 scores did not differ between internal cases and external cases.


The use of PAM50 (Prosigna®) as molecular classifier in breast cancer - the experience of 106 consecutive cases analysed at the Royal Surrey Hospital (RSH), Guildford

C. Spencer*, S. Di Palma, B. Ping, N. Collins, T. Irvine

*Department of Histopathology, Royal Surrey Hospital, United Kingdom

Background & objectives: Hormone receptor status in breast cancer confers vital prognostic and therapeutic implications. It is currently defined using immunohistochemistry, a subjective test which may not represent true genotype. We explore discrepancies between immunohistochemical and molecular profiles using the molecular classifier Prosigna.

Methods: Since 2019 RSH has used Prosigna clinically to evaluate 106 consecutive cases of ER/PR positive, Her2 negative early breast cancer predicting risk of recurrence (ROR) and likely benefit of chemotherapy. Analysis of the Prosigna genetic signature also defines the “intrinsic” molecular subtype; luminal A, luminal B, Her2-enriched and basal-like, which we compared with immunohistochemical hormone receptor status.

Results: Of 106 cases, 101 cases had strong positive immunohistochemistry for ER (Allred score 7-8/8). PAM50 confirmed the ER positive genetic profile in all cases by categorising 66 as luminal A and 35 as luminal B. No cases were classified as Her2-enriched.

There were 5 cases with “weak” positive ER immunohistochemistry (Allred score 3-6/8). Prosigna defined these as a “basal-like” molecular profile.

Conclusion: Since eligibility for case selection was immunohistochemical ER positivity, all cases were expected to be categorised Luminal A or Luminal B as per molecular intrinsic subtype. However, Prosigna defined 5% of our cases as “basal-like”. Further investigation of the “basal like” cases demonstrated that they had weak ER positivity by immunohistochemistry. This corroborates recent ASCO/CAP guidelines proposing a new reporting category “ER Low Positive” which have a gene expression profile more similar to ER-negative cancer.

Funding by: BDIAP Fellowship


Multiplex immunohistochemistry/immunofluorescence (mIHC/if) for PD-L1 testing in triple negative breast cancer: validation of a translational assay with conventional IHC

J. Yeong*, T. Tan, H. Li, J. Lim, J. Lim, A.A. Thike, J. Iqbal, R. Dent, P.H. Tan

*Singapore General Hospital, Singapore

Background & objectives: There are several immunohistochemical (IHC) assays approved as companion diagnostics to select for treatment with specific programmed death (PD)-1/PD-ligand-1 (L1) inhibitors. There is no uniformity in assessing PD-L1 across trials in triple negative breast cancer (TNBC).

Methods: Recently, we reported a comparison study (JCP 2020) demonstrating that mIHC/IF represents a promising tool in the era of cancer immunotherapy, as it can simultaneously detect and quantify PD-L1 labelling with multiple antibody clones, allowing accurate evaluation of tumour and immune cells. Hereby we conducted a validation study with bigger cohort, aiming to translate to routine diagnostic setting.

Results: Our cohort comprised of 37 TNBC cases, 20 non-small cell lung carcinomas, 9 colorectal and 29 other cancers. Three in-vitro diagnostic PD-L1 antibody clones (22C3, SP142 and SP263) were used to perform multiplex immunohistochemistry/immunofluorescent (mIHC/IF) staining. To detect tumour, EpCAM and cytokeratin staining were applied. This was followed by digital pathological evaluation to report the combined positive score (CPS), tumour proportion score (TPS) and immune count (IC) on a single tissue section. We compared the automated scoring results to standard, manual PD-L1 scoring by pathologists using conventional IHC staining.

Conclusion: Moderate-to-strong correlations in PD-L1 positivity were found between results obtained through mIHC/IF and IHC. Individual concordance rates in the study ranged from 69-98%, with Spearman’s rank correlation coefficient values up to 0.91. Further analysis within the context of therapeutic trials is required to develop PD-L1 testing as a complementary diagnostic platform in TNBC and other cancers.


Assessment of the usefulness of selected methylation features in different tissues of breast cancer patients for preoperative risk stratification

K. Kankava*, E. Kvaratskhelia, G. Burkadze, E. Abzianidze

*Tbilisi State Medical University, Georgia

Background & objectives: Epigenetic changes have long been believed to reflect tumour biology. We tested some of them in different tissues of breast cancer patients to analyse epigenetic background and the value of these parameters in predicting clinicopathological characteristics of tumours.

Methods: 100 Patients with breast cancer were included in the study along with age-matched controls and patients with benign breast lesions. Methylation of global DNA, repetitive elements (LINE-1 and Alu) and BRCA1 gene were studied in blood, tumour tissue and normal breast ductal epithelial cells. These parameters were analysed in comparison to clinical, histologic and phenotypic characteristics of tumours.

Results: Alu, LINE-1 and global DNA methylation showed markedly low levels in all tissues of cancer patients compared to healthy controls, with extreme hypomethylation in tumour tissue. Changes in these parameters were not always concordant. BRCA1 promoter methylation was detected exclusively in tumour tissues and in only two cases, both of which had aggressive phenotype. Methylation levels detected in blood didn't allow stratification of patients with benign and malignant breast lesions and showed rather weak relationship with clinicopathological characteristics of tumours.

Conclusion: Epigenetics signatures show distinct pattern in tumour tissue and are not homogenous in different tissues of the same patient. Given the uniform picture of DNA methylation in blood in some of investigated groups, additional data and very careful approach are required when considering using it as a diagnostic or predictive tool.

Funding: The research was supported by Shota Rustaveli National Science Foundation Of Georgia (SRNSFG) [Grant Number - PhDF2016_85].


Comparison of different pathological complete remissions in neoadjuvant breast cancer based on different assessment systems

Y. Liu*, Y. Ma

*The Fourth Hospital of Hebei Medical University, China

Background & objectives: Differences in pathological complete remission (pCR) achieved after neoadjuvant therapy (NAT) of breast cancer patients based on Miller–Payne (MP) classification, residual cancer burden (RCB), and Pinder assessment system.

Methods: 276 patients with breast cancer who underwent neoadjuvant therapy after surgical diagnosis of preoperative puncture pathology from January 2014 to January 2015 in the Fourth Hospital of Hebei Medical University were retrospectively studied. Chi-square test was used to analyse the relationship. ROC curves to analyse respective diagnostic efficacy. COX regression model was used to analyse the significance in prognostic evaluation.

Results: In this study, we found that tumour size, ER expression state, PR expression state, Ki67 index, and molecular classification of HER2 overexpression type and three negative type breast cancer were statistically significant in terms of achieving pathological complete response after neoadjuvant chemotherapy (P<0.05); Chi-square test showed that there was no statistically significant difference in pCR rate (P>0.05); ROC curve analysis shows that RCB has higher diagnostic efficiency than Pinder; COX survival analysis shows that the RCB index classification is more instructive for the prognosis of breast cancer patients after neoadjuvant treatment.

Conclusion: Evaluation of pathological response after neoadjuvant, the RCB index evaluation system has better performance than MP and Pinder evaluation system.


AI-assisted interpretation of Ki-67 standard comparison cards for different types of breast cancer

Y. Liu*, l. Li

*The Fourth Hospital of Hebei Medical University, China

Background & objectives: Compare the difference between the reference breast cancer standard comparison card and conventional microscope interpretation and explore the clinical applicability of breast cancer standard comparison card.

Methods: 311 breast cancer surgical resection specimens from our Hospital were selected, Ki-67 immunohistochemical staining was performed by 3 senior pathologists with conventional interpretation and comparative artificial standards. Comparison card interpretation method, by selecting two regional interpretation methods, high value-added area and average value. Intraclass correlation coefficients were used to assess reproducibility between observers.

Results: The reference standard comparison card showed excellent consistency between Ki-67 values in different types of breast cancer by selecting the average value (a high value-added area and a low value-added area, AVE) (ICC: 0.797, CI 0.753-0.834).

Conclusion: The reference standard comparison card interpretation is expected to become a common method for breast cancer Ki-67 interpretation.


ZNF703 copy number and protein expression in breast cancer

E. Klæstad*, M. Valla, A.M. Bofin

*Department of Clinical and Molecular Medicine, Faculty of Medicine and Health Sciences, Norwegian University of Science and Technology, Norway

Background & objectives: ZNF703 is associated with proliferation and is thought to be most common in luminal breast cancer. We aimed to study ZNF703 copy number and protein expression in breast cancer and elucidate associations with proliferation, molecular subtype and prognosis.

Methods: Using fluorescence in situ hybridization (n=702) and immunohistochemistry (IHC) (n=829), we examined ZNF703 copy number and protein expression in primary breast cancers. We assessed associations between ZNF703 copy number and proliferation, molecular subtype and prognosis, and between protein expression and molecular subtype. In the prognostic analyses, we estimated cumulative incidence of breast cancer death and hazard ratios.

Results: We found that 7% of primary tumours had increased copy number (mean copy number ≥6), and 76% of primary tumours were IHC positive (≥50% tumour cell nuclei positive). The highest proportion of tumours with mean copy number ≥6 was found in the Luminal A and Luminal B(HER2-) subtypes. Luminal subtypes also had the highest proportion of IHC+ cases. We found a positive association between ZNF703 copy number increase and proliferation. Patients with ZNF703copy number ≥6 had poorer prognosis compared to cases without copy number increase (HR 1.6 (95% CI 1.1 -2.5)).

Conclusion: ZNF703 copy number increase is associated with the Luminal A and Luminal B (HER2-) subtypes. Protein expression is associated with all luminal subtypes. There is an association between ZNF703 copy number increase and proliferation, and a poorer prognosis.


The association of clinicopathological parameters and the extent of neuroendocrine differentiation with microRNA 21 and microRNA let7f expression in primary invasive breast carcinomas with neuroendocrine features

G. Usul, C. Kelten Talu, I. Yilmaz, T.C. Savli*, S. Bektas, D. Can Trablus

*Health Science University, Istanbul Training and Research Hospital, Department of Pathology, Turkey

Background & objectives: To compare primary Breast Carcinoma with Neuroendocrine Features (BCNF) cases with Invasive Ductal Carcinoma (IDC-NOS) cases without neuroendocrine differentiation matched for molecular subtype and stage as regards the clinicopathological features and the expression levels of two miRNAs.

Methods: The slides and paraffin blocks of the 29 cases with BCNF diagnosis and the 29 cases with IDC-NOS diagnosis as reported in our clinic between 2012 and 2017 were obtained from the archives and retrospectively re-evaluated. The miR-21 and miR-let7f expression levels were detected with the qRT-PCR method in paraffin blocks containing invasive tumour and normal tissue in each case.

Results: The mean age in the groups with neuroendocrine differentiation was almost 10 years higher than in the IDC-NOS group. The Ki-67 proliferation index level, nuclear grade and histological grade were higher in the group with 10% to 50% neuroendocrine differentiation than in the group with ≥ 50% neuroendocrine differentiation.

Comparison of the groups for miR-21 expression revealed that the increased expression in the IDC-NOS group was more prominent than in the other two groups with neuroendocrine differentiation. The decrease in the miR-let7f expression level in the group with 10% to 50% neuroendocrine differentiation was less than seen in the IDC-NOS group.

Conclusion: BCNF is seen at more advanced ages than IDC-NOC cases. Cases with widespread neuroendocrine differentiation within tumour tissue are associated with a lower nuclear/histological grade and Ki-67 levels. Decreased miR-let7f expression is more prominent in BCNFs while increased expression of miR-21 is more prominent in IDC-NOS cases without neuroendocrine differentiation. Our results let us to consider various treatment options (miRNA based therapy) for BCNF.


Germline mutations and pathological features of breast and female genital cancers in increased-risk patients of Asian origin

M. Zaakouk*, A. Longworth, J. Hoffman, A. Sharma-Oates, C. Huber, K. Ong, R. Hart, Y. Wallis, A. Considine, P. Dasani, R. Ganesan, I. Tomlinson, S. Sundar, A. Shaaban

*Institute of Cancer and Genomic Sciences, University of Birmingham, University Hospitals Birmingham NHS Foundation Trust Queen Elizabeth Hospital Birmingham, United Kingdom

Background & objectives: Patients of Asian ancestry comprise 10.8% of UK population in West Midlands. Breast and ovarian cancers in non-Caucasian women tend to present at earlier age with more aggressive disease. We aim to analyse patient genetics and pathologic features of those cancers.

Methods: We identified 164 increased-risk patients of Asian ancestry, out of 5618 patients genetically tested between 2006-2018 at a large reference genetics centre. All patients were screened for mutations of BRCA1/2, and a subset were also tested for (PALB2, BRIP1, RAD51D & others) as per the clinical guidelines. Histopathological and clinical data were collected and correlated with the genetic results.

Results: 51 mutations in 46 patients (median age 38) were identified of which 20/26 were BRCA1-pathogenic and 13/22 were BRCA2-VUS. Two rare pathogenic-BRCA1 variants were found repeated. BRIP1 & PALB2 mutations were pathogenic, and RAD51D was VUS. The 46 patients presented mostly by breast cancers. Breast cancers were predominantly grade3(75%), >15mm in 57.9%(11/19) and with 43% lymph node positivity and high proportion of the triple-negative phenotype (TNBC)(36.4%).

The 118/164 patients (negative for germline mutations) presented at median age of 40 years. The breast cancers were mostly grade3(52.7%), of invasive size (>15mm) in 64.6%(31/48) and node negative(55.3%;26/47). There was a higher proportion of the luminal phenotype(63.8%) and lower proportion of the TNBC(25.5%).

Conclusion: Increased-risk Asian patients presented with high grade breast cancers, at young age with larger proportions of the unfavourable triple negative molecular subtype (TNBC). Patients with pathogenic mutations had a higher proportion of TNBC. Two cases were negative for BRCA1/2 mutations but later tested positive for BRIP1 (pathogenic) and RAD51D (VUS). Extended panel testing is warranted and informs genetic counselling and optimal management in this patient group.

OFP-03 Gynaecological Pathology


Multiplexed immunohistochemistry for immune cell phaenotyping, quantification and spatial localisation in a rare tumour, clear cell ovarian cancer

C. Genestie*, C. Klein, L. Laot, A.S. Lemaire, E. Vaz, S. Gouy, A. LeFormal, A. Maulard, P. Morice, P. Pautier, M. Kfoury, E. Colomba, E. Yanis-Galende, A. Leary

*Gustave Roussy, France

Background & objectives: Little is known about the unique immuno-phenotypical features of clear cell ovarian cancer (CCOC).We conducted multiplexed IF/IHC to 1) describe the tumour microenvironment (TME) of CCOC and 2) compare CCOC to high grade serous OC (HGSOC).

Methods: Tissue micro-arrays with triplicate tumour samples from 29 CCOC and 30 HGSOC were stained using multiplex IF/IHC panels including: CD3, CD4, CD8, FOXP3, Granzyme B (GrzB), CD68, CD163 and CK. IC topography was described as intraepithelial if <20uM from tumour cells (TC), or stromal (>20uM from TC). Image analysis (Visiopharm) performed to quantify IC in number/mm2 or % surface positive

Results: There were significantly fewer CD8+ IC in CCOC vs HGSOC (median 15 vs. 48 cells/mm2; p=0.01) and 34% (10/29) of CCOC were “immune-excluded” with CD8+ IC present only in the stromal compartment. However, despite lower CD8+ IC, CCOC showed significantly greater GrzB expression (marker of effector T cell cytotoxicity) (p=0.0007), and significantly reduced FOXP3+ T cells (p=0.03) resulting in favourable effector/suppressor ratio compared to HGSOC (median GrzB+/FoxP3+: 10.7 vs 0.04; P<0.0001). CCOC also showed significantly higher levels of CD68+ macrophages (M1) (<0.0001), resulting in a significantly more favourable M1/M2 ratio than HGSOC (median CD68+/CD163+: 3.6 vs. 0.8; p=0.0001).

Conclusion: One third of CCOC are “immune excluded”. However, when present, the effector to suppressor balance of both T cells (GrzB+/FoxP3+) and macrophages (CD68+/CD163+) is in favour of anti-tumour immune response.


Micropapillary variant of serous borderline tumour (MSBT) with BRAF mutations, a molecularly distinctive and less aggressive precursor of low-grade serous carcinoma (LGSC)

M. Martini*, M. Bilotta, A. Santoro, M. Dall'Acquila, S. Capodimonti, T. Cenci, G.F. Zannoni, L.M. Larocca

*Università Cattolica del Sacro Cuore, Rome, Italy

Background & objectives: Despite the MSBT has been recently identified as a “synonymous” of non-invasive LGSC (niLGSC; WHO, 2014), on the base of the clinical-prognostic reports, few molecular studies are carried out on this pathological entity.

Methods: KRAS, NRAS and BRAF mutational analysis and HER2 and p53 expression were assessed on 50 LGSCs, 42 MSBTs, 50 Serous Borderline Ovarian Tumours (SBT). On these groups, we performed the Human Cancer Pathway- and miRNA- Finder PCR array (Qiagen). Altered miRNAs and pathways were confirmed by real-time PCR or immunohistochemistry. Results were correlated with the clinical and pathological features.

Results: We found a higher rate of BRAF mutation in SBT (65%) than in MSBT (30%) and LGSC (5%). Conversely, RAS mutations rate significantly increased from SBT (6%) to MSBT (25%) to LGSC (40%). We did not find HER2 and p53 alterations. MSBTs with BRAF mutations had a miRNAs and cancer pathway alterations significantly similar to those SBT cases with BRAF mutations, but different to those MSBT group with RAS mutations. Contrariwise, MSBT with RAS mutations had a miRNAs and cancer pathway alterations significantly similar to those RAS mutated LGSC cases. Finally, MSBT with RAS mutations are significantly associated to a worse PFS respect to MSBT cases with BRAF mutations (p=0.003).

Conclusion: Our data suggested the idea that MSBTs is a heterogeneous group of patients that when associated to RAS mutations represent a more aggressive malignant precursor of the LGSC. Conversely, MSBTs with BRAF mutations are molecularly and clinically more similar to SBTs.


How much tumour do we need to submit during grossing to correctly evaluate Silva pattern in endocervical adenocarcinomas?

M. Souto Moura*, R. Vieira, H. Pereira, L. Moço, S. Salta, C. Bartosch

*Department of Pathology, IPO-Porto, Portugal

Background & objectives: Endocervical adenocarcinoma(EA) Silva pattern stratifies tumours based on morphology and correlates with clinical outcome. It’s unknown if entire tumour submission is necessary to assign the appropriate Silva pattern. Our aim is to clarify EAs gross submission to correctly diagnose Silva pattern.

Methods: We retrospectively reviewed a cohort of 37 patients that underwent surgery and were diagnosed with HPV-related EAs at IPOP between 2005 and 2019. Macroscopic information and histological slides were re-evaluated, particularly regarding tumour size and submitted sections. Silva pattern was evaluated in each tumour section. Probability models were created using random sampling without replacement.

Results: Patients underwent hysterectomy(n=29) or conization only(n=8). Mean tumour size was 17mm (range:2,55mm). Twenty-one tumours were macroscopic. Twenty-seven tumours were totally submitted (mean size=12mm) and 10 were partially submitted(mean size=29mm). Mean tumour sections was 6 (range:1,13). Tumour’s final Silva pattern included: 7 pattern-A, 7 pattern-B, 23 pattern-C. Intratumoral comparison of Silva pattern between sections showed 15 homogeneous(8-pattern C; 7-pattern A) and 22 heterogeneous tumours. Larger tumour size is associated with pattern C(p=0.006). In large tumours(~2cm) this pattern composes in median 67% of sections. Probability models showed that submission of at least half the tumour identifies the correct Silva pattern in 88% of all cases, and in 81% of heterogenous larger tumours.

Conclusion: Our findings support the current practice of partially including large tumours, since they are most frequently extensively pattern C. Total inclusion is recommend for small tumours and for larger tumours that are heterogeneous.


Integrated histopathologic and molecular classification of endometrial carcinoma: identification of prognostic subgroups

A. De Leo*, D. de Biase, G. Dondi, P. De Iaco, A.M. Perrone, D. Santini, G. Tallini, C. Ceccarelli

*University of Bologna, Italy

Background & objectives: The TCGA project discovered four distinct prognostic endometrial carcinoma (EC) classes: POLE-mutant, mismatch repair deficient-MMRd, p53-mutant and no specific molecular profile-NSMP groups. Our aims were to integrate the histological and molecular classifications, and to identify markers relevant for risk stratification.

Methods: Comprehensive clinicopathologic and follow-up data including immunohistochemical (IHC) analysis and/or Next-Generation Sequencing (NGS) were analysed in a retrospective cohort of 110 ECs. IHC and/or NGS were used to assign TCGA groups and to investigate molecular alterations of multiple target genes including POLE, PTEN, ARID1A, ß-catenin, TP53, MLH1, PMS2, MSH2, MSH6 and L1CAM.

Results: TCGA class assignment of EC cohort: 8 (7.3%) POLE, 34 (30.9%) MMRd, 23 (20.9%) p53abn and 45 (40.9%) NSMP. Molecular subgroups were significantly associated with different grade, tumour-infiltrating lymphocites (TILs), mitoses, Ki-67 index, pattern of invasion, L1CAM expression. NSMP class was further subdivided based on ARID1A and on ß-catenin alterations. ARID1A loss was associated with higher grade, increase of TILs, necrosis, higher mitotic and Ki-67 index, specific patterns of invasion. Ten of 110 (9.1%) ECs recurred: 5 NSMP with ARID1A mutation, 2 MMRd, 3 p53abn. NSMP cases with ARID1A mutation showed the worst outcome with early recurrence (log-rank p<0.01).

Conclusion: An integrated EC classification may better define risk assessment, representing a step towards precision medicine. The analysis of ß-catenin and ARID1A alterations in NSMP class identified subsets of ECs statistically correlated with specific histopathological parameters and different disease recurrence.


Correlation of MMR protein deficiency with histopathologic parameters in endometrial carcinoma

T. Soylemez*, G. Kir, R.B. Girgin, Z.C. Olgun, A. Aydin, S.R. Dur, M.I. Tosun, H. Ankarali, A. Karateke

*Istanbul Medeniyet University / Pathology Department, Turkey

Background & objectives: The aim of the study was to analyse the correlation between DNA mismatch repair (MMR) proteins status and clinicopathological characteristics of endometrial cancers (ECs).

Methods: The relationship between clinicopathologic parameters and MMR proteins status was investigated in 198 consecutive patients who underwent surgery for ECs.

75 out of 198 (33%) cases showed immunohistochemical alteration of MMR proteins: 65=(32.8%) cases lost MLH1&PMS2, 10(5.1%)cases lost other proteins(8MSH2 & MSH6, 1MSH6, 1PMS2).On univariate analysis, we more frequently observed high grade endometrioid carcinoma in cases with MLH1&PMS2 losses, low grade endometrioid carcinoma in cases with other losses (p=0.021).

Results: Larger tumour size and increased tumour infiltrating lymphocytes(TILs) were more frequently observed in patients with MLH1 & PMS2 losses than other losses (p=0.004, p=0.009, respectively). On multivariate binary logistic regression analysis, loss of MLH1 & PMS2 revealed statistically marginal significance for larger tumour size than MMR protein intact and other losses (odds ratio [OR]=3,052, 95% confidence interval [CI]=0.817–11.396,p=0.097,OR=0,135, 95% CI=0.014–1.263,p= 0.079, respectively).TILs was independently correlated with MMR protein loss, there was a marginal significance between infiltrative glands type myoinvasion and MMR protein loss(OR=3.104, 95% CI=1.431–6.736, p=0.011,OR=0.307, 95%CI=0.116-0.808, p=0.064, respectively).

Conclusion: The presence of infiltrative glands type myoinvasion showed predisposition to be associated with MMR protein loss. Cases with MLH1&PMS2 losses tend to be larger and higher-grade tumours than cases with MMR intact status and other losses. These results have to be proven in larger cohorts.


Correlation of PD-L1 expression with MMR protein subtype deficiency in endometrial carcinomas

G. Kir*, Z.C. Olgun, T. Soylemez

*Istanbul Medeniyet University / Pathology Department, Turkey

Background & objectives: Programmed death-ligand 1 (PD-L1) was studied in several cancer types, however there is stil limited data about endometrial cancer (EC). The purpose of this study was to evaluate PD-L1 expression of tumour cells(TC) and immune cells(IC) at different cut-off values.

Methods: By using immunohistochemical staining, in 176 resection specimens of EC patients, PD-L1 results were correlated with mismatch repair (MMR) protein status and several prognostic parameters including myometrial invasion patterns.

Results: On both univariate and multivariate analysis, TC and IC PD-L1 positivity with a 5% cut-off were significantly associated with MMR protein loss(univariate: p=0.015, and p=0.001; multivariate: p=0.027, CI=0.003-0.583, and p=0.002, CI=1.498-5.557 respectively). MLH1&PMS2 loss subgroup had higher rates of positivity for IC PD-L1 expression, however other MMR loss subgroup(MSH2 and/or MSH6,PMS2) tended to have higher rates of positivity for TC PD-L1 expression. MELF type myoinvasion were marginally significant for TC PD-L1 expression at 5% cut-off (univariate:p=0.089; multivariate: p=0.062, CI:0.022-10.101).

Conclusion: This study demonstrated that tumoral PD-L1 expression was more common in patients with other MMR loss subgroup, IC PD-L1 expression was more common in patients with MLH1&PMS2 loss subgroup. Also, the presence of MELF type myoinvasion showed predisposition to be associated with TC PD-L1 positivity. These results have to be proven in larger cohorts.

This research was funded by Istanbul Medeniyet University Scientific Research Project Fund.


Increased FOXJ1 protein expression is associated with improved overall survival in high-grade serous ovarian carcinoma: an ovarian tumour tissue analysis consortium study

E.Y. Kang*, A.L. Weir, N.S. Meagher, P.D. Pharoah, S.J. Ramus, M. Kӧbel

*University of Calgary, Alberta, Canada

Background & objectives: NanoString mRNA analyses have identified FOXJ1 (ciliated cell transcription factor) and GMNN (DNA replication inhibitor) as prognostic markers in tubo-ovarian high-grade serous carcinoma (HGSC). We aimed to evaluate FOXJ1 and GMNN expression by immunohistochemistry and survival associations within ovarian carcinomas.

Methods: Ovarian Tumour Tissue Analysis Consortium tissue microarrays containing high-grade serous, low-grade serous, mucinous, endometrioid, and clear cell carcinomas were stained for FOXJ1 (N=5,544) and GMNN (N=3,787). Nuclear staining was scored in 5%-increments then categorized (FOXJ1: 0%, 5%, 10-15%, 20-45%, 50-100%; GMNN: 0%, 5%, 10-15%, 20-25%, 30%, 35-100%). The log-rank test and Cox proportional hazards regression compared expression levels and survival.

Results: FOXJ1 and GMNN expression was heterogeneous across histotypes. Increased FOXJ1 expression was associated with improved overall 5-year survival in HGSC: 47.1% in patients with high (50-100%) FOXJ1 expression versus 34.7% in those with absent FOXJ1 expression (HR= 0.78, 95%CI 0.66-0.93, p<0.0001). However, GMNN expression was not significantly associated with survival. No significant association with overall survival was observed in any other histotype, for both markers.

Conclusion: FOXJ1, but not GMNN, can be translated into an immunohistochemical biomarker to stratify prognosis in HGSC.


Technical and interpretation performance characteristics of P16 immunohistochemistry: a joint project by the British Association of Gynaecological Pathologists (BAGP) and United Kingdom External Quality Assessment Service (UKNEQAS)

N. Singh*, B. Gilks, J. Won, G. von Schalkwyk, R. Ganesan, S. Arif, P. Mukonoweshuro, B. Tanchel, S. Parry

*Department of Cellular Pathology, Barts NHS Trust, United Kingdom

Background & objectives: Immunohistochemistry for the biomarker p16 is a surrogate for high-risk Human Papillomavirus (HPV)-mediated anogenital precancerous and malignant lesions. The accuracy of this biomarker is dependent on the technical quality of the immunostaining as well as well as correct interpretation.

Methods: BAGP members were invited to participate. Unstained sections from a tissue microarray containing 74 cores from anogenital neoplasms, non-invasive and invasive, as well as control cases, were sent to each of 40 laboratories. Participating laboratories were asked to submit details of their staining protocol as well as interpretation results electronically, the latter for each core as “Normal/reactive”, “Abnormal” or “Other/Uninterpretable”.

Results: Results were received from a total of 37 laboratories and 109 individuals (93 pathologists and 16 Biomedical Scientists). Two antibody clones were used; BDPharmingen: G175-405 (n=4) and Ventana CINtec: E6H4 (n=26). (No methods from 7 laboratories). IHC staining platforms from 3 suppliers were represented: Ventana Benchmark: 70%; Leica Bond: 27%; Dako autostainer: 3%.

Slides were returned by 35 laboratories enabling assessment of results of 74 cores from 102 individuals (n=7548). 30/35 laboratories (86%) showed >95% concordance with the reference result for 52 assessable cores. Excluding uninterpretable cores, there was 94% (5331/5673 core results) absolute agreement between individual and central review results.

Conclusion: This study shows excellent technical performance of p16 immunohistochemistry using different antibody clones and automated staining platforms. Interpretation accuracy for abnormal results was high overall, and discrepant results are explained largely through technical reasons.


Indicators of the intensity of intoxication of the term placenta by tobacco smoke

I. Novosel*, M. Rastovac, Ž. Josić, M. Kos, D. Babić

*Østfold Sykehuset Kalnes, Norway

Background & objectives: Intrauterine exposure to tobacco smoke can cause not just metabolic, immunological and endocrine diseases but also epigenetic changes in new-borns DNA.

Our objective was to establish an indicator of toxic changes in the form of Placental intoxication index (PII).

Methods: 290 postpartum women were divided into four groups with respect to smoking habits during pregnancy: NS, non-smokers; PS, passive smokers; AS, active smokers; APS, combined smokers. Placenta samples were analysed pathohistologically (H/E), immunohistochemically (p53, bcl-2, Hsp-70 and Metallothionein) and statistically.

PII was established for each placenta. It incorporates the sum of Placental morphologic index (PMI) and Placental immunohistochemical index (PIHI).

Results: Statistical comparison of PII for each group enabled the distinction of placentas exposed to tobacco smoke during the intrauterine period from those that were not exposed; the PII of placentas exposed to passive smoking differed statistically significantly from PII of placentas exposed to active smoking, while there was no difference between PII of placentas exposed to active smoking and combined forms of smoking.

PII in placentas exposed to tobacco smoke indicated intensity of intoxication since it showed a statistically significant correlation with the level of tobacco smoke exposure regardless of the mode of exposure.

Conclusion: PII detected intoxicated placentas that would otherwise be considered unexposed to the effect of tobacco smoke during 40 weeks of pregnancy because neonatal and macroscopic placental parameters did not indicate significant changes. Therefore, PII enables the early detection of risky new-borns who require enhanced care up to adulthood because of the harmful intrauterine effects of tobacco smoke.


Inter-observer variability in differentiated vulvar intraepithelial neoplasia

S. Dasgupta*, E. de Jonge, L.S. Alcala, L. Makkus, S. Wilhelmus, S. Smits, K. Schelfout, K.K. Van de Vijver, M.R. Van Bockstal, E. Marbaix, L. Libbrecht, S. Koljenović, F.J. van Kemenade, P.C. Ewing-Graham

*Erasmus MC, University Medical Center Rotterdam, The Netherlands

Background & objectives: Differentiated vulvar intraepithelial neoplasia (dVIN) progresses rapidly to carcinoma but manifests a subtle histologic appearance. We assessed the variability of the diagnosis of dVIN amongst pathologists and sought to identify the most reproducible histologic features.

Methods: Two pathologists selected a set of dVIN (n=30) and no-dysplasia (n=10) slides with a range of histologic appearance and prepared a check-list of histologic features of dVIN. Nine external participants independently judged these slides as ‘dVIN’ or ‘no-dysplasia’ and noted which features they used for the diagnosis. ‘Consensus diagnosis’ was defined as 70% agreement. Inter-observer agreement (Fleiss’ Kappa) was calculated.

Results: Consensus was reached for 23 (77%) dVIN cases and for all the no-dysplasia cases. For the diagnosis of dVIN moderate agreement (ĸ=0.46) was obtained; pair-wise agreement ranged from slight to substantial (mean ĸ=0.51; range: 0.14–0.71). Among all histologic features, moderate agreement was obtained only for parakeratosis (ĸ=0.51). Fair agreement was obtained for nuclear features such as atypia discernable under 100X magnification (ĸ=0.39), nuclear angulation (ĸ=0.38), abnormal chromatin (ĸ=0.38), and suprabasal mitoses (ĸ=0.32). Histologic features related to disturbed maturation and architectural changes, such as deep squamous eddies (ĸ=0.39), elongated/anastomosing rete-ridges (ĸ=0.33), altered cellular alignment (ĸ=0.32), cobblestone appearance (ĸ=0.29), and individual cell keratinization (ĸ=0.27), also had fair agreement.

Conclusion: Histologic diagnosis of dVIN suffers from considerable inter-observer variability. Moderate agreement was obtained only for parakeratosis, and there was only fair agreement for most histologic features. Future studies should focus on ancillary immunohistochemical and molecular biomarkers to facilitate accurate diagnosis.

OFP-05 Digestive Diseases Pathology - Liver / Pancreas


A new marker for intrahepatic cholangiocarcinoma: cystic fibrosis transmembrane conductance regulator

F. Pedica*, F. Falcinelli, F. Invernizzi, N. Ahmed, F. Filipello, C. Doglioni

*Pathology Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy

Background & objectives: Biliary tract cancer are aggressive and heterogeneous neoplasms and a specific marker for the biliary epithelium is missing. We performed immunohistochemical analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) in a series of 74 biliary and non biliary tumours.

Methods: We collected a cohort of 40 intrahepatic cholangiocarcinomas and 7 combined hepatocellular-cholangiocarcinomas, then compared them with 8 perihilar adenocarcinomas, 5 hepatocellular carcinomas, 1 case of biliary adenofibroma, 1 case of colorectal liver metastasis, 5 pulmonary adenocarcinomas and 7 pancreatic ductal adenocarcinomas. 3-μm-thick tissue sections were incubated with monoclonal antibody anti-CFTR (NeoBiotechnologies, mouse monoclonal antibody, CFTR/1643, diluition 1:100).

Results: Intrahepatic cholangiocarcinomas were 10 “large duct type” and 30 “small duct type”. Of the "small duct type" 6 cases partially resembles biliary adenofibroma and 6 had a cholangiolocellular carcinoma component. CFTR was expressed in 67% of "small duct type", whilst never expressed in the"large duct type", nor in the perihilar adenocarcinomas (p=0.009). Moreover, CFTR was positive both in the biliary adenofibroma and cholangiocellular components of the intrahepatic cholangiocarcinomas (p=0,011) and was positive in the case of biliary adenofibroma. In the combined cancers, CFTR was expressed in 4 of 7 cases. HCCs, colorectal liver metastasis and pulmonary adenocarcinomas were negative; 1 of the 7 cases of pancreatic adenocarcinoma was positive.

Conclusion: CFTR is a reliable marker for intrahepatic biliary tumours and can be useful both in routine practice and for understanding their biology, which is fundamental to understand the results coming from genomic studies and for the personalization of therapies.


Histological and molecular profiling of intrahepatic cholangiocarcinomas in patients with a history of asbestos exposure

F. Vasuri*, A.G. Corradini, M. Deserti, S. Tavolari, G. Brandi, A. D'Errico

*Pathology Unit, S.Orsola-Malpighi Hospital, Bologna University, Italy

Background & objectives: There are recent evidences of a role of asbestos in the cancerogenesis of intrahepatic cholangiocarcinoma (iCC). Here we present the preliminary results of a study on the histopathological features and mutational status of iCC in asbestos-exposed patients.

Methods: We retrospectively selected the iCC patients from our Institution with a proven history of asbestos exposure according to modified RENAM questionnaire. Clinical and histological features were revised. Immunohistochemistry for CK7, CK19, pCEA and CD56 was automatically performed. Next-Generation Sequencing was performed for mutational status.

Results: Until now, 19 patients were selected; 12 cases with concomitant risk factors for liver disease (alcohol, steatosis/steatohepatitis, HBV/HCV infection) and 7 cases with only asbestos exposure in anamnesis.

Six out of 7 asbestos-only exposed cases were iCC of the small-duct subtype, 1 case was an undifferentiated peripheral iCC, without a specific morphological subtype for small or large bile ducts.

Some gene mutations were more typical of multifactorial iCCs: BAP1 was mutated in 6/12 (50.0%) multifactorial cases and 1/7 (14.3%) asbestos-only cases (p=0.004, t-test); MUC genes were mutated in 6/12 (50.0%) and 1/7 (14.3%) cases respectively (p=0.004). Conversely, ATN1 was mutated in 4/12 (33.3%) and 3/7 (42.9%) cases respectively (p=0.05).

Conclusion: These preliminary results suggest an association between asbestos exposure and small-duct subtype iCC: thus, asbestos could be considered a “non-biliary” risk factor. Moreover, the different incidence of some gene mutations in presence of other risk factors suggests different carcinogenetic mechanisms.


Combined hepatocellular-cholangiocarcinoma (CHCC-CC): are the cellular components important?

F. Yilmaz*, H. Koc, P. Savas, O.V. Unalp, D. Nart, M. Zeytunlu

*Ege University, Faculty of Medicine, Department of Pathology, Turkey

Background & objectives: Primary liver carcinomas (PLC) with unequivocal presence of both hepatocytic and cholangiocytic differentiation within the same tumour are classified as combined hepatocellular-cholangiocarcinoma (cHCC-CC). In this study the clinicopathologic characteristics and immunophenotypes of combined CC-HCC cases were analysed.

Methods: Clinicopathologic data of 19 combined CC-HCC cases were retrospectively analysed. Thirty tumour nodules from 19 cases were retrospectively reviewed for the morphological features of their hepatocytic, cholangiocytic and stromal components based primarily on routine stains.

Results: There were 12 male, 7 female patients (mean age 57, range 34-76). Tumours were, multifocal in 3 (15.79%) cases. Histopathological analyses showed presence of combinations of classical-Hepatocellular carcinoma, classical-cholangiocarcinoma (cCC) and components composed of intermediate cells, intermixed/transitional nests and immature (embryonal-like) cells. Twelve cases were classical cHCC-CC, two were combined intermediate carcinoma-CC, 3 were Intermixed/Transitional Nests combined with cCC and two had embryonal liver-like hepatocytic/cholangiocytic components. Classical HCC-CC was more multifocal (25% vs 0%), smaller (mean diameter 6,38 cm vs 8.64 cm), had more major vascular invasion, satellite nodules, intrahepatic metastases and perineural inavasion (33.33% vs. 14.48%, 16.66% vs. 0%, 33.33% vs. 0%16.66% vs. 14.28 %, respectively) than other subtypes.

Conclusion: Histopathological components of cHCC-CC may have an influence on the biological behaviour of the tumour and merits analysis in larger series


Importance of recipients’ clinical profile in the allocation of liver grafts with fibrosis from dyslipidemic donors

M. Riefolo*, D. Malvi, G. Germinario, M. Ravaioli, M. Cescon, A. D'Errico, F. Vasuri

*Pathology Unit, S.Orsola-Malpighi Hospital, Bologna University, Italy

Background & objectives: A novel role of graft fibrosis and dyslipidemic profile in extended-criteria donors (ECD) in predicting early allograft dysfunction (EAD) was previously demonstrated.

Aim: to validate our previous results on a prospective cohort, matching graft histology to recipients’ characteristics.

Methods: We prospectively enrolled all ECD whose liver grafts underwent to transplant after biopsy in 2 years.

Histological variables of the graft, donors' and recipients' clinical data, and follow-up data were recorded, including EAD (at 7 days).

Multivariate and univariate analyses were applied. ROC curves were built to calculate bilirubin and creatinine cut-offs.

Results: We enrolled 103 ECD and corresponding recipients. Fourteen (13.6%) had portal fibrosis ≥2 (according to Ishak) and dyslipidemia (dyslipidemic donors with fibrosis, DDF); 42.8% DDF recipients experienced EAD versus20.9% of the other recipients (p=0.076); in particular, DDF recipients had more episode of AST raise within 7 days (p=0.048).

At multivariate ad univariate analyses, EAD correlated with pre-OLT bilirubin levels (p=0.030) in non-DDF recipients, and with pre-OLT creatinine and INR in DDF recipients (p=0.024 and p=0.016).

In particular, DDF recipients with >0.85 mg/dL pre-OLT creatinine had 71.4% EAD episodes, compared to 14.3% in DDF recipients with <0.85 mg/dL creatinine (p=0.009).

Conclusion: Our prospective cohort confirmed the importance of DDF model in predicting EAD. Moreover, the choice to transplant DDF grafts likely depends on recipients’ clinical profile, in particular creatinine level, according to our model.


Histopathologic evaluation of resected colorectal cancer liver metastases

J. Santos*, R. Machado-Neves, T. Amaro, M. Honavar

*Unidade Local de Saúde de Matosinhos, Portugal

Background & objectives: Colorectal liver metastasis (CRLM) presents histopathologic patterns with prognostic implications. The objective of this study is to evaluate the influence of pathological findings on mortality rate (MR).

Methods: Analysis of clinical and outcome data from 30 patients who underwent CRLM resection, 25 of whom were treated with chemotherapy before hepatic resection. Pathological review: resection margins; tumour regression grade (TRG); growth pattern (GP); necrosis and mucinous pattern.

Results: Thirty patients: 12 women and 18 men. Mean age: 65 years. Median survival following hepatic resection: 41 months. Resection margins: positive in nine specimens, associated with higher mortality rate (MR) (56% vs 29%). TRG: fourteen showed complete/partial response (TRG1-3) and eleven showed poor response (TRG4-5). TRG1-3 had lower MR (21%) vs TRG4-5 (54%). GP: five pushing, twelve desmoplastic, seven replacement and six mixed. Replacement GP showed higher MR (57%). Oxaliplatin-based chemotherapy was associated with desmoplastic GP (56%). Necrosis: sixteen had >10% and fourteen <10% (MR 63% vs 8%). Mucinous pattern: present in six, all four with mucinous pattern >50% died.

Conclusion: The pathological features associated with higher MR were positive resection margins, TRG4/5, replacement GP, >10% of tumour necrosis and mucinous pattern >50%. Prospective studies of patients with CRLMs are warranted to evaluate the prognostic value of histopathological features.


Role of cellular senescence in the natural history of primary sclerosing cholangitis

S. Sarcognato*, N. Cazzagon, G. Corrà, S. De Martin, V. Guzzardo, A. Floreani, F.P. Russo, M. Guido

*Dept of Medicine, University of Padova, Italy

Background & objectives: Cellular senescence (CS) represents an irreversible cell cycle arrest to prevent replication of injured cells, whose role in chronic liver diseases was recently suggested. We aimed to evaluate hepatic CS-marker expression in primary sclerosing cholangitis(PSC) and its correlation with clinical-pathological features.

Methods: Thirty-five PSC patients with at least one liver biopsy(LB)/explant at diagnosis and/or during follow-up were enrolled. Clinical/laboratory data(including prognostic models) at the time of LB were collected. Endpoint was survival without liver transplantation or cirrhosis decompensation. Grading/staging according to Nakanuma were performed. Immunohistochemistry for CS-markers(p16,p21) were performed and scored based on positivity extent in native bile duct(NBD) and ductular reaction(DR).

Results: p16 and p21 expression in NBD and DR was directly correlated with hepatitis activity, cholestasis-related histological lesions, fibrosis, and disease stage (p<0.05 for all). By multivariate analysis, p16 expression in DR was independently associated with advanced disease stages [p=0.002,HR 13.6 (95%CI 2.6-72.6)]. p16 and p21 expression in DR was directly related to Mayo Risk Score (p=0.02 for both) and Amsterdam-Oxford Model (AOM;p<0.001 and p=0.007, respectively). p16 and p21 expression in NBD was directly related to AOM (p=0.03 for both). p21 expression in DR was independently associated with adverse outcome-free survival [p=0.005,HR 4.6 (95%CI 1.6-13.6)].

Conclusion: In PSC, CS marker expression in cholangiocytes is related to clinical and histological severity, disease progression and patients’ prognosis, suggesting a role for CS in the natural history of the disease.


Biomarkers to predict response to immunotherapy identify pancreatic cancer patient subgroups with prolonged progression-free survival

M. Wartenberg*, E. Vassella, S. Eppenberger-Castori, B. Gloor, A. Perren, E. Karamitopoulou-Diamantis

*Institute of Pathology, University of Bern, Switzerland

Background & objectives: Immunotherapy alone or in combination with chemotherapy can improve treatment response in pancreatic ductal adenocarcinoma (PDAC). Therapy success has been linked to microsatellite instability (MSI), inflamed tumour microenvironment (TME), high tumour mutational burden (TMB) and PD-L1 expression.

Methods: The study cohort comprised 115 resected PDACs, TNM stage I-III, including 25 long-term survivors (progression-free survival>60 months). TMB was assessed using the oncomine mutation load assay. PD-L1, immune cell densities and MSI-status were assessed immunohistochemically (PD-L1, MLH1, PMS2, MSH2, MSH6, CD3, CD4, CD8, FOXP3, CD68) on tissue microarrays.

Results: MSI-H was present in 2.6% of all patients and more frequent among LTSs (8%). 75% of MSI-H-cases were also TMB-H (i.e. >10), showing T-cell-inflamed TME. TMB-H was found in 15% of PDACs; 60% also expressing PD-L1. PD-L1-positivity was found in 37% (20% were also TMB-H). PD-L1-high (expression-score >4; 7%) was associated with an inflamed TME. Better overall (OS) and progression-free survival (PFS) was found for TMB-H cases (p=0.01817 and p=0.00664).

Conclusion: The morphologic/microenvironmental and clinicopathologic features of MSI-H and TMB-H patients indicate a better anti-tumour immune response. Based on concurrent biomarker expression, TMB-H patients might further profit from checkpoint inhibition, while other strategies inducing immune response might be an option for the remaining majority of patients. Combinations of biomarkers indicating potential immunotherapy response along with TMB can assist the selection of target groups as part of an individualized, precision oncology approach.


Predicting morphological classifications of pancreatic ductal adenocarcinoma (PDAC) using deep learning

S. N Kalimuthu*

*Toronto General Hospital, Canada

Background & objectives: We recently proposed a revised, pattern based morphological classification for PDAC, associated with transcriptional subtypes and overall survival1.We aim to assess if deep learning can automate our system to enhance reproducibility and accessibility to pathologists for patient care.

Methods: 365 H&E slides from 58 patients (range 3-11 slides/patient) with tx-naïve PDAC were digitized. Tiling was automated to generate a total of 1.6 x 105 image 524 um2 patches tiles and tumour-containing image patches extracted. Five-fold cross validation approach was used to assess the performance of our classifier in predicting the A and B morphologic subtypes of our entire cohort.

Results: Using unsupervised hierarchical clustering and dimensionality reduction, histological features, defined by 512 deep learning feature vectors (DLFV) generated by convoluted neural network (CNN) developed in our department2, were used to subgroup PDAC that mirrored our morphological subtypes. When both stromal and epithelial components were included, 68% were classified as Group A and 78% were classified as group B accurately. However, when stromal elements, to only include epithelial elements, it was able to classify 80% of Group A and 85% of Group B patients accurately, with an area under the receiver operator characteristic (ROC) curve (AUC) of 0.83.

1. N Kalimuthu S et al 2019

2. Faust et al 2019

Conclusion: We have demonstrated a proof of concept that deep learning, when applied to PDAC can objectively classify the morphological subtypes previously described on H&E slides. This could provide rapid survival data and act as a surrogate to tailor targeted therapy.


Reliability of EUS-guided fine-needle biopsy specimens to assess ATRX/DAXX and PDX1/ARX by immunohistochemistry in pancreatic neuroendocrine tumours: a proof of concept study

M.G. Mastrosimini*, S. Crino, A. Parisi, L. Bernardoni, M. De Bellis, E. Torresani, S. Ammendola, S. Marletta, A. Gabrielli, M.C. Conti Bellocchi, E. Manfrin

*Department of Diagnostics and Public Health, University of Verona, Italy

Background & objectives: Somatic inactivating mutations in ATRX/DAXX genes and altered expression of transcription factors ARX and PDX1 are associated with poor outcomes in PanNETs. We sought to assess ATRX/DAXX and PDX1/ARX expression by immunohistochemistry in EUS-FNB and paired surgical specimens of PanNETs.

Methods: EUS-FNB and corresponding surgical specimens of twenty-six patients with PanNETs were retrieved at the University Hospital of Verona (2017-2018). ATRX, DAXX, ARX and PDX1 protein expression and Ki-67 proliferation index were detected by immunohistochemistry on biopsy and paired surgical specimens. Tumour grade was classified according to the WHO 2019.

Results: All EUS-FNB cases provided adequate material for immunohistochemistry examination. ATRX, DAXX, PDX1, and ARX concordance between biopsy and surgical specimens was 92% (K = 0.705; p < 0.001), 96% (K = 0.866; p < 0.001), 100% (K = 1.000; p < 0.001), and 82% (K = 0.400; p = 0.050), respectively. Likewise, tumour grade concordance was 92% (K = 0.752; p < 0.001), since 2 cases were under-staged on biopsy.

Conclusion: The assessment of ATRX/DAXX and PDX1/ARX protein expression by immunohistochemistry can be accurately performed on EUS-FNB. The concordance between EUS-FNB and surgical specimens is substantial for ATRX and tumour grade, and almost perfect for DAXX and PDX1.


Through the needle biopsy (TTNB) specimens. A new procedure in the setting of pre-operative histological diagnosis of the cystic lesions in the pancreas and retroperitoneum

S. Ammendola*, E. Cavallo, S. Crino, L. Bernardoni, A. Parisi, M.G. Mastrosimini, A. Gabbrielli, M.C. Conti Bellocchi, E. Manfrin

*Department of Diagnostics and Public Health, University of Verona, Italy

Background & objectives: Endoscopic ultrasound guided through the needle biopsy (TTNB) with microforceps procedure ecruits specimens from the cyst wall for the histological evaluation.

Hereby, we describe our experience at the University of Verona Hospital trust with TTNB of pancreatic and retroperitoneal cysts.

Methods: Electronic files were searched for TTNBs performed between January 2016 and January 2020. The clinical, serological, imaging and cyto-histological data were collected for the selected cases.

Results: 99 TTNB procedures were performed and the histological yield was 99% (98/99 cases). The histological diagnosis was performed in 83/99 (84.7%) cases. The aspecific histological characteristics of the biopsy prevented the conclusive diagnosis in 15 cases (15.3%).

In 30 cases, the patients underwent surgery and TTNB diagnosis was confirmed in 27/30 cases (90%). Grading of epithelial dysplasia between biopsy and surgical specimen was discordant in 2/30 cases (6.7%).

Conclusion: TTNB is a reliable diagnostic tool for the pre-operative diagnosis of cystic lesions in the pancreas and retroperitoneum and it allows to avoid unnecessary surgery. However, due to the histological heterogeneity of the cyst wall, pathologists must be aware that TTNB microforceps could miss epithelial dysplasia or sample non diagnostic areas, favouring misdiagnosis. A multidisciplinary approach helps reduce misdiagnosis.

OFP-06 Digestive Diseases Pathology - GI


Serrated polyposis syndrome: morphologic and molecular heterogeneity of dysplasia-carcinoma progression

J. Gibson*, A. Barbieri, Z. Walther

*Yale University School of Medicine, USA

Background & objectives: Serrated polyposis syndrome (SPS) predisposes to colorectal cancer (CRC) and is characterized by multiple serrated polyps (SP) according to WHO criteria proposed in 2010 and revised in 2018. We aim to characterize the pathologic and molecular progression of SPS neoplasia.

Methods: 32 SPS patients were identified using WHO criteria in 2345 consecutive patients with at least 1 SP. Clinicopathologic assessment of advanced neoplasia (AN) in SPS patients was performed, including CRC and/or SP with dysplasia of any grade (SSPD), intestinal (I) or serrated (S) type. 10 AN were subjected to next generation sequencing (NGS) using a 50 gene hot spot panel.

Results: SPS incidence was 1.4% (18:14 female:male, mean age 67 years). 9 SPS patients had AN, including 7 CRC, 8 SSPD-I and 3 SSPD-S. BRAF V600E mutation was detected in 9 cases (5 SSPD-I, 1 SSPD-S, and 3 CRC) and KRAS Q61H in 1 CRC. Additional mutations were found in CRC cases in the following genes: p53 (2 cases), FBXW7 (2), and SMAD4, PTEN, RB1, and RET (1 each).

Conclusion: In our cohort, 13% SPS patients had AN, including CRC and SSPD. All AN had activating mutations involving the RAF-RAS -MAPK signalling pathway, with majority with BRAF mutations and one case with a KRAS mutation. Our study confirms that while BRAF V600E is a common early mutation in SPS neoplasia, analysis of CRC in SPS patients demonstrates a diverse mutational profile. Our results support the conclusion that progression of carcinogenesis in SPS involves heterogenous molecular pathways.


Computer-assisted hot-spot selection for tumour budding assessment in colorectal cancer

J. Bokhorst*, F. Ciompi, I. Zlobec, A. Lugli, M. Vieth, R. Kirsch, J. van der Laak, I. Nagtegaal

*Radboud University Medical Center, The Netherlands

Background & objectives: Tumour budding (TB) is an established prognosticator for colorectal cancer. Detection of the hot-spot to score TB is based on visual inspection, hindering reproducibility of this important factor. We present an algorithm that can potentially assist pathologists in this task.

Methods: We used a previously developed algorithm for the detection of tumour buds in pan-cytokeratin stained whole slide images, calculating the number of buds for each location using a circle with 0.785mm2 surface area. From these numbers, density heatmaps were produced. The algorithm was applied to 270 slides from Bern University hospital, in which hot-spots and tumour buds were visually identified.

Results: Heat maps were created and we located the hand-selected hotspot and noted the associated TB number. The differences and similarities between computer identified and manually selected hot-spots were visually assessed as well as via histograms. Preliminary results show that the heatmaps are helpful, as locations with the highest TB density (the top 15%) also include the hand-selected hotspots. The full results will be presented during the conference.

Conclusion: The presented algorithm can assist the pathologist in selecting the hot-spot with the highest tumour bud count with more ease at low magnification and can help to reduce the high inter-observer variability among pathologists in scoring tumour budding.

This project has received funding from the Dutch Cancer Society, project number 10602/2016-2.


Prognostic value of tumour deposits for disease-free survival in patients with stage III colon cancer: a post hoc analysis of the idea France phase III trial (prodige-gercor)

M. Svrcek*, J. Delattre, R. Cohen, J. Henriques, J. Emile, F. Bibeau, J. Taieb, C. Louvet, D. Vernerey, T. Andre

*Sorbonne Université, AP-HP, Hôpital Saint-Antoine, Department of Pathology, France

Background & objectives: Tumour deposits (TD) seem to impact the prognosis of patients with colon cancer (CC). In the

TNM7 CC staging, their presence is only considered in the absence of lymph node metastases

(LNM). In the era of personalized duration of histopathological criteria-based adjuvant therapy, this could potential lead to a loss of the prognosis prediction accuracy.

Methods: A post hoc analysis of all pathological reports from stage III CC patients included in the IDEA

France phase III study (NCT00958737) investigating the duration of adjuvant FOLFOX or CAPOX therapy (3 vs 6 months) was performed. The primary objective was to determine the prognostic impact of TD on disease-free survival (DFS). The effect of the addition of TD to LNM count on pN restaging was also evaluated. A multivariable analysis was performed to establish the association between TD and DFS.

Results: Of 1942 patients, 184 (9.5%) had TD. The pN1a/b and pN1c populations showed similar DFS. TD-positive patients had worse prognosis compared with TD-negative patients, with the 3-year DFS rate of 65.6% (95% CI 58.0 to 72.1) and 74.7% (95% CI 72.6 to 76.7; P=.0079). On multivariable analysis, TD were associated with the higher risk of recurrence or death (HR=1.36; P=.0201). Other pejorative factors included pT4 and/or pN2 (HR=2.21; P<.001), the 3-month adjuvant treatment (HR=1.29, P=.0029), obstruction (HR=1.28; P=.0233), and male gender (HR=1.24; P=.0151). pN2-restaged patients (n=35, 2.3%) had similar DFS than those initially classified as pN2

Conclusion: TD is an independent prognostic factor for DFS in stage III CC patients. The addition of TD to LNM may help to better define the duration of adjuvant therapy.


Deep neural networks and supervised machine learning for the screening and classification of dysplasia in inflammatory bowel disease

J. Kantonen*, A. Knuuttila, S. Blom, N. Sjöblom, R. Samaletdin, M. Färkkilä, J. Arola

*University of Helsinki, HUS/Pathology, Finland

Background & objectives: Algorithms based on deep neural networks (DNN) have been shown to outperform humans in specialized tasks in pathology. Here we assess the performance of a pathologist-trained algorithm in the search for and grading of dysplasia in inflammatory bowel disease (IBD).

Methods: HE-slides from a cohort of 391 IBD patients were used. A training set of 50 slides was annotated by a pathologist, subsequently used to train a DNN using the Aiforia® cloud platform. The resulting algorithm was applied to a validation set of 250 slides and results were compared to pathologist performance. Discrepant results were reviewed by an expert panel.

Results: The algorithm was consistently able to find areas suspicious for dysplasia. The algorithm could also differentiate between the classes 'indefinite for dysplasia', 'LG dysplasia', 'HG dysplasia' and 'Carcinoma budding', with the diagnosis for carcinoma being aided by the immediate vicinity of a class for 'reactive stroma'. Review of the discrepant diagnoses revealed the inconsistencies of human performance on the one hand and limitations of the trained algorithm on other hand. In a speed test with 100 random IBD slides, the algorithm was able to outperform a pathologist using a conventional microscope, finishing within 1/3 of the time needed by the pathologist.

Conclusion: Our algorithm outperforms pathologists in both speed and consistency, but is limited in scope, and requires a pathologist to make the definitive diagnosis for carcinoma. Nevertheless, with the number of samples continuing to grow, it provides a useful tool for the screening and classification of dysplasia in IBD. With such tools, training pathologists to become 'diagnostic machines' is questionable as a vision for the future of pathology.

Funding by: HUS (Valtion tutkimusrahoitus)


Markers of epithelial-mesenchymal transition in early colorectal carcinoma compared to adenoma and adenoma with pseudoinvasion

B. Ranković*, E. Boštjančič, M. Žlajpah, N. Zidar

*Institute of Pathology, Faculty of Medicine, University of Ljubljana, Slovenia

Background & objectives: Distinguishing malignant adenoma/early colorectal carcinoma (CRC) from adenoma with pseudoinvasion can be challenging. Our aim was to investigate the expression of markers of epithelial-mesenchymal transition (EMT) in malignant adenoma/early CRC compared to adenoma and adenoma with pseudoinvasion.

Methods: Thirty-two cases of formalin-fixed paraffin-embedded tissue were included (10 adenomas, 10 malignant adenomas/early carcinomas and 12 adenomas with pseudoinvasion). We analysed the expression of miR-200 family and their target genes CDKN1B, ONECUT2, PTPN13, RND3, SOX2, TGFB2 and ZEB2 using qPCR.

Results: We found statistically significant down-regulation of miR-141, miR-429 and PTPN13 and up-regulation of CDKN1B in malignant adenomas/early carcinomas compared to adenomas with pseudoinvasion. We also observed down-regulation of miR-200c and miR-429, and up-regulation of CDKN1B and RND3 in malignant adenomas/early carcinomas compared to adenomas, and down-regulation of TGFB2 and up-regulation of RND3 and CDKN1B in adenomas with pseudoinvasion compared to adenoma.

Conclusion: Down-regulation of the miR-200 family and PTPN13, and up-regulation of CDKN1B in malignant adenomas/early carcinomas compared to adenomas and adenomas with pseudoinvasion is consistent with the postulated role of EMT in the progression of adenoma to early CRC.


Inhibition of LPS-mediated TLR4 activation abrogates gastric adenocarcinoma-associated peritoneal metastasis

M. Alzahrani*, C. Janeiro, L. Almarzouki, V. Stavrakos, L. Ferri

*McGill University, Saudi Arabia

Background & objectives: Curative-intent-surgery remains the most beneficial treatment-option for localized gastric-adenocarcinoma(GC),the majority of patients experience cancer recurrence, with peritoneum as the most common site. Clinically, there is an observed link between post-operative infection and metastasis of primary(GC)to the peritoneum, however the mechanisms behind are poorly-understood.

Methods: We aimed to show the effects and mechanisms of LPS-induced TLR4-signaling on peritoneal metastasis of gastric adenocarcinoma, as a proposed mechanism for post-surgical infection induced cancer recurrence. in vitro, ex vivo and in vivo activation of TLR4 by LPS or heat inactivated E. coli using both murine- and human-derived gastric cancer cell lines to human peritoneal mesothelial cells(HPMCs) were done.

Results: Up to a six-fold significant increase was seen in the LPS and E. coli conditions compared to control (P<0.05). Use of neutralizing TLR4 antibody and TLR4 signal cascade blockades, notably TAK1 and MEK1/2 blockades, significantly abrogated adhesion (P<0.05). A similar effect was seen using a novel ex vivo model of peritoneal metastasis employing peritoneal surface harvested from both C57BL/6 wild type and TLR4–/– knockout mice. In vivo LPS infection potentiates the development of gross metastases in NSG mice after intraperitoneal injection with MKN-45 human gastric cancer cells. Furthermore, murine peritoneal carcinomatosis index (PCI) scores are highest in mice that received LPS injection with MKN-45 cells LPS stimulation.

Conclusion: These studies have shown the relevance of TLR4 and the TLR4 signalling cascade in potentiating metastatic adhesion and spread to the peritoneum upon activation.

Funding by: McGill University


Integrated clinicopathologic and immunohistochemical analysis of α-foetoprotein-producing gastric carcinoma: an exploration of histogenesis, molecular features and prognostic markers

F. He, Y. Fu, Q. Sun, X. Fan*

*Nanjing Drum Tower Hospital, China

Background & objectives: Typical a-fetoprotein–producing gastric cancer (AFPGC), which is characterized by histological resemblance to hepatocellular carcinoma and a poor prognosis. The aim of this study is to elucidate the clinicopathological, histogenesis and molecular characteristics as well as prognostic markers of AFPGC.

Methods: Fifty-five patients with AFPGC were screened out by morphological features for hepatoid adenocarcinoma (HAC), gastric cancer with enteroblastic differentiation (GCED) and common tubular/papillary adenocarcinoma (COM). The immunohistochemical diagnostic markers including AFP, GPC3 and SALL4, mucin phenotypes, molecular features and Immunoscore were assessed. Finally, the relationships between each factor and clinicopathologic features as well as the overall survival time were analysed.

Results: In 55 patients, mixtures of 3 types (COM+GCED+HAC) were most commonly observed. The transition between COM and GCED, GCED and HAC, and sometimes transition between COM and HAC were could frequently see. Hyaline globule and pink amorphous substance were often present. Of the three diagnostic indictors, SALL4 was the most sensitive marker (90.9%). AFPGC showed relatively frequent expressions of HER2 (38.2%). Besides, 38 patients (69.1%) showed pure intestinal phenotype and the remained 17 patients were mixed gastric/intestinal phenotype. Cases with prominent hyaline globules had a significantly poor prognosis. SALL4, HER2 and Immunoscore had an independent influence on OS. In addition, HER2low + SALL4low+ Immunoscore high group have the best prognosis.

Conclusion: AFPGC is a genetically heterogenous group. It might be developed from intestinal type gastric adenocarcinoma and progress to GCED, finally HAC. CIN/GS subtypes could be where they belong. SALL4, HER2 and Immunoscore are independent influence on OS of AFPGCs.


A pathological risk score based on poorly differentiated clusters, tumour budding and number of harvested loco-regional lymph nodes predicts prognosis in stage II colorectal cancer

S. Ammendola*, G. Burato, S. Pecori, A. Tomezzoli, C. Pedrazzani, V. Barresi

*Department of Diagnostics and Public Health, University of Verona, Italy

Background & objectives: Use of adjuvant chemotherapy is controversial in patients with stage II colorectal carcinoma (CRC), because of good overall prognosis. This study aims to investigate the prognostic value of several clinical-pathological variables in stage II CRC.

Methods: A cohort of 149 surgically resected stage II CRC patients who did not receive neoadjuvant or adjuvant treatment was selected. Data on cancer specific survival (CSS) was available in all cases. All cases were revised to assess histological grading, tumour budding (TB) grade, poorly differentiated clusters (PDC) count, lymphovascular/perineural invasion and tumour border configuration.

Results: Multivariate logistic regression analysis showed that high TB grade (Bd3), presence of PDC and a number of lymph nodes <12 were significant and independent variables for prediction of cancer related death. A risk score, by assigning 1 point to the presence and 0 points to the absence of each of these variables was formulated. A risk score ≥ 1 was significantly and independently prognostic of shorter CSS.

Conclusion: Examination of an inadequate number of locoregional lymph nodes, high TB grade and presence of PDC were associated with shorter CSS and cancer-related death in patients with stage II CRC. These features may be helpful to select high risk patients who could benefit from adjuvant chemotherapy.


Mucinous appendiceal neoplasms: a 10 year review with reclassification and outcomes

A. Colmenares Lozada*, M. Oliveira, C. Ivanova, P. Luís, S. Ortiz

*Hospital Distrital de Santarém, Portugal

Background & objectives: Appendiceal mucinous neoplasms (AMN) tend to occur in adults in the sixth decade, and usually present with features of appendicitis. In recent classifications two new concepts for AMN were introduced: LAMN (Low-grade AMN) and HAMN (High-grade AMN).

Methods: Retrospective study, in a 10-year period, of 29 cases of AMN.

The cases were analysed for gender, age, initial symptoms, TNM classification when applicable, surgical approach, complications, survival rate and recurrence.

Histologic review of all cases was made, to demonstrate how many would now fulfil the criteria for LAMN or HAMN, and how it could have changed treatment and/or prognosis.

Results: 15 were initially diagnosed as mucinous cystadenoma [after review 12 were reclassified as LAMN (80%), 2 as HAMN (13,3%) and 1 as sessile serrated lesion]; 5 were LAMN, 2 HAMN and 5 mucinous adenocarcinomas [no change after review].

Global 2-year survival rate was 95% [LAMN 100%; HAMN 100%] and 5-year survival rate was 66% [LAMN 81%; HAMN 100%]; global recurrence index was 11,1% [LAMN 6,2%; HAMN 0%] and the global rate of disease progression was 14,8% [LAMN 5,8%; HAMN 0%].

Conclusion: In reclassified cases there was no significant change in both prognosis and disease-free survival (despite pT stating), however follow-up is short.

Most LAMN and all HAMN have a 2-year to 5-year follow-up free of disease or signs of recurrence.

Those with adenocarcinoma, as described in the literature, had worse prognosis.


p53 and p16 combined immunohistochemistry for the prognostic stratification of adenocarcinomas of cardias and gastroesophageal junction: A multicentric study

D. Malvi*, M. Riefolo, F. Vasuri, E. Bonora, L. Mastracci, H. Söderström, J. Räsänen, S. Krishnadath, M. Seri, R. Fiocca, S. Mattioli, A. D'Errico

*Pathology Unit, S.Orsola-Malpighi Hospital, Bologna University, Italy

Background & objectives: Adenocarcinoma of the gastroesophageal junction (AdGEJ) is rare and aggressive, and still lacks of bio-pathological prognostic markers. Aim of the present study was to establish the usefulness of p53 and p16 immunohistochemistry (IHC) in AdGEJ prognosis.

Methods: Eighty-three patients resected for non-treated AdGEJ were enrolled in 5 European centres. Histological variables included: tumour classification according to WHO and Lauren, concomitant intestinal metaplasia, tumour stage, vascular invasion.

IHC was performed for Cytokeratin 7 and 20 and CDX2 for diagnosis and tumour classification, as well as for p53 and p16. p53 mutational status was confirmed by NGS.

Results: p53 was hyperexpressed in 46 (55.4%) cases, and completely null in 5 (6.0%), for a total of 51 p53-mutated cases (p53+), normoexpressed in the remaining cases (p53wt). p16 was hyperexpressed in 42 (50.6%) cases (p16+), applying a cut-off of 7.5% of p16-positive cells, calculated by ROC curve.

Patients were sorted in: p53wt/p16- (N=11), p53+/p16- (N=30), p53wt/p16+ (N=21) and p53+/p16+ cases (N=21). Kaplan-Meier curves of the 4 patients groups showed a significantly different cancer-specific survival (p=0.032): in particular, 90% of patients with p53wt/p16- AdGEJ were alive after 80 months of follow-up, versus 20% of cancer-specific survival recorded among patients with p53+/p16+ AdGEJ. Patients with p53+/p16- and p53wt/p16+ AdGEJ showed intermediate survivals.

Conclusion: IHC for p53 and p16 seems to be an independent prognostic factor in patients with AdGEJ, and it could be proposed in the pathological report.

This work was partially funded by Associazione Italiana Ricerca sul Cancro (AIRC), grant Nr. IG1769.

OFP-07 Joint Oral Free Paper Session: Dermatopathology / Soft Tissue & Bone Pathology


Data mining on transcriptomic data unmasks AXL, TYRO3 and MERTK receptors as new potential prognostic markers for advanced cutaneous melanoma: an ongoing project

L. Nicolè*, R. Cappellesso, F. Cappello, T. Sanavia, D. Serafin, G. Cortese, P. Del Fiore, S. Mocellin, A.P. Dei Tos

*Department of Medicine, University of Padova, Italy

Background & objectives: TAM receptor (TAMr) tyrosine-kinase family plays a significant role in oncogenesis and in immune-modulation, but their role in melanoma is largely unknown. TAMr prognostic relevance and correlation with CD8+ T-cells infiltration (Tils8) was assessed in melanoma patients.

Methods: AXL, TYRO3 and MERTK gene expression profiles regarding 235 primary melanomas along with clinico-pathological and follow-up records were downloaded from TCGA repository. Overall and disease-free survival analyses were performed considering clinico-pathological data, TAMr gene expression and Tils8. Tils8 was estimated by a consensus ranking obtained with TIMER and CIBERSORT tools. P-values<0.05 were considered for statistical significance.

Results: After stepwise selection based on Akaike Information Criterion, low AXL and TYRO3 expressions resulted significantly correlated with worse disease-free survival, while low AXL and high MERTK expressions significantly decrease overall survival in patients with high tumour stage. Significant positive Spearman correlation was observed between AXL and Tils8 score (p<0.001).

Conclusion: TAMr are promising prognostic markers for advanced melanoma. To assess our results, we are validating AXL, TYRO3 and MERTK expression by immunohistochemistry in a large cohort of patients with III-IV stage melanoma. Moreover, studies reported the molecule UNC2025 as a specific MerTK inhibitor, suggesting that MerTK receptor could also represent a potential therapeutic target for advanced melanoma.


T-cell receptor expression in cutaneous benign and malignant lymphoid infiltrates in comparison with T-cell receptor gene rearrangement and its diagnostic utility in borderline cases

K. Kamarádová*, K. Hrochova, D. Belada

*University Hospital Hradec Kralove, The Fingerland Department of Pathology, Czech Republic

Background & objectives: Cutaneous T-cell lymphoid infiltrates can represent benign lesion or an infiltration with T-cell lymphoma. Histopathological appearance can overlap and T-cell receptor gene rearrangement evaluation is not always accessible. Expression of different TCR antibodies is studied in cutaneous lymphoid infiltrates.

Methods: Representative cases of reactive and malignant lymphoproliferations were collected. Separate group of lesions with borderline morphology was selected for comparison. Immunohistochemical expression of TCR-beta-F1 (TCR-BF1), TCR-C-beta1 (TCR Jovi.1), TCR gamma/delta (TCRg/d) and TCR delta (TCRd) was performed in all cases. TCR gene rearrangement evaluation was performed in all cases using PCR BIOMED-2 assay.

Results: Benign lymphoid infiltrates were all negative in TCRd and TCRg/d, expression of TCR-BF1 in one. T-cell lymphomas were positive for TCR-BF1 and TCRg/d in 60% and 30% of cases, respectively. TCR gene rearrangement was confirmed in 90% of lymphoma cases. All benign lesions were polyclonal. Borderline lesions showed expression of TCR-BF1 in 6/10 cases and TCR gene rearrangement in 4/10 cases.

Conclusion: Expression of TCR-BF1 and TCR-gene rearrangement was significantly associated with malignant infiltrates. Re-evaluation of the cases and clinical correlation led to the change of the diagnosis and confirmation of T-cell lymphoma in 4/10 of borderline cases. TCR-BF1 positivity in borderline cutaneous lymphoproliferations can raise or support the suspicion of malignancy, nevertheless, confirmation by TCR gene rearrangement and careful clinical correlation is still advisable.

Supported by MH CZ - DRO (UHHK, 00179906), BBMRI-CZ: No: EF16 013/0001674, PROGRES Q40/11 and BBMRI-CZ LM2018125.


ROCK1 and ROCK2 expression is decreased in late-stage skin melanomas

M. Kaczorowski*, P. Biecek, P. Donizy, M. Pieniążek, R. Matkowski, A. Hałoń

*Wroclaw Medical University, Poland

Background & objectives: Rho-associated coiled-coil kinases (ROCK) 1 and 2 are controllers of cytoskeleton dynamics and are implicated in several features of carcinogenesis, e.g. adhesion, invasiveness, migration or proliferation. We aimed to determine clinicopathological significance of ROCK1 and 2 expression in skin melanoma.

Methods: ROCK1 and ROCK2 expression was assessed in immunohistochemically stained sections of 129 primary skin melanomas. Obtained results were statistically analysed together with other clinicopathological characteristics.

Results:We found negative correlations between melanocytic expression of either ROCK kinase and Breslow thickness, Clark level, AJCC stage as well as mitotic rate. Moreover, there was a predilection for low ROCK1 and 2 reactivity in ulcerated tumours and nodular melanomas. Decreased ROCK2 immunoexpression in melanoma cells was associated with shorter melanoma-specific and melanoma-free survival in Kaplan-Meier analysis, but did not independently predict prognosis in a multivariable model.

Conclusion: Our results indicate that ROCK signalling may be involved in the pathogenesis of melanoma and that ROCK1 and 2 are downregulated in aggressive and late-stage tumours, which is contrary to most reports concerning other cancers. Therapeutic inhibition of ROCK kinases in skin melanoma may thus be ineffective.

Funding by: statutory subsidy by the Polish Ministry of Science and Higher Education as part of grants STM.B131.17.008, SUB.C280.19.050 and ST.B130.18.030; National Science Centre grant Opus 2016/21/B/ST6/02176


Clinical relevance of tumoral indoleamine 2,3-dioxygenase 1 and PD-L1 co-expression in Merkel cell carcinomas

P. Donizy*, C. Wu, J. Kopczynski, M. Pieniazek, D.M. Miller, J. Rys, M.P. Hoang

*Department of Pathomorphology and Oncological Cytology, Wroclaw Medical University, Poland

Background & objectives: The prognostic role of combined tumoral indoleamine 2,3-dioxygenase 1 (IDO1) and PD-L1 expression in Merkel cell carcinomas (MCCs) has not been studied in a large cohort of patients.

Methods: IDO1 and PD-L1 immunostains were performed on tissue microarrays constructed from paraffin-embedded tissues of 132 MCCs. PD-L1 expression >1% was considered positive. The cytoplasmic IDO1 staining was done using the H-score. We correlated IDO1 and PD-L1 expression with clinicopathologic variables and patient outcomes.

Results: High tumoral IDO1 expression was observed in 48,5% of cases and PD-L1 expression >1% in 48% of cases. There was co-expression of IDO1 and PD-L1 in 25% of cases. By Fisher’s exact tests, combined high tumoral IDO1 expression and tumoral PD-L1 expression >1% significantly correlated with favourable survival status (p=0.0002). By univariate analysis, combined high tumoral IDO1 and tumoral PD-L1 >1% expression revealed a trend with improved overall survival (p=0.057).

Conclusion: The co-expression of IDO1 and PD-L1 in MCC suggests a potential therapeutic role of combined IDO1 inhibitor and PD1/PD-L1 inhibitor. Understanding the clinical and pathologic features associated with IDO1/PD-L1 co-expression in MCC may provide insights into effective strategies for therapy.


Systems biomarker discovery implicates TEAD3 in cutaneous squamous cell carcinoma perineural invasion

I. Katsyv*, G. Niedt

*Columbia University, New York Presbyterian Hospital, USA

Background & objectives: Cutaneous squamous cell carcinoma (cSCC) is the second most common primary skin malignancy. Although resection/radiation is generally curative, a significant minority of patients develop recurrence. Perineural invasion (PNI) is a significant risk factor for recurrence, however detection can be limited.

Methods: We apply Weighted Gene Coexpression Network Analysis (WGCNA) to 24 human cSCC transcriptomes (GSE86544) to identify gene clusters associated with PNI. Clusters are ranked by their correlation with PNI, and candidate biomarkers in top clusters are identified based on intracluster centrality, differential expression between tumours with and without PNI, and expression in normal tissues from the Genotype-Tissue Expression (GTEx) project.

Results: We identify 56 gene clusters ranging in size from 31 to 4166 genes. Sixteen clusters show significant correlation with PNI (p < 0.05). These clusters are enriched for genes involved in transcriptional regulation, epithelial-mesenchymal transition (EMT), and extracellular matrix (ECM) remodelling (p < 0.05). The transcription factor TEAD3 has high network centrality in a gene cluster enriched for transcriptional regulators (p < 0.05), is significantly upregulated in cSCC with PNI (fold change = 4.57, p < 0.05), and is preferentially expressed in skin compared to nerve, adipose, and salivary gland tissue. TEAD3 expression significantly correlates with expression of the EMT/ECM gene cluster (p < 0.05).

Conclusion: Our work suggests TEAD3 contributes to transcriptional regulation of cSCC invasiveness, and TEAD3 expression may be useful in identifying high-risk lesions. Further work must be done to characterize TEAD3 protein expression in cSCC.


Secondary syphilis: three-case with different clinico-pathological presentations

S. Lashin*, S. Elsheikh, A. Sharma

*University of Nottingham, United Kingdom; Menoufia University, Egypt

Background & objectives: Syphilis, the old friend, continues in re-emerging with heterogenous presentations which represents a diagnostic dilemma to dermatologist and dermatopathologists. The UK was one of five countries that shows more than double the number of cases over the last 10 years.

Methods: This study aims to highlight the increasing challenge in the diagnosis of secondary syphilis by presenting three cases with different presentations

Results: The first case was a 56-year-old gentleman complaining of fever, chest pain and hearing loss. Followed by the eruption of extensive papular rash over the trunk, palms and soles. Pulmonary scan showed bilateral lung nodules. Histology showed localised granulomatous inflammation with plasma cells and few eosinophils.

The second case was a 43-year-old lady presented with scaly sarcoidal-like nodular lesions on face and chest without mucous membrane involvement. Biopsy showed non-specific perifollicular granulomatous lympho-histiocytic infiltrate with plasma cells.

The third case was a 32-year-old homosexual man developed oral thrush and juicy scaly nodular rash on trunk sparing palms and soles with generalized lymphadenopathy. HIV test was negative and histopathology was non-specific.

Conclusion: Secondary Syphilis can present with non-specific clinical and histological features. Clinical, histological and serological correlation is pivotal for early diagnosis.


Indolent cutaneous T-cell lymphoid proliferations of the nose region - a novel diagnostic algorithm and molecular description

O. Kuczkiewicz-Siemion*, K.A. Seliga, K. Błachnio, Z. Bystydzieński, A. Tysarowski, M. Prochorec-Sobieszek, G. Rymkiewicz

*Institute of Haematology and Transfusion Medicine, Diagnostic Haematology Department, Maria Sklodowska-Curie National Research Institute of Oncology, Department of Pathology and Laboratory Medicine, Poland

Background & objectives: Primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder (pc-CD4+TCLPD) and primary cutaneous acral CD8+ T-cell lymphoma (pc-acral-CD8+TCL) are indistinguishable morphologically and could pose a challenge on histopathological and immunohistochemical examination. The study presents a novel diagnostic approach for these rare entities.

Methods: Two patients (p1, p2) present solitary nasal skin lesions. Skin punch biopsies were obtained with histopathological and immunohistochemical evaluation, which were inconclusive. Subsequently fine-needle biopsies (FNB) of the lesions were performed and flow cytometry (FCM) analysis of immunophenotype and T-cell clonality (IO Test Beta Mark) were done. Finally, both lesions underwent next-generation sequencing (NGS) molecular testing (Archer® FusionPlex Lymphoma panel).

Results: Histopathologically, both cases were characterized by a diffuse focal-epidermotropic dermal proliferation of small to medium-sized lymphocytes. Immunohistochemically neoplastic cells were: CD3(+)/CD4(+)/CD8(-)/CD7(-/+)weaker/CD20(-) (p1) and CD4(+/-)/ CD8(+)/CD7(-)/CD30(-)/granzymeB(+)/perforin(-) (p2).

Subsequent FCM showed distinct origin of pathological lymphocytes. Memory CD4(+): CD45RO(+)/CD45RA(-)/CD4(+)/CD27(+)moderate-high/ CD26(+)low/CD7(-)/CD8(-) (p1) and naive CD8(+): CD45RO(-)/CD45RA(+)/CD8(+)/CD27(-)/CD26(+)/CD7(-/+) /CD4(-) (p2). The monoclonal nature of the pathologic T-lymphocyte population was confirmed by demonstrating the lack of clonality of TCR Vbeta on their surface in both cases. The diagnosis of pc-CD4+TCLPD (p1) and pc-acral-CD8+TCL (p2) was made. Next-generation sequencing analysis revealed the EIF4E3[7]-FOXP1[3] fusion in pc-acral-CD8+TCL sample.

Conclusion: FNB/FCM analysis of immunophenotype and clonality are useful in the final diagnosis of cutaneous T-cell lymphoid proliferations. Study presents the first molecular description of pc-CD4+TCLPD and pc-acral-CD8+TCL. We report a novel FOXP1 fusion, which requires confirmation in a larger study.


Use of pan-TRK immunohistochemistry for identification of NTRK fusions in mesenchymal neoplasms – real life experience

I. Brcic*, T.M. Godschachner, M. Bergovec, J. Igrec, S. Scheipl, K. Kashofer, A. Leithner, J. Szkandera, B. Liegl-Atzwanger

*Diagnostic and Research Institute of Pathology, Medical University of Graz, Austria

Background & objectives: Fusions involving NTRK1-3 are oncogenic drivers occurring in a spectrum of mesenchymal neoplasms. To get further insights into the staining profile with the pan-TRK antibody we performed immunohistochemistry on various soft tissue sarcomas and correlated our results with molecular testing.

Methods: We retrospectively reviewed soft tissue sarcomas diagnosed from 1999 until 2019 at the Institute of Pathology, Medical University of Graz retrieved from the surgical pathology files and consultation cases of one of the authors. Tissue micro arrays from 409 cases were performed and analysed immunohistochemically with pan-Trk rabbit monoclonal antibody (clone EPR17341, Roche/Ventana). Positive cases underwent next generation sequencing (NGS).

Results: Immunohistochemical staining was observed in 18 (4.4%) cases. Five cases showed diffuse nuclear and/or cytoplasmatic staining and each harboured an NTRK-fusion (LMNA-NTRK1, IRF2BP2 -NTRK1, TMB3-NTRK1, ETV6-NTRK3, RBPMS - NTRK3). Thirteen cases (3/31 (9,7%) synovial sarcomas, 2/73 (2,7%) leiomyosarcomas, 2/2 (100%) extraskeletal myxoid chondrosarcomas, 2/116 (0,2%) myxofibrosarcomas, 1/34 (0,3%) atypical lipomatous tumour, 1/33 (0,3%) dedifferentiated liposarcoma) showed weak cytoplasmic/membranous staining and harboured no NTRK Fusion.

Pan-TRK (clone EPR17341, Roche/Ventana) immunohistochemistry is a reliable, highly sensitive but less specific diagnostic marker that can be expressed in non-NTRK-rearranged mesenchymal neoplasms.

Conclusion: Pan-TRK (clone EPR17341, Roche/Ventana) can be used as a surrogate marker for an identification of NTRK fusion, nevertheless, an RNA-based NGS for detection of the specific fusion should be performed to confirm the rearrangement if patients are undergoing targeted therapy.


Human sarcoma nude mice xenografts

A. Llombart-Bosch*, C. Carda, E. Mayordomo, F. Giner, R. Noguera, S. Navarro, I. Machado

*Department of Pathology, University Valencia, Spain

Background & objectives: Biological and molecular studies of human sarcomas require fresh tumour tissue. Nude mice xenografts offer a valuable method to maintain tumours, retaining the histological and genetic characteristics. Since 1990 we have produced a tissue bank of 438 xenografted human tumours.

Methods: Tumour tissue is inoculated subcutaneously in the back of the animal, under sterile conditions (1–2 mm3 of tumour tissue). Nude mice are kept in germ-free conditions (complying with all ethical regulations). The tumour is followed until it reaches 1–2 cm in diameter, and then transferred to new animals. Material obtained is kept for histology, cell culture, EM, and frozen.

Results: Positive grafts (Average 40%, Range 27-60%). By histology and frequency: Osteosarcoma and Chondrosarcoma (44/100), Es/PNET (30/80), MFH (18/48), Rhabdo-Leiomyosarcomas (18/41), Synovial sarcomas (17/28), Liposarcomas (12/39), Fibrosarcomas (4/11), GIST (6/22), Vascular sarcomas (5/8) and miscellaneous tumours (22/61). Total 176 positive grafts out of 438 xenografts (40%). Several cell lines in vitro have been established. Frozen tissue and PFETB are available in most cases.

Conclusion: Sarcoma xenografts in nude mice provide a suitable model for the study of tumour morphology, biology and molecular genetics.

Funding by: Grants PI040822 Instituto Carlos III de Madrid, Spain Contract nº: 018814 (EuroBoNet) 6thFP of the EC and Instituto Valenciano de Oncología Valencia Spain.


Primary and metastatic dedifferentiated malignant melanoma is significantly under-recognized: analysis of 22 cases

A. Agaimy*, R. Stöhr, A. Hartmann

*Institute of Pathology, University Hospital Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Germany

Background & objectives: Dedifferentiated melanoma (primary or metastatic) lacks histological and immunophenotypic melanocytic features; hence cannot be confirmed as melanoma by these conventional tools alone. To date, 38 cases have been reported. We describe 22 cases expanding their phenotypic and genotypic spectrum.

Methods: Cases were identified in our pathology (n=2) and consultation files (n=20). Histological and immunophenotypic findings were reviewed. All cases were negative for melanocytic markers we use routinely (SOX10, S100, pan-melanoma, Melan A, HMB45) except for minimal foci (<5%) of differentiation or immunoreactivity observed in a few cases. Molecular testing was performed (TST15 gene panel; Illumina® Inc., San Diego, CA, USA).

Results: Age range was 31-86 years (median, 62). Fourteen patients were males. Twenty tumours were metastases and two were primary (subungual, anorectal). Initial diagnosis was undifferentiated pleomorphic sarcoma (10), unclassified malignancy (5), other sarcomas (2), metastatic melanoma (2) and reactive (1). In only two cases was dedifferentiated melanoma mentioned. History of melanoma was positive in 13 cases. Sites of metastases were axilla, inguinal, shoulder, gastrointestinal, bone, neck, lung/pleura. Histology was predominantly undifferentiated pleomorphic sarcoma (11). Unusual patterns include osteo-chondroblastic, tenosynovial giant cell tumour-like and low-grade fibromyxoid sarcoma-like. Melanoma-typical mutations were detected in 17/21 (81%) cases (NRAS=8, BRAF=8 and KIT=1). Of 4 wild type tumours, one showed NF1 mutation by extended testing.

Conclusion: This extended follow-up study highlights the phenotypic plasticity of dedifferentiated malignant melanoma and indicates significant underrecognition of this aggressive disease among general surgical pathologists. UPS and other unclassified high-grade sarcomas occurring at unusual sites such as the axilla, inguinal area and the lateral neck should alert to the possibility of dedifferentiated melanoma, even in the absence of positive clinical history. Genotyping represents a valuable adjunct to confirm diagnosis.


Atypical pleomorphic lipomatous tumours: a clinicopathologic analysis of 55 cases

W. Anderson*, C. Fletcher, V. Jo

*Brigham and Women's Hospital, USA

Background & objectives: Atypical pleomorphic lipomatous tumour is a recently described adipocytic neoplasm. Diagnosis and distinction from pleomorphic liposarcoma can be challenging due to its wide-ranging morphology. We present the clinicopathological features of a large case series to further characterise this emerging entity.

Methods: 55 cases were identified from the consultation files. Clinical and follow up information were obtained from referring institutions. Immunohistochemistry for MDM2, CDK4, CD34, Rb, S100 and desmin was performed in cases with available material.

Results: Patients were 20 women and 35 men; median age was 59 years (range: 20-89). Tumours arose in upper limb (33%), trunk (31%), lower limb (29%), head and neck (7%) with size range 2.3-11.5 cm (median: 5.9). Tumours were variably composed of atypical and pleomorphic cells, adipocytes, and lipoblasts, often showing infiltrative growth, myxoid or collagenous stroma, and multinucleated floret cells. Mitoses were infrequent. Tumours expressed CD34 (65%), S100 (64%), and desmin (30%). 86% (38/44) showed Rb loss. Rare cases expressed MDM2 (2%) or CDK4 (13%); FISH for MDM2 amplification was negative (0/11). Follow up in 15 patients so far (median: 33 months) revealed no local recurrences or metastases.

Conclusion: Despite its frequently infiltrative growth, nuclear pleomorphism, and hypercellularity which can mimic sarcoma, atypical pleomorphic lipomatous tumour behaves in an indolent fashion. Immunohistochemistry for CD34, S100, desmin and Rb (demonstrating loss) can support the diagnosis.

OFP-08 Joint Oral Free Paper Session: Endocrine Pathology / Head and Neck Pathology


Diagnostic findings and clinical behaviour of non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) in a large Iberian retrospective series

M. Bella-Cueto*, C. Zafon, E. Anda, N.M. Sanchez-Gomez, G. Carnero, A. Póvoa, C. Blanco, I. Mancha, L. García-Pascual, A. Lázaro, V. Alcázar, I. Capel, J. Maravall, J. Sanz-Gallur, M. Paja

*Parc Taulí Hospital Universitari, I3PT, Universitat Autònoma de Barcelona, Sabadell, Spain

Background & objectives: Providing diagnostic and clinical data about the challenging entity Non-invasive Follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) is of interest.

Objective: To record the diagnostic, management and follow-up data of NIFTP in a large multi-centre Iberian retrospective series.

Methods: Surgical cases of Papillary Thyroid Carcinoma (PTC) and tumours of Uncertain Malignant Potential (UMP) over 5 mm (years 2005 to 2015) were recorded from pathological reports of 16 institutions, and slides reviewed if needed. Diagnostic and clinical data from cases finally considered as NIFTP were recorded.

Results: From 3134 PTC and 14 UMP nodules, 175 (5.55%) cases were considered to meet the original criteria of NIFTP (141F/34M, age range 18-78, average 49). Ultrasound report was available in 142, 56 of them with suspicious findings (39.4%). Pre-surgical fine needle (FN) cytology or biopsy was performed in 150 cases (85.7%), being its result the first reason for surgery in 102 (58.3%). Undetermined Bethesda categories were the most prevalent (52.66).Total thyroidectomy was performed initially in 112 (64%), and deferred in 45 (25.7%), receiving radio-iodine therapy 132 (75.4%), with an average dose of 96.7 mCi. Follow-up disclosed 10 patients with biochemical +/- structural persistence, but no recurrences.

Conclusion: In our series of NIFTP prevailed non-suspicious ultrasonography (60.6%), undetermined Bethesda categories (52.6%) total thyroidectomy procedure (89.7%), and radioiodine therapy (75.4%). No recurrence was identified.


Incidence and clinicopathological features of non-invasive follicular neoplasm with papillary-like nuclear features (NIFTP) in a large Iberian retrospective series

M. Bella-Cueto*, J.M. Cameselle-Teijeiro, A. Ugalde, P. Fernandez-Seara, H. Quiceno, I. Gomez de la Riva, M.V. Ortega, Á. Castaño, C. Gonzalez, S. Lopez-Agulló, M. Chao, E. Roselló, P. Meseguer, J.J. Paricio, C. Iglesias

*Parc Taulí Hospital Universitari. I3PT, Universitat Autònoma de Barcelona, Sabadell, Spain

Background & objectives: Incidence of Non-invasive Follicular Neoplasm with papillary-like nuclear features (NIFTP) varies between countries. Furthermore, histological diagnostic criteria have changed recently.

Objective: To determine the incidence and histological features of NIFTP in Iberian countries applying the original and new diagnostic criteria.

Methods: Sixteen institutions from Spain and Portugal joined the study. Surgical cases of Papillary Thyroid Carcinoma (PTC) and tumours of Uncertain Malignant Potential (UMP) over 5 mm (years 2005 to 2015) were recorded from pathological reports, and slides reviewed if needed. Incidence and histopathological findings of cases considered finally as NIFTP following the original and the new diagnostic criteria were recorded.

Results: From 3134 PTC and 14 UMP lesions, 175 (5.55%) cases were considered to meet the original criteria of NIFTP. Overall incidence among PTC was 4.67% (range: 0-12.12% depending of the institution), and 50% among tumours of UMP. Thirty-five cases measured between 5 and 10 mm. Average size was 21.8 mm (range 5-100 mm). In 95 cases the whole lesion was studied, and in 97 the whole nodular margin. If new diagnostic criteria were applied, 39 cases (22.28%) must be removed (38 with papillae, 1 BRAF V600E mutated), with a final incidence of NIFTP of 4.32%. There were no significant differences regarding the nuclear features when applying the different diagnostic criteria.

Conclusion: In our Iberian retrospective series, incidence of NIFTP was low (5.55%), with moderate variability among institutions. The lesion was studied completely in more than half of the cases. Applying the new diagnostic criteria, 22.28% of them had to be excluded from the series, with a final incidence of 4.32%. Although that, no differences in nuclear features were observed.


Chromophobe renal cell carcinoma-like thyroid carcinoma – expanding spectrum of thyroid cancer with a distinct entity

A. Bychkov*, R. Katoh, T. Amano, K. Ito

*Kameda Medical Center, Nagasaki University, Japan

Background & objectives: Chromophobe renal cell carcinoma (cRCC)-like thyroid carcinoma was first described in 2017 as a distinctive clinicopathological entity of thyroid carcinoma. However, there have been no additional cases reported so far.

Methods: We retrieved four cases of primary thyroid tumours with cRCC-like appearance from the files of Ito Hospital, Tokyo, a large-volume thyroid centre.

Results: There were 3 males and 1 female (mean age 46 years) without history of genetic disorders. Two cases were initially diagnosed as invasive HTT, one as PTC, solid variant, and one as PDTC. All tumours were invasive and showed trabecular/alveolar pattern. IHC revealed TTF1+, PAX8+, Tg-, CD117-, and low Ki67 index. Histochemistry showed weak cytoplasmic colloidal iron staining. One patient developed locoregional recurrences and probable lung metastasis (late after surgery).

Conclusion: cRCC-like thyroid carcinoma revealed to be distinctive in histology and could be misdiagnosed as Hurthle cell carcinoma, HTT, and metastatic carcinoma in thyroid tumour pathology. Our cases were not in association with any finding of tuberous sclerosis complex. Further characterization by immunohistochemistry and genetic analysis in this rare tumour is required to define its position in the histopathological classification.


Distinct morphological and immunophenotypical features of cribriform-morular tumour of thyroid: towards a separate entity

B. Boyraz*, V. Nose

*Department of Pathology, Massachusetts General Hospital, USA

Background & objectives: Cribriform-morular variant of papillary thyroid carcinoma (CMv-PTC) is an extremely rare variant of PTC with cribriform architecture and squamoid morules. In this study, we investigated morphology, immunoprofile and genetics of these tumours to further our understanding of this biologic entity.

Methods: We reviewed twenty-three CMv-PTC cases in this study. Representative blocks from these cases were subjected to immunohistochemical studies using antibodies against B-Catenin, BRAF, HBME1, TTF-1, p63, CD5 and CK5. Molecular studies to identify APC variants were performed in certain cases.

Results: All patients with CMv-PTC were female and ranged in age from 18 to 59 (n=23).

All cases showed strong nuclear and cytoplasmic staining of B-Catenin (23/23+).

Molecular studies revealed pathogenic APC variants (12/12+).

HBME1 (15/15-) and BRAF (4/4-) showed negative staining.

TTF-1 showed positive staining in cribriform areas and negative staining in morules (n=2).

CD5 (8/8+) and CK5 (5/5+) staining highlighted the morules, and the cribriform areas were negative. Morules were negative for p63 (2/2-).

Conclusion: Nuclear B-Catenin positivity and APC mutations are characteristic features of CMv-PTC.

Negative HBME-1 and BRAF staining, TTF-1 staining pattern suggest a different pathogenesis than classical PTC.

The morules are positive for CK5 and CD5, and negative for p63. These findings suggest a different biological pathway for morule formation and can be used to aid diagnosis of this tumour.

Further immunohistochemical and genetic studies would increase our understanding of this tumour leading to reclassification as a separate entity as cribriform-morular tumour of thyroid.


Invasion matters also in encapsulated conventional papillary thyroid carcinoma

E.D. Kuz*, D. Akbulut, S. Dizbay Sak, N. Kurşun

*Ankara University Faculty of Medicine, Pathology Department, Turkey

Background & objectives: We aimed to investigate the histopathological features and BRAF status of understudied encapsulated (conventional) papillary thyroid carcinoma (EC-PTC).

Methods: Among 823 PTC cases diagnosed at our institution between 2015-2018, 121 tumours in 105 patients(12,75%) were reclassified as EC-PTC. Size, localization, cystic changes, background thyroiditis, presence/amount of psammoma bodies(PB), cervical lymph node metastasis(LNM), capsular/lymphovascular invasion(CI/LVI), immunohistochemical BRAF-VE1 expression were evaluated. 92 non-encapsulated conventional PTC cases were chosen as control group. Pearson chi-square and Fischer’s exact tests were performed for analyses.

Results: EC-PTC cases were predominantly women(73,3%), 47% were microcarcinomas, 97,5% had cystic changes, and LNM was positive in 55,2%. 20.7% of the tumours showed total encapsulation, whereas, CI was detected in 79,3%(partial:57,9%, extensive:21,5%). LVI was rare(5,8%). Thyroiditis(48.8%) and PB (52.9%) were detected in nearly half of cases. Relationship between LNM and CI was statistically significant (p=0.008) and was more prominent in cases with extensive CI(p=0.011). PB positive cases showed a higher rate of LNM(p=0.009). BRAF was positive in 81,1% of EC-PTC and 85,7% of nonencapsulated PTC. In nonencapsulated cases, patients older than 40 showed more BRAF positivity (p=0.016), whereas in EC-PTC, there was no difference in terms of age and BRAF(p=0.15).

Conclusion: As in encapsulated follicular-variant PTC, invasion status is important in EC-PTC in predicting clinical behaviour, such as LNM.

Cystic changes are very common, deserves mentioning in morphological characteristics of EC-PTC.

BRAFV600E mutation determined by immunohistochemistry isn't different than non-encapsulated PTC.


The enigmatic mixed medullary-follicular carcinoma of the thyroid: a comparison of 2 cases and a review of the literature

A. Varelas*, M. Farinha, P. Lopes, M. Jácome

*Department of Pathology, IPO-Porto, Portugal

Background & objectives: Mixed medullary-follicular thyroid carcinomas(MMFTC) comprise a partially understood group, including collision tumours, synchronous/metachronous follicular and medullary carcinomas and “true” MMFTC.There are many theories of possible origins of MMFTC, none indisputably proved.

We relate two cases followed at our institute, while reviewing the literature.

Methods: A 65year-old female and 60year-old male patients were followed at our institute since 2011 and 2019 for thyroid nodules. Analytically, only calcitonin levels were increased (1949pg/mL and 428,5pg/mL, respectively). Total thyroidectomy and cervical lymph node dissection were performed in both cases.

Results: Histology showed predominantly medullary carcinomas(calcitonin+, neuroendocrine+).In the first case, there was a slightly juxtaposed population of morphologically-papillary features(thyroglobulin+); the second case had also a confirmed papillary component, deeply intermingled. Both lymph node metastasis had a mixture of papillary and medullary-like features. The final report diagnosis was collision tumour(pT1bN1b) and “true” MMFTC(pT2(m)N1b) respectively, both patients being alive and without evidence of the disease.

Conclusion: While some authors defend a common origin for the components of MMFTC using a “stem cell” or a “divergent differentiation” theory, others suggested a “field effect“, a “collision effect” or a “hostage” theory based on a dual origin. It has been recently questioned whether C-cells are endodermal-derived rather than neural-crest-derived, suggesting the ultimobranchial stem-cell as a possible common cell lineage for MMFTC.

We report two cases, one of which was considered a collision tumour and other one a “true” MMFTC. The etiopathogenesis remains uncertain.


Prognostic significance of oral dysplasia features – analysis of a preliminary cohort

H. Mahmood*, S.A. Khurram

*Academic Unit of Oral & Maxillofacial Surgery, School of Dentistry, University of Sheffield, United Kingdom

Background & objectives: Grading of oral epithelial dysplasia (OED) remains the gold standard for malignant change prediction. However, little is known about the prognostic significance of individual histological features. This study aims to determine the prognostic significance of OED features in a preliminary cohort.

Methods: OED cases with varying grades (n=51) were identified (2005-2013) from the local tissue archive after ethical approval. New H&E sections were obtained, and five-year clinical follow-up data collected. Blind histological examination was undertaken to review the original diagnosis and identify the most prominent features from the WHO Classification for each case. The prognostic significance of specific histological features was measured.

Results: 17/51 (33%) lesions showed malignant progression; 10 (59%) of these were originally graded as moderate dysplasia and 7 (41%) as severe dysplasia. The most significant features predictive of malignant transformation were: loss of epithelial cohesion/acantholysis (RR 3.9, CI 1.4-10.4, p=0.0057), hyperchromatism (RR 4.0, CI 1.6-9.6, p=0.0018), irregular and/or verruciform surface keratination (RR 0.44, CI 0.2-0.9, p=0.0282), dyskeratosis/individual cell keratinisation (RR 4.4, CI 1.1-17.3, p=0.0316) and presence of suprabasal mitotic figures (RR 7.8, CI 1.9-30.6, p=0.0033).

Conclusion: Our preliminary findings reveal feature-specific risk of malignant progression in OED for the first time. These findings need to be explored on bigger cohorts to establish their true significance and provide useful prognostic information to aid management.


Head and neck squamous cell carcinoma of never-smokers and never-drinkers show frequent PD-L1 expression and association of tumour infiltrating lymphocytes with favourable survival

F. Mulder, E. de Ruiter, T. Gielgens, F. van Voorst van Beest, R. de Bree, M. van den Hout, B. Kremer, S. Willems, E.J. Speel*

*Maastricht UMC, The Netherlands

Background & objectives: PD-L1 expression is a predictive biomarker for immunotherapy in head and neck squamous cell carcinoma (HNSCC). Our objective was to assess if PD-L1/2 expression and tumour infiltrating lymphocytes (TILs) in HNSCC of non-smokers and non-drinkers (NSND) have prognostic value.

Methods: Clinical data and tumours of 114 NSND HNSCC patients were collected retrospectively. Immunohistochemistry was performed for PD-L1, PD-L2, and lymphocyte markers PD-1, CD45, and CD8 on Tissue MicroArrays. Difference in 5-year survival of patients with and without expression of these biomarkers was determined using Kaplan-Meier and log rank analysis.

Results: The patients were on average 71.6 years old and predominantly women (77%) with a tumour of the oral cavity (78%). PD-L1 (tumour proportion score ≥1% and ≥50%) and PD-L2 (≥1%) expression was detected in 70%, 18%, and 10% of the tumours, respectively. A high number of CD45 and CD8-positive TILs (≥150/mm2) was a predictor for a significantly better 5-year disease free (HRCD45=.50, p=.010; HRCD8=.51, p=.013) and overall survival (HRCD45=.40, p=.001; HRCD8=.50, p=.014). This significant difference persisted, regardless of which cut-off value for TILs/mm2 was applied.

Conclusion: PD-L1 expression is present in a large percentage of HNSCC in NSND. This might have implications for the application of immunotherapy in this patient group. A high number of CD45 and CD8-positive TILs is predictive for a better disease free and overall 5-year survival.


A subgroup of oropharyngeal squamous cell carcinoma with unfavourable prognosis shows oxidative stress signatures and a mesenchymal-like phenotype

C. Huebbers, O. Siefer, U. Drebber, M. Odenthal, B. Akgül, M. Hufbauer, D. Beutner, J. Klussmann, E.J. Speel*

*Maastricht UMC, The Netherlands

Background & objectives: A subgroup of oropharyngeal squamous cell carcinoma (OPSCC) with integrated HPV 16 shows oxidative stress signatures and an unfavourable prognosis. The objective was to further investigate these oxidative stress signatures and their possible relation to a mesenchymal like (EMT) phenotype.

Methods: 51 OPSCCs including 28 HPV-positive cases (FFPE and frozen tissue) were available. Expression of EMT, retinoic acid and oxidative stress pathway components including NRF2, AKR1C1/3, ALDH1A2, Frizzled 10, b-Catenin, E-Cadherin, Vimentin, miR-9 and miR-16-2 were assessed by immunohistochemistry, RT-qPCR and/or in-situ hybridization. The impact of viral oncogenes on EMT-relevant components was addressed in primary human keratinocytes overexpressing HPV16-E6 and/or -E7.

Results: Expression analysis revealed that subgroups of OPSCC predominantly related to HPV-infection exhibit an increased oxidative stress response (NRF2, AKR1C1/3) as well as activating EMT pathway signatures (Frizzled 10, b-Catenin, E-Cadherin, Vimentin, miR-9 and miR-16-2), frequent metastasis, and downregulated retinoic acid synthesis (ALDH1A2). Moreover, in vitro experiments showed that HPV16-E6 expression results in induction of miR-9, miR-16-2 and b-Catenin expression.

Conclusion: Our data show, that OPSCC presenting with upregulation of oxidative stress response signatures have a higher tendency to undergo EMT. Frizzled 10 expression known to be regulated by retinoic acid was highly correlated to ALDH1A2 expression and inversely correlated to EMT and oxidative stress. Our data implicate that subgroups of tumours might benefit from adjuvant treatment with retinoids, which should be further studied.


Secretory carcinoma of the parotid gland a new entity: a case series

L.E. Salazar Huayna*, C. Alborch-Gil, E. Dacosta, O. Moscoso Miranda, S. Ramón y Cajal, M. Alberola

*Vall d´Hebron University Hospital, Spain

Background & objectives: Secretory carcinoma, first described in 2010 by Skálová et al, is a low-grade salivary gland carcinoma characterized by sharing morphological features with mammary secretory carcinoma. It harbours a recurrent translocation t(12;15)(p13;q25) which causes ETV6-NTRK3 fusion.

Methods: It activates RAS-PI3K-AKT pathway, promoting cell proliferation and survival. Moreover, it’s also known that first generation TRK inhibitor can be used as a treatment.

We report three patients diagnosed with secretory carcinoma at the Vall d´Hebron University Hospital Pathology department from 2017 to 2020.

Results: In our series, two patients were female with a median age of 43 years (range 32-62).The tumour was located in parotid gland. All cases were treated with parotidectomy. Histologically the tumour showed solid, tubular and microcystic structures with abundant eosinophilic bubbly secretion. Neoplastic cells had eosinophilic vacuolated cytoplasm, occasionally with condensed material (mucin) and uniform, round nuclei with small nucleoli. Immunohistochemical stains were performed, the tumour cells were positive for CK7, mammaglobin and S100. In all the cases pan-NTRK immunohistochemistry was positive. The presence of ETV6-NTRK3 translocation was confirmed by Fluorescent in-situ hybridization (FISH) break-apart for ETV6. In only one patient distant metastases and local recurrence have been reported.

Conclusion: Secretory carcinoma is a recently described tumour with distinctive morphology, immunohistochemistry and genetic profile. It is the only salivary gland tumour related to ETV6-NTRK3 gene fusion. Nowadays, its presence is useful for diagnosis and specific treatment.


Revisiting a case series of glomangiopericytomas: reassessing STAT6 value in differential diagnosis

D. Gigliano*, M. Farinha, J. Lobo, P. Lopes, C. Pinto, M.R. Teixeira, M. Jácome

*Portuguese Institute of Oncology - Porto, Department of Pathology, Portugal

Background & objectives: Glomangiopericytoma is a rare sinonasal neoplasm. Despite contrary evidence in literature, we came across a STAT6-equivocal and beta-catenin+ glomangiopericytoma. This prompted a thorough review of all published reports and re-evaluation of histology and immunohistochemistry in all cases from our Institute.

Methods: Four cases were retrieved from our pathology archive. All the histological material and corresponding clinical files were re-examined. New STAT6, beta-catenin and CD34 immunostains were performed on freshly cut formalin-fixed paraffin-embedded sections. Older stains were also reviewed. Furthermore, one case required additional search for mutations of the CTNNB1gene by Sanger sequencing. Literature review was conducted using PubMed.

Results: Two patients were male. The age range was 40-79 years. All cases displayed typical cytoarchitectural and immunohistochemical features of glomangiopericytomas, except for one showing moderate STAT6 nuclear positivity, both in tumoral and peritumoral tissues. Being described as consistently negative in glomangiopericytomas and positive in solitary fibrous tumour (SFT), our STAT6 yielded differential diagnosis issues. Nevertheless, our literature review disclosed rare STAT6-equivocal glomangiopericytomas. Furthermore, Ghaffar et al. described non-specific STAT6 positivity in the nasal mucosa of patients with chronic rhinitis, which our patient had. This phenomenon might lead to misdiagnosis. A mutation in exon 3 of the CTNNB1gene was found in this last case, confirming the diagnosis of glomangiopericytoma.

Conclusion: Glomangiopericytomas represent a diagnostic challenge. Differential diagnosis especially includes SFT, and STAT6 immunostaining plays a key role in discriminating the two entities. Cautious interpretation of STAT6 in patients with chronic rhinitis is advised.


IDH2 R172 mutated sinonasal carcinoma: pathological and molecular assessment of 32 cases

S. Dogan*, S. Gloess, B. Xu, V. Vasudevaraja, A. Jungbluth, M. Snuderl, D. Capper

*Memorial Sloan Kettering Cancer Center, New York, USA

Background & objectives: IDH2 R172 mutations are present in >80% sinonasal undifferentiated (SNUC) and large-cell neuroendocrine carcinomas (LCNEC), while their presence in olfactory neuroblastoma (ONB) remains controversial. Here, we explored pathological features of a sizable cohort of IDH2 mutated sinonasal carcinomas (SNC).

Methods: Thirty-two primary IDH2 R172 mutated SNC were studied by light microscopy and by immunohistochemistry (IHC) including S-100, cytokeratins, synaptophysin and chromogranin. IDH2 mutation status was assessed by targeted exome sequencing or by IDH2 IHC (clone 11C8B1) in most cases. DNA methylation profiles were analysed by semisupervised hierarchical clustering and IDH2 wild-type sinonasal tumours were used for comparison.

Results: IDH2 variants included R172S (61%), R172T (14%), R172K (14%), R172G (7%) and R172M (4%). All cases were high-grade with a median 21 mitoses/10 high-power-fields, frequent necrosis (85%), apoptosis (100%) and prominent nucleoli (97%). Most (94%) were SNUC or LCNEC. Two cases showed glandular/acinar differentiation. While no tumour had definitive features of ONB, two ethmoid carcinomas focally labelled for S-100 (sustentacular pattern) suggesting the olfactory bulb origin. Surface and/or minor salivary glands involvement was detected in 39% cases supporting the Schneiderian mucosal origin. All cases, irrespective of subtle differences as to their putative cell/tissue of origin, formed one distinct methylation cluster and clearly separated from IDH2 wild-type tumours.

Conclusion: IDH2 R172 mutated SNC are phenotypically, genetically and epigenetically remarkably similar suggesting that their biology and clinical behaviour might be primarily defined by their unique molecular signature. A confirmation of these findings in further studies may impact future (re)classification of the current WHO categories: SNUC, LCNEC and ONB.

Funding by: MSKCC Support Grant of the National Institutes of Health/National Cancer Institute - award number P30CA008748 (to SD).

OFP-09 Joint Oral Free Paper Session: Pulmonary Pathology / Thymic and Mediastinal Pathology


Clinical significance of ephrin receptors (Ephs) type-a and -b expression in thymic epithelial tumours

C. Masaoutis*, P. Sarantis, M. Bobos, I. Theochari, N. Tsoukalas, P. Alexandrou, D. Rontogianni, S. Theocharis

*Department of Pathology, Evangelismos General Hospital of Athens, First Department of Pathology, School of Medicine, National and Kapodistrian University of Athens, Greece

Background & objectives: Ephrin receptors (EPHs) are receptor tyrosine kinases (RTKs) implicated in tissue development and homeostasis and are aberrantly expressed in tumours. Here, immunohistochemical EPHs (type-A and -B) expression in thymic epithelial tumours (TETs) was assessed and correlated with clinicopathological parameters.

Methods: TMAs from 98 FFPE TETs excised in 2009-2019 (55 females, 43 males; aged 29-85; histologic types: 12 A, 22 AB, 49 B, 2 micronodular, 13 carcinomas) were stained for EPH-A1, -A2, -A4 -A6, -B1, -B2, -B4, -B6. The relationship between neoplastic and lymphoid cell immunoreactivity score (IRS), histopathological parameters (Pearson’s test) and survival of 35 patients (Mantel-Cox model) was explored.

Results: Epithelial-rich subtypes showed higher EPH-A6 cytoplasmic IRS (B2, B3, carcinoma) (P<0.001) and stronger EPH-A4 IRS (B3, carcinoma) (P=0.011). The immature T-cells, especially in subtypes AB and B1, had a higher EPH-B6 IRS than carcinoma-associated mature lymphocytes (P<.001); carcinomas had a higher lymphocytic EPH-B1 IRS (P=0.026). High lymphocytic EPH-A6 IRS was overall rare, but more common in B1 thymomas (P=0.015). Higher lymphocytic and lower epithelial EPH-B6 IRS correlated with low Masaoka stages (stages ≤II) (P=0.043, P=0.010 respectively). All cases showed variable epithelial and lymphocytic EPH-A2 expression, but clinicopathological associations with EPH-A2 were not reached.

Conclusion: Our study confirms the expression of EPHs type-A and -B in TETs, which is associated with established prognostic parameters, i.e. tumour subtype and Masaoka stage, although correlation with patient survival was not reached. Such findings suggest involvement of these RTKs in thymic neoplasia, as well as their potential utility as treatment targets.


Primary mediastinal germ cell tumours with high prevalence of somatic malignant transformation: an experience from a single tertiary care cancer centre

A. Beg*, A. Sahay, R. Kumar

*Tata Memorial Hospital, India

Background & objectives: Primary mediastinal germ tumours(PMGCT) constitutes 3-4 % of all germ cell tumours(GCT).They account for approx. 16% of mediastinal tumours in adults and 19-25% in children as per western literature. There is hardly any large series on PMGCT from India.

Methods: We have retrospective analysed clinic-pathological features of 101 PMGCT cases, diagnosed over a period 10years(2010-2019) from a single tertiary care oncology centre. Metastatic tumours to mediastinum were excluded.

Results: The study group(n=101) comprised predominantly of males(n=95),with mean age=25yrs(range 3months to 57 year).Tumours were predominantly located in anterior mediastinum(n=99).Non-seminomatous germ cell tumours (NSGCT)(composed embryonal, yolk sac tumour(YST) &teratoma) was the most common histological subtype(n=26), followed by seminoma(n=25),mature teratoma(n=21),YST(n=13),immature teratoma(n=6) , mixed tumours (n=4) and GCT;NOS(n=6).Secondary somatic malignancy was seen in 11 cases and six out of them either died or had progressive disease.

Conclusion: Interestingly, in this series, PMGCT was seen predominantly in young adult males and somatic malignancies was noted in as high as in 10% of cases. Patient with somatic malignancy have aggressive clinical course, hence, extensive sampling and careful histopathological evaluation is recommended for definitive characterization.


Clinicopathological correlations of PD-L1 expression in thymic epithelial tumours using two immunohistochemical assays

C. Masaoutis*, D. Rontogianni, P. Sarantis, M. Bobos, E. Dana, N. Tsoukalas, S. Theocharis

*Department of Pathology, Evangelismos General Hospital of Athens, First Department of Pathology, School of Medicine, National and Kapodistrian University of Athens, Greece

Background & objectives: Thymic epithelial tumours (TETs) range from locally aggressive to frankly malignant. In our study, PD-L1 expression in TETs was assessed by two immunohistochemical assays and associations with clinicopathological parameters, as well as the concordance of the two assays were evaluated.

Methods: TMAs from 98 FFPE TETs excised in 2009-2019 (55 females, 43 males; 29-85 years old; histologic types: 12 A, 22 AB, 49 B, 2 micronodular, 13 carcinomas) were stained using SP143 and SP263 assays. The relationship between epithelial PD-L1 expression at 1%, 5%, 10%, 25%, 50% cut-offs, histopathological parameters (Pearson’s test) and survival of 35 patients (Mantel-Cox model) was explored.

Results: The percentage of PD-L1+ TETs at ≥1%, ≥5%, ≥10%, ≥25% and ≥50% cut-offs was respectively: 45%, 34%, 29%, 19% and 14% for SP143; 54%, 46%, 40%, 36% and 29% for SP263. PD-L1 expression was more frequent in type B thymomas, especially in the epithelial-rich subtypes B2 (67-78%) and B3 (93-100%) [P<0.001 (all assays and cut-offs)]. A suggestive relationship between PD-L1 ≥50% expression and shorter survival [P=,077 (SP143); P=0,068 (SP263)] was noted. Cohen’s agreement (SP143 vs SP263) was: substantial for 5% (k=0.662), 10% (k=0.753) and moderate for 1% (k=0.590), 25% (k=0.575) and 50% (k=0.586) cut-offs.

Conclusion: Epithelial PD-L1 expression in TETs is frequent, variable, significantly associated with histological subtype and less strongly with survival, suggesting a role in TET pathobiology. About half [45% (SP143) vs 54% (SP263)] of TETs are PD-L1 positive (≥1% epithelial expression), therefore potentially eligible for anti-PD-L1 treatment. Discrepant scoring (SP143 vs SP263) is noted and varies with cut-off values. Clinical trials of anti-PDL1/PD1 agents should take into account variability in PD-L1 expression.


Utility of methylthioadenosine phosphorylase (MTAP) immunohistochemistry in malignant pleural mesothelioma: correlation with CDKN2A genomic status and inter-assay agreement

Y.Z. Zhang*, J. Hughes, A. Nastase, A. Mandal, C. Brambilla, A. Rice, J.L. Robertus, W.O. Cookson, M.F. Moffatt, A.G. Nicholson

*National Centre for Mesothelioma Research, National Heart & Lung Institute, Imperial College London, United Kingdom; Department of Histopathology, Royal Brompton And Harefield NHS Foundation Trust, London, United Kingdom

Background & objectives: Deletion of CDKN2A/p16 is one of the hallmark genomic events in the pathogenesis of malignant pleural mesothelioma (MPM). Methylthioadenosine phosphorylase (MTAP) has emerged as a potential cost-effective surrogate marker. Inter-assay agreement and correlation with genomic status however are currently uncertain.

Methods: This is a single-institutional validation study of MTAP IHC for routine diagnostics using whole exome sequencing (WES) and single nucleotide polymorphism (SNP) genotyping as gold standard. Twenty-seven histologically confirmed MPM with matched frozen and FFPE tissue were included. Inter-assay agreement was performed for two commercial clones (EPR6893, Abcam & 2G4, Abnova). Inter-observer agreement was performed between two scorers.

Results: CDKN2A deep deletion was detected in 56% (15/27) of cases. Overall concordance between sequencing and MTAP IHC (EPR6893) was 73%. Focal staining was seen in 22.2% (6/27). By treating focal staining as positive, sensitivity, specificity, positive predictive and negative predictive values were 81.8%, 66.7%, 64.3% and 83.3% respectively, similar to previous studies. No significant difference was observed between the two clones (p=0.180). Inter-observer agreement was good (Cohen’s kappa = 0.713).

Conclusion: MTAP IHC showed reasonable concordance with CDKN2A genomic status. Based on available evidence, EPR6893 and 2G4 clones could be used interchangeably and inter-observer agreement was good. Given detection of CDKN2A status is currently hampered by inadequate assay sensitivity (p16 IHC), limited access to assay platforms (PCR, NGS) or high cost (CDKN2A FISH), our findings support MTAP IHC to be used as a reliable surrogate marker. Financial and logistic considerations need to be taken into account prior to local implementation.

Funding by: National Centre for Mesothelioma Research (NCMR), established via a Department of Health (United Kingdom) grant awarded to National Heart & Lung Institute, Imperial College London.


Atypical mesothelial proliferation (amp) of the pleura: multidisciplinary approach, prognostic stratification and proposal of minimally invasive malignant pleural mesothelioma

Y.Z. Zhang*, C. Brambilla, P.L. Molyneaux, A. Rice, J.L. Robertus, S. Jordan, E. Lim, L. Lang-Lazdunski, S. Begum, M. Dusmet, V. Anikin, S. Popat, W.O. Cookson, M.F. Moffatt, A.G. Nicholson

*National Centre for Mesothelioma Research, National Heart & Lung Institute, Imperial College London, United Kingdom; Department of Histopathology, Royal Brompton And Harefield NHS Foundation Trust, London, United Kingdom

Background & objectives: Atypical mesothelial proliferation (AMP), along with cases limited by sampling, also likely includes those exhibiting morphological features of malignant pleural mesothelioma (MPM) yet showing insufficient invasion. We reviewed these cases with a view to improve early diagnosis of MPM.

Methods: This is a retrospective study including 131 consecutive cases of histologically confirmed AMP diagnosed at our institution (2005-2019), of which 35 (26.5%) had subsequent biopsies showing definitive MPM. Clinicopathological data were retrieved from an institutional mesothelioma database. Prognostic variables predictive of overall survival (OS) were identified using univariate and multivariate Cox regression analyses. Statistical significance was denoted as p<0.05.

Results: AMP had comparable demographic profile to invasive MPM. Multivariate analysis showed lack of subpleural structure sampling (p=0.010) and severe nuclear atypia (p=0.001) were independent predictors of worse OS. Cases fulfilling the criteria of (i) adequate subpleural sampling either without invasion or invasion limited to fibrous pleura, (ii) no severe nuclear atypia, (iii) no necrosis, were associated with significantly improved median OS compared with those without (24.4 vs 12.8 months, p<0.001).

Conclusion: AMP poses challenges to diagnosis and patient management as it encompasses a spectrum of benign and malignant mesothelial lesions. We propose morphology-based criteria to stratify AMP into high and low risk groups. It is likely the former was the result of insufficient sampling whilst the latter represented minimally invasive MPM. This allows MPM to be diagnosed earlier in its natural history. Further refinement using BAP1/MTAP immunohistochemistry as part of a multidisciplinary approach is currently being investigated.

Funding by: National Centre for Mesothelioma Research (NCMR), established via a Department of Health (United Kingdom) grant awarded to National Heart & Lung Institute, Imperial College London.


Congenital pulmonary airway malformations with K-RAS mutations may have malignant potential

J. Wolf*, S.M. Hermelijn, D. den Toom, R.M. Wijnen, R.J. Rottier, J.M. Schnater, J. von der Thüsen

*Department of Pathology, Erasmus University Medical Center, Rotterdam, The Netherlands

Background & objectives: Potential malignant degeneration is often argued as a reason to resect asymptomatic congenital pulmonary airway malformations (CPAM). In this study, we analysed immunohistochemical expression patterns and molecular findings of adult lung tumour markers in CPAM.

Methods: CPAM tissue samples were morphologically evaluated, classified, and subsequently analysed by immunohistochemistry using a panel of lineage differentiating (TTF1/CDX2/MUC2/MUC5ac/CC10) and tumour (p16/p53/Dicer1) markers. Intensity of immunohistochemical staining was quantitatively assessed in normal lung tissue, CPAM and mucinous proliferations (MP) in each sample. Next generation sequencing of adult tumour markers (KRAS/BRAF/EGFR/ERBB2) was also performed separately for the aforementioned categories.

Results: We analysed 50 CPAM samples (Types 1 and 2; n=25 each). Irrespective of the type of CPAM, mucinous cell clusters were detected in 11 cases all characterized by a lack of CDX2, MUC2 and to a lesser extent TTF1 expression, whereas MUC5ac was strongly expressed. P16, DICER1 and p53 showed no aberrant expression. All 11 samples with mucinous proliferations harboured a K-RAS mutation. The normal, surrounding lung tissue showed no genetic alternations.

Conclusion: Immunohistochemistry using TTF1 and MUC5ac may help to identify small MP in the pathological assessment of CPAM. These proliferations were found in both CPAM type 1 and 2 and harboured a K-RAS mutation in all cases. These findings support the notion that a subset of CPAMs may be considered potentially pre-malignant lesions.

Funding by: Educational Fellowship BDIAP


Can pulmonary sarcoidosis with severe fibrosis be considered as a severe form of lung sarcoidosis?

E. Kogan*, S. Demura

*Sechenov University, Russia

Background & objectives: 0,3 - 7,8 % of patient population with lung sarcoidosis (SL) has "malignant course" which leads to the rapid development of honeycomb lung. Target: to study morphological and molecular biological particularities of lung sarcoidosis with severe fibrosis.

Methods: A retrospective clinical and morphological analysis of open transthoracic (46) and transbronchial (10) lung biopsies and autopsy (2 cases) from 58 patients with diagnosed lung sarcoidosis. Serial paraffin sections were stained with hematoxylin and eosin, picrofuchsin Van Gieson. Immunohistochemical analysis was performed with the following markers: MMPs, Dsm, Vim, SMA, Apo-Cas, IGF1, TNF-α, TGF-β, Oсt-4, СD 117.

Results: SL with severe fibrosis - 15 cases (16%). were characterized by multiple sarcoid granulomas with apoptosis of epithelial cells, lymphohistiocytic infiltration, vasculitis and sclerotic changes, the destruction of the basement membranes in the bronchioloalveolar transition zone (BATZ) with the replacement of alveolocytes by cells with a myofibroblastic immunophenotype (SMA, Dsm, Oсt-4 & СD 117 positive). It differed from SL without fibrosis by increased expression of profibrotic molecular markers in the in the epithelium of bronchioles, myofibroblast foci: and granuloma cells (p˂0,05).

Conclusion: Pulmonary sarcoidosis with severe fibrosis can be considered as a severe form of lung sarcoidosis according to the differences of molecular profibrogenic activity of granuloma cells.


Evaluation of pathologic response and immune microenvironment in “Pancoast tumour” series after induction chemo-radiotherapy

F. Fortarezza*, F. Pezzuto, A. Pavan, G. Pasello, S. Frega, R. Polverosi, L. Evangelista, A.S. Fraia, C. Giraudo, M. Mammana, F. Rea, F. Calabrese

*University of Padova, Italy

Background & objectives: The analysis of pathologic response (PR) is crucial for the evaluation of the response to chemo-radiotherapy treatments. The histological analysis of PR is coded for other malignancies in which it seems to play a prognostic role.

Methods: We retrospectively analysed eight consecutive samples of Pancoast tumour surgically resected after induction chemo-radiotherapy. The PR was evaluated according to the last IASCL recommendations [Travis WD, 2020]. Microenvironment assessment included immunostaining for several tumour-infiltrating immune cells (TIICs) (CD3, CD4, CD8, CD68) and PD-L1 expression (tumour proportion score). Radio-metabolic responses and radiomic evaluation were also reported and correlated with our data.

Results: Histologically the tumours were six adenocarcinomas, one squamous cells carcinoma and one non-small-cell lung cancer-not otherwise specified (NSCLC, NOS). Two cases showed a complete pathologic response (CPR) and two other a major pathologic response (MPR); the rate of residual tumoral cells (RTCs) was 10% (median value; from 0 to 60%). No oncogene addiction was detected, PD-L1 was < 1% in all cases. All TIICs had a score >2+, except for CD4+ that scored 1+ in 88%. Tumours with a greater metabolic response showed a higher CD68+ TIICs and lower RTCs (p=0.020 and p=0.042, respectively). No other statistically significant differences were found.

Conclusion: Based on our data, the histological evaluation of the PR does not correlate with the radiological, metabolic and radiomic findings, highlighting the crucial value of morphological analyses. Data from radiomic analyses require larger case series. A precise immune microenvironment assessment with focus on macrophages (CD68+) could represent a marker to include in the evaluation of PR.


Revised MET-FISH evaluation algorithm: a simplified method

R. Castiglione*, N. Duerbaum, B. Holz, M. Wittersheim, E. Binot, S. Merkelbach-Bruse, N. Friedrichs, A. Bosse, R. Buettner, A. Schultheis

*Institute of Pathology, Klinikum Stuttgart, Germany

Background & objectives: MET is altered in ~5 % of non-small-cell lung cancer (NSCLC) and according to the newest NCCN guideline, evaluation of MET should be performed for every advanced-stage NSCLC. FISH-analysis reliably identifies MET-amplified tumours. With a steadily increasing number of cases, currently applied FISH-criteria can be time-consuming for the evaluating pathologist.

Objectives: to propose a faster and equally reliable MET-FISH evaluation algorithm.

Methods: In the present two-centred study, we re-evaluated the MET-amplification status obtained by FISH of n=497 NSCLC. For each case, after counting the first 20 cells, the possible results (negative/low-/intermediate-/high-level amplification as previously defined by us) were compared to the original results based on 60 cells.

Results: Of all 497 cases, 471 (94%) showed identical results when counting 20 or 60 cells. Only 26 cases (5%) showed discrepancy: n=19 (4%) would have been upgraded in a higher diagnostic category when counting 20 cells only and n=6 (1%) would have been downgraded. Most importantly, all high-level amplified and negative cases remained the same. To prevent scoring-errors, we propose the following evaluation algorithm: After thorough examination of the tumour, 20 continuous nuclei are counted in the area with most MET-signals. If the case is negative or high-level amplified, it can be signed out. If it fulfils the criteria for low- or intermediate-level amplification, another 40 cells in two different areas must be counted to reach a total of 60 cells.

Conclusion: We propose a modified FISH-evaluation algorithm for MET-amplification in NSCLC that provides a much faster, yet equally reliable method.


Overexpression of p14/ARF in PD-L1 positive malignant epithelioid pleural mesotheliomas with high nuclear grade

F. Pezzuto*, F. Lunardi, F. Fortarezza, S.E. Vuljan, M. Mammana, A. Boscolo, L. Urso, G. Pasello, F. Rea, F. Calabrese

*University of Padova, Italy

Background & objectives: CDKN2A gene plays a central role in the pathogenesis of malignant pleural mesothelioma (MPM). It encodes for two tumour suppressor proteins, p16/INK4A and p14/ARF. We aimed to determine the correlation between p14, morphology and immune microenvironment, to date still unknown.

Methods: Diagnostic biopsies from 76 chemonaive MPM (54 epithelioid, 18 biphasic, 4 sarcomatoid) were evaluated. Pathological assessment of histotype, necrosis, inflammation, grading, and mitosis was performed. p14/ARF positivity (nuclear and cytoplasmic), PD-L1 (tumour proportion score), Ki-67 (percentage) were evaluated by immunohistochemistry. Inflammatory cells (CD3, CD4, CD8 T lymphocytes; CD20 B-lymphocytes; CD68 macrophages) were quantified as percentage distinguishing intratumoral and peritumoral areas.

Results: P14/ARF was evaluable in 68 patients that showed a sufficient number of tumour cells. A strong nuclear and cytoplasmic positivity was detected in 14 patients (21%) (11 epithelioid and 3 biphasic MPMs). P14/ARF positive epithelioid MPMs showed higher nuclear grade (G3) (p=0.002), higher peritumoral CD4 positive T-lymphocyte percentage (p=0.04) and PD-L1>1% (p=0.04). No other statistically significant differences with morphological findings were found.

Conclusion: p14ARF positive epithelioid MPMs seem to mark a more aggressive histological phenotype, being related to a higher nuclear grade and PD-L1 expression. Larger case series are mandatory to validate our preliminary results thus carrying out more complete statistical tests also with clinical data.


Revised usual interstitial pneumonia/idiopathic pulmonary fibrosis diagnoses after the 2018 International Guideline: a 17-year bicentric experience

F. Lunardi*, F. Pezzuto, C. Giraudo, E. Balestro, A. Fattori, M. Ohana, A. Labani, E. Cocconcelli, R. Polverosi, E. Faccioli, M. Chenard, R. Kessler, S. Hirschi, F. Rea, F. Calabrese

*University of Padova, Italy

Background & objectives: Improved knowledge has highlighted the importance of additional histological and clinico/radiological features for the diagnosis of non-Idiopathic Pulmonary Fibrosis (IPF)/Usual Interstitial Pneumonia (UIP) patients. We present findings of bicentric revised UIP/IPF cases after multidisciplinary discussions(MDDs) according to the 2018 ATS/ERS/JRS/ALAT Guideline.

Methods: Histological and radiological review were performed, categorizing cases as “UIP”, “Probable UIP”, “Indeterminate for UIP”, “Alternative diagnosis” following the guideline. Experienced pathologists reviewed all cases considering also additional morphological features suggestive of connective tissue diseases (CTD):germinal centres,>4 lymphoid follicles/cm2, lymphocyte/plasma-cell ratio≥1, lymphocytic pleuritis, vessel remodelling and of chronic hypersensitivity pneumonitis (CHP): bridging fibrosis, peribronchial metaplasia, centrolobular fibroblastic foci, granulomas/giant-cells. Cinical data were also reviewed.

Results: All 2002-2018 cases with diagnosis of IPF, complete clinical information and available HRCT images (101 patients: 81 explanted lungs and 20 VATS) were independently evaluated by pathologists, radiologists and clinicians; they were then re-categorized after MDDs. In 44% of patients the final diagnosis was IPF, in the remaining 56% it was not a “high-confidence” IPF diagnosis: 16% IPF likely, 10% indeterminate for IPF, 8% IPF likely/non-IPF and 22% non-IPF. After MDDs, 62% of cases were finally categorized as IPF, 13% as CHP, 7% as CTD, 12% as other diseases and 6% remained unclassifiable. Pathological diagnosis of “UIP” showed the best performance in term of sensitivity and specificity than radiological assessment (67%-92% vs 40%-97%).

Conclusion: In conclusion, the rate of IPF diagnoses appears to have been overestimated in the last decade. The current knowledge and good practice of MDDs have significantly improved the diagnostic accuracy. In MDDs, experienced pathologists play a key role to establish a final diagnosis.


Association of genetic variants of glutathione S-transferase with P53 phenotype and DNA damage in non-small cell lung carcinoma

N. Husain*, A.K. Pathak, S. Kant, L. Bala, S. Shukla

*Dr Ram Manohar Lohia Institute of Medical Sciences, Lucknow, India

Background & objectives: Glutathione S-transferases (GSTs), including GSTM1, GSTT1 and GSTP1 are phase II metabolic enzymes which regulate cell signalling and cell death. They may enhance occurrence of mutations in critical tumour suppressors, such as p53 increasing risk of Non-Small Cell Lung cancer(NSCLC).

Methods: A case series of histologically diagnosed NSCLC (n=74) were tested for GSTM1 (Fuc./Del.),GSTTI(Fuc./Del.) and GSTP1 (Ile 105 Val) variants using polymerase chain reaction(PCR) followed by restriction digestion. P53 phenotype was categorized as mutant type with >50% 2-3+ intensity in IHC. DNA damage (n=48) in lymphocytes was assessed by comet assay. Statistical analyses were performed with SPSS version 16 (Chicago, USA).

Results: Age was 54.4±11.4; male:female ratio was 2.36; Study sample included 91.8% adenocarcinoma, and 8.1% squamous cell carcinoma. Mutant type P53 was seen in 62.2 % NSCLC and was significantly associated with GSTM1 del genotype RR1.97(p=0.002) and GSTP1 Val/Val genotype RR1.98(p=0.03). GSTT1 genotype showed insignificant RR1.03(p=1.0), Lymphocyte DNA damage was significantly associated with GSTM1 (Fuc/Del) (Mean±SD 5.50±1.62/8.74±3.46;p=0.001) and GSTT1(Fuc/Del) genotype (Mean±SD 5.60±2.09/8.76±3.20; p=0.001); and with GSTP1 Val/Val genotype (Mean±SD 7.24±2.81;p 0.001).

Conclusion: GSTM1 and GSTP1 genes could play a role in carcinogenesis in NSCLC, possibly through increased frequency of mutations in p53 independently or through known mechanisms of decreased detoxification of carcinogens including tobacco smoke. Mutant type GSTs genotype are associated with increased post chemotherapy DNA damage evidenced in circulating lymphocytes possibly affecting susceptibility of tumour to chemotherapy. Association between mutant p53 phenotype and DNA damage was not seen.

OFP-10 Joint Oral Free Paper Session: IT in Pathology / Autopsy Pathology / Pathology in Favour of Developing Countries / Cardiovascular Pathology / History of Pathology / Electron Microscopy / Other Topics


Deep learning artificial intelligence-based image analysis algorithm for Ki-67

T. Vesterinen*, J. Säilä, J. Arola

*HUSLAB, Department of Pathology, University of Helsinki and Helsinki University Hospital, Finland

Background & objectives: Analysing of immunohistochemical stains is often time consuming, non-reproducible, and prone to subjectivity. The aim of this study was to determine, how a deep learning artificial intelligence (AI)-based image analysis algorithm performs in analysing Ki-67 labelled pulmonary carcinoid tumour samples.

Methods: Study material consisted of 127 pulmonary carcinoid tumours labelled with Ki-67. AI was trained to recognize diaminobenzidine stained Ki-67 positive cells as well as all cells negative for Ki-67. The results retrieved from AI’s Ki-67 algorithm were compared with a conventional area-based image analysis software as well as with the analysis of the pathologist that served as the ground truth.

Results: Altogether 11 658 tumour cells positive or negative for Ki-67 were annotated to train the algorithm. After training, the specificity of the algorithm was 96.8% and sensitivity 96.1%. F1-score was 96.4%. The correlation between the AI model and manual analysis performed by the pathologist was 0.94 (P=0.0001). The correlation between the AI model and the conventional image analysis software was 0.66.

Conclusion: This study showed that AI-based image analysis algorithm yields similar results compared with a human observer. It was superior compared with a conventional image analysis software. Thus, AI-based Ki-67 algorithm can assist pathologists to perform Ki-67 analysis in PC tumours.

Funding by: Finnish Cancer Foundation and Helsinki University Hospital Research Fund


Development of semi-automatic interactive algorithm for annotating histological images of colon epithelial neoplasms

I. Mikhailov*, A. Khvostikov, A. Krylov, P. Malkov, N. Oleynikova, O. Kharlova, N. Danilova

*Lomonosov Moscow State University, Russia

Background & objectives: Complete manual labelling is extremely time-consuming process which limits possibility of developing deep learning algorithms for histological images analysis (e.g. the problem of segmentation of mucous glands). Development of semi-automatic interactive annotation tools helps a lot to solve this problem.

Methods: The proposed algorithm for semi-automatic image segmentation works with scribble annotations and is based on a graph model with 2-stage label propagation. The weights of graph edges are predicted with a CNN, that was trained on a fully labelled dataset.

Results: The developed algorithm was trained and tested on the Warwick-QU dataset as well as PATH-DT-MSU dataset ( containing 80 full-size annotated images of colon epithelial neoplasms. The proposed algorithm works in real time and allows to add new scribbles during the annotation which makes the labelling process interactive. Despite the high accuracy of the proposed algorithm we also offer a “classical” set of manual annotating tools to postprocess the results of the algorithm thus allowing annotator to finalize the annotation down to the smallest details. The developed algorithm allows to perform interactive labelling with scribbles and reduce the annotation time of one image from 150 minutes to 25-30 minutes.

Conclusion: Using this semi-automatic interactive tool will significantly increase the number of fully annotated images of colon epithelial neoplasms in PATH-DT-MSU which is necessary for the development of real diagnostic algorithms. The work was supported by RFBR grant 19-57-80014 (BRICS2019-394).


Giant cell interstitial pneumonia: value of scanning electron microscopy coupled with energy dispersive X-ray spectroscopy analysis

F. Fortarezza*, M. Della Barbera, F. Pezzuto, F. Lunardi, E. Faccioli, F. Rea, F. Calabrese

*University of Padova, Italy

Background & objectives: Hard metal lung disease (HMLD) is an occupational disease caused by exposure to hard metal particles histologically characterized by giant cell interstitial pneumonia (GIP). We present a case series of HMLD studied by histology and scanning electron microscopy with microanalysis.

Methods: Surgical pathology files from 2006 to 2019 were searched for lung biopsies and explanted lungs with morphological diagnosis of GIP. A detailed clinical history was collected in all cases. Sections from formalin-fixed paraffin-embedded (FFPE) of two transbronchial biopsies, one cytoblock of bronchoalveolar lavage and from native lung tissue were analysed at Scanning Electron Microscopy and Energy Dispersive X-ray spectroscopy (SEM/EDX).

Results: All patients had clinical history of occupational exposure (metal workers, miner, railway maintenance worker), none reported a clear-cut hard metal exposure (tungsten carbide or cobalt). At histology all lung samples showed features of GIP with multinucleated giant cells of both alveolar macrophage and type II pneumocytes. SEM/EDX analysis was successfully performed also in small transbronchial biopsies and cytoblock. Only one patient (with both biopsy and cytology) had detectable tungsten level. Other metals and inorganic chemical compound were detected in the remaining cases.

Conclusion: SEM coupled with microanalysis, the gold standard tool for the detection of environmental dusts, may be performed also in FFPE tissue of small samples or cytology. SEM/EDX is ongoing on additional FFPE fragments of explanted lung and the final data will be presented at the next ECP-IAP congress.


Evolution of pathology repositories – from kunstkammer to digital pathology

J. Gulczyński*, S. Goertz, T. van de Wetering, L. Ventura, P. Paluchowski, E. Izycka-Swieszewska

*Dept. of Pathology and Neuropathology, Medical University of Gdansk, Pathomorphology Dept. Copernicus Hospital, Poland

Background & objectives: Description how collections of anatomy/ pathology specimens changed in time. Serving at the beginning multiple purposes like art or medicine, slowly became didactic aid. Nowadays we face the computer revolution on massive scale and pathology also entered the digital world.

Methods: We analyse early anatomical drawings and models accompanied by digital collections of microscopic slides of contemporary and ancient tissue samples. Through evolution of recreating nature, we move to collections of specimens in new dimension. We present the consecutive steps to contemporary microscopy supported by digital equipment with efficient and accurate representation of specimen collections from everyday work to ancient research.

Results: We present the evolution of pathology representations. From collections of objects showing gross pathology, through the world of art to science, with specimens as didactic help. Presented in special cabinets, then dedicated rooms and museums. Those collections started with basic figurative sculptures presenting anatomy and pathology, through drawings to spatial models. At the beginning made of wood, bone up to wax resembling human tissues. It was necessary when preserving real tissue was impossible. The invention of fixatives let show real-life pathology. Development of microscopes let observe the cellular pathology and stained microscopic slides could also be stored. Development of collections with consecutive evolution to digital pathology and scanned image databases.

Conclusion: Analysing the timeline we observe that the quality and amount of information carried by the collection’s changes accordingly to the of development of science, including all aspects of presentation. From organized rooms and buildings, we move into digital realm not limited by any physical boundaries. We go through macroscale to microscopy, augmented by 3D representation, even on the cellular level, but also to virtual images of anatomopathological collections.


Encephalitis lethargica from the autopsy reports of the University of Turin: a brief history full of doubts

L. Ferrari*

*ASL AT Asti, Italy

Background & objectives: Since the greatest virulence of Encephalitis lethargica was during the Spanish flu pandemic, an archive search was carried out on the old autopsy reports to investigate the relationship between Encephalitis lethargica and Spanish flu in Turin.

Methods: The cases were selected based on the diagnosis of “Encephalitis lethargica” which was clearly indicated on all the selected reports. The time period considered was that of the spread of Encephalitis lethargica, between the years1915 and 1926 according to the spread of the pandemic.

Results: The first case of Encephalitis lethargica in Turin dates back to 20th January 1920. The autopsy was performed on a 30-years-old male who died at St. John Hospital. No special autopsy findings were reported except a red colour of the meninges. There were a total of 14 cases all in the year 1920. None of them were affirmed by the pathologists of Turin. Indeed, many question marks were written near the diagnosis.

Conclusion: The autopsy reports show the presence of the Encephalitis lethargica in Turin at the end of the pandemic of Spanish flu. A big doubt seems to emerge from these reports due to the scepticism of the pathologists.


Comparison of antemortem clinical diagnosis and post-mortem findings in intensive care unit patients

S. Rusu*, P. Lavis, V.S. Domingues Salgado, M. Van Craynest, J. Creteur, I. Salmon, A. Brasseur, M. Remmelink

*Department of Pathology, Erasme Hospital, Free University of Brussels, Belgium

Background & objectives: The autopsy is an important tool to advance medical knowledge and an excellent assurance indicator of patient management.

The objective is to analyse the discrepancies between premortem clinical and post-mortem pathology findings in adult patients deceased in Intensive Care Unit(ICU).

Methods: We retrieved the clinical and post-mortem findings of all patients who died in the ICU of Erasme University Hospital, Belgium from January 1st 2016 to December 31st 2018 and underwent autopsy. Afterwards the comparisons between clinical and histological findings were classified in five classes according to the classification proposed by Goldman et al.

Results: The 2016’ series included 141 complete autopsies. Post-mortem examinations revealed unexpected findings in 63 cases (47%), of whom 20 (14.2%) were classified as major discrepancies. The first two major misdiagnosis were malignancy/metastasis (9 [6.4%]) and pulmonary embolism (4 [2.84]). There is no statistical difference between major-minor discrepancies and the ICU stay and age respectively. Compared to the study conducted by C. Maris et al. in 2007, a discrete decrease of the frequency of major discrepancies was observed (14% vs 19%) and the absence of statistical difference in discrepancies with regard to length of stay in the ICU was observed (p=0.21 vs p=0.008).

Conclusion: Although the incidence of major discrepancies identified is low and in discrete decrease compared to our previous study, autopsy remains a valuable tool to asses the causes leading to death, to assess the accuracy of clinical diagnosis and for training.


Cardiac amyloidosis in the elderly is not related to Alzheimer’s disease or cerebral amyloid angiopathy

M. Martins Garcia*, J. R. Stone

*Massachusetts General Hospital, USA

Background & objectives: Cerebral amyloid angiopathy (CAA), Alzheimer's disease (AD) and cardiac amyloidosis due to transthyretin are all age-related diseases characterized by amyloid deposition. In this study, the relationship between cardiac amyloidosis and AD/CAA was investigated in autopsies of elderly patients.

Methods: Autopsies with cardiac amyloidosis in patients 80 years old or older were matched with control patients without cardiac amyloidosis based on age and gender. Patients with AL amyloidosis were excluded. The association between cardiac amyloidosis and CAA or severe AD (Braak stage V or VI) was assessed by Fisher test. P values less than 0.05 were considered significant.

Results: Sixty-five patients with cardiac amyloidosis met the study criteria and were matched with 65 control patients without cardiac amyloidosis. CAA was present in 17 (26%) of the cardiac amyloidosis cases and in 13 (20%) of the controls (p=0.53). Severe AD was present in 11 (17%) of the cardiac amyloidosis cases and in 14 (22%) of the controls (p=0.66). The amyloid stained positively for transthyretin by immunohistochemistry or immunofluorescence.

Conclusion: Some recent studies have suggested a relationship between AD and cardiac amyloidosis in the elderly, potentially resulting from the release of amyloidogenic peptides from the brain into the bloodstream in AD. However, in this series of patients, there is no significant association between cardiac amyloidosis in the elderly and severe AD or CAA. The cardiac amyloid in these elderly patients is derived from transthyretin.


Implementation of a cardiac proforma to autopsy practice

K. Tilley*, S. Wright

*Queen Elizabeth University Hospital, Glasgow, United Kingdom

Background & objectives: There is considerable variation in the way in which pathologists approach heart dissection. To improve the quality of reporting, a proforma was introduced. A subsequent audit was conducted to see if there was improvement in the recording of heart measurements.

Methods: Training was given on how to use the cardiac proforma, specifically on how to undertake the measurements required. Following this, the proforma was introduced. We subsequently extracted specific data items from our departmental post-mortem reports before and after the introduction of the cardiac proforma.

Results: Undertaking heart examination in a systematic way with reproducible results helps to inform clinico-pathological correlation and the need for genetic testing. Since introducing the proforma, septal wall thickness measurement increased by 30%, left ventricular wall thickness by 12% and right ventricle wall thickness by 10%. The ventricular diameters measurements increased by 8% and 10% more cases commented upon the pattern of ventricular hypertrophy. Measurement of aortic valve circumference/leaflet number rose by 53%.

Conclusion: Following the introduction of the proforma, there was an improvement in the number of measurements recorded when examining the heart during autopsy. User satisfaction has also increased. We recommend the use of cardiac proforma in the autopsy setting.


Valid diagnostics from autopsy samples: proteomical analysis of the post-mortem interval-dependent human tissue degradation

E. Kocsmar*, I. Kocsmár, M. Cosenza-Contreras, M. Schmid, G. Rácz, A. Kiss, M. Werner, P. Bronsert, O. Schilling, G. Lotz

*2nd Department of Pathology, Semmelweis University, Hungary

Background & objectives: Comprehensive analyses of changes in the composition of proteins using high resolution mass spectrometry-based proteomics might provide insight into post-mortem decomposition processes. Here we characterize the dynamics of protein degradation in human autopsy samples of the liver, kidney and lung.

Methods: Snap-frozen tissue samples were processed for liquid chromatography-tandem mass spectrometry with label-free quantification. Representation pattern of the proteins in the mass-composition were identified by comparative analysis of co-expression networks of the targeted tissues. Stable and unstable proteins were coupled with the Gene Ontology (GO) database and analysed according to molecular functions.

Results: Patients were included with predefined exact post-mortem time intervals (6-12-18-24-48-96 hours; 78 tissue specimens of 26 patients). Dynamic changes in representation of proteins in the mass-composition were different in the investigated organs. The kidney tissue cytoskeletal proteins remained stable by the increasing time intervals whereas proteins of the mitochondrial respiratory chain complex became underrepresented. In the liver, proteins of the lipid metabolism became underrepresented while pigment storage-related proteins accumulated by the time. Contrary to the liver and kidney, proportion of the lung cytoskeletal proteins decreased while proteins involved in activation of neutrophil granulocytes and lipid metabolism increased by the time in the mass-composition.

Conclusion: Protein degradation is one of the major processes in tissue decomposition after death. Understanding the biology behind the post-mortem interval-dependent changes of the mass-composition offers breath-taking opportunities for diagnostic and therapeutic improvements and paves the way for proteomic quality analyses of snap-frozen and formalin fixed-paraffin embedded tissue specimens.

This study was supported by the Start-up Grant of Semmelweis University (11722).


Review of water-related deaths in Oxfordshire over the last 22 years

A. Julai*, K. Collis, A. Srinivasan, I.S. Roberts

*Department of Cellular Pathology, John Radcliffe Hospital, Oxford, United Kingdom

Background & objectives: The UK National Water Safety Forum reported 585 water-related deaths in 2018. Most water-related deaths are presumed to be accidental drownings. Autopsy reports on water-related deaths in Oxfordshire over 22 years was reviewed to identify factors contributing to these deaths.

Methods: Water-related deaths (n=194) in Oxfordshire (1997-2019) were identified by search of the electronic adult post-mortem database and the Coroner’s autopsy reports were reviewed.

Results: The mean age of individuals dying in water was 57 years (range 15-96 years). 70% of the deaths occurred in men. The month of August witnessed the most deaths. Water-related deaths were more likely to occur in the summer for individuals under 30 years-old and in the winter for those above 60. 71% were deaths due to drowning of which 32% were directly contributed to by medical conditions. Toxicology results were available for 107 autopsies; 28% of deaths due to drowning were associated with alcohol and/or drug intoxication. 22% of individuals had a history of psychiatric illness; 10% of deaths were attributed to suicide and occurred mostly in the spring.

Conclusion: A majority of water-related deaths occurred in men and the most common cause of death was drowning. Alcohol/drug intoxication and pre-existing medical conditions frequently contributed to drowning.


Implementing digital pathology in a large-scale laboratory in Brazil: one year of experience

L. Maia*, L. Berriel, R. Salomão, V. Krauss Silva, C. Scapulatempo Neto

*DASA, Brazil

Background & objectives: The implementation of digital pathology in Brazil is an opportunity to provide diagnostics to a larger number of people. It has the potential to overcome some of the urgent demands of serving a population of 210 million and 3,500 pathologists.

Methods: DCP was implemented at the main laboratory that processes 50% of the cases. Five Philips UFS scanners were installed and operate 24/7. Eighteen pathologists sign out using glass slides and 14 pathologists report 100% digitally from distant locations.

Results: 260,000 cases were reported digitally in 2019, which represents >65% of the case volume of the main laboratory. The cases have been signed out by pathologists in 8 different cities from 6 different states.

Conclusion: The implementation of digital pathology in large scale is technically challenging and costly. The adaptation of the laboratory, pathologists and technical staff is a complex process. DP implementation allowed the reference lab to greater a network of specialized pathologists.


How does it feel to be a pathologist? Results of a nation-wide survey on job satisfaction of pathologists in Turkey and comparison with Minnesota satisfaction questionnaire

B. Pehlivanoglu*, H. Hassoy, G. Gul, U. Aykutlu, B. Doganavsargil

*Adiyaman University, Turkey

Background & objectives: Job satisfaction has a direct impact on workplace performance and there is limited research regarding pathologists’ job satisfaction.

Methods: We conducted a 59-item web-based survey questioning respondents’ institutional background, history of training (HT), workplace (technical and physical) conditions (WPC), professional well-being (PWB) and job satisfaction level. Likert-type and open/close ended questions were asked and cored. The participants were also asked to complete Minnesota Satisfaction Questionnaire-Short Form (MSQ).

Results: Of the 321 participants, 75% were female and median age was 41±8.82 (range 28-71 y.o.). The respondents’ experience as a pathologist ranged between 0.12 years and 44 years (mean 11.4±9.16 years). Academic pathologists had significantly higher scores of MSQ, HT, WPC and PWB. Senior pathologists working at larger laboratories were significantly more satisfied of their WPC, with higher scores of MSQ and HT. Overall, 83% of participants were happy to have chosen pathology, but 45% reported to experience the feeling of burnt out. Most (98.4%) agreed that pathologists have a critical impact on patient management, but 92% thought that patients barely know pathologists’ importance.

Conclusion: Our findings suggest that pathologist’s job satisfaction increases with years of experience, physical and technical quality of the pathology laboratory/institution, and professional well-being, i.e., to maintain work/life balance. Pathologists seem to be aware of their important role in patient management although pathology remains to be invisible to the patients.


Evaluation of two-photon fluorescence microscopy for sectioning-free virtual H&E imaging of different tissues

M. Strauch*, J.P. Kolb, W. Draxinger, N. Merg, J. Hundt, S. Karpf, R. Huber

*Institute for Biomedical Optics, University of Lübeck, Germany

Background & objectives: The pathology workflow relies on preparing paraffin sections with a thickness of one cell layer. We develop a two-photon fluorescence microscope based on optical fibre lasers to create H&E images from bulk tissue samples without sectioning in less than 1h.

Methods: A bulk tissue sample is stained with acridine orange (AO) and sulforhodamine 101 (SR) for <10min. The two-photon fluorescence microscope creates an image of the sample within ~20 min/cm2. The obtained data is transformed to a virtual H&E image in 10min. Two-photon fluorescence can be used to scan a section or a 3D volume of a bulk tissue sample.

Results: We measured a set of different tissues with our self-built two-photon fluorescence microscope to show a proof of concept for a digital and fast sectioning-free H&E imaging technique with similar quality to paraffin sectioning. It allows staining with AO&SR as well as H&E. By improving the microscope scanner and data processing, we expect to increase the speed of our two-photon fluorescence microscope to a few minutes per square centimetre.

Conclusion: Virtual H&E imaging provides an alternative to conventional bright field microscopy of stained paraffin sections in a fraction of time. We will further investigate the usage of fluorescent immunostains and potential combination with other imaging modalities (e.g. Two-photon fluorescence lifetime imaging) for in vivo application.

Funding by: European Union (ERC CoG no.646669), German Research Foundation (HU1006/6,EXC 306/2), German Federal Ministry of Education and Research (BMBF no.13GW0227B: “Neuro-OCT”), European Regional Development Fund CELLTOM.

OFP-11 Joint Oral Free Paper Session: Cytopathology / Neuropathology / Ophthalmic Pathology


Primary sarcomas of the orbit: 15-year experience from the Queen Elizabeth University Hospital, Glasgow

K. Kinch*, F. Roberts

*Department of Pathology, Queen Elizabeth University Hospital, Glasgow, United Kingdom

Background & objectives: In adults, primary orbital sarcomas are rare, limited to individual case reports and small case series in the literature. Here we review our experience with these tumours at the Department of Pathology, Queen Elizabeth University Hospital, Glasgow.

Methods: Seven cases were identified from the institutional database. Three were classified as post-radiation sarcoma (PRS) using the following diagnostic criteria: 1. the sarcoma arose in a previously irradiated field; 2. it was histologically distinct from the original neoplasm; 3. it was not present at the time of radiotherapy; and 4. there was a latency period of at least 2 years.

Results: A variety of histological types were encountered, including malignant peripheral nerve sheath tumour (MPNST) (2); atypical lipomatous tumour/well-differentiated liposarcoma (ALT/WDLS) (2); malignant solitary fibrous tumour (SFT) (1); rhabdomyosarcoma (1); osteosarcoma (1); and low-grade sarcoma of uncertain histogenesis (1). PRS accounted for 43% of cases, with an average age at diagnosis of 40 years and a median latency period of 15 years (range 12­-45).

Conclusion: This review represents one of the largest series to date, with seven cases identified over a 15-year period between January 2005 and January 2020. Primary orbital sarcomas are rare and difficult to treat. Whereas in children embryonal rhabdomyosarcoma predominates, in adults they comprise a variety of histological types. Irradiation is a significant risk factor and patients who receive radical radiotherapy require long-term follow-up.


The genetic background of conjunctival melanoma and correlation with recurrences and metastasis

J. van Ipenburg*, N. van Poppelen, Q. van den Bosch, J. Vaarwater, T. Brands, B. Eussen, D. Paridaens, N. Naus, E. Kilic, A. de Klein, R. Verdijk

*Erasmus MC-University Medical Center, Rotterdam, The Netherlands

Background & objectives: Conjunctival melanoma have limited treatment options once a lesion has metastasized. The aim of this study is to elucidate the genetic background of conjunctival melanoma within the spectrum of ocular melanoma, correlated with the development of recurrences and metastasis.

Methods: Twenty-eight conjunctival melanoma from the Rotterdam Ocular Melanoma Study group were collected and DNA was isolated from formalin fixed paraffin embedded tissue using lysis buffer and Chelex. DNA from fresh tumour tissue was isolated using the QIAmp DNA-mini kit. Targeted next-generation sequencing was performed using a panel covering GNAQ, GNA11, EIF1AX, BAP1, BRAF, NRAS, c-KIT, PTEN, SF3B1 and TERT.

Results: Preliminary data show recurrences and metastasis in ten (36%) and nine (32%) conjunctival melanoma cases respectively. BRAF mutations were most common, but also NRAS, c-KIT, PTEN, TERT and SF3B1 mutations were observed. No mutations in GNAQ, GNA11 and EIF1AX were found. There was no significant association with the development of recurrences or metastasis.

Conclusion: Conjunctival melanoma and uveal melanoma are both ocular melanomas. Yet, based on molecular characteristics our data suggest a different pathogenesis in conjunctival melanoma compared to uveal melanoma, with involvement of BRAF, NRAS, c-KIT, PTEN, SF3B1 and TERT mutations occurring mainly in conjunctival melanoma and less frequent in uveal melanoma, although there are overlapping gene mutations. In our study no association was found between specific mutations and recurrences or metastasis.

Funding by: Stichting Nederlands Oogheelkundig Onderzoek Stichting Wetenschappelijk Oogheelkundig Onderzoek


Morphological study of perivascular interstitial cells in glioblastoma

L. Mitrofanova*, A. Hazratov, B. Galkovsky, A. Gorshkov, D. Gulyaev, D. Bobkov

*The Almazov National Medical Research Centre, Russia

Background & objectives: Telocytes (Tcs) and pericytes (Pcs) are two types of perivascular interstitial cell known to be widespread in various organs and tissues, including the brain. Objective: morphological study of Tc and Pc role in glioblastoma (GBM) neovascularization.

Methods: Samples from 15 GBM, 10 diffuse astrocytomas and 5 tumour-free frontal lobes (control samples) were studied. We used immunohistochemistry and double immunohistochemical staining with antibodies to GFAP, Ki-67, CD117, NeuroD1, NG2, CD34, SMA, connexin43; confocal laser scanning microscopy of glioma cultures and frozen and paraffin embedded GBM sections. Electron microscopy of GBM was performed in 4 cases.

Results: The presence of Tcs and Pcs was shown in GBM sections and glioma cultures. The Tc immunophenotype was CD117+/CD34+/connexin43+/NeuroD1+. The Pc immunophenotype was SMA+/NG2+/CD13+. The number of Tcs in GBM specimens was 10 times more than in astrocytoma. We also identified CD13/CD117 and CD34/NG2 co-expressing cells in GBM blood vessels.

Conclusion: Four immunophenotypes were found in GBM vessels, corresponding to endotheliocytes, Pcs, Tcs, and a mixed Pc/Tc immunophenotype. Study of these cell types and their roles in brain tumour oncogenesis will likely enable improved targeted therapies.


Copy number alterations of chromosome 1p could be helpful to differentiate spindle cell oncocytoma from pituicytoma

V. Barresi*, M. Simbolo, M. Gessi, S. Rossi, M. Caffo, A. Eccher, F.F. Angileri, S. Cannavò, M. Brunelli, A. Scarpa

*Department of Diagnostics and Public Health, Section of Pathology, University of Verona, Italy

Background & objectives: Spindle cell oncocytoma (SCO) and pituicytoma are tumours of the posterior pituitary with overlapping morphology. They are both classified as grade I, but SCO has higher local invasiveness and more frequent recurrence rate. This study aimed to analyse which histopathological, immunohistochemical and genetic features may be helpful in the differential diagnosis between these two tumours.

Methods: We reviewed the histological and immunohistochemical features of 7 SCOs and 4 pituicytomas. In addition, we analysed the mutational and copy number variation (CNV) profile of all cases using the Oncomine Tumour Mutational Load assay, which covers 409 cancer-related genes, and Fluorescent In Situ Hybridization (FISH) for chromosomes 1p and 1q.

Results: All SCOs, but not pituicytomas, had lymphocytic infiltrate. 3 SCOs and 1 pituicytoma had sufficient DNA for sequencing and had no mutations in any of 409 genes analysed. At CNV or FISH analysis 7/7 SCOs, but none of 4 pituicytomas, had loss or gains of chromosome 1p.

Conclusion: CNV of chromosome 1p could be potentially used in the differential diagnosis between these SCO and pituicytoma.

This study was supported by Research and Mobility 2017 Funding of the University of Messina to MC and VB.


Cytokeratin 18-negative somatotroph pituitary neuroendocrine tumours (adenomas) are not clinically distinct tumour subset and often express internexin-alpha

J. Soukup*, T. Cesak, M. Manethova, L. Michnova, D. Netuka, L. Popovska, J. Cap, F. Gabalec

*The Fingerland Department of Pathology, University Hospital and Faculty of Medicine Hradec Kralove, Charles University, Czech Republic

Background & objectives: Low molecular weight cytokeratin (LMWK: CAM5.2 or cytokeratin 18) expression is used for subclassification of somatotropinomas. A small subset is LMWK immunonegative and these have not been well studied: we decided to explore clinicopathological features of this subgroup.

Methods: 105 somatotroph tumours with known clinical features (tumour size, levels of IGF1, GH, prolactin, TSH) were analysed using antibodies against cytokeratin 18, prolactin, growth hormone, βTSH, Ki67 and p53. E-cadherin, SSTR2 and SSTR5 expression was recorded in form of H-score. In cytokeratin-negative tumours (<10% of immunoreactive cells), additional antibodies (AE1/3, CK8/18, vimentin, neurofilament and internexin-alpha) were used for further characterisation.

Results: In total, 10 tumours (9,52%) stained negatively for CK18. Compared to the rest of tumours (n=95), we observed no difference with respect to the tumour size, biochemical or other pathological features. The results were similar when compared with subgroups of sparsely granulated (SGT, n=39) and non-sparsely granulated (nSGT, n=46) tumours. CK18-negative tumours showed lower E-cadherin expression compared to nSGT but not SGT (p<0,01), while SSTR2 expression was comparable to nSGT group and significantly higher than in SGT (p<0,01). One tumour stained for cytokeratin 8/18 and AE1/3 and 6 tumours stained for internexin-alpha in more than 10% of cells (mean 74% positive cells, SD±26,1%) in morphological patterns similar to LMWK.

Conclusion: Cytokeratin 18-negative somatotroph tumours are not a distinct clinicopathological subgroup. They may show lower E-cadherin expression and majority of these tumours express internexin-alpha, while vimentin and neurofilament light chain are not expressed at all.

Supported by MH CZ NV19-01-00435.


Metastatic tumours of the central nervous system and their prognostic outcomes: a clinicopathological evaluation of 221 cases

E. Gun*, F. Cakalagaoglu, A. Kahraman Akkalp, I.E. Sevin, N. Yuceer

*Izmir Katip Celebi University Ataturk Training and Research Hospital, Department of Pathology, Turkey

Background & objectives: The involvement of the central nervous system (CNS) with metastatic tumours is seen frequently since it has a strong neurovascular network. We aimed to evaluate the histopathological characteristics and the prognostic outcomes of the patients with metastatic tumours of the CNS.

Methods: All patients who had a diagnosis of a CNS tumour between January 2006 and January 2020 were re-evaluated with the help of clinical, morphological, immunohistochemical, radiological and prognostic findings. The age, gender, primary tumour, histopathological diagnosis, survival time of the patients, the duration between the metastasis and the primary tumour, the location and number of metastases in the brain were recorded and analysed.

Results: Of all 936 patients operated with a CNS tumour, 221(23.6%) patients were diagnosed with metastasis.34.8% was female and 65.2% was male. The mean age was 58.3.The most common primary site was the lungs(55.7%) followed by breast(14%) and colon(7.7%). There were multiple metastases in 37.6%.In 50.2%, first diagnosis was made with the metastasis. In patients with a previously known primary tumour, the median interval between the diagnosis of the primary tumour and CNS metastases was 13.2 months. Mean overall survival time was 6.9 months. There wasn't a statistically significant correlation between the primary origin of the metastases and the survival time. The patients with multiple metastases had a significantly worse prognostic outcome (p=0.002).

Conclusion: Clinical symptoms and radiological features can be diagnostic in CNS tumours, however; as our study shows, brain metastasis can be the first onset of previously unknown primary tumour. Histopathological evaluation is crucial if the differential diagnosis includes a primary tumour or unknown primary site.


Implementing diagnostic digital cytology into routine clinical practice

A. Bychkov*, T. Hori, Y. Koyama, S. Yoshida, A. Yoshikawa, Y. Masuzawa, J. Fukuoka

*Kameda Medical Center, Nagasaki University, Japan

Background & objectives: Adoption of digital cytology into routine pathology workflow is challenging due to technical limitations. There are only few reports on successful implementation available, mainly limited to remote rapid onsite evaluation. Other case uses of digital cytology are largely unexplored.

Methods: Kameda Medical Center with approximately 22.000 cytopathology cases annually served as a model institution. The routine cytologic workflow included two-step screening by cytotechnologists followed by a sign out by pathologist. Available equipment suitable for digital cytology were digital microscope with live video output (Olympus), robotic microscope (Sakura), Panoptiq microscopic digital imaging platform, and slide scanner with Z-stack mode (Motic).

Results: We introduced sign out of cytologic cases using live digital microscope operated by cytotechnologist, which allowed reviewing slides remotely by pathologist via video streaming (1800 cases in 16 months). We also provided cytologic correlation to support the virtual slide-based sign out of histopathological specimens (2560 cases in 32 months). In addition, positive cytology cases are archived for integration into LIS and for prospective AI studies (1300 cases in 32 months).

Conclusion: Since 2017 several case uses of digital cytology, including remote sign out, providing cytologic correlations, and archiving virtual slides are routinely used at our hospital and proved to be successful for practical and educational purposes. This unique experience may serve as a role model for other institutions.


Accuracy of pleural and peritoneal effusion cytopathology using the newly-proposed international system for reporting serous fluid cytopathology

J. Costa*, S. Petronilho, P. Monteiro, L. Leça, F. Schmitt, C. Lobo

*IPO-Porto, Portugal

Background & objectives: Serous effusion cytology is an important tool in the evaluation of effusions. Currently, there is no framework for reporting serous effusion cytology. The recently proposed International System for Reporting Serous Fluid Cytopathology (ISRSFC) aims to standardize reporting and increase reproducibility.

Methods: Pleural and peritoneal effusion samples admitted at our institution between 2012-2016 for cytological analysis were reviewed and reclassified according to the five categories proposed by the ISRSFC. Accuracy of serous effusion cytology was measured against histological analysis, imaging, surgical findings and clinical follow-up.

Results: 1496 pleural effusion samples were recategorized: 12(0.8%) non-diagnostic(ND)|944(63.1%) negative for malignancy(NFM)|9(0.6%) atypia of undetermined significance(AUS)|54(3.6%) suspicious of malignancy(SFM)|477(31.9%) malignant(M). 763 peritoneal effusion samples were reclassified: 5(0.7%) ND|457(59.9%) NFM|12(1.6%) AUS|37(4.8%) SFM|252(33%) M. Risk of malignancy was, for the aforesaid categories, 57.1%;23.9%;50%;76.2%;100% in pleural effusions and 100%;26.3%;62.5%;91.7%;100% in peritoneal effusions. Sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy were, respectively, 61.6%;100%;100%;73.3%;81.3% for pleural effusions, and 61.2%;100%;100%;70%;79.7% for peritoneal effusions.

Conclusion: Serous effusion cytology has a confirmatory role in the diagnosis of malignancy, corroborated by a high positive predictive value and specificity and a modest negative predictive value and sensitivity. The tiered classification system proposed (ISRSFC) will increase standardization, consistency and reproducibility in reporting, with an aim to improve communication and clinical decision-making.


Moesin (MSN) as a novel proteome-based diagnostic marker for early detection of muscle-invasive bladder urothelial carcinoma in liquid-based cytology

H.S. Ryu*

*Department of Pathology, Seoul National University Hospital, Republic of Korea

Background & objectives: Treatment for Bladder urothelial carcinoma (BUC) highly depends on the invasiveness of cancer cells. A predictive biomarker needs to be identified for invasive BUC. Here, we employed proteomics on urine liquid-based cytology samples to demonstrate a biomarker for invasive BUC.

Methods: Proteomics methods were employed on 16 urine liquid-based cytology (LBC) samples and a set of BUC cell line library to verify candidate biomarker for invasive BUC. Furthermore, in vitro 2-dimensional (2D) and 3-dimensional (3D) invasion study and validation of immunocytochemistry were performed on an independent liquid-based cytology cohort.

Results: The proteomic analysis suggested moesin (MSN) as a potential biomarker to predict the invasiveness of BUC. Moreover, an in vitro 3D invasion study showed that inhibition of MSN significantly decreased invasiveness in BUC cell lines. Further validation of the slide-based moesin immunocytochemical test ultimately confirmed moesin (MSN) as a potential biomarker to predict the invasiveness of BUC (p = 0.042). The predictive ability was more powerful in the dichotomous comparison between BUC groups without invasion and the other group with invasion (p-value = 0.023, moesin immunoreactive rates, 38.5% vs. 82.4%, respectively).

Conclusion: In conclusion, we suggest moesin as a potential diagnostic marker for early detection of BUC with invasion in LBC and as a potential therapeutic target.


EUS guided FNA in diagnosing pancreatic lesions according to Papanicolaou Society of Cytopathology for pancreaticobiliary system, a single centre experience

I. Guvendir*, I.E. Zemheri

*Health Sciences University, Umraniye Education and Training Hospital, Pathology Department, Turkey

Background & objectives: Early diagnosis of pancreatic cancers is important for therapeutic efficacy, disease management. EUS-FNA cytology is helpful for early diagnosis, significant for cost-effectivity. Our aim is to present EUS-FNA cases diagnosed according to Papanicolaou Society of Cytopathology for pancreaticobiliary system in one centre, evaluate clinicohistopathological correlation.

Methods: This retrospective study includes 84 EUS‑guided pancreatic FNA cases between 2015-2019 years in our hospital. Clinical data, laboratory tests, cytopathology, histopathology and imaging reports were included in assessment. Final diagnosis were given according to the findings of EUS‑FNA cytology, cell block and/or histopathology of surgical specimens. All results were compared with the final diagnosis to calculate the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV).

Results: 84 EUS-FNA procedures were performed.7 cases were excluded from study. Lesions were grouped as cystic 16(21%)and solid 61(79%).Median age was 52;%37 (n=28 ) of patients were female,%63 (n=49) were male. According to Papanicolaou classification,5 cases were category I(12%),19 were II(24%),15 were III(19 %),4 were IVB (5%),8 were category V(10%),26 were category 6(33%).Cytological evaluations were compared with final clinical and histopathological diagnoses. Consequently, sensitivity, specificity, PPV and NPV of EUS-FNA cytology to predict final clinicohistopathology were 91%,60%,80%,80%, respectively.

Conclusion: The EUS-FNA guided cytology with Papanicolaou Society Classification is a very helpful diagnostic tool for accurate diagnosis and interpretation of pancreatic lessions with high sensitivity (91%) and PPV (80%).


Cytomorphologic and immunophenotypic characterisation of tumour-associated circulating atypical cells in prostate adenocarcinoma

S.C. Bhakdi*, P. Suriyaphol, P. Thaicharoen, S.T.K. Grote, B. Chaiyaprasithi, K. Charnkaew

*Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok, Thailand; X-ZELL, Singapore

Background & objectives: Systematic classification of tumour-associated circulating atypical cells (tCAC) in solid-tumour patients has been hampered by the inherent challenges of rare-cell detection. We present a first classification of tCAC obtained from patients diagnosed with prostate adenocarcinoma.

Methods: The study enrolled 170 patients with suspected prostate cancer scheduled for transrectal ultrasound-guided tissue biopsy. Blood was drawn immediately before the biopsy, thereby avoiding cross-contamination with cells released into circulation by the procedure. Atypical cells were isolated using hMX high-gradient magnetic cell separation and characterised by highly-multiplexed Cryoimmunostaining on standard laboratory slides in a seven channel widefield fluorescence microscope.

Results: Atypical circulating cells or cell clumps were observed in 49 out of 74 patients with a positive tissue biopsy for prostate adenocarcinoma, as well as in 22 out of 72 patients with a negative biopsy. Cells from patients with positive biopsy showed classical structural changes such as nuclear enlargement, indentation, irregularity, increased chromatin, multinucleation, and high nuclear-to-cytoplasmic ratio. The immunophenotype of atypical cells was predominantly endothelial (CD31, CD34) with diffuse overexpression of vimentin.

Conclusion: Papanicolau first systematized general criteria for cellular malignancy. The Bethesda classification, among others, later refined and elaborated such criteria. The present study demonstrates that morphologies of tCAC in prostate adenocarcinoma patients correspond well to these general criteria, and, in addition, takes advantage of highly multiplexed immunophenotyping. The present classification may improve the clinical utility and reproducibility of cell-based liquid biopsies in diagnosing prostate cancer and, possibly, other cancers.

The study was jointly funded by the Thailand Research Foundation and X-Zell Biotech Pte. Ltd. (Singapore).

OFP-12 Joint Oral Free Paper Session: Molecular Pathology / Haematopathology


Genomic and histological characterisation of gallbladder cancer identifies potentially actionable therapeutic targets in the majority of patients

T. de Bitter*, E. de Savornin Lohman, E. Vink-Börger, S. van Vliet, M. Hermsen, L. Kroeze, D. von Rhein, E. Jansen, I. Nagtegaal, P. de Reuver, M. Ligtenberg, R. van der Post

*Department of Pathology, Radboud University Medical Center, Nijmegen, The Netherlands

Background & objectives: Gallbladder cancer (GBC) is a highly aggressive malignancy with a low incidence in Western countries and a 5-year survival rate highlighting the need for more effective therapies. We aimed to identify potentially actionable therapeutic targets for GBC.

Methods: We collected specimens and clinicopathological data of >800 GBC patients between 2000 and 2019 using the Dutch Pathology Registry (PALGA) and the The Netherlands Cancer Registry. Pathology review was performed for 500 cases. A subset (N=79) was subjected to the Illumina TruSight™ Oncology (TSO)500 assay. We assessed variants in a panel of 54 actionable genes, tumour-mutational burden (TMB) and microsatellite instability.

Results: Histopathological subtypes comprised biliary-type (66%), intestinal-type (20%), poorly cohesive-type including signet ring cell (1%) and other (neuroendocrine carcinoma and (adeno)squamous carcinoma) (14%). Most cases were of pT1/2 stage (70%), followed by pT3/4 (29%) and pTx (1%). Vascular-, lymphatic-, and perineural invasion were observed in 47%, 53% and 37% of cases, respectively. Most tumours (58%) carried at least one (likely) pathogenic variant in an actionable target gene including KRAS(11%), ERBB2(10%), CDKN2A(9%) and PIK3CA(9%) and with varying frequency in numerous other genes. One case showed microsatellite instability. The median TMB was 5.5 mutations/Mb (range: 0 – 161.1 mutations/Mb) and 12 cases (15%) had a TMB of >10 mutations/Mb.

Conclusion: The majority of GBC patients carry a potentially actionable variant, increasing the potential for targeted therapy selection and highlighting the importance of molecular testing in this population.

Funding by: Foundation ADP


Next-generation-sequencing based molecular profiling of low-cellularity cytological specimens

P. Jermann*, I. Alborelli, B. Calgua de Leon, D. Gozuyasli, L. Quagliata, M. Matter

*University Hospital Basel, Switzerland

Background & objectives: Cytological specimens represent up to 50% of diagnostic cancer samples, but are seldomly used for molecular profiling, as they often contain <20% tumour cells. We use a novel NGS assay to accurately profile low-cellularity cytological specimens within a single day.

Methods: The Oncomine Precision Assay (OPA) covers 50 genes and uses unique molecular identifiers to maximize sensitivity. Libraries from of 15 previously characterized cytological specimens were prepared and sequenced on the Ion Torrent Genexus Integrated Sequencer. Sensitivity and specificity of variant detection was assessed.

Results: To assess the limit of detection we used serial dilutions as well as samples with a tumour cell content ranging from 5% – 20%. Clinically relevant cancer hotspot mutations as well as copy number alterations in HRAS; EGFR; PIK3CA and TP53 were accurately detected at <1% variant allele frequency. Preliminary analysis shows 100% concordance with previously characterized matched laser-microdissected samples. Turnaround time from sample to results was as short as one day. Comprehensive analysis of the complete dataset will be presented at the conference.

Conclusion: The OPA allows for sensitive and specific detection of variants in low-cellularity cytological samples, obviating the need for laser-capture microdissection. Using the Genexus Sequencer, NGS results can be reported to clinicians as quickly as immunohistochemical analyses.

Funding by: Thermo Fisher Scientific


Pervasive lesion segregation shapes cancer genome evolution

S. Aitken*, C. Anderson, F. Connor, L.C.E. Consortium, C. Semple, N. López-Bigas, P. Flicek, D. Odom, M. Taylor

*Cancer Research UK - Cambridge Institute, University of Cambridge; Department of Pathology, University of Cambridge, United Kingdom

Background & objectives: Cancers arise through the acquisition of oncogenic mutations and grow through clonal expansion. Sequencing and analysis of cancer genomes have identified a wealth of driver mutations and mutation signatures, illustrating how environmental mutagens cause genetic damage and elevate cancer risk.

Methods: We chemically induced liver tumours in fifteen-day-old male C3H/HeOuJ inbred mice and performed whole genome sequencing and histological analyses of of 371 independently-evolved tumours from 104 individuals.

Results: We revealed that most mutagenic DNA lesions are not resolved as mutations within a single cell-cycle. Instead, DNA lesions segregate unrepaired into daughter cells for multiple cell generations, resulting in the chromosome-scale phasing of subsequent mutations. We characterised this process in mutagen-induced mouse liver tumours and show that DNA replication across persisting lesions can produce multiple alternative alleles in successive cell divisions, thereby generating both multi-allelic and combinatorial genetic diversity.

Conclusion: The phasing of DNA lesions enables the accurate measurement of strand biased repair processes, the quantification of oncogenic selection, and the fine mapping of sister chromatid exchange events. Finally, we demonstrate that lesion segregation is a unifying property of exogenous mutagens, including UV light and chemotherapy agents in human cells and tumours, which has profound implications for the evolution and adaptation of cancer genomes.

Funding by: S.J.A. received a Wellcome Trust PhD Training Fellowship for Clinicians (WT106563/Z/14/Z) and is now funded by a National Institute for Health Research (NIHR) Clinical Lectureship.


Influence on cancer cell energy metabolism by pharmacological inhibition of the histone modifying lysine-specific-demethylase-1

M. Schmiel*, L. Wang, J. Wang, X. Yu, P. Dalvi, R. Buettner, M. Odenthal

*Department of Pathology, University Hospital Cologne, Center for Molecular Medicine Cologne, Germany

Background & objectives: The lysine-specific-demethylase-1 (LSD1) is overexpressed in different cancer types. Additionally, to its effects on cell cycle, our studies revealed LSD1 function in cancer metabolism. To investigate the metabolic effects of LSD1 inhibition in tumours we used the novel inhibitor HCI-2509.

Methods: RNA sequencing followed by pathway analysis and qPCR validation was performed on non-treated versus HCI-2509 treated cancer cell lines. The mitochondria were investigated by mitochondrial membrane potential staining, flow cytometry quantification of the mitochondrial membrane potential and analysis of the respiratory chain, using the Seahorse device. Besides, public available patient datasets were assessed regarding differential expression of metabolic genes.

Results: Expression profiling revealed altered regulation of many metabolic genes upon pharmacological LSD1 inhibition. Especially, transcription of genes related to mitochondrial metabolism and in particular, expression of genes encoding subunits of the electron transport chain (ETC) complex I were repressed. Accordingly, we proved a decrease of the mitochondrial membrane potential, lower ATP production and lower maximal respiration upon LSD1 inhibition in cancer cells. Interestingly, the expression of various mitophagy effectors was increased. Additionally, public available datasets confirmed altered expression of metabolic genes in tumours compared to non-tumour specimens.

Conclusion: Our data emphasizes the value of LSD1 inhibition in cancer therapy since LSD1 inhibition does not only disrupt cell cycle progression but also leads to energy depletion by ETC complex I dysregulation and upregulation of mitophagy inducers.


The expression levels of microRNA 21 and microRNA 210 in renal cell carcinoma subtypes and renal cell carcinoma metastases and their relationship with prognosis

T.C. Savli*, I. Yilmaz, S. Baykal Koca, C. Kelten Talu, T. Bolme Savli, G. Narli, H. Koseoglu, E. Tural

*Health Science University, Istanbul Training and Research Hospital, Department of Pathology, Turkey

Background & objectives: MicroRNAs are non-coding RNA groups consisting of approximately 20 nucleotides with single strands. They play roles in many biological events such as cell proliferation and differentiation, apoptosis, angiogenesis and also in cancer pathogenesis and metastasis.

Methods: Cases diagnosed between 2012-2018 were re-evaluated. 35 ccRCC, 11 CRCC, 9 PRCC and 9 metastatic ccRCC were included.

Tissues were studied using real-time PCR. Fold change, which is the change in the expression level of the target microRNA in tumour and metastatic tissue compared to normal tissue, was calculated with the formula 2-ΔΔCT. Significance was determined by P value (p<0.05).

Results: Expression levels of microRNA-21 and microRNA-210 are statistically significant between histological types (p<0,001). Only the effect of microRNA-210 expression level on survival time is statistically significant. (p=0,034). There was no statistically significant difference between microRNA-21 and microRNA-210 expression levels between primary and metastatic tumours (p=0,053; p=0,237). microRNA-210 has a statistically significant diagnostic accuracy for metastatic tumours (p=0.04). Both microRNA-21 and microRNA-210 have a statistically significant diagnostic accuracy for clear cell RCC versus choromofob RCC and papillary RCC versus choromofob RCC (p<0,001). microRNA-210 has a statistically significant diagnostic accuracy for clear cell RCC versus papillary RCC (p=0,001).

Conclusion: MicroRNAs show different ELs in kidney tumours according to histological types. This feature can make the use of microRNA-21 and microRNA-210 significant in the subtype diagnosis process. With treatment to be developed against microRNA-210, which can be used in determining survival times, patients can survive longer.


Anti-arthritic effect of hesperidin loaded lecithin NPs coated with folic acid silver nanoparticles via modulating immune mediators of inflammation and bone damage

A. Razzak*, T. Roome, S. Aziz, T. Jabri, M. Imran, M. Raza Shah

*Section of Molecular Pathology, Department of Pathology, Dow Diagnostic Reference and Research Laboratory; Cell Signalling Pathways & Immunopathology Lab, Dow Research Institute of Biotechnology and Biomedical Sciences; Department of Laboratory Animal Sciences, Dow International Medical College, Dow University of Health Sciences, Karachi, Pakistan

Background & objectives: Activated macrophages by expressing Folate receptor (FR) play significant role in rheumatic inflammation. Presently, Hesperidin loaded lecithin NPs coated with folic acid silver nanoparticles (FA-L HP-AgNPs) was investigated against CFA-induced arthritis model targeting downstream inflammatory mediators of macrophages signalling.

Methods: FA-L-HP-AgNPs characterized through UV–vis, zetasizer, FT-IR and AFM. During CFA-induced arthritis rat model, oxidative stress markers (NO & PO), M1/M2 cytokines (TNF-α, IL-1β, IL-6, IL-23, IL-10 and TGF-β1), and TLRs expressions were tested in serum, peritoneal macrophages and splenocytes, respectively. Additionally, mRNA levels of TLR-2/-4, RANK, RANKL & ROR-γt and percentage of Th17 cells were performed in spleen tissues.

Results: Oral administration of FA-L-HP-AgNPs (1 & 3mg/kg) exhibited potent anti-arthritic activity with minimal arthritic score, reduced degenerative changes and lesser influx of inflammatory cells in macroscopic, radiographic, and histological examination, respectively. The treatment not only inhibited TLRs, RANKL, MMP-9/-2 and IL-17 levels in synovium but also suppressed the serum NO and HO (p <0.001) production demonstrated its immunomodulatory effects. Interestingly M1 and M2-derived cytokines was also disrupted including TNF-α, IL-1β and TGF-β1 (p <0.005).

Conclusion: FA-L-HP-AgNPs (1&3mg/kg) had profound therapeutic effect on arthritic rats, consistent with reductions in bone damage and Th17-associated IL-17 and ROR-γt expressions. Moreover, FA-L-HP-AgNPs suppressed RANK/RANKL and MMPs production, which supported its anti-osteoclastic effects. Conclusively, FA-L-HP-AgNPs acted as antiarthritic agent in a pleiotropic manner in CFA rats by not only reducing the clinical signs of arthritis, inflammatory cytokines and free radical production but also attenuating the M1/M2 and RANK/RANKL signalling pathway.


Glutathione-S-transferase (GSTT1, M1 and P1 ) genetic polymorphisms among Sudanese acute lymphoblastic leukaemia patients

N.E. Husain*, A. Abodlaa, H. Idris, M. Masri

*Omdurman Islamic University, Sudan

Background & objectives: Glutathione S-transferase (GST) is an enzyme involved in metabolic activation and detoxification of carcinogens. This study examined differences in the frequencies of selected single nucleotide polymorphisms in GST (P1, T1 and M1) genes in Sudanese ALL patients.

Methods: This case-control study included extracted DNA from 280 blood samples (150 patients attending Khartoum Oncology Hospital and 130 healthy matched volunteered controls). PCR-RFLP assay was used for genotyping of the GSTP1 (Ile105Val) polymorphism. A multiplex PCR was used to amplify both GSTM1 and GSTT1 in a single PCR reaction together with ß-globin gene primer as an internal control.

Results: The risk of ALL in patients with GSTT1 null genotype was not significant (OR= 1.23, 95% CI=0.74 -2.0, P-0.44). The GSTM1 null genotype was significantly higher in the ALL group (79, 60.3 %) compared to controls (52, 39.78%) and increased the risk to ALL by two folds (OR=1.72, 95% CI=1.1-2.7, p=0.03). The frequency of GSTP1 genotypes was significantly different between the two groups (P=0.000). AG, GG and AG+GG genotypes were found to increase the risk of ALL by more than three times (P<0.000) compared to the AA genotype. Allele frequencies were significantly different (P= 0.001, OR= 1.79, CI= 1.247-2.58). High frequencies were reported in Kordofan and Khartoum states.

Conclusion: GTM1 null allele and GSTP1 mutant genotype exhibits significant association with the risk of developing ALL among Sudanese patients, either alone or with environmental factors related to some Sudan states. Accompanied environmental studies are needed to highlight possible substrate carcinogens.


Brain tumour diagnostics using a DNA methylation-based classifier as a diagnostic support tool

W. de Leng*, L. Priesterbach-Ackley, H. Boldt, J. Petersen, D. Scheie, B. Ulhøi, M. Gardberg, T. Brännström, S. Torp, E. Aronica, B. Küsters, W. den Dunnen, F. Vos, P. Wesseling, B. Kristensen

*Department of Pathology, University Medical Centre Utrecht, Utrecht University, The Netherlands

Background & objectives: Methylation profiling is increasingly incorporated in the diagnostic process of central nervous system tumours at our centres in The The Netherlands and Scandinavia. We aimed to identify the benefits and challenges of MP as a support tool for CNS tumour diagnostics.

Methods: 502 CNS tumour samples were analysed using (850k) methylation profiling (MP). Profiles were matched with the DKFZ/Heidelberg CNS Tumour Classifier. For each case the final pathological diagnosis was compared to the diagnosis before MP.

Results: In 54.4% (273/502) of analysed cases, the suggested methylation class (calibrated score ≥0.9) corresponded with the initial pathological diagnosis. The diagnosis of 24.5% of these cases (67/273) was more refined after incorporation of the MP result. In 9.8% of cases (49/502) the MP result led to a new diagnosis, resulting in an altered WHO grade in 71.4% of these cases (35/49). In 1% (5/502) of cases the suggested class based on MP was initially disregarded/interpreted as misleading, but in retrospect the MP result predicted the right diagnosis for 3 of these cases. 33.7% of cases (169/502) had a calibrated score <0.9, including 7.8% (39/502) without class indication (calibrated score <0.3).

Conclusion: MP is a powerful tool to confirm and fine-tune the pathological diagnosis of CNS tumours, and to avoid misdiagnoses. However, it is crucial to interpret the results in the context of clinical, radiological, histopathological and other molecular information.


Explanation of unexpected IHC and MSI results of Lynch syndrome diagnostics

W. de Leng*, H. Ma, I. Geurts-Giele, A. Jansen, M. Koudijs, T. Letteboer, W. Dinjens, F. Morsink, J. Offerhaus, L. Brosens

*Department of Pathology, University Medical Centre Utrecht, Utrecht University, The Netherlands

Background & objectives: During our 10 year experience in performing Lynch Syndrome (LS) diagnostics we have encountered 28 patients where MMR IHC and microsatellite instability (MSI) analyses showed unexplained contradictory results. This study is to clarify these non-corresponding molecular observations.

Methods: Tumours of 2335 patients were tested for MMR-deficiency and MSI between 2007 and 2017 in the UMC Utrecht. Diagnostic MMR IHC and MSI analyses were repeated for patients with unexplained contradictory results. Two MSI assays were used; the Bethesda markers (mono- and di-nucleotide repeats) and the Pentaplex assay (mononucleotide repeats). Germline and somatic mutation analysis and protein modelling were performed.

Results: Repeated IHC analysis resulted in a different outcome in 7 out of 17 patients. Repeated MSI analysis with only mono-nucleotide markers resulted in a different result in 9 out of 10 patients. Somatic MMR mutations were identified in 10 patients. Protein modelling could explain preserved protein expression in some tumours as mutations did not cause major conformational changes. In total, in 15 patients the unexplained results could be clarified (e.g. bi-allelic somatic mutations, LOH, minor conformational changes), while 5 patients were partially explained.

Conclusion: Mononucleotide markers for MSI analysis were more sensitive than dinucleotide markers and a combined analysis including germline and somatic mutations proved crucial for identifying a definitive diagnosis as somatic mutations were frequent in this study population.


Abdominal involvement of Erdheim-Chester disease: clinical, histological and molecular characteristics of 22 cases

I. Ungureanu*, F. Cohen-Aubart, F. Charlotte, S. Valmary Degano, Z. Hélias-Rodzewicz, D. Cazals-Hatem, P. Dartigues, M. Delage-Corre, J. Selves, P. Tas, L. Veresezan, T. Kuopio, C. Prokopiou, J. Haroche, J. Emile

*Pathology Department, Ambroise-Paré Hospital, France

Background & objectives: Peritoneal infiltration rarely occurs in Erdheim-Chester disease and other histiocytoses and may be misdiagnosed when presenting as a single-organ involvement. We aimed to analyse clinical, histological, and molecular features of peritoneal manifestations of patients diagnosed with histiocytosis.

Methods: Twenty-two patients were retrieved from the file of Ambroise-Paré Hospital. All slides were stained with H&E, and immunohistochemistry was performed for the following markers: CD163 (or CD68), CD1a, S100 protein and phosphoERK. DNA was extracted from histiocyte-rich areas and analysed using real-time PCR, Picodroplet Digital PCR and NGS. Clinical information was obtained for each case.

Results: The final diagnosis of seventeen patients was ECD. For twelve patients, the abdominal symptoms revealed the disease, and nineteen underwent surgical biopsies. Four patients had a unique abdominal mass, while the others had diffuse peritoneal infiltration. Sixteen had subserosal histiocytic infiltration, ten of whom also had deep infiltration of adipose tissue. Histiocytes were mainly mononucleated, with multinucleated cells in seven cases. Eighteen cases contained foamy histiocytes. Most cases with low or moderate density of histiocytes had ECD. Cytosteatonecrosis was present in one patient with ECD. All cases were positive for CD163 or CD68 and negative for CD1a. Twelve cases contained phosphoERK positive histiocytes. Thirteen patients had BRAF or MAP2K1 mutations.

Conclusion: Abdominal symptoms may reveal unsuspected Erdheim-Chester disease. In more than half of these referred cases the diagnosis of ECD was not initially proposed. Molecular biology is a helpful diagnostic tool.


Prognostic value of histone modifying enzyme EZH2 and histone mark H3K4me3 in R-CHOP-treated diffuse large B-cell lymphoma

S. Petronilho*, J.P. Sequeira, S. Paulino, S. Chacim, J. Lobo, C. Jerónimo, R. Henrique

*Pathology Department, IPO-Porto, Portugal

Background & objectives: DLBCLs are aggressive, heterogeneous lymphomas. Under RCHOP-treatment, 30% of patients progress or ultimately relapse. Histone modifying enzymes’ alterations are frequent, but their prognostic value is still controversial.

Aim: To ascertain the prognostic value of EZH2/H3K4me3 immunoexpression in RCHOP-treated DLBCL.

Methods: Retrospective cohort study including 127 patients with RCHOP-treated DLBCL (2008-2017). EZH2/H3K4me3 immunoexpression was evaluated using a digital imaging system for H-score calculation and dichotomized in high/low expression. Clinicopathological variables (sex/age/stage/R-IPI/bone marrow status/Hans molecular group/BCL2/CD5 positivity) were re-evaluated/collected.

Primary outcome was event free survival (EFS); secondary outcomes were lymphoma specific (LSS) and overall survival (OS). Subgroup analysis by molecular group was performed.

Results: EZH2 expression levels did not differ between molecular groups (p=0.749). Exclusively in the non-Germinal Centre (nonGC) subtype, high EZH2 expression was associated with poor EFS, both in univariable (HR 2.29; 95%CI[1.08- 4.85]; p=0.031) and multivariable analysis (HR 2.78; 95%CI[1.18- 6.54]; p=0.019). EZH2 expression was not significantly associated with LSS (p=0.046*) or OS (p=0.031*). [*alpha=0.01 for secondary outcomes]

We did not find an association between H3K4me3 expression levels and survival.

Conclusion: High EZH2-expression was associated with worse EFS in nonGC-DLBCL, supporting its role as prognostic biomarker in this subgroup.

Although EZH2-mutations are almost exclusively found in GC-DLBCL, both subgroups shared similar expression levels, suggesting EZH2-antagonists might also be beneficial in nonGC-DLBCL.

Oral Free Paper Session: One-Day Computational Pathology Symposium

CP-03 One-Day Computational Pathology Symposium - Selected Abstracts


Deep learning-based tumour bud detection in pan-cytokeratin stained colorectal cancer whole-slide images

J. Bokhorst*, I. Nagtegaal, I. Zlobec, A. Lugli, M. Vieth, R. Kirsch, J. van der Laak, F. Ciompi

*Radboud University Medical Center, The Netherlands

Background & objectives: Tumour budding (TB) is an established prognosticator for colorectal cancer. Deep learning-based TB assessment has the potential to improve diagnostic reproducibility and efficiency. We developed an algorithm that can detect individual tumour buds in pan-cytokeratin stained colorectal cancer slides

Methods: Tumour-bud candidates (n=1765, collected from 58 whole slide images; WSI) were labelled by seven experts as either TB, poorly differentiated cluster, or neither. The 58 slides were randomly split into a training (49) and test-set (9). A deep learning (DL) model was trained using the buds identified by the experts in the training set.

Results: The algorithm was tested on the nine remaining WSI and 270 WSI from pan-cytokeratin stained slides from Bern University hospital, in which hot spots and TB were manually scored. An F1 score of 0.82 was found for correspondence at the bud level between experts and DL. A correlation of 0.745 was found between the manually counted buds within the hot-spots and the automated method in the 270 WSIs.

Conclusion: Assessment of tumour budding as a prognostic factor for colorectal cancer can be automated using deep learning. At the level of individual tumour buds, correspondence between DL and experts is high and comparable to the inter-rater variability. However, compared to the manual procedure, the algorithm yields higher counts for cases with relatively high bud densities (>15). Follow-up studies will focus on the assessment of TB in H&E stained slides.

Funding: This project has received funding from the Dutch Cancer Society, project number 10602/2016-2.


Optimised tumour infiltrating lymphocyte assessment for triple negative breast cancer prognostics

M. Balkenhol*, F. Ciompi, Ż. Świderska-Chadaj, R. van de Loo, M. Intezar, I. Otte-Höller, D. Geijs, J. Lotz, N. Weiss, T. de Bel, G. Litjens, P. Bult, J. van der Laak

*Radboudumc, The Netherlands

Background & objectives: The tumour microenvironment was shown to harbour prognostic value for breast cancer. For instance, tumour infiltrating lymphocytes (TILs) are studied using a variety of methods. This study aims to establish the most optimal (marker, region) and objective TIL assessment method.

Methods: Multiplex immunohistochemistry (mIHC) was applied on 94 triple negative breast cancer cases, using CD3, CD8 and FOXP3. Multispectral images were co-registered with serial H&E sections, to combine deep learning based automated tissue delineation with lymphocyte detection. Densities of positive lymphocytes were analysed in different regions (peri- and intra-tumoral, within tumour stroma etc) and correlated to relapse free and overall survival.

Results: We found that for all three markers of TILs, the presence of a high density of positive cells correlated with an improved survival, with none of the markers being superior to the others. The results of TILs assessment in the various regions did not show marked differences: TILs assessed within the tumour stroma, in the entire tumour area or at the tumour periphery all show similar prognostic value.

Conclusion: We showed that completely automatic assessment of TILs in different regions of the tumour is feasible. The correlation between TILs and survival in our cohort are in line with previous studies. Our results provide directions for optimizing TILs assessment methodology and will be validated in larger series.

Funding: This study was funded by a Junior Researcher grant from the Radboud University Medical Center Institute for Health Sciences (RIHS), Nijmegen, The Netherlands.


An AI system for predicting ER/PGR/HER2 status from H&E slides in breast cancer

P. Gamble, R. Jaroensri, F. Tan, M. Moran, D.F. Steiner, C.H. Mermel, P.C. Chen*

*Google Health, United States

Background & objectives: Existing ER/PgR/HER2 guidelines require IHC interpretation by experts. Prior research suggests histological features in H&E images correlate with biomarker status. Here, we develop deep learning systems for predicting ER/PgR/HER2 status directly from H&E slides.

Methods: Paired H&E and IHC slides were prepared using representative tissue blocks from 260 cases from two independent institutions. Board-certified pathologists annotated H&E images using the IHC images for reference. Using these annotations, we developed convolutional neural networks to predict ER/PgR/HER2 status directly from H&E slides.

Results: Each model was trained on 200 cases and evaluated on 60 held-out cases. Rates of biomarker positivity in our sample were 0.76/0.72/0.32 for ER/PgR/HER2, respectively. The deep learning system demonstrated an accuracy of 0.87, 0.93, 0.79 (95% CIs: 0.85-0.89/0.91-0.94/0.77-0.81) and an area under the receiver operating characteristic curve (AUC) of 0.87, 0.82, 0.76 (95% CIs: 0.89-0.91/0.81-0.87/0.73-0.79) on slide-level status of validation cases for ER/PgR/HER2, respectively.

Conclusion: Histopathological features present in H&E slides can be used to predict ER/PgR/HER2 status as demonstrated by our deep learning system. Recent guideline updates recommend repeated testing when biomarker results are discordant with the histopathological review, highlighting the value of understanding biomarker status’ association with morphological features. Further research into the features learned by our models may provide novel insights into biomarker status in breast cancer.


Grading nuclear pleomorphism in breast cancer using deep learning

C. Mercan*, M. Balkenhol, J. van der Laak, F. Ciompi

*Radboudumc, The Netherlands

Background & objectives: Nuclear pleomorphism is defined as the variability in size and shape of tumour cells as compared to normal epithelial cells. Objective of this study is to train a deep neural network that can achieve pathologist-level pleomorphism grading performance.

Methods: We collected 29 whole slide images (WSI) of breast cancer resections in which we manually selected 90 regions of interest, ensuring grade homogeneity of tumour cells within a region. Subsequently, we cropped regions of ~0.38 mm2 at 40X magnification (0.25 um/px) and asked six pathologists to grade each region independently.

Results: We used an epithelial cell detector network to detect the epithelial cells in each region and extracted fixed-size patches from these regions with high tumour density. For the task of pleomorphism grading, we trained a densenet model on those patches with the majority voting of the grades of the pathologists (majority grades). The variation of kappa scores of the pathologists with the majority grade was very high, ranging between 0.37 and 0.69 on the standalone test set consisting of 18 regions from 7 WSI. On the same test set, our densenet model had a kappa score of 0.47 with the majority grades.

Conclusion: We demonstrated that our network trained only on tumour cells achieved performance on the task of pleomorphism grading of breast cancer around the low mid-range of the inter-pathologist variability where the inter-observer variability of pathologists was very high. Future research will include scores of a larger panel of pathologists, and study alternative deep learning strategies to improve the performance.


Artificial intelligence to aid the diagnosis of head and neck precancerous and cancerous lesions: a systematic review

H. Mahmood*, M. Shaban, I. Indave, A. Santos-Silva, N. Rajpoot, S.A. Khurram

*Academic Unit of Oral & Maxillofacial Surgery, School of Dentistry, University of Sheffield, United Kingdom

Background & objectives: This systematic review aims to evaluate the published literature on AI/machine/deep learning applications for detection, grading and classification of head and neck precancerous and cancerous lesions using digitised human tissue.

Methods: Electronic database search was conducted using MEDLINE via OVID, Scopus and Web of Science to retrieve relevant articles (2009 and 2019). A tailored search strategy using database specific search terms and text words were used, and studies screened against an eligibility criteria. Modified QUDAS-2 tool was used for risk of bias assessment.

Results: 11 articles met the inclusion criteria which used a combination of heuristics, supervised and unsupervised learning methods, including more than 10 different classification and segmentation techniques. Automated detection was based on analysis of specific histological markers for oral precancer (n=1), oral submucous fibrosis (n=5), oral cancer (n=4) and oropharyngeal cancer (n=1). Most studies used small unicentric datasets (range 40-270 images) comprising small visual fields. Detection accuracy ranged from 79%-100%.

Conclusion: Most studies have shown a high risk of bias which is likely to have contributed to high accuracy rates. Traditional supervised learning methods were most commonly used highlighting the need for modern state-of-the-art deep learning techniques in future research.


Validation of computer-assisted tumour-bud and T-cell detection in pT1 colorectal cancer

L. Studer*, J. Bokhorst, I. Zlobec, A. Lugli, A. Fischer, F. Ciompi, J. van der Laak, I. Nagtegaal, H. Dawson

*Institute of Pathology, University of Bern, Switzerland

Background & objectives: Tumour budding, and T-cells are robust prognostic biomarkers in colorectal cancer. A combined analysis is complex and can be greatly expedited and automated using deep learning. The implementation of computer-based analysis in diagnostics is challenging and necessitates extensive validation.

Methods: Randomly selected (n=61) double-stained immunohistochemical slides (AE1-AE3 pancytokeratin for tumour buds and CD8 for cytotoxic T-cells) from our pT1 cohort from 3 different institutions were used to validate the deep learning algorithms for tumour budding and CD8 T-cell detection developed by the International Budding Consortium Computational Pathology Group. Staining and scanning were performed in a single laboratory.

Results: In the visually identified tumour budding hotspot (0.785 mm2), tumour buds were manually annotated, and the output of the T-cell algorithm manually corrected by a single observer. For budding, 645 out of the 1’306 buds were correctly identified by the algorithm. Recall and precision were 49.4% and 61.4%, respectively. For the T-cells, 89.3% were correctly detected (from a total of 16’296). The recall was 90.3% and the precision was 87.3%. Reasons for misclassified T-cells included staining intensity, suboptimal tissue recognition and slide artifacts.

Conclusion: Our preliminary data demonstrates satisfactory results for T-cell detection. Automated budding detection is more difficult, as inter-observer variability of bud calling is high among experts. These issues merit consideration when developing reliable deep learning algorithms examining the tumour/host interface.

Funding: The work presented here has been supported by the Rising Tide foundation with the grant number CCR-18-130.


Characterisation of the tumour-host interface as a prognostic factor through deep learning systems

T. Haddad*, J. Bokhorst, L. van den Dobbelsteen, F. Zimmer, F. Ciompi, I. Zlobec, A. Lugli, J. van der Laak, I. Nagtegaal

*Radboud University Medical Center, The Netherlands

Background & objectives: Interpretation of the tumour-host interface can provide relevant prognostic information. Through H&E and IHC double staining in combination with deep learning, we set out to find a novel way of characterizing features of the invasive margin in colorectal carcinoma (CRC).

Methods: Formalin-fixed-paraffin-embedded human CRC tissue obtained from the Radboudumc (Nijmegen, Netherlands) was automatically H&E stained and digitized. A double staining procedure using anti-CD3 and pan-cytokeratin was conducted on the same slides and scanned, generating a 2nd whole slide image (WSI) per sample. WSIs were processed using a series of deep learning neural networks (automated lymphocyte detection, tissue classification, tumour budding detection).

Results: Our method was successfully implemented as the tumour budding and lymphocyte detection algorithms were able to automatically detect peritumoral budding in pan-cytokeratin and CD3+ lymphocytes respectively in the immuno-stained WSIs. The tissue classifier segmented the H&E-stained WSIs into 13 classifications. This technique allows us to combine the relevant data and link the density of peritumoral budding with immune infiltration, tumour cell ratio, mismatch repair (MMR) status and clinical outcome. Data correlations on a cohort of 150 colorectal cancer patients with varied MMR status will be presented at the meeting.

Conclusion: Automated classification of the invasive margin in combination with clinical outcome can lead to an improved understanding of patient prognosis. This unbiased and transferable method could improve diagnostic accuracy as well as broaden our understandings of tumour budding and other poor prognostic factors.

Funded by the Dutch Cancer Society.

Oral Free Paper Sessions: One-Day Molecular Pathology Diagnostics and Translational Research Symposium

MD-02Joint Session One-Day Molecular Pathology Diagnostics and Translational Research Symposium, Molecular Pathology & Trainees: Case Presentations


First ESWR1-SMAD3 rearranged tumour arising in bone: expanding the clinical and histological spectrum of this emerging tumour type (case presentation)

S. De Noon*, R. Tirabosco, P. O'Donnell, A. Flanagan, F. Amary

*UCL Cancer Institute, United Kingdom

Background & objectives: EWSR1-SMAD3 fibroblastic tumours are an emerging group of rare soft-tissue neoplasms, defined by characteristic genetic alteration, similar fibroblastic morphology and predilection for hands and feet. We describe the first case of an EWSR1-SMAD3 rearranged tumour arising in bone.

Methods: We reviewed the imaging, clinical records, histopathology and genomic findings of a patient diagnosed at our institution with an unusual bone tumour. Whole genome sequencing (WGS) was performed on fresh frozen tissue as part of the 100,000 genomes project, and data was analysed using standard bioinformatics pipelines in the Genomics England research environment.

Results: A 44-year old male presented with a 9-month history of a painful right knee swelling, with no significant functional defects. MRI revealed a partly ossified mass involving the proximal tibia with a large extraosseous component. The pathology showed a biphasic tumour comprising cellular regions of atypical spindled to oval cells and interspersed hyalinised nodular areas, in places resembling cartilaginous differentiation. The differential diagnosis at the time included a mesenchymal chondrosarcoma and a dedifferentiated chondrosarcoma. Subsequently in the analysis of WGS data, an EWSR1-SMAD3 fusion was discovered.

Conclusion: We present, to our knowledge, the first case of an EWSR1-SMAD3 fibroblastic tumour occurring in bone. This case highlights the role of next generation sequencing in the accurate classification of mesenchymal tumours, particularly in difficult cases showing atypical histological features.


A case report of ROS1 fusion detection by FISH - a possibility of false positive results

J. Siemanowski*, C. Heydt, A. Schultheis, S. Merkelbach-Bruse

*University Hospital Cologne, Institute of Pathology, Germany

Background & objectives: While fluorescence in situ hybridization (FISH) is commonly used for the detection of drug-targetable fusion events, false positive results can hamper profitable treatment decisions. This case report visualizes the possibility of false positive ROS1 FISH results.

Methods: ROS1 FISH and ROS1 immunohistochemistry (IHC) were performed using a SPEC ROS1-Dual Colour Probe (ZytoVision) and a ROS1 antibody Clone D4D6 (Cell Signalling Technology). RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) material. Gene fusion detection by parallel sequencing was done using the FusionPlex CTL panel (Archerdx) on a MiSeq (Illumina).

Results: A 44 –year-old male, diagnosed with an NSCLC adenocarcinoma showed a ROS1 translocation with aberrant signals in 79% of nuclei in FISH analysis. Further evaluation revealed a pattern of 78 nuclei with isolated 3’ extra green signals and 1 nucleic signal with break apart signal. Follow up IHC showed no ROS1 protein expression. Parallel sequencing was also negative for ROS1, indicating a false positive FISH result. Analysing a larger cohort of FISH positive cases by isolated 3’ green signals demonstrated that 48% were not confirmed by alternative methods.

Conclusion: This case report showed that isolated 3’ green signals in ROS1 break-apart FISH should be confirmed by alternate methods. It seems that isolated 3’ green signals could indicate a chromosomal break albeit not generating a functional fusion product.


Molecular characterisation of high grade papillary urothelial carcinoma with divergent glandular differentiation reveals the clonal origin of both components

I. Carretero-Barrio*, T. Caniego, E. Moreno Moreno, M.T. Martínez, I. González, A. Saiz Gonzalez, J. Palacios-Calvo

*Hospital Universitario Ramon y Cajal, Servicio de Anatomia Patologica, Spain

Background & objectives: Urothelial carcinoma with divergent glandular differentiation is a recognised variant of urothelial carcinoma. There is no clear evidence about the clinical significance of this neoplasm, although some studies associate it with worse prognosis. There are studies about the immunophenotype of tumours with divergent differentiation, but there are no publications to date about the possible different genomic profiles of the urothelial and the glandular components.

Methods: We present the case of a high grade papillary urothelial carcinoma with divergent glandular differentiation, on which we performed immunohistochemical and molecular analyses of the two areas. For the molecular analysis, both components were sequenced with next generation sequencing (NGS) techniques, using a 59 gene custom panel.

Results: The high grade papillary urothelial component was only positive for GATA3, p63 and CK7. The glandular area was only positive for CDX2 and CK20.

Both components shared the same mutations on FGFR2, CDH19, KMT2C, MKI67 and PMS2. The glandular component had additional mutations on TP53, ARID1B, ARID5B, CDH19, CTNNB1 and PMS2. Immunohistochemical analysis showed p53 loss of expression (mutated “null” pattern) only in the glandular area, confirming the NGS result.

Conclusion: The molecular analysis, showing the same mutations on both components and the acquisition of new mutations on the glandular component, suggest a common clonal origin. To our knowledge, this is the first study reporting this finding.


A case of Burkitt-like lymphoma with 11q aberration arising in Waldeyer’s ring

C. Masaoutis*, V. Pantelaion, D. Rontogianni

*Department of Pathology, Evangelismos General Hospital of Athens, First Department of Pathology, School of Medicine, National and Kapodistrian University of Athens, Greece

Background & objectives: A 35-year-old female with Crohn’s disease under azathioprine and mesalazine presented with a right rhinopharyngeal tumour causing local symptoms over the last 4 months, without B-symptoms, lymphocytosis, lymphadenopathy or organomegaly. LDH levels were normal. A tumour biopsy was performed.

Methods: H&E: Mucosal involvement by a diffuse population of large cells with vesicular nuclei and conspicuous nucleoli and medium-sized, relatively monomorphic cells with a “starry sky” pattern. Immunohistochemistry: Strong, uniform co-expression of CD20, PAX5, CD10, BCL6; c-MYC>80%, MUM1/IRF4-, BCL2-, CD5-, CD23-, cyclin-D1-, CD30-, Ki67>99%. EBER-ISH: negative. Break-apart FISH for MYC, BCL2, BCL6: negative. Triple-colour FISH revealed 11q23.3 gains and 11q24.1-q25 losses.

Results: A Burkitt-like lymphoma with 11q aberration [BLL-11q] diagnosis was rendered. The demonstration of 11q aberrations without detectable MYC, BCL2 or BCL6 rearrangements helped exclude high grade B-cell lymphoma, NOS (see mixed Burkitt-like and DLBCL cytology) and a morphologically "atypical" Burkitt lymphoma (BL) with a cryptic MYC translocation (see c-MYC>80% and “BL immunophenotype”).

The patient received R-DA-EPOCH including CNS prophylaxis, switched to R-CHOP, and is disease-free 5 months after the diagnosis.

Conclusion: BLL-11q explains some “MYC-negative BLs” or BLs with presumed cryptic translocations. Copy number arrays are the gold standard, but no false positive cases have been reported with FISH. The azathioprine-induced immunosuppression is noteworthy, as cytogenetically similar lymphomas arise in post-transplant patients. Transformed follicular lymphoma may have similar 11q aberrations, but this case lacked such evidence. 11q22-q24 and 11q12.3-q25 gains without telomeric losses, seen in DLBCL and LBCL-IRF4 respectively, are nonspecific.


Spindle cell tumour of the liver with an ETV6-NTRK3 fusion

D. Nann*, I. Bonzheim, M. Scharpf, F. Fend

*Institute of Pathology and Neuropathology, University Hospital Tübingen and Comprehensive Cancer Center, Tübingen, Germany

Background & objectives: A 31-month-old girl suffered from abdominal pain, sickness, and fever. Radiologically, she showed a 7.9 cm large tumour of the liver.

Methods: Hematoxylin and eosin, and immunohistochemical staining was done on a formalin-fixed, paraffin-embedded biopsy. For mutation and fusion analysis, the Oncomine Focus Assay (ThermoFisher Scientific, Waltham, MA, USA) was performed and for confirmation of fusions the Archer FusionPlex solid tumour panel (ArcherDX, Boulder, CO, USA) was used.

Results: The tumour composed of a spindle proliferation with ovoid or elongated nuclei and medium sized cytoplasm undermixed with inflammatory cells. Immunohistochemically, the cells showed a weak and heterogenous positivity for SMA, but were negative for desmin, myogenin, CD23, S100, ALK, pancytokeratin, CD21, lysozyme, CD1a, glypican 3, HepPar1, and didn’t show loss of INI1. MIB1 staining indicated 10% proliferation activity. Molecular analysis detected an ETV6-NTRK3 inframe fusion gene.

Conclusion: The tumour was diagnosed as a spindle cell tumour with ETV6-NTRK3 fusion with differential diagnoses infantile fibrosarcoma and ALK negative inflammatory myofibroblastic tumour (IMT). The morphology, the interspersed inflammatory cells and the localization are more in line with IMT.


Fusion and mutation-negative inflammatory myofibroblastic tumour in bladder: a case report

A. Vernemmen*, I.V. Samarska, V. Winnepenninckx, R.M. Sciot, A.P. de Bruïne, S. Wouda, A.M. van Marion, A. zur Hausen

*Department of Pathology, GROW School for Oncology and Developmental Biology, Maastricht University Medical Center, The Netherlands

Background & objectives: A 71-year old patient underwent a radical cystoprostatectomy due to a rapidly growing bladder tumour with bilateral hydronephrosis and infiltrative growth on imaging. The bladder contained a 7.4 cm polypoid lesion, invading the detrusor muscle and the perivesical tissue.

Methods: The lesion was examined with hematoxylin-eosin (H&E) and immunohistochemical stainings, including ALK-1. The morphology showed a spindle cell lesion, organised in fascicles, with an intervening mixed inflammatory infiltrate. The ALK and ROS1 fluorescent in situ hybridisation (FISH) assay was performed, followed by a DNA analysis and an RNA gene fusion panel assay (Archer) using next-generation sequencing.

Results: Using the H&E and immunohistochemical stainings, the morphology of the tumour was consistent with an inflammatory myofibroblastic tumour (IMT), although ALK-1 was negative. No ALK and ROS1 gene alterations were detected in the FISH assays. The Archer gene fusion panel assay (ALK, CAMTA1, CCNB3, CIC, EPC1, EWSR1, FOXO1, FUS, GL1, HMGA2, JAZF1, MEAF6, MKL2, NCOA2, NTRK3, PDGFB, PLAG1, ROS1, SS18, STAT6, TAF15, TCF12, TFE3, TFG, USP6 and YWHAE) did not reveal any gene fusion either and the DNA mutation analysis detected no mutations. Despite extensive molecular testing, no gene alterations or mutations were encountered. External consultation by Prof. Dr. J.I. Epstein (JHH, Baltimore, USA) was confirmative.

Conclusion: Inflammatory myofibroblastic tumour is a rare tumour with potentially aggressive biologic behaviour. Subset of IMT can be ALK-negative and shows no molecular alteration. Additional molecular research remains necessary for potential future targeted therapy.


Vulvar pilomatrix carcinoma: morphological and molecular features

D. Bueno Sacristán*, M.T. Martínez, I. González, T. Caniego, J. Palacios-Calvo, B. Pérez

*Hospital Universitario Ramón y Cajal, Department of Pathology, Spain

Background & objectives: Pilomatrix carcinoma (PC) is the rare malignant counterpart of pilomatrixoma, a skin adnexal tumour originating from hair matrix cells. Mutations in CTNNB1, the encoding gene of beta-catenin, have been found in both entities, but other molecular alterations are still unknown.

Methods: We present the morphological, immunohistochemical and molecular features of a PC of the clitoris, the third known reported case located on the external genitalia, which developed multiple metastases in a 53-year-old woman. Next generation sequencing (NGS) was performed with a custom-panel including 59 genes. Sanger sequencing was also carried out.

Results: The tumour showed an atypical neoplastic proliferation with scattered abrupt keratinization and shadow cells upon tumour areas. Negative immunohistochemical stain for p16 and p53 excluded the diagnosis of squamous cell carcinoma. Neoplastic cells showed nuclear and cytoplasmic beta-catenin overexpression, characteristic of pilomatrix carcinoma. Sanger sequencing identified S37F mutation in CTNNB1 and NGS also evidenced mutations in ARID1A, PTEN and PIK3CA. Posterior negative immunostaining for ARID1A confirmed NGS result.

Conclusion: PC is a rare neoplasm usually located in head and neck region. Metastasis only occur in 10% of cases. In contrast, this study presents a vulvar PC with multiple metastasis. To the best of our knowledge, this is the first case analysed by NGS, which demonstrated not only the common initiating mutation in CTNNB1, but also other mutations that could have contributed to the dismal evolution in this patient.


Sino-nasal Ewing family tumour – a morphomolecular case report of a “small round blue cell tumour”

H. Morris*, G. Dowds, C. McKeeve, S. Wright

*Department of Pathology, Institute of Cancer Sciences, University of Glasgow, United Kingdom

Background & objectives: An elderly female presented with right eye pain, epiphora and proptosis. CT and MRI showed a 7.7cm mass involving the right nasal cavity, ethmoid sinus and orbit with bilateral frontal sinus involvement and early intracranial extension.

Methods: Biopsy showed a small round blue cell tumour. Focally, the lesional cells formed whorls around small blood vessels. There was no keratinization or gland formation. An extensive immunohistochemistry (IHC) panel was performed and supplemented by FISH for EWSR1 translocation, RT-PCR for EWSR1-FLI1 and SS18 fusion products and multiplex PCR for HPV-16 and HPV-18 genotypes.

Results: IHC demonstrated staining with antibodies to broad spectrum cytokeratins and focal staining with squamous markers p63 and p40. Antibodies to p16 were positive. Of note, CD99 was negative. Neuroendocrine, neural, melanocytic, skeletal and smooth muscle, lymphoid and vascular markers were negative. EBV-ISH was negative. There was no loss of INI-1 expression. There was focal cytoplasmic PAS-positive material. Molecular testing revealed EWSR1-FLI1 fusion.

A diagnosis of Ewing family tumour (EFT) was made. Sinonasal EFTs are very rare and have a wide differential diagnosis. EWSR1 translocation can also be seen in anaplastic myoepithelial carcinoma but EWSR1-FLI1 fusion is specific to EFT and is present in 85% of cases.

Conclusion: This report raises awareness of sinonasal EFT, a rare entity which has a wide differential diagnosis, and demonstrates the use of molecular analysis as an adjunct to traditional histology, histochemistry and IHC to reach a definitive diagnosis.

Hayley Morris is a NHS Education for Scotland funded Clinical Lecturer in Pathology.

MD-04 One Day Molecular Pathology Diagnostics and Translational Research Symposium - Selected Abstracts


Evaluation of 1429 lung tumour samples analysed by targeted RNA-Seq

S. Wagener-Ryczek*, C. Heydt, R. Pappesch, J. Siemanowski, J. Fassunke, J. Rehker, M.A. Ihle, S. Merkelbach-Bruse, R. Buettner

*University Hospital Cologne, Institute of Pathology, Germany

Background & objectives: Molecular assays for fusion detection in lung tumours are widely used in routine diagnostics. However, conventional methods like fluorescence in situ hybridization (FISH) can come with several drawbacks. In this study, 1429 lung tumour samples were evaluated with targeted RNA-Seq.

Methods: For the detection of known and novel gene fusions, the Archer FusionPlex Lung panel (Archerdx, Boulder, CO) was used. In brief, 35 to 200 ng of tNA was target enriched by single primer extension. Prepared cDNA libraries were sequenced on a MiSeq (Illumina). Data analysis and fusion detection were done using Archer Analysis software versions 5.0.4, 5.1.3 and 6.2.1 (Archerdx).

Results: 1429 lung tumours were evaluated in routine diagnostics. 98% of these samples were analysable. Novel fusion partners, for example EGFR – VSTM2A or NIPBL-NRG1 were found as well as rare but clinically relevant lung fusions like ST7-MET and the EGFR VIII variant were identified. Furthermore, the assay was found to be a useful tool to verify, whether mutations and deletions in close proximity to characterized MET exon 14 skipping mutations will lead to a real exon skipping event. This assay is only hampered by the fact that fusions involving genes both outside the selectively captured region cannot be detected and false-negative results due to poor quality samples can be encountered.

Conclusion: This method has demonstrated excellent diagnostic utility for fusion detection in lung tumours. Novel and rare fusions partner can be identified by targeting one gene fusion partner only. Mutations/Deletions leading to exon skipping events can be verified.


Participation in external quality assessment (EQA) for BRCA testing in ovarian cancer: lessons learned and the need for continued quality improvement

M. Cheetham*, V.S. Williams, J.A. Fairley, N.L. Wolstenholme, Z.C. Deans, S.J. Patton

*EMQN, United Kingdom

Background & objectives: Ovarian cancer is the eighth most common cancer in women for which PARP inhibitors have proved effective therapeutic agents in eligible patients. We present data from BRCA-based EQA schemes over a 3-year period (2016-2018) involving over 300 laboratories globally.

Methods: EMQN and GenQA sent identical clinically relevant samples to each EQA participant to be tested for BRCA1 and BRCA2 gene variants using their routine methodologies. The anonymised results were assessed, peer reviewed and individual laboratory and overall summary scheme reports were produced to help laboratories improve their performance and to enable comparison (benchmarking) of results and reporting.

Results: Over the three years, there has been significant improvement in the quality of testing, clinical reporting of results and standardisation with respect to the nomenclature used for the reporting of sequence variants. Improvement in the clinical interpretation is also evident but there are areas where further work is required.

Conclusion: Although errors persist, we conclude that annual participation in these EQA schemes can improve the quality of diagnostic testing for ovarian cancer and considerably contribute to the achievement of the promise of accurate personalised medicine.

The EQA schemes in question were sponsored by an educational grant from AstraZeneca.


Diagnosis of microsatellite instability in routine practice in colorectal adenocarcinoma: what we learn from the rereading of discordant cases

P. Cervera*, O. Lascols, M. Svreck, S. Breton, R. Cohen, D. Tougeron, T. Andre, A. Duval, J. Fléjou

*APHP Sorbonne Université INSERM, France

Background & objectives: In the era of immunotherapies in Microsatellites Instability, efficiency of diagnostic is challenged as routine screening is not standardized. The two methods used, immunohistochemistry and molecular testing, give discordances.

These discordances are studied in a retrospective series of colorectal adenocarcinomas.

Methods: In 2128 colorectal patients, MMR and MSI status were established between 2013 and 2018. MSI testing was carried out using the pentaplex panel and MMR immunohistochemistry testing two MMR proteins (MLH1, MSH2). The primary endpoint was the rate of real discordance between MSI and MMR IHC tests. Each discordance case was thoughtfully examined to be confirmed and explored.

Results: This cohort initially presented 28 (1.3%) discordant cases. After rereading of 2 tests (immunohistochemistry and molecular testing) and/or new tests, 75.0% of discordant cases (n=21/28) were reclassified as non-discordant. After rereading and addition of MSH6/PMS2 IHC, 32,1% of discordant cases, initially pMMR (n=9/28) were reclassified as non-discordant. Among the 19 remaining discordant cases (0,68%), 4 were MSI/pMMR and 16 MSS/dMMR. For 9 of them, the discordance was the result of sampling error (47% of the remaining cases). The genetic profile of the three cases pMMR that are MSI, has to be explored. The 7 MSS/dMMR tumours demonstrated loss of any of the four MMR protein.

Conclusion: Our study emphasises the importance of accurate and careful material sampling and the advantage of combination of two complementary techniques. Discordant cases must be reviewed, since it is a major issue to select patient for appropriate treatment.


Real-world challenges of EGFR mutation testing in advanced non-small cell lung cancer: complementary roles of next generation sequencing and Idylla

L. Habran*, H. Piron, S. Pisvin, P. Delvenne

*CHU Liege, Anatomopathology Department, Belgium

Background & objectives: This real-world study aims to weigh up benefits/challenges of next generation sequencing (NGS) versus the Idylla™ Epidermal growth factor receptor mutation (EGFRm) Test in non-small cell lung cancer (NSCLC), regarding pathology lab applicability, turnaround-time (TAT) and EGFRm test performance.

Methods: A central lab analysed 193 routine diagnostic NSCLC samples in parallel with in-house validated NGS and Idylla™ EGFR Mutation Test (CE-IVD, Idylla™).

Results: Ease-of-use and short TAT were confirmed for Idylla, with shortest TAT of 1 versus 13 calendar days with NGS. 19 samples (10%) contained <5% tumour cells and were excluded from EGFRm analysis. Extracted DNA quantity was insufficient for NGS in 17 samples (10%), but 65% of those had a valid Idylla result. 27 contributive samples (16%) gave an Idylla result with reservation because Cq>26.0: 15 of those had a NGS result with reservation suggesting inferior DNA quality; 6 had insufficient DNA for NGS; another 6 had a conclusive NGS result. If sample/test quality criteria were met, 7 concordant EGFRm (5%) were detected with NGS and Idylla for known actionable variants.

Conclusion: This regional study reveals pre-analytical challenges of NSCLC biopsies. NGS and Idylla demonstrate a complementary role to improve clinically relevant molecular results. Both have distinct advantages and labs will have to determine locally the appropriate EGFRm test algorithm.

Funding: Unconditional grant provided by AstraZeneca for the purchase of Idylla cartridges, Idylla demo instrument provided by Biocartis.

Poster Sessions

PS-01 Breast Pathology


Comparing histological grading of breast cancer on core biopsy and excision specimen

S. Appukutty*, M. Jimenez-Linan, B. Mahler-Araujo, A. Duckworth, P. Wright, E. Provenzano

*Addenbrooke's Hospital, United Kingdom

Background & objectives: Histological grade of breast cancer is one of the key variables in treatment decisions following a breast cancer diagnosis. Concordance in breast cancer grade between core biopsy and excision specimens is reported as around 75%.

This study aims to look at the concordance rate of primary breast cancer grading on core biopsy and excision specimens from three centres over a period of two years reported at Addenbrooke's Hospital.

Methods: A retrospective computer search for all breast core biopsies with a B5b classification diagnosed between April 2017 and March 2019, and subsequent surgical excision specimens identified. Overall grade and the three individual grade components were extracted from the paired reports. Concordance for overall grade and each of the grade components was determined, along with percentage of cases upgraded or downgraded.

Results: A total of 980 primary surgery and 89 post-neoadjuvant cases were included.

The grade concordance following primary surgery was 82.8%, with 12.8 % upgraded and 4.4% downgraded. The concordance rates for tubule formation, pleomorphism and mitoses were 84.4%, 80.5% and 76.7% respectively.

Amongst the neoadjuvant cases overall concordance was only 45%, with upgrading and downgrading in 1.1% and 53.9% of cases respectively. Mitotic count reduction accounted for most downgrading (56.2%).

Conclusion: The concordance rate of primary breast cancer grading on core biopsy and excision specimens is in line with published literature. Reduced mitotic count following neoadjuvant chemotherapy is well described, and has been associated with improved survival, reflecting response to therapy.


Adenomyoepitheliomas of the breast: a 15-year case series

X. Ara Mancebo*, I. Miranda-Gómez, E.M. Fuentes-Camps, C. Alborch-Gil, S. Ramón y Cajal, V. Peg-Cámara

*Department of Pathology, Hospital Universitari Vall d'Hebron, Spain

Background & objectives: Adenomyoepitheliomas (AME) of the breast are uncommon biphasic tumours composed of prominent myoepithelial cells around glandular spaces. Rarely, the epithelial or myoepithelial component, or both, becomes malignant (malignant AME). AME should be considered in the differential diagnosis of several lesions.

Methods: We present a retrospective study reviewing the main clinical-pathological findings diagnosed in our institution in the last 15 years. All AME from 2004 to 2019 were collected, recording the relevant information from the pathology reports and medical histories. 13 cases of AME were identified, which corresponded to 24 biopsies.

Results: Patients' age ranged from 20 to 88 years (mean of 59 years). Most presented with a single tumour on imaging, but in three cases (23.1%) synchronous lesions were found.

In all the cases a core biopsy and a lumpectomy were performed. In 9 cases (69.2%) the first diagnosis was confirmed. 2 cases (15.4%) were initially misdiagnosed as juvenile secretory carcinoma and complex sclerosing lesion. 2 cases first diagnosed as AME finally appeared to be solid papillary carcinoma and complex fibroadenoma.

In one single case (7.7%) malignant criteria were detected, as both the myoepithelial and the epithelial component presented cytologic atypia and increased mitotic activity.

Conclusion: Due to its highly morphological variation, AME should be considered in the differential diagnosis of both benign and malignant breast lesions when a biphasic pattern with extensive adenosis and a prominent myoepithelial component is observed, especially in small biopsies.


Molecular profiling of breast carcinoma in patients of African origin

N. Badr*, O.A. Ajayi, M. Haruna, J. Steven, A. Daramola, N. Asaad, A. Abdou, A. Shaaban

*Institute of Cancer and Genomic Sciences, The University of Birmingham, United Kingdom; Department of Pathology, Faculty of Medicine, Menoufia University, Shebin El-Kom, Egypt

Background & objectives: Breast carcinoma represents a major health problem in Africa. Natural history and molecular profile of the disease remain poorly understood. We aim to study the molecular profile of African breast cancer at protein level as compared to published European data.

Methods: One North African (Egyptian, n=84) and two West African (Nigerian, n=176) breast cancer cohorts were assembled into tissue microarrays. Sections were immunohistochemically stained for ER, PR, Androgen receptor (AR), CK14, ki67, GATA3 and scored by two pathologists.

Results: 37.2% of Egyptian and 47.7% of Nigerian patients were younger than 50 years. Grade 3 carcinomas were diagnosed in 22.8% and 31.7% of Egyptian and Nigerian cases respectively. 61% of the Egyptian cases showed luminal and 23.4% triple negative phenotype. 68.3% of Nigerian patients were triple negative and only 24.2% of them were of luminal phenotype. AR was positive in 53.3% and 45.5% of Egyptian and Nigerian cancers respectively. GATA3 was positive in 79.2% of Egyptian and only 8.6% of Nigerian cases. 21.5% of Egyptian and 25.6% of Nigerian patients were of the basal phenotype. High Ki67 expression (>10%) was seen in 46.1% of Egyptian and 12.8% Nigerian patients.

Conclusion: Breast cancer in Nigerian was predominantly of triple-negative phenotype compared to Caucasians. GATA3 expression was remarkably low in the Nigerian cohort. The proportions of luminal and AR positive cancers were low in both cohorts. Basal subtype was more common in African in comparison to Caucasian patients. Unexpectedly, Ki67 showed very low expression in Nigerian patients. Our data highlight differences in the immunohistochemical profile between the African and European breast cancer.


Immunohistochemical expression of tetraspanin 6 (TSPAN6) in inflammatory breast carcinoma and correlation with tumour microenvironment

N. Badr*, J. McMurray, I. Danial, N. Asaad, N. Sharma, Y. Horimoto, F. Hoar, D. Rea, J.L. Jones, J. Steven, R. Merard, P. Lewis, K. Hunter, F. Berditchevski, A. Shaaban

* Institute of Cancer and Genomic Sciences, The University of Birmingham, United Kingdom; Department of Pathology, Faculty of Medicine, Menoufia University, Shebin El-Kom, Egypt

Background & objectives: Inflammatory breast cancer (IBC) is an aggressive form of breast cancer. Tumour immune microenvironment and tetraspanins are thought to play role in tumorigenesis of various malignancies. We aim to correlate Tspan6 expression with clinical parameters and characteristics of IBC microenvironment.

Methods: 124 IBC core biopsies were immunohistochemically stained for Tspan6. Multiplex immunofluorescent staining of CD4, CD8, CD68, CD20, and FOXP3 was performed on 75 cases. Machine learning with Inform 2.2.1 software was used to analyse images. Clinicopathological parameters including tumour characteristics, neoadjuvant pathological response, oestrogen receptor, HER2 status, and overall survival were collected.

Results: Half of the cases were ER positive and 29.5% were HER2 positive. 53.3% showed high grade tumour morphology. Pathological complete response was achieved in 20.9% of cases. Survival ranged from one to 138 months with a median of 32.5 months. 58.9% of cases were Tspan6 positive. Positive Tspan6 expression correlated with pathological response (p=0.04) and increased intratumoral CD20+ cell infiltration (p=0.02). Stromal CD20+ cell infiltration correlated with grade I and II (p=0.04) while high stromal CD68+ cells correlated with grade III tumour (p=0.03). High intratumoral CD68+ density correlated with no pathological response (p=0.02). Intratumoral co-localization of CD20+ and CD68+ cells correlated with positive Tspan6 expression (p=0.04).

Conclusion: The expression of Tspan6 was significantly associated with increased B-lymphocyte (CD20+) cell counts and increased response rates. B-lymphocytes were shown to be closer to other immune subsets in those patients with higher Tspan6 expression. We show for the first time Tspan6 role in tumour response to neoadjuvant chemotherapy and the ability of tumour cells to interact with the immune cells in different locations of the IBC microenvironment.


Neoadjuvant chemotherapy for breast cancer – analysis of changes in breast cancer phenotype, hormonal and HER2 expression

N. Badr*, I. Danial, D. Rea, J. Steven, A. Shaaban

*Institute of Cancer and Genomic Sciences, The University of Birmingham, United Kingdom; Department of Pathology, Faculty of Medicine, Menoufia University, Shebin El-Kom, Egypt

Background & objectives: Neoadjuvant chemotherapy (NACT) is being used for primary breast carcinoma with the aim of down-staging and facilitating conservative surgery. We assessed the effect of NACT on pathological characteristics, hormone receptors and HER2 expression and whether post-treatment re-testing is warranted.

Methods: Patients who received neoadjuvant chemotherapy for primary and operable invasive breast cancer were selected from the database of a large UK tertiary referral centre in the period between 2006 and 2019. Comprehensive clinical, histopathological and follow up data were collected. Statistical analysis of the data was performed using SPSS V.26.

Results: A total of 351 cases were identified. 68.7% patients were Caucasians and 50.4% were younger than 50 years. No special type carcinoma was diagnosed in 80.3% and 51.3% were grade 3 tumours. Pathological complete response was achieved in 23.1% of cases particularly in HER2 positive group (35.8%) (p=0.043). 39.9% of cases with pre-treatment positive lymph nodes showed complete response. There was a change in the histological type in 20.1% (p<0.001). Significant down-grading was seen in 59% (p<0.001). ER status changed in 3% of cases. PR and HER2 status showed significant changes post-treatment in 11.2% and 5.9% of the cases respectively, (p<0.001). Patients with luminal cancer showed better overall survival (p<0.001).

Conclusion: Significant changes in tumour morphology, grade and marker expression occur following NACT. Pathological complete response was achieved in more than fifth of cases especially with HER2 positive status. We recommend o repeat testing of hormone receptors and HER2 on residual tumours. This can provide alternative treatment options for those patients.


Correlation between PD-L1 expression and clinicopathological characteristics in triple-negative breast cancer patients

N. Basheska*, B. Ognenoska-Jankovska

*Department of Histopathology & Clinical Cytology, University Clinic of Radiotherapy & Oncology, Ss Cyril and Methodius University Faculty of Medicine, North Macedonia

Background & objectives: While immunotherapy is emerging as an effective treatment option for advanced triple-negative breast carcinoma (TNBC) patients, the clinicopathological significance of PD-L1 expression in TNBC remains unclear. Our objective was to investigate the association between PD-L1 expression and clinicopathological characteristics in TNBC.

Methods: The study group comprised 47 TNBC patients in which PD-L1 status was evaluated by immunohistochemistry with SP142 assay on the Ventana BenchMark. All PDL1(+) tumour-associated immune cells (IC) were quantified as % of the tumour area. Tumours were classified as PD-L1(+)(≥1%) or PD-L1(-)(<1%). The statistical significance of the correlation between PD-L1 status and clinicopathological characteristics was determined by chi-square test.

Results: PD-L1(+) were 24(51.1%) of the 47 TNBC patients whose median age at diagnosis was 59 (range, 39-79). 53.5%(23/43) of the primary and 25%(1/4) of the metastatic TNBC cases were PD-L1(+). 21(87.5%) of the PD-L1(+) TNBC had IC1(≥1 and <5%), 2(8.3%) had IC2(≥5 and <10%), and 1(4.2%) had IC3(≥10) score. The PD-L1(+) status significantly associated with high histological grade (G3, P=0.022), and higher proliferative index (Ki-67>35%, P=0.004), while the correlation with larger tumour size (>2 cm, P=0.055) did not reach statistical significance. No significant relationship was found between PD-L1 status and other variables such as patients` age, postoperative stage, tumour status, lymph nodal status, tumour type, vascular invasion, and p53 expression.

Conclusion: Our preliminary results suggest that PD-L1 expression is associated with several high-risk clinicopathological parameters in TNBC patients. Further larger studies are warranted to clarify the clinical relevance of PD-L1 expression in TNBC patients.


The significance of tumour budding in breast carcinomas and its relationship with E-cadherin, CD44 and CTLA-4 expressions

T. Bolme Savli*, H. Pasaoglu, A. Muhammedoglu, T.C. Savli

*Health Science University, Bagcilar Training and Research Hospital, Department of Pathology, Turkey

Background & objectives: Tumour budding(TB) (isolated <5 tumour cells at invasive edge)are thought to be effective in prognosis in breast carcinomas(BC).We aimed to investigate TB in BC using E-cadherin that provides cell-cell interaction,CD44 as stem cell marker and CTLA-4 receptor as self-tolerance marker.

Methods: 179 BC patients (172 invasive ductal, 7 metaplastic) included. For each case, buds were counted at x200 magnification. 90% for E-Cadherin and 10% for CD44 were regarded as positive staining cut-offs. Staining intensity and percentage of CTLA-4 in bud and bud microenvironment lymphocytes were evaluated. Tumours were separated into low (<5) and high bud groups (≥5) according to median bud number.

Results: High bud tumours were likely to have lymphovascular (p= 0.001), perineural invasion (p<0.001), higher pT stage(p=0.025). Tumour bud number is correlated with higher metastatic lymph node number (p<0.001) and tumour size (p<0.01). No significant relation was found between number of buds and peritumoral lymphocytic reaction, immunohistochemistry based molecular subtyping, E-cadherin, and CD44 staining (p>0.05 for all). E-cadherin was significantly lost in buds in regard to corresponding tumour (p<0.001), while CD44 staining pattern is preserved (p = 0.76). In high bud tumours, CTLA-4 staining percentage of lymphocytes in bud microenvironment was found to be significantly higher (p =0.026). Every increase in tumour bud number, decreases overall survival risk 1.07 times (1.01-1.12 95%CI, p=0.013).

Conclusion: Tumour bud is a poor prognostic factor in breast carcinomas. Increased CTLA-4 can block antitumour response, causing an increase in the number of buds. Anti-CTLA-4 immunotherapies may be beneficial in patients with high bud detected breast carcinoma.


A dynamic macrophagic environment in xanthogranulomatous mastitis: a broader clue to xanthomatous diseases pathophysiology and clinical evolution?

L. Campos Clemente*, M. Marques-Piubelli, M. Balancin, L. Reis, V.L. Capelozzi

*University of São Paulo, Brazil

Background & objectives: Xanthogranulomatous mastitis is an inflammatory condition. Usually no immunohistochemistry (IHC) characterization is performed in routine surgical pathology. In this study, we explored by IHC the macrophagic polarization status of M1 and M2 subtypes.

Methods: A retrospective single-institution case retrieve was performed from the archives. IHC was performed for CD68, Arginase I, Arginase II, NOS-2, and TGF-Beta. Sequential 400x images were acquired through a microscope coupled camera and quantified through an optical threshold method in ImageJ for each marker. Statistics were executed on SPSS 25 (ANOVA and Spearman's test), p-value less than 0.05 was significant.

Results: We reviewed 11 cases. All patients were female, median age of 56,5 years (29-72). Seven (63%) cases were radiologically described as an irregular nodule/asymmetry, while 4 (36%) as circumscribed nodule. The mean ratio of IHC stain was: Arginase I (0.80), Arginase II (0.70), NOS-2 (0.85), and TGF-B (0.60). M1 polarization elements (ArgI, II and TGF-B) were correlated (p=0.005 for ArgI-ArgII and p=0.001 for TGF-B-ArgI/II). Both M1 and M2 surrogate markers were expressed in all cases, characterizing a bipolar macrophagic activation status. No significant expressed in all cases, characterizing a bipolar macrophagic activation status. No significant correlation was evidenced between macrophage status and radiological presentation, necrosis or epithelioid giant cells.

Conclusion: XM is an uncommon diagnosis and this is the first attempt at classifying MC in indexed English-language literature. The bipolar status may translate as the destructive and reparative dynamic of lesions, evoking a slow-progressing evolution until reparative pole is reached.


Synchronous phyllodes tumour and ductal carcinoma in situ of breast

R. Carp*, C. Suciu, D. Anderco, D. Grujic

*Timisoara County Emergency Hospital, Romania

Background & objectives: We present a case of a phyllodes tumour coexisting with ductal carcinoma in situ (DCIS). Rarely, those two lesions are part of the same tumour, but in this case they represent two different neoplasms.

Methods: A 70-year-old female patient presented in our hospital complaining of a lump within the left breast. The lesion appeared 5 months before and it didn’t increased in size until lumpectomy was performed. For the histological examination we used hematoxylin&eosin stained slides and immunohistochemical markers: CK5, ER, Ki67 and p-53.

Results: Histology analysis of the lumpectomy specimen showed phyllodes tumour with a 20% proliferative index and approximately 10 mitoses/10 HP, p-53 positive stromal cells. Following the histopathological result, modified radical mastectomy with lymph node dissection (Madden technique) was effectuated. The mastectomy specimen revealed multifocal high grade DCIS with comedo and non-comedo pattern; CK5 was expressed focally or in a continuous layer in the myoepithelial cells, excluding an invasive component, the tumour cells being diffusely and intensely positive for ER.

Conclusion: The coexistence of phyllodes tumour and ductal carcinoma in situ as different lesions is a rare occurrence which complicates the evolution and treatment. As far as we know, this is the first case of synchronous malignant phyllodes tumour and ductal carcinoma in situ diagnosed in our department.


PD-L1 status by SP142 and sp263 immunohistochemistry in triple-negative breast cancer

A. Cordoba*, M.D.R. Mercado, I. Amat, R. Beloqui, B. Aguiar, Y. Rodriguez, D. Guerrero-Setas

*Complejo Hospitalario de Navarra, Spain

Background & objectives: Triple-negative breast cancer (TNBC) is the most immunogenic subtype with a higher proportion of tumour-infiltrating lymphocytes (TILs). TNBC is very aggressive but the choice of Immunotherapy based on PD-L1 expression seems to be a promising therapeutic option for this subtype.

Methods: PD-L1 expression was evaluated with SP142 and SP263 antibody clones in 100 TNBCs included in tissue microarrays. We assessed the presence of evaluable PD-L1 staining in inflammatory cells (ICs) in the intratumoral stroma, considering positive if present in ≥1% of the area occupied by ICs. The presence of TILs were also evaluated.

Results: The percentage of patients with PD-L1 positive ICs was 46% and 82% by using SP142 and SP263 antibody, respectively. SP142 is the approved diagnostic test to identify patients who most likely would benefit for the treatment with PD-L1 inhibitor (atezolizumab).The overall percentage agreement between both systems was 55%.The SP263 PD-L1 assay (IC ≥1%) identified a larger number of TNBC than SP142 PD-L1 assay (IC ≥1%).

Conclusion: All SP142-positive cases were also SP263-positive.SP142 and SP263-positive cases displayed higher number of TILs.Additional studies about PD-L1 expression with different clones and the identification of improved biomarkers to predict clinical benefit are needed.


Adenoid cystic carcinoma of the breast, 20-year experience

A. Cordoba*, I. Fernandez, M.D.R. Mercado, I. Amat, B. Aguiar, A. Larrea, R. Beloqui, D. Guerrero

*Complejo Hospitalario de Navarra, Spain

Background & objectives: Adenoid cystic carcinoma (ACC) is an uncommon breast cancer (< 0.1%). ACC is typically triple negative with good prognosis. Due to its low frequency there's no therapeutic consensus. We report a series of ACC collected during the last 20 years.

Methods: A retrospective study of all breast ACC cases diagnosed in Complejo Hospitalario de Navarra, between 1998-2018 was performed. Clinical and pathological characteristics were assessed and compared with current literature. We identified twelve cases of ACC, from a total of 18,241 patients. Mean age patients years was 63.5. All of them were clinical stage I-II at diagnosis.

Results: Average tumour size of 23.84 mm. According to Nottingham grading system, eight cases were grade 1, two grade 2 and two grade 3. When compared with Ro classification three were grade 1, four grade 2 and five grade 3. One out of 12 cases was ER positive, the rest of them were triple negative. One case presented a lymph node micrometastases. None of them presented local recurrences (77.5 months mean follow-up), and one developed metastases 5 months after diagnosis. None of them were BRAF-V600E immunoreactive.

Conclusion: Prognosis of breast ACC remains uncertain.Seldom usually ACC shows an indolent course, we report one case with an aggressive behaviour.Sentinel node biopsy can't be avoided according to our study (one micrometastases).Despite of previous reports of BRAF-V600E expression in ACC, all our cases were negative.


Predictive factors of pathological complete response to neoadjuvant therapy in triple-negative breast carcinoma

J. Costa*, N. Coimbra, P. Lopes, L. Antunes, M. Souto Moura, C. Leal

*IPO-Porto, Portugal

Background & objectives: Triple-negative breast carcinoma (TNBC) is a heterogeneous disease, both clinically, pathologically and molecularly. There is increasing evidence that response to neoadjuvant therapy (NAT) may be affected by certain molecular and morphological features.

Methods: Core-needle biopsies of TNBC patients submitted to NAT in our institution (2016-2018) were reviewed. Clinico-pathological features and expression of biomarkers (Ki67|vimentin|androgen receptor (AR)|SOX-10|p63|PD-L1) were recorded and correlated with pathological response to NAT (complete response/near complete response with ≤10% residual tumour (pCR/pNCR) group versus partial response with ≥10% residual tumour/no evidence of response (pPR/pAOR) group). Statistical significance was considered as p<0.05.

Results: Biopsies of 45 patients with a median age of 51 years were reviewed. 27(60%) presented as stage I-II and 18(40%) as stage III-IV. 20(44.4%) presented pCR and 6(13.3%) pNCR, after NAT. 35(77.8%) were diagnosed with invasive carcinoma NST, 40(88.9%) had histological grade 3. When comparing the pCR/pNCR group versus the pPR/pAOR group, p63 was positive in 12(46.2%)vs.4(21.1%), vimentin in 19(73.1%)vs.11(57.9%), SOX-10 in 20(76.9%)vs.10(52.6%), AR in 13(50%)vs.11(57.9%) and PD-L1 in 17(65.4%)vs.11(57.9%). The pCR/pNCR group had a significantly higher proportion of cases with Ki67>50% when comparing to the pPR/pAOR group (23(88.5%)vs.6(31.6%),p<0.001), corresponding to almost 17 times more odds of having pCR/pNCR in patients with Ki67>50% (OR=16.61(95%CI=[3.55;77.78])).

Conclusion: TNBC express a wide range of myoepithelial markers, as well as variable expression of AR and PD-L1, with potential therapeutic applications. High values of Ki67 are significantly associated with pCR/pNCR. The size of the cohort may have impacted our results.


Breast myofibroblastoma – a benign rarity on biopsy

R. Cruz*, I. Alves

*Department of Pathology, Hospital de Santa Maria, CHULN, Instituto de Histologia e Biologia do Desenvolvimento, Faculdade de Medicina da Universidade de Lisboa, Portugal

Background & objectives: Breast myofibroblastoma is a rare benign mesenchymal tumour, with <90 cases described in the literature. We report a case of myofibroblastoma of the breast. The purpose of this case report was to review the characteristics of this rare neoplasm.

Methods: A 72-year old woman with a clinical history of HIV infection under antiretroviral therapy and depressive syndrome, which during her routine breast ultrasonographic screening exam was revealed a well-circumscribed hypoechogenic breast nodule with 8 mm, biopsied under the clinical and radiographic suspicion of being a fibroadenoma.

Results: Histologically, the nodule was composed by a proliferation of spindle cells arranged in haphazardly intersecting fascicles, interspersed by bands of collagen. The cells had abundant eosinophilic cytoplasm with oval nuclei, occasionally with small nucleoli; necrosis was absent, mitoses were rare (1/10 HPF), without atypia. With this morphological aspects, the differential diagnosis should include nodular fasciitis, fibromatosis, leiomyoma, spindle cell metaplastic carcinoma and spindle cell lipoma. The neoplastic cells showed diffuse and strong staining for CD34, BCL2 and oestrogen receptors, focal staining for desmin, smooth muscle actin and h-caldesmon, and were negative for cytokeratins, β-catenin, S100 and p63. The morphological and immunohistochemical features were compatible with myofibroblastoma.

Conclusion: Myofibroblastoma of the breast is a benign mesenchymal tumour composed of fibroblasts and myofibroblasts, which could have a wide variety of morphological features, frequently similar to spindle cell lipoma. The molecular study of this entities has revealed the same genotype.


Comparison of the hormone receptor status, HER2 status and Ki67 index between preoperative core needle biopsy and surgical specimens in invasive breast cancer

A. Dobriakov*, K. Opalenov, V. Salaeva, E. Poputchikova, N. Shvets, M. Antonov, A. Tokmakov, Y. Cherkasova

*Moscow Bakhrushin Brothers Hospital, Russia

Background & objectives: Accurate analysis of hormone receptor status, HER2 status and Ki67 of breast core needle biopsy (CNB) tissue is important for determining neoadjuvant systemic therapies for invasive breast cancer.

Methods: We aimed to evaluate the concordance rate (CR) of oestrogen receptor (ER), progesterone receptor (PR), HER2, and Ki67 between CNBs and surgical specimens. The study was conducted with invasive breast cancer patients who underwent surgery after CNB without preoperative systemic therapy at Moscow Bakhrushin brothers Hospital between December 2013 and December 2019.

Results: ER, PR, HER2, and Ki67 were analysed using immunohistochemistry. ER and PR were evaluated by Allred score (0-8). HER2 was graded from 0 to +3, and all 2+ cases were tested with in situ hybridization. The labelling index of Ki67 was counted manually. The cutoff value for Ki67 was 20%. In total, 239 patients were evaluated. The median age was 65 (range, 29 to 88 years).The CRs of ER, PR, HER2, and Ki67 were 97.1% (kappa, 0.805; p<.001), 89.5% (kappa, 0.589; p<.001), 97.4% (kappa, 0.81; p<.001), and 79.1% (kappa, 0.555; p<.001), respectively.

Conclusion: CNB was reasonably accurate for determining ER, PR, HER2 and Ki67.


Clinical features and pathological findings in a series of MRI-guided breast biopsies

A. Doyle*, A. Maguire, M. Courtney, S. O'Keefe

*St James's Hospital, Ireland

Background & objectives: Breast magnetic resonance imaging is used for high-risk screening and in evaluation of extent of disease in newly diagnosed breast cancer patients. We wanted to correlate the pathology results of MRI-guided biopsies to MRI findings and patient clinical history characteristics.

Methods: We retrospectively reviewed clinical history, MRI features and pathology outcomes of MRI-guided vacuum needle biopsies performed at our institution between September 2015 and July 2019.

Results: 45 patients were biopsied. 48% had newly diagnosed breast cancer, the strongest predictor of malignancy (malignancy rate 56%). 46% of biopsies were from women on surveillance (malignancy rate 16.7%) and 6% in women with conventional imaging and suspicious symptoms (malignancy rate 33%). 18 (40%) lesions were malignant: 12 invasive carcinomas; 6 DCIS. Non-mass like enhancement was the most frequent descriptor of both malignant (8/19, 42.1%) and benign lesions (11/26, 42.3%).

Conclusion: MRI detects malignancies undetected by other imaging modalities but also detects a wide variety of benign lesions and MRI features of both can be similar necessitating biopsy for histological evaluation. There should be a low threshold for proceeding to MRI-guided biopsy particularly in women with newly diagnosed breast cancer.


Two cases of intramammary metastases from cutaneous melanoma

D. Dzhenkov*, L. Petkova, N. Yanilova, P. Ghenev

*Medical University Varna, Bulgaria

Background & objectives: We present here two cases of histologically diagnosed metastatic small cell achromatic malignant melanoma. Each of these cases presented a challenge for the pathologist in a different way.

Methods: The first case is of a 49-year-old woman with subsequent sectoral resections of both breasts (2017 and 2019), histologic findings interpreted as triple negative mammary carcinoma. Later on, pulmonary lesions appeared and transthoracic thick-needle biopsy was performed. Histology and immunohistochemistry revealed small cell achromatic malignant melanoma. Meanwhile, regressive melanoma of the skin over the sternum was suspected.

Results: So, previous biopsies were revised and the diagnosis of achromatic malignant melanoma, producing bilateral mammary and pulmonary metastases was confirmed. Finally, BRAF V600 mutation was proven in the breast metastasis. The second case is a 45-year-old woman, with a malignant melanoma of the breast skin 13 years ago. Because of the staging (pT1, Clark 1) treatment with Calgevax was performed only. In 2020 intramammary lesion was detected, clinically suspected for mammary fibroadenoma. The lesion was surgically removed and the histology and immunophenotyping were consistent of a small cell malignant melanoma - a metastasis of the primary skin tumour.

Conclusion: Achromatic melanoma, “the great imitator” has always been a challenge for pathologists. In the first case metastases were interpreted as a primary tumour, and in the second – late metastases appeared. Careful interpretation of histological findings with correlation of clinical data is of pivotal importance.


High HER2 signals and HER2/CEP17 ratio tumours achieve better responses in breast cancer

I.J. Expósito Afonso*, T. del Pino Sedeño, I. González Villa, J.F. Loro Ferrer, J.J. Cabrera Galván

*Hospital Universitario de Canarias, Spain

Background & objectives: HER2 molecular subtype is one of those which achieves most pCR rates in breast cancer. The objective of this study is to evaluate the relationship between HER2 signals and pCR and HER2/CEP17 ratio and response to neoadjuvant chemotherapy.

Methods: 37 HER2-overexpressing early breast cancer patients who had received neoadjuvant trastuzumab were included in this study. We performed in situ hybridization and measured HER2 and chromosome 17 centromere (CEP17) copy numbers. HER2/CEP17 ratio was calculated. Analysis between that and response to neoadjuvant chemotherapy assessed by RCB were examined.

Results: In trastuzumab treated patients, 83,8% were HER2-Luminal and 16,2% HER2 hormone receptor (HR) negative. 46% of patients were Nottingham grade 2 and 54% were grade 3. From 37 patients, 29,7% achieved pCR. 43,5% of patients with high-level amplification (HER2/CEP17 ratio ≥ 6) obtained pCR compared to 2,7% of patients with low-level amplification (HER2/CEP17 <6 ratio) (p=0.030). Regarding to HER2 number of signals, 27% of patients with HER2>6 signals per nuclei obtained pCR compared to 2,7% of patients with HER2<6 signals per nuclei (p = 0.04852).

Conclusion: An HER2 copy number>6 in the pretherapeutic tumour biopsy is associated with a significantly higher pCR rate. Also HER2/CEP17 ratio>6 is related to pCR in HER2+ tumours treated with neoadjuvant trastuzumab.


Columnar cell lesion in breast tissue biopsies: an often underreported diagnosis

U. Ezenkwa*, A. Rahman, A. Olusanya, R. Anyanwu, G. Ogun

*University College Hospital Ibadan, Nigeria

Background & objectives: Columnar cell lesion (CLL) of the breast belongs to the spectrum of proliferative breast disease with premalignant potential, although it is often underreported. This study investigated the frequency of CCL in non-malignant breast biopsies in a single general pathology practice.

Methods: H&E stained slides of non-malignant breast biopsies received over a four-year period were reviewed for the presence of columnar cell features (columnar cell change with/without atypia, columnar cell hyperplasia with/without atypia, and flat epithelial atypia) following established protocol. Information on patient age at diagnosis was obtained from the surgical pathology records. Data generated was presented as percentages.

Results: Columnar cell lesions were present in 22 (7.7%) of 286 cases reviewed. Of these, 18 (81.8%) were columnar cell change, whilst 2 (9.1%) each were columnar cell hyperplasia and flat epithelial atypia. The median age was 39 years (18-64years) with modal age at 5th decade of life. Eleven (50%) cases occurred in individuals who were aged 40 years and below. Flat epithelial atypia was seen at 4th and 5th decades.

Conclusion: This study suggests that CCL occur in about 7% of benign proliferative breast lesions in our practice and are predominantly non-atypical. The observed median age of occurrence suggests a frequent occurrence in the young. This might influence the earlier occurrence of breast cancers in our population.


Interaction between the expression of mTOR and PD-L1 signalling pathway in triple-negative breast cancer

J.P. Flores Gutierrez*, F. Lopez Lopez, S. Treviño Garza, N. Vilches Cisneros, O. Barboza Quintana, R. Garza Guajardo, M. Molina Ayala

*Universidad Autónoma de Nuevo León, Mexico

Background & objectives: Programmed death ligand 1 (PD-L1) and mammalian target of rapamycin (mTOR) are pathways expressed on tumour cells that interact with the immune system function. We investigated the relationship between their expression in Triple-Negative Breast Cancer (TNBC) based on clinicopathological variables.

Methods: 50 consecutive TNBC cases were collected, the histopathological report and paraffin block were obtained from each case. The expression of mTOR and PD-L1 on tumour cells was evaluated by immunohistochemistry using their respective antibodies. The results were correlated with the age, clinical stage, metastatic lymph nodes and tumour size.

Results: The results from the immunohistochemistry show a positivity for mTOR in 38 cases (76%) and for PD-L1 in 11 cases (22%), a joint expression of 21.05% was demonstrated. PD-L1 and mTOR expression did not correlate with clinical stage, age, metastatic lymph nodes or tumour size, however the analysis of the number of metastatic lymph nodes shows a slight significant tendency in tumour cells when both markers were shown positive (p=.068). The results of PD-L1 and mTOR appears to have a correlation with the lymph nodes status based on the TNM staging system.

Conclusion: mTOR pathway has been poorly studied in TNBC and seems to favour PD-L1 expression, the results suggest new scenarios with drugs that inhibit both pathways. More clinical response-related TNBC cases are needed to assess the correlation between mTOR and PD-L1.


A case of tall cell carcinoma with reversed polarity of the breast with a recent history of gastric carcinoma

M. Gündüz*, S. Önder, B. Yeni, A. Bayram, M. Tükenmez, E. Yavuz

*Istanbul Faculty of Medicine, Pathology Department, Turkey

Background & objectives: Tall cell carcinoma with reversed polarity (TCCwRP) is a rare breast tumour. Despite the triple negative profile, it has an indolent behaviour. However, cases with lymph node and bone metastasis were published in literature.

Methods: The patient was a 62-year-old female who complained of multiple lumps in both breasts and had a history of surgery and chemotherapy for gastric carcinoma, 12 months before mastectomy. Grossly, there were two tumours, measured 14 and 13-milimeters, which were partly and totally well-demarcated respectively. Both had brown and white cut surfaces.

Results: Microscopically, both tumours were composed of nests surrounded by artefactual empty space and separated by dense fibrous stroma. Both exhibited no sign of chemotherapy-induced regression. The tumour cells were columnar to cuboidal with abundant eosinophilic cytoplasm and oval, bland-looking nuclei which were located at the apical rather than basal pole of the cells. Foci of ductal carcinoma in situ were also found. Lymphovascular invasion and necrosis were absent. One of 22 axillary lymph nodes showed metastasis. The neoplastic cells were positive for GATA-3 and mammaglobin and negative for PAX-8 and TTF-1, excluding metastasis of thyroid carcinoma. They displayed diffuse expression of cytokeratin 7, cytokeratin 5/6 and 14, and were negative

Conclusion: for oestrogen receptor, progesterone receptor and HER2. Ki67 proliferation rates were 18% and 15%, respectively. EMA highlighted the apical membranes of neoplastic cells and pointed out the apical positioning of nuclei, therefore was helpful to designate the term reversed polarity. TCCwRP is a rare breast neoplasm, may mimic thyroid carcinoma metastasis. Hence, pathologists should keep it in mind and be aware of its morphological and immunohistochemical features.


High molecular weight cytokeratin expression in ductal carcinoma in situ of the breast: a diagnostic pitfall

T. Heaton*, A. Elhag, R. Mihai, E. Rakha, S. Bakhiet

*Hull University Teaching Hospitals NHS Trust, United Kingdom

Background & objectives: In breast pathology, the distinction between intraductal epithelial hyperplasia of usual type (HUT) and neoplastic lesions is challenging and often requires immunohistochemistry for confirmation. However, deviations from well-known patterns of expression exist in routine practice making such diagnoses challenging.

Methods: We present two cases of mammary Paget’s disease (PD) that underwent central excision of the nipple-areolar complex.

Histology confirmed PD with underlying intraductal epithelial proliferations difficult to differentiate between HUT and intermediate grade DCIS. Basal cytokeratins (CK5/6 and CK14) and oestrogen receptor (ER) immunohistochemical staining were performed to differentiate between the two entities, as well as HER2.

Results: Immunohistochemistry showed mosaic positivity of both basal cytokeratins akin to HUT. ER immunohistochemistry was heterogeneous in one case and negative in the other. However, the diagnosis of DCIS was confirmed in both cases by strong HER2 positivity and by extension of the proliferating cells into the epidermis as PD. Further morphological evaluation revealed nuclear hyperchromasia and occasional prominent nucleoli, features not seen in HUT.

Conclusion: These cases demonstrate the importance of cellular morphology in diagnosing intermediate and high-grade DCIS and the potential diagnostic pitfall of relying on immunohistochemistry alone. The mosaic pattern of expression for basal CKs may represent basal-like differentiation of the neoplastic cells and this pattern of immunostaining should be interpreted in the appropriate context.


Comparison of the pam50 intrinsic subtypes with immunohistochemistry in breast cancer patients

E. Honrado*, S. Sáez Álvarez, A. López González, B.I. Távara Silva, L. Prieto Domínguez, J. Martínez Martínez, M.A. López Flores, A. García Palomo

*Complejo Asistencial Universitario de León, Spain

Background & objectives: PAM50 classifies breast tumours, predicts risk of recurrence and evaluate the need of adjuvant chemotherapy. Clinical subtyping, however, is based on immunohistochemistry (IHC) biomarkers. We aimed to analyse the discordance between immunohistochemistry based surrogate subtyping and PAM50 intrinsic subtypes.

Methods: PAM50 intrinsic subtypes were determined according to 50 cancer genes expression in a total of 74 breast tumours, oestrogen receptor positive, HER-2 negative, T<=30 mm, pN0, pN1mi or pN1. Subtype Luminal A was defined by IHQ as Ki67 < 14% and Luminal B as Ki67 >=14%. Clinicopathological parameters were collected, including grade and stage in all tumours.

Results: A 33% of tumours, showed discrepancy between IHC subtype and PAM50 intrinsic subtype in our study. Out of 41 Luminal B tumours subtype performed by IHC, PAM50 reclassified 7 (17%) as Luminal A and low risk of recurrence, indicating that no adjuvant chemotherapy was needed. On the other hand, 7 (20.5%) Luminal A tumours classified by IHQ, changed to high risk by PAM50, being able to benefit from chemotherapy. The tumours reclassified by PAM 50 as low risk of recurrence were grade 1 and stage IA (71.4% Vs 17.7%, p=0.036) while the tumours reclassified as high risk, were grade 2 and stage IIA (60.0% Vs.47.8% p=0.048).

Conclusion: A substantial portion of tumours showed discrepancy between IHC subtype and PAM50 subtype in our study. Gene expression-based classification shifted some tumours categorized as low risk by IHQ criteria to higher-risk subtypes and vice versa. Current IHC-based classification could mislead the treatment and result in poor outcome in 20% of breast cancers. Subtypes from PAM50 seem better able to predict tumours with poorer outcomes compared with using IHC markers.


A rare case of tumour-to-tumour metastasis from invasive lobular breast carcinoma to ovarian cystadenoma

R. Ibrahim*, Z. Elalfy

*Department of Pathology, Faculty of Medicine, Ain Shams University, Egypt

Background & objectives: Tumour-to-tumour metastasis is an unusual phenomenon. The most frequently reported donor tumours are lung and breast carcinomas, whereas meningiomas represent the most common recipients. We hereby describe a rare case of metastasis from invasive lobular breast carcinoma to ovarian cystadenoma.

Methods: A 60-year-old female presenting with a 9 cm ovarian mass and a right breast lesion. Ultrasonography report of the breast lesion revealed features suggestive of malignant mass (BIRADS 4b). Fine needle aspiration of the breast tumour yielded malignant smear. Patient underwent right modified radical mastectomy, right oophorectomy and infracolic omentectomy.

Results: Histopathologic evaluation of the breast mass revealed invasive lobular carcinoma, positive for oestrogen, progesterone receptors and Her2-neu negative. The ovarian mass exhibited features of benign ovarian cystadenoma harbouring a distinct malignant component, histologically similar to invasive lobular carcinoma of the breast. Immunohistochemical assessment of the aforementioned invasive component using GCDFP15 and E-cadhrin confirmed the breast origin of the tumour and the lobular carcinoma diagnosis. Other markers were used to exclude metastasis from different sites such as Napsin-A and CDX-2

Conclusion: This is the first reported case of invasive lobular breast carcinoma metastasis to ovarian cystadenoma. The possibility of tumour-to-tumour metastasis rises if any tumour exhibits dimorphic histology, which is diagnostically challenging. This indicates poor outcome, disease dissemination and aggressiveness.


Audit of histopathology reports for mastectomy specimens in a tertiary hospital, south west Nigeria

V. Iyawe*, O. Qassid, K. Adeleke, A. Agboola

*Olabisi Onabanjo University and Teaching Hospital, Sagamu, Nigeria

Background & objectives: Mastectomy remains the mainstay of the treatment of breast cancer in Nigeria. Pathologic diagnosis and reporting is one of the basic available resources. It is therefore critical that reports contain key diagnostic and prognostic indicators guide the management of patients.

Methods: Copies of histopathology reports were retrieved from the histopathology department for all mastectomies reported between January 2016 and December 2019. Completeness for reporting of the mastectomies was examined for 7 key items: tumour size, tumour type, histological grade, margins of excision, vessel invasion, nodal status and TNM stage. The items were indicated as present or absent from the report.

Results: The histological type had the highest frequency of 100%, while the lymph node status and tumour size were reported in 92.3% and 82.1% respectively. The least frequently reported items were the TNM staging and the vessel invasion at 23.1% and 25.6% respectively. Only 2.6% of reports had all the variables reported. In addition, histological grade was reported in 76.9% while statements about margins of excision were contained in 71.8% of the report.

Conclusion: There is currently no local guidelines to adhere to in the reporting of mastectomy specimens, therefore having discussed the results with the local team, the royal college of pathology datasets have been adopted for use. Synoptic reporting will allow for more consistent and complete reports. A similar study will need to be repeated after the synoptic reporting is introduced to evaluate its effectiveness in improving the quality of pathology reports.


Neuroendocrine neoplasms of the breast showing peculiar forms of recurrence and/or metastasis

T. Kawasaki*, A. Sapino, M. Ishida, T. Tada, H. Matsuya, H. Nakagomi, T. Oyama, C. Muramatsu, J. Ichikawa, M. Saitoh, T. Kondo, R. Katoh, T. Sugai, Y. Nakamura, K. Kaira

*Saitama Medical University, Japan

Background & objectives: This is the first report of a mammary neuroendocrine tumour (NET) showing a relapse related to needle implantation (Case 1). Furthermore, we describe the first case of a NET resulting in a huge tumour embolization in the breast (Case 2).

Methods: [Case 1] A 60-year-old Japanese woman presented with skin retraction in the right breast. Ultrasound-guided core needle biopsy (CNB) of the irregular, hypoechoic breast mass yielded a pathological carcinoma diagnosis. [Case 2] A 42-year-old Thai woman presented with a palpable mass in the left breast. Ultrasonography revealed a sharply-marginated, hypoechoic tumour, with a cranially-extending lumen-like structure filled with solid tumour.

Results: [Case 1] A poorly-delimited tumour (12x10mm) was composed of a trabecular growth of polygonal cells with a highly-vascular stroma. We noted small subcutaneous scars with disseminated neoplastic cell clusters, causally-related to the preoperative CNB. Neoplastic cells were immuno-positive for chromogranin A and NCAM. She had become aware of a mass at the skin puncture site for the previous CNB 47 months after surgery. We pathologically confirmed the recurrent NET. [Case 2] The lesion was histopathologically diagnosed as a mammary NET, measuring 17x15mm, with extensive intravenous spread. She postoperatively received adjuvant chemotherapy. However, bone metastases were identified 36 months after surgery and liver metastases were detected 47 months after surgery.

Conclusion: [Case 1] To minimize the clinical impact of tumour displacement, we need to consider preventive radiation therapy for needle pathways, their excision and/or use of vacuum-assisted biopsy devices (Virchow Archiv, 2015). [Case 2] Our patient, who had a relatively-small, well-differentiated NET with no nodal involvement and a low MIB-1 index (6.7%), followed a rather aggressive clinical course with distant metastases, probably associated with the extraordinary intra-mammary tumour emboli (Histopathology, 2014).

Funding: Grants‐in‐Aid for Scientific Research (No. 16K08654 & No. 16H00668) from the Japanese Ministry of Education, Culture, Sports, Science and Technology National Hospital Organization Grant (H29‐NHO‐01)


Immune microenviroment in triple negative breast carcinoma

H. Kaya*, H. Sahin Ozkan

*Department of Pathology, Marmara University School of Medicine, Istanbul, Turkey

Background & objectives: Immune microenviroment has been researched in many cancer types including breast carcinoma, especially the triple negative subtype. Our aim is to evalaute immune microenviroment in triple negative breast carcinoma and to understand its relationship with prognosis.

Methods: Fifty-one cases of triple negative breast carcinoma with no history of neoadjuvant therapy, whose resection specimens were examined in our department between 2012 and 2017, were retrospectively evaluated using CD8 (C8/144B, monoclonal, mouse, Dako), FOXP3 (EP340, monoclonal, rabbit, Epitomics), PDL1 (SP142, monoclonal, rabbit, Ventana) antibodies.

Results: Median age was 49. Majority of the cases (54.9%) were diagnosed as invasive breast carcinoma, no special type; while 35.3% were lymphocyte-predominant breast carcinoma and 9.8% were metaplastic carcinoma. All cases were histological grade 3. Nine (17.6%) cases ended with exitus. Five-year overall survival rate was 75.6%. Immunexpressions of all three antibodies were found to be correlated with each other (p<0.01). Lymphovasculary invasion and axillary lymph node metastasis were associated with lower FOXP3 expression (p<0.05). Higher expression of PDL1 was found in lymphocyte-predominant breast carcinoma cases (p<0.01). PDL1 expression was related to prolonged overall survival (p<0.01), whereas neither CD8 nor FOXP3 was associated with survival.

Conclusion: Tumour immune microenviroment is an important prognostic parameter in triple negative breast carcinoma. Further investigation is needed to better understand the prognostic role of immune microenviroment and to manage medical therapy.


Expression of WNT, Hedgehog, NOTCH, PI3K, NF-kB, PTEN signalling molecules in HER-2 overexpressed and triple negative breast cancer with positive and negative ALDH1A1 expression in cancer cells

N. Kazantseva*, A. Brilliant, Y. Brilliant, S. Sazonov

*Sverdlovsk Regional Oncological Center, Russia

Background & objectives: We investigated the expression of NF-kB, PI3K, PTEN molecules, as well as WNT, Hedgehog, NOTCH in cells of triple negative and HER-2 overexpressed breast cancer with high and low content of cancer stem cells (positive and negative ALDH1A1 expression).

Methods: We studied 110 cases of invasive breast cancer. To determine stem cells in tumour population, the presence of ALDH1A1 protein in cancer cells was investigated. In all cases, expression of oestrogen, progesterone receptors, HER-2 and Ki-67 was studied by immunohistochemistry to determine a subtype of breast cancer. The expression of molecules PI3K, NF-kB, PTEN, WNT, Notch, Hedgehog was also explored.

Results: All cases, that were investigated for the expression of ALDH1A1, were divided into two groups - with a low content of cancer stem cells (cases with expression of ALDH1A1 0 and 1+), and with a high content of cancer stem cells (cases with expression ALDH1A1 2+ and 3+). The results are shown in the Table.

Low level ALDH1A1 Triple negative 100% 98% 20% 27% 33% 14%
HER-2 overexpressed 100% 100% 11% 30% 29% 7%
High level ALDH1A1 Triple negative 100% 100% 33% 11% 11% 0%
HER-2 overexpressed 100% 100% 9% 36% 36% 0%

Conclusion: Activation of Notch and WNT signalling pathways was more common for cells of HER-2 overexpressed than Triple-negative subtype (p<0.05) in the group with high level of ALDH1A1. Expression of Hedgehog signalling molecule was only positive in the group of low level of ALDH1A1 expression, it was higher in cancer cells of Triple negative subtype than HER-2 overexpressed subtype (p<0,05). PTEN signalling was found more often for Triple negative cases (p<0.05).


The value of templates to histopathology trainees in cut-up of breast specimens

M. Kelleher*, A. Hayward, N. Mungalsingh

*High Wycombe Hospital, United Kingdom

Background & objectives: Junior histopathology trainees often find cut-up daunting. Cut-up is often protocol driven, and especially for similar specimens like breast, it suits template usage. Wycombe ST1 to ST4 histopathology trainees were not using cut-up templates. They reported a lack of confidence in cut-up of mastectomies and wide local excisions (WLEs) and problems with dictation due to lost recordings. 88% of the trainees felt templates would be helpful.

This project aimed to assess whether cut-up templates help trainee confidence and dictation problems.

Methods: The authors wrote templates for breast WLE and mastectomy cut-up using Standard Operating Procedures and Royal College of Pathologists datasets. They were reviewed by departmental consultants, trainees and the lead biomedical scientist (BMS) and trialled in cut-up for 6 weeks. The BMS completed the printed template as requested by the trainee performing cut-up. Trainees answered pre and post-trial questionnaires.

Results: All trainees used both templates during the trial. 75% found them very helpful. For 39% it helped as a memory aide, 33% it made dictation easier and 11% it helped with a lost dictation. Trainee confidence improved from 37% to 75% post-trial. For 75% it was quicker or time equivalent to complete the case. 100% would continue using the templates. 75% would like templates for other specimens, especially renal.

Conclusion: Wycombe Hospital histopathology trainees found templates for breast WLE and mastectomy specimens valuable and helped with dictation problems. Trainee confidence improved. Most wanted further templates for other specimens and a template for renal specimens is currently being trialled with trainees.


Fibulin-2 expression associates with stromal elastosis and is inversely related to vascular invasion in breast cancer

T.A. Klingen*, Y. Chen, H. Aas, E. Wik, L. Akslen

*Centre for Cancer Biomarkers CCBIO, University of Bergen, Department of Pathology, Vestfold Hospital Trust, Norway

Background & objectives: Stromal elastosis relates to good prognosis in breast cancer, and fibulin-2 helps to stabilize elastic fibres in basement membranes. We explored perivascular fibulin-2 expression in relation to elastosis content, vascular invasion, molecular subtypes, tumour detection mode, and breast cancer survival.

Methods: We performed a population based retrospective study of invasive breast cancers from the Norwegian Breast Screening Program, Vestfold County (2004-2009) including 200 screen-detected and 82 interval cancers. Perivascular fibulin-2 staining was semi-quantitatively graded and dichotomized using immunohistochemistry. Elastosis content was graded and dichotomized as high and low expression, whereas lymphatic (LVI) and blood vessel invasion (BVI) were recorded with immunohistochemistry.

Results: High perivascular fibulin-2 expression was strongly related to stromal elastosis (p<0.001), and inversely associated with BVI and LVI (p<0.001 for both). High perivascular fibulin-2 was associated with luminal breast cancer subgroups (p<0.001) and inversely with interval cancers compared with screen-detected tumours (p<0.001). By univariate analysis, low perivascular fibulin-2 was associated with reduced recurrence-free survival (p=0.002) and disease specific survival (p=0.019) compared to high perivascular fibulin-2 expression.

Conclusion: Presence of low perivascular fibulin-2 expression was strongly related to vascular invasion, low stromal elastosis, non-luminal breast cancer subtypes, interval presentation, and adverse prognosis.


A new method based on internal extractive electrospray ionization analysis of breast cancer samples for detection metastases in sentional lymph nodes

V. Kometova*, V. Frankevich, V. Chagovets, N. Starodubtseva, M. Rodionova, V. Rodionov

*National Medical Research Center for Obstetrics, Gynaecology and Perinatology named after Academician V.I. Kulakov of Ministry of Healthcare of Russia, Moscow, Russia

Background & objectives: Breast cancer is accompanied by metastases in lymph nodes in about 30% of cases. Pathological methods is used to verify metastatic lymph nodes. Faster and specific methods are required for the identification of metastatic lymph nodes directly during the surgery.

Methods: New method based on internal extractive electrospray ionization (iEESI) combined with high-resolution mass spectrometry analysis was developed. It allows to perform rapid (less than in 5 minutes) molecular profiling of the breast cancer tissue and the sentional lymph nodes tissue with regard to presence of metastatic lesion. Distinctive feature of proposed method allows direct extraction of whole-volume tissue samples rather than their surface.

Results: Rapid diagnosis of the metastatic lesion of the signal lymph nodes upon invasive breast cancer on the biopsy tissue samples was performed in 50 patients. The signals of differential metabolites associated with the occurrence of lymphatic lesion were identified using high resolution tandem MS analysis and reference LC-MS analysis. Bioinformatics approaches were developed in order to study the complex relationships between the identified metabolites. It makes it possible to substantially increase the sensitivity, accuracy and reproducibility of analysis compared with existing analogues. New analytical advancements were implemented which allow the sequential ionization of lipids, metabolites and proteins from the same single biopsy tissue sample.

Conclusion: A fast method, which allows to avoid injury of lymph nodes, has been developed for the verifying breast cancer metastases in sentional lymph nodes based on direct mass spectrometric analysis. A mechanism for metastasis to sentional lymph nodes in breast cancer was proposed.

The reported study was supported by RFBR grant, research project No.19-515-55021


Methylation of genes CDO1 and MEST in primary ER-positive breast cancer with metastases to lymph nodes

V. Kometova*, A. Krasnyi, A. Sadekova, S. Kurevlev, M. Rodionova, V. Rodionov

*National Medical Research Center for Obstetrics, Gynaecology and Perinatology named after Academician V.I. Kulakov of Ministry of Healthcare of Russia, Moscow, Russia

Background & objectives: Aberrant methylation of genes is associated with the development of malignant tumours; however, there is insufficient data on the relationship between methylation and metastasis to regional lymph nodes.

Methods: 116 patients with breast cancer were enrolled into the study. Of these, 61cases had Luminal A subtype and 49 cases Luminal B subtype by immunohistochemistry. To assess the level of methylation MS-HRM (Methylation Sensitive High-Resolution Melting) method was used on breast cancer samples taken immediately after surgery. The results were confirmed by pyrosequencing.

Results: The aim of this work was to access the relationship between the level of genes CDO1 and MEST methylation in the primary breast tumour and metastasis to lymph nodes. It was found that in the tumours of LumB subtype there was a statistically significant increase in the level of gene CDO1 methylation and a decrease in the level of gene MEST methylation. In the tumours of LumA subtype there was an increase in the level of both CDO1 and MEST genes methylation. The differences were close to statistical significance (for Mest gene p=0.12, for CDO1 gene p=0.09).

Conclusion: The results obtained expand our knowledge about pathogenesis of breast cancer development. Along with other molecular events associated with the primary tumour they can predict metastasis to lymph nodes.

The reported study was supported by governmental grant № НИОКТР АААА-А18-118053190016-7


Antibody clone-dependent thyroid transcription factor-1 expression in a primary breast carcinoma

L. Korsa*, A. Lukac, L. Kovacevic, M. Prutki, Z. Marusic

*Clinical Department of Pathology and Cytology, University Hospital Centre Zagreb, Croatia

Background & objectives: Thyroid Transcription Factor-1 (TTF-1) can be a useful marker for lung and thyroid primaries. However, TTF-1 expression has been reported in up to 2.5% of breast carcinomas. There are two main commercially available TTF-1 antibody clones: 8G7G3/1 and SPT24.

Methods: A 60-year-old woman presented with a large tumour involving the entire right breast together with multiple pulmonary and mediastinal tumour masses. Due to the aggressive course, the working diagnosis was a lung primary producing breast metastasis. Breast and lung core needle biopsies both revealed a TTF-1, ER and GATA3 positive adenocarcinoma, while napsin A, PR and HER2 were negative.

Results: Endocrine therapy was administered, leading to significant reduction in the size and number of pulmonary and mediastinal metastases. Two years later, a sanitary mastectomy was performed. The breast tumour was positive for ER and GATA3, and negative for napsin A and PR. This time, TTF-1 was performed with both commercially available clones, showing a positive reaction with SPT24 and a negative reaction using the 8G7G3/1 clone.

Conclusion: Breast metastases from extramammary malignancies are rare (0.2-2% of all tumours), with lung being one of the most frequent primary sites. TTF-1 clone selection can have clinical significance in the workup of a potential metastatic lung adenocarcinoma, especially in settings with limited amount of tumour tissue. In the setting of breast vs lung primary, an antibody panel including napsin A and GATA3 should be used, as occasional ER-positive lung carcinomas as well as TTF-1 positive breast carcinomas have been documented.


The CD8 / FOXP3 ratio of tumour infiltrating lymphocytes (TILs) predicts the effect of adjuvant radiotherapy in breast carcinomas; an immunohistochemical analysis in the SweBCG91RT trial

A. Kovács*, A. Stenmark Tullberg, H. Puttonen, M. Sjöström, E. Holmberg, D. Lundstedt, F. Killander, E. Nimméus, P. Karlsson

*Department of Clinical Pathology, Sahlgrenska University Hospital, Gothenburg, Sweden

Background & objectives: The aim was to analyse the predictive role of the CD8/FOXP3 ratio of tumour infiltrating lymphocytes (TILs) on effect on radiotherapy (RT) in the SweBCG91RT trial with the primary endpoint of breast cancer recurrence (BCR) within 10 years.

Methods: Patients with stage I and IIA breast cancer were randomised to breast-conserving surgery and postoperative radiotherapy (n=444) or surgery only (n=483) between 1991-1997. Median follow-up was 15.2 years. Primary tumours were scored for TILs on whole slides. CD8 and FOXP3 were scored on tissue microarrays using immunohistochemistry and the absolute stromal area occupied by the respective cell type was calculated.

Results: Among unirradiated patients, a high absolute CD8:FOXP3 balance (CD8High/FOXP3Low) was associated with a decreased risk of BCR (HR 0.40, CI 95% 0.21-0.78, p=0.005) compared to the CD8Low/FOXP3Low group (HR 1.0). RT produced a significant risk reduction of BCR among CD8Low/FOXP3Low tumours (HR 0.49, CI95% 0.35-0.70, p<0.001), but not among tumours with CD8High/FOXP3Low (HR 1.60, CI 95% 0.71-3.6, p=0.26). The interaction between RT and immophenotype was significant for any recurrence (p=0.024).

Conclusion: As the values (CD8High/CD8Low and FOXP3High/FOXP3Low) were combined to create groups reflecting the absolute balance between CD8+ T cells and FOXP3+ T regulatory cells, it showed that a favourable immune response (CD8High/FOXP3Low) was protective for breast cancer recurrence, moreover it was predictive of less benefit from adjuvant radiotherapy. These results may have an impact on therapy choice regarding postoperative radiotherapy in early breast cancer.


Evaluation of angiogenesis as a marker of progression of invasive carcinoma of no special type

D. Kushch*, M. Mnikhovich, T. Bezuglova, N. Maluygin, V. Balayants, A. Romanov, P. Bliganov, G. Ushanov

*Pirogov Russian National Research Medical University, Russia

Background & objectives: Activation of angiogenesis is considered a key step in the invasion and metastatic spread of malignant tumours.

Methods: We studied 86 cases of invasive carcinoma of no special type (IC NST). The material was studied using light microscopy using IHC. We used antibodies CD 34, CD 31, VEGF, MMP2. Cases were divided into 3 groups depending on the degree of differentiation - G1 21 cases (24.4%), G2 22 cases (25.5%) and G3 43 (50%).

Results: The endothelium of atypical capillaries was unevenly stained with CD34, marked expression of VEGF and negative expression of CD31 were noted. The dependence of the degree of vascularization on the degree of differentiation of carcinoma was established. In low-grade carcinomas, the number of microvessels in the field of view increased. A significant difference in vascular density was detected in groups of tumours with moderate and low differentiation (p = 0.019). The largest difference in the number of vessels was determined when comparing neoplasias in groups with the degree of differentiation G1 and G3 (p = 0.011).

Conclusion: Establishing the number and density of tumour microvessels can help establish the degree of differentiation and determine the prognosis of the course of non-specific breast carcinoma, and continue to be used as an additional criterion in the classification of non-specific breast carcinomas.


1% ER positive is not any positive cells

V. Kushnarev*, P. Krivorotko, A. Kudaybergenova, A. Artemyeva

*N.N Petrov Cancer Research Center, Russia

Background & objectives: ASCO/CAP recommends that ER status should be considered positive if 1% or more of tumour cells demonstrate positive nuclear staining. We tested the validity of digital image analysis algorithms for the assessment low-positive ER staining in breast carcinoma.

Methods: Eighteen ER+/HER2- invasive carcinomas of NST with 1-10% expression by visual assessment were scanned (Pannoramic 250, 3D Histech) and assessed by digital image analysis (DIA) algorithms Qupath and QuantCenter (3D Histech) of whole slides images (WSI). The degree of inter observer variation was determined by using Kappa statistics (less than 1% and 1%⩾) and intraclass correlation coefficient (ICC).

Results: In 40% DIA showed ER negative compared to visual positive assessment in same cases. Agreement between DIA and VA was weak (weighted kappa =0,46). ICC between DIA and VA was weak (ICC=0,58).

Conclusion: One percent as cutoff is based on the premise that patients derive benefit from endocrine therapy even with minimal receptor activity. False positive results lead to the fact that patients do not receive the necessary therapy as with triple negative carcinomas. Digital image analysis algorithms need to consider in group of low ER expression. It can be an instrument to decreases inter raters variability.


Characterisation of PD-L1 expression in triple negative breast carcinoma (TNBC) in a comprehensive cancer centre in Mexico City

C. Lara Torres*, J. Reyes Carrasco, J.F. Martínez Herrera, G. Molinar Flores, R. Gerson

*Surgical and Molecular Pathology Department, The American British Cowdray Medical Center, Mexico

Background & objectives: Breast cancer represents one of the main causes of cancer-related death. TNBC has few therapeutic options, recently the combination of chemotherapy plus immunotherapy (nab-paclitaxel+atezolizumab) showing advantage for PD-L1 positive patients. The use of PD-L1 immunohistochemistry as a biomarker shows numerous

Methods: difficulties making hard to extrapolate its analytical performance across populations and tumour histologies. We present the first study in Mexico to evaluate PD-L1 expression in TNBC using the SP142Ventana companion diagnostic-assay We reviewed the registries of the Pathology Department for TNBC. Inclusion criteria were tissue availability and medical information. IHC was performed and evaluated

Results: using the PD-L1 SP142 Ventana-Assay criteria for TNBC. Fifty cases were identified fulfilling inclusion criteria from 2009-2019. Average age at diagnosis was 54.9 years (+/- 15.9 yo). The tissue available for study was: primary breast tumour (84%), metastasis (14%) and 2%(NA). Fifty-two percent of our cases were PD-L1 positive, (IC1-22%, IC2-10%, IC3-20%). Different histologies were represented(NOS(30%), medullary-like(8%), apocrine(6%), metaplastic( 4%), and pleomorphic lobular (2%), with a trend for PD-L1 positivity in higher grade tumours(84% of G3 PD-L1+ vs 64% G2 PDL-1+). We found no difference on age at presentation, smoking history, or association with previous treatment on PD-L1(positive/negative) groups.

Conclusion: We found a slight increase of PD-L1 positivity compared to the IMPASSION130 study(52%vs41%), although sample bias is a potential issue due to the number of cases studied. TNBC is a group with different immunogenicity profiles and histologic characteristics


Expression of melanoma antigen - as in peripheral blood circulating tumour cells (CTCs) of breast cancer patients

Y. Liu*, X. Fan

*The Fourth Hospital of Hebei Medical University, China

Background & objectives: The purpose of our study is to detect the expression of MAGE-A genes, including MAGE-A1,-A2,-A3,-A4and-A6, in peripheral blood circulating tumour cells of breast cancer patients.

Methods: Multiplex nested RT-PCR was used to detect the level of MAGE-A mRNA in peripheral blood circulating tumour cells of 106 breast cancer patients and 30 health donors. Restriction endonuclease treatment was used to detect the expression of each member of MAGE-A family, including MAGE-A1,-A2,-A3,-A4 and -A6 genes.

Results: The expression rate of MAGE-A gene was 40% (43/106) in breast cancer peripheral blood. The frequency of MAGE-A expression in breast cancer peripheral blood was the following order: A2>A3>A4>A1>A6.

Conclusion: MAGE-A gene expression in peripheral blood of breast cancer may be as a important maker for detection of breast cancer CTCs. The expression of MAGE-A1, -A2 and -A4 may be correlated with prognosis of breast cancer, may be an important marker for monitoring the treatment and prognosis of breast cancer.


Correlation between HER2/NEU protein overexpression on immunohistochemistry and flourescence in situ hybridisation in breast carcinoma: problems in a developing country

N. Mamoon*, Z. Yaqoob, G. Mudassir

*Shifa International Hospital, Islamabad, Pakistan

Background & objectives: The aim of this study was to correlate the FISH results with the IHC results and also document the problems we faced in performing FISH in our particular scenario as a developing country with limited and nonuniform medical facilities.

Methods: It was a retrospective study conducted at Shifa International Hospital spanning a period of four years from 1st Jan 2015 to 31st December 2019. A total of 451 cases of breast carcinoma who underwent florescence in situ hybridization (FISH) were analysed for correlation between Her2/Neu over expression on IHC and its amplification on FISH. Reasons for unacceptable results were documented.

Results: Out of 451 cases submitted for HER2/Neu testing by FISH from 2015 to 2019, acceptable results were obtained in 391 (86.7%) cases. Among 348 HER2/Neu 2+ cases, 97 cases (27.9%) showed HER2/Neu gene amplification on FISH and 251 cases (72.1%) were negative. Out of 39 cases (10.1%) which were scored 3+ on IHC, 36 cases (92.3%) were amplified and 3 cases (7.7%) were negative. All 4 HER2/Neu 1+ cases (1.0%) were non- amplified. Overall 63 cases (13.9%) did not yield acceptable results; 20 due to fixation artifacts (31.7%), 15 due to scanty tumour (23.8%), 15 due to eosin dye application (23.8%) and 13 due to crushing artifacts (20.63%).

Conclusion: HER2/Neu gene amplification was seen in 27.9% and 92.3% cases scored as 2+ and 3+ on IHC respectively. In order to get adequate results on FISH, optimal fixation, adequate amount of tumour without crushing and dye application are important pre-requisites.


Cytotoxicity potential of tetragunola laeviceps bee propolis on breast cancer cells

R. Masadah*, D. Ikram, S. Ekawardhani, R. Eka Putra

*Hasanuddin University Makassar, Indonesia

Background & objectives: The resistance of breast cancer chemotherapy triggered the researcher to study herbals alternative therapy that have been known to have anti tumour potential. Propolis has been recognised to have more than 300 chemical compounds, has pro-apoptosis effect and selective to target cancer cell, thus could be a candidate for therapy of breast cancer. Tetragunola laeviceps is a stingless bee that produce propolis, it was found a lot in Southeast Asia include Indonesia.

This study was aimed to analyse the potential of Tetragunola laeviceps propolis as a pro-apoptosis agent to MCF-7 breast cancer cell lines.

Methods: Propolis Tetragunola laeviceps was extracted by ethanol extraction (EEP extract). MCF-7 breast cancer cells, treated by EEP extract, supplemented by FBS10%, penicillin-streptomycin 1%, amphotericin 1% in CO2 5% 370C. Cytotoxic effect of EEP to MCF-7 and Vero cells was examined by MTT test 48 hours after treatment. Absorbance was measured by microplate reader (450nm) to get the viability presentation of cells.

Results: There was a decrease of MCF-7 cells viability and an increase of apoptosis cells after 48 hours of propolis treatment in 200ppm concentration (IC50 79,45 ppm). One-way Anova test showed a significant difference of mean percentage of MCF- 7 viability (< 0,0001) than control cells group.

Conclusion: Propolis Tetragunola laeviceps and its bioactive compound has a selective pro-apoptosis effect to dMCF-7 breast cancer cell lines. This was an early results of alternative therapy for breast cancer, and needs more investigations in the future

Key words: Propolis, Tetragunola laeviceps, breast cancer.


High tissue inhibitor of matrix-metalloproteinase 2 expression correlates with poor prognosis in breast cancer

T. Heyman, C. Joseph, M. Craze, A. Green, C. Nolan, O. Rueda, E. Provenzano, E. Rakha, I.O. Ellis, A. Mukherjee*

*University of Nottingham, United Kingdom

Background & objectives: TIMP2, tissue inhibitor of matrix-metalloproteinase 2, inhibits the matrix-metalloproteinase, MMP2, but may activate pro-MMP2. Hence its correlation with prognosis in breast cancer (BC) is contradictory. This study investigated the correlations of TIMP2 expression in BC with clinicopathological variables.

Methods: Differential expression analysis of TIMP2 was assessed in the lymphovascular invasion (LVI) positive versus negative cohorts in the METABRIC BC dataset. Immunohistochemical analysis for TIMP2 (1:50 dilution) expression was conducted on BC tissue microarrays (n=1048) and clinicopathological correlations were assessed, including VI and expression of MMPs, 2, 14 and 15.

Results: TIMP2 was associated with positive LVI in the METABRIC cohort (p= 0.002). On immunohistochemistry, significant positive correlations were found between TIMP2 protein expression and higher grade (p=0.019), including its components nuclear pleomorphism (p=0.004), mitotic count (p=0.000087), LVI (p=0.014) and Nottingham prognostic Index (p=0.024). TIMP2 expression showed a significant correlation with negative ER status (0.014) and Triple negative status (p =0.008). TIMP2 expression also showed a positive correlation with cytoplasmic MMP2, MMP14 and MMP15 (p<0.0001) expression. A significant difference in 10-year BC specific survival (BCSS) was seen between high and low TIMP2 expression (p= 0.018).

Conclusion: Overall, the study indicates that higher TIMP2 expression correlates with poor prognostic factors in BC including high grade, negative ER status, poor NPI and LVI. These effects may be explained through its activation of pro-MMPs, reflected by positive associations with MMP expression. Further studies of the ratio between MMPs and TIMP2 may help delineate its functional role in BC.

Funding: Academy of Medical Sciences and Pathological Society of Great Britain and Ireland (CDF).


Low FOXA1 expression in aggressive breast cancer associates with ER negativity, BRCA1 germline mutations, and gene expression programs for cellular plasticity and immune cell responses

A.K. Myrmel Sæle*, A.A. Svanøe, C. Askeland, G. Knutsvik, B. Davidsen, T. Aas, K. Collett, I.M. Stefansson, L. Akslen, E. Wik

*Centre for Cancer Biomarkers CCBIO, Department of Clinical Medicine, Section for Pathology, University of Bergen, Norway, Department of Pathology, Haukeland University Hospital, Bergen, Norway

Background & objectives: FOXA1 is linked to oestrogen receptor signalling, suggested as a factor upstream of the ER-complex, and proposed to play a role in immune responses. Here, we assessed FOXA1-associated phenotypes in breast cancer (BC) and explored related gene expression patterns.

Methods: We analysed three BC cohorts. Cohort-I: Hordaland County; <70 years; n=612. Immunohistochemical staining of FOXA1 on tissue microarrays of formalin-fixed paraffin-embedded BC tissue. Cohort-II-III: Global mRNA data; METABRIC (n=997) and TCGA (n=505; including BRCA1 germline mutations). Analyses of genes differentially expressed between FOXA1 high and low cases and gene set enrichment analyses (GSEA) were performed on global transcriptomic data.

Results: Low FOXA1 associated with higher histologic grade and tumour cell proliferation (Ki67), triple- negative phenotype, and reduced survival (P≤0.008). ESR1, GATA3, AGR2, AGR3, and AR were downregulated in FOXA1-low tumours. Further, FOXA1-low cases were enriched for signatures of low ER-signalling, stemness, progenitor cells, EMT, and BRCA1 mutations. Triple FOXA1/GATA3/ER-negative status strongly predicted a basal-like phenotype (OR≥198; P<0.001) and associated with higher expression of PD1, PD-L1, and CTLA4 (P<0.001).

Conclusion: Low FOXA1 is a marker of aggressive BC and associates with BRCA1 germline positive cases. Associated transcriptional programs reflect increased cellular plasticity and stemness. A triple FOXA1/GATA3/ER negative phenotype strongly predicts a basal-like phenotype and relates to immune cell responses.


Development of a novel clinical trial immunohistochemistry (IHC) assay using Ki-67, clone MIB-1, monoclonal antibody for Dako Omnis

G. Nielsen*, Y. Gu, A. Weaver, G. Gegg, S. Tabuena-Frolli, A.M. Gruver, M. Cajaiba, C. Gottstein

*Agilent Technologies, USA

Background & objectives: Lack of standardized Ki-67 assay performance remains an impairment to global clinical research. Therefore, a novel IHC prototype assay and scoring algorithm were developed for the Dako Omnis platform to detect Ki-67 expression in formalin-fixed paraffin-embedded human breast carcinoma.

Methods: The Ki-67 IHC prototype assay is based on EnVision FLEX technology using Ki-67, clone MIB-1, primary antibody validated for Dako Omnis. A scoring guide was developed and optimized for high inter-observer precision. The assay has been analytically validated for sensitivity, specificity, precision (inter-day, inter-instrument, inter-lot and repeatability: intra-instrument/intra-rack/intra-day) and robustness.

Results: Ki-67 IHC detected a relevant range of Ki-67 expression in 148 breast carcinoma specimens, including resection and core needle biopsy specimens. All precision and robustness studies achieved 95% confidence interval lower bounds (LBCI) of >90% for negative percent (NPA), positive percent (PPA) and overall (OA) agreement. Specifically, observer reproducibility results demonstrated high agreement with 95% LBCI values for NPA/PPA/OA of: 97.2%/91.7%/95.4% for inter-observer and 98.3%/94.4%/96.8% for intra-observer.

Conclusion: Our studies demonstrate that the standardized Dako Ki-67 IHC assay for Dako Omnis is sensitive, specific, precise, and robust for reproducible detection of Ki-67 expression in breast carcinoma. The prototype assay is the first Ki-67 IHC in vitro diagnostic developed on the Dako Omnis platform for global use in support of patients participating in the monarchE study (NCT03155997).


Identification of potential markers of aggressive behaviour in young women with breast cancer through transcriptomic analysis

F.J. Obiajulu*, M. Allen, A. Anthony, E. Gadaleta, J. Gomm, C. Chalala, I. Goulding, S. Duffy, J.L. Jones

*Barts Cancer Institute, Queen Mary University of London, United Kingdom

Background & objectives: Breast cancer in young women (BCYW) is usually aggressive with devastating consequences. Evidence suggests that BCYW has a distinct biological behaviour from those of older women. The study aim is to identify biological markers of aggressive behaviour of BCYW.

Methods: The young (≤40 years) and age independent reduction mammoplasty samples in the GEO datasets (GSE29431, GSE42568, GSE61304) were analysed with GEO2R. Survival analyses were performed with R survival and prodlim packages on TCGA cohorts. Statistical significance was defined as p ≤ 0.05 or False Discovery Rate (FDR) ≤ 0.05. The identified genes were validated by qPCR, Western blotting and Immunohistochemistry.

Results: High median expression of Wntless (WLS) (HR=1.927, p0.05), low median expression of Kinesin Family KIF4A (HR=0.246, p0.006), Glycosylphosphatidylinositol Specific Phospholipase D1 (GPLD1) (HR=0.294, p0.004) and Structural Maintenance of Chromosome (SMC4) (HR=0.331, p0.04) were associated with overall poor survival. Further analysis showed that SMC4 and KIF4A mRNAs were upregulated in cancer vs reduction mammoplasty, while WLS and GPLD1 mRNAs were downregulated. qPCR and western blot revealed higher expression of SMC4 and KIF4A and low expression of WLS in breast cancer cell lines vs normal breast epithelial cells. SMC4 immunostaining showed high expression in young female breast cancer, low expression within this cancer cohort correlates with Luminal B, TNBC and higher grade.

Conclusion: SMC4, KIF4A, WLS and GPLD1 could be important biological markers of the aggressive behaviour of BCYW through unknown pathways. This provides a basis to further investigate the functional significance of these pathways in a model system.

Funding: Commonwealth Scholarship Commission UK The British Division of the International Academy of Pathology (BDIAP)


Micropapillary variant of mucinous breast carcinoma: a report of two cases

T. Pikivaca*, L. Korsa, Z. Marusic

*Clinical Department of Pathology and Cytology, University Hospital Centre Zagreb, Croatia

Background & objectives: Breast cancer is a heterogeneous disease composed of various histological types that differ in prognosis and response to therapy. Micropapillary variant of mucinous carcinoma (MPMC) is a subtype with more aggressive behaviour than classic, pure mucinous breast carcinoma.

Methods: We present two cases of MPMC in female patients of identical age (51-year-olds). The first patient underwent neoadjuvant therapy for an extensive right breast tumour, with partial response and extensive residual disease within the lymph nodes. The second patient underwent tumourectomy for a recurrence of mucinous carcinoma, 10 years following right mastectomy with adjuvant endocrine therapy.

Results: Both cases were characterized by micropapillary clusters of tumour cells in abundant extracellular mucin, with specific 'inside out' positive staining for EMA and E-cadherin. The first case presented with extensive lymphovascular invasion. Both tumours were positive for ER and PR, with an intense basolateral membranous staining for HER2 in the first case. Micropapillary carcinoma and micropapillary mucinous carcinoma are the only breast tumours in which basolateral HER2 staining is considered positive.

Conclusion: Greater awareness and recognition of the micropapillary variant of mucinous breast carcinoma has significant prognostic and therapeutic implications by avoiding underestimation of malignant potential for individuals whose tumours may have been diagnosed as classic mucinous carcinoma.


Clinical impact of microscopic margin status on invasive breast cancer treated with breast-conserving surgery plus adjuvant radiation therapy at a specialised cancer hospital in Portugal

L.P. Queiroz Rei*, K. Pereira, C. Abrantes, R. Félix, D. Roda, S. Gonçalves, P. Alves, P. Figueiredo

*Pathology Department, Portuguese Oncology Institute, Coimbra, Portugal

Background & objectives: Most early-stage breast cancers (BC) are treated conservatively with breast-conserving surgery plus adjuvant radiation therapy (BCSART). We evaluate the clinical impact of microscopic margin status in a sample of conservatively treated invasive breast cancer (IBC) patients, compared to worldwide meta-analyses.

Methods: Unicentric, retrospective, descriptive study including all female invasive breast cancer (IBC) patients who concluded BCSART treatment in 2012 at a specialized cancer hospital in Portugal. Microscopic margins, as evaluated and measured by histopathology, were considered positive with “ink on tumour”, and clinical data was collected from follow-up records. Analysis was performed with IBM’s SPSS software.

Results: 92 patients were included, 3 with bilateral disease. Of the 95 surgical specimens, 8 had positive margins – 6 for IBC and 2 for ductal carcinoma in situ (DCIS) – and, of these 8, all but one were submitted to further re-excision or mastectomy surgery, prior to adjuvant radiotherapy. After a mean follow-up time of 84 months, wider margins did not prove protective against relapse, nor otherwise beneficial.

Conclusion: Breast cancer is the most common type of cancer worldwide in women. For IBC, treatment outcomes with BCSART are comparable to mastectomy, and the current consensus is to consider “no ink on tumour” as a negative margin. In patients programmed to undergo adjuvant radiation therapy, wider surgical margins are not clinically justified. Our results tend to concur with this consensus, but longer follow-up time may be of value.


Deployment of a multi-tissue AI-based quality control system in routine clinical workflow

I. Krasnitsky, I. Yazbin, I. Gross, G. Sebag, J. Sandbank*

*Ibex Medical Analytics, Institute of Pathology, Maccabi Healthcare Services, Israel

Background & objectives: Maccabi is a large healthcare provider with centralized pathology institute handling >140,000 accessions/ year, (700 PCNBs and 6850 breast biopsies). IBEX Medical Analytics develops Galen Prostate (CE-IVD) & Breast AI-based diagnostic solutions that detect cancer in prostate & breast biopsies.

Methods: The underlying algorithms utilize state-of-the-art Artificial Intelligence (AI) and Machine Learning techniques and were trained on many thousands of image samples, obtained from slides from multiple labs and geographies, and manually annotated by senior pathologists. Galen Prostate CE-IVD detects and grades prostate core needle biopsies, and Galen Breast detects invasive and in-situ carcinomas in breast biopsies.

Results: Both algorithms were assessed for performance on independent data from various labs and demonstrated high specificity and sensitivity, including identification of cancers missed by pathologists. Maccabi has deployed both Galen Prostate and Galen Breast as a QC system on all new prostate and breast biopsies entering the lab. The system raises an alert whenever it encounters a discrepancy between the automated analysis and the original diagnosis, prompting a second human review. In this talk, we will discuss the workflow in the lab and the performance of the algorithms. To the best of our knowledge, these are the first AI-based prostate and breast diagnostic systems running in a live clinical setting.

Conclusion: The importance of accurate diagnosis in prostate and breast biopsies, together with the growing shortage of pathologists, makes a QC system like this extremely useful for diagnostic accuracy and safety.


Crosstalk between programmed death ligand 1 (PD-L1) expression, Ki-67 labelling index and tumour infiltrating lymphocytes (TILs) in invasive breast cancer: a tertiary care centre study

R. Sharma*, P. Elhence, M. Rao, S. Khera, R. Chaudhary, P. Pareek, J.R. Vishnoi

*Junior Resident, Department of Pathology, AIIMS Jodhpur, India

Background & objectives: Breast cancer is leading cancer among Indian females (25.8 per 100,000 women) and accounts for approximately one fourth of cases worldwide. Present study aimed to ascertain and correlate PD-L1 expression with Ki-67 labelling index and TILs in Invasive breast carcinoma

Methods: It was a two year (2016-2018), descriptive observational study including all the diagnosed cases of Invasive breast carcinoma. All Core biopsy, wedge biopsy, lumpectomy and MRM specimens were examined. PD-L1 expression was assessed in tumour and stromal TILs and its correlation was done with hormone receptor status and with Ki-67 labelling index. Appropriate statistical analysis was done using SPSS 23.0v.

Results: Total 114 cases were included in present study, of which 33.33% (38) showed PD-L1+ expression in tumour cells and 47.37% (54) showed PD-L1+ expression in stromal TILs. Higher Ki-67 labelling index was observed in 96 cases and low in 18 cases. 49 cases were ER- and 65 were ER+. Of these 49 cases, 22 cases showed PD-L1+ expression and from 65 cases, 49 showed PD-L1+ expression.

Present study highlighted a statistically significant relationship between expression of PD-L1 in tumour cells and stromal TILs. Significant concordance between Ki-67 labelling index and PD-L1 expression was noted in tumour cells and stromal TILs. An inverse correlation was noted between PD-L1 expression and ER.

Conclusion: PD-L1 expression in tumour and TILs with high Ki-67 index may have role in targeted chemotherapy. Also, inverse relationship with ER suggests scope for targeted therapy in ER- Invasive breast cancer cases. However, further research is required in this area.


Clinical value of PD-L1 (SP142) expression on immune cells (IC) in small biopsy specimens (SBS) from untreated triple-negative breast cancer (TNBC) patients - a single institution retrospective study

O. Stanowska*, W. Olszewski, J. Owczarek, J. Barańska, M. Prochorec-Sobieszek

*Maria Sklodowska-Curie National Research Institute of Oncology, Department of Pathology and Laboratory Medicine, Poland

Background & objectives: This study analyses the prognostic significance of PD-L1 positivity in SBS from untreated TNBC patients, as illustrated by complete pathological response(cPR) and aims at targeting patients for PD-L1 screening by correlating its expression with tumour infiltrating lymphocytes(TILs) and MIB1 rates.

Methods: We evaluated TILs by HE staining, assessed PD-L1(SP142 assay) expression on IC and MIB1 on tumour cells by immunohistochemistry in 99 FFPE SBS of untreated TNBC patients diagnosed in 2018-2019. Cases with ≥1%PD-L1 expression were considered positive. MIB1 and TILs were evaluated according to the WHO guidelines(table 1-2). The association of PD-L1 status with TILs,MIB1 and cPR was statistically tested.

Results: 29(67,4%) of 43 so far tested cases were PD-L1-positive. PD-L1 positivity was correlated with TILs>30% and MIB1index>70%. PD-L1 positivity was seen in 75% (6/8) cases with pCR.


  Pd-L1(+) Pd-L1(-)
MIB1 <40% 4 6
MIB1 40-70% 7 7
MIB1 >70% 18 1


   Pd-L1(+) Pd-L1(-)
TILs <5% Negative 2 3
TILs 5-30% Low 13 10
TILs 35-50% High 9 1
TILs >50% Predominant* 5 0

*IC > tumour cells

Conclusion: Our study confirmed that PD-L1–positive IC have a positive predictive impact in TNBC cases without PD-L1 therapy and reflect a trend for better pathologic response to neoadjuvant chemotherapy.

Therefore, PD-L1 could act as a promising marker to predict neoadjuvant chemotherapy response in TNBC patients. TNBC patients with MIB1index>70% and/or TILs>30% should be screened for PD-L1 expression by immunohistochemistry as they may benefit from hybrid neoadjuvant treatment with PD-L1 inhibitor.


Imaging-pathology correlation: concordance analysis between sonomammographic findings BI-RADS 4 and histopathologic result in Filipino women in a single institution tertiary university hospital

R. T. Yolo*, P.A.M. P. Paulino, D.P. delos Angeles, R.C. B. Cabalfin, E.M. N. Bañares, M. B. Geslani, L.G. L. Lim

*Dept of Pathology, Faculty of Medicine and Surgery; Dept of Medical Technology, Faculty of Pharmacy, Dept of Anatomic Pathology, The University of Santo Tomas Hospital, Manila, Philippines

Background & objectives: Objectives: To correlate between the sonomammographic findings and pathologic results of BI-RADS 4 lesions; compare imaging-pathologic concordance between BI-RADS 4A, 4B and 4C according to age group; Assess concordance of BI-RADS 4 category in palpable and non-palpable breast lesions.

Methods: A retrospective study in Filipino women had mammogram and/or breast ultrasound with BI-RADS 4 score underwent biopsy or surgery. Patients were classified as: BI-RADS 4 subcategory, histology, age groups, and if lesions were palpable or not. Mammogram and breast ultrasound were performed using mediolateral oblique and craniocaudal projections. Comparison was done using Chi-square or Fisher’s exact test.

Results: A total of 203 BI-RADS 4 patients were included with mean age of 51.18 years old, out of which more than half were ≥50 years old. Majority were categorized as BI-RADS 4A (n=77). The results indicate similar likelihood rates of malignancy at 69%, 38% and 9% for BI-RADS 4C, BI-RADS 4B, and BI-RADS 4A, respectively, compared to the computed likelihood rate by the ACR which is validated and followed worldwide.

Conclusion: Malignancy rate of BIR-ADS 4 lesions were comparable to the likelihood of malignancy worldwide. There is positive correlation between BI-RADS 4 malignancy rates supporting the current recommendation for BI-RADS 4 category regardless of subcategory to acquire histologic diagnosis through biopsy.


Intratumoral heterogeneity in pure ductal carcinoma in situ of the breast does not affect the representativity of a biopsy

M.R. Van Bockstal*, C. Stanciu-Pop, M. Berlière, F. Duhoux, M. Nollevaux, C. Galant

*University Clinics Saint-Luc, Brussels, Belgium

Background & objectives: Ductal carcinoma in situ (DCIS) of the breast, a non-obligate precursor of invasive breast cancer, is a heterogeneous disease in terms of morphology and genetics. Active surveillance for low risk DCIS is currently explored as a valid treatment option in randomised clinical trial setting. The current study investigated the impact of morphological intra-tumoral heterogeneity in a series of 51 large DCIS lesions by comparing biopsies with their subsequent resection specimen.

Methods: Nuclear atypia, DCIS architecture, necrosis, calcifications, stromal architecture and stromal inflammation were assessed in one biopsy slide and three slides from the resection specimen. For each histopathological feature, a histoscore was determined and compared between the biopsy and the resection. Statistical analysis comprised Friedman tests, Wilcoxon tests with Bonferroni corrections and Spearman’s correlations.

Results: Despite substantial morphological heterogeneity in up to 50% of DCIS, the assessment of each histopathological feature in the biopsy correlated significantly with the assessment of the histopathological features in the resection specimen, with the exception of necrosis. Morphological heterogeneity was not associated with patient age, DCIS size or type of surgery, except for a weak association (p=0,048) between heterogeneous stromal inflammation and smaller DCIS size.

Conclusion: Histopathological features assessed at the biopsy level correlated well with the surgical specimen, except for necrosis. Overall morphological intratumoral heterogeneity has limited impact. However, caution is warranted for the assessment of comedonecrosis in biopsies, as its presence can be underestimated.

Funding: Postdoctoral clinical mandate (2019-089) of the Foundation Against Cancer (Brussels, Belgium).


The diverse molecular landscape of breast cancers with heterogeneous HER2 gene amplification

M.R. Van Bockstal*, M.C. Agahozo, R. van Marion, P.N. Atmodimedjo, H.F. Sleddens, W. Dinjens, L.L. Visser, E. Lips, J. Wesseling, C.H. van Deurzen

*University Clinics Saint-Luc, Brussels, Belgium

Background & objectives: Heterogeneous HER2 amplification is noted in 5-41% of breast carcinomas, depending upon its definition. Intra-tumour heterogeneity drives cancer progression and may enable circumventing HER2-targeted therapies, thereby causing primary and acquired resistance. Here, we performed an in-depth molecular analysis of admixed HER2-positive and HER2-negative breast cancer components.

Methods: We micro-dissected formalin-fixed, paraffin-embedded breast cancer tissue of ten patients. Each tumour contained at least one HER2-negative and at least one HER2-positive component. Targeted next-generation sequencing was performed using a 53-gene panel. Somatic mutations were analysed. Copy number variations were investigated, based on the available coverage data.

Results: The following (likely) pathogenic molecular anomalies were identified in 26 genes: 3 splice site alterations, 32 missense variants, 7 nonsense variants, 9 insertions and 12 deletions. Copy number gains were identified in the CCND1, EGFR, ERBB2, FGFR1, MYC and PVT1 genes. Overall, most somatic mutations and copy number variations were heterogeneously distributed within the different HER2-negative and HER2-positive tumour components. The HER2-negative cancer components did not harbour common alternative drivers.

Conclusion: Breast cancers with heterogeneous HER2 gene amplification display several molecular anomalies that might act as alternative or collaborative drivers of carcinogenesis. However, these genetic aberrations display a heterogeneous distribution as well, and likely contain a large number of ‘passenger’ mutations.

Funding: Dr. Mieke Van Bockstal is supported by the Belgian not-for-profit organisation 'Foundation Against Cancer' (Grant 2019-089) and by the Mathilde Horlait-Dapsens Foundation (Brussels, Belgium).


HER2-positive apocrine carcinoma of the breast: a population-based analysis of incidence, treatment, and outcome

F. Skenderi, M.A.M. Alahmad, Y.M. Alahmad, I.H.E. Abdelhafez, Z. Gatalica, S. Vranic*

*College of Medicine, QU Health, Qatar University, Qatar

Background & objectives: Most of the published clinical studies on apocrine carcinoma of the breast focused on the triple-negative variant. In the current study, we investigated clinical, pathological and outcome characteristics of HER2-positive apocrine carcinomas of the breast.

Methods: We searched Surveillance, Epidemiology, and End Results (SEER) program database for HER2+ apocrine carcinomas of the breast and compared HER2+ enriched (ER-/HER2+) (n=167) and Luminal B (ER+/HER2+) (n=85) molecular subtypes. The categorical variables were compared by Chi-squared test. The mortality difference was evaluated using the Kaplan-Meier, Cox regression, and competing risk regression analyses.

Results: 75% of the patients with HER2+ apocrine carcinoma presented at an early stage (stages I/II). The average survival by the end of the study period was 31 months [range, 12.2-48.8 months]. Both HER2+ enriched and luminal B subgroups showed excellent short-term survival (~90%). After performing mortality-specific risk analysis, only early-stage patients that were treated by surgery were found to have better survival. Other clinicopathological parameters did not reach statistical significance.

Conclusion: Our study indicates that the majority of the patients with HER2+ apocrine carcinoma presented at early stages (I or II). Only the tumour stage and surgery exhibited a significant impact on overall survival. HER2+ apocrine carcinoma patients had an excellent short-term (first 2-3 years) survival, which is similar to the clinical behaviour of luminal breast carcinomas (NOS). Further studies are required to explore the long-term outcome among the HER2+ apocrine carcinomas.


External quality assessment for PD-L1 testing in triple negative breast cancer

N. Warrick*, S. Khalid, A. Dodson, S. Parry

*UK NEQAS ICC & ISH, United Kingdom

Background & objectives: PD-1/PD-L1 inhibitors are first line treatment options for patients with advanced disease in several tumour types. They are effective in a subset of patients with advanced triple negative breast cancer (TNBC). Immunocytochemistry (ICC) assays predict which patients may benefit.

Methods: Unstained composite slides consisting of tonsil, and both TNBC tissues and cell lines of known PD-L1 expression levels were distributed. Participants stained these for PD-L1 by the same method used in their clinical testing; following return, slides were centrally assessed by a panel of experts. Methodology details and participants own interpretation scores for the TNBC cases were also collected.

Results: The UK National External Quality Assessment Scheme for Immunocytochemistry and In-Situ Hybridisation (UK NEQAS ICC & ISH) assesses the technical quality of laboratory testing. Here we present results from the first round of a pilot EQA for PD-L1 staining in TNBC.

The data will be examined for associations between methodological parameters and test accuracy. In particular, results produced by the recommended companion assay (SP142, Roche Tissue Diagnostics) will be compared with non-assay results. Participants own interpretive scores will be examined and compared with those of the expert panel.

Conclusion: This is the first report presenting EQA results for PD-L1 testing in TNBC.

Funding: A generous educational grant was provided by Roche Tissue Diagnostics, 1910 E. Innovation Park Dr., Tucson, Arizona 85755.


Significance of S100A8-positive immune cells in relation to other immune cell subset infiltration in pre-invasive and invasive breast cancer

J.W. Woo*, Y.R. Chung, M. Kim, C. Hye Yeon, S.Y. Park

*SNUBH, Republic of Korea

Background & objectives: Myeloid-derived suppressor cells (MDSC) suppresses anti-tumour immunity. The purpose of study is to evaluate expression of S100A8, one of well-known MDSC markers, and see its relation to other factors including immune cell (IC) subsets in pre-invasive and invasive breast cancers.

Methods: A total of 765 cases of breast ductal carcinoma in situ (DCIS) and invasive carcinoma were included in this study. S100A8 expression in tumour cells (TCs) and ICs, and infiltration of other IC subsets (CD4+, CD8+, and FOXP3+ T cells and PD-L1+ ICs) were detected by immunohistochemistry using tissue microarrays.

Results: S100A8 expression in either TCs or ICs did not differ in frequency between DCIS and invasive carcinoma. In both DCIS and invasive carcinoma, high-S100A8 in TCs and ICs were commonly associated with aggressive pathologic features, such as histologic grade. High-S100A8-IC status positively correlated with infiltration of CD8+ T cells, and presence of PD-L1+ ICs, in both DCIS and invasive carcinoma. In survival analyses, high-S100A8 in TCs and ICs was associated with decreased overall survival in patients with invasive breast cancer, and the difference in survival was most remarkable in HR-positive tumours. In subgroup analysis, tumours containing high-S100A8 and low-PD-L1 IC showed worst overall survival compared to other groups.

Conclusion: S100A8+ ICs are already found in pre-invasive stage as much as in invasive carcinoma and are correlated with CD8+ T cells and PD-L1+ ICs. S100A8+ ICs exert immunosuppressive effect in breast cancer, especially in HR-positive and low-PD-L1 IC subgroup.


Histopathological characteristics and outcome of breast cancer in non-Caucasian women - a large single institution’s experience

M. Zaakouk*, A. Longworth, M. Evans, A. Shaaban

*Institute of Cancer and Genomic Sciences, University of Birmingham, University Hospitals Birmingham NHS Foundation Trust Queen Elizabeth Hospital Birmingham, United Kingdom

Background & objectives: Breast cancer in non-Caucasian females is under-studied and its management is based on Caucasian data. 17 % of the West Midlands females are non-Caucasian. We aimed to elucidate the pathologic features, molecular profile and outcome of non-Caucasian breast cancer.

Methods: We identified 7553 breast cancers of different ethnic origins diagnosed at a large Birmingham tertiary referral hospital between 2000 and 2016. Histopathological and clinical data were collected and statistically analysed.

6804 cases were excluded; British/white ancestry(n=6191), unconfirmed ethnicity/diagnosis(n=511), patients with sparse histological/clinical data(n=94) and male patients(n=8). The remaining 749 cancers comprised 735 non-Caucasian patients, of whom 14 had bilateral disease.

Results: 47 in-situ and 702 invasive carcinomas of predominantly (86.2%) symptomatic presentation and median age of 51 years were identified. Only 31.2% of invasive tumours measured <15mm and 11.98% were >50mm. Cancers were predominantly of grade3(45%), followed by grade2(42.4%). Median NPI was 4.35. 65.1% of the carcinomas were of luminal subtype, 18.6% were Her2 positive and 16.2% triple-negative. Median overall survival was 62 months. Five and ten year survival was 81% & 65.5% respectively.

Ethnicity correlated with higher NPI(p<0.001), larger tumour size(p=0.001) and larger number of positive axillary nodes(p=0.007). Negative correlations were found between age at diagnosis and both invasive tumour size & grade(p<0.001) and between tumour grade and overall survival(p=0.006).

Conclusion: Compared with Caucasian breast cancer, Non-Caucasian tumours presented predominantly symptomatically at younger age, were of larger size, higher grade with more unfavourable phenotypes and shorter survival. This is important in counselling, planning management and follows up of those patients.


Multiplex immunophenotyping of breast cancer tumour microenvironment

M. Zaakouk*, A. Longworth, K. Hunter, A. Shaaban

*Institute of Cancer and Genomic Sciences, University of Birmingham, University Hospitals Birmingham NHS Foundation Trust Queen Elizabeth Hospital Birmingham, United Kingdom

Background & objectives: Tumour microenvironment in breast cancer has become recently recognised as important for tumour prognosis and response to immunotherapy. We aimed to evaluate tumour microenvironment immune cells of breast carcinoma and their relationship to clinico-pathological parameters.

Methods: 54 core biopsies of grade 2-3 ductal NST carcinoma were selected and immuno-stained using multiplex immunofluorescence for CD4, CD8, CD20, CD68 and FOXP3. Cases included were of luminal (n=28), HER2 positive (n=14) and triple negative, TNBC (n=12) molecular subtypes. Detailed quantitative tumour microenvironment immune cell count and density were correlated statistically with patient and tumour characteristics and overall survival.

Results: The most frequent immune cell phenotype was CD4 (35/54;64.8%), followed by CD68 (11/54;20.4%) and CD8 (5/54;9.3%). TNBC & grade-3 cancers had higher total median counts than other molecular subtypes & grade-2 cancers, respectively. In luminal cancers; CD20, CD4 (stromal/total), tumoral CD8 & FOXP3 positive cells correlated positively with survival, whereas CD68 & FOXP3 (stromal/total) positive cells, correlated negatively with survival in HER2 positive tumours. A statistically significant negative correlation was found between tumour FOXP3 and number of positive axillary lymph nodes (p=0.036) and between intratumoral cellular density and age at diagnosis (p=0.026). Stromal cellular density correlated positively (p=0.018) with survival.

Conclusion: CD4 was the dominant immune cell phenotype, followed by CD68 and CD8. The profile of microenvironment and its correlation with survival, varied according to the molecular subtype. Triple negative & grade 3 cancers had the highest median counts of all immune cells. Tumour microenvironment immune-cell characterization could therefore provide prognostic information. The work is being applied to a larger sample to further elucidate the role of microenvironment in breast cancer.

PS-02 Gynaecological Pathology


Quality of pathology reports for epithelial ovarian cancers at a university teaching hospital in Nigeria, West Africa: an audit of the surgical pathology reports

K. Adeleke*, V. Iyawe, A. Agboola

*Olabisi Onabanjo University and Teaching Hospital, Sagamu, Nigeria

Background & objectives: Ovarian cancer is a major cause of morbidity and mortality among women. Therefore, pathology reports must contain important diagnostic and prognostic information. The objective of this study is to assess the quality of pathology reports for epithelial ovarian cancers.

Methods: Pathology reports for the years 2006-2019 were assessed for their conformity with standard universally accepted pathologic diagnostic and prognostic features sign-out checklist. This involved assessing for the presence of the following quality indicators on the pathology reports: histologic type, size, grade, state of the ovarian capsule and stage of disease.

Results: Majority of the tumours (77.8%) were serous carcinomas. The histologic type was stated on 96.3% of the reports while information on the grade of the tumour was also available on 66.7% of the reports. However, information on size, state of ovarian capsule (intact or ruptured) and stage of disease was available for only 14.8%, 7.4% and 3.7% of the reports respectively.

Conclusion: This audit on surgical pathology report of epithelial ovarian cancers shows that a substantial number of reports lacked information on important diagnostic and prognostic indicators. This can have negative consequences on quality of care offered to patients. The use of synoptic reporting can lead to improvement in quality of surgical pathology reports. A follow up study to evaluate quality of the pathology reports after adoption of synoptic reporting is necessary.


Lymphoepithelioma- like carcinoma of the uterine cervix; tumour microenvironment, tumour budding and p16 expression

K. Adoke*, A. Adoke

*Department of Pathology, FMC KEBBI, Nigeria

Background & objectives: Lymphoepithelioma-like carcinoma (LELC) of the uterine cervix is a rare tumour of the cervix composed of poorly defined islands of undifferentiated squamous cells in a background intensely infiltrated by lymphocytes. It has striking similarity with its counterpart in the nasopharynx and studies have shown that LELC of the cervix has a good prognosis. We retrospectively study three cases of LELC of the uterine cervix over a three years period (2016-2018) with immunohistochemistry (IHC).

Methods: Three cases of LELC of the uterine cervix were retrieved from our departmental archives. H&E blocks were cut and (IHC) using CD3,CD20, AE1/AE3 and P16 antibodies were done. CD3 and CD20 for IM. Tumour buds were defined according to ITBCC, counted at x20 magnification at the invasive front. P16 stain was defined according to 8Th edition of AJCC guidelines.

Results: The patient’s ages were 50, 60 and 65 years respectively. Histology showed a tumour composed of polygonal cells with prominent nucleoli and moderate eosinophilic cytoplasm. Intersperse these cells are numerous lymphocytes. IHC analysis shows the lymphocytes were both intratumoral and peritumoral CD3+ T cells, while CD20+ B cells were mainly peritumoral. Tumour budding was less than 5 buds in all cases which were also P16 positive.

Conclusion: LELC of the uterine cervix good prognosis may be explained by its low level of tumour buds, tumour microenvironment and P16 expression


BCOR immunohistochemistry is a useful tool to evaluate high-grade endometrial stromal sarcomas but BCOR negative high-grade ESS cases are also present

N.E. Alkanat*, A. Uner, A. Usubutun

*Hacettepe University Faculty of Medicine, Department of Pathology, Turkey

Background & objectives: High grade endometrial stromal sarcoma (HG-ESS) is a malignant mesenchymal tumour of uterus which frequently demonstrates YWHAE-NUTM2 fusion. Previous studies identified HG-ESS cases with BCOR gene translocations. Therefore, immunohistochemistry for BCOR could be useful in identifying such HG-ESS cases.

Methods: One hundred seventy-two uterine sarcomas diagnosed between 2000-2019 were re-evaluated. Tissue microarray blocks were constructed from 3 HG-ESS, 19 LG-ESS and 15 uterine sarcomas with high-grade morphology which were included in this study. Fluorescent in situ hybridization (FISH) studies using YWHAE and BCOR brake-apart probes and immunohistochemical studies for BCOR were performed. Patients’ files were used for clinical information.

Results: Five patients, 4 of which had tumours extending beyond uterus, had translocations involving YWHAE or BCOR. Four tumours showed high-grade morphology and revealed YWHAE translocation. One patient with myxoid morphology revealed BCOR translocation. In immunohistochemistry, all YWHAE translocated tumours showed BCOR positivity, however, BCOR translocated tumour was negative. One patient is alive after 18 months, three patients died 4, 18 and 34 months after diagnosis, one was lost to follow-up.

Conclusion: HG-ESS is an aggressive tumour with poor clinical outcome. Our study showed that BCOR immunohistochemistry could be a useful diagnostic tool to identify HG-ESS cases with translocation of YWHAE gene. However, FISH analysis is essential for definitive diagnosis. We also report that molecular analysis and conspicuous myxoid morphology can help identifying HG-ESS with translocation of BCOR gene which also seems to have aggressive clinical behaviour. Another important finding is that BCOR translocated HG-ESS was negative with BCOR immunohistochemistry.

This research was supported by Hacettepe University - Coordination Unit of Scientific Research Projects.


Mismatch repair protein deficiency in the endometria of the general population: an immunohistochemical study

K. Allen*, N. Gahir, E. Crosbie, N. Orsi, N. Wilkinson

*Leeds Teaching Hospitals NHS Trust, United Kingdom

Background & objectives: There is little literature looking into mismatch repair deficiency in endometrial biopsies of women from the general population. This study aims to assess the frequency of mismatch repair protein deficiency in this group within non-neoplastic and hyperplastic endometrium by immunohistochemistry.

Methods: 200 biopsies of polypoid and flat endometrium were anonymised and allocated to four different diagnostic groups (n = 50 each) for analysis of mismatch repair protein deficiency: non-hyperplastic, disordered proliferation, hyperplasia without cytologic atypia and hyperplasia with cytologic atypia. Each biopsy was assessed by immunohistochemistry for MLH1, PMS2, MSH2 and MSH6.

Results: One (2%) biopsy within the hyperplasia with cytological atypia group showed glandular MSH2 and MSH6 deficiency in areas of atypical hyperplasia and adjacent carcinoma. In the complex hyperplasia group, two (4%) showed glandular MLH1 and PMS2 deficiency, one with focal and the other with widespread loss of staining. The disordered proliferation group contained one biopsy with focal glandular loss of MLH1 and PMS2. The non-hyperplastic group contained no cases of mismatch repair protein deficiency.

Conclusion: Mismatch repair deficient glands are observed in morphologically benign endometrium from women not known to have Lynch syndrome. Glandular loss of mismatch repair protein expression may be an early event in endometrial carcinogenesis and its detection may identify high-risk women in whom endometrial surveillance and/or progestin treatment is justified.

Funding: BDIAP Glasgow 2020 Educational Fellowship


L1 cell adhesion molecule expression in endometrioid endometrial carcinoma, its prognostic significance, and correlation with survival

S.D. Altindag*, S. Yigit, L. Sen, S. Sen

*Izmir Katip Celebi University Ataturk Training and Research Hospital, Department of Pathology, Turkey

Background & objectives: L1 cell adhesion molecule (L1CAM) is a biological marker that can help identify endometrial cancers with poor prognostic features, and worse survival. This study aims to analyse the relationship between L1CAM expression, clinicopathological parameters, and survival in endometrioid endometrial carcinoma.

Methods: Patients diagnosed with endometrioid carcinoma were selected. Grade, presence/depth of myometrial invasion, invasion patterns, lymphovascular invasion, cervical stromal invasion were re-evaluated. Age, stage, lymph node metastasis, peritoneal cytology positivity, recurrence, overall and disease-free survival were recorded. Risk classification was made according to ESMO-ESGO-ESTRO consensus. The relationship between L1CAM expression and clinicopathological parameters and its effect on survival was calculated statistically.

Results: A total of 264 cases were included in the study. L1CAM positivity was observed in 22 cases (8.3%). A statistically significant relationship was found between L1CAM expression and advanced age, high grade, deep myometrial invasion, high-intermediate with high-risk cases and pelvic recurrence. Kaplan-Meier survival analysis showed that L1CAM positive patients had poorer overall survival and disease-free survival. L1CAM expression was found to affect overall and disease-free survival in univariate analysis.

Conclusion: L1CAM expression was significantly associated with advanced age, high grade, deep myometrial invasion, high-intermediate, and high-risk cases. Moreover, L1CAM expression predicts pelvic recurrence and poorer survival. Although L1CAM is not routinely used in treatment management algorithms, it seems to be a promising marker for managing patient follow-up algorithms.


Myoinvasion pattern evaluation in endometrioid endometrial carcinoma

C. Amalinei*, A.M. Aignatoaei, R. Balan, L. Lozneanu, R. Avădanei, S. Giusca, I.D. Caruntu

*"Grigore T. Popa" University of Medicine and Pharmacy, Institute of Legal Medicine, Romania

Background & objectives: Endometrioid endometrial carcinoma has variable five-year survival rates in FIGO stage I, suggesting the involvement of other factors related to tumour behaviour. Our study is evaluating the immunoexpression of epithelial-mesenchymal transition markers (EMT) in different types of myoinvasion patterns.

Methods: The study has been performed on a study group of 20 cases selected from the files of “Elena Doamna” Obstetrics and Gynaecology University Hospital of Iasi, between 2013 and 2017, in patients of 43-78 year-old. The hysterectomy specimens have been evaluated by routine histology and immunohistochemistry for CK7, E-cadherin, N-cadherin, β-catenin, and Vimentin.

Results: Clinicopathological parameters have been evaluated. The myoinvasive pattern has been identified as: MELF (multicystic, elongated and fragmented glands) (45%), diffusely infiltrative (25%), broad front (expansile, pushing border) (15%), adenomyosis-like (10%), and adenoma malignum (5%).The evaluation of different myoinvasive patterns of endometrioid endometrial carcinomas has been challenging, mainly in appreciation of the depth of myometrium invasion in adenomyosis-like and adenoma malignum patterns. MELF pattern has been correlated to age >50 years, deep myometrium invasion (>50%), recurrences (22.22% of cases), and partial immunoexpression of EMT markers.

Conclusion: The evaluation of myometrium invasion types may open new perspectives for endometrioid endometrial carcinoma prognosis. Although our study had been limited, our results are supporting the possibility of EMT phenomena in MELF. Further studies would validate our preliminary results.


Prostatic metaplasia of the vagina and uterine cervix: an androgen-associated glandular lesion of surface squamous epithelium

W. Anderson*, D. Kolin, G. Neville, D. Diamond, C. Crum, M. Hirsch, S. Vargas

*Brigham and Women's Hospital, USA

Background & objectives: Prostatic-type differentiation in the lower female genital tract is rare and its causes not well established. Herein, we characterize a distinctive form of prostatic-type differentiation that primarily involves surface squamous epithelium and is associated with androgen exposure.

Methods: Superficial prostatic glandular proliferations involving the vagina or cervix were sought over a 26-year period (1993 – 2019). Cases were also identified from patients clinically suspected to have either exogenous or endogenous androgen excess. Clinicopathological features were reviewed and immunohistochemistry was performed for PSA, NKX3.1, CK7 and AR.

Results: Fourteen cases were identified involving the vagina (n=13) and exocervix (n=1). The most common clinical context was gender dysphoria with long-term testosterone therapy (7/14). Five other patients had congenital disorders of sexual development associated with endogenous androgen excess. Two had no known exposure to androgen excess. Immunohistochemically, glands stained for NKX3.1 (93% of cases), androgen receptor (92%), CK7 (92%), and PSA (64%). Follow-up (median: 11 months) showed no masses or neoplasia.

Conclusion: We propose the designation ‘androgen-associated prostatic metaplasia’ for this distinctive form of prostate tissue not previously described within the vagina. It is strikingly prevalent among patients undergoing gender-affirming surgery. Recognition is important to distinguish it from other glandular lesions.


A proposed prognostic panel of immunohistochemical markers for endometrial carcinoma

M. Arafa*, A. Salama, E. ElZahaf, A. Shebl, A. Awad

*Pathology Department, College of Medicine and Health Sciences, Sultan Qaboos University, Oman

Background & objectives: Histopatholological prognostic parameters in endometrial carcinoma (EC) include tumour grade, stage and depth of myometrial invasion. This study aimed to assess the value of immunohistochemistry (IHC) "ER, PR, HER2, Ki67 and p53" in assessing the prognosis of EC.

Methods: Archival material of 105 EC were retrieved. Sections were IHC stained and data were evaluated in relation to 3-year overall survival (OS) and disease-free survival (DFS). Analysis were done using Kaplan Meier test and log rank Chi-Square test to detect effect of measurements on survival duration. Cox regression analysis was to detect predictors of survival using forward wald technique.

Results: The median follow-up duration for the included patients was 38 months (range, 1 - 87 months). Twenty-six patients (24.8%) died during the 3-year follow-up period. Fourteen patients had local recurrence in the rectal wall (5 patients), vagina (4 patients) and bladder (2 patients) with distant metastasis in 3 patients. It was found that PR, HER2 and P53 expression had significant effect on the overall survival, unlike ER and ki-67. Regarding disease-free survival, PR is the only marker found to have significant effect on DFS.

Conclusion: ER, PR, HER2, Ki-67 and p53 are not independent prognostic factors in endometrial carcinoma regarding OS and DFS. However, a combination of low ER and PR expression together HER2 overexpression, high ki-67 and aberrant P53 expression are associated with more aggressive behaviour of endometrial carcinoma.


Cross talk between TP53, MDM2 and CDKN1A expression and progression-free survival in patients with ovarian cancer after platinum-based chemotherapy

A. Asaturova*, T. Zavarikina, A. Tyulandina, S. Hohlova, J. Nosova, G. Khabas, A. Burdenny, M. Kapralova, V. Loginov, P. Brenner, M. Atkarskaya, D. Hodirev

*FSBI 'National medical research centre for obstetrics, gynaecology and perinatology named after V.I. Kulakov', Russia

Background & objectives: The goal of the investigation is to assess the link between multiform markers of cell cycle control genes (Arg72Pro of ТР53, T(-410)G of MDM2 and Ser31Arg of CDKN1A) and progression-free survival (PFS) in ovarian cancer patients after platinum-based chemotherapy (CT).

Methods: we analysed 49 tumour tissue samples from ovarian cancer patients (high-grade serous carcinomas) obtained before platinum-based CT. All patients were treated with standard protocols and examined till disease progression. Multiform markers of cell cycle control genes were investigated with Полиморфные маркеры генов были исследованы методом Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and real time PCR.

Results: Results We demonstrated the trend to decrease of PFS median in allele G of T(-410)G MDM2 carriers. We also have shown the increase of PFS median in allele Pro of ТР53 (р = 0.045). This phenomenon was more prominent in minor homozigota Pro/Pro carriers in compare to patients with Arg/Arg genotype (р = 0.007). In group of patients with optimal or total debulking minor allele Arg of Ser31Arg СDKN1A carriers had the decreased PFS mediana (р = 0.004).

Conclusion: Thus, we revealed the interconnection between investigated multiform gene markers and remission duration after platinum-based CT. Further investigation should be conducted to assess prognostic value of these markers for ovarian cancer patients before CT application.

The investigation was supported by RFF grant 18-08-01258


Steroid receptors isoforms disbalance in endometrial polyps pathogenesis

A. Asaturova*, G. Chernukha, I. Ivanov

*FSBI 'National medical research centre for obstetrics, gynaecology and perinatology named after V.I. Kulakov', Russia

Background & objectives: Steroid receptors directly impact on endometrial polyps (EPs) development, although the role of their isoforms play even more significant role. The goal of our investigation was to assess the proportion between proliferative and anti-proliferative steroid receptors in endometrial polyps

Methods: We assessed ER-alpha, ER-beta, PgR-A and PgR-B immunohistochemically in 20 EPs and 20 proliferative endometrium. We also calculated ER-alpha/ER-beta and PgR-B/PgR-A.

Results: We demonstrated higher PgR-B expression in EP in compare to normal endometrium (NE) (р=0.003).We shew the decrease of glandular and stromal ER-beta expression (p=0.01 and p=0.04 respectively) and stromal PgR-A expression (p=0.003) in EPs in compare to NE. The ER-beta/ER-beta was higher in glands of EPs glands then in NE (1.4 and 1.2 respectively, p=0.006). We did not reveal difference in ER-beta/ER-beta in stromal components of EPs and NE (p>0.05) . The PgR-BE/PgR-A was higher in glands of the EPs then in NE (1.2 and 1.1 respectively, p=0.04) and in stroma of the EPs then in stroma of NE (1.4 и 1.1 р<0.001).

Conclusion: In endometrial polyps we revealed the increase of PgR-B expression which accelerate the proliferation. In addition we demonstrated the decrease of ER-beta and PgR-A expression (these isoforms facilitate decidualization and atrophy of the endometrium). This disbalance can promote the proliferation, angiogenesis, apoptosis deactivation, cell cycle disturbance leading to endometrial polyps growth.


Ovarian morphology in patients treated with one-step surgical procedure for ovarian function activation

A. Asaturova*, L. Adamyan, V. Dementieva

*FSBI 'National medical research centre for obstetrics, gynaecology and perinatology named after V.I. Kulakov', Russia

Background & objectives: Patients with primary ovarian insufficiency (POI) have few remaining follicles, their only chance for pregnancy is through egg donation. We tried to reveal morphological features of ovarian in patients treated with surgical procedure for ovarian function activation using ovarian fragmentation.

Methods: We histologically investigated 52 samples of ovarian cortex obtained during surgical operation from 33 patients (19 with POI and 14 with ‘poor’ response to ovarian stimulation. We assess from 1 to 11 fragments of ovarian tissue 0,2х 0,4 to 1,5х1,5 cm in size.

Results: in 26,9% we demonstrated primary or secondary follicules, in 15,4% - follicle cystic atresia, in 53,8% - corpus fibrosum. Totally we revealed any of folliculogenesis signs in 82,7% samples. This date proved that operated patients had functional active ovaries and supposed an efficacy of ovarian surgical activation. In addition, we revealed serous surface proliferation (in 7,7%) and two types of inclusion cysts (ovarian surface epithelium (OSE)-type and fallopian tube-type). Moreover, we demonstrated samples with preserved OSE so we can suppose that POI can result in fibrous stroma of ovarian cortex and in basal membrane changes leading to tighter junction between these two histological structures.

Conclusion: Detailed assessment of a deliberately reduced ovarian reserve is required to overcome infertility in patients with POI who are resistant to standard f treatment strategy (including assisted reproduction methods). The histological examination ovarian cortex let us characterize the follicular apparatus and surrounding tissue, which is of undoubted significance in patients stratification and their effective treatment.


The immunoexpression of COX2, HIF-1α , and VEGF-C in the cervical cancer with neoadjuvant therapy (NAT)

R.A. Balan*, M.M. Gavrilescu, N. Ioanid, I. Scripcariu, I.D. Caruntu, C. Amălinei, S. Giusca, L. Lozneanu, T.A. Balan, V. Scripcariu

*U.M.F. "Grigore T. Popa", Romania

Background & objectives: Cervical cancer can be clinically staged prior to surgery, chemoradiotherapy being the elected treatment for locally advanced disease. Our study aims to assess key biomarkers in NAT cervical cancer, in order to evaluate their diagnostic and prognostic potential.

Methods: We evaluated 20 patients with IIB to IVA cervical carcinoma diagnosed and treated with NAT and subsequent surgery at the Regional Institute of Oncology of Iasi, between 2016 and 2018. The preNAT cervical biopsies and postNAT hysterectomy specimens have been assessed by routine histology and immunohistochemistry for COX2, HIF-1α, and VEGF-C, the results being correlated with clinicomorphological parameters.

Results: The histological type of the tumours included in our study were as follows: keratinized and nonkeratinized squamous cell carcinoma (16/80%), mucinous adenocarcinoma (1/5%), mixed tumours, as carcinosarcoma (1/5%), as well as mixed neuroendocrine and mucinous adenocarcinoma (2/10%). Of all patients, none presented a complete response to chemotherapy, the majority presenting a partial response (19/95%) and a single one (5%) not responding at all to NAT. The imunoexpression of VEGF-C was found significantly higher in preNAT carcinoma (16/80%) than in postNAT malignant cells (11/55%). HIF-1α positivity was higher in preNAT cervical carcinoma (17/85%) than in postNAT patients (13/65%). COX-2 expression was more significant in preNAT carcinoma (18/90%) than in postNAT (9/45%).

Conclusion: The validation of these elements will enable adjusted therapeutic strategies to improve the prognosis and reduce the morbidity of uterine cervical patients, and will identify the molecular variables that predict either sensitivity or resistance to chemotherapy.


Seromucinous cystadenomas and adenofibromas: first report of a case series

A. Ben-Mussa*, G. McCluggage

*Department of Pathology, Belfast Health and Social Care Trust, Northern Ireland, United Kingdom

Background & objectives: In the 2014 WHO Classification of Tumours of the Female Reproductive Organs, a category of seromucinous cystadenoma/adenofibroma was introduced as a benign counterpart of seromucinous borderline tumours and carcinomas. However, there is minimal literature regarding such benign ovarian neoplasms.

Methods: The cases were derived from the Department of Pathology, Belfast Health and Social Care Trust archives between 1/1/2010 and 19/11/2019 (n=18). Additional prospective cases were added from 20/11/2019 till 7/1/2020 (n=3). Most (n=19) were reported by the second author (W.G.M). All the reports and slides were retrieved and reviewed. Clinical information was obtained from the electronic care record.

Results: 21benign ovarian seromucinous neoplasms. Mean age 61years. 19unilateral and 2bilateral. 18cystadenomas and 3cystadenofibromas. Mean tumour size 9cm. The epithelium was predominantly single layered and consisted of admixture of cell types:serous and mucinous(14cases)and serous, mucinous and endometrioid (7cases).12had endometriosis, most commonly in the same ovary. 3had an ovarian borderline tumour or carcinoma; a grade1 endometrioid adenocarcinoma within the same ovary and a contralateral serous borderline tumour and borderline clear cell adenofibroma.

Conclusion: This represents by far the largest case series of benign ovarian seromucinous neoplasms and we show a close relationship with endometriosis and endometrioid differentiation.


Role of six levels in cervical biopsy - is it really required?

D. Bhattacharjee*, A. Irvine, D. Arora

*St James' Hospital, Leeds, United Kingdom

Background & objectives: Currently, there is no consensus as to the optimum number of levels that should be examined in cervical biopsies. We therefore investigated whether it is necessary to take six levels or taking three would be sufficient

Methods: We collected prospective data over a four month period at Leeds. The parameters included: the last smear result, colposcopy impression, HPV status, histological abnormality. Six pathologists independently recorded on which level abnormality was noted and which was the worst level.

Results: Majority of cases (72.67%) showed abnormality in all six levels. No particular worst level was seen in 54% (81/150). The level showing worst changes was L6 - 36.2% (25/69). Level 1 was worst in a single case (0.67%). Level range with worst changes were L4-6 - 71% (49/69). Levels 1-3 were worst in only a minority (2.67%) of cases (4/150). L1-3 were shallow in 12.6% (19/150) in decreasing order from 1 to 3 (12.6, 10.6 and 8% respectively). Abnormality is seen in increasing order from L1 to 6 (78%, 82%, 86%, 94%, 94 and 94% respectively).

Conclusion: No significant benefit is seen in examining all six levels as majority of cases show similar changes in all levels. Three levels may be sufficient to examine. Although worst levels are more seen in later levels (L4-6), the range needs to be wide as minority of cases had worst changes even in earlier levels (L1-3). It may be optimal to cut L1-7,examine alternate HE keeping coated spares for further work


Cytology versus endocervical curettage for the detection of HSIL persistence after loop electrosurgical excision procedure (LEEP)

R. Bishop*, I. Torras, M. Munmany, A. Torne, M. Del Pino, A. Saco, J. Ordi

*Department of Pathology ISGlobal, Hospital Clínic Barcelona, University of Barcelona, Spain

Background & objectives: Endocervical curettage (EC) and cytology are the common methods to determine the risk of persistence/recurrence after LEEP procedure for high-grade squamous intraepithelial lesions (HSIL) of the uterine cervix, but there is controversy on which method provides more accurate information.

Methods: Prospective study that included 307 women treated for HSIL by LEEP between 2013 y 2018. In all cases, EC and liquid-based cytology (LBC, Thinprep) were obtained immediately after the procedure. The follow-up controls were scheduled every 6 months for at least 24 months and included LBC, HPV testing and colposcopy with biopsy if indicated.

Results: Mean age of the patients was 40.3 ± 10.1. HPV testing was positive in 128 (41.7%) women, cytology in 46 (15.0%) and EC in 18 (5.9%) of the patients. The samples obtained after LEEP were insufficient/inadequate in18 (5.9%) of the LBC and in 71/307 (23.1%) of the EC. HSIL persistence/recurrence was identified in 24/307 patients (7.8%). The sensitivity and specificity of the LBC were 23.9% and 95.0%, respectively. The sensitivity and specificity of the EC were 16.7% and 92.7%, respectively.

Conclusion: Conclusions:

Cytology provides more accurate information on persistent/recurrent than EC.


Application of the promise molecular classification in endometrial carcinomas: clinical, pathological, metabolical and molecular characterisation

C. Caral*, A.P. Caresia, C.M. Blazquez, L. Nebot, R. Carrera, J.A. Vazquez, M.I. Agustin, L. Ribot, Y. Garcia, À. Casalots, J.C. Ferreres, I. Costa

*Parc Taulí Hospital Universitari. I3PT. Universitat Autònoma de Barcelona, Spain

Background & objectives: ProMisE molecular classification recognizes four groups of endometrial carcinoma (EC), improving morphological and treatment approaches. The objective of the study is to confirm the contribution of ProMisE in EC, adding metabolical analyses and test pyrosequencing (PSQ) in POLE gene study.

Methods: 54 EC diagnosed in our institution were evaluated: histological type, pathological stage, "MELF" and vascular invasion, metabolical activity by PET/TAC and clinical behaviour. Molecular classification was performed in ProMisE sequential order (MMR-D, POLE-EDM, p53abn and p53wt): MMR and p53 proteins by immunohistochemistry and codons 286/411 POLE by PSQ. p53 mutational status was analysed by NGS (TST15) in 7 selected cases.

Results: We collected 21 MMR-D, 1 POLE-EDM, 13 p53abn and 19 p53wt CE: 10 serous carcinomas and 2 carcinosarcomas in p53abn; 3 in/dedifferentiated carcinomas in MMR-D and 2 clear cell carcinomas in p53wt. MMR-D and p53wt harboured 34/35 endometrioid carcinomas. POLE-EDM exhibited high-grade histology. Vascular invasion: 6/21(28,6%)MMR-D, 6/13(46,2%)p53abn, 8/19(42,1%)p53wt. "MELF" invasion: 1/21(4,8%)MMR-D, 1/13(7,7%)p53abn, 5/19(26,3%)p53wt. Extrauterine disease: 4/21(19%)MMR-D, 4/13(30,8%)p53abn, 6/19(31,6%)p53wt. POLE-EDM was IA and disease free survival(DFS). 14/21(66,7%)MMR-D, 5/13(38,7%)p53abn and 17/19(89,5%)p53wt are DFS. SUVmax/SUVpic were higher in MMR-D than p53abn. p53abn MTV and TLG were the lowest (p<0,005). Only one p53abn(negative pattern) did not correlate with NGS results. PSQ was restricted to two codons, with frequent contaminations, but easy and quantitative interpretation.

Conclusion: ProMisE classification exhibits pathological correlation that improves diagnoses in ambiguous cases, displays strong prognostic value, above classical ones, and guides treatment. p53 immunohistochemistry correlates with molecular status of the gene. Paradoxically, the lowest metabolical activity is observed in p53abn group. PSQ is a sensitive technique, allowing an easy interpretation and quantitative results. However it exhibits some technical limitations. A comparative study with Sanger approach would have to be carried out.



Possible etiologic role of human papillomavirus in vulvar seborrheic keratosis: a study combining whole tissue section-PCR, immunohistochemistry (p16 and e4), and laser capture microdissection-PCR

S. Dasgupta*, R. van Eersel, B. Morrel, H.A. van den Munckhof, V.A. de Geus, N.M. van der Hoeven, M.M. van de Sandt, W.G.V. Quint, I.A. van der Avoort, P.C. Ewing-Graham, F.J. van Kemenade

*Erasmus MC, University Medical Center Rotterdam, The Netherlands

Background & objectives: HPV-DNA has been previously detected in vulvar seborrheic keratosis (VSK) by performing polymerase chain reaction (PCR) on whole-tissue sections (WTS). However, this has been refuted as evidence of an etiologic involvement of HPV, as WTS-PCR does not inform on HPV-integration.

Methods: We explored the association of HPV and VSK by performing WTS-PCR, immunohistochemistry with p16 and E4, and laser capture microdissection (LCM)-PCR. p16 and E4 are surrogate markers of HPV-oncogene expression, and LCM-PCR allows HPV-detection from specific lesional cells, with a higher precision than WTS-PCR. VSKs were selected following strict histologic criteria to avoid including the common differential, HPV-related condyloma acuminata.

Results: HPVs were detected in 73% (11/15) of VSKs through WTS-PCR (SPF10-PCR-DEIA-LiPA25); these included HPV44 (n=4), HPV6 (n=4), HPV42 (n=2), HPV53 (n=1), and an untypable genotype (n=1). p16-positivity was noted in 82% (9/11), and E4-positivity was noted in 36% (4/11) of HPV-positive VSKs. LCM-PCR was performed on six lesional areas (two p16+/E4+ and four p16+/E4-) and one area of adjacent normal epithelium (p16-/E4-) selected from four HPV-positive VSKs. HPVs were detected from all lesional areas by LCM-PCR and the results were concordant with WTS-PCR. HPV could be detected in the basal/intermediate epithelial layers of the lesional areas, thereby ruling out surface contamination. The area from normal epithelium was negative for HPV.

Conclusion: These results imply a pathogenic involvement of HPV in a proportion of VSKs. p16 and E4-positivity indicated HPV-integration, and through LCM-PCR, HPVs could be localized to the lesional cells. The natural of history of these VSKs deserves further study.


Analysis of endometrial carcinoma with subclonal P53 expression

A. Dashora*, E. Thompson, J.V. Broek, A. Lum, J. Senz, S. Leung, J. Huvila, N. Singh, M. Koebel, J.N. McAlpine, B. Gilks

*Royal Free Hospital, London, United Kingdom

Background & objectives: To study endometrial carcinoma (EC) cases showing subclonal p53 expression on immunohistochemistry (IHC) to determine which molecular category these should be assigned to; in addition, to develop practical guidance for the interpretation of this novel and poorly understood expression pattern.

Methods: 278 EC’s were classified into molecular categories using p53 and MMR IHC on whole sections, together with pathogenic POLE mutation testing using targeted next generation sequencing (NGS). Cases with subclonal p53 IHC expression involving ≥5% of tumour underwent directed sampling [a single 1mm core for each differentially staining area] and TP53 sequencing through NGS.

Results: Subclonal p53 expression was observed in 19/278 (6.8%) cases at levels ranging from 0.5% to 95% (median 11%). 7/19 cases (37%) were classified as MMRd EC. An additional 5/19 cases (26%) harboured pathogenic POLE mutations (POLEmut). The remaining 7/19 tumours (37%) were MMR proficient (MMRp) and POLE wild type (POLEwt). Of these, 6/7 contained a TP53 mutation in both the areas with mutant pattern and wild type p53 expression, the only exception being a tumour where the subclonal loss was only present in 3% of the tumour cells, and below the limit of detection of the assay used.

Conclusion: Subclonal p53 IHC reflects underlying TP53 mutation. In MMRd and POLEmut EC this is secondary and unlikely to show adverse prognosis. In MMRp and POLEwt EC, however, this pattern appears to indicate a driver TP53 mutation, ie p53 abnormal EC.


An audit of histopathology reports of carcinoma endometrium: assessment of different histology parameters from specimens operated at a cancer centre in india, between January 2013 and March 2018

K. Deodhar*, D. Singh, A. Budukh, B. Rekhi, S. Menon

*Tata Memorial Hospital, India

Background & objectives: To review histopathology reports of patients operated for carcinoma endometrium, at our institute between Jan 2013 to March 2018, and to see our compliance to minimum data sets.

Methods: -After obtaining approval from the Institutional Review Board (IRB), our hospital records showed a total of 457 reports of carcinoma endometrium, operated between January 2013 to March 2018. Various parameters from the reports were noted, and their frequencies were calculated with the help of SPSS software (version 21).

Results: The mean age was 57.5 years, mean tumour size was 3.78 cm. The depth of myometrial invasion and tumour type was mentioned in 100% reports, commonest tumour type being endometrioid-87.3%. The tumour was stage pT1a in 63.9%, stage pT1b in 36.1% cases. Distance of tumour from uterine serosa was mentioned in 86.4% cases. The FIGO tumour grades were grade II- 56.2%; grade III- 28.7%; grade I-14.9%; mentioned in 99.8% cases. Lymphovascular invasion was present in 15.5% cases. The cervix was reported in 99.8% cases, involved in 10.7% cases. Lymph nodes were removed in 78.6% cases ( mean lymph node yield 14.01). TNM stage was mentioned in only 56.2 % cases.

Conclusion: -Overall compliance to minimum data sets in carcinoma endometrium reports by our team is good. pTNM stage, distance to serosa need to be mentioned.


MYB rearrangement is frequent in adenoid cystic carcinomas of the Bartholin’s gland: a series of 5 cases

D. Doutel*, D. Venda, A. Felix, C. Martins, J. Ferreira

*Instituto Português de Oncologia de Lisboa Francisco Gentil, Portugal

Background & objectives: Primary vulvar adenoid cystic carcinoma(ACC) is very rare. In most anatomic locations, MYB-NFIB and MYBL1-NFIB fusions are oncogenic drivers in ACC. In two prior series, MYB was rearranged in 2/9 and 3/3 vulvar ACC cases.

Methods: We report the clinicopathologic features of five vulvar ACCs diagnosed between 1963 and 2020, and we describe the prevalence of MYB and MYBL1 rearrangements in this series.

All HE slides from these cases were reviewed, as well as their medical records. FISH using MYB and MYBL1 BAC-clones break-apart probes was carried out.

Results: Patients’ mean age at diagnosis was 52 years. Tumour size ranged from 0.5-5cm. Microscopic examination revealed tubular and cribriform patterns. A minor solid component was identified in one case. No high-grade components were detected. Perineural invasion was seen in all cases. All patients were treated with surgery, followed by postoperative radiation in one of them. Positive surgical margins were reported in four patients. During follow-up (median:21 years), three patients (all with positive margins) developed local recurrences treated with surgery and radiation. Currently, all patients are disease-free.

FISH analysis was successful in 4/5 cases. MYB rearrangement was present in 3/4 cases, including one with concurrent MYB amplification. No MYBL1 rearrangements were found.

Conclusion: We corroborate the histological and genetic similarity between vulvar ACC and ACCs elsewhere. We also confirm that MYB rearrangement is frequent in vulvar ACC.


Prognostic role of mismatch repair protein defect in endometrioid type of endometrial carcinoma

N. El Anwar*, A. Eid, S. Ezzeldin

*Pathology Department, Faculty of Medicine, Tanta University, Egypt

Background & objectives: Microsatellite instability, associated with mismatch repair proteins deficiency, is a frequent alteration in endometrial cancer(EC) that has been associated with prognosis.

evaluate the state of MLH1 and MSH2 as a potential prognostic factor in endometrioid type of endometrial cancer(EEC).

Methods: the current work was carried out on 80 cases of EEC retrieved [with clinical data] from the department of Pathology, Faculty of Medicine, Tanta University in the period from June 2017 to December 2018. H&E staining and immunohistochemical staining with MLH1and MSH2 was done for each case.

Results: among 80 cases, 29 (36.3%)showed abnormal MMRP expression (loss of MLH1 expression was detected in 19 cases (65.5%) and loss of MSH2 expression was detected in (34.5%) of cases. Loss of MMRP expression was closely related to some clinicopathologic features (patient’s age, histopathological tumour grade, and tumour stage) with a statistically significant relation . No significant relation was found with myometrial and lymphovascular invasion (p value= 0.054 and 0.028)

Conclusion: a subset of endometrioid type demonstrates MMRP defect; the MMRP deficient EEC often displays adverse clinicopathological parameters as poorly differentiated or undifferentiated histology as well as advanced stage with early age of onset at presentation.


Non squamous malignancies of vagina and vulva: 33 year experience at a tertiary centre in UK

R. El-Shennawy*, C. Aird, W. Boyle, J. Yap, A. Williams, R. Ganesan

*Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, The University of Birmingham, United Kingdom; Pathology Department, Faculty of Medicine, University of Alexandria, Egypt

Background & objectives: Under 10% of gynaecological cancers are diagnosed in the vulva and vagina; mostly squamous cell carcinomas. Melanoma, Paget disease, basal cell carcinomas and other cancers can present with vulval/vaginal symptoms.

Methods: The pathology information system of a tertiary referral centre for vulvo-vaginal cancers was searched for cancers of the vulva and vagina from 1996 to 2019. Squamous carcinomas were excluded and the remaining entities were catalogued.

Results: 135 vaginal and 86 vulval cases of non-squamous cancers were found. 108 cases of metastatic carcinomas from endometrium, cervix, ovary, bowel, bladder, kidney and breast formed the largest category. Basal cell carcinomas constituted the 2nd largest category. Others included melanomas, Paget disease, and adenoid cystic carcinomas. Primary adenocarcinomas included porocarcinoma, mammary type carcinoma, enteric type carcinoma, clear cell carcinoma, Bartholin gland adenocarcinoma and malignant transformation of hidradenoma papilliferum.

Conclusion: The vulva and vagina can harbour a wide range of non-squamous malignancies. The most challenging of these are adenocarcinomas which can be metastatic from other sites. The dominance of metastatic carcinomas in this series is likely to reflect consultation practice of specialist pathologists


Vulva carcinoma: an overview of a rare disease and summary of experience from King Hussein Cancer Center (KHCC)

M.A.A. Erashdi*, M. Al-Hussaini

*King Hussein Cancer Center, Jordan

Background & objectives: Carcinoma of the vulva constitutes only 5% of female genital malignancies, of which the most common type is squamous cell carcinoma (SCC). There is global variation in the prevalence, with only few epidemiologic studies were conducted in the Arab world.

Methods: This is a retrospective chart review of all vulva SCC cases referred to KHCC between 2004-2018. Phase 1; demographic data, changes in the trends over the years, pathology findings, treatment and outcome are summarized. Phase 2 (in progress); include immunohistochemical testing for (P16, P53, Ki-67), for detection of Vulvar intraepithelial neoplasia (VIN), and RFLP-PCR for detection of HPV infection/serotyping, respectively.

Results: The total number of patients is 62.There is a dramatic increase in the number of cases (59.7%; 2016-2018). The mean age at diagnosis was 56 years. The average tumour size and depth of invasion were 30.1mm and 7.73mm, respectively. According to FIGO staging, stage I, II, III and IV were 46.2%, 5.8%, 28.8%, and 19.2%,respectively. Surgical resection was offered to 82.8% of cases, with or without chemotherapy/radiotherapy. With mean follow up duration of 18.4 months, 64.5% are alive; 90% of them are in complete remission. The 5 years event free survival, and overall survival rates are 67.65% and 55.51%, respectively. At this time, prevalence of VIN and HPV infection are under investigation.

Conclusion: Carcinoma of the vulva is a rare disease that is witnessing an increased trend in the last few years. It is associated with a favourable outcome when treated properly. The underlying predisposing and/or associated risk factors need to be further indicated.

Funding: Phase 2 of this project is currently funded by King Hussein cancer centre-Jordan.


Plexiform tumourlets of the uterus: clinicopathological and molecular analysis of a series

R. Erber*, R. Stoehr, N. Oks, F. Haller, M.W. Beckmann, A. Hein, B.P. Rubin, A. Hartmann, A. Agaimy

*Institute of Pathology, University Hospital Erlangen, Friedrich‐Alexander University Erlangen‐Nürnberg (FAU), Comprehensive Cancer Center (CCC) Erlangen-EMN, Germany

Background & objectives: Plexiform tumourlets of the uterus (PTU) are extremely rare, tiny, multifocal epithelioid mesenchymal lesions thought to represent variants of epithelioid leiomyoma. They are detected as incidental histological findings in hysterectomy specimens for other diseases. However, their pathogenesis remains unknown.

Methods: Seven cases of PTU were identified in routine pathology files at the Institute of Pathology, Erlangen (2012-2017), and in consultations files of two authors. Clinical and pathological characteristics were reviewed. Four cases with sufficient material were investigated for gene fusions using TruSight-RNA Fusion Panel (Illumina® Inc., San Diego, CA, USA) and three were assessed for FOXL2 mutations by direct sequencing.

Results: Age at diagnosis ranged between 44 and 56 years. Patients received curettage (1 case) or hysterectomy due to perimenopausal bleeding, endometrial carcinoma, symptomatic adenomyosis uteri or uterine leiomyomas. PTUs presented as solitary lesions (n=2), multiple tiny nodules (n=3) or as tiny lesions extensively involving the myometrium (size range: 0.15-0.5 mm). None of three cases each successfully tested for gene fusions by RNA testing or FOXL2 mutations revealed positive results.

Conclusion: The molecular pathogenesis of uterine plexiform tumourlets remains elusive.


PD-L1 expression in different molecular subgroups of endometrial carcinomas

A. Evsei*, A. Birceanu, N. Copca, A. Dumitru, M. Sajin

*Saint Mary Clinical Hospital, Romania

Background & objectives: New oncological treatment guidelines for endometrial carcinomas (ECs) are constantly evolving with the recent emphasis on immunotherapy options for distinct subsets of ECs. Our purpose was to identify specific molecular categories and analyse PDL-1 expression on these different subtypes.

Methods: We included in our study 50 cases of ECs in a Romanian cohort over 5 years (2014-2019). For each case, we evaluated a vast set of pathological parameters and tested p53, MSH6 and PMS2 for MMR status. PDL-1 expression was quantified in four distinct molecular groups: MMR-D, MSS, CNH (abnormal expression of p53) and CNL (wild-type p53).

Results: According to our findings, the mean age for diagnosis was 63 years, with endometrioid subtype being the most frequent. We identified 34 cases - MMR-D, 16 cases - MMR-S, 13 cases - CNH and 37 cases - CNL. PDL-1 positivity was more than 50% in two MMR-D cases and one CNH,1-49% in 7 MMR-D cases, 4 CNH cases and 6 CNL cases and negative in 30 cases.Five-year OS was 84%.

Conclusion: Recent advances in immunotherapy have opened options for women with the MSI subset of tumours and current trials are attempting to expand the benefits of immune checkpoint inhibition to a wider group of patients with ECs. For that reason, we set out to define the molecular landscape of ECs in Romania and establish what other molecular groups would be suitable for PDL-1 testing. To our knowledge, this is the first attempt in our country.


PD-L1 expression in high grade serous carcinoma of the female genital tract correlates with favourable prognosis - experience within an immunohistochemically re-classified large clinical cohort

A.K. Fischer, N. Neudeck, M. Grube, B. Ney, H. Boesmueller, J. Pasternak, K. Greif, C. Beschorner, S. Brucker, D. Wallwiener, F. Fend, S. Kommoss, A. Staebler*

*Institute for Pathology, University of Tübingen, Germany

Background & objectives: High-grade serous tubo-ovarian carcinoma (HGSC) is the most lethal carcinoma of the female genital organs. In recent studies, PD-L1 expression correlates with prolonged overall survival. We investigated a large consecutive cohort of immunohistochemically re-classified HGSCs for PD-L1 expression status.

Methods: A consecutive series of 420 cases diagnosed as HGSC and treated at the Womens’ Hospital of the University Tübingen from 2000 until 2016 was reviewed by two independent pathologists. Tissue microarrays (TMAs) were stained for a panel of four markers including WT1, p53, progesterone receptor and Napsin-A. PD-L1 expression was analysed using Combined Positivity Score (CPS).

Results: In 94.0% of cases (n= 395/420), the diagnosis of HGSC was confirmed. Within the re-classified 395 (100%) HGSCs, 131 presented PD-L1 expression (36.7%), 250 were negative (63.3%). Median overall survival (OS) for mean CPS showed significant differences: 906 days for patients with PD-L1-negativity (n=250, 63.3%), 1347 days for CPS <1 (n=85, 21.5%) versus 1412 days median OS for CPS 1-25 (n=60, 15.2%)(p<0.001). We found significant association with complete resection (p=0.049).

Conclusion: Within our cohort of re-classified HGSCs of the female genital tract, PD-L1 expression was found in 36.7%, whereas the majority showed negative results (63.3%). In summary, PD-L1 is expressed in a subgroup of HGSCs associated with a favourable prognosis and presents a significant overall survival benefit. These results suggest an important role in the tumour microenvironment and provide a potential therapeutic target.


Tumour budding and cell nest size did not prove useful as prognostic factors in a series of 121 squamous cell carcinomas of the uterine cervix

A. Flora*, A. Felix

*Rede D'Or São Paulo, Brazil

Background & objectives: Squamous cell carcinoma of the cervix (SCCC) grading has no prognostic value. Recently, Jesinghaus, M. et al, proposed a new grading system based on the evaluation of tumour budding and cell nest size, that could help in prognostic patient stratification.

Methods: Two pathologists reviewed a series from 2002 to 2016 of H&E whole slides from SCCC, radical hysterectomies. The clinical files and tumour grading were evaluated accordingly to the parameters proposed by Jesinghaus, M. et al. This grading score was correlated with the staging, treatment and overall survival of the patients.

Results: We studied 121 cases of SCCC. Patients’ mean age was 47 years-old (29-80yrs). Tumour stage was pT1(n=99), pT2(n=21) and pT3(n=1) and 31%(n=37) had regional lymph node metastases. 77 patients received adjuvant therapy. Average follow-up was 112 months (5-210mo). 81% of the patients are alive without disease, 8% died of the disease or are alive with disease. 11% of the patients died from other causes or were lost for follow-up. 5%(n=6) had recurrence. Accordingly to the proposed grading system, 11 cases were grade 1, 35 grade 2 and 75 grade 3. We found no correlation between the new grading scores and metastasis, staging, follow-up or recurrence.

Conclusion: In our series, the new grading system showed no correlation with the patient prognostic factors (recurrence, metastasis or follow-up). Poor reproducibility or different patient characteristics might explain this. Although the proposed system seems promising and is useful in other topographies, our results imply that it must be validated in other SCCC cohorts and institutions.


Clinical and pathologic variables associated to cervical conizations without high grade intraepithelial lesions: a series of 399 cases

B. Fuertes Negro*, G. Muñiz, S. Gracia, F.J. Queipo, J.M. Ramón y Cajal

*Pathology Department, Hospital San Jorge, Huesca, Spain

Background & objectives: Between 10-34% of conizations lack ≥CIN-2 (conization without disease, CWD). Our aims are to estimate our CWD rate, to evaluate the usefulness of deeper morphological reassessment plus p16 to ensure a CWD and to assess clinic-pathologic variables associated to CWDs.

Methods: All conizations and colposcopy biopsies from 2010-19 were reviewed. In CWDs, three deeper extra hematoxylin-eosin slides and one p16 were performed.

The association of clinical (age, colposcopic extension of lesion, only or multiple colposcopy biopsies) and pathological variables (lesion size, lymphocytic reaction in the colposcopy biopsy, conization size; rate of CWD initially and after extra levels) to CWD were studied.

Results: A total of 399 conizations were performed, with a mean age of 38.59 years. 96% of colposcopic lesions were ≥CIN-2, with mean size of 3.23 mm, occupying in a 60.55% of cases a single quadrant. In a 60.55% the colposcopy biopsy was unique.

The initial 81 (20.40%) CWDs were reduced to 63 (15.79%), a decrease of almost a quarter, after applying deeper hematoxylin-eosin levels and extra p16. Six cases (7.41%) were reclassified as low grade dysplasia after reviewing the colposcopy biopsies.

CWD was significantly associated to younger age, single quadrant lesion and a smaller lesional size.

Conclusion: 1.- Our CWD rate is within the published range.

2.- Performing extra hematoxylin-eosin levels and p16 would be a recommended practice to ensure a CWD diagnosis.

3.- There are scarce clinic-pathological variables associated to CWD.


Uterine rhabdomyosarcomas: 8 cases, a 10-year retrospective study in a tertiary institution

J. Gama*, J. Madeira, R. Almeida, H. Moreira, F. Ramalhosa, V. Almeida, C. Faria, M.B. Pimentão, A. Lai, J. Fraga, R. Oliveira, T. Simões Silva, M.A. Cipriano

*Centro Hospitalar e Universitário de Coimbra, Portugal

Background & objectives: WHO defines rhabdomyosarcoma as a rare and malignant, heterologous mesenchymal tumour showing evidence of skeletal muscle differentiation. The cervix is the most common localization, followed by the uterine corpus.

Methods: We conducted a 10-year-retrospective transversal study at Coimbra University Hospital, a tertiary hospital in Portugal (2009-2019) and reviewed all uterine and cervix sarcomas (n=45). Relevant clinical and pathological data was extracted from the hospital database.

Results: A total of 8 cases of rhabdomyosarcomas were found, representing 17.8% of uterine and cervix sarcomas. The tumours consisted of 6 pleomorphic and 2 embryonal rhabdomyosarcomas. No spindled and alveolar variants were found. In this cohort, 2/8 of the cases were diagnosed in a biopsy and 6/8 in the surgical specimen. The most common site was the uterine corpus with 62.5% of cases; the remaining arose in the cervix.

In our cohort, the median age at diagnosis was 69 years (range:40-86years) and the median survival was 21.8 months (range:1-86,7 months). Tumour size ranged from 4 to 13 cm.

Conclusion: Uterine rhabdomyosarcoma is a rare entity. In our series, pleomorphic rhabdomyosarcomas were the most common subtype followed by the embryonal variant.


First presentation of Mullerian serous carcinoma as nodal metastases: a case series

R. Ganesan*, W. Boyle, D. Midha, J. Vella, J. Pascoe, A. Williams

*Birmingham Women's Hospital, United Kingdom

Background & objectives: More than of 50% women with serous carcinoma of Mullerian origin present at advanced stage. Dissemination typically occurs via transcoelomic spread. Tubo-ovarian carcinoma presenting as nodal metastases in the absence of clinical features of pelvic disease is rare.

Methods: We present a summary of findings in a series of serous carcinomas of Mullerian origin, diagnosed on lymph node biopsies. These cases were seen as a part of the diagnostic and consultation service of a specialist gynaecological pathology department.

Results: 12 women, 4 each with enlarged axillary and neck nodes, 1 each with enlarged nodes in the supraclavicular fossa, inguinal, peripancreatic and retroperitoneal sites; were diagnosed with metastatic serous carcinoma of Mullerian origin (10 high grade and 2 low grade). 9 presented with mass related symptoms, others with nausea, backache and dysphagia. Pelvic masses were found in 6 cases, 3 patients had lymphadenopathy only, 3 were lost to follow up.

Conclusion: This case series demonstrates that Mullerian serous carcinomas can present as distant nodal metastases in the absence of presenting symptoms of pelvic disease. It is postulated that malignant cells gain access to lymph nodes via peritoneal lymphatics or that carcinomas can arise from Mullerian nodal inclusions. Investigations may demonstrate a pelvic mass in some cases. Accurate and confident diagnosis of Mullerian origin can be made by a combination of morphology and immunohistochemistry.


Evaluation of spontaneous abortion samples following the Amsterdam Placental Workshop Group consensus statement: study in a single tertiary hospital centre

N. Gonzalez-Ortega*, M.J. Isorna-Porto, B. Sopeña, A. González-Quintela, F. Gude-Sampedro, J.M. Cameselle Teijeiro

*Mateu Orfila Hospital, IB-Salut, Spain

Background & objectives: In daily clinical practice, the study of spontaneous abortion is usually limited to the morphological confirmation of pregnancy. In this study we investigated whether the Amsterdam Placental Workshop Group (APWG) recommendations could provide relevant information concerning placental lesions, in comparison to routine study (RS).

Methods: We carried out a 9-month retrospective study of spontaneous abortion samples collected in our institution. All samples coded as “spontaneous abortion” and/or “product of conception” were selected from the Pathology Department database. Maternal clinicopathological and demographic variables were collected from the electronic records. All histopathological samples were reviewed a second time by two pathologists simultaneously using the APWG criteria.

Results: 408 samples were analysed. Mean age 35.5 years (17-48 years), mean gestational age 9.2 weeks (4-18 weeks). Samples were normal in 405 cases in the RS compared to 352 following our systematic revision. Relevant data were identified in 3(1%) cases of the RS [mola(1), acute inflammation(2)] vs. 56(14%) cases with APWG criteria [mola(1), distal villous hypoplasia(1), accelerated villous maturation(1), infarction(12)]. Fibrin was identified in the intervillous space in 41 samples.

Conclusion: The use of the APWG recommendations for the study of spontaneous abortion or placental samples resulted in our identifying 18.6 times more pathology than the RS. Most findings suggest poor placental perfusion, but the intervillous fibrin has uncertain significance.


Complement system involved in endothelial injury in preeclamptic placenta

M.T. Gonzalez-Serrano*, M. López-Andreu, A. Sanz-Zorrilla, I. Ruíz-Núñez, R. Ortega-Salas

*Dept. of Pathology, Hospital Universitario Reina Sofía, Spain

Background & objectives: Preeclampsia is a potentially severe condition of pregnancy, characterized by severe hypertension, proteinuria and vascular placental lesions. Links between preeclampsia and oxidative stress in trophoblast and endothelium were described and have investigated the role of complement system in these placenta

Methods: 34 placentas from patients with severe preeclampsia (PE) diagnosed before week 32 of pregnancy were obtained. The control group was 10 placentas of women without a diagnosis of hypertension or preeclampsia. Placental specimens obtained were processed and microscopic section were stained for H&E and PAS, as well as for immunohistochemical stains for CD61 (platelet thrombi) and C4d (complement component).

Results: PE group revealed thickened wall of foetal blood capillaries, crowded degenerating villi with decreased intervillous spaces, intravillous and perivillous fibrinoid deposition. Numerous syncytial knots and there was thickened layer of subchorionic fibrinoid. In addition, PE group showed atherosis, swollen endothelial cells and intracapillary thrombi with CD 61 immunomarking and C4d deposit.

Conclusion: Our results show a relationship between PE, lesions in vascular lesions with activation of the complement cascade and C4d deposit. Identification of mechanisms involved in the triggering of endothelial dysfunction may be a promising therapeutic approach in management of PE.


Co-expression of GATA-3 and PAX-8: a diagnostic pitfall

N. Herlihy*, R. Arora

*UCLH, United Kingdom

Background & objectives: Immunohistochemistry is invaluable in establishing the origin of a poorly differentiated primary tumour or metastatic carcinoma. Overlapping/atypical staining patterns are a common pitfall. Our objective was to ascertain the frequency of tumours co-expressing Müllerian marker PAX-8 and breast/urothelial marker GATA-3.

Methods: We performed a search on our internal pathology I.T. system (CoPath) for all specimens from 2015-2019 on which PAX-8 and GATA-3 immunostains were performed. We then collated the cases which expressed both markers and reviewed the final diagnoses on the pathology reports.

Results: PAX-8 and GATA-3 immunostains were performed on 228 surgical and cytological specimens. 22 tumours expressed both markers. The final histological diagnosis in 12/22 cases (54.5%) favoured tumours originating in the gynaecological tract; most of these were high-grade (8/12) and of tubo-ovarian origin (6/12), while 2/12 were uterine, 1/12 was cervical and for 3/12 the particular site of gynaecological origin was uncertain. 8/22 cases (36.5%) were diagnosed as poorly differentiated/high grade carcinoma where the site of origin could not be determined; 3/8 of these cases had a background history of previous ovarian or endometrial cancer. 1/22 (4.5%) was diagnosed as high-grade urothelial carcinoma and 1/22 (4.5%) as malignant epithelioid tumour.

Conclusion: Immunohistochemistry is commonly used to establish the origin of poorly differentiated or metastatic tumours. PAX-8 is positive in approximately 80% of Müllerian tract-derived carcinomas. GATA-3 is positive in 94% of breast and 86% of urothelial carcinomas. Our study highlights potential diagnostic pitfalls when both markers are positive, particularly in the diagnosis of gynaecological tract malignancy, and supports previous findings of GATA-3 positivity in a subset of endometrial and ovarian carcinomas.


Mismatch repair deficiency in uterine carcinosarcoma - a 20 year retrospective review: 18 cases tested

S. Hernandez Bonilla*, L. El Bouayadi, T. Rivera García, M.D. Rodríguez Garnica, R.M. Álvarez López, E. Jareño Dorrego

*Hospital Universitario Santa Cristina, Spain

Background & objectives: Immunohistochemistry for mismatch repair (MMR) proteins is recommended in endometrial carcinomas as a screening test for Lynch Syndrome. Carcinosarcomas are staged and treated like other epitelial malignancies, however few studies have evaluated the rate of MMR loss in uterine carcinosarcomas.

Methods: A 20 year retrospective database search of uterine carcinosarcomas was performed at our institution. The histologic diagnoses were confirmed by a gynaecologic pathologist. One tissue section from each case was stained with the 4 MMR proteins (MLH1, PMS2, MSH2, MSH6) and p53.

Heterologous elements and lymphovascular invasion were noted if present.

Clinical features were collected.

Results: 18 cases of uterine carcinosarcoma were identified. 16 cases showed intact expression and 2 mismatch repair deficiency (MMRd) with loss of MSH6 and aberrant p53 expression. 12 of the total number of cases showed aberrant p53 expression. 3 cases showed wild-type p53 expression. 2 cases of Lynch Syndrome were identified among

carcinosarcoma patients. Nowadays 17 patients are alive and 1 dead and 1

patient with loss of MSH6 had colorrectal carcinoma.

Conclusion: The rate of MMRd is lower in uterine carcinosarcoma when compared with endometrioid carcinoma. In the setting of MMR loss, a diagnosis of dedifferentiated carcinoma should be considered. Understanding how MMRd contribute to carcinosarcoma pathogenesis is relevant not only for identifying Lynch síndrome and prevent the development of colorectal cancer but also for identifying candidates for immunotherapy, as defects in MMR have been shown to impart vulnerability to checkpoint inhibition.


Peculiarities of vascularisation of serous adenocarcinoma of fallopian tubes

N. Hyriavenko*, M. Lyndin, V. Sikora, L. Karpenko, R. Moskalenko, A. Piddubnyi, A. Romaniuk

*Sumy State University, Ukraine

Background & objectives: Sometimes it is difficult to estimate the degree of neoplastic vascularization. It can be achieved by using the immunohistochemical studies of CD31 receptors.

To study the peculiarities of the serous adenocarcinoma of fallopian tubes (SAFT) vascularization.

Methods: The study was carried out on 66 samples of tumour tissue of serous adenocarcinoma of fallopian tubes. For study the CD31 expression, the rat monoclonal antibodies 1A10 were used.

Results: In well-differentiated tumours, the structured focal localization of the vessels in papillary formations, which were observed gradually disappeared in the papillae branched. While neoplasia dedifferentiation, the disorders of tumour tissue vascularization with chaotic vessels localization were found. Despite the data on the degrees of microvascular density in different types of carcinoma in case of malignant process development, areas with pronounced microvascular density as well as nonvascular tumour lesions were observed.

Conclusion: The tissue of SAFT is characterized by the structured vascularization of neoplastic tissue with its gradual disorganization under carcinoma dedifferentiation. These results can be used to predict the tumour course, considering the influence of the increased angiogenesis on cancer metastasis and the possibility of using the vascular-suppressant treatment in antitumour therapy.


Antenatal foetal vascular malperfusion is a placental factor for term preeclampsia

M. Irfan*

*Karaganda Medical University, Kazakhstan

Background & objectives: Currently, there is evidence that late preeclampsia (PE) in full-term pregnancy is more due to metabolic and cardiac dysfunction of the mother’s body than to placenta pathology, therefore PE in full-term pregnancy should be considered as a pathological condition different from early preeclampsia.

A comparative histopathological evaluation of the foetal-placental and uteroplacental compartments of term and preterm placentas from pregnancies with preeclampsia was performed.

Methods: Retrospective histological examination of 81 placentas from abnormal pregnancies complicated by preeclampsia at 32-35 weeks (n-52) and full-term (37-41 weeks, n-29) gestation was performed. Histopathological criteria of maternal vascular malperfusion follows: acceleration of villi development and placental infarction (more than 10% the placenta volume); criteria for foetal vascular malperfusion follows: fibromuscular sclerosis, vascular ectasia and thrombosis of placental stem villi.

Results: Histopathological signs of maternal vascular malferfusion in all placentas with preeclampsia were detected in 71 cases (88%). Among them, with preterm PE in 52(100%) cases, in term gestation - in 19 (66%) cases. Histopathological signs of FVM were detected in 27 (93%) placentas with term preeclampsia and only in 11(21%) placentas with preterm preeclampsia.

Conclusion: We hypothesize, that FVM is important feto-placental factor in the development of PE. PE in a full-term pregnancy may be associated with compensatory activation the tone of feto-placental large vessels in conditions of clinically latent fetoplacental blood flow disturbance (FVM).


MRI investigation in preeclampsia for severity diagnostic purposes

M. Irfan*, S. Zhuravlev, D. Kossitsyn, Y. Kotov, O. Kostyleva

*Karaganda Medical University, Kazakhstan

Background & objectives: Over the past 5 years, the number of brain haemorrhages in patients with hypertensive pregnancy disorders has increased 6-fold in the world.

MRI usage prospective in patients with preeclampsia (PE) complaints of headache for reducing the frequency of its complications.

Methods: 36 delivery cases in patients with preeclampsia that had undergone brain MRI were studied.

Results: Brain MRI indications: headache – 35 (97.2%), ophthalmic lesion – 1 (2.8%). PE intensity before the MRI: severe – 24 (66.7%), mild – 12 (33.3%). MRI done during pregnancies – 16.5 (31.3%) of which have revealed abnormalities that prohibit continuing pregnancy: 3 (60%) – posterior reversible encephalopathy syndrome (PRES), 2 (40%) – vascular focal lesions in the brain; a patients (68.7%) had no pathology.

Conclusion: According to MRI data, changes were revealed in 58.3% patients with PE and complaints of headache. Data obtained from the objective assessment of the brain condition helped to avoid unreasonable pregnancy continuation and contributed to the timely therapy correction, which helped reducing the frequency of complications. in 2 patients with PRES and 1 with vascular focal lesions arterial blood pressure and proteinuria levels matched mild PE, but regarding all the revealed changes, all the patients undergone emergen c-section;


To evaluate the presentation and time of diagnosis of cervical cancer and its precursor lesions in a tertiary care hospital of a low resource country like Pakistan lacking a national screening programme

M. Javed*, R. Rafi, H. Saleem

*Cantonment General Hospital, Pakistan

Background & objectives: The purpose of the study is to evaluate the most common presentation and diagnosis of cervical cancer and its precursor lesions in a tertiary care hospital of Pakistan in the absence of a local or national level screening program.

Methods: A retrospective descriptive study was carried out in which all the cases of cervical cancer and cervical intraepithelial lesions received in the Histopathology Department of Foundation University Islamabad (FUI) were retrieved from January, 2010 to December 2014. All the surgical specimens were included including biopsies and hysterectomy specimens. The data was then analysed for clinical presentation and age of diagnosis.

Results: A total of 47 cases of cervical cancer and precursor lesions were identified in the 5 year study period.out of which 7 patients were diagnosed with cervical intraepithelial neoplasia(14.89%)while the rest of the women were diagnosed with different types of cervical carcinoma(85.1%).the most common clinical presentation was postmenopausal bleeding(90%) followed by dysfunctional uterine bleeding.(10%).the most common colposcopic findings were cervical growth/mass(89%).the mean age of diagnosis of CIN1,CIN2 and CIN3 was 44.57 and the mean age of diagnosis of carcinoma was

Conclusion: The burden of disease will continue to increase with more frequent presentation with invasive carcinoma therefore education of the public,political support and awarenes is important to combat the disease


Association of CCL2 (monocyte chemoattractant protein-1) expression with obesity in endometroid endometrial cancer

P. Jolugbo*, M. Cummings, G. Mappa, R. Hutson, N. Orsi

*Women's Health Research Group, Pathology & Data Analytics, Leeds Institute of Medical Research at St James's, University of Leeds, United Kingdom

Background & objectives: Despite the epidemiological association between endometrial cancer and obesity, the underlying pathophysiological link remains ill-defined. We investigated the potential association between obesity and the tumour-associated inflammatory microenvironment.

Methods: 48 cytokines were profiled by multiplex immunoassay in endometrial cancer (N=93, including 53 endometroid endometrial cancers (EEC) and 40 non-EECs). Sample-matched cytokine mRNA expression data were obtained by microarray analysis of laser-captured microdissected tumour cells. Patient demographics including BMI were also collected for these women. Non-parametric statistical tests were carried out with Benjamini-Hochberg correction for multiple comparisons, as appropriate.

Results: There was no significant difference in BMI between patients with EECs and non-EECs. No significant association between cytokine profiles and BMI were identified in the non-EEC group. However, in the EEC group, there was a significant association between CCL2 expression and obesity, where CCL2 mRNA was higher in patients with BMI >30 (corrected P=0.008). There was a trend for increased CCL2 protein expression (uncorrected P=0.008). CCL2 mRNA and protein levels significantly correlated with each other (SRC=0.551, P<0.00001), as well as positively with BMI (SRC=0.518 and 0.336; P=0.0001 and P=0.017, respectively). There were no significant differences in CCL2 expression between EECs and non-EECs.

Conclusion: We identified an association between tumour epithelial CCL2 expression and BMI in EEC. These findings suggest a potential link between obesity-associated systemic inflammation and its localised effects in the tumour microenvironment.

Funding: Wellbeing of Women (RG1210), Yorkshire Cancer Research (LPP053), Pathological Society of Great Britain and Ireland (Career Development Fellowship awarded to NMO; 1090)


Pap smear and primary HPV tests results over a recent 3-year period from a routine laboratory setting in Kenya

A. Kalebi*, R. Mukadam, L. Muchiri

*Pathologists Lancet Kenya

Background & objectives: Cervical cancer is a leading cancer in Kenya where a National Cancer Screening Guideline was recently released recommending HPV Testing as the primary screening method. We hereby share experience on Pap smear and HPV tests done at our laboratory.

Methods: We retrospectively retrieved data from our laboratory information system (LIS) focusing on conventional cervical cytology done over a recent 3-year period from July 2016 to June 2019 corresponding to the period HPV test on Cobas 4800 platform (Roche) was introduced in our laboratory, which is ISO15189 accredited for both tests and one of the busiest laboratories in the country.

Results: A total of 27,447 Pap smears were reported with a median age of 38 years, of whom 97.31% had NILM,1.58% HSIL, 0.57% LSIL, 0.33% ASC-H, 0.02% ASCUS and 0.04% SCC. Up to 43.3% had significant cervicitis on conventional Pap smear and 13.6% had bacterial vaginosis, while 8.9% had atrophic changes.

During the corresponding period, a total of 3523 HPV tests were done, with an over two-fold year-on-year increase in numbers tested. High-risk HPV (HR-HPV) was detected in 26.6% of the cases, of whom 24.5% tested positive for HPV non-16/18 type, 6.2% type 16 and 3.9% type 18. Infection with multiple HPV types was noted in 6% of cases.

Conclusion: The rate of HR-HPV is noticeably high in these women tested in a routine setting. ASCUS seems unusually, low likely due to dual reporting by cytologists and pathologists; HSIL and LSIL rates are comparable to the published literature from Africa


Clinicopathological features, including immunohistochemical profile of 14 malignant peritoneal mesotheliomas diagnosed at a single institution

S. Karmarkar*, B. Rekhi, K. Deodhar, S. Menon

*Department of Surgical Pathology, Tata Memorial Hospital, HBNI University, Mumbai, India

Background & objectives: Malignant peritoneal mesotheliomas (MPMs) are rare tumours with overlapping clinical and histopathological features, especially with epithelial ovarian carcinomas (EOCs). There is no substantial documentation on these rare tumours from our country.

We studied clinicopathological features, including the immunohistochemical profile and clinical outcomes of 14 MPMs, diagnosed at our Institution.

Methods: This was a retrospective study, wherein, 14 cases of MPM in female patients,diagnosed at our Institution, between January 2008 and May 2019 were included, after critical review.

Results: Median age was 54.5 years. Microscopically,most cases(11, 78.6%) displayed epithelioid morphology, followed by biphasic(2, 14.3%) pattern.Sensitivity and specificity of calretinin was 100%(13/13) and 85.%; HBME1 was 100%(5/5) and 100%, and of podoplanin(D2-40) was 60%(2/5) and 100%. Other positively expressed immunomarkers were EMA (n=2/5, 40%) CK5/6 (n=4/4, 100%) and WT1 (n=9/10, 90%). Most patients(5/12)(41.7%) were treated with chemotherapy.The 3-year disease-free and overall survival rates were 25.7% and 54%, respectively.

Conclusion: MPMs can be diagnosed with a combination of clinicopathological features and optimal immunohistochemical markers. Their differentiation from EOCs and other metastatic carcinomas is imperative in view of significant treatment implications.


Hypoxic damage and endometrial cell antiapoptosis in endometrial hyperplasia

E. Kazachkov*, E. Voropayeva, E. Kazachkova, A. Zatvornickaya

*South Ural State Medical University, Russia

Background & objectives: Hypoxia stabilizes hypoxia inducible factor-1 alpha (HIF-1 alpha) leading to cell hyperproliferation and antiapoptosis. However, hypoxic damage and antiapoptosis in endometrial hyperplasia (EH) remain little studied. The aim was to compare hypoxic damage and endometrial cells antiapoptosis in EH.

Methods: Endometrial biopsy was obtained from 25 patients with EH without atypia and 25 healthy women. Expression levels of HIF-1 alpha and Bcl-2 were determined by monoclonal antibodies (Epitomics, Clone EP118 and Dako, Clone 124). Videotest - Morphology 5.2 (Russia) was used for morphometric analysis, and nonparametric statistical methods were applied.

Results: In the group of patients with EH, the mean area of HIF-1 alpha immunopositive structures was 41.7% (35.8%; 47.1%). The mean area of Bcl-2 expression was 68.6% (58.4%; 76,2%). In endometrial samples of healthy patients, HIF-positive cells were not recorded, and the average area of immunopositive structures was 0%. The average area of Bcl-2 positive structures was significantly lower in this group compared to patients with EH and was 22.53% (15.6%; 29,8%).

Conclusion: Significantly increased HIF-1 alpha and Bcl-2 expression in EH samples indicates hypoxic damage and raised antiapoptotic activity of intracellular endometrial systems. Increased antiapoptotic activity and HIF-positive cells presence may be an adverse factor leading to endometrial oncotransformation.

The reported study was funded by RFBR, project number 19-315-90101.


Vitamin D receptors as an additional marker of implantation in ART programmes

E. Kazachkov*, E. Chuhnina, E. Voropayeva, E. Kazachkova

*South Ural State Medical University, Russia

Background & objectives: Vitamin D receptors (VDR) play an important role in endometrial receptivity and implantation. The objective is to assess the impact of VDR expression levels in stroma and endometrial glands on the outcomes of ART programs.

Methods: A prospective study of IVF outcomes was performed in 70 women of advanced reproductive age with tubal-peritoneal infertility. An endometrial pipe-biopsy was taken during the supposed implantation window. The endometrial samples were formalin-fixed and embedded in paraffin. For immunohistochemistry, rabbit polyclonal Vitamin D receptor antibodies were used. The percentage of VDR-positive stromal cells was calculated.

Results: VDR expression in the endometrial stroma was significantly higher in the case of successful implantation. The threshold value was 8.7%, since clinical pregnancy was not observed if the percent of VDR positive cells was equal to or higher than 8.7%. Therefore, the endometrium was favourable for implantation in values of less than 8.7%. There were no significant differences in VDR expression in the endometrial glands.

Conclusion: The obtained results expand the range of endometrial receptivity markers. The identified reference value of VDR expression in the endometrial stroma may optimize the preparation of the endometrium for blastocyst implantation.


The role of epithelial-to-mesenchymal transition in the development of deep endometriosis

E. Kogan*, Т. Demura, A. Mursalova

*Sechenov University, Russia

Background & objectives: Pathogenesis of endometriosis (EM) is not completely studied. According recent researches epithelial-mesenchymal transition (EMT) is involved in progression of EM. Purpose. Identifying the role of epithelial-to-mesenchymal transformation in the development of endometriosis.

Methods: Materials and methods. The study was performed on the surgical materials and from 70 women with deep endometriosis: The control group consisted of 20 women with local peritoneal endometriosis. Expression levels of panCK, E-cadherin, Vimentin, CD34, SMA were identified by immunohistochemistry analysis of epithelium and stromal cells of heterotopic endometrium. Semi-quantitative assessment of the immunohistochemical results was done.

Results: EMT was found in foci of vascular invasion of deep EM in 47 patients. A hallmark of EMT were the loss of E-cadherin and panCK expression together with arising of vimentin and SMA in epithelial cells.

Conclusion: Progression of deep EM may be associated with EMT. Identifying and understanding the signalling mechanisms, promoting EMT may lead to novel therapeutic strategies, which will inhibit this cellular transformation


Study of the effect of gestabutonoil on endometrial proliferation in female rats

M. Kostyaeva*, I. Kastyro, Y. Gushchina

*Peoples' Friendship University of Russia (RUDN University), Russia

Background & objectives: Progesterone analogues are used to preserve pregnancy, treat endometrial hyperplastic processes and endometriosis in combined oral contraceptives. Objective: to evaluate the effect of gestabutonoil on endometrial proliferation in female rats when taken orally.

Methods: All groups included 15 female Wistar rats. Microcapsules with 0.002 g gestobutanoil were intragastrically administered to the experimental groups for 30 days: group 1 at a dosage of 2.5 mg/kg, group 2 - 25 mg/kg. 10 rats made up the control group. Endometrial sections were stained with H&E. Endometrial decidualization was evaluated.

Results: In the first group, the endometrium was in the phase of secretion, an increase in the number, size and tortuosity of the glands, vacuolization of the gland epithelium, loosening of the subglandular zone with pronounced decidualization of the cells was noted. In group 2, the proliferation of glandular epithelium was too intense until the appearance of glandular hyperplasia in 4 females of this group.

Conclusion: Gestabutonoil in small doses causes a therapeutic effect, but at high concentrations it contributes to the development of hyperplastic processes in the endometrium. The use of high doses of gestobanoyl can provoke further proliferation of glands and melignation against the background of endometrial hyperplasia


Expression of mismatch repair proteins, hormone receptors, and HER2 in metastatic/recurrent endometrial carcinoma

W. Lai*, C. Lai

*Taipei Veterans General Hospital, Taiwan

Background & objectives: Endometrial carcinoma is the most common uterine corpus cancer. Recurrence/metastasis occurs in 1/5 of cases. Molecular markers tailor adjuvant therapies. We aimed to characterize therapy-related marker expression including mismatch repair (MMR) proteins, hormone receptors, and HER2 in metastatic/recurrent endometrial carcinoma.

Methods: A retrospective search in our pathology archive for metastatic/recurrent endometrial carcinoma subsequent to a staging surgery from 2008 to 2019 was performed. Tumours of epithelial origin including carcinoma and carcinosarcoma were included. Slides were retrieved for morphology review. Cases with sufficient formalin-fixed paraffin embedded materials were constructed into tissue microarray for immunohistochemical stains.

Results: Thirty-six cases were identified. The median age at diagnosis was 60 years (34-76) and the median interval to 1st metastasis was 76.4 weeks (1.1 to 795.6). Loss of MMR protein expression was found in 22.2% of cases (seven paired loss of MLH1 and PMS2 and one paired loss of MSH2 and MSH6). Oestrogen receptor, progesterone receptor, and HER2 was positive in 65.6%, 43.8%, and 6.3% of cases, respectively. Thirteen had more than one metastatic event, and paired specimens were available in seven. Four had discrepant marker expression, with one developing MMR protein loss, one oestrogen and progesterone receptor loss, one HER2 gain and one HER2 loss at the last metastasis.

Conclusion: Expression of therapy-related molecular markers is common in metastatic/recurrent endometrial carcinoma. More than half may benefit from hormone therapy and some from immune checkpoint or HER2 inhibitors. Marker expression alters in consecutive metastasis, warranting re-assaying each time to guide treatment.

Funding: Institutional funding


Case series: p57 discordant hydatidiform moles, case report series

S.H. Lee*, L. Mcmahon, K. Gillespie, A. Alder, G. Moffat, N. Andrew, L. Cuthill, P. Chien, L. Christie

*Ninewells Hospital, NHS Tayside, United Kingdom

Background & objectives: Diagnosis and subclassification of hydatidiform moles (HM) is critical to assess the risk of gestational trophoblastic neoplasia (GTN). Rare cases of HM with aberrant p57 immunohistochemistry necessitate genotyping for diagnosis. Here, we present a case series of p57 discordant villi.

Methods: 675 cases of possible HM referred to the Scottish HM service between 2016 and 2019 were analysed using haematoxylin and eosin (H&E) light microscopy examination, ploidy analysis and p57 immunohistochemistry. DNA from 5 cases with discordant p57 staining pattern in cytotrophoblasts and stromal cells were extracted by laser microdissection and genotyped for further characterisation.

Results: Three cases showed aberrant morphology in keeping with HM and a discordant p57 pattern being negative in villous stromal cells while being positive in cytotrophoblasts. Flow cytometry showed a diploid DNA complement. Stromal cells and cytotrophoblasts were separately genotyped using laser microdissection and molecular genotyping which showed mosaicism. Two further cases demonstrated a homozygous and heterozygous complete mole.

Conclusion: This study emphasizes that a multidisciplinary approach using light microscopy, immunohistochemistry and molecular studies is invaluable in subclassifying HM. In particular, rare cases of mosaicism were identified using such approach which is necessary for understanding their risk of developing GTN.


Immunohistochemistry of P16 and P53 in vulvar cancer

M.D.L.M. Lewkowicz*, M.E. Paradeda, F. Gomez Cherey, M.F. Falcon, F. Garcia Kammermann, D. Sciaccaluga, V. Maldonado, L. Palaoro, L. Diaz, L. Cardinal

*Hospital de Clínicas "José de San Martin", Universidad de Buenos Aires, Argentina

Background & objectives: Squamous cell carcinoma of the vulva may develop in association or independently of HPV infection. We aim to confirm the association of keratinizing carcinomas with absence of HPV infection, and warty and basaloid carcinomas with presence of this virus.

Methods: We reviewed clinical manifestations, histological morphology and immunophenotype of 39 cases. We performed immunohistochemistry for p16 and p53, and PCR to evaluate HPV status. Statistical analysis was performed using Fisher´s exact test and t/ANOVA test. Histologically we determined 30 classic keratinizing squamous carcinomas, 5 warty and 4 basaloid carcinomas.

Results: In the statistical analysis, diffuse expression with p16 was significantly associated with younger age (p = 0.0025), presence of high-grade intraepithelial lesion (p < 0.0001), koilocytosis (p = 0.02), and morphological subtype (p = 0.02), and was inversely associated with the expression of p53 (p < 0.0001) and the presence of lichen sclerosus (p = 0.0051). It is curious that 4 keratinizing carcinomas of the cases studied presented coexpression of p16 and p53. Only 1 warty tumour was negative for p16 and positive for p53, and 9 keratinizing tumours were positive for p16 and negative for p53. Four of them were PCR positive for high risk HPV.

Conclusion: Although these findings show that the use of hematoxylin and eosin could correctly define tumours associated with HPV, we strongly suggest the performance of immunohistochemistry, especially in squamous keratinizing classic carcinomas in young patients with a history of HPV.


Cross-talking of two apoptotic molecules in ovarian cancer

L. Lozneanu*, S. Giusca, I.D. Caruntu

*U.M.F.” Grigore T. Popa” Iași, Romania

Background & objectives: Objective: Our study aimed to analyse the immunoexpression of p53 and c-FLIP in ovarian carcinoma in relationship with the clinicopathological features.

Methods: The study group consisted of 63 cases of ovarian carcinomas histopathologically diagnosed as serous (44 cases), and non-serous (19 cases); 24 cases were classified as stage I-II and 39 as stage III; 12 cases were assessed as G1, 24 cases as G2, 27 cases as G3 and G4. Tissue fragments were immunohistochemically processed by using anti-p53 and c-FLIP antibodies.

Results: c-FLIP+/p53+ expression was noted in 15 cases and negative in 14 cases. 17 cases exhibited c-FLIP-/p53+ and c-FLIP+/p53- respectively. Cases with c-FLIP+/p53- profile had the following distribution according to tumour stage: 8 in stage I and 9 in stage III, and tumour grade: G1 in 6 cases, G2 in 8 cases and G3 in 3 cases. Cases presenting c-FLIP-/p53+ profile were framed as follows: 2 in stage I, 2 in stage II and 13 in stage III; 1 case was graded as G1, 6 cases as G2, 9 cases as G3 and 1 case as G4. Statistical analysis revealed significant differences between c-FLIP+/p53- and, respectively, c-FLIP-/p53+ expression, and tumour grade.

Conclusion: The study of FLIP and p53 molecules provides integrated images of the apoptotic mechanism based on a cross-talk between the intrinsic and extrinsic pathways


Hsp70 and Hsp90 expression in normal and tumour endometrial tissues

M. Lyndin*, V. Sikora, O. Kravtsova, A. Romaniuk

*Sumy State University, Ukraine

Background & objectives: Endometrial cells acquire new peculiarities during malignant transformation and progression, which plays a significant role in the behaviour of neoplastic cells.

The objective of our study presented here was to characterize hsp70 and hsp90 expressions in normal and tumour endometrium.

Methods: The investigation was conducted on 50 samples with different types (endometrioid, serous and clear-cell) endometrial carcinoma. Ten cases of normal endometrium were used for comparison. The presence of hsp70 and hsp90 was detected by the immunohistochemistry utilizing the mouse mAb W27 and rabbit pAb (0.1μg/ml), respectively.

Results: Normal endometrium is characterized by the focal nuclear-cytoplasmic expression of hsp70. Endometrial carcinomas showed an increase in its expression in tumour cells with the appearance of hsp90. The de-differentiation of tumours was accompanied by increase chaperone response. Both were found in a high proportion of cancer cells. It should be noted, that most neoplasias had a heterogeneous expression of chaperons in tumour tissue.

Conclusion: The occurrence and progression of endometrial carcinomas are accompanied by the change hsp70 and hsp90 expression in tumour cells. They acquire additional resistance due to the synthesis of chaperones which increase their survival.


A histological analysis of the placenta for the diagnosis of chronic abruption-oligohydramnios sequence

S. Minamiguchi*, A. Ishida, S. Sumiyoshi, Y. Chigusa, E. Kondoh, H. Haga

*Department of Diagnostic Pathology, Kyoto University Hospital, Japan

Background & objectives: Chronic abruption-oligohydramnios sequence (CAOS) is characterized by diffuse chorioamniotic hemosiderosis (DCH). We compared the degree and distribution pattern of hemosiderin deposition (HD) on the chorionic plate (CP) and free membrane (FM).

Methods: We selected 20 CAOS patients, 21 non-CAOS patients as control group A (CA) matched by gestational weeks and 21 non-CAOS patients as control group B (CB) with bloody amniotic fluid. Iron staining of CP and FM was performed for every case. HD was evaluated by a histological score (HS) determined as positivity (0-3) multiplied by the staining area extent (0-4).

Results: HD was found in 100% (20/20) of CAOS patients and 14% (3/21) of CA and 9.5% (2/21) of CB patients. In both FM and CP, CAOS patients showed a significantly higher HS than control patients (CAOS, HS=4-12; CA, HS=0-1, p<0.0001; CB, HS=0-3, p<0.0001). In three CAOS patients, HD was seen only in the CP. The HS of the CP was significantly higher than that of the FM (p=0.0003).

Conclusion: CAOS was histologically characterized by DCH with an HS ≥4. The CP was better suited for the evaluation of DCH than the FM.


A review of cases submitted for molecular genotyping by the Scottish Hydatidiform Mole Service over a 3-year period

S. Mohan*, L. McMahon, N. Andrew, L. Cuthill, A. Alder, K. Gillespie, P. Chien, L. Christie

*Ninewells Hospital, United Kingdom

Background & objectives: In Scotland, suspected molar pregnancies are referred by the local pathologist to the Scottish Hydatidiform mole service (SHMS) in Dundee. This is a review of those cases that were diagnostically challenging and were submitted for molecular genotyping.

Methods: Submitted slides and blocks are booked in and ploidy analysis undertaken on all cases. If diploid, P57 immunohistochemistry (IHC) performed. Slides are reviewed by the pathologist and a final diagnosis of complete or partial mole or non-molar pregnancy reported. Molecular studies were undertaken if the diagnosis was equivocal. Genotyping was performed on DNA extracted from chorionic villi and maternal decidua.

Results: 689 cases received between 2017 and 2019. Histology, ploidy analysis and p57 IHC sufficient for diagnosis of 95% of referrals. The remaining 5% (34/689) were submitted for molecular genotyping to confirm the diagnosis. Where required, specific populations of chorionic villi or regions of discordant P57 expression were microdissected. Results showed, 56% (19/34) of cases were associated with a molar genotype including complete heterozygous or homozygous complement, or diandric triploidy. This included 5 cases of complex results including mosaicism and triandric tetraploidy. Biparental inheritance was reported in 15% (5/34) of cases. 18% (6/34) of cases were uninterpretable and the remaining 12% (4/34) of cases were associated with trisomy 21 or digynic triploidy.

Conclusion: Only a small number of cases submitted to the SHMS required further investigation by molecular genotyping with several cases showing complex results that could not be reliably established by other methods. This work reviews the range of diverse and complex cases seen by a national referral centre.


Correlation of core needle biopsy to diagnose breast phyllodes tumours – experience of a tertiary hospital

H. Moreira*, R. Almeida, J. Gama, J. Madeira, C. Faria, M.B. Pimentão, A. Lai, F. Ramalhosa, T. Simões Silva

*Centro Hospitalar e Universitário de Coimbra, Portugal

Background & objectives: A retrospective study of diagnosis was performed in patients with phyllodes tumours (PT) of the breast who were submitted to preoperative core needle biopsy(CNB) and had breast surgery at Centro Hospitalar e Universitário of Coimbra, comparing accuracy of both diagnosis.

Methods: Data from 13 patients with PT who underwent preoperative CNB and breast surgery were retrospectively analysed. We reviewed the CNB and surgical specimen diagnostic data. A diagnostic test was used to evaluate the sensitivity and specificity of CNB in diagnosing benign, borderline, and malignant phyllodes tumours.

Results: All patients were female with a mean age of 50.3 years (range 27-70 years). The accuracy of CNB for diagnosing PT was 38.5% (5/13). Of the remaining patients, were diagnosed with fibroadenoma or high-grade sarcoma. The sensitivity of CNB to diagnose benign, borderline, and malignant phyllodes tumours were 50% (2/4), 25% (1/4), and 20% (1/5), respectively, whereas the corresponding specificity were 40%, 100%, and 100%, respectively.

Conclusion: CNB provides a pathological basis for the preoperative diagnosis of PT of the breast, but it’s accuracy is poor and guidance is limited for surgical decisions. Considering CNB along with multiple histologic features may improve the ability to accurately diagnose PT. Literature recommends an integrated assessment using CNBs in combination with clinical data and imaging features as a reliable strategy to assist PT diagnosis.


Predictive assessment and follow-up strategy for patients with CIN 1 based on the use of colposcopic indices and immunohistochemical markers

S. Mozgovoi*, E. Abrosimova, S. Veremeeva, O. Lazareva

*Omsk State Medical University, Russia

Background & objectives: There are currently no clear criteria for patient management with CIN 1. It is important to find diagnostic tests and approaches that can predict the following development of CIN and justify the choice of patient management tactics.

Methods: 84 biopsy specimens from patients with a histologically verified diagnosis of CNS 1 were examined: HPV 16/18 status, colposcopic features, routine histopathology with koilocytosis evaluation, expression of Ki-67, P53 and P16INK4a by immunohistochemistry. The control study was carried out 12 months later. The patients were divided into 3 groups according to the results: regression, CIN permanence, progression to CIN 2.

Results: Differences in the colposcopic characteristics using Reid and Coppleson indexes have been identified. In the group with CIN persistence a well-established increase in the number koilocytosis changes were revealed, as well as differences in the level of P16ink4a expression (p<0,05). .After calculation of the quantitative influence of factors and using the Wald test (non-uniform serial analysis) diagnostic coefficients for these factors were calculated with the following validity parameters: sensitivity - 92,7% and specificity - 86,1%. Immunohistochemical detection of Ki-67 and P53 correlate with the dynamics of morphological changes but was not relevant to the prediction for the evolution of CIN 1.

Conclusion: CIN 1 is a heterogeneous group with different colposcopic characteristics and parameters of cell renewal which determine the prognosis. The following characteristics are significant for the forecast: colposcopic indexes Reid and Coppleson, detection of koilocytosis after haematoxylin and eosin stain of fixed tissue, evaluation of expression level of P16ink4a. The proposed method of CIN 1 prognosis assessment with estimation significant colposcopic, morphological and immunohistochemical indicators is effective.


Evaluation of PTEN protein expression in normal endometrium and endometrioid carcinoma surgical specimens

Y. Nishijima*, S. Kobayashi, M. Saio

*Laboratory of Histopathology & Cytopathology, Department of Laboratory Sciences, Gunma University Graduate School of Health Sciences, Japan

Background & objectives: In the present study, we examined PTEN protein expression patterns in normal endometrium (NE) and endometrioid carcinoma (EC).

Methods: Thirty cases each of FFPE NE and EC samples were examined by immunohistochemistry for PTEN expression. Specimens were evaluated manually by scoring the staining intensity of the strongest stained part for glands and stroma separately. The score was 0 to 4; negative, very weak, weak, medium, and strong. For statistical analysis, Wilcoxon rank-sum test was used.

Results: Distribution of gland score in NE was 76.6%, 20%, 3.3%, 0%, 0%, (average: 0.266). While, the one in EC was 50%, 16.7%, 20%, 10%, 3.3% (average: 1.000). The average gland score of EC were significantly higher than that of NE (P = 0.0104). Distribution of stroma score in NE was 10%, 0%, 60%, 30%, 0% (average: 2.1). While, the one in EC was 20%, 0%, 60%, 20%, 0% (average: 1.8). The stroma score was not significantly different in both groups. In both NE and EC, the stroma score was significantly higher than the gland score (NE: P<0.0001, EC: P = 0.0065).

Conclusion: PTEN expression in NE is supposed to be weak because PTEN is not mutated. Indeed, in our analysis, PTEN expression was mostly negative or very weak, which results were identical to the previous report. While, in EC, strong expression and no expression was sometimes observed in the same specimens. Our results indicated that heterogeneous PTEN gene mutation would happen during progression to cancer and resulted in a patchy staining pattern.


Histological pattern of endometrial biopsies in women with abnormal uterine bleeding in a hospital in North Central Nigeria

O. Olaofe*, I.M. Asuzu

*Obafemi Awolowo University Teaching, Nigeria

Background & objectives: Varied morphological patterns have been reported in Endometrial biopsies in various parts of our country. We aim to document fnding from a one-year review of cases seen in a community group private practice.

Methods: The study is a retrospective cross-sectional study carried out in the Department of Pathology of Premier Hospital, Abuja, on specimens received over a one-year period. Four hundred and eighty-six samples of endometrial biopsies and curettings from women presenting with abnormal uterine bleeding sent to the histopathology laboratory were analysed.

Results: Four hundred and eighty-six samples of endometrial biopsies and curettings from women presenting with abnormal uterine bleeding sent to the histopathology laboratory were analysed. The most common biopsies were those of product of conception which accounted for 304 cases (62.6%). Most of the cases of endometrial hyperplasia were typical. Endometritis and chorioamnionitis were the infammatory conditions seen. Twenty-three women had molar pregnancies. The most common cause of abnormal uterine bleeding in this population is retained products of conception.

Conclusion: The most common cause of abnormal uterine bleeding in this population is retained products of conception. There may be need to retrain some of the staffs involved in the management of pregnancy related complications. There is need to further evaluate pregnancy related complications to ascertain the causes and circumstances responsible for them so as to appropriately direct interventional protocols.


High risk HPV detection in formalin fixed paraffin-embedded (FFPE) cervical tissue with Aptima HPV assay

G. Kir, Z.C. Olgun*, H. Gunel

*Istanbul Medeniyet University Pathology Department, Turkey

Background & objectives: High risk-HPV(HR-HPV) is implicated in the development of intraepithelial neoplasia and carcinoma of uterine cervix. Several liquid based HPV detection techniques are available, and approved by FDA, however there are no FDA approval and enough experience for HPV detection in FFPE tissues.

Methods: One hundred and ninety-six cervical tissue specimens, each from a patient, were retrieved for HR-HPV detection. One-hundred and fifteen of these samples were diagnosed as HSIL and 46 as cervical squamous cell carcinoma (SCC). 36 of these FFPE cervical samples were normal. Histopathologic diagnosis was accepted as the golden standard.

Results: 189 of the assays provided informative results. HR-HPV was detected in 99.35% of the HSIL and cervical SCCs. Only one tissue sample was resulted as false negative and there were no false positive results. Sensitvity and specifity of the test were 99.35% and 100% (95%CI: 96.41%-99.98% and 90.26%-100% respectively). Positve predictive value and negative predictive value was 100% and 97.30% (95%CI: 83.62%-99.61%). Accuracy for this technique was 99.47% (95%CI: 97.09%-99.99%)

Conclusion: This HPV detection technique with Aptima HR-HPV Assay provides a reliable method for HR-HPV testing in FFPE tissue specimens however there is still need for larger studies.

Funding: HPV detection test cost covered by Hologic


New approaches in study of pathomorphological aspects of diabetes on background of pregnancy

T. Pavlova*, A. Kaplin, L. Zemlyanskaya

*Belgorod State University, Russia

Background & objectives: About 422 mln. people are suffered from diabetes. The birth of children in women with such pathology is very important question. In this connection, the aim of our research is the exploration of pathomorphological features of diabetes in pregnant.

Methods: The pregnant with diabetes of 1 type (25), diabetes of 2 type (22), gestational diabetes (20) were explored. The way of modelling of pancreatogenic diabetes with relevant insulin insufficiency at partial resection of pancreas in rats was designed. The samples of placenta and uterus of rats and women of line "Wistar" (30) were studied in light and scanning electron microscope.

Results: Plethora, diapedesis, thrombosis, fibrin were revealed in vessels of miometrium. Their extension at gestational diabetes was 1,9±0,6μm, at diabetes of 1 type was 1,4±0,2μm, at diabetes of 2 type was 1,7±0,6μm (3,0±0,4). The surface of endotheliocytes was altered. Villi of intermediate type were prevailed in placenta. The vast majority of blood vessels are full-blooded, with area 33,6±4,3, 40,2±5,0, 49,3±6,6μm (28,7±2,4). The sites of sclerosis were revealed in stroma.

Conclusion: Necessary correction for improving of pregnancy and childbirth indexes’ may be conducted by clinicians due to received data


Analysis of intraoperative consultation in ovarian tumours: an 11-year retrospective study of 321 cases

F. Pereira*, A. Pereira, C. A. Padrão, J. Tinoco, A. T. Alves

*Hospital Prof. Doutor Fernando Fonseca, EPE, Portugal

Background & objectives: The intraoperative consultation evaluation in ovarian tumours is crucial for surgical management. Our purpose was to assess the concordance, discordance, and deferred rates of intraoperative diagnosis, along with the agreement in the behaviour of the tumour.

Methods: A retrospective, 11-year review of consecutive intraoperative consultations done between January 2009 and December 2019 at the Department of Pathology, Prof. Doutor Fernando Fonseca Hospital, was compared with definitive histology reports. The sensitivity, specificity and predictive values of each category (benign, borderline/uncertain behaviour and malignant) were determined.

Results: On a total of 321 intraoperative consultations, 261 reports (81.3%) were concordant with the definitive histology, 34 reports (10.6%) had minor discordances, 3 reports (0.9%) had major discordances and 23 cases (7.2%) had a deferred report to paraffin sections. The diagnosis that resulted in most inconsistencies were borderline tumours that turned out to be carcinomas and benign mesenchymal lesions, highlighting the importance of some limitations, including the sampling errors. Agreement between the behaviour of the tumour in the frozen section and definitive histology was observed in 98.3%, yielding a sensitivity and a positive predictive value for malignant tumours of 94.9% and 98.2%, respectively.

Conclusion: This retrospective study shows that frozen section evaluation of ovarian tumours represents a highly sensitive and specific technique that can be used to guide the surgeon to perform the appropriate surgical procedure. The authors present the main diagnostic challenges.


Histopathological spectrum of ovarian tumours- a prospective study at CMS-TH - Bharatpur - Chitwan - Nepal

H. Regmi*

*Manmohan Memorial Medical College and Teaching Hospital, Kathmandu, Nepal

Background & objectives: Ovary is the third most common site of neoplastic lesions in female genital tract. Ovarian tumours occur in any age group. Ovarian neoplasms have become increasingly important because they have gradually increased mortality rate due to female genital cancers.

Methods: This study was carried out on 75 cases of surgically resected ovarian tumour specimens fulfilling inclusion criteria at Department of Pathology in CMS-TH, from January 2016 to June 2017. Clinical data was recorded in predesigned proforma. The specimens were grossed, processed, embedded and stained using standard procedures and were analysed using light microscopy. Statistical analysis was done using SPSS 20.0

Results: Age range of the patients was from 10 to 70 years with maximum cases 32.0 in the age group of 21-30 years. Histopathological diagnosis was mature cystic teratoma 38.8%, serous cystadenoma 29.4%, mucinous cystadenoma 8.0%, borderline mucinous tumour 5.3%, serous cystadenocarcinoma 4.0%, dysgerminoma 4.0%, steroid cell tumour 2.7%, and borderline serous tumour, mucinous cystadenocarcinoma, mature cystic teratoma transforming into SCC, immature teratoma, Sertoli Leydig cell tumour and fibroma 1.3%.

Conclusion: Benign tumours were more common than malignant tumours for all age group. Most of the tumours were of surface epithelial cell origin. Mature cystic teratoma was the most common ovarian tumour as well as the most common benign tumour. Serous cystadenocarcinoma and dysgerminoma were most common malignant tumours. Malignant surface epithelial tumours usually occurred in older age whereas malignant germ cell tumours occurred in younger age. There was significant statistical Clinico-pathological correlation.


Clinicopathological features, immunohistochemical profile and clinical outcomes of 27 primary peritoneal carcinomas: a single institutional study

B. Rekhi*, S. Karmarkar, K. Deodhar, S. Menon, A. Maheshwari, S. TS, J. Ghosh, S. Gulia, S. Gupta

*Tata Memorial Centre, India

Background & objectives: Primary peritoneal carcinomas (PPCs) are rare tumours, with overlapping clinic-pathological features with epithelial ovarian carcinomas(EOCs) and mesotheliomas. We studied clinicopathological features, including immunohistochemical profile and clinical outcomes of PPCs diagnosed at our Institution.

Methods: This was a retrospective study, wherein 27 PPCs, diagnosed between January 2008 and May 2019 were included, after review, as per established criteria. Various clinicopathological features were analysed with Median and average age of patients being 60 and 55 years, respectively.

Results: Microscopically, 93% tumours (25/27) were of high-grade serous type. Sensitivity and specificity of PAX8 was 100% (13/13) and 87.5%, and for oestrogen receptor (ER) was and 100% (7/7) and 100%. Most patients (14/23, 60.9%) were treated with neoadjuvant chemotherapy (NACT), interval debulking surgery (IDS) and adjuvant chemotherapy. Median disease-free survival (DFS) was 32 months. Estimated 3 year-DFS and overall survival was 47.3% and 69.8%. There were lesser recurrences in cases of NACT and IDS (4/14) vs. surgery and adjuvant CT (4/8) (p=0.59).

Conclusion: This constitutes the largest series on clinicopathologic profile of PPCs from our subcontinent. PAX8, ER and calretinin constitute as useful diagnostic immunostains. It is crucial to differentiate these tumours from their mimic, mesotheliomas, in view of associated treatment implications.


CEACAM1 expression in the normal uterus of rats

K. Sikora, M. Lyndin, O. Kravtsova, V. Sikora, A. Romaniuk*, B.B. Singer

*Sumy State University, Ukraine

Background & objectives: The carcinoembryonic antigen-related adhesion molecules (CEACAM) play a significant role in numerous physiological processes, such as cell-cell and cell-ECM adhesion, angiogenesis, proliferation, etc.

The objective is characterization of the CEACAM1 expression pattern in normal tissues of rat uterus.

Methods: Uterus sections were taken from intact female wild-type Wistar rats. The immunohistochemical investigation was performed utilizing mAb Be9.2 (α-rat-CEACAM1, N domain binding, mouse IgG1 kappa), mAb 11-1H (α-rat-CEACAM1, not-N domain binding, mouse IgG1 kappa) and isotype matched control antibodies kindly provided by B.B.Singer. Goat anti rat-HRP coupled antibody and a DAB substrate were used for visualization of the CEACAM1 expression.

Results: CEACAM1 expression was found on the apical cellular polarity of the luminal and glandular columnar cells along the surface of normal endometrium and endocervix. This localization was coinciding with the location of the cellular microvilli. Interestingly, the use of mAb 11-1H allowed to detect week CEACAM1 expression only in the luminal and single glandular epithelium, although mAb Be9.2 was expressed by all endometrium. As expected, single leukocytes diffusely scattered in the underlying stroma were CEACAM1-positive as shown by Be9.2 and 11-1H binding and thus served as internal staining control for both antibodies.

Conclusion: The luminal and glandular epithelium of normal endometrium and endocervix express significant amounts of CEACAM1 on the apical cell surface. However, mAb Be9.2 showed a higher sensitivity for CEACAM1 in uterus epithelial cells during immunohistochemical investigation than mAb 11-1H.


Differential proteomic analysis between low grade, early stage endometrioid endometrial carcinoma and benign endometrium

I. Ruz-Caracuel*, A. López-Janeiro, L. Yébenes, A. Berjón, J.L. Ramón-Patino, A. Hernández, A. Redondo, M. Mendiola, D. Hardisson

*Ramon y Cajal University Hospital, Department of Pathology, Spain

Background & objectives: Low grade, early stage endometrioid endometrial carcinomas are the major proportion of endometrial carcinomas. As a previous step to identify diagnostic biomarkers, we aimed to elucidate the differences at the proteomic level between tumour tissues and matched uterine non-tumour tissues.

Methods: Tumour and non-tumour tissue including endometrium and myometrium from 16 patients was analysed. Proteins were extracted from formalin-fixed paraffin-embedded tissue. Quantitative proteomic analysis was done by isobaric labelling with tandem mass tags (TMT). Tryptic peptides were performed using a Q Exactive mass spectrometer coupled to a nEasy-nLC 1000 nano system (ThermoScientific). MS data were analysed with Maxquant using standardised workflows.

Results: A total amount of 3,113 proteins were quantified and 730 were differentially expressed between both conditions. Relevant pathways affected included integrin signalling and inflammation mediated by chemokine and cytokine signalling. The main biological processes altered were cellular and metabolic processes.

Conclusion: Integrin signalling and inflammation mediated by chemokine and cytokine signalling pathways are promising pathways to identify diagnostic biomarkers in low grade, early stage endometrioid endometrial carcinomas.

Funded by the Instituto de Salud Carlos III (ISCIII) (PI17/01723), co-financed by the European Development Regional Fund ‘A way to achieve Europe’ (FEDER).


A morphological and immunohistochemical comparison of primary low grade, early stage endometrioid endometrial carcinomas and their relapses

I. Ruz-Caracuel*, A. López-Janeiro, A. Berjón, L. Yébenes, J.L. Ramón-Patino, A. Hernández, M. Mendiola, A. Redondo, A. Pelaez-Garcia, D. Hardisson

*Ramon y Cajal University Hospital, Department of Pathology, Spain

Background & objectives: Low grade, early stage endometrioid endometrial carcinomas fall into copy number low and microsatellite unstable molecular groups. Our main objective was to analyse concordance between morphological features and immunohistochemical markers used for molecular classifying in primary tumours and their relapses.

Methods: A total of 19 relapse biopsies from 16 patients were identified from a single hospital cohort comprising 258 low grade, early stage endometrioid endometrial carcinomas. Morphological features such as grade, squamous and mucinous differentiation were evaluated in primary and relapses. Tissue microarray were constructed and immunohistochemical markers for mismatch repair proteins (MLH1, PMS2, MSH2 & MSH6) and p53 were performed.

Results: There were 16 biopsies from locoregional relapses and 3 from lung metastases. Concordance was poor for morphological features such as grade (kappa=0.023), squamous (kappa=-0.203) and mucinous differentiation (kappa=0.215). Tumour grade at relapse was the same in 8 cases, upgraded in 8 and downgraded in 3. Microsatellite instability interpretation derived from mismatch repair proteins expression and p53 expression had a perfect concordance (kappa=1).

Conclusion: There is a perfect surrogate molecular group concordance between relapses and primaries low grade, early stage endometrioid endometrial carcinomas. Morphological features of primaries are poor predictor of their relapses.

Funded by the Instituto de Salud Carlos III (ISCIII) (PI17/01723), co-financed by the European Development Regional Fund ‘A way to achieve Europe’ (FEDER).


HPV-independent intraepithelial lesions of the vulva mimicking HPV-positive squamous intraepithelial lesions

A. Saco*, N. Rakislova, L. Alemany, O. Clavero, B. Lloveras, J. Ordi

*Department of Pathology, ISGlobal Hospital Clínic Barcelona, University of Barcelona, Spain

Background & objectives: There are two etiopathogenic types of vulvar squamous cell carcinoma (VSCC), HPV-associated and HPV-independent, each one associated with a specific intraepithelial lesion. However, unusual patters have been described. We aimed to explore these unusual histological patterns of intraepithelial precursors.

Methods: We analysed 779 DNA HPV negative, p16-negative VSCCs, with at least 1 cm of skin adjacent to the invasive tumour available. We evaluated the neighboring skin and performed p53 immunohistochemistry.

Results: 254 tumours (33%) had adjacent intraepithelial lesions. Of them, 22 (9%) had vulvar acanthosis with abnormal differentiation /differentiated exophytic intraepithelial lesion, and 232 had differentiated vulvar intraepithelial neoplasia (dVIN), which was of conventional type in 184 cases (72%), and mimicked squamous intraepithelial lesions (SIL) in 48 cases (19%)). Four of these SIL-like lesions resembled LSIL and 44 simulated HSIL (24 basaloid-like, 13 warty-like and 7 mixed basaloid/warty features). Thirty-five of 48 (73%) of these HPV-negative intraepithelial lesions mimicking HSIL showed p53 abnormal staining.

Conclusion: A small, but significant percentage of intraepithelial precursors associated with HPV-independent VSCC mimic SIL, mostly HSIL. Approximately one-third of these lesions will arise in absence of TP53 mutations.


An audit of histopathology reports of carcinoma cervix: assessment of different histology parameters from specimens operated at a cancer centre in India, between 2013 and 2018

D. Singh*, K. Deodhar, S. Menon, B. Rekhi, A. Budukh

*Tata Cancer Hospital, India

Background & objectives: To review histopathology reports of patients operated for carcinoma cervix, at our institute between 2013 to 2018, and to see our compliance to minimum data sets.

Methods: After obtaining approval by institutional review board (IRB), our hospital records showed a total of 243 reports of carcinoma cervix, operated between 2013 to 2018. Various parameters from the reports were noted down, and their frequencies were calculated with the help of SPSS software (version 21).

Results: The mean age was 50.5 years (range 15-82). The mean tumour size being 3.03 cm. The depth of cervical stromal involvement was mentioned in 98.8% cases; It was more than half In 67.5% cases. Distance of tumour from cervical stromal adventital margin was mentioned in 94.2% cases.The vaginal cut margins were mentioned in all but one case ( 99.6%). Right and left parametria were free in 96.7% and 96.3% cases respectively. The commonest tumour type was squamous (74.5%), followed by adenocarcinoma (17.6%) followed by adenosquamous carcinoma (5.35%). Lymphovascular emboli present in 40.3% cases.The mean lymph node yield was 18.54. The stage was not mentioned in 127 cases (52.3%).

Conclusion: Compliance to minimum data sets for carcinoma cervix reports in our hands was generally good. Lymph node yield improved comparative to previous audit. Stage need to be mentioned.


Tumour size and mTOR pathway activation assesed by immunohistochemistry as predictor of Silva pattern of invasion in biopsies of HPV-associated endocervical adenocarcinomas (HPVA)

S. Stolnicu*, S. Segura, M. Boros, K. Park, P. Ramirez, G. Salvo, R.A. Soslow

*University of Medicine and Pharmacy Targu-Mures, Romania

Background & objectives: We aimed to use small cervical biopsies to determine whether markers of mTOR pathway activation and tumour size associate with aggressive Silva patterns of invasion B and C.

Methods: 34 HPVA biopsies were evaluated for size and expresion of pS6, pERK and HIF1a. Immunohistochemical stains were scored semiquantitatively, ranging from 0-4+ with scores 2-4+ considered positive, and Silva pattern was determined in follow-up excisional specimens.

Results: 8 cases were pattern A, 4 pattern B and 22 pattern C. Statistically significant associations were found comparing pS6 and pERK immunohistochemistry with Silva pattern (p=0.034 and 0.05, respectively). pERK was the most powerful for distinguishing between patterns A and B/C (p=0.026). Large tumour size (≥2 cm) correlated with Silva pattern (p=0.027) with a moderate association when comparing pattern A with B/C (r: 0.404, p=0.027, R square: 0.163).

Conclusion: Both tumour size and pERK immunohistochemistry can be used to predict Silva pattern using only small biopsies of HPV-associated endocervical adenocarcinomas.


Comparison between P53 immunohistochemical staining pattern and molecular characteristics of tubo-ovarian high-grade serous carcinoma by next-generation sequencing examination

K. Suh*, K.H. Kim

*Chungnam National University Hospital, Republic of Korea

Background & objectives: Strong p53 immunohistochemical staining is one of the hallmarks of tubo-ovarian high-grade serous carcinoma (HGSC). The aim of this study was to compare between the p53 staining patterns and the molecular characteristics of HGSC cases identified by next-generation sequencing (NGS).

Methods: A commercial NGS cancer panel comprising 143 genes, including TP53, was used to analyse the genetic profiles of tubo-ovarian HGSC cases. Eleven HGSC cases were sequenced using formalin-fixed paraffin-embedded (FFPE) sections. Immunohistochemical stain for p53 was performed for each case.

Results: Among 11 HGSC cases, nine novel TP53 somatic mutations from nine patients (81.8%) were identified; missense mutation in 3 cases, frameshift mutation in 5 cases, and nonsense mutation in one case. Among 9 TP53 mutant cases, p53 IHC staining revealed strong nuclear overexpression in 3 cases and complete absence in 6 cases. The remaining 2 cases showed no TP53 mutation; p53 overexpression in one and complete absence in the other.

Conclusion: Genomic sequencing in the tubo-ovarian HGSC cases revealed mutations of TP53 in 81.8%. p53 immunohistochemical staining revealed overexpression in 4 cases (36.4%) and complete absence (abnormal/aberrant/mutation-type) in 7 cases (63.6%). The p53 IHC staining patterns should be considered in diagnosis of HGSC cases.


Reproducibility of interpretation of P53 immunostaining in vulvar squamous cell carcinomas using a pattern-based approach

E. Thompson*, L. Hoang, J. Huvila, K. Talia, N. Tchrakian, A. Palicelli, L. Horn, C.B. Gilks, N. Singh

*Vancouver General Hospital, Department of Pathology and Laboratory Medicine, Canada

Background & objectives: p53 immunostaining is a surrogate for TP53 mutation assessment in vulvar squamous cell carcinoma (VSCC) and allows biomarker-based subclassification into prognostically significant subtypes, with HPV-independent/p53abn VSCC having the worst prognosis. We assessed the reproducibility of p53 interpretation in VSCC.

Methods: p53 immunostaining (N=69) was performed and evaluated independently at two institutions, by five pathologists, and classified as abnormal (basal overexpression, basal and parabasal overexpression, absent expression, or cytoplasmic staining), wild-type(scattered or mid-epithelial staining with basal sparing) or indeterminate.

Results: In 51/69 cases (73.9%) there was complete agreement between all 5 raters. In 12 cases (17.4%) one or more raters considered the p53 staining indeterminate, while the remaining observers agreed that it was either abnormal or wild-type. In 5 cases (7.2%) there was disagreement regarding whether the p53 staining was aberrant or wild-type and in the final case the range of opinions included abnormal, wild-type, and indeterminate. The overall Fleiss’ kappa for the five observers was 0.78. If all indeterminate results were to be resolved by performing sequencing of TP53, there were only 6 cases with a disagreement (Fleiss’ kappa = 0.91).

Conclusion: There was excellent inter-observer agreement in the interpretation of p53 immunostaining in VSCC, with staining performed independently at two different laboratories. The main issue identified was variability in the number of cases considered to show indeterminate staining i.e. not possible to conclude whether it was abnormal or wild-type. Although such cases can be resolved through TP53 sequencing, refined p53 interpretation criteria may help reduce the frequency of indeterminate results.


P53 immunohistochemical patterns in HPV-related neoplasms of the female lower genital tract can be mistaken for TP53 null or missense mutational patterns

E. Thompson*, J. Chen, J. Huvila, J. Pors, H. Ren, J. Ho, C. Chow, M. Ta, L. Proctor, J.N. McAlpine, D. Huntsman, C.B. Gilks

*Vancouver General Hospital, Department of Pathology and Laboratory Medicine, Canada

Background & objectives: We recently encountered cases where p53 IHC in HPV+ tumours were confused with mutational-pattern staining. Herein, p53 IHC were assessed in a cohort of HPV+ invasive and in-situ squamous cell carcinomas (SCC) and adenocarcinomas (EDAC) of the cervix and vulva.

Methods: Only cases with block-like p16 staining were included. p53 was scored as “wild-type”, “overexpression” (strong diffuse staining), “null” (absent staining), “markedly reduced” (reduced staining, >70% of cells, mimicking null staining) and "midepithelial (basal sparing)” (absent staining in basal cells juxtaposed with strong staining in parabasal cells). TP53sequencing and HPV in-situ hybridisation (ISH) were performed in select cases.

Results: “Markedly reduced” staining was present in 14/25 SCCs, 7/14 HSIL and 18/20 EDACs. This mimicked null-pattern staining. There was “mid-epithelial (basal sparing)” in 10/25 SCC and 7/14 HSIL. This mimicked overexpression-pattern staining. Wild-type was seen in 1/25 SCC and 2/20 EDAC. No cases showed overexpression. One EDAC had a TP53 missense mutation and markedly reduced staining. HPV ISH results showed an inverse association with p53 immunoexpression, cells positive for HPV mRNA were negative for p53.

Conclusion: p53 IHC patterns in HPV+ neoplasms can mimic the null mutational patterns (“null-like”) and overexpression mutational patterns (“overexpression like”). Knowledge of these patterns can help pathologists avoid misinterpreting p53 status in the setting of HPVA cancers.


Role of epigenomics in ovarian serous carcinomas

M.M. Ustaioglu*, G. Erdil, M.E. Ercin

*Karadeniz Technical University, Faculty of Medicine, Department of Pathology, Turkey

Background & objectives: We aimed to determine the difference in terms of epigenetic modifications between the primary tumour cells, ascites tumour cells and metastasis tumour cells of patients with high-grade and low-grade serous ovarian cancer (HGSOC and LGSOC) by bioinformatic analysis.

Methods: GSE73168 gene’s microarray data were downloaded from the "Gene Expression OmniBus" data base to investigate the association of serous ovarian carcinomas with epigenetic modifications. Differentially expressed genes were generated by re-analyzing them RNA transcripts from tissues obtained from 5 HGSOC patients and 3 LGSOC patients. "Biobase", "Limma" and "Geoquery" libraries were obtained with bioinformatics analysis and R program.

Results: Cytoscape with Search Tool for the Retrieval of Interacting Genes and Molecular Complex Detection plug-in was utilized to visualize protein-protein interaction of these differentially expressed genes. In comparing the expression profiles of transcripts, "log2 fold change >1" and P ≤ 0.05 were considered statistically significant. Statistically significant differences were found in genes related to DNA methylation and histone modifications ( acetylation, methylation, phosphorylation, ubiquitination, sumoylation) by using the DAVID (Database for Annotation, Visualization and Integrated Discovery) functional annotation system.

Conclusion: A greater understanding of the role of epigenetics in high grade and low grade serous ovarian carcinomas will provide for improved therapeutic interventions. Moreover, this study can shift the focusing on the K-RAS, B-RAF and p53 mutations to genes involved in epigenetic modifications.


Concerns of the histological phenotype of the placenta during physiological pregnancy and antenatal foetal death of unknown aetiology

S. Yessen*

*Karaganda Medical University, Kazakhstan

Background & objectives: Unfavourable conditions for foetal development are associated with the development of diseases in the postnatal period. Cause of perinatal death is latent or clinically expressed placental dysfunction, which developed under the exposure of various damaging factors at any stage of pregnancy.

Methods: A prospective histological study of 106 placentas (37-42 weeks) during physiological pregnancy (n-82) and prenatal death of the foetus (n-24) was carried out. All placentas were examined in accordance with Vogel principles (1996). The relative number of immature forms of villi in 100 peripheral grooves of the representative placental area was determined.

Results: In 28 placentas, the delayed development of chorionic villi was determined. Of these, 22 placentas (79%) showed expressed (> 50% immature villi) and 6 placentas (21%) moderate delay (30-50% of immature villi) of the development of chorionic villi. In 92% of the placenta, with antenatal asphyxia of the foetus and in 15% of the placenta during physiological pregnancy was found an immature histological phenotype (54.2 ± 12% and 34.3 ± 5.8% of immature villi, respectively). Therefore, postnatal evaluation of placental phenotype can help in the timely identification of adverse foetal conditions and early stratification of the heterogeneous population of new-borns, with identification of individual risk of disease in post-natal life.

Conclusion: Physiological pregnancy may be accompanied by a delay in the maturation of chorionic villi and thereby latent chronic placental insufficiency. In the case of antenatal foetal death,the placenta becomes the main reliable source of information for making a diagnosis.


Dedifferentiated endometrioid adenocarcinoma of uterus

Ö.N. Yildiz*, M. Yilmaz, C.S. Topal

*Health Sciences University, Turkey

Background & objectives: Dedifferentiated endometrioid adenocarcinoma is recently described and rare uterine neoplasm which contains both low grade(FIGO grade 1 or 2) endometrioid adenocarcinoma and undifferentiated carcinoma. This tumour is a new entity and may pose diagnostic challenges.

Methods: We discuss a case of 28-year old women who had severe stomach ache with palpable mass in abdomen. By the surgical examination we learned that the tumour was mainly located in uterus and invaded colon, bladder, liver, paraaortic lymph nodes. Hysterectomy specimen showed polypoid tumour filling endometrial cavity, invading full thickness and exceeding serosa.

Results: Tumour was described by the coexistence of undifferentiated carcinoma(%85) and low grade endometrioid adenocarcinoma(%15).Undifferentiated component was characterized by the proliferation of noncohesive, medium size, monotonous epithelial cells growing in a patternless solid fashion. The transtion between the two tumour components was abrupt with a sharp border. Undifferentiated cells were negative for epithelial markers, oestrogen, and progesterone receptors although others were positive. There were no loss of mismatch repair gene proteins. Metastases contained only undifferentiated component.

Conclusion: Dedifferentiated endometrioid adenocarcinoma has poor prognosis and aggressive behaviour. It can be misdiagnosed as grade 3 endometrioid adenocarcinoma. Accurate diagnosis requires more awareness of this entity. Morphological, immunohistochemical and molecular features should be considered for better clinical outcome and patient survival.

PS-03 Head & Neck Pathology


Ladinin-1, overexpressed in oral squamous cell carcinoma adjacent to non-cancerous epithelium, is involved in cell motility by mediating actin and focal adhesion dynamics

T. Abe*, M. Yamazaki, S. Maruyama, J. Tanuma, Y. Ajioka

*Division of Molecular and Diagnostic Pathology, Niigata University Graduate School of Medical and Dental Sciences, Division of Oral Pathology, Department of Tissue Regeneration and Reconstruction, Japan

Background & objectives: Oral squamous cell carcinomas (SCCs) often form the interface against non-cancerous epithelium. We identified several proteins, including ladinin-1 (LAD1), overexpressed in cancer tissue at the interface by tissue proteomics. This study aimed to examine LAD1 functions in oral SCCs in-vitro.

Methods: Oral SCC cell lines, HSC-2, 3, and 4, were used for in-vitro functional and immunofluorescence analyses with siRNA knockdown methods.

Results: LAD1-knockdown cells showed decreasing of cell proliferation in MTS assay and increasing of numbers of migrated cells in the transwell migration assay. Morphologically, LAD1 was localized in the peripheral area of the cytoplasm. High-resolutional morphological analysis using structured illumination microscopy revealed that LAD1 was co-localized with actin filaments forming “actin arc”. LAD1-kockdown cells showed decreasing of elongated filopodial filaments and ruffling of cell borders compared with controls. In addition, LAD1-kcockdown cells overexpressed WNT5a and showed prominent accumulations of phosphorylated focal adhesion kinases in the cytoplasm.

Conclusion: LAD1 is potentially involved in cell motility by modulation of actin dynamics and focal adhesion formation in oral SCC cells and the non-canonical WNT pathway may play a pivotal role in LAD1 functional association.


Effectiveness of rapid onsite evaluation of thyroid fine needle aspiration cytology

F. Ali*, U. Bashir, F. Rehman, S. Shahid, S. Ahmad

*King Edward Medical University, Lahore, Pakistan

Background & objectives: FNA is the first line diagnostic modality in the evaluation of the thyroid lesions. However, the specificity is variable which can be improved by the implementation of ROSE, thus, to evaluate the improvement in adequacy of thyroid cytology with ROSE.

Methods: A comparative cross-sectional study was conducted on 166 patients with thyroid masses (83 in each of the two groups). After the standard FNA procedure, ROSE was performed by staining the slides with rapid stains. For inadequate smears, repeat FNA was done at the same time thus improving the adequacy and reducing the number of patients’ visits to the hospital for repeated attempts.

Results: RESULTS: The study showed that there was no dramatic increase in the diagnostic rate of thyroid FNA with ROSE (86.7%) as compared to that without ROSE (72.3%). However, ROSE assisted to reduce the rate of false negative cytology and the degree of patients’ inconvenience due to delay in the diagnosis ,owing to repeated visits.

Conclusion: Rapid Onsite Evaluation of thyroid cytology smears enhances the effectiveness of Fine Needle Aspiration by reducing the rate of false negative cytology and alleviating the degree of inconvenience to the patients.


Assessment of tumour-infiltrating lymphocytes (TILs) in head and neck squamous cell carcinoma: inter-observer agreement and visual versus digital image analysis comparison

J.A. Baena-Del Valle*, M.A. Palau-Lázaro, C. Buriticá-Cifuentes, A.M. Baldión-Elorza, A. Escallón-Cubillos, J.A. Hakim-Tawil, S.P. Perdomo-Velásquez, P.A. Rodriguez-Urrego

*Fundacion Santa Fe De Bogota, Colombia

Background & objectives: TILs correlate with patient outcome in many cancers. The evaluation of TILs in HNSCC has not been yet standardized. The aim was to evaluate the inter-observer agreement of visually-assessed TILs and a visual-versus-digital image analysis (DIA) evaluation comparison.

Methods: 40 samples from HNSCC patients from the Colombian cohort of the InterCHANGE study (IARC) were included. TMAs were constructed and Hematoxylin and eosin (H&E) and Immunohistochemistry for CD3, CD4 and CD8 were performed. TILs average score was assessed independently by three pathologists. Their scores were compared to DIA-scores using QuPath.

Results: The inter-observer agreement coefficients were 0.64, 0.71, 0.55 and 0.70 (ICC p=0.0001) for H&E, CD3, CD4 and CD8, respectively. When the DIA-scores were compared to visual estimates, the average scores of the pathologists (H&E: 0.71, CD3: 0.73, CD4: 0.4, CD8: 0.68, weighted-kappa) had better agreement than comparing individual scores (H&E: 0.52-0.66, CD3: 0.54–0.79, CD4: 0.33–0.39, CD8: 0.5–0.66, weighted-kappa). When scores were grouped into categories (low-moderate-severe) the agreement decreased compared to evaluation as continuous parameter.

Conclusion: We show a substantial inter-observer agreement between pathologists, these scores have moderate agreement with DIA ones, but improve when evaluated as an average. We consider that visual estimation can be used with an acceptable level of agreement in routine practice and research. However, in cases when the scores are close to a limit of a category/range, DIA can be very useful to proper classify the density of the infiltrate.

Funding: Interchange - HEADSpace (IARC); Researchers own funds


PIK3CA gene mutations in human papilloma virus 16 head and neck scamous cell carcinoma

D. Cochicho*, A. Felix, R. Gil Da Costa, L. Martins, M. Cunha, M. Magalhães

*Clinical Pathology Department, Virology Lab. IPOLFG, Portugal, NOVA Medical School, NOVA University of Lisbon, Portugal

Background & objectives: Human Papillomavirus (HPV)-associated head and neck squamous cell carcinomas (HNSCC) typically arise in the pharynx. HPV active infection may lead to alterations in key signalling pathways that promote carcinogenesis. Different studies identified PIK3CA mutations more commonly in HPV positive tumours (37%).

Methods: We evaluated PIK3CA gene mutations associated with HPV16+HNSCC patients from the Portuguese Institute of Oncology between 2007 and 2019. HPV DNA status was evaluated by PCR and genotyped (INNOLIPA/ALLPLEXHPV28). PIK3CA mutation analysis was performed by real-time PCR for the qualitative detection of four mutations (H1047L; E542K, E545K, E545D) in human genomic DNA extracted from tumour tissue (AmoyDx).

Results: PIK3CA mutations were evaluated in 50 squamous cell carcinomas, all HPV16 positive. Co-infection with HPV18, HPV53, HPV58 was present in 2 cases. Ten cases harboured PIK3CA mutations (20%). Three different mutations H1047R (10%), E545D (50%), E545K (20%) were identified. Combined E545D/E545K mutations were present in 20% of cases. Mutations in PIK3CA gene were present in cases from the oral cavity (20%) and the oropharynx (80%).

Conclusion: In our series, a lower frequency of PIK3CA gene mutations was found among HPV16+ HNSCC compared with other series. These results may be related to the method used and to clinical aspects and should be confirmed by other series in Portugal. Multiple PI3K/AKT/mTOR inhibitors are under investigation as potential therapeutic options for HNSCC, adding relevance to regional variations in PIK3CA mutations and their identification methods.


The effect of E6 and E7 knockdown and PARP inhibition on cisplatin sensitivity in oropharyngeal squamous cell carcinoma

H. Crane*, K. Hunter, S. El-Khamisy

*University of Sheffield, United Kingdom

Background & objectives: Clinical outcomes in HPV- OPSCC are poor, with a 3-year survival of 57.1%. Although HPV+ has an improved response to therapy, a subset of patients suffer from loco-regional recurrences and distant metastases, with a poor prognosis.

Methods: The effect of E6 and E7 on cisplatin sensitivity in HPV+ OPSCC was investigated by knockdown of E6 and E7 using siRNA in a HPV+ cell line. A HPV+ and HPV- cell line were exposed to long term cisplatin treatment to generate resistant models. Immunofluorescence and MTS assays were used to assess DNA double strand breaks and cell viability respectively.

Results: Knockdown of E6 and E7 in a HPV+ cell line did not significantly effect cell viability in response to cisplatin. Following long term treatment with cisplatin, the HPV+ and HPV- resistant cells had a significantly higher half maximal inhibitory concentration (IC50) compared to the parental cells (HPV- resistant cells IC50: 47.5 compared to 10.3 and HPV+ resistant cells IC50: 84.9 compared to 40.0) and showed fewer DNA double strand breaks following cisplatin treatment. Treatment with Olaparib partially resensitised both HPV+ and HPV- resistant cells to cisplatin.

Conclusion: Although knockdown of E6 and E7 did not appear to sensitise HPV+ cells to cisplatin, Olaparib was able to resensitise both HPV+ and HPV- resistant cells to cisplatin. These findings have implications for future research in this area.

Funding: Funded by a CRUK/Pathological Society Predoctoral Research Bursary


Status of HPV-16 in cervical lymph node metastatic squamous cell carcinoma of unknown origin - a study of three cases

R. Dolz Gaitón*, R. Dolz, M.J. Roca

*Hospital Vithas 9 de Octubre, Spain

Background & objectives: In head and neck pathology 5% of malignancies present as lymph node metastasis of unknown origin. In up to 45-80% of cases the primary site can be identified. In identifying the origin, the study of HPV-16 status can be useful.

Methods: We have studied three patients that presented cervical lymph node metastases of unknown origin. All three of them were middle-aged women with no relevant medical history. The lymph node study was done with hematoxylin eosin slides and immunohistochemistry techniques including CK5/6, p63 and p16. The HPV status was studied through DNA-PCR amplification and microarray hybridization.

Results: All cases showed infiltration in the lymph node of poor-differentiated squamous cell carcinoma. Two of the three cases showed positivity for p16 and also for HPV-16. The third case was negative for p16 and HPV. Gynaecological exams were performed in all these women, with absence of cervical dysplasia. In one of the patients the origin was identified, being oropharyngeal. The other two patients still remain with no primary tumour detected.

Conclusion: The presence of HPV seems to be important in this patients but appears to not have direct relationship with cervical dysplasia or anogenital origin. Also, in oropharyngeal carcinomas it has been associated with a more favourable prognosis. This shows that identifying the presence of HPV should be a routine procedure in all cervical lymph node metastases.


PD-L1 expression in head and neck squamous cell carcinomas (HNSCC) and its correlation with clinical variables

M. Espínola Benza*, A.B. Enguita Valls, L. Iglesias Docampo

*University Hospital 12 de Octubre, Spain

Background & objectives: Immune checkpoint inhibitors have created a revolution in oncology. Despite a higher likelihood of response to immunotherapy in PD-L1 positive tumours, the cut-off remains unclare. The objective is to describe PD-L1 expression related to clinical variables, disease-free survival and toxicity.

Methods: Case reports of HNSCC diagnosed between 2014 and 2019, treated with immunotherapy. PD-L1 IHC using the 22C3 antibody was performed. The scores were compared at both the 1% and 20% cutpoints, using the CPS result obtained from core biopsy or resected specimen. Information regarding clinical characteristics was retrieved as documented by the treating physicians.

Results: 67 patients were identified, both in clinical trials and assistance. PD-L1 expression was related to gender, smoking, age and histologic status. Only 11 of them were women. 48 of them received nivolumab alone, 3 pembrolizumab alone, 7 durvalumab alone. 3 patients received durvalumab in combination with tremelimumab, another 3 nivolumab with ipilimumab and 3 combination chemotherapy with pembrolizumab.

Conclusion: To identify more suitable HNSCC cases for anti-PD-1/PD-L1 therapy, PD-L1 expression in HNSCC should be combined with clinicopathologic features.


Small round blue cell tumours of the sinonasal tract: histopathological evaluation of 34 cases

E. Gun*, D. Etit, F. Cakalagaoglu

*Izmir Katip Celebi University Ataturk Training and Research Hospital, Department of Pathology, Turkey

Background & objectives: Histopathology of the small round blue cell tumours of the sinonasal tract (SRBCT-SN) remain challenging because they consist of diverse malignancies including epithelial, haematolymphoid, neuroectodermal and mesenchymal origin. We aimed to evaluate the histopathological and immunohistochemical characteristic of these tumours.

Methods: All the patients who were diagnosed with a SRBCT-SN between January 2006 and January 2020 in our department were evaluated retrospectively. The demographical, clinical, radiological, histopathological and immunohistochemical findings were recorded.

Results: There were a total of 34 cases with 13 females and 21 males. The mean age was 58 years. Histopathologically, there were 10 sinonasal undifferantiated carcinoma, 9 malignant melanoma, 3 rhabdomyosarcoma, 3 olfactor neuroblastoma, 3 lymphoma, 2 plasmocytoma, 2 ectopic pituitary adenoma, 1 Ewing sarcoma and 1 sinonasal neuroendocrine carcinoma cases. Twenty-two of these cases were located inside the nasal cavity, whereas 6 were in the paranasal sinuses and 6 were in the nasopharynx. When diagnosing these challenging lesions, clinical and radiological findings should be taken into account since they have overlapping histologic and immunohistochemical findings.

Conclusion: With the help of newly described entities and increased usage of molecular testing,these tumours can be diagnosed better.A precise diagnosis is importat especially because some of these tumours behave aggresively and the pathological diagnosis will help identifying the correct treatment.


Metastatic, non-cutaneous squamous cell carcinoma to the salivary gland: a cases series observed at the Royal Surrey Hospital

G. Gupta*, S. Di Palma

*The Royal Surrey County Hospital, United Kingdom

Background & objectives: Metastasis to the salivary glands tends to occur from primary squamous cells carcinoma (SCC). Rarely metastases may be from more distant sites or of non squamous cells cutaneous tumour. Given the histological variety of salivary tumours, diagnosis can be difficult.

Methods: Tumours of salivary gland origin discussed at Head and Neck MDT meeting at the RSH have been reviewed searching from metastasis versus primary neoplasms with the exception of squamous cell carcinoma of skin origin. Here we present our experience and differential diagnoses.

Results: Metastasis to the salivary glands included basal cell carcinomas, a rare merkel cell carcinoma and distant neoplasm such as renal cell carcinoma and Upper GI tract adenocarcinoma. A metastasis from an undiagnosed olfactory neuroblastoma to the submandibular gland was initially interpreted as unusual primary neuroendocrine malignancy. The primary manifested 6 months after submandibular gland resection. All cases were diagnosed and confirmed by appropriate immunohistochemistry and additional clinical information.

Conclusion: Non squamous cell cutaneous carcinoma metastasizing to salivary glands is uncommon and difficult to diagnose especially without sufficient clinical history and familiarity with the long list of primary malignant tumours of salivary glands.

We propose a high index of suspicion in cases with unusual histological or immuno-histochemical features and inadequate clinical information when assessing salivary gland neoplasms.


The tumour immune microenvironment and its implications for clinical outcome in patients with oropharyngeal squamous cell carcinoma

M. Hermanova*, D. Gurin, M. Slavik, M. Hendrych, I. Selingerova, T. Shatokhina

*First Department of Pathology, St. Anne's University Hospital and Faculty of Medicine, Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic

Background & objectives: To analyse the PD-L1 expression on tumour cells (TC) and immune cells (IC), and densities of CD3+ and CD8+ tumour-infiltrating lymphocytes (TILs) in oropharyngeal squamous cell carcinoma (OPSCC), and to correlate them with radiotherapy treatment outcomes and clinicopathological parameters.

Methods: The study included 65 OPSCC treated by definitive intensity-modulated radiotherapy (IMRT) in curative intent. Immunohistochemical analysis of PD-L1 expression on TC and IC, and TILs subtyping was performed on primary biopsy tumour tissues, followed by prognostic evaluation of these immune response related parameters including classification into four tumour immune microenvironment (TIM) types. To evaluate HPV status, p16 immunohistochemistry was performed.

Results: Densities of CD3+ and CD8+ TILs and PD-L1 expressions on TC and IC were significantly higher in p16+/HPV-mediated OPSCC. Patients with high densities of stromal CD8+ T cells displayed significantly better overall survival (OS) and progression-free survival (PFS). PD-L1 expression neither on tumour cells (TCs) nor immune cells (ICs) affected survival outcomes. Distribution of TIM types based on combination of PD-L1 expression on TC and densities of CD8+ TILs is significantly different in p16+ compared to p16- OPSCC. In type III TIM OPSCC (TC-PD-L1+/low CD8+ TIL density), significantly better OS was showed in p16+ group compared to p16- OPSCC.

Conclusion: The role of tumour immune microenvironment was confirmed and combining HPV status with evaluation of densities of CD8+ TILs and PD-L1 expression including TIM classification might provide valuable predictive and prognostic information for patients with OPSCC.


Perineural invasion of squamous cell carcinoma of the head and neck – ten years of experience

P. Hurník*, V. Zidlik, D. Ziak, M. Sporkova, J. Stembirek, Z. Cermakova, B. Putnova, M. Buchtova

*Department of Pathology, University Hospital of Ostrava, Department of Pathology, Faculty of Medicine, University of Ostrava, Czech Republic

Background & objectives: Perineural invasion has been defined as the ability of cancer cells to invade through, in or around nerves and is clinically associated with elevated recurrence and diminished survival. It is morphologically described in many solid tumours (head and neck, prostate)

Methods: We have retrospectively analysed cases of 532 patients with squamous cell carcinoma of head and neck in age from 33 to 86 years. In all cases, cervical block dissection was also perfomed with the resection of the main tumour. Patients without cervical block dissection were excluded. All cases were paraffin embedded and 3 um sections were stained by the hematoxylin/eosin.

Results: The tumour size, depth of invasion, the presence and morphology of perineural invasion and the presence of lymfangioinvasion and hemangioinvasion were evaluated. From the total number of 532 cases, 70 (13,15%) cases with perineural invasion, 22 (4,13%) with hemangioinvasion and 60 (11,27%) with lymphangioinvasion were encountered. Metastases in ipsilateral lymph nodes were found in 249 (46,80%) cases, contralateral metastases were present in 39 (7,33%) cases. These factors were further compared to other morphological features (depth of invasion, tumour budding).

Conclusion: We focused mostly on perineural invasion, which is probably based on the chemotropism of tumour cells, that can be stimulated by nerve tissue to further growth. The interactions between tumour and neural cells is not only limited to cell migration and tumour growth from the primary location but such interaction can also stimulate axonogenesis or extend the nerves themselves together with increasing number of axons.

Supported by Ministry of Health Of Czech Republic, grant nr. AZV NV19-08-00383. All rights reserved.


Variants in SDHAFx genes in vagal paragangliomas

A. Kobelyatskaya*, V. Pavlov, D. Kalinin, A. Golovyuk, M. Fedorova, Z. Guvatova, E. Pudova, A. Poloznikov, E. Slavnova, A. Polyakov, G. Razmakhaev, G. Krasnov, A. Snezhkina, A. Kudryavtseva

*Engelhardt Institute of Molecular Biology, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia

Background & objectives: SDHAFx encode assembly factors of the succinate dehydrogenase (SDH) complex; variants in these genes are associated with the SDH complex deficiency. The objective of this study was to analyse the pathogenic/likely pathogenic variants in SDHAFx genes in vagal paragangliomas (VPGLs).

Methods: In the study, we used a set of eight VPGLs collected in the Vishnevsky Institute of Surgery, Ministry of Health of the Russian Federation. Exome libraries were prepared using the TruSeq DNA Exome Kit (Illumina, USA) and were sequenced on the Illumina NextSeq 500 System in pair-read mode (76x2). Immunohistochemistry (IHC) was performed using anti-SDHx antibodies (Abcam, USA).

Results: We revealed two patients with VPGLs characterized by likely pathogenic mutations in SDHAFxgenes. Variant NM_145267 (SDHAF4): c.C223T, p.P75S (chr6:71298323, rs146446063) in SDHAF4was detected in both patients. In one patient, the variant co-occurred with a pathogenic mutation in SDHB(NM_003000 (SDHB): c.541-2A>G (chr1:17350571, rs786201161)), in another patient – with likely pathogenic variant in SDHFA3(NM_020186 (SDHAF3): c.T157C, p.F53L (chr7:96747192, rs62624461)). IHC revealed negative and weak diffuse expression of SDHB protein in these cases.

Conclusion: Variants in SDHAFx are less frequent than mutations in SDHx in paragangliomas. We revealed likely pathogenic variants in SDHAF3 and SDHAF4 genes in two patients with VPGLs. According to the IHC results, we revealed changes in SDHB protein expression in patients with SDHB/SDHAF4 and SDHAF3/SDHAF4 variants. Thus, co-occurring variants in SDHAFx genes or present mutations in SDHAFx and SDHx together can lead to disruption of SDH complex stability and function.

This work was financially supported by the Russian Science Foundation, grant no.19-15-00419.

This work was performed using the equipment of EIMB RAS “Genome” centre (


Germline mutations in metastatic carotid paraganglioma

A. Kobelyatskaya*, A. Snezhkina, V. Pavlov, O. Stepanov, D. Kalinin, A. Golovyuk, A. Poloznikov, Z. Guvatova, A. Sadritdinova, M. Fedorova, E. Pudova, M. Savvateeva, G. Krasnov, A. Kudryavtseva

*Engelhardt Institute of Molecular Biology, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia

Background & objectives: Carotid paraganglioma (CPGL) is a rare neuroendocrine tumour of the head and neck that is characterized by the variable metastasis potential. Mechanisms and biomarkers of this process are unclear. In the study, we performed a genetic analysis of metastatic CPGL.

Methods: We carried out the exome analysis of tumour, lymph node and metastatic tissues derived from a patient with metastatic CPGL. Exome libraries were prepared using the TruSeq DNA Exome Kit (Illumina, USA) and were sequenced on an Illumina NextSeq 500 System (paired-end reads, 76x2). We performed the search for pathogenic/likely pathogenic germline variants in the patient using GATK HaplotypeCaller.

Results: Base on in silico prediction tools (PolyPhen2, LRT, SIFT, and others) and ClinVar database, we analysed the pathogenicity of germline variants identified in the patient. We found germline pathogenic variants in ALDH7A1and CBSgenes. Likely pathogenic variants were determined in a wide range of genes, including HK2, HYDIN, GALC, ZNF717, MMP28, EIF2AK3, and others.

Conclusion: We revealed a number of germline pathogenic/likely pathogenic variants that can be associated with the metastatic CPGL. Interestingly, no variants were found in any genes, which are frequently associated with paragangliomas/pheochromocytomas (SDHx, RET, NF1, etc.). However, likely pathogenic variant was identified in the HK2 indicating alterations in glycolysis. Mutation in HYDIN can also play an important role because a high mutation frequency of this gene in CPGLs was previously shown.

This work was financially supported by the Russian Science Foundation, grant no. 17-75-20105.

This work was performed using the equipment of EIMB RAS “Genome” centre (


Immunohistochemical expression of programmed death ligand-1 (PD-L1) in oral squamous cell carcinoma and its clinicopathological correlation

M. Kumar*, S. Babu, S. Ratnakar

*King George's Medical University, India

Background & objectives: Oral squamous cell carcinoma is responsible for tremendous cancer related deaths and tumour load in the Indian sub-continent. To assess the immunohistochemical expression of programmed death ligand-1(PD-L1) in oral squamous cell carcinoma and correlation of PD-L1 expression with clinico-histopathological parameters.

Methods: All 106 cases histopathologically and programmed death ligand-1(PD-L1) expression in tumour cells were evaluated separately by two observers. Only membranous positivity was considered as positive. Immunoreactivity score for PD-L1 expression was calculated based on staining intensity as well as percentage. The statistical analysis was done using SPSS version 21.0 statistical analysis software. P value of <0.05 was considered statistically significant.

Results: Age group 40 to 60 years was most affected. Tongue was the most commonly involved site. Tobacco smoking was reported in 86.8% of cases. No statistically significant association of histological grade, ulceroproliferative pattern, and patient survival could be seen with PD-L1 immunoexpression scores, however, tumour stage, lymph node metastasis, tumour size (p=0.030), depth of invasion(p=0.005), lymphovascular and perineural invasion (p=0.008) were found to be statistically significantly associated with immunohistochemical scores.

Conclusion: We conclude that total of 106 cases were included in this study. PD-L1 expression was evaluated in tumour cells by two observers, interobserver agreement of 97.1% was present which is in the acceptable range.PD-L1 pathway plays a significant role in tumour immune evasion in oral SCC patient. Role of targeted therapy can be predicted by evaluating PD-L1 expression and suggesting the OSCC patients that may be benefited by immunotherapy.


P62/sequestosome1(SQSTM1) and GATA3 expression in salivary duct carcinoma: in comparison with pleomorphic adenoma

K. Kusafuka*, J. Itakura, Y. Otsuki, N. Kuroda, I. Ito, R. Wada, A. Muramatsu, M. Suzuki, K. Arai

*Shizuoka General Hospital, Japan

Background & objectives: SDC is immuno-phenotypically akin to mammary apocrine carcinoma (MAC), due to the expression for AR and GCDFP-15. GATA3 is a well-known biomarker for breast and urothelial cancers. We aim to elucidate the status of SQSTM1 and GATA3 expression in SDC.

Methods: We collected 53 cases of SDC, including 30 cases of carcinoma ex pleomorphic adenoma (CXPA), and 30 cases of pleomorphic adenoma (PA), and performed the immunohistochemistry for SQSTM1 and GATA3 on their FFPE sections, adding GCDFP-15 and AR. Finally, we estimated the positive rates and the pattern for these molecules in SDC and PA cases, respectively.

Results: The positive rates for GCDFP-15, AR, SQSTM1, and GATA3 in SDC cases were 91%, 85%, 81% and 89%, respectively. The signals for AR and GATA3 were limited to nuclei (N), whereas the signals for GCDFP-15 were observed in the cytoplasm (CY). The signals for SQSTM1 were seen in N and/or CY. Although approximately 27% of PA cases were positive for GATA3, a few cases of PA were positive for SQSTM1.

Conclusion: SQSTM1 is a molecule related to the autophagy, whereas it was recently reported that its expression was up regulated in MAC. As SQSTM1 is also a marker for apocrine differentiation, it is better that SDC is called “apocrine carcinoma of the salivary gland”. Although GATA3 has been also reported the consequential positivity in secretory carcinoma of the salivary gland, it could be one of the sensitive diagnostic markers for SDC, adding to AR, GADFP-15 and SQSTM1.

Funding: A Grant-in-Aid for Medical Research Support Project of Shizuoka Prefectural Hospital Organization in 2019 of Japan (to KK).


PD-L1 IHC 22C3 pharmDx helps determine first-line pembrolizumab eligibility in head and neck squamous cell carcinoma patients

C. La Placa*, B. Watts, M. Vilardo, M. Polewski, S. Tabuena-Frolli, M. Jansson, J. Juco, K. Emancipator, F. Jin, B. Gumuscu, J. Ge, K. Kulangara

*Agilent Technologies, Inc., USA

Background & objectives: PD-L1 IHC 22C3 pharmDx is a CE-IVD-marked assay intended for use in detection of PD-L1 protein in formalin-fixed, paraffin-embedded non-small cell lung cancer, urothelial carcinoma, melanoma, and recently, head and neck squamous cell carcinoma (HNSCC) tissues.

Methods: For the HNSCC indication, the device has been validated at Dako North America on the performance of precision and robustness using the Combined Positive Score (CPS) ≥ 1 and CPS ≥ 20 cutoffs; external validation studies were performed at three external laboratories. CPS ≥ 1 and CPS ≥ 20 cutoffs were evaluated in KEYNOTE-048, a phase 3 clinical trial.

Results: Analytical validation studies supporting the companion diagnostic indication (CPS ≥ 1) achieved point estimates of > 85% for negative, positive, and overall percent agreement. Clinical validation studies show that patients treated with pembrolizumab as a single agent had an overall survival (OS) of 12.3 months at CPS ≥ 1 (95% CI, 10.8-14.3) compared with patients receiving cetuximab, platinum, and 5-fluorouracil (CPS ≥ 1 OS of 10.3 months (95% CI, 9.0-11.5)).

Conclusion: Analytical and clinical validation studies demonstrate that PD-L1 IHC 22C3 pharmDx is a robust and precise companion diagnostic assay, allowing for selection of eligible HNSCC patients for treatment with pembrolizumab.


New entities of sinonasal tract tumours: a single centre experience

K. Liyanaarachchi*, C.W. Lee, S. Sunkaraneni, S. Di Palma

*Royal Surrey County Hospital, United Kingdom

Background & objectives: Sinonasal tract tumours have been newly classified according to molecular and immuno-histochemical studies leading to an expansion of the category of sinonasal undifferentiated carcinoma (SNUC). Our goal here is to familiarize the new entities with the pathologists and clinicians.

Methods: We have searched our files to see if there were cases initially called SNUC and now reclassified according to the recent WHO recognized sinonasal tract carcinoma, classification, which include NUT mid-line carcinoma, SMARCB 1 deficient sinonasal carcinoma and HPV related carcinoma with adenoid cystic like features. Participation to clinical trial and follow up was arranged.

Results: When immunohistochemistry and molecular tests were performed, the new tumour entities were identified. Molecular analysis including the NUT gene (t15 q14) and immunohistochemistry for NUT protein was necessary to identify NUT midline carcinoma. T6:9 translocation, hallmark of adenoid cystic carcinoma, was performed to confirm HPV related carcinoma with adenoid cystic like features which has better prognosis. Deletion of SMARCB1/22q was observed in a case of SMARCB1 deficient sinonasal carcinoma.

Conclusion: Our study confirms that sinonasal carcinoma can be reclassified using immunohistochemistry and molecular testing leading to new entities with prognostic, diagnostic and therapeutic implications. Our reclassification allowed a patient with NUT midline carcinoma to be enrolled in clinical trial for BET inhibition with good clinical response.


Histopathological processing of sentinel lymph nodes in head and neck cancer

D. Mooney*, S. Wright

*Queen Elizabeth University Hospital, Glasgow, United Kingdom

Background & objectives: Sentinel node biopsy (SNB) can be offered to patients with early-stage squamous cell carcinoma (SCC) of the head and neck. There is currently no agreed protocol for histopathological processing of SNBs – an audit was undertaken to inform local practice.

Methods: Seventy seven SNBs from 2013-2016 were retrieved from our records. We examined each report, recording specimen size, dissection method, and total number of histological levels and immunohistochemical stains requested. The positive SNBs were and the histological level at which metastatic SCC was first seen was recorded. The results and clinical outcomes for the positive and negative SNB cases were compared. Results: In all positive SNB cases, metastatic SCC was present on the first H&E level. Additional histopathological levels and immunohistochemical staining did not contribute to the identification of SCC. Histological levels were required in one case to demonstrate conversion from a micrometastatic to a macrometastatic deposit and in one case to demonstrate extracapsular spread. There was no documented clinical recurrence in any of the negative SNBs.

Conclusion: We propose examining a single H&E section of each block without pre-emptive ordering of levels, blanks and immunohistochemistry. When a micrometastasis is identified or extra-capsular spread is suspected, then additional levels may be warranted. In our institution, examining a single H&E as has a potential cost saving of £25 per block.


Evolutionary epigenetics: DNA methylation modifications in ultra-conserved non-coding elements from squamous cell carcinoma of different species revealed a common epigenetic behaviour

L. Morandi*, S. Sabattini, A. Renzi, A. Rigillo, G. Bettini, E. Dervas, A. Schauer, D. Gissi, A. Tarsitano, S. Evangelisti, M. Morandi, C. Tonon

*Alma Mater Studiorum - University of Bologna - Department of Biomedical and Neuromotor Sciences, Functional MR Unit, IRCCS Istituto delle Scienze Neurologiche di Bologna, Italy

Background & objectives: Ultra-Conserved-Non-coding Elements(UCNEs) represent genomic sequences that exhibit >95% sequence identity between human, mammals, birds, reptiles, fishes. Recent findings reported their functional role in cancers. Aim of this study was to evaluate DNA methylation modifications in oral-squamous-cell-carcinoma (OSCC) from different species.

Methods: 50 OSCC, 14 feline stomatitis and 63 normal tissue were enrolled in this study. DNA purification followed by bisulfite Next Generation Sequencing protocol were used to quantify the DNA methylation level of each CpG from seven UCNE(uc.160, uc.283, uc.416, uc.339, uc.270, uc.299, uc.328). Bioinformatic data analysis was performed in cloud computing using galaxyproject tools, Methylation Plotter and ClustVis.

Results: 50 OSCC, 26 from human, 17 from cats, 3 dogs, 1 badger, 1 horse, 1 porcupine, 1 bovine, 1 chicken were investigated. Moreover, 14 feline stomatitis and normal tissue from 42 healthy human donors, 7 cats, 5 dogs, 5 horses, 2 caws, 1 badger, 1 iguana were collected as normal controls. DNA methylation analysis revealed consistent epigenetic modifications able to correctly stratify OSCC vs feline stomatitis and vs normal tissue. A common DNA methylation pattern was observed in OSCC of all the species evaluated in this study with an increasing trend of hypermethylation starting from normal mucosa, through stomatitis to OSCC. 57/59 CpGs were statistically significant in Kruskal-Wallis test (P<0.05).

Conclusion: Our findings indicate that UCNEs are hypermethylated in human OSCC, and this behaviour is also conserved among different species of mammals and also in reptiles. These similarities at both clinical and molecular level have led to the proposal that feline and other mammal species may serve as a spontaneous model for human disease.

This study was supported by an academic grant (ALMAIDEA) from the University of Bologna.


The diagnostic utility of RAS Q61R mutation-specific immunohistochemistry in epithelial-myoepithelial carcinoma

T. Nagao*, M. Nakaguro, M. Tanigawa, H. Hirai, Y. Yamamoto, Y. Tada

*Tokyo Medical University, Japan

Background & objectives: Epithelial-myoepithelial carcinoma (EMC) is a rare salivary gland tumour characterized by biphasic tubular structures. The HRAS Q61R mutation is frequent in and specific to EMC. We evaluated the usefulness of immunohistochemical staining for detecting this genetic alteration in EMC.

Methods: We investigated 74 EMC cases and 63 cases of salivary gland tumours with an EMC-like component, including pleomorphic adenoma, adenoid cystic carcinoma, basal cell adenoma, and myoepithelial carcinoma. Immunohistochemical staining was conducted using the monoclonal antibody specific to the RAS Q61R mutation (SP174; Abcam, Cambridge, MA, USA). Sanger DNA sequencing was also performed for HRAS, KRAS, and NRAS.

Results: The diffuse and membranous RAS Q61R immunohistochemical expression was observed in 51 of 74 EMC cases (68.9%), in which 50 cases (98.0%) harbored the HRAS Q61R mutation. The immunoreactivity was largely restricted to the myoepithelial cells in EMC. Conversely, only one of the EMC cases lacking the HRAS Q61R mutation and no EMC-like salivary gland tumours showed the abovementioned immunopositivity. None of the cases examined carried KRAS or NRAS mutations.

Conclusion: Immunohistochemistry for RAS Q61R is highly sensitive and specific for detecting the HRAS Q61R mutation in EMC. Further studies will be needed to clarify the molecular mechanisms underlying the differences in the immunoexpression between the ductal and myoepithelial cells in HRAS Q61R mutation-positive EMCs. Since significant immunopositivity was exclusively identified in more than two-third of EMCs but not in the histologic mimics, the immunohistochemistry of RAS Q61R is a useful tool for diagnosing EMC in general pathology laboratories.


BRAF V600E mutations in odontogenic tumours with ameloblastic epithelium: clinicopathological significance and immunohistochemical validation

K. Oh*, S. Cho, H. Yoon, J. Lee, S. Hong

*Seoul National University, Republic of Korea

Background & objectives: The BRAF V600E mutation is an oncogenic mutation that can be a therapeutic target. We determined the prevalence and clinicopathological significance of BRAF V600E mutations in benign and malignant odontogenic tumours and validated immunohistochemistry using a BRAF V600E-specific antibody (VE1).

Methods: BRAF V600E detection was performed using Sanger sequencing in a total of 20 odontogenic tumours: 5 ameloblastic carcinomas (ACs), 1 metastasizing ameloblastoma, 1 clear cell odontogenic carcinoma, 7 ameloblastic fibromas (AFs), 2 ameloblastic fibro-odontomas (AFOs), and 4 developing odontomas. Associations between BRAF V600E and clinicopathological factors were statistically analysed. VE1 immunohistochemistry was conducted and its validity was evaluated.

Results: BRAF V600E mutations were identified in 40.0% of AC, 42.9% of AF, and 50.0% of AFO, although no clinicopathological factors were significantly associated with the mutation status. VE1 immunohistochemistry showed diffuse cytoplasmic staining in AC, but no VE1 expression was found in BRAF V600E-mutant mixed odontogenic tumours. Compared with the sequencing results, VE1 immunohistochemistry yielded a sensitivity of 33.3% (2/6) and a specificity of 100% (14/14).

Conclusion: Odontogenic tumours with ameloblastic epithelium have BRAF V600E mutations in common, which suggests the utility of BRAF-targeted therapy for AC and supports the notion that AFO is a true neoplasm. Considering the low sensitivity of VE1 immunohistochemistry in odontogenic tumours, molecular tests should be performed to determine the presence of BRAF V600E mutations.


Orofacial tumours and tumour-like lesions in Birnin-Kebbi metropolis: histopathological analysis

O. Oluwole*

*University of Abuja, Nigeria

Background & objectives: Orofacial tumours can occur at any age. An increasing occurrence has made these tumours a significant cause of morbidity and mortality.


To determine the histopathological pattern of orofacial tumours and tumour-like lesions in Birnin-Kebbi, North-West, Nigeria.

Methods: This is a 9-year retrospective histopathological analysis of orbital tumours diagnosed between 2004-2012 in the Department of Histopathology, Federal Medical Centre, Birnin-Kebbi, Kebbi State, North-West, Nigeria. All the Haematoxylin &Eosin stained slides and paraffin embedded blocks were retrieved and studied.

Results: A total of 23 patients were studied, 11 males and 13 females. The age range is 7-80 years with the mean age of 43.5 years. There is a bimodal peak age at the fourth and fifth decades. The most common benign tumour was pleomorphic adenoma, PA (43.5%), while the most common malignant tumour was adenoid cystic carcinoma, ACC followed by squamous cell carcinoma, SCC.

Conclusion: Orofacial tumours and tumour-like lesions are common in our environment. A very high-index of suspicion, good clinical acumen, adequate histopathologic sampling and reporting can go a long way at making diagnosis.


The cytokeratin 7/α-smooth muscle actin immunohistochemical combination is sufficient to distinguish adenoid cystic carcinoma from polymorphous adenocarcinoma in small biopsies

R. Ottaviani*, S. Souza, B. Sedessari

*Nove de Julho University, School of Medicine, Brazil

Background & objectives: Adenoid cystic carcinoma (ACC) and polymorphous adenocarcinoma (PAC) represent one of the most troublesome examples of morphological overlap seen in salivary gland pathology. We aimed to demonstrate the combined CK7/α-SMA immunohistochemical combination as a reliable method to make this distinction.

Methods: 100 PACs and 79 ACCs samples diagnosed in small incisional biopsies were submitted to CK7 and α-SMA immunohistochemical reactions. Results were described as positive or negative.

Results: ACC group of patients consisted of 53 women and 26 men, with an overall mean age at presentation of 45.6 years. Cases affected the palate, upper lip, buccal mucosa, retromolar trigone and paranasal sinus. All cases were infiltrative biphasic tumours composed by basaloid cells showing a concordant CK7+/α-SMA+ immunoprofile. The PAC patients were represented by 80 women and 20 men, with a mean mean age of 58.7 years. Cases affected the palate, upper lip, buccal mucosa and retromolar region. All were represented by invasive tumours with diverse architectural growth patterns, displaying a consistently discordant CK7+/α-SMA- profile.

Conclusion: application of the combined cytokeratin 7/α-SMA antibodies is a trustworthy and cost-effective method to separate ACC from PAC, even in small biopsy samples.


Multi-parametric analysis of interferon γ transcription as assessed by in situ hybridization in head and neck human papillomavirus-related squamous cell carcinomas

S. Outh-Gauer*, M. Rassy, M. Mandavit, C. Lépine, J. Augustin, C. Gasne, T. Denize, E. Tartour, C. Badoual

*Georges Pompidou European Hospital, Inserm U970, France

Background & objectives: Patients with Human Papillomavirus (HPV)-related head and neck (HN) squamous cell carcinoma (SCC) have long been considered as having a better outcome. However, they still exhibit variability in their prognosis, that could be linked to micro-environment heterogeneity.

Methods: Sixty-three patients diagnosed with HPV-positive HNSCC and confirmed by HPV DNA PCR, were included. For each of them, one whole slide was stained by Interferon γ (IFNγ) RNAScope® coupled with pancytokeratin, PD-L1 and CD8. Tyramide signal amplification and immunofluorescence revelation followed. IFNγ and/or CD8 expressing cells were quantified in 10 representative high-power fields (distinctively 5 tumoral and 5 stromal).

Results: More than half of HPV-positive oropharyngeal SCC showed IFNγ transcription. The latter was localised in stromal and tumour infiltrating immune cells, as well as in carcinomatous cells. Analysis by log-rank test showed that intra-tumoral IFNγ high expression was associated with a better overall survival (p=0.035), unlike stromal IFNγ expression (p=0.806). Furthermore, HPV-positive oropharyngeal SCC showed enrichment of CD8+IFNγ+ lymphocytes, in both tumoral and stromal compartments (respectively p=0.037 and p=0.048). Of note, the results were independent of PD-L1 expression (positive in 92.1% of all cases).

Conclusion: In conclusion, intra-tumoral IFNγ expression distinguishes two prognostic groups of oropharyngeal HPV-positive SCC. The associated enrichment in CD8+IFNγ+ lymphocytes suggests an underlying mechanism involving the immune micro-environment.

Funding: Fonds de Recherche Société Française de Pathologie


Foetal parotid gland morphology in cases of toxaemia of pregnancy

O. Reshetnikova*, S. Morozov

*Immanuel Kant Baltic Federal University, Russia

Background & objectives: Salivary gland's adequate function is important in maintaining an oral environment. Saliva’s composition alterations may cause periodontal status and be responsible for the caries development. The aim of the study was to examine the foetal parotid gland morphology under toxaemia of pregnancy,

Methods: 20 foetal parotid glands of 18-22 weeks of gestation were examined after spontaneous abortions in pregnancies complicated by toxaemia of pregnancy. Salivary glands of 15 human foetuses with the same gestational age in cases of induced abortions due to social and psychological reasons were in control group(CG). Histological study of H&E stained slides was followed with morphometry and statistical analysis.

Results: The delay of acini and intralobulae ducts differentiation within the parotid gland tissue were revealed in the study. Dystrophic changes of parenchymal elements of large salivary glands were also determined. Morphometry investigation have shown a reduction of parenchymatous elements volume fraction, decreased vascularity and decline of parenchyma/stroma index. The connective tissue volume fraction in parotid gland tissue increased.

Conclusion: The results of the study present a structural impairments in foetal parotid gland anternatal development in cases of pregnancies complicated with toxaemia. These alterations perhaps cause functional disorders in salivary gland's activities and may contribute to the pathology of the oral cavity in childhood and later life.


Intraosseous adenoid cystic carcinoma – a case series

M. Ryan*, S. Wright

*Queen Elizabeth University Hospital, United Kingdom

Background & objectives: Intraosseous adenoid cystic carcinoma (ACC) is a rare neoplasm with only a small number of cases described in the literature. This report describes two cases of intraosseous ACC arising in the mandible, and discusses their presentation, imaging, histopathology and management.

Methods: The slides and case reports were examined and data was extracted including demographics, clinical presentation, histological and immunohistochemical features of each case. Further clinical information including radiological findings, clinical management and prognosis was obtained from our regional electronic patient record system. A literature review was performed.

Results: Both cases are those of middle-aged females, presenting with large lytic lesions involving the mandible. Histopathology of both cases showed the typical features of a solid adenoid cystic carcinoma and immunohistochemical stains and molecular studies were supportive of this. Imaging, including PET CT, highlighted metastatic disease at presentation in both cases. MDT discussion highlighted poor prognosis in each case.

Conclusion: Intraosseous salivary gland neoplasms, although rare, should be considered in the differential diagnosis for tumours within the mandible. They have a propensity for local recurrence and metastasis, and complete surgical resection therefore offers the best outcome. Their clinical and radiological features can be similar to odontogenic tumours. Accurate histological diagnosis is therefore crucial as well as close clinical and radiological correlation to delineate the most appropriate treatment for these patients.


Protocols of lymph node sampling in 95 cases of head & neck squamous cell carcinoma: is excessive concern for cost control leading to understaging and under treatment?

S. Sancheti*, P. Somal

*Homi Bhabha Cancer Hospital, India

Background & objectives: 1. To assess the sampling of large lymph nodes (LN) (>15mm) in Head and neck cancer as per recommendation & evaluate its effect on LN stage and prognostication.

2. Does under-sampling of larger grossly uninvolved LN compromise the patient care.

Methods: 95 cases of head -neck SCC with metastatic lymph nodes(MLN) were categorized into four groups as small nodes(submitted entirely), grossly positive LN, bisected LN and both halves show metastasis and LN which were bisected or trisected and only one slice shows metastatic disease. LN level and the 'N' stage were noted. Retrospective analysis of its impact on staging was done.

Results: Nine cases out of ninety-five revealed that under-sampling of the MLN would have missed the detection of metastasis. Surprisingly, there would have been under-staging of LN stage in five out of these nine cases if the LN were not sampled entirely. This also changes the prognosis of the patient and will lead to false staging.

Conclusion: The gross and microscopic examination provides information about the number of nodes involved, size of the deposit and presence of extracapsular extension (ECE) for staging of the disease. Accurate sampling and thorough examination of LN is perhaps the most important contribution of a pathologist in the management of head and neck cancers. Adequate sampling should be the standard of practice to avoid compromise in patient care.

Larger grossly uninvolved LN should be submitted entirely for adequate staging.


Focal melanocytic lesions of the oral mucosa: an 18-year retrospective study

T. Santana*, A. Queiroz, L.M. Cerioni Gonçales, A. Fraga Costa Samos Paris, M. Trierveiler

*School of Dentistry, University of Sao Paulo, Brazil

Background & objectives: This study aimed to compile the focal lesions of melanocytic origin diagnosed at the Service of Surgical Pathology of the School of Dentistry of the University of São Paulo and to analyse their respective clinical and histopathological characteristics.

Methods: A cross-sectional study was conducted over an 18-year period. Patient information was collected from medical charts, and all archived histopathological specimens with diagnoses of oral melanocytic lesions were retrieved and reviewed.

Results: We identified 341 melanocytic lesions among 64,695 samples received for histopathological analysis. Of these, 191 were melanotic macule; 112 melanocytic nevi; 14 mucosal lentigo simplex; 12 oral melanomas; nine actinic lentigines and one melanoacanthoma. Lesions occurred mostly in women (n=218; 65.5%), with white skin colour (n=217; 74%). The main reported clinical aspect was macular (n=124; 67.6%). Melanocytic lesions occurred mainly in lip vermilion (n=83; 25.6%), followed by palate (n=74; 22.8%). The age of the patients was higher in melanoma patients and these cases had a shorter average duration until the time of diagnosis. Most lesions were small and only oral melanomas were larger in size when compared to other lesions.

Conclusion: Melanocytic lesions are an uncommon diagnosis in oral pathology routines. The most frequent lesions are melanotic macule and nevus. Patients with these lesions are usually white-skinned women presenting a small, long-lasting, macular lesion on lip vermilion or palate.


Expression of microRNA-31 in saliva-liquid biopsy of oral squamous cell carcinoma patients

P. Kumari, S. Syed*, M. Wahid

*Department of Oral Pathology, Dow Dental College, Dow University of Health Sciences, Pakistan

Background & objectives: Aberrant expression of microRNA-31 in saliva-liquid biopsy has been reported in various cancers. However, little is known about its expression in oral-squamous-cell-carcinoma (OSCC). We aimed to investigate microRNA-31 expression in pre-and post-surgical OSCC patients and its association with clinicopathological features.

Methods: We investigated salivary microRNA-31 expression in pre-surgical and post-surgical (6 weeks after surgery) patients with oral squamous cell carcinoma (n=16) and control (n=2) by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) after ethical approval from institutional review board (IRB).

Results: Salivary microRNA-31 expression was differentially regulated in pre- and post-surgical patients with oral squamous cell carcinoma and controls. MicroRNA-31 was significantly upregulated in pre-surgical cases (p<0.05). However, the expression was significantly reduced after 6-weeks of surgical excision of tumour, indicating that most of the microRNA-31 derived from oral squamous cell carcinoma tissues. Moreover, no significant association was found between pre-surgical microRNA-31 expression level and age, gender, site, habits, pathological TNM staging and grading.

Conclusion: Our findings showed increased expression of microRNA-31 in saliva-liquid biopsy, suggesting microRNA-31 an important player in pathogenesis of oral squamous cell carcinoma.


HRAS mutation is a surrogate diagnostic marker in challenging cases of epithelial-myoepithelial carcinoma

C. Taverna*, A. Franchi, A. Agaimy

*Division of Pathological Anatomy, Department of Health Sciences, University of Florence, School of Human Health Sciences Florence, Italy

Background & objectives: Epithelial-myoepithelial carcinoma (EMC) is a challenging diagnosis for the wide variety of patterns. We analysed HRAS status in 3 cases of EMCs with predominant solid-oncocytic and sebaceous features, as Urano et al (2019) demonstrated HRAS mutation in 82.7% of EMCs.

Methods: Patients were 2 males and 1 female aged 69-75 years. Two cases presented prominent solid oncocytic pattern and 1 was characterized by extensive sebaceous differentiation (initially diagnosed as sebaceous carcinoma). HRAS sequencing were analysed in 3 cases using a single base primer extension approach (SNaPshot assay) on an ABI Prism 3500 genetic analyser. Fragment analysis was performed using GeneMapper software.

Results: All 3 cases analysed for mutations in HRAS showed positive results. The site of mutation was exon 2 (p.Gly13Arg; c.37G>C) in one oncocytic EMC, while the same mutation in exon 3 (Q61R; c.182A>G) was found in the second oncocytic case and in the sebaceous EMC.

Conclusion: EMCs with prominent unusual variants of histologic features can be frequently confused with other entities with different behaviour and prognosis. We demonstrated that HRAS mutations are present in these variant EMCs. Thus assessment of HRAS status is useful in salivary gland neoplasms that do not entirely fulfil clear diagnostic criteria for EMC and cannot otherwise be classified as other entities.


PD-L1 score and CD8 heterogeneity in head and neck squamous cell carcinomas

S. Tzorakoleftheraki*, V. Kotoula, K. Markou, S. Chrisafi, E. Daskalaki, E. Sgouramali, P. Hytiroglou, T. Koletsa

*Department of Pathology, School of Medicine, Aristotle University of Thessaloniki, Greece

Background & objectives: In an effort to further characterise head and neck squamous cell cancer (HNSCC) phenotypes relevant to the currently applied immunotherapy with checkpoint inhibitors (ICIs), we profiled PD-L1 status, CD8+ lymphocytic infiltrate density and their heterogeneity in these tumours.

Methods: We evaluated p16 protein expression (positive/negative), combined PD-L1 positivity score (CPS, clone 22C3) and CD8+ lymphocyte density/mm^2 in 228 HNSCC (52.5% laryngeal supraglottic/glottic/subglottic; 19.7% oral cavity; 12.2% oro-hypopharyngeal; 15.6% other) informative for all 3 markers. CD8+-density was analysed as continuous variable. Immunohistochemistry was performed on tissue-microarray sections with multiple cores per tumour, allowing for the assessment of intra-tumour heterogeneity.

Results: Out of 228 HNSCC, 16 were p16 positive (7.0%), half of them oro-hypopharyngeal; 37 (16.3%) were PD-L1 positive with CPS ≥1, while 7 of them with CPS ≥20 (high PD-L1 expression level); median CD8+-density was 204.9 (interquartile-range 105.0-437.2). Twenty-two (9.6%) and 48 (26.7%) tumours exhibited heterogeneous PD-L1 status and CD8+-density, respectively. p16-positive tumours demonstrated higher CD8+-density (Wilcoxon rank-sum p=0.0022), but none of them expressed PD-L1 at high levels (Pearson’s p=0.0008). PD-L1-positive tumours had higher CD8+-density (Wilcoxon rank-sum p<0.0001); the latter did not differ for PD-L1 scores 1-19 and ≥20. PD-L1-positivity was associated with CD8+-heterogeneity (Fisher’s exact p=0.0005), particularly concerning tumours with high PD-L1 expression level (p<0.0001), independently of tumour location.

Conclusion: PD-L1/CD8 profiles may distinguish previously described immunologically “hot” and immunosuppressed HNSCC. CD8+-heterogeneity may be related to different immunological priming by tumour subpopulations. PD-L1/CD8 assessment may further aid in the distinction of tumour immunological classification, particularly for predicting efficiency to ICIs.


Evaluation of high-risk human papillomavirus mRNA silver in situ hybridisation diagnostic assays in oropharyngeal squamous cell carcinomas

M. Volavšek*, M. Poljak, P. Strojan, D. Velkavrh, L. Hošnjak, K. Fujs Komloš, N. Zidar, L. Bolha, A. Aničin, N. Gale

*Institute of Pathology, Faculty of Medicine, University of Ljubljana, Slovenia

Background & objectives: Testing is required to distinguish human papillomavirus (HPV) driven oropharyngeal squamous cell carcinomas (OPSCC) with favourable treatment response and prognosis from HPV negative tumours. Recently developed RNA ISH probes complementary to HPV E6/E7 mRNA enable direct visualization of viral mRNA.

Methods: Sensitivity/specificity of high-risk (HR) HPV mRNA silver in situ hybridization (SISH) for HPV16, HPV18 and HPV33 was tested for the first time. 67 FFPE OPSCC and 3 papillomas from 62 patients diagnosed between 2006-2014 were tested with SISH, Abbott RealTime High Risk HPV test, in-house GP5+/6+/68 PCR test and p16 immunohistochemistry (IHC).

Results: Using HR-HPV PCR, 46 (69%) OPSCC were HPV+, with single HPV type in 44/46 (38 HPV16+, 2 HPV33+, 3 HPV35+, 1 HPV58+), 2/46 with co-infections (HPV16/X, HPV16/35). Of HR-HPV PCR+ OPSCC, 45/46 overexpressed p16, 43/46 were SISH+. Others were PCR HPV16+/p16-/SISH-, HPV35+/p16-/SISH-, HPV58+/p16-/SISH-.

Sensitivity/specificity/AUC with 95% confidence interval of combined SISH+ (HPV16/33) and p16 IHC compared to PCRs were 100.0%, 100.0% and 1.00. HPV+ group had favourable overall survival (p<0.001).

Conclusion: mRNA SISH methodology for FFPE tissue reliably detects HPV-driven OPSCCs without need for additional tests in most cases, with clearly positive signals easily detectable at 10x/20x magnification, enabling visualisation of viral transcripts required to recognise clinically relevant HPV infection. Compared to PCR, clear signals reliably exclude false-positivity, but rare/tiny signals require experienced pathologist or a team for consensus interpretation of results.

Funding: Roche Ventana provided mRNA probes for SISH


An audit to compare the turn-around times of oral and maxillofacial resection specimens including and excluding bone

H. Walsh*, D. Brierley

*Unit of Oral and Maxillofacial Pathology, School of Clinical Dentistry, University of Sheffield, United Kingdom

Background & objectives: To identify the turnaround times (TATs) for resection specimens and to assess for compliance with The Royal College of Pathologists Key Performance Indicators that 80% of resection cases (excluding those with bone) should be reported within 10 calendar days.

Methods: Two cycles of the audit were completed. All resection specimens (including and excluding bone) over 3 separate and comparable 3-month periods were assessed. The average TATs and percentage of cases reported in both 10 working days and 10 calendar days was recorded.

Results: There have been significant improvements between cycles one and two with the average TAT for resection cases without bone decreasing from 12.9 to 10.4 calendar days. The percentage of resections without bone reported within the recommended timeframe increased from 31.4% to 57%. The average TAT for resections including bone decreased from 29.2 to 20.4 calendar days, although in both cycles 0% of cases were reported within 10 calendar days.

Conclusion: TATs for specimens including bone are significantly higher with decalcification processes being a major contributory factor. To improve patient care, work is required to achieve the target within the recommended timeframe. A possible solution includes use of alternative decalcification techniques.

PS-04 Dermatopathology


Acral melanomas in Singapore

I. Busmanis*, P.Y. Tang, L. Oon

*Singapore General Hospital, Singapore

Background & objectives: Singapore population has higher incidence of acral melanoma compared with Caucasians, with fewer BRAF mutations compared with cutaneous melanomas. Our Singapore study correlates molecular findings in acral melanomas with clinicopathological features and compares with other Asian data.

Methods: 195 cases of melanoma with molecular analysis for mutations in exon 15 of the BRAF gene and exons 9, 11, 13, and 17 of the ckit gene in genomic DNA by polymerase chain reaction amplification and direct Sanger sequencing were retrieved from Singapore General Hospital files (2007-2020

Results: Of 195 cases, 72(37%) were cutaneous, 69(35%) acral and 54(28%) mucosal. Of 69 acral tumours, 16/53(30%) were BRAF positive. Of 28 acral cases with ckit mutational analysis, 11/43(26%) were ckit positive. The majority of BRAF positive cases were of Chinese ethnic group (12/16), female (11/16), mean age 59 years, located at acral non-pressure sites (12/16) and foot (12/16). No significant differences in the incidence of ulceration were seen between the BRAF positive (52%) and BRAF negative (42%) tumours, and also ckit positive (55%) and ckit negative (63%) tumours. The incidence of nodal metastasis was markedly different between ckit positive tumours (55%) compared with ckit negative tumours (33%).

Conclusion: The BRAF positive mutation rate in Singaporean ALM was comparable with data from China, Korea, Taiwan and Japan (range 25-42%). Our findings suggest a possible relationship between ckit mutation and the incidence of nodal metastasis.


Histopathologic features of dermatologic eruptions due to immune checkpoint inhibitors [monoclonal antibodies targeting cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), programmed cell death protein 1 (PD-1) or programmed death ligand 1 (PD-L1)]

C. Cabañuz Rocatallada*, V. Baena Romero, M.J. Beato-Merino

*Hospital Universitario La Paz, Spain

Background & objectives: Checkpoint inhibition induces immune-related adverse events. Skin toxicity is one of the most common, mostly as a maculopapular rash, less frequently as vitiligo, psoriasis and auto-immune diseases. This review describes the histopathologic aspects of dermatologic irAEs induced by checkpoint inhibitors.

Methods: We reviewed19 biopsies, obtained from 16 patients who were referred to the Dermatology Department of “La Paz” University Hospital, a tertiary care hospital. All patients had developed dermatologic toxicity while receiving either anti-PD-1, anti-PDL1, or anti-CTLA4 agents, from January 2017 to January 2020.

Results: Patients were 9 males and 7 females, with a medium age of 64 years. Lung carcinoma was the most common tumour being treated, followed by gastric adenocarcinoma and melanoma. In twelve biopsies the patient was on anti-PD-1 agents, in 4 on anti-PDL1, and in 3 on anti-CTLA4 plus anti-PD1 or anti-PDL-1 treatment.

The most common histopathologic pattern was “interface dermatitis”, in 10 biopsies (52,6 %), which was lichenoid in 6 of them. Other patterns observed (sometimes overlapped) were: psoriasiform (15,3%), spongiotic,vesiculobullous (bullous pemphigoid and Grover’s disease) and urticarial (10,5% each). One patient with melanoma, showed loss of melanocytes, which was interpreted as vitiligo.

Conclusion: The results reveal that histopathologic features of reactions secondary to check point inhibitors therapy arequite heterogeneous. Clinical correlation is needed for definitive diagnosis. Despite interface dermatitisis the most common pattern, florid lichenoid pattern was observed in just 6 biopsies (31,6%). It is important that doctors are familiar with these increasing adverse effects, quite unknown to date, for the correct management of patients.


Subcutaneous sarcoidosis: a series of 8 cases

M.I. Cevallos*, Í. Martínez de Espronceda Esquerro, Y. Rodriguez, M. Bronte, G. De Lima Piña, S. Oscoz Jaime, C. Llanos, A. Cordoba

*Department of Pathology, Complejo Hospitalario de Navarra, Spain

Background & objectives: Subcutaneous involvement has been described in up to 16% of patients with cutaneous sarcoidosis. It consists of sarcoid granulomas affecting mainly subcutaneous tissue and manifests clinically as erythematous, flesh-colored, violet or hyperpigmented nodules, mostly in upper limbs.

Methods: We reviewed our institutions' archives from 2013 to 2019 and found eight cases. Histopathological diagnosis of subcutaneous sarcoidosis was demonstrated by the presence of non-casefying granulomas in subcutaneous tissue while excluding other causes of granulomatous panniculitis.

Results: Patients’ ages ranged between 33 and 76 years old. Six had nodules in limbs, one in scalp and one in face and breast. Seven patients presented with subcutaneous sarcoidosis at the beginning, being the only sign of disease in four of them. Two cases showed other sarcoid cutaneous manifestations and two others, bilateral hilar adenopathies. Two cases had history of dermatomyositis under treatment and another, prostate adenocarcinoma with hormone therapy.

Conclusion: Our series' results are consistent with current literature. It is important to recognize subcutaneous sarcoidosis since this would allow earlier diagnosis and treatment of systemic disease. It is remarkable that three patients had history of immunosuppressive therapy; also reported as cases of subcutaneous sarcoidosis related to this type of treatment.


Nevus sebaceous with associated uncommon pathologies

Q. Chundriger*, M.U. Tariq, S. Moeen, S. Fatima

*Aga Khan University Hospital, Pakistan

Background & objectives: The obectives were to study clinicopathological features of Nevus Sebaceous and associated other pathologies – benign and malignant, reported from 2010 till February 2020 in the section of Histopathology at our University Hospital.

Methods: We retrieved ten year data from our archives, using our Integrated Laboratory management system software, by using words “Nevus Sebaceous”. Demographics including age, gender, site and presence of other pathologies was recorded. Frequencies of benign and malignant lesions were calculated.

Results: 111 cases of Nevus Sebaceous were reported from 2010 till February 2020. Age ranged from 3-85 years (mean age 31.2 years). 74.7% of patients were above 20 years. 57.6% were male and 40.5% females. 45.9% were located on scalp, face (42%), ear (4.5%), back (1.8%) and neck (0.9%). Site was unknown for 8 cases (7.2%). 23/111 cases (20.7%) showed associated lesions. These included Syringocystadenoma Papilleferum (39.1%), Epidermal Nevus (17.4%) and Trichoepithelioma (8.7%). One patient each had Syringoma, Solar Lentigo, Actinic Keratosis, Tumour of Follicular Infundibulum, Intradermal Nevus and Hemangioma. Two showed metaplastic bone formation and Amyloidosis respectively. Two patients (1.8%) had Basal Cell Carcinoma, one of them below 20 years.

Conclusion: Knowledge of the probability of encountering other benign or malignant lesions arising within a nevus sebaceous is essential for correct diagnosis, as they might be missed due to inadequate sampling, which may adversely affect patient management and follow up.


Primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder - the presence of dual lineage rearrangements

A. Cordoba*, M.D.R. Mercado, C. Llanos, A. Larrea, I. Yanguas, J. Mitxelena, I. Fernandez, D. Guerrero-Setas

*Complejo Hospitalario de Navarra, Spain

Background & objectives: The Primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder (PCSMP-TLPD). It is included in the Peripheral T -cell lymphoma with follicular T helper phenotype; which are the providers of T- cell help to B cells for the development of germinal centres

Methods: We have studied the cases diagnosed in our hospital from 2012 until now. We have reviewed 16 cases and analysed them with CD3, CD4, CD8, CD7, CD10, Bcl6, PD-1, CD20, CD38, Kappa and lambda. Polymerase chain reaction (PCR) was performed to analyse clonal expansion of T and B cells.The mean age at diagnosis was 58.6 years (range 31-85).

Results: There was a women predominance (10 women out of 16 cases). Histologically, the lymphoid infiltrated shows nodular arrangement; a very focal epidermotropism and extended folliculotropism. By immunohistochemical analysis, all cases showed a mixed population of T-cells that expressed T helper phenotype, and B cells.

All are cases were positive for TCR gene arrangements, but 4 of 10 cases tested were also positive for IgH gene rearrangements and one was pseudoclonal.

Conclusion: -The mixed nature T and B could make a challenging diagnosis in PCSMPTLPD.

-The presence of dual lineage rearrangements in cutaneous lymphoproliferative disorders is documented but rare phenomenon.

-The presence of two distinct monoclonal populations could confirm the reactive nature.


A 5-year retrospective study reviewing the histopathological features of cutaneous and subcutaneous malignant melanoma metastases

M. Craescu*, T. Georgescu, A.C. Lisievici, M. Leventer, T. Tebeica

*Colentina Clinical Hospital, Pathology Department, Romania

Background & objectives: Melanoma can metastasize either by lymphatic or haematogenous route and may develop either satellite, in-transit, regional lymph node or distant metastases. Cutaneous and subcutaneous metastases can be difficult to diagnose in the absence of clinical suspicion or hallmark histopathological characteristics.

Methods: In this study, we reviewed the medical records of 21 patients who presented in our Dermatopathology unit with cutaneous and subcutaneous metastatic melanoma during a period of five years (2014-2019). We thoroughly re-examined clinical photos, Hematoxylin-Eosin and immunohistochemistry slides, aiming to identify characteristic histological features.

Results: Most tumours presented as nodular proliferations of atypical epithelioid cells with abundant clear or eosinophilic cytoplasm, hyperchromatic, atypical nuclei with prominent nucleoli and high mitotic index. Approximately half of them presented with pigment formation. Four of them presented necrosis and only two of them presented epidermotropism. One particular case, had a blue nevus-like appearance, with round-oval and spindle cells, subtle atypia, fibrous stroma, varying pigmentation and extremely rare mitoses. This study included patients with various clinical presentations, from a 24-year-old male with no history of melanoma, to a 74-year-old male with two concurrent metastases.

Conclusion: Although they are a common occurrence in Dermatopathology, up to 10% of melanoma metastases appear in the absence of a previously diagnosed or currently detectable primary tumour. We identified common histological hallmarks and rare features such blue nevus-like metastasis.


Malignant melanoma arising in tattooed areas: report of two cases

M. Craescu*, T. Georgescu, A.C. Lisievici, G. Dodan, T. Tebeica

*Colentina Clinical Hospital, Pathology Department, Romania

Background & objectives: Malignant melanoma is an aggressive type of skin cancer, with a high risk of metastasis in late stage disease, making early diagnosis essential. The relationship between melanomas and tattoos is still unclear.

Methods: We report two cases of a 33-year-old and a 35-year-old patient, both males, each presenting with a cutaneous melanocytic lesion on a tattooed area, located on the upper extremity. We examined the Hematoxylin-Eosin slides and clinical photos of the lesions.

Results: We present two extremely rare cases, both from a clinical perspective (tattooed area) as well as a histopathological one (nevoid melanoma and completely regressed melanoma), both of them historically documented with clinico-pathological images.

First case was a naevoid melanoma with 2.9 mm Breslow index (pT3a) and Clark level IV. Low-power appearance was that of an intradermal nevus with hypercellular dermal papillae featuring “puffy-shirt-sign” with 3 mitoses/mm², anisomorphic nuclei, 1-3 nucleoli and no maturation. Second case was a completely regressed melanoma which presented as tumoral melanosis, with dense fibrosis and lymphoplasmacytic inflammation. Breslow maximum depth was of 8.1 mm, corresponding to Clark level V.

Conclusion: Despite paucity of literature data regarding malignant melanoma in tattooed areas, establishing a clinico-pathological connection between the two could increase chances of early detection and therefore, effectiveness of treatment. Both lesions presented in young patients and were challenging to diagnose.


Primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder: one-year experience in one dermatopathology unit

M. Craescu*, T. Georgescu, A.C. Lisievici, A.M. Stanciu, G. Dodan, T. Tebeica

*Colentina Clinical Hospital, Pathology Department, Romania

Background & objectives: Primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder is characterized by a solitary cutaneous lesion on the face, neck or trunk, with excellent prognosis. According to 4th Edition of the Blue Books it is a provisional entity, with uncertain malignant potential.

Methods: In this retrospective study, we examined the clinical records of 4 patients diagnosed with Primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder in our Dermatopathology unit. We thoroughly reexamined clinical photos, Hematoxilyn-Eosin and immunohistochemistry slides (CD3, CD20, CD4, PD-1, CD 8, BCL6, Kappa and Lambda).

Results: Each patient (median age 44-years-old), presented with a single lesion: three on the trunk and one on the forearm. In two cases, the lymphoid infiltrate featured a lichenoid pattern, predominantly affecting the papillary and upper reticular dermis. In the other cases, the lymphoid infiltrate was nodular and mostly affected the reticular dermis. The lymphoid proliferation was constituted primarily of small-sized lymphocytes, with fewer medium-sized lymphocytes and peripherally situated plasma cells. One of the cases presented with epidermotropism, and in all of them, the infiltrate surrounded cutaneous adnexa. Immunohistochemically, all cases were positive for CD3, CD4, PD-1 and negative for CD20 and CD8.

Conclusion: We highlight the importance of recognizing this entity in order to avoid misdiagnosis as lymphoma and subsequent over-treatment. In the case of Primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder, intralesional steroids or simple excision are sufficient, with rare recurrences.


Slow Mohs micrographic surgery in dermatofibrosarcoma protuberans: 6 years experience in one dermatopathology unit

M. Craescu*, T. Georgescu, A.C. Lisievici, A.M. Stanciu, M. Leventer, T. Tebeica

*Colentina Clinical Hospital, Pathology Department, Romania

Background & objectives: Dermatofibrosarcoma protuberans is a soft tissue sarcoma with aggressive local behaviour and high recurrence risk. Due to its seemingly benign cytological appearance and irregular pattern of invasion, some cases may raise difficulties in determining whether the surgical margins are clear.

Methods: We present 12 cases of dermatofibrosarcoma protuberans which were surgically removed using Slow Mohs micrographic surgery. All surgical specimens were intraoperatively marked by the surgeon and then photographed, inked and sectioned in our pathology lab, according to our slow Mohs protocol. All sections were examined using Hematoxylin-Eosin and CD34 immunolabeling.

Results: Patient age was 15-55 years, with male predominance (58%). Tumours presented either on the torso or head and neck area, apart from one, located on the foot. Two cases presented with clear margins after first excision, the rest having positive deep excision margins. The highest number of interventions was 5, the average being 3. One case presented with fibrosarcomatous transformation, featuring hyperchromatic nuclei and 21 mitoses per 10 HPF.

Our experience to date demonstrates that Mohs microsurgery combined with immunohistochemical evaluation is a reliable method of providing patients with complete surgical excision, especially when facing tumours in challenging locations, which are growing between nerves and/or tendons.

Conclusion: Slow Mohs micrographic surgery offers the surgeon a histological confirmation before closing the wound. If the resection margins are positive, the surgeon can further remove the areas marked by the pathologist, sparing as much healthy tissue as possible.


Melanocytic nevus and osseous metaplasia: pathological features of 5 cases

C. D'Angelo*, C. Torre Carrera, A.L. De la Guardia Rodriguez, E. Sobrino Reig, P. Gomez Iglesias, M.d.l.Á. Muñóz Fernández

*Hospital Universitario de Mostoles, Spain

Background & objectives: Wide variety of histopathological changes may be found in melanocytic nevi. Osseous metaplasia represents an unusual finding, sometimes misunderstood or overlooked, characterized by foci of bone formation adjacent to or surrounded by melanocytic nests exhibiting a normal maturation pattern.

Methods: We conducted a retrospective review, using our database from the last 15 years. Clinicopathologic features were gathered: age, sex, location, nevus type, intra or extralesional ossification and presence of dermal inflammation. Five cases were identified, four osteonevi of Nanta and one blue osteonevus.

Results: There were three men and two women. The ages ranged from 32 to 81 with a mean age of 53 years. Of these nevi, only one had congenital features, four showed presence of fatty marrow, three were intradermal and two compound, and all were on the upper part of the body. There were two of them with inflammation associated, and one with foreign-body granuloma. Four cases presented intralesional bone formation.

Conclusion: To our knowledge, this represents the largest series of such cases in Spanish literature, and the fourth example of blue ostenevus ever reported. Bone formation in nevi represents an unusual finding, and only small series have been reported. We confirmed the upper body location, intradermal type predominance and age span, similar to literature. In contrast to previous series, we found a slight predominance in males. The pathogenesis of osseous metaplasia in nevi is not fully understood and requires further study.


Neuronevus of masson: four cases with typical clinicopathological features

F.J. Díaz de la Pinta*, C. Santonja Garriga, M.D. Suarez Massa, C. Saus, I. Eraña Tomás, L. Carrasco Santos, L. Requena Caballero

*Pathology Department Hospital Fundacion Jimenez Diaz, Spain

Background & objectives: The so-called “blue neuro-nevus of Masson” or "Masson neuronevus" (MN) is a benign variant of the cellular blue nevus that due to its cellularity and abundant pigment is sometimes misdiagnosed as melanoma.

Methods: We present four cases obtained from the files of our hospitals.

Results: The patients were three women and a man, whose ages were 8, 19, 35 and 50 years. All lesions were localized on gluteal areas, consisting of asymptomatic, brownish to greenish nodules, one of them presumed to be since birth.

Histologically all cases were circumscribed tumours situated in medium and deep dermis, with intact epidermis, growing in a fascicular pattern, with a “hourglass” configuration.

Cytologically they were composed of ovoid and/or spindled cells, surrounded by fibrous tissue containing sparse dendritic cells and melanophages, without mitotic figures. All of them where immunoreactive for S100, SOX10 and HMB45 (the latter only focally at the periphery). Betacatenin was negative in all of them.

Conclusion: MN can mimic melanoma because of the size, the involvement of deep dermis and the abundance of melanophages. The absence of mitosis (or <1/mm²), necrosis and atypia, without infiltrative margins supports the diagnosis. Unlike deep penetrating nevus, Beta-catenin is absent.


Blastic plasmocytoid dendritic cell neoplasm: a rare cutaneous lymphoma - analysis of 3 cases

C. Faria*, M.J. Julião, J. Fraga, F. Ramalhosa, J. Gama, J. Madeira, J.C. Cardoso, M.A. Cipriano

*Centro Hospitalar e Universitário de Coimbra, Portugal

Background & objectives: Blastic plasmocytoid dendritic cell neoplasm represents less than 1% of cutaneous lymphomas, being more common in males. Although some cases are indolent, the course is often aggressive with involvement of the bone marrow, peripheral blood and lymph nodes.

Methods: We report the cases of three male patients with ages between 57 to 85 years old, with diagnosis from 2013 to 2019, that presented multiple purpuric, painless and non-ulcerated nodules on the trunk, extremities and face. All patients didn't present systemic symptoms.

Results: Histopathological examination revealed a dermal infiltrate by a population of medium-sized cells with nuclear pleomorphism, several nucleoli and a moderate to scant cytoplasm. The mitotic count was high, and the epidermis was spared. In one case there was a lobular pattern extended to the subcutis.

Immunohistochemistry study showed positivity for CD4 and CD56 in all cases; however, in one case it revealed focal positivity for LCA and CD43 that could lead to a differential diagnosis of cutaneous infiltration by myelomonocytic leukaemia. CD3, CD20, TdT, CD138, CD30 and CD8 were negative.

All patients had involvement of the bone marrow and there was affection of the cervical lymph nodes in two cases.

Conclusion: The expression of CD56 and CD4 favoured the diagnosis of blastic plasmocytoid dendritic cell neoplasm in all cases and immunophenotyping by flow cytometry was concordant. The median survival varies from 10 to 20 months. In our study, one patient died three months after the diagnosis and the others showed initial response to chemotherapy; however, advanced age, negative expression of TdT and bone marrow involvement have a negative impact on prognosis.


Cutaneous leukemic infiltrates: unusual manifestation of haematological diseases - study of 4 cases

C. Faria*, M.J. Julião, J. Fraga, M.B. Pimentão, V. Almeida, A. Lai, R. Almeida, H. Moreira, J.C. Cardoso, M.A. Cipriano

*Centro Hospitalar e Universitário de Coimbra, Portugal

Background & objectives: Leukaemias are neoplastic proliferations in the bone marrow and peripheral blood that rarely involve the skin as non-specific lesions like purpura, vasculitis and vesicular eruptions that may simulate acantholytic dermatoses. Reports of specific involvement have been documented in several subtypes.

Methods: We describe the cases of three males and one female patients with ages between 44 to 78 years old, that had previous diagnosis of chronic myeloid leukaemia and myelomonocytic leukaemia. Now they present multiple non-symptomatic purpuric to reddish cutaneous lesions in the scalp, trunk, arms and legs.

Results: Histopathological examination revealed a diffuse and interstitial dermal infiltrate composed by atypical round to polygonal cells with slight nuclear pleomorphism with dispersed chromatin and small nucleoli; cytoplasm was moderate to scant. High mitotic count, apoptosis and epithelial hyperplasia with mild acanthosis were present in all cases.

Immunohistochemistry study showed positivity for LCA, CD34, CD117 and myeloperoxidase in two cases, favouring the cutaneous involvement by chronic myeloid leukaemia. Diffuse positivity for CD4, CD56, CD68, CD163 and lysozyme with negativity for CD34 and CD117 in the other two patients, suggesting cutaneous infiltrate by chronic myelomonocytic leukaemia. CD3, CD5, CD20, CD79 and CD30 were negative in all cases.

Conclusion: Most cutaneous involvement by myeloid leukaemia follow an aggressive clinical behaviour. In our analysis, two patients died in less than six months following the appearance of the cutaneous lesions despite systemic chemotherapy. Both cases were from elderly patients (>70 years old): one with chronic myelomonocytic leukaemia that had transformation to acute myeloid leukaemia and other with chronic myeloid leukaemia. Advanced age is also associated with shortened survival.


Kaposi sarcoma: a 3-year retrospective study with emphasis on epidemiology in a non-endemic country

T. Georgescu*, A.C. Lisievici, M. Craescu, A. Stanciu, A. Stancu, B. Simona, O. Voinea, T. Tebeica

*Department of Pathology, "Carol Davila" University of Medicine and Pharmacy; Department of Pathology, "Dr. Leventer Centre", Bucharest, Romania

Background & objectives: Kaposi sarcoma (KS) is a malignant vascular proliferation usually encountered in elderly middle eastern men, young African-American and patients with iatrogenic immunosuppression or AIDS. Patients usually present with multiple coalescent red macules or nodules located on the distal lower extremities.

Methods: This is a retrospective study including 32 cutaneous KS diagnosed in our unit during a period of four years (2016-2019). We aimed to analyse the epidemiologic features and establish clinico-pathological correlations regarding KS diagnosed in Romania. Clinical information was retrieved from the virtual database. We thoroughly re-examined all Hematoxylin-Eosin and immunohistochemically (HHV-8, CD34, D2-40, SMA) stained slides.

Results: Patient age ranged from 25 to 85 years. Lower extremities were frequently involved (55.2%), followed by upper extremities (27.6%), face (10.3%) and buttocks (6.9%). The overwhelming majority of the patients were males (72%) and nodular stages of the disease represented 65.5% of cases. Clinical diagnosis varied from Kaposi’s sarcoma (58%), haemangioma (10.3%), carcinoma (20.7%), sarcoma (7%) and melanoma (3.4%). “Promontorium sign” was present in 60% of patches and plaques cases. Clinical diagnosis accuracy increased in patients who already developed nodules.

Conclusion: Most cases in our study consisted of classic Kaposi’s sarcoma developing on acral sites, with only one exception encountered on the face of a young male. In our experience, KS should be considered when encountering vascular lesions on acral sites.


Slow Mohs micrographic surgery in extramammary paget disease: 3-year experience in one dermatopathology unit

T. Georgescu*, A.C. Lisievici, M. Craescu, S.A. Barbu, G. Dodan, M. Leventer, T. Tebeica

*Department of Pathology, "Carol Davila" University of Medicine and Pharmacy, Department of Pathology, "Dr. Leventer Centre", Bucharest, Romania

Background & objectives: Extramammary Paget disease (EMPD) is known to have a local recurrence rate of 34-40% after surgical excision, while the recurrence rate after Slow Mohs micrographic surgery is only 12%. This way, the wound is left open, awaiting the pathological report.

Methods: We re-evaluated 4 cases of extramammary Paget disease which were cured in our unit (2016-2019) using this technique. All specimens were received with marking sutures. Excision margins were inked, sectioned, mapped and submitted in separate blocks, in regard to the suture mark. All resulting slides were evaluated using Hematoxylin-Eosin staining and CK7 immunolabeling.

Results: All patients were females in their sixties and most lesions were located on the labia (75%) or in the perianal area (25%). In 50% of cases, the lesion spread in opposite directions, requiring multiple excisions per intervention. The total number of blocks used ranged from 18 to 38. First interventions required larger excisions (40/21/3 mm to 115/85/10 mm), while the last ones were much smaller (16/9/2 mm to 42/8/2 mm). Patients required 3 to 5 reinterventions in order to achieve curability. No case featured dermal invasion or signs of recurrence to date. This technique combined with immunohistochemistry is a reliable technique which increased the curability of EMPD in our unit.

Conclusion: Slow Mohs micrographic surgery allowed the clinician to remove a targeted area with residual tumour located between specific hour intervals, thus sparing healthy tissue. In this manner, many reinterventions can be done, in order to achieve curability.


The MANIAC: pagetoid spread in benign acral nevi

T. Georgescu*, A.C. Lisievici, M. Craescu, G. Dodan, M. Leventer, T. Tebeica

*Department of Pathology, "Carol Davila" University of Medicine and Pharmacy, Department of Pathology, "Dr. Leventer Centre", Bucharest, Romania

Background & objectives: Melanocytic acral nevus with intraepidermal ascent of cells (MANIAC) was first described by LeBoit et al. in 1997. Although the lesion is characterized by suprabasal scatter of single melanocytes, it is considered benign and has good outcome.

Methods: In this study we re-evaluated the clinical records of 8 patients diagnosed with MANIAC in our Dermatopathology unit within the last 3 years. Average age was 20.5 years. Upon follow-up, none of the lesions recurred or progressed. We thoroughly re-examined the slides and analysed the clinical data retrieved from the virtual database.

Results: Most lesions were located on the toes, followed by the lateral aspects of the foot and palms. Histologically, 75% were compound and 25% were junctional nevi. 50% were well-circumscribed, 25% were moderately well-circumscribed, and 25% were poorly circumscribed. No cytological atypia or lymphocytic infiltrate were noted. All lesions featured intraepidermal ascent of melanocytes throughout the entire epidermis, and 25% of the cases even showed rare melanocytes within the cornified layer as pigmented columns above the sulci or crista profundae intermediae. Well-circumscribed and symmetric lesions occurring in young adults which feature maturation and lack cytological atypia or brisk lymphocytic infiltrate usually manifest as benign nevi.

Conclusion: Practicing pathologists should be aware that suprabasal scatter of melanocytes is not pathognomonic for melanoma in acral nevi without other worrisome features like lateral confluence of junctional nests, cytological atypia or lymphocytic infiltrate.


Merkel cell carcinoma of an unknown primary origin, 5 case report

I. Guvendir*, I.E. Zemheri, M. Ozcelik

*Health Sciences University, Umraniye Education and Training Hospital, Pathology Department, Turkey

Background & objectives: Merkel cell carcinoma(MCC) is a rare neuroendocrine tumour of the skin. MCC of an unknown primary origin(MCCUP) is much rarer; approximately 200 cases have been reported in literature. In this study,5 patients diagnosed with MCCUP in the lymph node in our hospital between 2015-2019.

Methods: The age distribution was 69-84(mean=75.8),3 were female;2 were male.2 patients had axillary; 3 patients had involvement in the inguinal region lymph nodes. All patients had skin examinations; no primary skin lesion was found. Diagnosis was made from axillary in 2 patients and inguinal lymph in 3 patients. The diameter of the lymph nodes assessed macroscopically was 6.42 cm(average).

Results: In the examination after diagnosis,3 patients were identified as pT0NxM1a Stage 4 and 2 patients as pT0N1M0 Stage 3.2 patients with stage 4 received palliative chemotherapy+radiotherapy,1 patient received palliative radiotherapy.2 patients with stage 3 had definitive surgery. While 3 patients with stage 4 were exitus,2 patients with stage 3 didn’t develop locoregional recurrence or distant metastasis, one of them was followed for 3,5 years and the other for 9 months without disease.

Conclusion: Polyomavirus, accused in the aetiology of MCC, was detected more rarely in MCCUP (76% vs 31% P = 0.001), and these two entities were thought to use different biological pathways. MCCUP is a tumour that shows a better progress compared to metastatic MCC and has different views on treatment. We presented our cases reported in a single centre for the purpose of contributing to the literature with clinical, histological and treatment methods.


Study of the sebostatic effect of retinoic acid on rat sebaceous glands

M. Kostyaeva*, B. Viktor, I. Kastyro, E. Irina, I. Zhuk, Y. Ondar, M. Grinberg

*Peoples' Friendship University of Russia (RUDN University), Russia

Background & objectives: Morphological changes in the skin with acne are characterized by acanthosis, parakeratosis and intercellular oedema in the epidermis, lymphocytic infiltration of the dermis and sebaceous gland (SG) hypertrophy. Using retinoic acid reduces the synthetic activity of SG cells.

Methods: 10 Wistar male rats (ExpG) were applied a 0.05%solution of retinoic acid on the clipped surface of the skin daily, for 10 days in the form of an application. The control group (CG) included 10rats of the same line. Histological sections were stained with H&E. 5 section was examined from each animal, in which up to 30 SG profiles were measured.

Results: The histostructure of SG of ExpG was distinguished by polymorphism. The gland profiles are visually unevenly reduced. The cytoplasm of individual sebocytes loses the cellular structure, the cells look like undifferentiated. Morphometric studies confirmed the results of visual assessment. A retinoic acid solution significantly reduces the average area of SG profiles (3323.42±82.65) in the section compared to CG (3940.19±67.12) (p<0.001). The proportion of undifferentiated sebocytes in the area of SG increases(p<0.001).

Conclusion: Applications with retinoic acid have a pronounced sebostatic effect, manifested in the reduction of sebaceous glands and an increase in the area occupied by undifferentiated sebocytes. Sebaceous gland profiles are visually and morphometrically reduced. This minimizes the etiological factor of acne.


Pathohistological examination of early detection of skin melanoma by ytterbium porphyrin complex gel

A. Nesterova, O. Olisova, E. Orlova, L. Smirnova, E. Shtemplevskaya, D. Kusraeva*

*Sechenov University, Russia

Background & objectives: The optimum diagnostics is the melanoma development in situ.

The present study considers the potential of melanoma fluorescent diagnostics by ytterbium porphyrin complex (YbPC) and consequent pathohistological examination of its effectiveness.

Methods: The method of using the laser consists in the ability of YbPC to accumulate well in tumour cells, they also have intense luminescence. First the pharmaceutical product containing Yb-2,4-dimethoxyhematoporphyrin IX is applied to the lesions. 30 minutes later we measured the luminescence intensity in the near infrared range (900-1100 nm) using the Onkoflurometer” laser device.

Results: The study involved a group of 28 patients aged from 19 to 67 years with dysplastic naevus as provisional diagnosis. The examinees were examined by means of dermatoscopy, a diagnostic laser with use of fluored gel containing YbPC, with exposure time of 30 minutes and evaluation of the depigmentosus part, the hyperpigmentosous parts in the centre and in peripheral zones.

Among 28 biopsies, 3 melanomas were detected at stages 1 and 2. However, there were no signs of melanoma during dermatoscopic examination

Conclusion: This method is highly sensitive and specific and can be implemented directly into the routine practice of specialists.


Melanocytic matricoma: report of two cases of a rare entity

S. Lerias*, D. Tomas, J. Costa Rosa

*Instituto Português de Oncologia de Lisboa, Francisco Gentil; Instituto de Anatomia Patológica, Faculdade de Medicina da Universidade de Lisboa, Portugal

Background & objectives: Melanocytic matricoma is an extremely rare benign tumour derived from the hair matrix and may be misdiagnosed because of its peculiar morphological features.

Methods: We report two cases of melanocytic matricoma: one from a 77-year-old man presented with a pigmented papule, 5 mm across, on the nose and the other from an 85-year-old female presented with an ulcerated dark-brown tumour, 12 mm across, on the right lower leg. We also review the literature.

Results: CASE 1: Microscopically, it was a nodular polypoid dermal proliferation of atypical basaloid cells, with prominent nucleoli. Some shadow cells were present. A conspicuous melanocytic proliferation was colonizing matrical areas. Mitosis: 18/10hpf(400x).

CASE 2: Histology showed a nodular dermal tumour, with prominent shadow cell formation. A conspicuous scattered proliferation of the melanocytes was also seen. Mitosis: 32/10hpf(400x).

Basaloid cells were cytokeratins+, p63+ and β-catenin+. Melanocytes were S-100+ and SOX-10+.

Conclusion: Melanocytic matricoma is a very rare entity with about 25 cases described in the literature. Its morphology can be confused with pilomatricoma, pilomatrical carcinoma with melanocytic hyperplasia or other malignant epithelial tumours. A prominent melanocytic proliferation and melanin in a tumour with cytological atypia and high mitotic count may lead to the erroneous diagnosis of malignant melanoma. Its distinction is important to ensure adequate treatment and follow-up.


A 3-year retrospective study reviewing the spectrum of orofacial granulomatosis: granulomatous cheilitis and Melkersson-Rosenthal syndrome

A.C. Lisievici*, T. Georgescu, M. Leventer, D. Iordache, M. Craescu, T. Tebeica

*Department of Pathology, "Carol Davila" University of Medicine and Pharmacy, Department of Pathology, "Dr. Leventer Centre", Bucharest, Romania

Background & objectives: Granulomatous cheilitis or Miescher cheilitis is a rare condition often related to hypersensitivity or atopy, characterized by protuberant swelling of one or both lips, and is sometimes associated with facial nerve palsy and fissured tongue, condition known as Melkersson-Rosenthal syndrome.

Methods: In this study we examined the clinical records of six patients presenting with granulomatous cheilitis which were examined in our Dermatopathology unit during a 3-year period (2016-2019). We thoroughly re-examined the slides, analysed the clinical data retrieved from the virtual database and correlated all the findings.

Results: Patients ranged from 28 to 60 years. Lower lip and upper lip were equally often involved, and males were slightly more frequently affected (66% males, compared to 33.3% females). One patient also presented with facial nerve palsy, consistent with Melkersson-Rosenthal syndrome. None of the patients had any previous history of Crohn disease. Upon histologic examination, all patients had non-necrotising granulomas, with mild dermal oedema and no sign of epidermal ulceration. If the histologic examination reveals non-necrotizing granulomas in the lamina propria, often protruding into and obstructing the vessels, then also Crohn disease, sarcoidosis or granulomatosis with polyangiitis should also be considered.

Conclusion: Granulomatous cheilitis is a rare entity. Taking into consideration the numerous reports of patients with orofacial granulomatosis and no gastrointestinal symptoms which were discovered with colonoscopic features consistent with Crohn disease, further check-up should be advised in such cases.


Sentinel lymph node excision in malignant melanoma: 3-year experience from one dermatopathology unit

A.C. Lisievici*, T. Georgescu, M. Craescu, F.T. Bobirca, T. Tebeica

*Department of Pathology, "Carol Davila" University of Medicine and Pharmacy, Department of Pathology, "Dr. Leventer Centre", Bucharest, Romania

Background & objectives: Sentinel lymph node (SLN) technique is used worldwide in patients with invasive breast carcinoma or malignant melanoma. It is recommended for patients with melanoma stage pT2 or pT3 and it could be considered in patients with pT1b or pT4 melanomas.

Methods: In this study we retrospectively analysed 84 patients which underwent SLN excision between 2017 and 2019, after being diagnosed with malignant melanoma. Each lymph node received in our pathology department was sectioned at 2-3 mm intervals and entirely submitted. All resulting paraffin embedded sections were examined at multiple levels in Hematoxylin-Eosin, Melan A and Tyrosinase.

Results: Out of 84 cases, 26.5% had lymph node metastases, and 12.05% had incidental capsular nevi. Positive SLN stemmed from pT2 cases (14.3%), pT3 (52.3%) and pT4 (33.3%). Most of them were axillary (54.5%), followed by inguinal (31.8%). Patients age ranged from 30 to 77 with both genders equally involved. Most cases featured one affected lymph node (75%), while 21.8% had two involved and only 3.2% had more than 2 affected.

Conclusion: This technique can provide important information which should be considered in the follow-up and treatment of patients. In our study size of the metastatic deposit ranged from 0.1 mm to 12 mm and only 4.7 % had extranodal extension. Statistical analysis showed that the number of lymph nodes involved, size of the metastatic deposits and the presence of extranodal extension strongly correlated with more advanced stage of disease (pT4) and presence of ulceration in the primary tumour.


Nodulotumoral cutaneous involvement in adult T-cell leukaemia/lymphoma: report of two cases

A.C. Lisievici*, T. Georgescu, M. Craescu, M. Mihai, T. Tebeica

*Department of Pathology, "Carol Davila" University of Medicine and Pharmacy, Department of Pathology, "Dr. Leventer Centre", Bucharest, Romania

Background & objectives: Adult T cell leukaemia/lymphoma (ATLL) is a rare lymphoproliferative disorder, encountered in HTLV-1 carriers, which may be associated with cutaneous manifestations, ranging from patches, plaques, nodules to erythrodermia. Patients with the lymphoma-type variant of the disease have a worse prognosis.

Methods: We report two cases of ATLL, one in a 44-year-old male, known with systemic disease and HTLV-1 infection which developed cutaneous nodules, and one in a 66-year-old male, who presented with cutaneous manifestations, without any prior relevant medical history. Both patients had multiple red, tender nodules on the forearms and hands, which were clinically suspicious for cutaneous lymphoma.

Results: Both excision specimens revealed a diffuse intradermic lymphoid proliferation composed of medium-large cells, with marked epidermotropism and angiotropism. The lesions from the patient known with systemic disease also featured central ulceration. Immunohistochemistry revealed CD3, CD4 and CD5 positive cells, which were negative for CD8. Both lesions had a KI67 proliferative index of 50%. Unfortunately, both patients died within the next 4 to 6 months.

Conclusion: We report two cases of cutaneous manifestation in patients with lymphomatous and acute ATLL variants, respectively. The only difference between the two lesions was that the one originating from the patient with systemic disease also had central ulceration and the cells were immunoreactive for CD30. The patient with no prior medical history was positive upon subsequent HTLV-1 testing. Both cases had an aggressive clinical course, associated with nodulotumoral presentation of the disease.


Dermatomyofibroma: a short series of a great mimicker

A.C. Lisievici*, G. Tiberiu-Augustin, B. Simona, M. Craescu, G. Dodan, A. Stanciu, T. Tebeica

*Department of Pathology, "Carol Davila" University of Medicine and Pharmacy, Department of Pathology, "Dr. Leventer Centre", Bucharest, Romania

Background & objectives: Dermatomyofibroma is a rare benign neoplasm, first described by Kamino et al. in 1992, histologically composed of spindle cells of fibroblastic/myofibroblastic origin. Patients are typically young females presenting with a plaque on the neck, shoulders and upper trunk.

Methods: In this study we examined the clinical records of five patients diagnosed with dermatomyofibroma in our Dermatopathology Department during a 3-year period. We thoroughly re-examined the slides and analysed the clinical data retrieved from the virtual database. Histologic examination reveals fascicles of spindle cells arranged parallel to the epidermis. Ancillary studies included S100, SMA and CD34.

Results: All patients were females in their thirties, with tender plaques located either on the upper trunk (60%) or proximal extremities (40%). Clinical diagnosis ranged from granuloma annulare (40%) to dermatofibroma (40%) and lymphoma (20%). Histologic examination revealed a diffuse spindle cell proliferation involving the reticular dermis. The neoplastic cells featured abundant eosinophilic cytoplasm and elongated nuclei. All cases featured faint SMA positivity, while S100 and CD34 were completely negative.

Conclusion: Dermatomyofibroma is a rare benign neoplasm, which should always be considered when evaluating a spindle cell proliferation from the upper trunk of young females. Due to inconsistency of immunohistochemical results, one should establish this diagnosis after excluding other benign mesenchymal proliferations, like leiomyoma or dermatofibroma Early lesions tend to show immunoreactivity for SMA, while older lesions are negative for SMA, S100 and CD34. This diagnosis should be kept in mind, even when SMA shows no or only weak positivity.


Cutaneous involvement by diffuse large B-cell lymphoma: clinical-pathological correlation

G. Malheiros*, M. Araujo Fonte Boa, Y. Gonzaga, S. de Oliveira Romano, J. Leite de Oliveira

*National Cancer Institute / INCA, Brazil

Background & objectives: Cutaneous involvement by diffuse large B cell lymphoma (DLBCL) is uncommon and may be primary or secondary to systemic spread. Skin biopsy leads to diagnosis. Our main objective is to describe clinico-pathological correlation between lesions and the skin infiltrate.

Methods: We report four cases of DLBCL whose initial manifestation was the appearance of distinct cutaneous lesions diagnosed through skin biopsy, describing clinical, histopathological and immunohistochemical findings.

Results: Case 1 - a man with multiple tumour lesions affecting the entire right lower limb; case 2 - a woman with tumoral and ulcerated lesions in the breast, simulating breast carcinoma; case 3 - a man with infiltrative and arciform lesions on the back, with a non-neoplasic appearance; case 4 - a woman with an ulcerated and necrotic lesion in the right leg. All cases were diagnosed as DLBCL through histopathological and immunohistochemical examination of skin biopsy.

Conclusion: Although uncommon, skin lesions can be the first and sole manifestation of DLBCL and may have multiple clinical presentations. We report four cases of cutaneous involvement by DLBCL and describe the clinical-pathological correlation.


Making mountains out of molehills – a study of possible links between malignant melanoma genotypes and their Royal College of Pathologists dataset phenotypes

A. Mokhtari*, T. Heaton, B. Mathew

*Leeds Teaching Hospitals NHS Trust, United Kingdom

Background & objectives: With the rise of molecular analysis in histopathology, malignant melanoma can be classified based on its genotype to allow for targeted therapy. This study aims to find a link between BRAF and NRAS mutations in malignant melanoma and histological phenotype.

Methods: Data was gathered from 337 cases of metastasizing malignant melanoma occurring between 2015 and 2018 that were tested for BRAF and NRAS mutations. The Royal College of Pathologists core data items were recorded from the primary skin lesions in each case. Data from BRAF positive, NRAS positive and wildtype cases was compared to assess patterns between mutation and histological phenotype.

Results: On comparing each genotype and histological phenotype, several trends have been noted. The most noteworthy of these were seen in the following dataset items: tumour subtype, tumour location and mitotic rate. These trends, as well as those of the remaining dataset items will be discussed further in this poster, as well as an assessment of the statistical significance of the data gathered.

Conclusion: The results generated from this study highlighted trends which could indicate that tumour phenotype can be predicted by genotype testing and vice versa. Future studies could also incorporate non-core data items from the dataset such as age, gender and prognosis.


Evaluation of histopathologic prognostic variables in cutaneous melanoma in a black African cohort

O.A. Odigwe*, U. Ezenkwa, G. Ogun

*Department of Pathology, University College Hospital, Ibadan, Nigeria

Background & objectives: The aim of this study was to evaluate the clinico-pathological prognostic variables in cutaneous melanoma cases in a black population, where it is believed to be rare and to run a more aggressive course compared to Caucasians.

Methods: This was a 20-year retrospective study (1997-2016) in a tertiary institution. Thirty-one H/E-stained tissue slides were reviewed. Microscopic features documented included histologic subtype, tumour thickness, level of invasion, ulceration, mitoses and vascular invasion. The relationship between these variables in addition to patient's age, gender and tumour site were assessed using Spearman rank correlation. Statistical significance was set at p<0.05.

Results: The median tumour thickness was 7.00mm (2.1-19.5mm). Twenty-five (80.6%) of tumours had pT4 stage and also had Clark level IV and V. Ulceration was present in 30 (96.8%) cases and 20 (64.5%) had evidence of vascular invasion. Thirteen (41.9%) of the patients had metastasis at presentation. There was significant positive correlation between tumour thickness and level of invasion (r=0.7, p<0.001), mitotic rate (r=0.45, p=0.01) and vascular invasion (r=0.42, p=0.02). Furthermore, level of invasion showed significant correlation with mitotic rate (r=0.41, p=0.02), and gender (r=0.37, p=0.04).

Conclusion: The findings of this study showed that cutaneous melanomas in our population are characterized by very poor prognostic histologic factors at presentation. Achieving early detection and treatment of this malignancy is advocated.


WT1: a useful immunomarker for the diagnosis of dermatofibrosarcoma protuberans

E. Piombino*, G. Broggi, G. Fuccio Sanzà, W. Saliba, S. Martello, R. Caltabiano, M. Barbareschi, G. Magro

*Department of Medical and Surgical Sciences and Advanced Technologies, G.F. Ingrassia, Azienda Ospedaliero-Universitaria "Policlinico Vittorio Emanuele", Anatomic Pathology, School of Medicine, University of Catania, Italy

Background & objectives: The most common malignant soft tissue tumours of the dermis/subcutis is dermatofibrosarcoma protuberans. The aim of the present study was to investigate the immunohistochemical expression of WT1 in primary and recurrent dermatofibrosarcoma protuberans and in its main morphological mimickers.

Methods: The cases were retrieved from Anatomic Pathology of the University of Catania and Santa Chiara Hospital of Trento. The following tumours were collected: 57 cases of dermatofibrosarcoma protuberans; 15 cases of dermatofibroma; 8 cases of dermal scars; 5 cases of deep-seated fibrous histiocytoma. The antibody against the N- terminal portion of WT1-6F-H2, was used.

Results: The majority of dermatofibrosarcomas protuberans (54 out of 57), exhibited cytoplasmic staining for WT1. The immunohistochemical expression was diffuse, heterogeneous or focal, respectively, in 75%, 15% and 6% of cases. With the exception of 4 cases showing a weak to moderate staining in different areas of the same tumour, the staining intensity was diffuse and strong. All recurrent tumours showed diffuse and strong WT1 cytoplasmic immunoreactivity while the fibroblasts of the associated scar tissue were negative. None of the other tumour or tumour-like, bland-looking spindle cell lesions examined, were WT1-positive.

Conclusion: WT1 is an ancillary immunomarker, exploitable in combination with CD34, in confirming the diagnosis of dermatofibrosarcoma protuberans, including in the recurrent tumours.


The relationship between BRAF mutation status and certain clinical and pathological features in melanoma

J. Potts*, G. Feddern, Y. Zhang

*North Bristol NHS Trust, United Kingdom

Background & objectives: BRAF mutation in melanoma predicts response to BRAF and MEK inhibitor therapy. This study investigated the relationship between clinicopathological characteristics of primary and metastatic melanomas with BRAF mutation status, in cases sent for testing in the period 2012-2016.

Methods: A total of 519 samples were identified. Patient age, gender and melanoma reporting dataset items were gathered from the histology report. Each variable was analysed against BRAF mutation status.

Results: 58% of patients were male and 42% were female with similar mutation rates for both (37% and 38% respectively). 40% of the samples were primary tumours, 57% were metastases and 3% were recurrences. 74% of mutations were v600E and there was no significant difference in type of mutation between primary and metastatic tumours. BRAF mutations were more common in metastases than primary tumours (41% and 33% respectively). BRAF mutation was significantly associated with superficial spreading and nodular histological subtypes; younger age; and location of metastasis. High rates of BRAF mutation were seen in brain metastases (78% positive). BRAF mutation was not associated with mitotic count, Breslow thickness, or ulceration.

Conclusion: BRAF mutation in our cohort is more common in younger patients and in metastatic tumours, in keeping with the published literature. We found high rates of mutation in metastasis to the brain, the reasons for which are unclear.

Funding by: BDIAP educational fellowship for Glasgow 2020


A case series of granular cell tumour with malignant potential; a rare cutaneous tumour

A. Randhawa*, M. Elgoweini, S. Digby, S. Tavadia, D. Torley, S. Holmes, S. Lo

*Queen Elizabeth University Hospital, United Kingdom

Background & objectives: Granular cell tumour (GCT), is an uncommon soft tissue neoplasm of neural in origin. These tumours are usually slow growing and benign. The malignant counterpart is extremely rare in the skin with a potential to metastasize.

Methods: Histologically, this can be diagnosed using the Fanburg-Smith criteria. We report a series of 3 cases with a histological diagnosis of granular cell tumour with malignant potential and evaluate the clinical presentation, investigations and progress.

Results: Case1-7 year-old boy presents a growing lump on his right shoulder. Histology revealed an incompletely excised GCT with atypia. Wider excision was consistent with a malignant GCT. Case2- 67 year-old female with firm nodule on her abdomen. Excised lesion revealed an atypical GCT. Consensus was to manage as a potentially malignant GCT. Case3-72 year-old man presents with a rapidly growing nodule on his right eyebrow. Excision biopsy favoured malignant GCT.

Conclusion: Clinical diagnosis of GCTs are difficult and should be included in the differential diagnoses of head and neck cutaneous lesions. Clinicians should be aware that atypical and malignant variants exist. There is some degree of pathological debate regarding classification of these lesions, especially in borderline atypical/malignant cases. There is a lack of consensus regarding the optimal management of this tumour but in our experience, we recommend a wide local excision for all GCTs and discussion at the multi-disciplinary team level.


Ultraviolet impact on rat skin

G. Sulim, M. Lyndin, V. Sikora, K. Sikora, N. Hyriavenko, A. Romaniuk*

*Sumy State University, Ukraine

Background & objectives: The aim of our study was to establish a >model imitating of UV-B (wavelength peak is 311 nm) therapy for rat’s skin and to find match macroscopical and following histological skin changes including melanocytes spreading and melanin distribution.

Methods: We used 6 laboratory rats with white and 12 with black coating applying UV-B light source (9 W power) during minimal erythema dose. The changes were studied by macroscopical and histological methods.

Results: In 3 of 18 of observations transitional symptoms looking alike, actinic keratosis occurred (1 black, 2 white rats). Other passing side effects (exfoliation, erythema) occurred and were successfully removed with exposure correction. Typical histological changes (chronic inflammation, hyperkeratosis, epidermal cells dystrophy) are more noticeable in rats with side effects, but still present in the rest of rats. Expected changes of melanocytes and melanin distribution could not be displayed with routine histological staining.

Conclusion: UV-B therapy model is a valid method to investigate it itself or to investigate chronic UV-exposure effects. The typical histological effects following UV-B exposure and their depending on macroscopical changes were found.


Prognostic value of tumour-infiltrating lymphocytes and mitotic rate in melanoma

A. Stanek-Widera*, B. Nikiel, D. Lange, E. Chelmecka

*Maria Sklodowska-Curie National Research Institute of Oncology, Gliwice Branch, Poland

Background & objectives: The use of immunohistochemistry to assess the number of CD3 and CD8 lymphocytes and an objective assessment of mitotic index, using computer-assisted image analysis allows for a more accurate assessment of the values of these parameters.

Methods: Our study included primary tissues from 88 non-consecutive cutaneous melanoma patient who were retrospectively examined at Maria Sklodowska-Curie National Research Institute of Oncology, Gliwice in 2005-2008 years. We used to determine the mitotic index Phosphohistone H3, Rabbit Polyclonal Antibody (PHH3) and to determine TILs :Polyclonal Rabbit Anti-Human CD3 and Monoclonal Mouse Anti-Human CD8.

Results: Statistically significant differences were found in the number of CD8 lymphocytes depending on the depth of the infiltrate (p <0.01), in the number of lymphocytes depending on the stage (p <0.05). Differences in lymphocyte counts between T1 and T3 (p <0.05) and T1 and T4 (p <0.05) were demonstrated. Analysing the number of abnormal mitoses depending on the stage, differences between T1 and T3 (p <0.05) were found, similarly between T1 and T4, as well as T2 and T4. Statistical differences were found for the number of mitoses depending on the state of the lymph nodes (p <0.05).

Conclusion: Identifying CD3 and CD8 instead of together as TIL allows a better understanding of the significance of these factors as elements of good prognosis, and the use of PHH3 allows a reliable assessment of the mitotic index as a factor of poor prognosis. The use of computer image analysis for routine diagnostics would improve the reliability of the assessment and the associated significance of prognostic factors.


Persistent late reactions in decorative tattoos: a series of eight cases with immunohistochemical study

A. Vicente Cid, M. Ginarte, L. Casas Fernandez, C. Aliste Santos, J.M. Suárez Peñaranda*

*Department of Pathology, University Hospital of Santiago de Compostela, Spain

Background & objectives: Persistent late reactions (PLR) in decorative tattoos are difficult to treat and their nature is not completely understood. They show different, poorly characterized histopathological patterns.

Methods: Eight cases of PLR in decorative tattoos have been retrieved form he files of the Department of Pathology, University Hospital of Santiago de Compostela (Spain). Clinical and histopathological features have been reviewed and immunohistochemical study performed in all cases.

Results: Seven women and one man, 21 to 39 years, presented PLR in decorative tattoos. Seven cases were related with red colour and one with black. Lesions started from two weeks until 2 years after performing the tattoo and were refractory to topical treatments.

Biopsies showed dermal lymphoid infiltrate, reaching the reticular dermis. Moderate to severe interface lesion was present in all but one case, the only one with well-formed granulomas. Five cases showed interstitial inflammatory infiltrate, mimicking granuloma annulare, but extensive collagen degeneration was present in only two cases.

Immunohistochemistry demonstrated a clear predominance of T lymphocytes, balanced for CD4 and CD8. CD8 lymphocytes were predominant in the interface lesion.

Conclusion: Decorative-tattoos- related PLR are characterized by a combination of inflammatory patterns, interface dermatitis with CD8 lymphocytes being the most common. Grannuloma annulare.-like lesions are also common, but collagen degeneration is usually limited. Well-formed granulomas are not a usual feature.

PS-05 Digestive Diseases Pathology - Liver


Morphofunctional and histochemical changes in liver tissue with predominant copper (Cu-10%) content in the experiment

K. Abdikadirova*, S. Zhautikova, F. Abikenova, B. Chergizova, Y. Talaspekova

*Karaganda Medical University, Kazakhstan

Background & objectives: Liver is a barrier to toxic substance and is primarily affected by pathogenic effect. Xenobiotics are metabolized exactly in this organ.

Evaluation of morphofunctional and histochemical changes in liver tissue at the exposure of polymetallic copper (Cu-10%) content dust.

Methods: The experiment included outbred white male rats weighing 120-170 g for 30 days. The dust was injected once intratracheally (50 mg/1.0 ml of physiological saline). Method of cytophotometry was used for quantitative characterization of the enzymes activity in liver cells. All manipulations, including elimination, were carried out by «Rules for biomedical experiments conducting» of MH RK (12.11.2009 №697).

Results: A histochemical study determined a sudden decrease of hepatocyte glycogen level. Glycogen was absent in cells containing altered nuclei. The activity of the enzymes was reduced – up to a complete absence. The activity enzymes showed the expressed decrease in comparison with control group: acid phosphatase by 54.5% (P<0.001), succinate dehydrogenase by 46.79% (P<0.001), lactate dehydrogenase by 56.96% (P<0.001), glucose-6-phosphate dehydrogenase by 36.48% (P <0.05), glycogen by 46.31% (P<0.001).

Conclusion: The liver cells are the first target of non-synthesized toxins Dust exposure intensified the trophic-circulatory disorders and exacerbated the alterative-dystrophic and inflammatory changes in the organ. The free lipids level increased in the cytoplasm of both liver cells and stellate reticuloendotheliocytes, which indicates increased destructive changes in cytoplasmic membranes and membrane complexes. Simultaneously, glycogen utilization in liver hepatocytes is enhanced, that led to inhibition of enzymes synthesis.


Potential role of neutrophil extracellular traps in non-alcoholic steatohepatitis

S. Arelaki*, T. Koletsa, G. Germanidis, E. Sinakos, K. Ritis, E. Akriviadis, P. Hytiroglou

*National Center of Tumour Diseases, Heidelberg, Germany; Pathology Department, Faculty of Medicine, Aristotle University of Thessaloniki, Greece

Background & objectives: Recent experimental evidence suggests that there are elevated levels of neutrophil extracellular traps (NETs) in sera of patients with NASH. We investigated the presence of NETs in liver biopsy specimens with NASH and potential correlations with inflammation and fibrosis.

Methods: This retrospective study included 40 liver biopsy specimens from 21 patients with NASH. Fourteen patients underwent sequential liver biopsies according to clinical indications. We assessed the presence of NETs by double immunofluorescence using the markers neutrophil elastase (NE) and citrullinated histone 3 (citH3). Immunofluorescence signals and colocalization of these markers were visualized with a confocal microscope.

Results: NETs were detected in 60% of the biopsy specimens, as extracellular colocalization of NE with citH3. In 54% of the NET-positive biopsies significant production of NETs was apparent, in an aggregated pattern, within areas of portal and lobular inflammation. In contrast, 46% of the biopsies showed small numbers of NETs, despite the presence of inflammation and/or fibrosis. In most cases of mild inflammation or established cirrhosis NETs were absent.

Conclusion: Our findings suggest that neutrophils are involved in the pathogenesis of NASH through NET release. Further research will be needed to clarify whether production of NETs is involved in the progression of NASH to cirrhosis or the development of hepatocellular carcinoma as suggested by recent experimental studies.

Funding by: Hellenic Association of the Study of the Liver


Histopathological features of inflammatory pseudotumours of the liver on resection specimens

A. Baltan*, E. Stoica-Mustafa, C. Pechianu, A. Iorgescu, A. Procop, A. Bancu, D. Hrehoret, I. Popescu, V. Herlea

*Emergency University Hospital Bucharest, Romania

Background & objectives: Inflammatory pseudotumours of the liver are nonneoplastic masses that cause clinical confusion due to their capacity to mimic malignancy. The aim of this study was to identify the histopathological features of these lesions and their clinical correlates.

Methods: We have reviewed the slides of 15 hepatic inflammatory pseudotumours resected between 2013-2018. The localization, presence of a pseudocapsule, the type and distribution of inflammatory cells, the presence of bile ductules/ducts within the lesion and of perilesional occluded large vessels and the degree of fibroblastic proliferation and fibrosis were noted. These were analysed in conjunction with clinical and imaging data.

Results: The mean age was 63 years and there was a slightly male predominance among patients. The relevant clinical associations were chronic cholecystitis, HBV infection, obesity, type 2 diabetes mellitus and malignancy. The masses had an average maximum diameter of 5 cm. The most common histological features were lack of a pseudocapsule, presence of foamy cells, variable inflammatory cells and necrosis, in both perihilar and peripheral localization. Two cases were entirely composed of lobules of foamy cells with no atypia. One case lacked foamy cells and necrosis, but showed no association with IgG4-related disease and no periductal growth. No relapse was noted, although three cases were complicated by postoperative abscesses.

Conclusion: Inflammatory pseudotumours of the liver have similar clinical correlates to other infectious/inflammatory lesions. Their pathological spectrum may be related to various aetiologies or inflammatory responses among individuals. Further studies are needed to clarify the pathogenesis and to classify these lesions.


Paediatric and adult non-alcoholic fatty liver disease (NAFLD) pathogenia: lymphocyte populations characterisation in the liver microenvironment

V. Cairoli, E. De Matteo*, C.G. Giadans, P.C. Casciato, E. Mullen, A. Pedreira, C. Lezama, M. Galoppo, G. Bertot, M.V. Preciado, P. Valva

*Multidisciplinary Institute for Investigation in Paediatric Pathologies (IMIPP), CONICET-GCBA, Pathology Division, Ricardo Gutiérrez Children’s Hospital, Buenos Aires, Argentina

Background & objectives: The immune system is one of the main drivers of NAFLD pathogenesis. The role of each liver lymphocyte population of the infiltrate in the pathogenesis is uncertain. Aim: to characterize liver infiltrate in relation to histological damage in NAFLD patients.

Methods: Twenty-six paediatric [11.5 yrs (4-17)] and 34 adult NAFLD patients [50 yrs (28-72)] were enrolled. On liver biopsies histological changes as well as T helpers Lymphocyte (Th), Cytotoxic T Lymphocytes (CTL), Regulatory T lymphocytes (Treg) and Th17 localization and frequency were evaluated by immunohistochemistry. Statistical analysis were performed to assess the relation between the immune response and the observed damage.

Results: Portal-periportal-CTL and -Th frequencies were similar in paediatric and adult NAFLD cases. Th17 seemed to be nearly exclusive of this location. In children Treg and Th17 counts were comparable, but in adults Tregs were higher than Th17 cells. In intralobular area CTL>Th>Treg predominance was observed both age group. Comparison between them demonstrated higher portal-periportal-Treg (p<0.006), intralobullar-CTL, -Th and -Treg counts (p=0.025, p=0.0004 and p=0.013) and lower Th17/Treg ratio (p=0.041) in adults.

In children higher intralobular-Treg were associated with inflammation severity (p=0.026), while in adults more severe inflammation stages showed lower Treg and higher Th17 counts. Paediatric and adult cases with fibrosis ≥2 displayed portal-periportal lower Treg and higher Th17.

Conclusion: The composition of the inflammatory liver infiltrate differ between paediatric and adult NAFLD, but the interplay between Treg and Th17 seems to condition the progression of the damage in both groups.


Digital histopathological characterisation of b cell subsets in early and advanced biliary atresia

M. Elghobashy*, M. Harris, A. Patel, C. Lloyd, R. Brown, D. Kelly, G. Reynolds, Z. Stamataki

*Institute of Immunology and Immunotherapy, Centre for Liver and Gastrointestinal Research, University of Birmingham UK, United Kingdom

Background & objectives: Biliary atresia (BA) is a neonatal cholangiopathy of various potential aetiologies. The role of B lymphocytes in the pathogenesis of BA has been hypothesised through murine models. We aimed to characterise B cell subsets in BA using triple immunohistochemistry.

Methods: CD20, CD24 and CD38 immunohistochemistry was carried out on three samples (hilum and peripheral liver (wedge) samples at time of Kasai hepatoportoenterostomy and an explant sample) for each patient (n=28). The slides were scanned to allow digital appraisal. Semi-quantitative analysis of inflammatory cells and fibrosis was performed. Phenotypic groups were formed using inflammatory cell frequencies and correlated with clinical outcome.

Results: Three B cell subsets were identified in the wedge samples at Kasai and the explant samples. CD20+CD24+CD38-cells were present in both sample types in all 28 patients. CD20+CD24-CD38+cells were present in 1 of 28 wedge samples and 3 of 28 explant samples. CD20+CD24+CD38+cells were identified in 4 of 28 explant samples. Patient stratification based on inflammation at the hilum did not predict transplant-free survival. CD20+CD24+CD38-cell frequency correlated with fibrosis scores.

Conclusion: The histopathological analysis of three distinct tissue regions using triple immunohistochemistry in this study is a novel method in studying the evolution of the disease from the extrahepatic (hilum) to intrahepatic biliary system. This work is the first to report distinct B cell subsets in BA and hypothesises the possible role of CD20+CD24+CD38-B cells in biliary damage. This opens the scope for their potential use as therapeutic targets to halt the progression of this highly morbid disease.


Mesenchymal hamartoma of the liver - diagnostic difficulties

E. Iżycka-Świeszewska*, M. Murawski, K. Buczkowski, J. Gulczyński, J. Stefanowicz, P. Czauderna

*Medical University of Gdansk, Poland

Background & objectives: Mesenchymal hamartoma of the liver (MHL) is a rare paediatric lesion, usually multicystic, slowly growing, and accompanied by slight elevation of serum alpha-fetoprotein. Diagnosis of MHL depends on its radiological and clinical presentation, while histopathology shows wide spectrum of changes.

Methods: Eight children with mesenchymal hamartoma diagnosed radiologically or histologically, operated in one paediatric surgery centre during last 10 years are presented. The clinical data analysis and detailed pathological examination with wide panel immunophenotyping covering epithelial and stromal components was performed.

Results: There were two girls and six boys in the age ranged 4 – 19 months. All tumours presented with abdominal enlargement, AFP elevation from 80 up to 10000. Radiologically five lesions were problematic, showing some indefinite features of vascular lesion(2), hepatoblastoma(2) or inborn defect(1). Two tumours were mainly solid, while the rest were multicystic. Three cases primarily underwent needle biopsy, with erroneous hepatoblastoma diagnosis in one boy and inconclusive answer in the second child. The final histological diagnosis of MHL was based on morphology and immunophenotyping. Two cases diagnosed radiologically as mixed hamartomatous lesion, were cavernous haemangioma with defect in portal veins and bile duct proliferation.

Conclusion: Mesenchymal hamartoma of the liver can cause diagnostic and therapeutic problems, mainly due to its rarity and different presentation in pathological and clinical aspects. Core needle biopsies in MHL may not be informative to establish the final diagnosis. Some rare vascular hepatic lesions enter the differential diagnostic field of MHL.

Funding: ST02-0562/07 MUG


Primary biliary cholangitis: histological criteria to predict UDCA therapy response - a case series

D. Naranjo-Hans*, J. Gimeno Beltran, A. Puche Gallego, L. Serrano Munne, J. Masso Castella, M. Garcia-Retortillo, B. Lloveras Rubio

*Hospital del Mar, Spain

Background & objectives: Primary biliary cholangitis (PBC) is a prevalent hepatic chronic cholestatic autoimmune disease characterized by chronic non-suppurative cholangitis affecting small to medium sized interlobular bile ducts. Its progression to cirrhosis and liver failure is associated with the response to UDCA therapy.

Methods: Determine if there is a statistical association between any histological feature included in either the Ludwig stage, the Scheuer stage and the recently proposed FBI score and the response to UDCA treatment in our series of patients with PBC. All the biopsies were obtained before starting UDCA treatment.

Results: 57 patients were studied with 39 cases percutaneous liver biopsies collected retrospectively from the files of our Institution. The histological evaluation according the FBI system showed a bile duct ratio (BDR) index of 0.9 with only one case presenting ductopenia. Respect lobular interphase hepatitis (LIH), five cases showed no LIH, 21 cases showed mild to moderate LIH and three cases showed severe LIH. Regarding fibrosis, 37 patients were in early stages (0-1), and only one biopsy showed histological criteria of cirrhosis. Statistical analysis showed non-statistical association between any histological criteria nor stage and the absence of response to UDCA therapy. However, we detected a trend between UDCA-therapy response and LIH.

Conclusion: The classical scores for staging PBC do not include histological features such as LIH, which might be useful to detect those potential patients whom may benefit from a combined or alternative therapy.


Identification of differential immune circuits in preneoplastic and neoplastic stages of hepatocellular carcinoma using murine (ABCB4-/-) and human tissues

M.A. Qureshi*, M. Ashfaq Khan, A. Qureshi, S. Sharafat, D. Schuppan

*Dow International Medical College, Dow University of Health Sciences, Department of Pathology, Karachi, Pakistan

Background & objectives: While HCC is a potential candidate for immunotherapy, immune circuits that drive the inflammatory-to-tumour milieu remain to be explored. In this study, we have characterized immune landscape in preneoplastic and neoplastic microenvironment of HCC using murine and human HCC tissues.

Methods: In-vivo murine model of HCC was generated using 3-5 days old Mdr2(Abcb4)-/- mice with intraperitoneal-injection of DEN followed by administration of 0.05%phenobarbital. Moreover, a total of 42 patients diagnosed with HCC and undergoing biopsy/resection were recruited in the study. Mice without carcinogenic-regime and liver from transplant donors were included as controls. Tissues were subsequently analysed using immunohistochemistry, flowcytometry and PCR.

Results: Mdr2-/- mice receiving DEN-and-PB exhibited a significantly different pathology with high proliferative index, increased lymphocyte infiltration, increased neoplastic progression, and increased fibrosis. Microscopically, tumour phenotype ranged from pseudo-tubular to trabecular-type morphology. Importantly, alternatively-activated-macrophages (M2) were heavily infiltrated in in the intra-tumoral compartment as early as 6 weeks. Liver from these mice were heavily infiltrated by the recently identified immune cells ILC1 and ILC2. Moreover, mRNA transcripts of MMP12, MMP13, MMP9, TIMP1, TGFβ, IL1, IL6, iNOS, CD68 and YM1 were differentially expressed in murine tissues. In human HCC-tissues, there was increased infiltration of macrophages, neutrophils and B-cells, while infiltration of T-cells (CD4+ and CD8+) was not different compared to the controls.

Conclusion: We report detailed analyses of preneoplastic and neoplastic microenvironment of HCC in murine as well as human tissues. These differentially regulated cells may represent novel targets for HCC immunotherapy.


Differential morphologic characteristics between autoimmune hepatitis (AIH) and drug induced liver diease (DILI)

Y. Rodriguez-Gil*, L. Cuevas del Campo, M. Camara, P. Arribas, B. Agredano, C. Jimenez-Revilla, E. Gómez-Dominguez

*Hospital Universitario 12 Octubre, Surgical Pathology Department, Spain

Background & objectives: Distinguishing DILI from AIH can be a challenge. There aren´t pathognomonic features of AIH. We reviewed a series of cases to find the more useful morphological features to differentiate DILI and HAI and selected and standardized them in a score.

Methods: We selected a series of 32 patients with clinically well-characterized AIH and DILI (14 DILI and 18 AIH). CIOMS-RUCAM and evolution parameters (including steroids responsiveness) were used to discriminate the patients in the two groups.

Retrospective observational cohorts study was performed assessing 31 histologic variables and comparing them between the two groups. Chi Square and non-parametric tests were applied.

Results: Following histological evaluation of 31 variables, we performed a comparative analysis and only five variables showed statistically significant differences between the DILI and AIH biopsies (p<0,05). AIH more frequently presented portal plasma cells and intra-acinar plasma cells inflammation; meanwhile DILI presented more frequently canalicular and/or hepatocellular cholestasis, portal neutrophilic inflammation and intra-acinar neutrophils.

A model combining the five variables with detailed histological evaluation in four grades and a standardized score predicted the diagnosis of most of the cases (AUC 0,913) with high specificity and sensibility

Conclusion: There are some overlap of most histologic findings between AIH and DILI, five variables showed differences: Portal plasma cells, intra-acinar plasma cells, portal neutrophilic inflammation, intra-acinar neutrophilic inflammation and canalicular and hepatocellular cholestasis. We were able to develop a score that could simplify histological evaluation and with clinical data allow the differential diagnosis. To validate our results we are performing a second analysis in a blinded manner by 4 pathologist.


Role of set protein in hepatocellular carcinoma: an immunohistochemical study

M. Samara*, N. Asaad, M. Shaban, D. Taie, M. Gadallah, S. Elkholy

*National Liver Institute, Menoufia University, Egypt

Background & objectives: HCC targeting therapy still remains unsatisfactory. SET protein is a potent inhibitor of protein phosphatase 2A (PP2A) through its interaction with PP2A-regulated oncogenic pathways. We investigated the oncogenic role of SET in HCC tumorigenesis and clinical aggressiveness in Egyptian patients.

Methods: This study was carried out on 100 HCC Egyptian patients. For each case, paired representative samples were collected from the tumour tissue and the adjacent non-tumorous liver tissue of the same surgical specimens and analysed for the immunohistochemical expression of SET. Correlation between SET expression levels in HCC and the available clinicopathological parameters and overall survival was done.

Results: SET protein showed significantly higher expression levels in HCC than in the adjacent non- tumorous liver tissue (P<0.0001). 54% of HCC cases exhibited overexpression of SET protein that correlated significantly with large tumour size (P=0.012), lymphovascular invasion (p-value=0.028) and shorter overall survival (P<0.001).

Conclusion: These findings suggested the oncogenic role of SET protein and the adverse prognostic outcome of SET protein overexpression in HCC and the potential to be used as a new prognostic tool and a therapeutic target.


A clinico-pathological audit of the effect of medical liver biopsies on patient management

S. Seth*, G. Kohnen, P. Konanahalli, K.A. Oien

*University of Glasgow, Queen Elizabeth University Hospital (QEUH), United Kingdom

Background & objectives: Medical liver biopsies are used to investigate patients with hepatic dysfunction and diffuse disease on imaging, when non-invasive methods yield insufficient information. We aimed to evaluate our medical liver pathology practice and its influence on patient management, using national standards.

Methods: The UK’s Royal College of Pathologists (RCPath) produces “Tissue pathway” guidelines promoting good practice in specimen handling and reporting. We used RCPath audit proformas to collect data on specimen quality/reporting and clinico-pathological effect. The QEUH pathology database was searched for liver specimens in 2019. Reports were retrieved for review (by SS, academic trainee), alongside clinical information from electronic patient records.

Results: After excluding resections, targeted biopsies and referral cases, we identified 135 medical liver biopsies reported in 2019 between three consultant histopathologists (GK,PK,KO). Sixty consecutive cases were included for audit. 51/60 (85%) biopsies met RCPath criteria for adequacy (length>15mm or ≥6 portal tracts); three (5%) samples were inadequate and six reports (10%) lacked enough information to evaluate. Most reports (56/60;93%) were judged to have helped patient management. The clinical diagnosis changed in 23/60 (38%); the different diagnosis was previously unanticipated in nine (39%). Patient management altered in 48/60 (80%); in most of these (33/48; 69%), the decision would not have been possible without the pathology report.

Conclusion: This audit highlights the clinical value of medical liver biopsy in diagnostic work-up and follow-up of patients with liver disease. These results will help optimise the content of our reports and wider clinico-pathological communications.


Diagnostic utility of von Hippel-Lindau tumour suppressor gene product in intrahepatic/extrahepatic bile duct and gallbladder adenocarcinomas

A. Tanoglidi, N. Stavrinou, A. Sykaras*, G. Liadakis, C. Kouvidou, C. Vourlakou

*Evangelismos General Hospital, Athens, Greece

Background & objectives: Von Hippel-Lindau tumour suppressor gene protein’s (pVHL) loss of expression has been observed in many tumours. Our aim is to assess pVHL expression in intrahepatic cholangiocarcinoma (ICC), pancreatic ductal adenocarcinoma (PDAC), bile duct adenocarcinoma (BDAC) and gallbladder adenocarcinoma (GBAC).

Methods: We examined the immunohistochemical expression of pVHL in ICCs (n=10), PDACs (n=5), GBACs (n=10) and bile duct adenocarcinomas (BDACs) (n=5) in biopsies and surgical resection specimens. A cytoplasmic and membranous staining was treated as positive if at least 5% of the tumour cells exhibited immunoreactivity. Positive staining was further graded as 1+(5%-25% of the tumour cells), 2+(26%-50%), 3+(51%-75%) or 4+(>75%).

Results: All cases of ICCs were diffusely and strongly positive (membranous and cytoplasmic staining 3+ or 4+). One out of five cases of PDACs was focally positive (1+) whereas the remaining were negative. Two out of five cases of BDACs were negative, one showed 3+ and two 2+ positivity. Four out of ten cases of GBACs were negative, two showed 4+, two 3+ and two 2+ positivity. Non-neoplastic epithelium served as positive internal control. Our results for ICCs and PDACs are in accordance with published data, but positive percentages in BDACs /GBACs are higher than those in the literature. We observed reduced positivity in less differentiated areas especially the tumour front.

Conclusion: pVHL immunohistochemistry can be used as a diagnostic tool in differentiating ICC from PDAC but isn’t so helpful in distinguishing ICC from GBAC or extrahepatic bile duct adenocarcinoma. Its prognostic and diagnostic role needs further investigation.


Liver biopsy in non-neoplastic paediatric liver diseases: 10 year experience at National Liver Institute, Menoufia University, Egypt

T. Talab*, Z. Fayed, M. Soltan, N. Ehsan

*National Liver Institute, Menoufia University, Pathology Department, Egypt

Background & objectives: Liver biopsy despite being invasive technique is considered fundamental in diagnosis and management of paediatric liver diseases. The aim of this study was to assess the role of liver biopsy in diagnosis of non-neoplastic paediatric liver diseases.

Methods: Pathological reports of paediatric patients with non neoplastic liver diseases collected from January 2010 through January 2019 and sorted according to the diagnosis. Pathological diagnoses were sorted into five groups; cholestasis ( extrahepatic biliary outflow obstruction and intrahepatic cholestasis), inflammatory (chronic hepatitis and granulomatous inflammation) , metabolic/storage diseases , cirrhotic and finally others that include the remaining entities.

Results: A total 2157 histopathological studies were found; 52.2% were boys, and 47.8% were girls. The major age group was young infants which biopsied mainly for cholestasis (37.5%). Other categories diagnosed included inflammatory group (29%), group with metabolic/ storage diseases (10.6%), group with cirrhosis (5.5%) and the remaining percentages were for other entities (17.4%). Biliary outflow obstruction and intrahepatic cholestasis were the most common diagnoses in young infants while chronic hepatitis was the main diagnosis of elderly children.

Conclusion: Liver biopsy is a main integral component in setting the initial diagnosis of many non-neoplastic paediatric liver diseases. Biliary outflow obstruction, intrahepatic cholestasis, chronic hepatitis and metabolic/ storage diseases are the most common diagnosed diseases.


The influence of the chronic alcohol intoxication on the morpho-functional status of the liver of rats at the age of 6 months

M. Kozlova, Y. Kirillov, D. Areshidze, I. Chernov, E. Shtemplevskaya, S. Timofeev*

*Tyumen State Medical University, Russia

Background & objectives: The circadian rhythmicity of biological processes is one of the important properties of the mammalian organism. The aim of this work was to study the effect of chronic alcohol intoxication on the circadian rhythmicity of some micromorphometric liver parameters.

Methods: The 72 6-month-old male Wistar rats, divided into 2 groups, were kept under fixed light conditions for three weeks, the experimental group received ad libitum a 15% ethanol instead of drinking water. Animals were sacrificed at 9, 15, 21 and 3 hours. Micromorphometric liver parameters were measured. The amplitude-phase characteristics of circadian rhythms were investigated using cosinor-analysis.

Results: In the control, circadian rhythms were found for every parameter. Acrophases for the rhythms of nuclear area, cell area, and nuclear-cytoplasmatic ratio (NCR) were noted at 12.16, 10.22, 13.56 hours, amplitudes were 38.47 μm2, 99.23 μm2 and 0.030, respectively. In the experiment, only the nuclear area rhythm was detected - acrophase at 5.18 hours and an amplitude of 12.72 μm2.

The study testifies that chronic intoxication with ethanol leads to significant violations in the organization and synchronization of CR of the studied morphometric parameters, reflecting the morphological and functional state of the liver of male Wistar rats.

Conclusion: The destruction of the СR of cell area and NCR, as well as change in the amplitude-phase rhythm's characteristics of the nuclear area is evidence of desynchronosis, which is an important link in the pathogenesis of a many of diseases.


The influence of modification circadian rhythms during the chronic alcohol intoxication

E. Shtemplevskaya, M. Kozlova, Y. Kirillov, D. Areshidze, I. Chernov, S. Timofeev*, M. Bereza, V. Kukushkin

*Tyumen State Medical University, Russia

Background & objectives: The increased consumption of alcohol is a serious problem in modern society. The goal of this study is to find out the effect of circadian rhythms on alcohol intoxication in 72 six-month old male rats.

Methods: The first group was kept at a fixed light mode, light-dark 14:10 and the second in conditions of constant light-light for 3 weeks. The animals were fed with a 15% ethanol solution ad libitum. Animals were removed from the experiment at 9am, 3pm, 9pm and 3am. The material was examined histologically, histochemically and through an electron microscope.

Results: In liver samples, small and large droplet fatty degeneration of hepatocytes, expansion and cellular infiltration of portal tracts, focal necrosis in the liver lobules were seen. The increase in alcohol consumption for the able-bodied population operating in continuous cyclic enterprises is a particular danger, since under these conditions, the rhythmic disturbances in biological processes caused by levelling of the time of day can be aggravated by the use of ethanol.

Conclusion: Changes in the CR, through the introduction of continuous lighting, when modelling chronic alcohol intoxication are accompanied by an increase in the concentration of ethanol in the blood and liver parenchyma and an increase in its toxic effect.


Lymphocytic apoptosis among the criteria of the histological diagnosis of autoimmune hepatitis in acute-on-chronic patients: the experience of a tertiary referral Italian centre

F. Vasuri*, T. Franceschini, M. Riefolo, P. Muratori, D. Malvi, M. Lenzi, A. D'Errico

*Pathology Unit, S.Orsola-Malpighi Hospital, Bologna University, Italy

Background & objectives: Liver biopsy represents the diagnostic gold standard in autoimmune hepatitis (AIH), but histological diagnostic criteria lack consensus and reproducibility.

Aims: (i) to redefine the histopathological criteria for AIH; (ii) to validate portal apoptosis as a histopathological criteria for AIH diagnosis.

Methods: We retrospectively evaluated 65 biopsies from AIH patients at their first access to our Center. Forty-five (69.2%) had a previous diagnosis of AIH (follow-up patients, FUP), for 20 (30.8%) the AIH diagnosis was made at the moment of the present biopsy (first-diagnosis patients, FDP).

Fourteen histopathological variables were collected, including the count of lymphocytic apoptosis within the portal tracts.

Results: As expected, all variables of active hepatitis were higher in the 20 FDP compared to the 45 FUP (p<0.05), while fibrosis Ishak’s stage was >2 in 11 (55.0%) FDP and 8 (17.8%) FUP (p=0.005).

Mean number of apoptotic bodies in portal tracts was 5.00±4.45 and 9.45±6.95 in FUP and FDP respectively (p=0.002). Moreover, apoptosis count positively correlated with fibrosis and almost all variable of inflammatory activity.

Conclusion: Most FDP showed an acute-on-chronic histology, highlighting the key role of early diagnosis in AIH patients to prevent fibrosis progression. Moreover, the count of the lymphocytic apoptotic bodies within the portal tracts might reinforce the pathological criteria for AIH diagnosis.


The role of c-Met signalling pathway in hepatocarcinogenesis and its involvement in Sorafenib resistance

P. Korhan, Y. Oztemur Islakoglu, F. Yilmaz*, D. Nart, N. Atabey

*Ege University Faculty of Medicine, Department of Pathology, Turkey

Background & objectives: Development of resistance to conventional therapies in Hepatocellular carcinoma (HCC) is an important problem. This study aims to explore the role of c-Met signalling in the acquisition of aggressive phenotypes in HCC.

Methods: HCC cell lines and HCC cases were analysed using immunohistochemistry, Western Blot, motility and invasion assays for c-Met expression. In-vitro mechanistic studies using overexpression and silencing vectors for the demonstration of for c-Met activation were made. Specific c-Met inhibitors were used for the reversal of sorafenib resistance in HCC cells.

Results: Immunohistochemical analyses of HCC cell lines and HCC cases, showed that immunoreactivity of phospho-Met and c-Met are increased in HCC compared to normal and cirrhotic tissues. Our Western Blot, motility and invasion assays showed higher c-Met expression in poorly-differentiated, highly motile and invasive HCC cell lines than well-differentiated ones. Our further in-vitro mechanistic studies using overexpression and silencing vectors revealed that c-Met activation was occurred mostly independent of HGF in HCC cells, including receptor cross-talk, non-coding RNAs and environmental factors. We demonstrated that miR-181a-5p regulates c-Met signalling in HCC and played a role in the acquisition of sorafenib resistance. We reversed sorafenib resistance in HCC cells using c-Met specific inhibitors.

Conclusion: Our results provide mechanistic insight into c-Met signalling in HCC and sorafenib resistance. These findings suggest that c-Met might be used as a predictive biomarker to guide patient selection for clinical trials of Met-related therapies.

PS-06 Haematopathology


Determination of mean HbA2 levels on high performance liquid chromatography (HPLC) in known beta thalassaemia trait individuals

R. Akbar*

*Cantonment General Hospital, Pakistan

Background & objectives: Beta thalassaemia is one of the most common inherited haemoglobin disorders. The present study was conducted to determine mean HbA2 levels of known β thalassaemia trait individuals on HPLC in South East Asian population, taking cellulose acetate electrophoresis as gold standard.

Methods: A descriptive cross sectional study was conducted from Dec 2018 to Dec 2019. A total of 495 patients who were diagnosed as β thalassaemia trait on cellulose acetate haemoglobin electrophoresis were included in the study. The blood samples were run on High Performance Liquid Chromatography (HPLC) and HbA2 levels were recorded on the proforma.

Results: Mean and standard deviation were calculated for HbA2, MCV and MCH. Frequency and percentage were calculated for gender. Effect modifiers like age and gender were controlled by stratification and post stratification independent sample t test was applied. Mean HbA2 levels of β thalassaemia trait individuals on HPLC was 5.63%. This percentage was higher than the mean value of HbA2 on Cellulose Acetate Electrophoresis, which is generally taken as >3.5%. There was no effect of age (p=0.07) and gender (p=0.14) on mean HbA2.

Conclusion: Measurement of HbA2 level on HPLC is a reliable way of detection of β thalassaemia trait individuals. However, a higher threshold of HbA2 is needed to label an individual as Beta Thalassaemia trait in South East Asian population.


Histopathologic diagnosis of lymph node biopsies in paediatric population: a 5 year descriptive study in a tertiary hospital

V. Baena Romero*, C. Cabañuz Rocatallada, E. Garcia Fernandez

*Hospital Universitario La Paz, Spain

Background & objectives: Lymphadenoapathy is a relevant clinical problem in children and biopsies are usually needed to determine if the cause is neoplastic or non-neoplastic. The aim of this study was to determine the histopathological spectrum of lymphadenectomies undertaken in our hospital.

Methods: An amount of 580 lymph node biopsies of 196 patients were carried out in our hospital from 2015 to 2019 in children from 0 to 18 years old. We reviewed the histological diagnoses in our database for each patient.

Results: Normal lymph node (33%) and reactive lymphoid hyperplasia (29%) were more common. Lymphomas were 14 %, most of them Hodgkin lymphomas. Metastatic lesions make up 8 % which included neuroblastoma, papillary thyroid carcinoma and melanoma metastases. Necrotizing tuberculous granulomatous lymphadenitis was the most common diagnose among infectious pathology (9%). Other diagnoses were post-transplant (6%) or inmunodeficiency related pathology (6%).

Conclusion: Lymph node biopsy plays an important role in establishing the cause of lymphadenopathy. Among the biopsied nodes, one of the most common diagnoses was lymphoid reactive hyperplasia, that shares sometimes common features with neoplastic lesions. It is important to recognise the different patterns of lymph node pathology in children to avoid misdiagnosis.


Differential expression of BCL-2 and LEF1 in mature T-cell lymphomas and reactive hyperplasia

L. Brown*, S. Prakash, L. Wang

*University of California, San Francisco, USA

Background & objectives: T-cell lymphomas may lack morphologic atypia or T-cell antigen loss, making differentiation from reactive conditions difficult. The goal of our study is to evaluate BCL-2 and LEF-1 expression in reactive lymphoid hyperplasia versus mature T-cell lymphomas.

Methods: BCL-2 and LEF-1 immunohistochemistry was performed on 43 and 42 previously diagnosed cases of T-cell lymphoma and 18 and 17 cases of reactive lymphoid hyperplasia, respectively, confirmed by clinical follow up. Expression was recorded as positive (>90% of cells staining), partial loss (<50%), and negative (<10%).

Results: BCL-2 showed partial loss in 4 cases (9%) and complete loss in 3 cases (7%) of T-cell lymphoma. LEF-1 showed partial loss in 7 cases (17%) and complete loss in 17 cases (40%) of T-cell lymphoma. None of the 18 cases with reactive lymphoid hyperplasia showed complete loss of BCL-2 or LEF-1. Two cases of paracortical hyperplasia showed partial loss of BCL-2, one of which also showed partial loss of LEF-1.

Conclusion: Complete loss of BCL-2 or LEF-1 expression is often seen in T-cell lymphoma but not in reactive lymphoid hyperplasia and may be helpful in the diagnosis of challenging cases of T-cell lymphoma without other T-cell antigen loss.


Molecular characterisation of acute myeloid leukaemia among Filipino patients using comprehensive next-generation sequencing

F.V. Casimero*, L.K. Cabral, M.L. Enriquez, R.A. Salamat, D. Villegas, M.A. Rodriguez, P. Caguioa, F.V. Lopez, D.F. Natino, J.A. Malala, M.C. Abad, J.J. Andal, A. Arevalo, D. Ang

*St. Luke's Medical Center, Philippines

Background & objectives: AML is the most common adult acute leukaemia. New molecular technologies allowed prognostication & targeted therapy. To date, AML molecular characterization in the country is non-existent. The study determined the prevalence of somatic mutations in Filipino AML patients using NGS.

Methods: All 44 bone marrow aspirates or peripheral blood samples of patients diagnosed with AML between January 2018 to August 2019 were subjected for mutational analysis using a NGS platform (Miseq), to screen for mutational hotspots in 40 genes relevant to myeloid neoplasms (Ampliseq Myeloid Panel, Illumina). Clinical characteristics of patients were recorded.

Results: Of the 44 cases in our study, there were 23 males and 21 females with a median age of 41 (9 to 75 years old). At least one non-synonymous gene mutation was detected 42 our AML patients (95%). There was an average of 2.8 mutations (range 1-8) per case. KIT (27%) was the most common mutation, followed by RUNX1 (20%); CEBPA, NPM1, TET2, FLT3 (13%); and NRAS, IDH1, IDH2, ASXL1, WT1 (7%) . The remaining genes showed a frequency of <5%. In our study, the most frequently identified mutations involve signalling and kinase pathways followed by epigenetic modifiers. We detected NPM1 and FLT3-ITD mutations at frequencies similar to previous reports.

Conclusion: Our study underscores the heterogeneity of AML among Filipinos. Although it is challenging to implement molecular testing in resource-limited countries, our study highlights the necessity of implementing NGS testing in AML patients, to aid in risk stratification and treatment planning.


Epstein Barr virus (EBV) recruits PD-L1 + cells in paediatric Hodgkin lymphoma microenvironment

O. Jimenez, S. Colli, M. Garcia Lombardi, M.V. Preciado, P. Chabay, E. De Matteo*

*Multidisciplinary Institute for Investigation in Paediatric Pathologies (IMIPP), CONICET-GCBA, Pathology Division, Ricardo Gutiérrez Children’s Hospital, Buenos Aires, Argentina

Background & objectives: Introduction: In Hodgkin lymphoma (HL), PDL1 are expressed by tumour cells and its expression could be upregulated in the microenvironment as well. Aim: To evaluate PD-1 /PDL1 pathway in children with HL from Argentina, a population with increased EBV-associated lymphomas

Methods: Methods: Formalin-fixed paraffin-embedded biopsy samples (FFPE) of 94 patients with HL (2-18 years, median 9) were collected at Ricardo Gutiérrez Children's Hospital. PD1 and PDL1 expression was studied by immunohistochemistry (IHC). FISH for 9p24.1PDL1 gene amplification in HRS cells was performed in 38 patients. EBV status was defined by in situ hybridization for EBERs and IHC for LMP1.

Results: Results: 64 % of cases were EBV+. There were no statistical differences for the mean PD1 + cells in EBV + vs EBV- groups(p> 0.05). A 5% of cases displayed PDL1 amplification, 18% copy gain, 11% copy gain and amplification. There was no significant differences in the means of PDL1+ HRS cells (p> 0.05, MW test), or in PDL1 gene alterations among EBV + vs EBV- cases (p> 0.05). No differences were found in the PDL1+ cell count with and without 9p24.1 gene alterations (p> 0.05). A significant increase in the number of PDL1 + cells in the microenvironment was detected in EBV + cases (p= 0.042).

Conclusion: Conclusions: EBV positivity in HRS tumour cells may not influence PDL1 gene amplification or copy gain in paediatric HL. However, EBV presence would favour the recruitment of PDL1+ cells at the microenvironment in paediatric, possibly leading to a tolerogenic milieu.


Prognostic value of the immunoexpression of H3K4me3 and H2K27me3 epigenetic marks in follicular lymphoma

J. Gama*, S. Chacim, S. Paulino, C. Jerónimo, R. Henrique

*Centro Hospitalar e Universitário de Coimbra, Portugal

Background & objectives: Follicular lymphoma is the second most common subtype of non-Hodgkin lymphoma. Additional prognostic markers are under investigation. Epigenetic changes are now recognized as playing an important and early role in the lymphomagenesis process, contributing to the disease progression.

Methods: A retrospective transversal study was completed using archival biological material from 48 patients with follicular lymphoma, diagnosed between 2007 and 2012 at the Portuguese Oncology Institute Porto. The H3K4me3 and H3K27me3 marks were assessed by immunohistochemistry, using the Hscore. Relevant clinical and pathological data was extracted from the database of the Department of Oncohaematology.

Results: In this series, the median age at diagnosis was 60 years and recurrent/transformed disease was experienced by 34% of the patients. The median follow-up time was 71.5 months. The overall survival was estimated as 74.7% at 5 years. The disease-specific survival was estimated as 77.3% at 5 years. No difference in prognosis was observed concerning gender (p=0.755). The FLIPI score (p<0.001) and age at the time of diagnosis (p=0.019) were confirmed as prognostic factors. There was a statistically significant association between the immunoexpression of H2K27me3 and the FLIPI score (p = 0.038). When evaluating the epigenetic marks H3K4me3 and H3K27me3 no significant association with clinical outcome was found.

Conclusion: In this study, the immunoexpression H3K4me3 and H3K27me3 did not disclose prognostic value. A larger study is needed to fully determine the prognostic value of H3K4me3 and H3K27me3.


Morphology and histotopography of megakaryocytes in Ph-negative JAK2-mutated, CALR-mutated and triple-negative myeloproliferative neoplasms

D. Gogoleva*, G. Sychugov

*Chelyabinsk Bureau of Pathology, Russia

Background & objectives: The aim of study was to assess the quantity of megakaryocytes, histotopography (endosteal translocation), clusters formation, nuclear features (bulbous, staghorn-like, naked, dysmorphic, separated nuclei) in bone marrow biopsies (BMB) of patients with JAK2-positive, CALR-positive and triple-negative (TN) myeloproliferative neoplasms (MPN).

Methods: We investigated 62 BMB of patients with Ph-negative MPN. Patients were divided into the groups: JAK2 (N=31), CALR (N=17), triple-negative (N=14). Serial sections from BMB were stained with haematoxylin-eosin. A standard light microscope (х400) was used. The quantity of megakaryocytes, the nuclei and clusters were assessed in 10 HPF. The endosteal translocation were assessed on the full histological section.

Results: The biggest quantity of megakaryocytes was detected in JAK2-group (mean value (MV): JAK2=203,7±79,1; CALR=142,1±52,9; TN=134,4±48). The smallest number of clusters was in TN-group (MV: TN=7,5±4; JAK2=18,8±6; CALR=11,7±5,8). The bulbous nuclei were prevalent in JAK2-group (MV: JAK2=29,8±26,3; CALR=12,6±12,5; TN=3,2±2,9), the dysmorphic nuclei were prevalent in CALR-group (MV: CALR=14,5±2,5; JAK2=3,2±3; TN=5,2±6), the separated nuclei were detected only in TN-group (MV: TN=3,2±1). There was no significant difference in counting of other nuclear forms.

Tendency to the endosteal translocation was demonstrated by CALR-group in 82% (14/17) in comparison with JAK2-group - 58% (18/31) and TN-group - 43% (6/14).

Conclusion: Ph-negative MPN with various mutation status have differences in morphology and histotopography of megakaryocytes. According to the results, the MPN can be divided into additional molecular subtypes. It can be useful for supporting the diagnosis and classification.


Impact of accurate quantification of bone marrow plasma cells in the trephine core biopsy for the diagnosis of multiple myeloma patients

I. Hernández Alconchel*, S. Marcos González, M. Briz del Blanco, A. Bermudez Rodriguez, J. García Reyero, E. Ocio, S. Montes Moreno

*HUMV, Spain

Background & objectives: The presence of clonal PCin≥10% marrow cellularity is the threshold for diagnosis of PCM. Cellular counts differ between bone marrow aspirate (BMA)and bone marrow biopsy (BMB).We evaluated the concordance in the PC quantification by aspirate cell count and absolute quantification by IHC withMUM1 staining and the clinical correlation in cases with discordant results.

Methods: We analysed BMA and BMB samples at diagnosis from 48patients. Quantification of BM plasma cell infiltrates in the BMB was done by MUM1 immunohistochemistry. 3 representative images at HPF (40X) were acquired, the number of MUM1 positive and negative cells was quantified by visual enumeration. Semiquantitative estimation of the %of plasma cells was done by 2 pathologists in 28 cases with a BMA count below10%. A three-tiered score was used:<10%,10-60% and>60%MUM1+ cells.

Results: 71%of cases showed a concordant result between the BMA and BMB counts [20 cases with≥10%plasma cells(PC) and 14cases with<10%PC in both samples] 29% of cases were discordant with≥10% MUM1+PC in the BMB and<10%in the BMA.5 out of these 14cases fulfilled clinical criteria(CRAB)for MM and 9cases were considered smouldering MM. Cohen´s kappa coefficient for the semiquantitative categorization of cases according to the BMB cell counts was 0,862with only 2out of 28cases with discordant results among the 2 pathologists

Conclusion: Bone marrow trephine biopsy evaluation of plasma cell infiltrates is critical for the early identification of MM patients. The conversion rate of BMA PC<10% patients to ≥10% in BMB after absolute quantification of MUM1+PC was 29%.In 10%of the cases the patients fulfilled clinical criteria for overt MM. In cases with low BMA cell counts, semiquantitative evaluation of BMB plasma cell infiltrates with MUM1 immunohistochemistry is consistent among pathologists and may be used in the diagnostic setting when absolute quantification methods are not available


BCL2 in follicular lymphomas (FL): the overrated guy?

D. Marchiori*, F. Magnoli, C. Amaglio, D. Sabatino, V. Martin, M. Merli, L. Campiotti, F. Sessa, M.G. Tibiletti, S. Uccella

*Dept. of Medicine and Surgery, University of Insubria, Varese, Italy

Background & objectives: The t(14;18)(q32;q21) is considered the genetic hallmark of FL. However, some authors have observed a high proportion of FL lacking t(14;18), supposing geographic differences.

Our aim was to test the incidence of BCL2- FL and investigate alternative genetic aberrations.

Methods: We collected a series of 76 consecutive FLs from our Pathology Department between 2013 and 2016. All lymphomas underwent histopathological revision and were immunohistochemically characterised. Interphasic fluorescent in situ hybridization (FISH) was performed targeting BCL2, IGH, BCL6 and MYC on paraffin embedded (PE) and fresh frozen (FF) specimens. Conventional cytogenetic was applied to a subset of cases as well.

Results: Overall, BCL2 rearrangements and protein expression were detected in 54% and 87% of cases, respectively, with statistical correlation between the two dramatically increasing with increasing intensity of immunostaining (p<0.0001). BCL2 expression was related to a lower proliferative index, as assessed by Ki-67 (p=0.02).

Among cases lacking t(14;18), 6 showed IGH rearrangement, and were further tested: 1 was characterized indeed by a variant BCL2 translocation, 1 had a IGH/BCL6 rearrangement, whereas the other 4 were negative for both BCL6 and MYC. FISH performed on FF specimens detected small BCL2-rearranged clones in three BCL2-negative PE cases. Finally, karyotype reconstruction documented 3q27 and 1p abnormalities in 3 cases, respectively.

Conclusion: Our study suggests that t(14;18) is not a constant finding in FL, its incidence being probably affected by geographical factors. Alternative genetic aberrations exist in negative cases, and conventional cytogenetic may still represent a useful tool to investigate their role in lymphomagenesis.


Haematological indicators of Dengue illness and recovery: emphasis on white blood cells

A. Rai*, S. Sahu, N. Kulkarni

*MGM Medical College, Navi Mumbai, India

Background & objectives: In Dengue, as a norm platelet count guides the treatment and prognosis. This study assesses if WBC (white blood cell) count can be used as an equally sensitive parameter and it also analyses other haematological parameters in dengue.

Methods: This is a retrospective observational hospital-based study conducted over 6 months. 140 cases of dengue fever were included by complete enumeration method. Consecutive WBC count, Platelet count, WBC differentials and haematocrit were obtained from day of admission till discharge as part of treatment. Analysis was done using Spearman's rank correlation method and descriptive statistics to find for associations and patterns.

Results: Majority (67%) were males with mean age 30 years. On day of admission, 51% patients had leukopenia while 89% showed thrombocytopenia. By third day, leukopenia was seen in 33% patients while thrombocytopenia noted in 94%. On discharge, 12% patients showed leukopenia while 70% had thrombocytopenia. On day of admission, third day and day of discharge, mean WBC count obtained were 4568, 5547 and 6989 respectively and mean platelet count were 88217, 78479 and 131850 respectively. Mean WBC count and platelet count showed moderately positive correlation (R=0.6,p<0.05). Neutrophils showed decreasing trend while lymphocytes, monocytes, eosinophils and haematocrit showed an increasing trend post admission. Reactive lymphocytes were noted in conjunction with lymphocytosis.

Conclusion: In dengue patients, WBC count normalized with clinical improvement earlier than platelet count for majority making it an equally important parameter and a good indicator of recovery. The other studied haematological parameters also contributed in understanding the disease progress.


The study of the toxic effect of the heavy metals salts on the erythropoiesis in the rats

Y. Lyndina, V. Sikora, M. Lyndin, Y. Soroka, S. Romaniuk, L. Karpenko, A. Romaniuk*

*Sumy State University, Ukraine

Background & objectives: Heavy metals salts (HMS) are the most common pollutants that are proved to have the negative effect.

The objective is to determine the morphological features of the marrow in rats, caused by the combined effect of the heavy metals salts.

Methods: The study was carried out on the laboratory male rats (n=24), which were divided into 2 groups (control and experimental – the rats received HMS (zinc, copper, iron, manganese, lead, chromium). The animals were taken out from the experiment on the 30th and 90th day.

Results: The HMS intake leads to the significant changes among the precursors of the erythropoiesis. The islet location of its predictors has been disturbed, they were found as the indistinct assembles of the cells and separately in the areas of myxomatosis and oedema. The size of the survived islets and the number of the cells in their structure gradually reduce, the single precursors of the erythropoiesis with the features of dyserythropoiesis: the signs of karyorhexis, irregular shape of the nuclei, internuclear bridges, are observed among them. The morphological changes in the marrow increase with the extension of the experiment and reach the maximum on the 90th day.

Conclusion: The excessive entry of the HMS to the animals’ body leads to the qualitative (dyserytropoiesis) and quantitative (reduced number and size of the erythroid islets) changes in the erythropoietic tissue, which depend on the term.


Multiparametric flowcytometry in the diagnosis of plasma cell disorders

N. Stoyanov*, T. Dikov, P. Ganeva, G. Balatzenko, M. Guenova

*NSBALHZ, Bulgaria

Background & objectives: Plasma cell disorders present a spectrum from benign to aggressive course. The diagnosis involves clinical, laboratory, and imaging data. Multiparametric flowcytometry (MFC) is used to determine clonality and aberrant immunophenotypes.

Evaluate the contribution of MFC to the diagnostic work-up.

Methods: All patients with plasma cell disorders (n=203) diagnosed between 2008 and 2014 and followed up for a median of 46 months. A comprehensive flow cytometry panel was applied to bone marrow aspirates, supplemented by biopsies with immunohistochemistry. Antigen aberrations and clonality differentiated between normal and abnormal cells. Expression patterns and quantitative characteristics were further correlated with clinical and lab tests.

Results: MFC-confirmed clonality and abnormal antigen expression supported diagnosis. Antigens were frequently co-expressed (CD20, 29%, and CD28 43%, p<0,001 CD200, 90% and CD27, 41%, p=0,008), or appeared mutually exclusive (CD20, 29% and CD56, 72%, p=0.023). Phenotypically abnormal plasma cells showed correlation with tumour volume - ISS stage (p=0.002), haemoglobin (p=0.001) and platelet counts (p=0.01) and, expectedly, morphological bone marrow infiltration (p<0.001). Moreover, myeloma patients with abnormal/total plasma cell ratio of <95% at diagnosis had significantly lower tumour volume and excellent overall survival compared to other similarly treated patients. This correlation was maintained in patients receiving stem cell transplant (not reached vs. 51 months) or non-intensive therapy (73 vs. 16 months, p=0.003).

Conclusion: MFC supports the diagnosis of plasma cell disorders and contributes valuable prognostic data.


Primary cerebral lymphoma: clinicopathological analysis of 9 cases

R. Szodorai*, I. Egyed-Zs., L. Banias, E. Horváth

*"George Emil Palade" University of Medicine, Pharmacy, Science and Technology of Targu Mures, Romania

Background & objectives: Primary cerebral lymphomas are relatively rare neoplasms (represent < 4 % of all brain tumours) and in this context diagnostics and treatment are accordingly chal­lenging.

Methods: We enrolled newly diagnosed cases between 2015-2019. Patients who were diagnosed with secondary CNS lymphoma (n = 4) or acquired immune deficiency syndrome (n = 4) were excluded. Remaining cases were retrospectively analysed considering localization, tumour morphology and immunophenotype, prognosis and overall survival.

Results: Nine cases diagnosed as primary diffuse large B-cell lymphoma (DLBCL) were analysed. The patient’s age ranged between 33-74 years, predominantly females (F/M-7:2). On stereotactic biopsy samples, all of them were diagnosed as DLBCL. In four cases the histological differentiation was limited by the reduced tissue sampling, rich in necrosis, without distinct tumour mass. Microscopic examination mostly highlighted an angiocentric growth pattern of tumour cells. Applying the Hans algorithm, using CD10, BCL-6 and MUM-1, 6 cases belonged to non-germinal centre subtype, associated with high Ki67 (>70%). In the subgroup of elderly patients (over 65 years), a high Ki67 index and non-germinal centre subtype has been associated with short survival (three months).

Conclusion: Stereotaxic biopsy and histology of the affected area in most cases will provide a reliable diagnosis. The diagnosis of primary cerebral lymphomas is complex, the histological subtypes are difficult to evaluate due to reduced tissue sampling and requires the application of a large immunohistochemistry arsenal. Non-germinal centre subtype with a high Ki67 index in our patients has been associated with worse survival.


Molecular signatures of anaplastic large cell lymphoma, ALK-negative – pilot study with 125 genes panel and recurrent MSCE116K mutation testing

A. Szumera-Cieckiewicz*, B. Bikowska-Opalach, A. Dansonka-Mieszkowska, M. Łukasik, A. Tysarowski, M. Prochorec-Sobieszek

*Institute of Haematology and Transfusion Medicine, Diagnostic Haematology Department, Maria Sklodowska-Curie National Research Institute of Oncology, Department of Pathology and Laboratory Medicine, Poland

Background & objectives: Anaplastic large cell lymphoma, ALK-negative [ALCL-ALK(-)] is CD30-positive T-cell neoplasm which should be distinguished from primary cutaneous ALCL, other B-cell/T-cell lymphomas with anaplastic features and CD30(+) and classic Hodgkin lymphoma. Lately, a novel molecular signature [MSCE116K mutation] has been described.

Methods: We selected 4 cases with clinically and histopathologically confirmed ALCL-ALK(-). The immunohistochemical assessment included: pan-B/pan-T panel, ALK-1, the axis: CD30-IRF4/MUM1-MYC. All cases were tested with FusionPlex Lymphoma 125 genes panel. The MSCE116K mutation was evaluated in the Sanger technique.

Results: The patients were 2 males: 2 females with a median age of 68 years and generalized lymphadenopathy and advanced clinical stage of the disease. None of the patients had skin involvement. The histopathological characteristics and immunophenotype were concurrent: pan-T(+), ALK1(-), CD30(+), IRF4/MUM1(+), MYC(+) in >40% of cells. None of the cases were DUSP22 rearranged nor had ALK alteration; all lymphomas had splicing mutation of KMT2A[c.11321+2delC] gene and one case presented p63 fusion. Additionally, none of the cases harboured the MSCE116K mutation.

Conclusion: Molecular profiling of ALCL-ALK(-) started to be a key point in prognostication. The presented series of ALCL-ALK(-) could be categorized molecularly as 3 cases triple-negative, and 1 case p63 rearranged (poor prognostic factor). According to the literature, the MSCE116K mutation has been found among DUSP22 rearranged lymphomas only (none of our cases). The quality and validation of molecular testing from FFPE is relevant.

Funding: This work has been implemented using the Project infrastructure POIG.02.03.00-14-111/13.

PS-07 Infectious Diseases Pathology


Diagnosing congenital malaria in the eastern province of Rwanda - a comparison of infant peripheral blood smear, cord blood smear and placental histopathologic examinations

F. Byiringiro Mugabe*, J. Uwinkesha, P. Ntihinyurwa, P. Nkurunziza, D. Nyirahabimana

*Department of Clinical Biology, School of Medicine and Pharmacy, University of Rwanda, Kigali, Rwanda

Background & objectives: Congenital malaria is detected within a neonate’s first week of life and the diagnosis is made by detecting Plasmodium parasites in peripheral blood smear (PBS). We compared the results of placenta histology to blood smears for diagnosis of congenital malaria.

Methods: Peripheral and cord blood smears (CBS) were sampled, from neonates whose mothers were diagnosed with positive (+) malaria within the last 2 weeks of pregnancy, for light microscopic exam as well as placenta histology (PH) for detection of malaria parasites after consenting the mother. Regression analysis were done and results were considered significant if p-value <0.05, 95%CI.

Results: 111 neonates were recruited for 5 months. 86(77%) had at least one of the 3 tests positive. 58% had CBS+, 56% had PH+ and 44% had PBS+. Of those with PBS+, 61.6% had CBS+, while 50% had PH+. Neither the PH nor CBS alone was significant predictor of a positive PBS. However, when both were positive, there was a 95.5% chance that the PBS will be positive (p-value: 0.000, RR: 18.8).

Conclusion: There is high prevalence of congenital malaria in new-borns from mothers presenting malaria during the last two weeks of pregnancy. Combined PH and CBS can be more reliable in early detection of parasites and allow treatment without sample from neonate.


Strongyloides stercoralis: hyperinfection syndrome with periumbilical purpura

M.D.L.M. Lewkowicz*, A. Bruno, J. Rodriguez Catan, C. Waldblaun, A. Paes de Lima, M.A. Avagnina Iribarren, M.A. Allevato, M.d.l.A. Juarez

*Hospital de Clínicas "José de San Martin", Universidad de Buenos Aires, Argentina

Background & objectives: Strongyloides Stercoralis is a pathogenic filariform nematode which causes mostly an asymptomatic chronic disease. Under immunosuppression conditions its life circle is exacerbated. Massive multiplication of larvae develops clinical manifestations within the usual pattern of migration, which is known as hyperinfection syndrome.

Methods: We report two cases of strongyloides stercoralis hyperinfection syndrome with periumbilical purpurae.

Results: 25-year-old female, HTLV1 positive, presented with abdominal pain, loose stools, severe malnutrition and non-confluent petechial and purpura lesions in abdominal region, flanks and proximal limbs. Tomography evidenced alterations in the gastrointestinal tract. Esophagogastroduodenoscopy showed petechial involvement of the duodenum and jejunum.

Biopsies of skin and colon evidenced mature filariform larvae.

40-year-old male with history of smoking and cocaine addiction consulted for abdominal pain, diarrhoea, unquantified weight loss and non-confluent petechiae in the periumbilical abdominal region. Tomography evidenced a heterogeneous consolidation in right lung and parietal thickening of jejunum and ileum. We received a bronchoalveolar lavage cytology, where mature filariform larvae were encountered.

Stool culture revealed Strongyloides Stercoralis in both cases.

Conclusion: Petechial and purpuric lesions in abdomen, flanks and limbs have been documented in the literature as a rare form of cutaneous presentation of hyperinfection syndrome but they may be a valuable diagnostic sign. Skin biopsy is essential for accurate diagnosis.


Histoplasmosis presenting as a cecal mass: report of two cases

M.D.L.M. Lewkowicz*, R. Malerba, G.A. Daniela Fernanda, A. Wonaga, C. Waldbaum, G. Benchetrit, J. Hevia, C. Cerisoli, J. Sorda, M.A. Avagnina Iribarren

*Hospital de Clínicas "José de San Martin", Universidad de Buenos Aires, Argentina

Background & objectives: Histoplasma Capsulatum is a dimorphic fungus, worldwide disseminated. It presents as a self-limited respiratory infection or is asymptomatic. Disseminated disease occurs in immunocompromised individuals. Gastrointestinal involvement is rare. Ileum is the most common site and clinical manifestations are exceptional and unspecified.

Methods: We report two cases of disseminated Histoplasmosis syndrome initially presented as a cecal mass, one in a non immunocompromised patient.

Results: 67-year-old woman, treated with oral medication for type 2 diabetes mellitus presented with anorexia and weight loss (10kg). Videocolonoscopy showed a sessile lesion, with central ulceration in the cecum. Biopsy specimens were obtained.

26-year-old male, HIV positive, presented with abdominal pain, liquid bowel movements, chills and unintentional weight loss in the last two months. Videocolonoscopy showed deformed, ulcerated and indurated cecum.

Diagnosis of histoplasmosis was done in both cases by histological examination of cecum biopsies, which revealed multiple intracellular small organisms suggestive of Histoplasma capsulatum.

First patient was HIV negative with positive serology for Histoplasma Capsulatum and PCR negative bronchoalveolar lavage. Second patient PCR in bronchoalveolar lavage was positive.

Conclusion: Histoplasmosis with gastrointestinal presentation and without respiratory symptoms is rare, as well as disseminated disease in patients without immunosuppression. Symptomatology can mimic tuberculosis infection, malignant lesions and others. Biopsy during endoscopic evaluation should be obtained to achieve the correct diagnosis.


Abdominal actinomycosis simulating a colorectal neoplasia – 2 cases

J. Madeira*, J. Gama, F. Ramalhosa, J. Fraga, M.B. Pimentão, A. Lai, C. Faria, V. Almeida, H. Moreira, R. Almeida, R. Oliveira, M.R. Silva, M.A. Cipriano

*Centro Hospitalar e Universitário de Coimbra, Portugal

Background & objectives: Intra-abdominal actinomycosis (AIA) is a progressive, granulomatous and suppurative disease that can simulate a neoplastic process and consequent inadequate treatment. It is most commonly ileocecal, mimicking colorectal neoplasia, often requiring surgery.

Methods: We present two cases in women with 60 and 84 years, with abdominal pain and a palpable mass with 1.8 and 3.2 months of evolution, one located in the right iliac fossa and the other in the right hypochondrium. Imaging examination did not exclude neoplasia. Endoscopy revealed congestive mucosa and biopsies showed ischemic colitis.

Results: After an antibiotic cycle without improvement, both underwent segmental colectomy resection. The macroscopic evaluation did not exclude a neoplastic process, and histological study showed pseudotumoral lesions consisting of fibrous tissue with abscesses and colonies of actinomyces (Grocott), without evidence of malignancy.

Until the moment of submission, patients were well, without any complications or reinfection.

Conclusion: AIA diagnosis is difficult due to non-specific features. It is often clinically confused with neoplasia or other inflammatory diseases. AIA is rare and a diagnosis of exclusion, usually made after surgery and histological analysis. It should be considered in immunocompromised patients.


Histopathological and MPT64 immunohistochemical diagnosis of extrapulmonary tuberculosis at the Muhimbili National Hospital

C. Ngimba*, A. Mwakigonja

*Muhimbili National Hospital, Mutokiti Diagnostic Laboratory, Tanzania

Background & objectives: Extra-pulmonary Tuberculosis (EPTB) has diagnostic challenges in resource limited setting. Confirmation depends on histology and Ziehl Nielsen stain (ZNS). Immunohistochemistry using anti-MPT64 can detect mycobacterium TB complex proteins. We describe the histomorphology and anti-MPT64 immunoreactivity on Formalin fixed paraffin embedded (FFPE) biopsies.

Methods: FFPE tissue blocks and haematoxylin-eosin stained slides of biopsies which were signed out as EPTB (July 2015 - June 2017) were retrieved from the archive. Slides of included cases were reviewed to confirm the diagnosis prior to resectioning for ZN staining and IHC. Demographic, clinical and histopathologic data were analysed. Proportions of AFB positivity for ZNS and anti-MPT64 immunoreactivity were calculated.

Results: 110 biopsies signed out as EPTB were analysed and biopsies were from patients aged between 2-76 years with mean of 36.3 years. Female represented 60% (n=66), lymph node was more frequent (41.8%, n=46) site and necrotizing granuloma was the most (69%, n=76) frequent lesion. IHC and ZNS positivity were found in 71% and 8% of the biopsies respectively. All ZN positive biopsies were also positive to MPT64 by IHC.

Conclusion: EPTB is encountered frequently among biopsies submitted at our hospital. Despite the challenges faced in establishing EPTB diagnosis, the histomorphology of necrotizing granulomas should be regarded as a strong indicator of EPTB, particularly in TB endemic areas. Furthermore, IHC by anti-MPT64 appears to be useful particularly in controversial cases where ZN staining is negative. EPTB affects mostly young adults in reproductive age group and the female gender and TB lymphadenitis is the most common type of EPTB presentation at MNH.


Laboratory and anatomopathological aspects in the diagnosis of Dengue in Ceara in 2011 and 2012: the role of the Death Verification Service

D. Nunes de Melo*, L. Matias Albuquerque, G. Bitu dos Santos Ponte, S. André de Souza Júnior, D. Nunes Oliveira, J. Carneiro Melo, L. Pamplona de Góes Cavalcanti

*State Secretary of Health, Brazil

Background & objectives: In Brazil, has expressive epidemic outbreaks of dengue and not all the cases with fatal evolution are diagnosed by the public health service. Evaluate the impact of Death Verification Service (DVS) for detecting non-suspected dengue deaths in Ceara.

Methods: Post-mortem examinations were performed in the period from 2011 to 2012 in cases suspect of dengue and cases non-suspected by the clinician but that were suspected by pathologist. The test to IgM, NS1, viral isolation and PCR and immunohistochemical test were performed. Deaths due to dengue were confirmed only if they fulfilled the WHO criteria.

Results: Were performed 214 post-mortem examinations in which 121 (56.5%) were confirmed as dengue, however 90 of these were asymptomatic for dengue. Co-infections were detected in 46 cases in which bacterial organisms were the most prevalent (93.5%). Oedema and haemorrhage occurred in all exams and organs with more pronounced oedema in the lungs (79%) and central nervous system (71%). Haemorrhage was predominant in the adrenals (31%) and lungs (24%). Acute respiratory failure (47.1%) and shock (33.8%) were the most frequently reported causes of death.

Conclusion: The DVS ,Center for Epidemiological Surveillance and Central Public Health Laboratory helped to increase by 5.1 times the number of dengue’s related deaths. This is the largest historic series of autopsied deaths due to dengue in the world.


Diagnosis of tuberculous lymphadenitis: from morphology to molecular analysis

B. Pehlivanoglu*, B. Aydin Turk, S. Aksoz

*Adiyaman University, Turkey

Background & objectives: Culture is still the gold standard for diagnosis of tuberculosis (TB), however, molecular methods have also been shown to be effective diagnostic tools. We aimed to investigate the association between histopathological features and molecular TB positivity in this retrospective study.

Methods: Slides of 36 consecutive patients who had undergone lymph node resection with clinical suspicion of TB lymphadenitis (TBLA) were re-evaluated. Molecular TB testing was performed on formalin fixed-paraffin embedded (FFPE) tissues using a real-time polymerase chain reaction (Anyplex™ MTB/NTM, Seegene, Inc). The findings were statistically analysed.

Results: The majority (n=26) were female and median age was 44.50±14.42 (range 17-69 y.o.). All but 2 patients had granulomatous inflammation, however, TBLA was confirmed by molecular analysis in only 8 patients, 5 of which had pure caseous granulomas. 2 patients with confirmed TBLA had mixed caseous and sarcoid-like granulomas, and 1 had suppurative necrotizing granulomas. The bacilli could be demonstrated by acid-fast staining in only 1 TBLA patient. TB-DNA was negative in 14 patients with mixed caseous-sarcoid granulomatous inflammation, 4 with suppurative necrotizing granulomatous lymphadenitis and 8 with sarcoid-like granulomas. Molecular TB positivity was significantly associated with the presence of caseation (p=0.018). No other significant association was found.

Conclusion: Molecular testing seems to be more sensitive for diagnosis of TB in FFPE tissues compared to acid-fast staining. Morphological overlaps of different granulomatous patterns can be seen in TB, and although widely considered to be characteristic for TB, caseation is not always a sign of TB positivity.


Tissue reactions in cases of progression of tuberculosis in patients with HIV/AIDS

A. Sapargaliyeva*, B. Alibekov, S. Koybakov, I. Sergeyeva

*Pathology Bureau of the City of Almaty, Kazakhstan

Background & objectives: Development of the inflammatory response in pulmonary tuberculosis in patients with HIV/AIDS are of interest from the point of view of establishing mechanisms of resistance to infection, and, accordingly, finding ways to increase resistance to tuberculosis

Methods: We conducted a retrospective morphological analysis of 15 autopsies of patients with HIV/AIDS with progressing tuberculosis (2016-2018). All patients (3 women and 12 men) had a diagnosis of pulmonary TB, MBT (+), TBMLU. The duration of inpatient treatment varied from 7 to 100 days. The average age of patients was 37 years.

Results: The progression of TB varied: in 6 cases we observed the development of a tissue reaction in organs with the formation of giant cell granulomas with central caseous necrosis and limited foci of tuberculous inflammation from the surrounding tissue. In 9 cases, there was a predominance of the exudative type of tissue reaction with the formation of large fields of caseous necrosis, surrounded by perifocal oedema. In the latter case, the inflammatory process was widespread with the involvement of both lungs, parietal pleura, and peritoneum. The cause of death was pulmonary haemorrhage (14 cases), diffuse purulent peritonitis (1 case). In all cases, cachexia was established.

Conclusion: Tuberculous inflammation in patients with HIV/AIDS is characterized by a variety of morphological manifestations, including a predominance of productive tissue reactions and the development of exudative reactions with massive zones of caseous necrosis. Differences in tissue reaction can be considered as one of the cytological mechanisms of sensitivity and resistance of tissues to tuberculosis infection.


Immediate death causes in HIV infection

V. Zinserling*, O. Leonova, M. Vassilyeva

*VA Almazov Research Center, SP Botkin Infectious Hospital, Russia

Background & objectives: HIV remains important infection frequently revealed in the deceased. In spite of numerous clinical investigations, many questions can be discussed only basing upon the results of post-mortem autopsy findings.

Methods: Detailed analyses of pathology records since the first lethal cases occurring in S.P. Botkin infectious hospital and Cities’ centre for Diagnostics and treatment of AIDS. Minor discrepancies between clinical and pathological diagnosis are revealed in majority of cases. Practically in all observations several secondary diseases (till10) are diagnosed, in many cases they disappear from the statistical analysis.

Results: Analysing the spectrum of secondary infections and immediate death causes we noted not significant changes since 1996. Thus, recently we observed increase of PML ( 25 in 2018) and different neoplasms (35 in the same year). Among The number of cases with pneumocystosis (51) , cytomegaly (2), cryptococcosis (17) and toxoplasmosis (38) formed sinusoid like variations. In HIV encephalitis we started to see giant-cell reactions never noted before.

In rare lethal cases of the treated patients we considered the cases not directly associated with HIV (in 2016 - 348). In certain cases we still can’t exclude the indirect role of the virus.

Conclusion: Thus, the exact data concerning HIV lethality and better understanding of the disease mechanisms can be obtained only in complex studies including post-mortem morphological.

PS-08 Nephropathology


Morphological findings in frozen non-neoplastic kidney tissues of patients with kidney cancer from large-scale multicentric studies on genomics of renal cancer

B. Abedi-Ardekani*, D. Nasrollahzadeh, L. Egevad, A. Warren, C. Carreira, J. Viksna, J. Mckay, G. Scelo, P. Brennan

*Int Agency for Research on Cancer-WHO, France

Background & objectives: Analysis of non-neoplastic tissues helps to better understand the process of carcinogenesis. We performed microscopic examination of non-neoplastic kidney tissues from kidney cancer patients who were recruited to large-scale multi-centric kidney cancer genomic studies from countries with variable incidence rates.

Methods: By applying digital pathology, we performed microscopic examination of 1012 frozen non-neoplastic kidney tissues from kidney cancer patients from Czech Republic, Romania, Serbia, United Kingdom, and Russia, with variable incidence rates of kidney cancer. Renal parenchyma was evaluated and scored for the interstitial inflammation and fibrosis, tubular atrophy, glomerulosclerosis, and arterial wall thickening, globally called chronic renal parenchymal changes.

Results: Moderate or severe chronic renal parenchymal changes was observed in 54 (5.3%) with predominance of occurrence among patients from Romania (OR=2.67, CI 1.07-6.67) and Serbia (OR= 4.37, CI 1.20-15.96) in reference to Russia. To assess the potential confounders, we adjusted for age, sex, history of hypertension and diabetes mellitus, percentage of medulla, stage of tumour, tumour size, and type of nephrectomy, which rendered no significant effect on the risk estimates.

Conclusion: Frequency of chronic renal parenchymal changes in kidney cancer patients varies by country, significantly more frequent in countries located in central and southeastern Europe where the incidence of kidney cancer has been reported to be high. This is unlikely to be due to sampling method or known confounding factors. We suggest that these parenchymal changes, possibly linked to environmental exposures, may be relevant to renal carcinogenesis in these countries.

Funding: Part of the samples are from CAGEKID study that is funded by European Union FP7 241669.


Lymphocyte populations in lupus nephritis

I.D. Caruntu*, T. Azoicai, R. Avădanei, L. Lozneanu, A.D. Timofte, S. Giusca

*UMF "Grigore T. Popa", Iasi, Romania

Background & objectives: Studies targeting the profile and role of lymphocyte populations in lupus nephritis (LN) are limited, most of them being from experimental research. Within this context, our study aims to analyse the T lymphocytes (Ly) populations in different classes of LN.

Methods: 53 LN renal biopsies (2 cases class II, 4 cases class III, 19 cases class IV, 22 cases class V, 6 cases class VI) were immunohistochemically investigated by using anti-CD4 and anti-CD8 antibodies. CD4 and CD8 T Ly were quantified in three distinct territories: periglomerular, intraglomerular and interstitial, at 400X. The results were expressed as the number of positive cells/mm2.

Results: We registered significant differences (p<0.05) for: periglomerular CD4 T Ly, class II versus class III; interstitial CD4 T Ly, class IV versus class V and class V versus class VI; periglomerular and intraglomerular CD8 T Ly, class III versus class IV; periglomerular and interstitial CD8 T Ly, class IV versus class V. Significant differences between CD4 Ly and CD8 Ly were recorded in all three territories, in all LN classes.

Conclusion: Quantification of T Ly offers the possibility to analyse their involvement in the immune pathogenic mechanism. LN lymphocytic infiltrate is predominantly composed of CD8 T Ly. This finding is in agreement with the immunological theory that supports the effector role of CD8 T Ly and that of initiator and target for CD4 T Ly. Our data complement the limited number of published studies on this topic and support the different behaviour of the two T Ly populations in LN pathogenesis.


Expression of indoleamine 2,3-dioxygenase 1 in tubular epithelial cells in renal grafts as a key feature of maintaining the local immunosuppression

P. Donizy*, M. Banasik, A. Hałoń, O. Mazanowska, D. Janczak, M. Krajewska

*Department of Pathomorphology and Oncological Cytology, Wroclaw Medical University, Poland

Background & objectives: Indoleamine 2,3-dioxygenase 1 (IDO1) is an enzyme that contributes to inducing local immunosuppression. The aim of our study was to evaluate the expression of IDO1 in renal transplant biopsies and to analyse the correlations between IDO1 expression and clinical parameters.

Methods: Immunohistochemical expression of IDO1 was analysed in 121 renal transplant biopsies in three tissue compartments: tubular epithelium (TE), glomeruli (G), and in foci of interstitial immune infiltrates (III). The scores for TE and G were dichotomized into two categories: no or any expression. IDO1-positive cells in foci of immune infiltrates were dichotomized into negative or positive group (any IDO1-positive cells).

Results: Rejection was observed in 32% (25/76) of patients with IDO1 expression in tubules (TE 1 or 2), and in 62% (28/45) of patients without IDO1 expression in tubules (TE 0) (p=0.0017). Increased number of IDO1-positive interstitial immune infiltrate cells was correlated with the presence of histopathologic features of any type of rejection. Rejection was observed in 56% (45/80) of patients from IDO1-positive group vs 19.5% (8/41) in IDO1-negative group (p=0.0001).

Conclusion: The expression of IDO1 in tubules had a potential protective role but enhanced number of IDO1-positive interstitial immune cells was associated with the presence of rejection. The analysis of IDO1 expression in renal transplant biopsies might be considered for better transplant immunological risk assessment. Our preliminary results should be validated in a larger group of patients.


Dent’s disease: clinicopathologic and genetic findings of two patients with novel variants/mutations in the CLCN5 and OCRL genes

H. Gakiopoulou*, A. Stofas, E. Dermitzaki, D. Lygerou, E. Drosataki, S. Maragkou, E. Daphnis, K. Stylianou

*1st Department of Pathology, The National and Kapodistrian University of Athens, Greece

Background & objectives: Dent’s disease is a rare X–linked-disorder characterized by low-molecular-weight proteinuria and often considered a renal tubular disease. It is frequently underdiagnosed or diagnosed after patients have failed an immune suppression regimen. We present two well documented cases of Dent’s disease.

Methods: A Caucasian boy aged 9,5years and a young Caucasian man aged 30years, underwent a renal biopsy due to low molecular weight proteinuria (1.3-1.9gr/24h), without haematuria, with normal renal function.

Light microscope, immunofluorescence, electron microscope (EM) and genetic testing [Kidney Seq TM v3.0. and Whole Exome Sequencing (WES)] were performed.

Results: The main histologic finding in both biopsies was the presence of intratubular calcium salts (Von Kossa+).

Immunofluorescence and EM were negative or nonspecific.

Genetic testing revealed that the boy was hemizygous for a pathogenic variant in CLCN5, NM_000084:c1934-2A>G. Variants in this gene are responsible for X-linked Dent’s disease. This variant alters a canonical acceptor splice site and has not been reported by the Genome Aggregation Database. The young man had a new mutation in the OCRL gene (p.Asp631Glu) which was verified by Sanger sequencing. Further analysis showed that the patient was homozygous for the c.2986G>T polymorphism in the CASR gene and heterozygous for the c.2878C>T polymorphism in the same gene.

Conclusion: Two cases of Dent’s disease with novel variants/mutations in the CLCN5 and OCRL genes - corresponding to Dent’s disease 1 and Dent's disease 2, respectively- are presented. Dent’s disease must be kept in mind in the differential diagnosis of nephrocalcinosis and genetic testing should be performed.


Factors affecting outcome in pauci-immune crescentic glomerulonephritis: a single centre study

P. Gupta*, A. Bhalla, D. Rana, A. Gupta

*Sir Ganga Ram Hospital, India

Background & objectives: Pauci-immune crescentic glomerulonephritis (PICN) presents as rapidly progressive renal failure. Present study was undertaken to determine factors influencing renal survival based on Berden’s classification, renal risk score (Brix et al.) and individual risk factors.

Methods: Retrospective study done from January 2013 to December 2018. Cases were grouped into focal, crescentic, mixed and sclerotic categories based on histology. Serum creatinine, eGFR at biopsy and follow-up, were recorded. Renal biopsies were scored based on renal risk score [% of normal glomeruli (>25%,10-25% and <10%), percentage of tubular atrophy and interstitial fibrosis(≤25%,>25%) and eGFR(>15ml/min,≤15ml/min)] into 3 risk categories.

Results: 51 adult patients were included. Mean serum creatinine at biopsy was 7.05±4.57 mg/dl and eGFR was 13.6±12.16 ml/min/1.73m2. 33 cases were ANCA positive and 18 were negative. Based on Berden’s classification, cases were grouped as focal(4), crescentic(29), mixed(12) and sclerotic(6). Based on renal risk score cases were grouped as low(8),medium(23) and high(20). Univariate Cox regression analysis showed eGFR at biopsy (p 0.024), % IFTA (p 0.001) and % normal glomeruli(p 0.023) are predictors of ESRD. Multivariate Cox regression analysis confirmed IFTA (p<0.001) and % normal glomeruli (p 0.018) as significant predictors of ESRD. Kaplan-Meier survival analysis for histological categories (Log-Rank p=0.046) and the renal risk categories was done (Log-Rank p=0.002).

Conclusion: Percentage of normal glomeruli, IFTA and eGFR at time of biopsy were important risk factors influencing renal survival. Our study validate that renal risk score is a better predictor of survival as compared to histological classification proposed by Berden.


C4d deposition in lupus nephritis; clinicopathological correlation

S. Kiremitci*, R. Eren Sadioglu, S. Kutlay

*Ankara University Medical School, Pathology Department, Turkey

Background & objectives: C4d deposition in native kidney has been investigated increasingly in recent years. For lupus nephritis, there are few studies reported and the results are conflicting. We aimed to investigate the association of C4d deposition with histopathological and clinical parameters.

Methods: Renal biopsies of 51 biopsy-proven lupus nephritis patients were evaluated for C4d immunohistochemistry. Clinical information at the time of biopsy and follow up data were collected from hospital database. Glomerular C4d staining scores, separately for mesangial and capillary wall, were obtained considering the pattern, extent and intensity of C4d deposition. Spearman’s correlation analysis was used for nonparametric correlations.

Results: According to 2003 ISN/RPS classification system for lupus nephritis; 5 biopsies were classified as class II (9.8%), 16 as class III (31.4%), 16 as class IV (31.4%) and 12 as class V (23.5%) in the cohort. All biopsies except one showed glomerular C4d staining in mesangium and/or capillary wall. Glomerular staining scores were significantly higher in class V compared to class III (p=0.016), whereas it was not significant between class III and class IV. Glomerular capillary wall staining was positively correlated with levels of 24-h proteinuria (p:0,020), and the staining scores were significantly lower in patients in remission compared to patients with active lupus nephritis (p:0.006).

Conclusion: C4d immunohistochemistry is useful to show both the presence and the localization of immune deposits in lupus nephritis. Besides, glomerular C4d load is correlated with clinical disease activity in terms of proteinuria.


The value of DARC expression as diagnostic marker in renal transplant biopsies

J. Kläger*, F. Eskandary, G. Böhmig, S. Segerer, H. Regele

*Department of Pathology, Medical University of Vienna, Austria

Background & objectives: Duffy antigen receptor for chemokines (DARC) is one of the genes most upregulated in antibody mediated rejection (ABMR) and is associated with endothelial injury. We investigated whether immunohistochemistry (IHC) for DARC might serve as a diagnostic marker for active ABMR.

Methods: 82 renal allograft biopsies performed during a prospective clinical trial (BORTEJECT Study) with detailed patient and biopsy characteristics available were included. IHC for DARC was performed on 2μm paraffin sections using a mouse monoclonal anti-human DARC-Fy6 antibody. DARC-IHC positive peritubular capillaries (PTC) were quantified and the distribution of the cortical stained PTC were further described

Results: DARC that was mainly observed in peritubular capillaries (PTC) and in small venules and arterioles. 61 biopsies showed positive DARC staining in ≥5% of PTC, mainly located in areas of interstitial fibrosis or inflammation. Most of the ABMR cases were DARC positive (n=40 vs. n=7, p=0.01), but a substantial amount of biopsies without signs of rejection still showed DARC positive PTC. C4d positivity was not associated with DARC positivity (p=0.365). On a molecular level, we see a significant difference of DARC gene expression in DARC positive vs. negative biopsies with higher DARC gene expression linked to more DARC positive PTC in biopsies (log scale: 8.68(7.91-9.32) vs. 7.52(6.99-8.14), p<0.001; rs=0,546, p<0,001)).

Conclusion: DARC expression on endothelial cells generally matches DARC gene expression and is associated with ABMR. However, DARC expression could also be observed in biopsies without signs of rejection which limits its value as diagnostic marker for ABMR.


Co-occurrence of spherocytosis and UMOD disease causing variant in a three-generation family presenting with kidney failure in adulthood

N. Kojc*, A. Meglič, J. Kovač, M. Debeljak, A. Trampuš Bakija, V. Rajić, K. Podkrajšek Trebušak

*Institute of Pathology, Faculty of Medicine, University of Ljubljana, Slovenia

Background & objectives: Hereditary spherocytosis is clinically and genetically heterogeneous disorder, but patients rarely present with kidney diseases. Seven patients from the same family with spherocytosis were evaluated to assess the kidney failure presented in all affected adult patients.

Methods: Clinical, radiological and laboratory investigations were assessed to evaluate the spherocytosis and kidney disease. We performed genetics testing with next generation sequencing of genes related to hereditary spherocytosis, inherited glomerular disorders and tubulo-interstitial kidney disease.

Results: Two adults had end-stage kidney disease and one chronic kidney disease stage 4 with histopathological findings of interstitial fibrosis/tubular atrophy and glomerulosclerosis. There were no signs of kidney disease in four paediatric patients. Novel nonsense variant in SPTB gene (NM 001024858; c.4796G>A; p.Trp1599Ter) was detected in all family members with spherocytosis. Furthermore, all adult patients with kidney failure and two paediatric cousins of the index patients were heterozygous for the UMOD gene variant (NM 003361.3:c.552G>C, NP 003352.2:p.Trp184Cys) previously reported in patients with autosomal tubulo-interstitial kidney disease. UMOD variant was not present in the index patients. Renal biopsy in two adult patients revealed diffuse interstitial fibrosis, tubular atrophy and glomerulosclerosis.

Conclusion: Kidney failure in all adult patients was probably related to disease causing UMOD variant characterized by deficient production of functional uromodulin. It is not possibly to evaluate whether the haemolytic crises due to spherocytosis were influencing the progression of the UMOD related renal disease. The kidney disease in the family is warranting the regular monitoring in UMOD positive paediatric patients in order to recognize early signs of tubular injury.

The study was supported by the financial support from the Slovenian Research Agency (research core funding P3-0343 and P1-0170).


A histopathological study of childhood clinically complicated acute diffuse proliferative glomerulonephritis and correlation with serum creatinine and serum albumin. A study at a tertiary care hospital in Sri Lanka

K. Liyanaarachchi*, S. Thalgahagoda, N. Rathnathunga

*Royal Surrey County Hospital, United Kingdom

Background & objectives: Acute diffuse proliferative glomerulonephritis (ADPGN) is usually self-limiting. This study was conducted to document reversible and irreversible pathologies seen in complicated ADPGN in a paediatric population and to ascertain whether biochemical parameters would give a clue to the changes observed.

Methods: Forty-eight ADPGN proven renal biopsies were assessed. Severity of the glomerular injury and tubular injury were scored as GI 1+ and 2+ (reversible lesions), 3+ and 4+ (irreversible lesions), TI 0(no lesions), 1+ and 2+ (reversible lesions), 3+ (irreversible lesions) . Serum creatinine (SC) and serum albumin (SA) were correlated with above lesions using the “Spearman’s rank-order correlation”.

Results: All cases showed glomerular injury. 69% had reversible glomerular lesions while 31% were irreversible. Irreversible lesions included crescents and necrotizing lesions. Tubular injury was present in 46% and 92% were reversible and 8% were irreversible. Irreversible tubular injuries included tubulorrhexis. The group that showed reversible glomerular injury did not have crescents or necrotizing lesions at all. However, 42% had tubular injuries. A significant statistical correlation was not seen between the glomerular/tubular injuries and serum creatinine and serum albumin (P value was > 0.05).

Conclusion: Approximately 2/3 of the lesions in complicated ADPGN are reversible where the kidney is salvageable. More than a third have non-reversible lesions possibly culminating later on sclerosis of glomeruli. Determining the renal pathologies on the biochemical parameters is not possible.


Fibrillary glomerulonephritis concurrent with IgA nephropathy: a case series of 14 patients

S. Said*, S. Nasr

*Mayo Clinic, USA

Background & objectives: Fibrillary glomerulonephritis (FGN) is a rare glomerular disease characterized by glomerular deposition of randomly oriented fibrils that stain with IgG and DNAJB9. In contrast, IgA nephropathy (IgAN) is characterized by IgA-dominant glomerular deposits which appear granular by electron microscopy (EM).

Methods: The characteristics of 14 cases of FGN (defined by smudgy glomerular staining for IgG with corresponding randomly-oriented fibrils on EM, mean thickness 16 nm) concurrent with IgAN (defined by granular mesangial IgA by immunofluorescence with corresponding granular electron dense deposits by EM) are provided. This represents the first study on the clinicopathologic and outcome characteristics of this dual glomerulopathy.

Results: The cohort included 11 males & 3 females, mostly White, with median age of 56, who presented with haematuria (86%), proteinuria (100%), and renal insufficiency (71%). 1 patient had carcinoma, 1 hepatitis C, and 1 lupus. None had hypocomplementemia or monoclonal gammopathy. On follow up (median 27 months), 8 progressed to ESRD. Histologically,12 cases showed mesangioproliferative GN, 1 crescentic GN and 1 mesangial sclerosis. DNAJB9 IHC was positive in all.

Conclusion: Most cases of FGN concurrent with IgAN are idiopathic, and not associated with liver disease, hepatitis C, malignancy, autoimmune disease, or monoclonal gammopathy. The pathologic diagnosis can be established by correlating the findings on immunofluorescence and electron microscopy. DNAJB9 IHC is useful to establish the diagnosis of FGN in these cases, particularly in centres that do not perform electron microscopy. The prognosis of this dual glomerulopathy is guarded with >50% of patients progress to ESRD within 2 years.


Immunological prospective of glomerulonephritis

S. Sarfaraz*, S. Anis, R. Muzaffar

*Dow University of Health Sciences, Pakistan

Background & objectives: Glomerulonephritis (GN) is a manifestation of various immune mediated and inflammatory mechanisms. Autoantibodies play a significant role in the diagnosis and pathogenesis. Antinuclear antibodies (ANA) were reported in 33% patients. This research aims to determine various autoantibodies in glomerulonephritis patients.

Methods: Two hundred clinically diagnosed patients of glomerulonephritis were included who had ANA. These patients were further evaluated for presence of anti-dsDNA and anti-extractable nuclear antibodies (ENA). ANA (pattern and titer) and Anti-dsDNA were evaluated via Indirect immunoflourecence assay. Anti-ENA were determined by Immunoblot assay which included anti-Sm, -anti-SSA, anti-SSB, anti-Scl-70, anti-jo-1, anti-PCNA, anti-Histones, anti-nucleosomes, anti-CENP B, anti-PM-Scl and anti-Rib.P-protein antibodies.

Results: Main causes of glomerulonephritis were Lupus nephritis 126(63%), idiopathic membranous GN 10(5%), post infectious GN 7(3.5%), FSGS 6(3%), Overlap syndrome 8(4%), RPGN 5(2.5%), Systemic sclerosis 3(1.5%) in 35(17.5%) no underline cause was identified. Autoantibodies were detected in 111(56%) patients. Anti-dsDNA was found in 56(50.4%) whereas anti-ENA were detected in 94(84.7%) patients. Single antigen specificity was found in 55(27.5%) individuals. Frequencies of various anti-ENAs were anti-SSA 58(61.7%),-Sm 18(19.1%), - histones 18(19.1%),-Rib. P-protein 15(16%)-RNPs 13(13.8%),-SS-B 8(8.5%), -Scl-70 7(7.4%),PCNA 3(3.2%),-Jo-1 3(3.2%) -CENP B 2(2.1%) and-PM-Scl 2(2.1%). Single ANA patterns was observed in 181(91%),among them speckled pattern was most common 72(36%).Frequencies of ANA titer were 1:40 ;30(15%), 1:80 ; 28(14%), 1:160; 62(31%), and ≥1:320; 80(40%)

Conclusion: ANA are hallmark of autoimmune connective tissue disorders however they are also found in normal individuals (5%-10%) and in other diseases and infections. ANA profile (anti-dsDNA and anti-ENA) helps in identifying underlying diseases, hence improves management in patients primarily presenting with glomerulonephritis. In this study patients were identified having high ANA titer with no underline autoimmune disorder. This group needs close monitoring as autoantibodies develop earlier before clinical manifestation of disease


BK-virus associated nephropathy in native kidneys and BK-virus in urothelial carcinoma

J. Schmitz*, J. Gottlieb, J. Korth, C. Bara, M.A. Kuczyk, H. Haller, J.H. Bräsen, A. Schwarz

*Institute of Pathology, Nephropathology Unit, Hannover Medical School, Germany

Background & objectives: Patients with non-renal organ or stem cell transplantation (Tx) may suffer from BK-viral nephropathy (BKVN) of their orthotopic kidneys by reactivating their own viral population living in latency of urothelial cells. BK-virus might be underdiagnosed in such uncommon patients.

Methods: We identified yet unknown cases and aimed to know how patients with BKVN after non-renal organ transplantation and patients with urothelial tumours differ from renal transplant patients with BKVN. We screened the past 20 years of the pathology archives of Hannover Medical School for suspective cases (lung-, heart-, liver-, stem cell- tx, patients after solid organ tx with urothelial carcinoma).

Results: We evaluated SV40 immunohistochemical stains in >100 identified cases and followed retrospectively the clinical course of patients with BKVN. Six lung-Tx, two lung-Tx/HSCT, one heart-Tx, one leukaemia and two stem cell-Tx patients showed positivity for SV40 in their renal biopsies. Three other patients after heart respective combined heart/kidney respective kidney-tx developed urothelial carcinoma with SV40 positive tumour cells six respective five respective nine years after high BK-viruria. Currently, BKV genotypes are being identified by PCR from the formalin-fixed paraffin-embedded tissues.

Conclusion: BKVN in orthotopic kidneys is seen preferentially after lung transplantation. In patients with BKVN after non-renal organ transplantation diagnosis is often established late and with high viral load of long persistence. Patients develop severe renal insufficiency and often end-stage renal disease. The course of BKVN of orthotopic kidneys after non-renal transplantation might be more detrimental than BKVN in renal transplants. BKVN might be a risk for urothelial cancer in Tx-patients.


Computer-assisted measurement of glomerular size in IgA nephropathy: practical issues and correlation with the Oxford Classification

M. Shamassi*, S. Shi, E.S. Ayva, I.S. Roberts

*Department of Anatomical Pathology, Monash Medical Centre, Monash Health, Melbourne, Australia

Background & objectives: Glomerular size has been demonstrated as an independent prognostic marker in IgA nephropathy. Using digital pathology, this study aims to determine reproducibility of GD measurement, defining minimum number of glomeruli required for accurate measurement, and correlating GD with MEST-C scores.

Methods: The diameter of all non-sclerosed glomeruli from renal biopsies of 420 patients with IgA nephropathy was measured independently by 3 pathologists, using the ruler device on Aperio ImageScope software. Inter-observer and intra-observer variability was then assessed using interclass correlation coefficient (ICC). Mean and maximum GD were correlated with Oxford Classification MEST- C scores, using the independent samples T test.

Results: There was excellent interobserver (between 3 pathologists) and intra-observer (one pathologist measuring 2 levels from the same biopsy) correlation with ICC of >0.9. Accuracy of measurement of maximum and mean GD increased with number of glomeruli measured, levelling off between 12-15 non-sclerosed glomeruli. T score was the only MEST-C variable to significantly correlate with glomerular diameter.

Conclusion: Use of the ruler device on digital slide viewing software provides a rapid and reproducible estimate of glomerular diameter. Greater than 12 non-sclerosed glomeruli are required to provide an accurate measurement of the glomerular diameter. Glomerulomegaly results from adaptive changes associated with hyperfiltration that is in part determined by the severity of chronic renal injury, reflected by correlation of GD with Oxford Classification T score.


A clinicopathological study to determine the different morphological patterns of renal biopsy in adult patients of nephrotic syndrome

D. Vedant*, V. Kaushal, S. Vikrant, S. Thakur, S. Asotra

*Department of Pathology, AIIMS Jodhpur, India

Background & objectives: Nephrotic syndrome is pathognomonic of glomerular diseases. Examination of renal biopsies only by light microscopy is not sufficient. Immunofluorescence studies can modify the final diagnosis. Hence this study was undertaken to know the spectrum of histopathological changes and immunofluorescence pattern.

Methods: A prospective observational study over a period of one year. Ultrasound guided renal biopsies of newly diagnosed patients of nephrotic syndrome were included. 10% Neutral buffered formalin fixed biopsies were processed and stained with H and E, PAS, Jones Silver, Masson Trichrome and congo red. Direct immunofluorescence studies done on saline samples with IgG, IgM, IgA, C3 and C1q.

Results: Present study evaluated 87 cases. Membranous nephropathy was the commonest type. FSGS was more common in elderly age group. In Membranous Nephropathy IgG positivity was seen in 97% of the cases whereas IgM positivity was seen in 26.4% cases. Linear pattern was the commonest pattern for both IgG (44.8%) and IgM (24.1%) followed by the granular pattern. Final diagnosis was modified in 8.05 % of cases after immunofluorescence studies.

Conclusion: Immunofluorescence studies are necessary for the evaluation of renal biopsies as they can modify the final diagnosis in a significant number of cases. Many cases of nephrotic syndrome can be diagnosed without the aid of electron microscopy. This study may help to understand the regional geographical variations in various aetiologies of nephrotic syndrome.

PS-09 Neuropathology


PlexinA1 receptor and its ligand, axon guidance molecule semaphorin 6D are implicated in medulloblastoma progression

M. Alshemeili*


Background & objectives: Medulloblastomas (MBs) are the most common malignant childhood brain tumours. Axonal guidance molecules specifically activated during brain development such as Semaphorin pathways also participate to the molecular signature of SHH and subgroup 4 of medulloblastoma which act with plexins.

Methods: - Cohort study of patients for QRT-PCR mRNA and protein analysis of PlexinA1 / Sema6D expression(13 MBs biopsies).

- Animals and cerebellar granule precursor cells purification

- Immunoblotting PlexinA1 and Semaphorin 6D antibodies. (Abcam (ab 32960) and R&D systems MAB 2095)

- Apoptosis and Proliferation assay (BrdU immunofluorescence staining and caspase 3 activity).

- Transwell migration assay and Immunofluorescence.

Results: - Sema6D mean values tended to be higher in demsoplastic MBs.

- PlexinA1 transcript expression was increased in all classic MBs compared to desmoplastic MBs.

- mRNA & protein PlexinA1 expressions are restricted to DEV cell line.

- High expression of PlexinA1 found in classic MB which enhance migration process.

- Higher expression of PlexinA1 was found in group 4 MBs and in metastatic MBs (6D).

Conclusion: We underscored a new role for Sema6D/PlexinA1, promoting MB cell migration and further correlated PlexinA1 expression to metastatic status. Altogether these data showed that Sema6D/Plexin-A1 may provide new insights into MB metastatic potential. The striking in vitro blockade of cell migration resulting from PlexinA1 ectodomain deletion let envision new fields of research to target this receptor in MBs.

PS: Immuno-histochemistry study of plexinA1 in MBs in progress.


Clinical management and genomic profiling of paediatric low-grade gliomas in Saudi Arabia

M. Alzahrani*, N. Mobark, M. Alharbi, L. Alhabeeb, L. AlMubarak, R. Alaljelaify, M. AlSaeed, A. Almutairi, F. Alqubaishi, M. Ahmad, H. Al-Khalidi, D. Barakeh, M. Abedalthagafi

*McGill University, Canada

Background & objectives: Paediatric Low Grade Gliomas (PLGGs) display heterogeneity regarding morphology, genomic

drivers and clinical outcomes. The treatment modality dictates the outcome and optimising

patient management can be challenging.

Methods: In this study, we profiled a targeted panel of cancer-related genes in 37 Saudi Arabian patients with pLGGs to identify genetic abnormalities that can inform prognostic and therapeutic decision-making.

Results: We detected genetic alterations (GAs) in 97% (36/37) of cases, averaging 2.51 single-nucleotide-variations (SNVs) and 0.91 gene fusions per patient. The KIAA1549-BRAF fusion was the most common alteration (21/37 patients) followed by AFAP1-NTRK2 (2/37) and TBLXR-PI3KCA (2/37) fusions that were observed at much lower frequencies. The most frequently mutated-genes were NOTCH1-3 (7/37), ATM (4/37), RAD51C (3/37), RNF43 (3/37), SLX4 (3/37) and NF1(3/37).

Conclusion: To our knowledge this is the first report of GOPC-ROS1 fusion in PLGG. Taken together, we reveal the genetic characteristics of pLGG patients can enhance diagnostics and therapeutic decisions. In addition, we identified a GOPC-ROS1 fusion that may be a biomarker for pLGG.


Review of atypical teratoid/rhabdoid tumours in a tertiary hospital

F. Arias*, E.M. Pena Burgos, R.M. Regojo Zapata, M.I. Esteban Rodríguez

*Hospital Universitario La Paz, Spain

Background & objectives: Atypical teratoid/rhabdoid tumour (AT/RT) is a rare entity accounting for 1-2% of all paediatric brain tumours. It is characterized by inactivation of SMARCB1 (INI1) or SMARCA4 (BRG1) genes. Our aim is to review the AT/RTs diagnosed in our hospital.

Methods: We searched our database for every AT/RT diagnosed since the introduction of immunohistochemical staining for INI1 in our hospital (2013). For each case we studied age, sex, location and tumour extent, tumour size measured by magnetic resonance, histopathological patterns (including rhabdoid, small-cell, gland-like and spindle cell patterns), immunophenotype, follow-up time, treatment and outcome.

Results: We gathered 5 AT/RTs. Four of them were children (median age of 4,5 years; range 1-11 years) and one was an adult (46 years). Male-to-female ratio was 1:1,5. Median tumour size was 6,4 cm and cerebrospinal fluid dissemination was found in one case. Histopathological analysis always revealed a combination of patterns. Rhabdoid features were present in 4/5 cases, being the predominant pattern in one of them. Every case showed negative INI1 expression by immunohistochemistry. Three patients died (mean survival 43 weeks), one was lost to follow-up, and one of the paediatric patients (11 years) is still on follow-up after 72 weeks.

Conclusion: AT/RTs have a wide differential diagnosis. We emphasize the importance of considering AT/RT in heterogeneous tumours in paediatric population, especially in infants. Due to its low prevalence in adults, other INI1-negative tumours should be ruled out prior to diagnosing AT/RT and the performance of additional studies should be suggested. We present our experience willing to provide data for a better understanding of this uncommon entity generally associated with poor prognosis.


Diffuse gliomas with non-canonical IDH mutations

E. Bariani*, A. Eccher, C. Ghimenton, M. Simbolo, L. Piredda, G. Pinna, F. Sala, A. Scarpa, V. Barresi

*Department of Diagnostics and Public Health, Section of Pathology, University of Verona, Italy

Background & objectives: The majority (90%) of IDH mutated diffuse gliomas carry IDH1 R132H mutation, while other (non-canonical) IDH1 or IDH2 mutations are rare. In this study, we aimed to analyse the histopathological features of diffuse gliomas with non-canonical IDH mutations.

Methods: We sequenced by Sanger IDH1/IDH2 genes in 89 IDH1 R132H immuno-negative diffuse gliomas and validated our findings using data in TGCA database.

Results: Diffuse gliomas with non-canonical IDH mutations were rare (3.3%). Non-canonical mutations in IDH1 were exclusive to astrocytic tumours, while IDH2 mutations were significantly more frequent, though not exclusive to, oligodendrogliomas (P= 0.0019). In oligodendrogliomas, IDH2 mutation was significantly associated with frontal lobe localization (P=0.005). Similarly to diffuse gliomas with IDH1 R132H mutation, those with non canonical IDH mutations were significantly more frequent in patients < 55 years (P< 0.0001) and had significantly better prognosis than IDH wt gliomas (P< 0.0001).

Conclusion: Non-canonical IDH mutations are mainly found in diffuse gliomas of younger patients and have prognostic significance similar to that of IDH1-R312H mutation. Non-canonical IDH1 mutations are exclusive to astrocytic tumours, IDH2 mutations associate with oligodendroglial histotype and frontal lobe localization.


Clinicopathological characterisation of tumours of the pineal region (tpr): a case series

A.R. Coelho*, R. Pestana Silva, M. Honavar

*Department of Pathology, Centro Hospitalar Universitário de São João, Porto, Portugal

Background & objectives: TPR are rare and present most frequently in children. Variable biological behaviour and histological features makes tumour grading a challenge. This case series aims to study the clinicopathological features of these tumours diagnosed at a Portuguese tertiary centre.

Methods: All cases of TPR diagnosed in our institution from 1999 to 2019 were retrieved from our files. Their clinicopathological features, including imaging, histology and imunophenotype, treatment and follow up (six years on average) were reviewed.

Results: We identified 24 TPR (16 males, 8 females; 19.8 years on average at diagnosis): eleven tumours of pineal parenchyma (six pineoblastomas, five parenchymal tumours of intermediate differentiation); four papillary TPR; four germ cell tumours; two embryonal tumours; one rosette-forming glioneuronal tumour and a pilocytic astrocytoma. Nineteen patients received radiotherapy, fourteen of whom had additional chemotherapy. Three patients had disseminated disease at diagnosis; four had craniospinal spreading on follow up (after 21 months on average). One patient died 23 months after the initial diagnosis. In our case series, recurrence and mortality were associated with disseminated disease at the time of diagnosis and high grade histology irrespective of tumour type.

Conclusion: The prognosis of TPR depends on the clinical factors and response to therapy, but also on the pathological features. However, further molecular characterization is essential to make an accurate diagnosis, establish prognosis, define therapeutic targets and predict therapy response.


Embryonal tumour with multilayered rosettes; unusual stromal desmoplasia and extracranial extension; a case report and review of literature

M. El Mahdy*, K. El Bahy, E. Sloan, D. Solomon, A. Perry

*Pathology Department, Faculty of Medicine, Ain Shams University, Egypt

Background & objectives: Embryonal tumour with multilayered rosettes (ETMR), is a recently described very rare tumour entity included in the latest update of the WHO classification of tumours of the central nervous system.

Methods: A case of ETMR in the left frontal lobe in a 3-year-old girl with extensive extracranial extension and associated desmoplastic stroma. The child complained of seizure, vomiting and headache. MRI showed a huge heterogenous left frontal mass infiltrating the skull with extracranial soft tissue extension. The diagnosis of ETMR was established based on histopathologic, immunohistochemical and molecular features.

Results: Histopathologic examination showed a neoplasm composed of primitive small cells arranged in sheets with scattered multilayered rosettes with extensive foci of desmoplastic stroma. Immunohistochemistry was performed using panel of antibodies and revealed positive expression for synaptophysin, glial fibrillary acidic protein, Olig2, smooth muscle actin, and LIN28A. Molecular testing revealed focal amplification of the chromosome 19q13.42 microRNA cluster (C19MC) with breakpoints of the amplification immediately preceding the C19MC locus at the 5' boundary and within the TTYH1 gene at the 3' boundary, thus confirming the integrated diagnosis of ETMR, WHO grade IV. The other chromosomal copy number changes in the tumour were losses of interstitial 5q and portions of 10q.

Conclusion: Desmoplastic stroma can occur as a very rare and unusual histologic finding in ETMR due to tumour invasiveness with extracranial extension.


Bifocal, anaplastic cerebellar liponeurocytoma with ABL1-L292M mutation

M.A.A. Erashdi*, A. Tbakhi, Y. Al-Othman, W. Naser, M. Al-Hussaini

*King Hussein Cancer Center, Jordan

Background & objectives: Cerebellar liponeurocytoma (cLNC) is a rare tumour of adults with no sex predilection. MRI/CT scan, with fat-suppressed views can support preoperative diagnosis. It is a WHO grade II, neurocytic tumour with lipomatous component, minimal atypia and low proliferative index.

Methods: The specimen was routinely processed, H&E-stained, then immunohistochemicaly stained for glial fibrillary acid protein (GFAP), neuron-specific enolase (NSE), synaptophysin, neuronal nuclear antigen (NeuN), S-100, neurofilament (NF), and Ki-67. Furthermore, the specimen was submitted for Next generation sequencing (NGS) analysis using TruSight solid tumour panel.The generated reads were aligned against the hg19/Genome Reference Consortium Human Build GRCh37.

Results: Here is a 41-year-old lady presented with headache and fever. MRI highlighted two contrast enhancing masses in both cerebellar hemispheres ( 3.2 x 2.6 x 2.3 cm and 1.9 x 1.6 x 1.2 cm). Gross total resections were performed, two months apart. Histologicaly, both tumours were similar. They showed sheets of monotonous neurocytic cells, admixed with fatty component. The tumour cells are positive for GFAP, Vimetin, Synaptophysin and Chromogranin, with weak focal expression of Neu-N, Desmin, and negative for EMA, ATRX, CD34, NFP and P53 immunostains. Anaplastic features and high Ki-67 (10%) were seen. NGS revealed ABL1-L292M mutation with alternate variant frequency of 5%. TP53, PTCH, CTNNB1 abnormalities were not detected.

Conclusion: We are reporting the fifth case of a multifocal cLNC in English literatures. Only a few other reports are describing cLNC with anaplasia, either as a tumour recurrence or with history of chemotherapy exposure due to other cancers. Limited data is available on the molecular genetic background of cLNC. There was no enough evidence in the literature to support the pathogenicity of the ABL1-L292M mutation detected in our case.


Comparison of clinicoradiological and histological findings in 8 patients with radionecrosis following treatment of cerebral avms with stereotactic radiosurgery: a single institution case series

M. Helley*, K. Agyemang, A. Stan, E.J. St.George

*Department of Neurosurgery, Institute of Neurological Sciences, Glasgow, United Kingdom

Background & objectives: "Radionecrosis" is a poorly defined entity, occurring in 7.1% patients following stereotactic radiosurgery (SRS) for arteriovenous malformation (AVM).

We report an unmatched case series of 8 patients who underwent surgery for suspected Radionecrosis at our institution over a 15-year period.

Methods: Retrospective interrogation of prospectively captured records of eight patients that underwent symptomatic lesion resection following SRS treatment for AVM was carried out. Patient Demographics, AVM clinical/angiographic features and treatment modality prior to SRS were reviewed and compared with histopathological findings, paying note to presence of AVM, fibrotic response, necrosis and evidence of neoplasia.

Results: 75% of patients underwent embolisation prior to SRS, median time from embolisation to SRS was 18.5 months (1-45m). Median latency from SRS to Radionecrosis was 91.5 months (12-209m) Shorter latency in patients who underwent prior embolization. 100% had MRIT1 post-gadolinium enhancement. FLAIR-signal change was average 2.3 times the size of the enhancing lesion

All patients had angiographic confirmation of obliteration of AVM post SRS. 5 patients had evidence of AVM on histology. 2 patients were reported to have changes in keeping with suspicion of Radionecrosis, supported by presence of fibrinoid degeneration and gliosis. Gliosis was present in 5 patients, haemorrhage in 3. No patients had evidence of neoplasia.

Conclusion: The latency and histopathological classification of Radionecrosis following SRS for Cerebral AVM requires definition. Our case series demonstrates a latency of 7.5 years. The heterogenous nature of the histology and presence of active AVM despite radiological obliteration requires further interrogation.


Glioblastoma with extracranial metastasis – case report and genetic analysis

M. Hendrych*, P. Solar, H. Valekova, R. Jancalek, Z. Mackerle, M. Vecera, A. Kopkova, O. Slaby, R. Lakomy, T. Kazda, M. Hermanova

*First Department of Pathology, St. Anne's University Hospital and Faculty of Medicine, Masaryk University, Brno, Czech Republic

Background & objectives: Glioblastoma (GBM) is aggressive brain tumour with low rates of extracranial metastases, affecting only 0.4 – 0.5% of patients. Molecular alterations in GBM are well described but there is only limited understanding of alterations associated with metastatic potential of GBM.

Methods: A case report of 44-year-old woman with left frontal GBM (IDH wildtype), surgically resected followed by standard chemoradiation. Five month after surgery, the patient was diagnosed with extracranial intraspinal metastasis, with no signs of intracranial tumour recurrence. Next generation sequencing analysis of both primary and metastatic GBM tissues was perfomed using Illumina TruSight Tumour 170 assay and NextSeq 500 sequencer.

Results: The number of the single nucleotide variants (SNVs) observed in the metastatic sample was more than 2-times higher compared to the primary GBM. Mutations in TP53, PTEN and RB1 observed in the both samples are indicative for mesenchymal subtype of GBM. Among others, there were two inactivating mutations (Arg1026Ile, Trp1831Ter) in the NF1 gene and two novel NOTCH3 variants of unknown significance predicted to be damaging (Pro1505Thr, Cys1099Tyr) detected only in the metastatic sample.

Conclusion: Based on the literature evidence, inactivation of NF1 and NOTCH3 could explain, at least in part, acquired invasiveness and metastatic potential in this particular GBM case. Further data are warranted to confirm these findings.

This work was supported by Grant Agency of Masaryk University (MUNI/A/1429/2019) and the Czech Health Research Council project NV18-03-00398.


Apoptosis of neurons in the hippocampus in rats during septoplasty modelling

I. Kastyro*, M. Kostyaeva, V. Torshin, Y. Gushchina, A. Kovalenko, P. Pryanikov, N. Ermakova, I. Reshetov

*Peoples’ Friendship University of Russia (RUDN University), Russia

Background & objectives: Damage to the nasal septum when modelling septoplasty in rats blocks the peripheral link of the olfactory analyser is also damaged. Objective:to study the effect of modelling septoplasty in rats on the activation of apoptosis of neurons in the hippocampus.

Methods: The mucous membrane of the nasal septum in rats was scarified over its entire length in 20rats under anesthesia. Brain sections were stained 2&4days after surgery with mouse anti-p53 antibodies. Apoptotic neurons in each hippocampal subfield (CA1,CA2,CA3&DG)in the pyramid layer was calculated over a length of 574.3±13.5μm and a width of121.94±31.5μm. The results obtained were compared with a control group(CG,10rats).

Results: On the 2nd day, more apoptotic neurons were detected in the hippocampal subfields CA1, CA2, CA3 (35.54±2.31, 38.91±3.05, 24.09±3.65), respectively, compared with CG (10.77±1.68, 11.54±2.04, 9.66±2.12, respectively) (p <0.001). On the 4th day, p53-positive neurons decreased compared to the 2nd day (26.13±2.78, 31.27±2.43, 18.85±2.99) (p <0.01), but there were more of them than in the CG (p <0.001).

Conclusion: The septoplasty modelling in rats provokes an acceleration of triggering of apoptosis mechanisms of hippocampal neurons in the CA1, CA2, CA3 subfields.

This work was supported by a grant from the Medical Institute of RUDN University No. 031823-0-000.


Inflammation and macrophage imbalance as risk factors for rupture in saccular intracranial aneurysms

M. Koblížek*, M. Stratilová, A. Hejčl, A. Štekláčová, V. Beneš, J. Zámečník

*Department of Pathology and Molecular Medicine, 2nd Faculty of Medicine, Charles University, Prague, Czech Republic

Background & objectives: Mechanisms leading to rupture of saccular intracranial aneurysms (IA) are still insufficiently described. Based on our findings from preliminary study, we compared structural and inflammatory changes in the aneurysmal walls between unruptured and ruptured IAs from updated set of patients.

Methods: Samples of 41 aneurysms resected after clipping (ruptured n = 14; unruptured n = 27), and 11 control samples of analogous regions of the circle of Willis were included to this study. For each sample we evaluated structural changes, and quantified lymphocytes, as well as M1 and M2 subtypes of macrophages using immunohistochemistry (anti-LCA, anti-HLA-DR, and anti-CD163, respectively).

Results: The absence of the internal elastic membrane was observed in both, ruptured and unruptured aneurysms. The presence of organized thrombus and severer fibrosis of vessel wall was more frequent in ruptured IAs. The inflammatory infiltrate was significantly smaller and the M1 subset prevailed among the macrophages in unruptured IAs. In the ruptured IAs, there was significantly more intense lymphocytic infiltration, and ratio of M1/M2 was almost balanced. Neither structural nor inflammatory changes were observed in controls.

Conclusion: Our findings suggest that chronic inflammation along with decreasing of the M1/M2 macrophages ratio may play a role in the progression of cerebral aneurysms to rupture.

Funding: Supported by grant AZV 17-32872A.


Clinical relevance of BRAF V600E mutation status in brain tumours: a review of evidence and directions for the management

A. Kowalewski*, J. Durślewicz, D. Grzanka, Ł. Szylberg

*Department of Clinical Pathomorphology, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, Poland

Background & objectives: Despite limitations imposed by the blood-brain barrier, heterogeneity and adaptive tumour resistance, the possible application of BRAF-targeted therapy in brain tumours grows continuously. In this study, we explore the relationship between BRAF mutations and prognosis following the accesible treatment options.

Methods: We analysed clinical strategies that address BRAF activation in primary brain tumours. Next, we verified current recommendations regarding screening for BRAF mutations and discussed issues that need to be solved before implementation of the treatment.

Results: There is preliminary evidence for a range of positive responses in certain brain tumour types harboring BRAF V600E mutation. We selected groups of patients, in which the immunohistochemical screening should be considered. Finally, we portrayed how these patients might benefit from targeted therapy.

Conclusion: BRAF V600E mutation emerges as a promising molecular target. Herein, we propose workup and treatment algorithm for primary brain tumours with the particular emphasis on BRAF V600E mutation.


An embryonal tumour with NUT expression and CIC-NUTM1 rearrangement mimicking desmoplastic infantile astrocytoma (DIA) – unsolved diagnostic boundaries

T. Maia*, J. Passos, D. Gonçalves, M. Mafra

*Instituto Português de Oncologia, Francisco Gentil, Lisbon, Portugal

Background & objectives: DIA is a rare grade I central nervous system (CNS) tumour usually following a favourable course regardless of a possible poorly differentiated component. Conversely, grade IV poorly differentiated/embryonal tumours (ET) include the NOS category (ET,NOS) sometimes exhibiting limited glial differentiation.

Methods: We describe a case highlighting challenging differential diagnostic problems between DIA and ET,NOS. The tumour pertains to an 1 year-old child that presented with apathy and de novo strabismus associated with a large frontoparietal mass whose imagiologic features were consistent with DIA. Total gross ressection was performed.

Results: Histologic exam showed a tumour comprising: a) desmoplastic areas with mature spindle astrocytes (GFAP positive) surrounded by a dense reticulin network and pericellular collagen IV (DIA-pattern); b) a poorly differentiated component (GFAP negative) of small round blue cells. Mitotic figures were restricted to poorly differentiated areas; microvascular proliferation and necrosis were absent. A diagnosis of DIA was made. Recurrence with aggressive progression within few weeks led to comprehensive genomic profiling that revealed a CIC-NUTM1 rearrangement. Additional immunohistochemical study showed strong NUT expression within both DIA-patterned and poorly differentiated components. The tumour was reclassified as ET,NOS with extensive mature astrocytic differentiation in a DIA-like pattern, a feature not previously described.

Conclusion: ET,NOS harbouring CIC-NUTM1 rearrangements may exhibit extensive astrocytic DIA-like differentiation, blurring diagnostic boundaries with “true” DIA, a differential diagnostic problem not previously addressed. NUT immuno-expression may be diagnostically useful in identifying these cases. DIA-patterned tumours may need molecular workup for appropriate classification/management.


Retrospective observational study of gliosarcomas

S. Marcos González*, L. Ferreira Freire, A. García Castaño, M. Drake Pérez, E. Marco de Lucas, J. Martino González, E.E. Arrojo Alvarez, J. Freire, A. Azueta Etxebarria


Background & objectives: Gliosarcoma (GSM) is a variant of glioblastoma IDH-wiltype, characterized by a biphasic pattern, with areas of glial and mesenchymal differentiation. Our objective is to describe the population and results obtained in a cohort of patients with GSM.

Methods: We conducted a retrospective observational study from 2010 to 2019, of patients with GSM over the total glioblastomas (GBM) in that period, reviewing the clinical histories and histopathological characteristics. In one of them a molecular genetic analysis (NGS) is carried out.

Results: We reviewed 8 GSM out of a total of 238 GBM (3.3%). 5 women and 3 men with a mean age at diagnosis of 57 years. At debut, the most frequent symptoms were behavioural disturbance, motor and sensory deficit. The temporal localization was the most frequent (87%) and all tumours were more than 3 cm in size, necrosis and oedema in MRI. Complete resection in one of them. 100% IDH-wildtype and 75% EGFR-negative tumours. One patient completes the entire treatment and 63% have progression. The molecular genetic analysis is performed in one of the cases, in which the result was an imbalance in NTRX1.

Conclusion: There is a great shortage of studies on GSM due to its low incidence, hence the importance of molecular study to identify target alterations that help improve the prognosis and quality of life of these patients.


Globular glial tauopathy type I presenting as atypical progressive aphasia, with comorbid limbic-predominant age-related TDP-43 encephalopathy

R. Matej*, G. Kovacs, J. Keller, Z. Csefalvay, R. Rusina

*Department of Pathology and Molecular Medicine, Third Faculty of Medicine, Charles University and Thomayer Hospital, Czech Republic

Background & objectives: Globular glial tauopathies (GGT) have heterogeneous presentations with little available information regarding typical clinical manifestations. We report on a case of atypical primary progressive aphasia due to comorbid GGT and limbic TDP-43 proteinopathy.

Methods: The initial clinical phenotype of a 69-year-old, right-handed man was compatible with the nonfluent-agrammatical variant of primary progressive aphasia and early hippocampal amnesia. Progressively, parkinsonism and supranuclear oculomotor impairment occurred, and finally, late mutism with frontal type dementia, impaired comprehension, and behavioural manifestations developed.

Results: The neuropathology was characteristic of globular glial tauopathy type I with vascular changes and comorbid limbic-predominant age-related TDP-43 encephalopathy (LATE).

Conclusion: Our findings expand the clinical spectrum of globular glial tauopathies to include a complex progressive aphasia syndrome. The extraordinary feature, in this case, was the combination of two progressive aphasia subtypes, i.e., the early nonfluent-agrammatical variant and the late semantic variant. Our findings also expand the spectrum of neuropathological comorbidities in GGT.

Supported by the Grant Agency of Czech Ministry of Health (NV18-04-00346, NV19-04-00090) and Charles University (Progress Q27/LF1, Q35/LF3) and DRO (Thomayer Hospital – TH, 00064190).


Carbonic anhydrase IX expression in high grade astrocytomas

R. McLendon*, P. Healy, K. Rodriguez, N. Cort, E. Lipp

*Duke University, USA

Background & objectives: Carbonic Anhydrase IX (CAIX) expression has been associated with a negative overall survival (OS) in Grade III-IV astrocytomas (AA+GBM). The effect of IDH status on CAIX status with regards to OS in AA+GBM is relatively unexplored.

Methods: 748 AA+GBM were examined prospectively over 7 years by IHC for CAIX Labeling Index (LI%), Ki-67 LI%, MGMT promoter methylation (PM), EGFR LI%, EGFRvIII LI%, VEGF LI% and VEGF-R2 LI%; 602 Grade IV (GBMs) and 146 Grade III (AA) were studied.

Results: Mean age was 55 years. 502 AA+GBM were newly diagnosed (ND) and 246 were recurrent (RD). CAIX expression (defined as CAIX LI>5%) was identified in 456 GBM (76%) and 32 (22%) AA. IDH status performed on 222 AA+GBM (30%) revealed 197 wild type (AA+GBM IDH1 mutated n=25). IDH WT AA+GBM exhibited a higher median CAIX LI%. Only in GBMs was Ki-67 LI correlated with CAIX expression.

Conclusion: In AA+GBM, VEGF LI%, VEGFR2 LI%, and EGFRvIII LI% correlated with CAIX expression. The correlation between CAIX and VEGFR2 LI% was independent of grade. Among GBM, factors associated with an adverse OS were MGMT PM and age. Among AA, factors associated with an adverse OS were CAIX, Ki67 LI% and age. CAIX negative AA have a prolonged survival not seen with CAIX positive AA and all GBM. CAIX expression is useful in characterizing prognosis in AA+GBM.


Histomorphological spectrum and trend of neoplastic spinal lesions: a 40-year review

I. Nwadiokwu*, I. Iwuagwu, A. Salami, U. Ezenkwa, D. Mashor, M. Ajani

*Department of Pathology, University College Hospital, Ibadan, Nigeria

Background & objectives: Neoplastic spinal lesions cause significant morbidity with poor neurological outcome in many patients. Studies describing their spectrum are generally few. Our objective was to determine the histomorphological spectrum of neoplastic spinal lesions and the trend in occurrence over four decades.

Methods: We reviewed Patients’ age, gender and histomorphological data of all the neoplastic spinal lesions from the patients’ hospital records over a 40-year period (January 1st, 1980 to December 31st, 2019). All the cases seen were classified as either benign or malignant neoplasms with their spectrum, frequencies and trends in decades determined using Statistical Package for Social Sciences (SPSS), version 20.

Results: Ninety-four cases of neoplastic spinal lesions were seen over the four decades of study with the highest number of cases observed in the last decade. The male to female ratio was 1.2:1. The mean age was 34.8 years with a peak incidence in the 5th decade. Benign and malignant spinal neoplasms comprised 55.3% and 44.7% respectively. Meningiomas and peripheral nerve sheath tumours had the highest occurrences (16% each), while germ cell tumours had the least occurrence (2.1%). Other spectrum of spinal tumours seen include metastatic tumours (14.9%), soft tissue tumours (14.9%), haemato-lymphoid tumours (9.5%), glial tumours (8.5%), bone tumours (7.5%), Vascular tumours (5.3%) and neuronal tumours (5.3%).

Conclusion: This study showed that benign spinal tumours are more common than malignant spinal tumours, with a slight male predominance and most of the spinal tumours occurring below 50 years. The increased number of spinal tumours observed in the last decade of study may be due to an increase in skilled personnel and improved diagnostic facilities in our health institution in addition to rising awareness of disease among the populace.


Two rare cases of IDH1-mutated gliosarcomas arising in IDH1-mutated, 1p/19q non-codeleted diffuse gliomas with prominent oligodendroglial morphology

J. Soukup*, M. Bartos, V. Hobza, P. Kasparova, A. Kohout, T. Cesak

*The Fingerland Department of Pathology, University Hospital and Faculty of Medicine Hradec Kralove, Charles University, Czech Republic

Background & objectives: Gliosarcomas may arise de novo or represent tumour progression in previously diagnosed diffuse glioma. Majority of gliosarcomas are IDH1/2 wildtype and mutated tumours are rare. Here we present two such cases.

Methods: Two males (case 1 - age 49, case 2 -age 39) were diagnosed with IDH1-mutated diffuse gliomas in left temporal (case 1) and left frontal (case 2) area. They recurred as gliosarcoma after 6 and 5 years respectively. Gliosarcomas and concurrent/precursor gliomas were studied using immunohistochemistry (IDH1 R132H, ATRX, GFAP, Olig2, SOX10, p53) and FISH (1p/19q probes).

Results: Both tumours showed conspicuous oligodendroglial morphology in glioma component. In both primary gliomas, immunohistochemistry showed expression of IDH1 R132H and ATRX and these were also expressed in sarcoma components. In case 1, repeated FISH showed no 1p/19q codeletion. In case 2, partial deletion of 1p (in 40% of cells) without loss of 19q was detected. Sarcoma components showed positive reticulin stain, loss of Olig2 expression and loss of GFAP expression in majority of tumour cells. Mutant-type (strong and diffuse) p53 expression was observed in both gliosarcomas, but not in the glial component of case 2. In case 1, 50% of cells of original glioma showed p53 expression.

Conclusion: We report two IDH1-mutated gliosarcomas, arising in IDH1-mutated/ATRX-positive gliomas with oligodendroglial morphology and lack of 1p/19q codeletion. In one case, mutant-type p53 expression was acquired in sarcomatous component during progression of the tumour.

Supported by MH CZ NV19-01-00435 and European Regional Development Fund-Project BBMRI-CZ: No: EF16 013/0001674.


The role of skeletal muscle biopsy in the diagnostic algorithm of anti-signal recognition particle myopathy

R. Tanasa*, A. Bastian, G. Grigore, C. Constantinescu, M. Craescu, E. Manole

*Colentina Clinical Hospital, Pathology Department, Romania

Background & objectives: Myopathies with serum antibodies to the signal recognition particle (anti-SRP) represent a rare category of refractory immune-mediated necrotizing myopathies. Muscle biopsy, along with immunological and clinical data may provide key information in understanding the pathological mechanisms of these controversial diseases.

Methods: Open muscle biopsy was performed on 42 patients with suspected inflammatory myopathy during the year 2019 in our hospital. We evaluated the main pathological features using histological, immunohistochemical, enzyme histochemical techniques, routine and special stains on cryosections and paraffin-embedded tissue. Among them, we identified 3 patients (7%) with myopathology suggestive for anti-SRP myopathy. Anti-SRP antibodies were further tested positive.

Results: Muscle biopsies showed the picture of a severe active necrotizing myopathy with marked variability in size of the fibres, numerous necrotic and regenerating fibres and rare scattered mononuclear cells in all three cases. ATPase staining showed type 1 fiber predominance myopathic pattern with a reduced size of type 2 fibres. Immunomarking with MHC Class I antibody showed a rather unexpected prominent expression in non-necrotic fibres in two cases and normal expression in the third one. Differential diagnosis with other types of inflammatory myopathies, especially dermatomyositis, polymyositis, and anti-HMGCR myopathy, even with muscle dystrophies proved sometimes to be challenging because of the similarity of both clinical and morphological aspects.

Conclusion: Anti-SRP myopathy is a distinct entity in terms of clinical traits, biopsy changes and response to conventional combined immunosuppressive therapy. Histopathology of muscle, along with complementary investigation, may provide useful clues regarding the correct diagnosis, in deciphering the mechanisms of this disease with a major impact on clinical management of this potentially life-threatening disease.

PS-10 Paediatric and Perinatal Pathology


Composite hemangioendothelioma - a clinicopathological study of site, composition and immunohistochemistry of an additional series

K. Adoke*, D. Suleiman

*Department of Pathology, FMC KEBBI, Nigeria

Background & objectives: Composite Hemangioendothelioma (CHE) is a recently described new entity classified under the Hemangioendothelioma group of vascular tumours, it was first described in 2000 by Nayler et al, and it is a rare vascular neoplasm of low malignancy that exhibits a composite of hemangioendothelioma variants. It usually presents on the skin and soft tissue of upper and lower extremities, especially the lower leg and foot. We describe additional series of CHE in paediatric age group.

Methods: Three cases of CHE were retrieved from our archives. Blocks were cut and stained with H&E. Immunohistochemical stains were done using DB Biotech protocol. Antibodies used include CD34, EMA and vimentin

Results: All cases of CHE encountered in this study were in the paediatric age group that is 8, 10 and 14 years respectively. The sites of presentation of CHE were head and neck, upper and lower extremities. Histology showed composite of hemangioendothelioma variants that is, Dabska, retiform, epitheloid and spindle cell haemangioma in varying proportions with brisk mitosis seen. All were positive for CD34 and vimentin but negative for EMA.

Conclusion: CHE can present with composite of hemangioendothelioma variants. In our environment this vascular lesion of low malignant potential is mostly seen in the paediatric age group. No recurrence was seen after one year of follow up.


Correlation between histology and genetic testing in surfactant dysfunction disorders

X. Ara Mancebo*, A. Navarro-Jimenez, J. Camacho-Soriano, E.M. Fuentes-Camps, C. Alborch-Gil, S. Ramón y Cajal, M. Garrido-Pontnou

*Department of Pathology, Hospital Universitari Vall d'Hebron, Spain

Background & objectives: Genetic surfactant dysfunction disorders are rare paediatric conditions. Three major genes are involved in their pathogenesis (ie STFPB, STFPC, ABCA3). Histologically, several patterns may be observed, and although suggestive for one of these disorders, they are not specific.

Methods: 10 patients with a positive genetic testing were identified. We blindly reviewed their biopsies (H&E, trichrome, PAS, SMA, CK7 and CD68 stains), recording the main histological findings according to a previous set list of items. Electron microscopy (EM) findings were also assessed, when available. The collected information was correlated with the corresponding mutation. Finally, we revised the clinical outcomes.

Results: 6 patients were female and 4 male, aged 0 to 11 (mean 1.6 years). The most common mutation was SFTPC(50%), followed by ABCA3(40%) and SFTPB(10%). All the biopsies presented features of chronic pneumonitis of infancy (CPI), with alveolar simplification, thickened septa and type II pneumocyte hyperplasia. All the cases with ABCA3mutation showed the most prominent fibrosis and muscularization of the septa. The histology in SFTPCmutated cases was more variable. The only patient with a SFTPBmutation died shortly after birth (3 weeks), presenting an unspecific histology. Electron microscopy was available in 3 cases, yielding a definite diagnosis in 2 of them.

Conclusion: Surfactant dysfunction disorders present histologically as CPI, but the accompanying morphological findings are variable. The most relevant finding is the marked fibrosis and muscularization of the alveolar septa in ABCA3mutated cases. SFTPBis reportedly the most severe of the mutations, as seen in our series. EM is helpful in achieving a definite diagnosis.


The role of placental examination in evaluating perinatal death: a retrospective analysis of 192 cases

M. Boros*, E.M. Popescu, A. Judea, A. Pascalau, O. Pop, A.A. Barbis Vesa

*Department of Pathology, Faculty of Medicine and Pharmacy, University of Oradea, Romania

Background & objectives: Most common causes that lead to intrauterine foetal death are the placental abnormalities. The aim is to analyse how many of the intrauterine/ perinatal deaths present morphological changes in the placenta and which are the associated clinical-pathological factors.

Methods: We have retrospectively analysed all placentas sent to the Pathology Laboratory from the Maternity of Oradea (Romania) during December 2018 - June 2019 from a clinical, macroscopic and microscopic point of view. We reassessed and compared the “sick” placentas (those with worse foetal outcome) with the “healthy” ones.

Results: 197 placentas were sent for histopathological examination. Of the autopsies performed during the same period, 22 had the placenta examined. The analysis of the association between the sick and the healthy placentas reveals statistically significant differences regarding education (p = 0.01), the type of birth (p = 0.0002), the gestational age (P <0.0001), the aspect of amniotic membranes (p = 0.011) and placental anomalies (p = 0.0012).

Conclusion: The low socio-economic status and prematurity are the risk factors associated statistically with a higher rate of adverse foetal outcome. Placental abnormalities, infections, and placental abruption are most commonly noted. A systematic histopathological analysis of the placenta is imperative in order to more accurately assess the causes of death and to reduce the rate of unexplained deaths.


Identification of SRF-STAT6 fusion transcript in a cellular myofibroma of the forearm in a 15 years old boy: expanding the molecular spectrum of the recently described entity

C. Bouvier*, H. Nihous, J. Baud, M. Nicolas, A. Haffner, F. Tirode, J. Jouve, J. Gentet, F. Le Loarer

*Department of Pathology, Timone Hospital, France

Background & objectives: The spectrum of mesenchymal tumours keeps increasing with the input of NGS. Recently myofibroblastic tumours arising within deep soft tissue with high cellularity and a more differentiated immunophenotype towards myoid lineage have been described harbouring a recurrent SRF-RELA fusion.

Methods: We report the case of a 15 years-old patient who presented with a calcified soft tissue mass of the left forearm. Histological features and unpreviously reported genetic data are described.

Results: Microscopic examination showed a fascicular proliferation mainly composed of spindle cells with variable eosinophilic cytoplasm. Areas with higher cellularity with basophilic cells were also found giving a biphasic appearance. Tumour stroma contained calcifications. There were no necrosis and mitotic index was 1 mitosis/50HPF. Cells stained for alpha smooth actin, H-caldesmon and desmin. Whole RNA-sequencing identified SRF-STAT6 fusion that joined SRF exon 5 to STAT6 exon 17. STAT6 immunohistochemistry was positive.

Conclusion: We have described a new variant of SRF-rearranged neoplasm. This case shared histological features with the recently described group of SRF-rearranged myofibroblastic neoplasms but involved STAT6 as a 3’ partner, which resulted in a strong nuclear expression of STAT6, a potential diagnosis pitfall with solitary fibrous tumours. The clinical relevance of this novel fusion requires further investigations.


Histological chorioamnionitis in placentas of extremely premature neonates: the impact of maternal and foetal inflammatory responses on clinical findings and neonatal outcome

E. Budal*, J. Kessler, C. Ebbing, M. Bentsen, O.H. Haugen, S.M. Aukland, M. Vollsæter, G.E. Eide, K. Collett

*Department of Clinical Medicine, University of Bergen, Norway

Background & objectives: The aim of this study was to explore if placental histological chorioamnionitis (HCA) with or without a foetal inflammatory response (FIR) differ in clinical findings and neonatal outcomes in a group of extremely premature (EP) neonates.

Methods: Singleton neonates with gestational age (GA) < 28 weeks and HCA at placental examination were included in the study. Data on maternal history, delivery, neonatal course, and placental findings were recorded prospectively. Staging of the inflammatory responses was performed in accordance with the Amsterdam Placental Workshop Group Consensus. A multivariate logistic regression model was used for prediction of neonatal outcomes.

Results: Of 84 included cases, 59 (70%) had FIR. FIR was associated with preterm prelabour rupture of membranes (PPROM) (p=0.002), clinical chorioamnionitis (CCA) (p=0.003), use of antibiotics before/during birth (p<0.001), antenatal steroids (0.008), higher neonatal leucocyte levels (p<0.001), and bronchopulmonary dysplasia (BPD) (p=0.007). No significant association was found for other common neonatal complications of extreme prematurity. After adjusting for GA and birth weight (Z-score), babies of mothers with clinical chorioamnionitis (CCA) had increased odds of neonatal death (OR=10.75, p=0.007). Surprisingly, FIR seemed to have a protective effect (OR=0.15, p=0.036) on mortality.

Conclusion: In a group of EP neonates with HCA, FIR was significantly associated with PPROM, CCA and BPD. CCA increased the odds of infant death during the first year, but a foetal inflammatory response seemed to have a protective effect.

Funding: Helse Vest Research Fund


A histopathological study of paediatric teratomas in Khartoum state 2010-2018

N. El Dawi*, S.A.M. Tamimeldar

*Soba University Hospital, Sudan

Background & objectives: To study the histological spectrum of teratomas and clinical presentation in paediatric patients presenting in Khartoum State hospitals.

Methods: Paediatric teratoma cases that underwent surgery at the main state hospitals in Khartoum State during the period from 2010 to 2018 were enrolled in a retrospective analytical study. The case slides were reviewed and correlated with the clinical data submitted.

Results: A total of 60 cases were included of which 82% were females and 26.7% were less than one year of age and 56.6% were 6-16 years of age. The main presentation was a mass. The site of origin was the ovaries in 57% (49), sacrococyx in 25%, abdomen in 8.3%, pelvis in 8.3%, head and neck in 6.7%, chest in 3.3% and testis 0.9% (1). Mature teratomas accounted for 81.7%, mixed teratomas for 13.3% and 5% were immature. Altman class I was the main classification in 53.3% of sacrococcygeal teratoma cases. Immature grade 3 was the predominant (62.5%) with neural component the commonest (71.4%).

Conclusion: Presentations were diverse regarding age, location, gross features and degree of differentiation. Mature teratomas predominate and the majority of the sacrococcygeal teratomas were mature in females less than one year of age.


Epidemiology of children and adolescents served in burning ambulatory in Brazilian public hospital

M.d.F. Figueiredo*, T. Farias Batista, D. Palácio Cardoso, T. Praça Brasil, T. Maciel Valente, M.E. Maciel de Brito, R.L. Freitas de Almeida

*University of Fortaleza, Brazil

Background & objectives: Among domestic injuries in children and adolescents, skin burns deserve special attention because they can lead to serious residual injuries.In adolescence, alcohol burns are the most common. Describe the epidemiological profile of hospitalizations of children and adolescents who suffered burns.

Methods: A cross-sectional descriptive study analysed hospitalizations between 2013 and 2019, regarding gender, age, severity of injury and causative agent. The data on the care provided at the Burn Clinic in a reference hospital in the Northeast of Brazil.

Results: During the study period, 2.564 patients were admitted, of which 1.433 were children between 4 and 12 years of age and 1.131 adolescents between 12 and 19 years of age. The odds ratio among children and adolescents was (OR = 1.5; CI 1.3-1.7) and boys are (OR = 1.3; CI 1.2-1.4) times more likely to suffer burns than girls. Among adolescents, the prevalence of burns is higher among girls (59.6%) (OR = 1.2 CI 1.1 - 1.4). Liquids were responsible for most accidents and adolescent girls are more likely to suffer burns with liquids when compared to boys in the same age group (OR = 1.6; CI 1.32-2.0).

Conclusion: The epidemiological surveys provide objective information on dangerous agents and on the profile of the populations where burns are most likely to occur. They are essential for the development of appropriate prevention strategies.


Value of autopsy examination in early neonatal death: report of 4 unusual cases

J. Filipe*, L. Monteiro

*Serviço de Anatomia Patológica, Instituto Português de Oncologia de Lisboa Francisco Gentil, Lisbon, Portugal

Background & objectives: Early neonatal death (ENND) is defined as newborn´s death in the first week of life. We present the unexpected autopsy findings in four cases of ENND that can change the clinical tenders and the Genetic counselling.

Methods: Gross and histopathological findings of four ENND post-mortem examinations.

Results: The four cases included 3 newborn males and 1 newborn female, aged between 25’ and 3 days old. The causes of death were clinically unexpected and clearly assessed by the post-mortem examination: extralobar pulmonary sequestration, cardiac malformation, iatrogenic pericardial effusion and thymus hypertrophy with heart compression. None of them were previously diagnosed.

Conclusion: Although the main causes of ENND are perinatal asphyxia, low birth weight/prematurity, and infections, a few unexpected congenital malformations or other causes are detected when a systematic autopsy examination is performed. These unusual cases highlight the importance of a minicious post-mortem examination in ENND, its important role in the knowledge of neonatal pathology and its contribution for Genetic counselling.


Foetal autopsies, a retrospective study in a tertiary institution

J. Gama*, A. Lai, R. Almeida, J. Madeira, J. Fraga, F. Ramalhosa, C. Faria, M.B. Pimentão, H. Moreira, V. Almeida, R. Pina, M.A. Cipriano

*Centro Hospitalar e Universitário de Coimbra, Portugal

Background & objectives: Foetal neoplasias are rare and have a poor prognosis. They can be associated with death and are an indication for termination of pregnancy. A diverse range of tumours affecting foetuses have been described.

Methods: We conducted a retrospective study at a tertiary hospital in Portugal – Centro Hospitalar e Universitário de Coimbra, and reviewed 1482 cases of autopsied foetuses.

A description of each case correlating with the gestational age, placenta histology, karyotype and ecographic findings was made.

Results: We have identified 10 foetal autopsies with neoplasias out of 1482 autopsies (0.67%). 60% were benign and the remaining malignant. The following tumours were found: cervical lymphangioma (n=4), choroidal plexus papilloma (n=2), neuroblastoma (n=1), embryonal tumour with abundant neuropil and true rosettes (n=1), immature sacrococcygeal teratoma (n=1) and diffuse astrocytoma (n=1). 60% of the autopsies were result of termination of pregnancy and 40% resulted from intrauterine death.

Conclusion: In this study, we concluded that histologically benign tumours can also result in intrauterine death as the location, size and other factors influence the clinical outcome. Foetal tumours are different from childhood’ tumours in their distribution, incidence and clinical outcome.


Morphological defects in distal 18q deletion syndrome: a case report

T. Georgescu*, A.C. Lisievici, S.A. Barbu, A. Dumitru, O. Voinea, R. Bohiltea, O. Munteanu

*Department of Pathology, "Carol Davila" University of Medicine and Pharmacy, Department of Pathology, "Dr. Leventer Centre", Bucharest, Romania

Background & objectives: There are two groups of 18q deletions: within the bottom half of the centromere (distal deletions) and closer to the centromere (proximal deletions). Distal 18q deletion syndrome can lead to a wide variety of signs and symptoms among affected individuals.

Methods: We report the case of a 35-year-old female admitted to the Department of Obstetrics-Gynaecology with a 21-weeks pregnancy featuring multiple cerebral and cardiologic malformations detected on foetal MRI. The multidisciplinary commission recommended therapeutic abortion with subsequent genetic analysis and the patient agreed. The product of conception, placenta and umbilical cord were sent to the Department of Pathology for histopathological evaluation.

Results: The most striking morphological defects were the presence of right cleft lip, bilateral anophthalmia with wide nasal base and low implantation of the ears. The eyelids appeared normal, but the lachrymal puncta were absent. Palpebral apertures were sealed and no vestigial ocular globe was identified. Orbital cavities were smaller than normal. A perimembranous ventricular septal defect measuring 0.3 x 0.2 cm with overriding aorta was also present. A modified female profile with a loss of genetic material was identified on chromosome region 18q21.1-18q23 with the size of 30.5 Mb. According to the databases, this region is responsible for deletion syndrome 18q, which presents with a clinically variable picture.

Conclusion: The most common phenotypic aspects of distal 18q deletion syndrome are small stature, intellectual disabilities, learning difficulties, hypotonia, hearing problems, microcephaly, cleft lip and cleft palate, foot abnormalities and heart malformations.


Childhood and adolescent malignant solid tumours in Rwanda: distribution of tumour types and challenges to diagnosis

A.B. Igihozo Sindikubwabo*, R.R. Flores, B. Rugwizangoga, T.Z. Muvunyi, D. Ruhangaza

*School of Medicine and Pharmacy, University of Rwanda, Kigali, Rwanda

Background & objectives: Data on paediatric tumours are very scarce in Rwanda. The goal of this study is to determine the distribution of childhood and adolescent malignant solid tumours in Rwanda and to shed light on the pathology-specific limitations to their diagnosis.

Methods: In this retrospective, descriptive multicenter analysis, we collected cases of solid malignant tumours diagnosed in patients aged 0-18 years from four regional hospitals over a 3-year period (2016 to 2018). Tumours were classified using the International Classification of Childhood Cancers (ICCC). All cases were then analysed in terms of age, age group, gender, residence, morphological features and immunophenotype, where possible.

Results: This study revealed that the most common solid tumours in children and adolescents diagnosed in Rwanda were retinoblastoma, followed by nephroblastoma, rhabdomyosarcoma, and osteosarcoma. The most common tumours by category were retinoblastoma (25%), soft tissue sarcomas (20%), renal tumours (18.5%) and bone tumours (14%). These tumours were more frequently seen in children between 1 and 4 years of age and were less frequent in infants. There was a slight male predilection (M/F ratio = 1.18). Among the cases where staging information was available, most patients presented at early stages; however, staging was not applicable or not provided for most cases (83.5%). Diagnostic immunohistochemistry was not performed in 46.1% of cases.

Conclusion: In slight contrast to other studies of paediatric malignant solid tumours in the region, our study showed the most common paediatric tumour was retinoblastoma. Specialty-trained pathologists and diagnostic tools such as immunohistochemistry are paramount for accurate diagnosis of paediatric tumours.


Peripheral medulloepithelioma: two cases report

I. Beliamime, R. Karam*, F.N. Dasilva, M. Regragui, N. G.Bennani, S. Benayad, M. Karkouri

*Department of Pathology, Ibn Rochd University Hospital, Casablanca, Morocco

Background & objectives: Medulloepithelioma is an extremely rare, of high malignancy embryonic tumour, generally located in the eye or in the central nervous system; a peripheral location is even rarer and will be the subject of our report.

Methods: We report two cases of female children; 30 months and 8 years old with a pelvic mass. Imagery found a secondary hepatic nodules in one of the two cases.

Histologically, it showed a proliferation of embryonic appearance of papillary, tubular and rosette architecture expressing CKAE1/AE3; CD56; WT1 and synaptophysin, without expression of CD99, AFP, CD30, Oct3 / 4 and BHCG.

Results: Medulloepithelioma is an embryonic tumour classified in the subgroup of primitive neuro-ectodermal tumours. It is a rare and highly malignant tumours made up of undifferentiated cells resembling germ or matrix cells of the embryonic neural tube.

These tumours most often survive during the first decade of life and have specific radiological, histological features.

It is most often found in the nervous system and in the eye.

Peripheral locations are extremely rarely known.

Treatment is based on chemotherapy, surgery and radiotherapy, targeted anti-PDGFR therapies are being evaluated.

Conclusion: We report two paediatric cases of peripheral medulloepithelioma.

Since this entity is rare with an unusual location, peripheral medulloepithelioma requires a correct diagnosis and proper classification to predict the clinical behaviour and for optimal management.


Cardiomyopathy in paediatric autopsy: the importance in the diagnosis after death

A. Lai*, M.B. Pimentão, R. Almeida, H. Moreira, J. Fraga, C. Faria, V. Almeida, F. Ramalhosa, J. Gama, J. Madeira, R. Oliveira, C. Cerdeira, M. Venâncio, R. Pina

*Centro Hospitalar e Universitário de Coimbra, Portugal

Background & objectives: Cardiomyopathy is defined as a myocardial disorder in which the heart muscle is structurally and functionally abnormal, without a cardiac malformation.

Our objective was evaluate cardiomyopathies in paediatric autopsies (PA) and correlate with genetic studies performed for further counselling.

Methods: We retrospectively reviewed the PA performed in a third care centre in Portugal between 2007 and 2019, contemplating those with heart disease (excluding heart malformations) and correlate with ultrasonography results and with the genetics reports when possible.

Results: From 2007-2019, were performed 1482PA. Four have a cardiomyopathy (0,23%), including one female and three male; one was a stillbirth death (29weeks, with normal ultrasonographic evaluation at 21weeks), two neonatal deaths (one peri-partum, other at 7hours of life) and one with 5months old.

The autopsy findings describe two dilated cardiomyopathy (one endocardial fibroelastoses) and two hypertrophic, without any evidence that could point a diagnosis, principally storage diseases.

One case had performed echocardiographic study (consistent with the autopsy findings).

There was family history of heart related disease in two and only in one of them was found a deletion (c.94316_94317del (p.Val31439Alafs*), classified as probably pathogenic in TTN gene, in heterozygosity.

Conclusion: Cardiomyopathy in children can be a diagnostic challenge, even after the autopsy. But due to the possibility of genetic cause, it is important to put our efforts in the correct diagnosis to provide guidance for the family and future generations.


Expression of VEGF and its receptors in placental villi in early and late preeclampsia

O. Mishnev*, V. Lyapin, U. Tumanova, A. Shchegolev

*Pirogov Russian National Research Medical University (RNRMU), Moscow, Russia

Background & objectives: Preeclampsia is formidable complications of pregnancy. An important role in the development of preeclampsia is assigned to violations of the expression of angiogenic factors. A comparative analysis of VEGF expression and its receptors in early (EPe) and late (LPe) preeclampsia.

Methods: We performed complex morphological and immunohistochemical study of 9 placentas from EPe cases, 14 placentas from LPe cases and 10 term placentas from uncomplicated pregnancies (control group). VEGF expression and its receptors (VEGFRs -1 and -2) in syncytiotrophoblast and endothelium of capillaries of terminal villi were detected by immunohistochemical methods.

Results: As a result of an immunohistochemical study, we identified changes in the expression levels of VEGF and its receptors in the structures of terminal villi in EPe and LPe.In the EPe observations, expression levels of VEGF and VEGFR-2 in syncytiotrophoblast and capillary endothelium were lower than in the control group.VEGFR-1 expression levels were slightly lower than control values.Similar changes were detected in the LPe, but the degree of deviation from the control levels were less.

Conclusion: The detected changes of VEGF and its receptors expressions indicate violations of angiogenesis in the placental villi. More pronounced changes in EPe reflect the features of the damage pathogenesis and compensatory processes in the placenta with various forms of preeclampsia.


Unsuspected myocarditis presenting as sudden death in infants and children

O. Neagu*, S. Stenton, M. Cohen

*University Emergency Hospital Bucharest, Romania

Background & objectives: Acute myocarditis is an inflammatory disease of the heart mostly diagnosed in young people which can present as sudden death. The aetiology includes infectious agents, systemic diseases, drugs and toxins. We aim to characterise myocarditis in infants and children.

Methods: Retrospective evaluation of 813 paediatric post-mortems conducted at Sheffield Children’s Hospital between 2009-2019. Data retrieved included histological features, virology, microbiology and clinical history.

Results: 23c of 813c corresponded to acute myocarditis (2.7% incidence) and 1c to a dilated cardiomyopathy related to undiagnosed remote Parvovirus infection. Most cases were below 2 years age. Histological features showed diffuse myocardial inflammation in 15c, focal in 8c and none in dilated cardiomyopathy. PCR identified Enterovirus (7c), Parvovirus (7c, 2 also with HHV6 and 1c with EVB), Influenza A (1c), Parainfluenza type 3 (1c). 2c were hypersensitivity myocarditis, 1c was Group A Streptococcus related and 5 idiopathic myocarditis.

Enterovirus was frequent in infants (7/10), and in new-borns was associated to meningoencephalitis or congenital myocarditis.

Presenting symptoms included vomiting, abdominal pain (2-3 days) and breathing difficulties (up to 10 days).

Conclusion: Myocarditis represents almost 3% of all sudden deaths in infants and children. Enterovirus and Parvovirus represented the most common aetiologies. Although patients experienced preceding viral symptoms, all remained clinically unsuspected, highlighting the need for clinical awareness of this condition.


Malignant childhood solid tumours in Ile-Ife, Nigeria: a 10-year retrospective review

I. Olorunsola*, O. Omoyiola, A. Adefidipe, A. Komolafe

*Department of Morbid Anatomy and Forensic Medicine, Obafemi Awolowo University Teaching Hospitals, Ile-Ife, Nigeria

Background & objectives: Childhood cancer is fast becoming a global challenge especially in developing countries where morbidity and mortality is still very high. The review is to determine the pattern of malignant solid tumours and to compare this with previous studies.

Methods: A 10-year retrospective review in which all histologically diagnosed childhood malignant solid tumours between ages 0-15 years were analysed.

The surgical daybook and the histopathology request card were retrieved and the patient’s biodata, nature of specimen and site were extracted. The histopathology reports were also retrieved and the size of the tumour, diagnosis, grade and stage (where applicable) were retrieved.

Results: A total of 120 cases of malignant childhood solid tumours were analysed in the study period with an average of 12 cases per year. 75 (62.5 %) cases were male, while 45 (37.5%) cases were female, hence a male to female ratio of 1.7:1. The peak age group was 5-9 years accounting for 37.5 %, while 0-4 years and 10-15 years accounted for 33.3% and 29.2% respectively. The fIve commonest tumours were lymphoma (27; 22.5%), nephroblastoma (25; 20.8%), soft tissue sarcoma (20; 16.7%), central nervous system tumours (13; 10.8%) and retinoblastoma (10; 8.3%).

Conclusion: Lymphomas as a group is the still the commonest childhood solid tumour in our centre as previously reported. However, nephroblastoma is the commonest single entity. There seems to be a decline in the diagnosis of Burkitt lymphoma in our centre over the last 10 years, whether this translate to reduction in incidence of Burkitt lymphoma needs further clarification.


Placenta referrals in a teaching hospital

F. Auld, L. Kean, C. Platt*

*Nottingham University Hospitals NHS Trust, United Kingdom

Background & objectives:

• To document and quantify reasons for referral of placenta specimens for histological analysis.

• To establish whether referrals are often received in line with RCPath guidelines (October 2019) (Appendix A).

• To investigate reasons behind inappropriate or ambiguous referrals, and to consider steps to minimise these in the future.


• 215 sequential referrals were prospectively collected, in which placenta specimens were referred for histopathological analysis.

• Referrals were categorised according to which part of the Appendix A, RCPath (October 2019) guidelines they pertained to (some fell into more than one category).

• Inappropriate and ambiguous referrals were investigated further using WinPath but not rejected.

Results: The majority (97%) of referrals for placenta pathology are in line with RCPath guidelines. Common reasons for referral are; severe foetal distress, requiring neonatal unit admission (23%), prematurity of less than 32+0 weeks gestation (20%) and prematurity between 32+0 and 36+6 weeks (17%). No referrals were made for foetal hydrops, maternal coagulopathy and maternal substance abuse.

No referral information received in 1.9% specimens. Chorioamnionitis was sole indicator in 7 cases.

Conclusion: The overwhelming majority of referrals are appropriate.

No clinical information was offered in a small percentage

Chorioamnionitis and sepsis are not in the RCPath criteria, however maternal pyrexia is

Referral practice in Low Apgar Scores requires further study


Violation of immunological tolerance as a predictor of the development of complications of allogeneic pregnancy

E. Rudenko*, E. Kogan, Т. Demura, N. Trifonova, N. Zharkov

*Sechenov University, Russia

Background & objectives: The high frequency of complications in allogeneic pregnancy is presumably associated with a violation of the immunological privilege of uteroplacental region. It is necessary to study the expression of markers reflecting the formation of immunological tolerance in placenta and placental bed.

Methods: the study was performed on placenta material and placental bed biopsies from 89 women; a histological and immunohistochemical examination was performed using antibodies to CD8, CD138, CD56, CD4, CD 25. xpression was evaluated by a quantitative method for calculating the number of stained cells in 10 fields of view at SW 400.

Results: Placentas taken from women after allogeneic pregnancy had pronounced signs of immune alteration, such as chronic histiocytic intervillositis, lymphoplasmacytic deciduitis, chronic chorioamnionitis, chronic villitis, perivillous fibrinoid with lymphocytes (p (F) <0.05). Immunohistochemical study of the placentas showed accumulation of CD138 + plasma cells, CD8 + T-lymphocytes and uNK cells, and a decrease in the number of CD25 \ CD4 + Treg cells in the structures of the uteroplacental region (Kruskal-Wallis test, p <0.05)Placentas after IVF with donor oocytes are characterized by similar changes associated with the development of chronic inflammation in the structures of the placenta and immunohistochemical signs of impaired immunological tolerance at the maternal-foetal interface.

Conclusion: The obtained data allow us to classify pregnancies under the surrogate motherhood programs and oocyte donation as a risk factor for the development of pregnancy complications with immune pathogenesis.


Correlations between mismatches in HLA II in mother-recipient – child couples and immunomorphological characteristics of placentas in allogeneic pregnancy

E. Rudenko*, E. Kogan, Т. Demura, N. Zharkov, N. Trifonova

*Sechenov University, Russia

Background & objectives: The number of mismatches in the HLA II gene system during allogeneic pregnancy can affect the development of complications and be a selective factor in the selection of a donor blastocyst.

Methods: Allelic polymorphisms of class HLA II genes (DQA1, DQB1, DRB1) were studied in pairs of 89 mother-child pairs by PCR. In accordance with the results, all pairs were divided into 4 groups according to number of mismatches (from 0 to 3). Morphological and IHC studies using antibodies to HLA-DR, HLA-C were performed on the material of placentas and placental sites.

Results: It was shown that with an increase in the number of mismatches, the incidence of foci of chronic inflammation in the tissues of the placenta and placental bed increases: perivascular infiltrates of the radial arteries, chronic chorioamnionitis, basal deciditis (p (F) <0.05). In the absence of discrepancies and with their maximum number, a change in the optimal level of HLA-C expression by villi syncytiotropholastome occurs, as well as an increase in HLA-DR expression in deciduas basalis and the stroma of the placental bed (Kruskal-Wallis test, p <0.05)

Conclusion: Both a high degree of antigenic spillage and significant coincidence in the HLA gene system result in structural changes and an imbalance in the immunological balance in the structures of the placenta and placental bed, which can be a selective factor in the selection of donor oocytes and the recipient mother.


Assessment of umbilical cord tissue morphology with α-SMA and desmin expressions under circulatory hypoxia

L. Rudiuk*, O. Reshetnikova

*Immanuel Kant Baltic Federal University, Kaliningrad, Russia; Regional Clinical Hospital of the Kaliningrad region, Kaliningrad, Russia,

Background & objectives: The umbilical cord(UC) insertion site, anatomical abnormalities are associated with foetal growth potential and clinical outcomes for the newborn. The aim of present study is to elucidate UC structural features in gestations under conditions of congenital heart disease(CHD) in pregnant women.

Methods: 35 full term placentas were divided into group I(20 cases of maternal CHD) and 15 cases of physiological pregnancy(control group-CG).The length and diameter of the UC were measured. Histological sections stained with H&E were studied microscopically, then by computer morphometry. The immunohistochemical(IHC) staining with monoclonal murine antibodies to α-SMA and Desmin conducted. The differences between groups were elucidated by the non-parametric Mann-Whitney U-test,p<0.05.

Results: Results of the study showed a decrease in the length (31.37±1.43 cm) and the diameter (1.01±0.11 cm) of the UC during pregnancy with CHD compared to the control group (respectively: 34.40 ±1.76 and 1.55±0.22 cm, p<0.05). Microscopic examination revealed an enhanced fibrosis and lack of Warton’s jelly in the fiber- free stromal clefts. α-SMA and desmin expressions were higher and more diffuse in the group I than in the CG.

Conclusion: The decreased area of the jelly-filled UC stomal clefts, reduced hydrophilicity of the tissues is accompanied by enhanced sclerosis a reduction of its turgidity. This results in the advancement of the sensitivity of the vessel walls to damaging mechanical influences. The umbilical cord structure remodeling is considered in the aspect of placental adaptation to circulatory hypoxia due to hemodynamic impairments in women with CHD.


Structural profile of the placental terminal villi stroma remodelling with emphasis on oxidative stress during pregnancy

L. Rudiuk*, O. Reshetnikova

*Immanuel Kant Baltic Federal University, Kaliningrad, Russia; Regional Clinical Hospital of the Kaliningrad region, Kaliningrad, Russia

Background & objectives: Morphological alterations of placental terminal villi can cause organ’s dysfunction, leading to deficiency of a foetal oxygen and nutrients supply.

The aim of this study was to investigate the remodelling of the placental terminal villi stroma under oxidative stress conditions.

Methods: Morphological study was carried out in following groups: I- 20 placentas from mothers with non-operated congenital heart defects (CHD);II- 19 cases of operated CHD and 15 cases of physiological pregnancy-control group(CG).The immunohistochemical(IHC) staining protocol with monoclonal mouse antibodies to collagen IV and collagen III for placental tissue has been developed. The differences between groups were identified by Mann-Whitney U-test at p<0.05.

Results: Histological study revealed violations of the placental chorionic villi maturation in CHD groups, especially in the I group. Morphometry showed a decreased volume fraction (VF) of the terminal villi stroma in CHD cases(I– 20.0(11),II- 20,0(4),CG- 24.0(12)%,p<0.05). IHC analysis discovered the expression of the atypical collagen III type in the group I. The VF of the type IV collagen decoration area consistently decreased from I to the II group and to the CG(respectively:19,0(6)-16,0(5,75)-11,5(7,75)%,p<0.05).

Conclusion: Hypoxia during gestation impacts both maternal health and foetal development. Placental villi maturation disorder with enhanced villi immaturity may cause the disruption of foeto-maternal metabolism and gas exchange via placental membrane(PM). The decreased VF of the villi stroma contributes the thinning of PM, thus promoting to the enhancement of transplacental exchange. Placental villi stroma remodelling, the role of collagen types III and IV discussed in the aspect of placental adaptations to oxidative stress due to hemodynamic disorders in CHD cases.


Basal plate myometrial fibres: a lesion of shallow placental implantation

J. Stanek*

*Cincinnati Children's Hospital, Division of Pathology, USA

Background & objectives: Basal plate myometrial fibres (bpmf) with or without intervening decidua is the earliest and clinically asymptomatic lesion of abnormal placentation. This retrospective analysis is an expansion of our clinicoplacental studies combining bpmf and occult placenta accreta.

Methods: 169 most recent consecutive cases with placental bpmf with or without intervening decidua (Group 1) were compared to 1661 cases without bpmf (Group 2). Frequencies of 25 independent clinical and 40 placental phenotypes were statistically compared between the groups.

Results: Of 1830 placentas examined since 2009, 169 showed bpmf (11.2%). Placentas with bpmf as compared with placentas without bpmf were statistically significantly (p<0.05) more common in association with cesarean sections (11.2% vs 7.5%), antepartum haemorrhage (17.7% vs 11.6.%), gestational hypertension or preeclampsia (23.1% vs 6.0%), complicated 3rd stage of labor (18.9% vs 6.4%), villous infarction (14.2% vs 8.9%), uterine pattern (14.8%, vs 9.6%), massive perivillous fibrin deposition (9.5% vs 5.3%), chorionic disc chorionic microcysts (21.8% vs 15.9%), clusters of maternal floor multinucleate trophoblasts (27.8% vs 21.2%), excessive trophoblasts of chorionic disc (24.3% vs 17.3%), segmental villous avascularity (27.8% vs 19.9%), and foetal vascular ectasia (26.2% vs 15.2%).

Conclusion: Because of the association of bpmf with increased extravillous trophoblasts in the chorionic discand maternal floor trophoblastic giant cells, they should be included into the spectrum of placental lesions of shallow placental implantation rather than linked to decidual deficiency only.

PS-11 Pulmonary Pathology


Polytropic effect of polymetallic dust on the experimental rats lungs

K. Abdikadirova*, C. Zhienbayeva, S. Arinova, L. Bystrevskaya, I. Medvedeva

*Karaganda Medical University, Kazakhstan

Background & objectives: Polytropic effect of dust depends on a complex chemical composition, which impact with possible effects of summation or potentiation on the organism. To study the polymetallic dust effect on the lung tissue of experimental animals at intratracheally exposure.

Methods: The experiment included 30 outbred white male rats weighing 180-220g. for 30 days. Lung pieces were fixed in neutral formalin 10%, morphological studies were carried out according to standard methods. The object of the study was the lung tissue of experimental animals. The experiment was realized according to «Rules for biomedical experiments conducting» of MH RK (November 12, 2009 №697).

Results: The initial stages of desquamative bronchitis and a pathological tumour in the middle lobes of the lungs with compaction of the lungs stroma of oncological nature were revealed after intratracheal dusting. An accumulation of dust as rounded formations surrounded by fibroblasts, fibrocytes and macrophages was noted in the adventitial bronchi membrane.

The number of destroyed alveoli increased, the formation of pronounced emphysematous zones occurred subpleural in the lungs of rats.

Conclusion: It can be assumed that dust causes a polytropic effect on lung tissue, provokes the formation of a tumour of a malignant nature and degeneration of fibrous tissue, since it is associated with a violation of the parenchyma of the lungs and blood vessels with the subsequent formation of cell-dust foci and expressed subpleural emphysematous zones.

The preserved alveoli were different shape, the blood vessels were characterized by plethora and severe perivascular oedema.


Pathomorphological changes in the lung parenchyma at the exposure of multicomponent polymetallic dust

K. Abdikadirova*, B. Chergizova, Y. Talaspekova, B. Suleimenova, I. Baryshnikova

*Karaganda Medical University, Kazakhstan

Background & objectives: The clinical picture of dusty diseases indicates damage of the lung tissue and respiratory tract with the development of pneumosclerosis, chronic bronchitis and bronchial asthma.

To study the pathomorphology of the lung parenchyma after exposure of dust in various doses.

Methods: Experimental study was carried out on outbred white rats weighing 180-220 g for 30 days. Effective doses of the toxicant 10 mg/m3 and 25 mg/m3 were used. The experiment was carried out by «Rules for biomedical experiments conducting» of MH RK (November 12, 2009 №697).

Results: The initial stage of desquamative bronchitis was revealed in the bronchi; in the bronchi adventitia, cell-dust foci of polymetallic dust accumulation surrounded by fibroblasts, fibrocytes and macrophages were observed. The number of destroyed alveoli increased with dose increasing, and the formation of expressed emphysematous zones of pneumosclerosis increased.

The behavioural activity of rats was studied after dust inhalation. The motor activity decreased after dusting dose increase.

Conclusion: The metals of the dust cause a polytropic effect with an increase of the toxicant dose, have high bioactivity and accumulate in the stroma of the bronchi and alveolar tissue. A significant decrease in the weight and muscle strength was observed at the exposure of dust maximum dose, which was clearly expressed by the experiment end.

The behavioural activity of rats at a dose of 10 mg/m3 was moderate, at 25 mg/m3 – expressed behavioural reactions were reduced.


Congenital pulmonary airway malformation diagnosed at elderly age – experience from a tertiary hospital

V. Almeida*, R. Almeida, J. Madeira, J. Fraga, V. Sousa, L. Carvalho

*CHUC, IAP-PM, FMUC, Portugal

Background & objectives: Congenital pulmonary airway malformation (CPAM) is a nonhereditary lung anomaly mainly detected prenatally or during early life, but some remain asymptomatic during adulthood.

This study summarizes the clinicopathological features of CPAM diagnosed in elderly patients over a 10-year period.

Methods: We retrospectively identified all CPAM diagnosis between 2009-2019 and selected all the cases in which patient age was equal or superior to 70-years-old. These cases were reviewed and classified according to the Stocker system. Clinical and radiological data were obtained from Hospital registries.

Results: Five CPAMs were diagnosed on patients older than 70-years; two were men. Three patients had registries of previous pneumonia and two were former smokers.

Presentation symptoms were haemoptysis, cough and/or fever. TC scan showed nodular formations or caveated masses. All patients were submitted to lobectomy: four were diagnosed with Stocker type I CPAM, one with Stocker type II CPAM. Two patients were concurrently diagnosed with mucinous adenocarcinoma.

Conclusion: CPAM can be asymptomatic or course with nonspecific lung infections, so it must be considered at any age.

Two patients presented with mucinous adenocarcinoma, supporting the need to treat every CPAM as it can potentially evolve to carcinoma.


Results of intraoperative consultation in lung and mediastinal lesions

E. Kilic Bagir*, D. Gumurdulu, A. Acikalin, M. İnceman, A. Avci

*Cukurova University, Faculty of Medicine, Pathology Department, Turkey

Background & objectives: The determination of histopathological diagnosis during mediastinal surgery is important for proper surgical staging. The aim of our study is to determine the accuracy rate of our intraoperative consultation results and to review the diagnostic problems that may cause errors.

Methods: In this study, frozen results of 563 lung, pleural, mediastinal masses and lymph nodes diagnosed by intraoperative consultation between December 2013 and July 2018 at Çukurova University Faculty of Medicine Pathology Department were retrospectively evaluated. Frozen sections were compared with paraffin sections.

Results: One hundred fifty six (27.6%) cases were female and 407 (74.4%) were male. The mean age was 57.6 years (3-90). The diagnostic accuracy rate was 95.9% in the frozen section results. Of the 16 patients who were misdiagnosed, 6 (1.06%) had false positives and 10 (1.77%) had false negative results. In 7 (1,24%) cases, the diagnosis was left to permanent sections. In the re-evaluation of 16 cases with incorrect results in our series, we found that the causes of misdiagnosis were caused by macroscopic sampling error, technical reasons and interpretation error.

Conclusion: Intraoperative consultation is a procedure that effects to surgery in mediastinal and lung parenchymal lesions. In order to minimize misdiagnosis rates, it is important to have sufficient number of samples and to eliminate technical problems.


An AI-based tool to identify cancer areas in lung biopsies

L. Björk*, H. Elfving, S. Elwing, M. Andersson, C. Lindskog, L. Kajland Wilen, P. Micke

*KBH, Karolinska Institutet, Contextvision AB, Sweden

Background & objectives: Histopathology of lung-cancer forms the backbone of diagnostics but is dependent on the experience of the pathologist. Even skilled pathologist, has a challenge to evaluate biopsies. To better the process, we trained a deep-segmentation neural-network to identify cancer in lung-biopsies.

Methods: We retrieved 90 retrospective cases of lung cancer, containing 200 WSIs of H&E-stained biopsies including clinical metadata form Uppsala University Hospital. The slides were scanned and two pathologists made pixel-level multiclass annotations, one reviewing the other, using Contextvision´s INIFY viewer and annotation tool. A U-Net was trained on 160 of the WSIs and was evaluated on the remaining 40 WSI’s

Results: Visual comparisons using probability heat maps revealed striking agreements between manual annotations and the predicted cancer areas selected by the model. In addition, the model correctly assigned areas to the Benign, Necrosis, or Cancer histological subgroup.

The table shows the median pixel-level performance statistics on the evaluation images:

  Benign Necrosis Cancer
Precision 0.95 0.97 0.81
Recall 0.93 0.73 0.72

Conclusion: We present a deep learning network that could identify and outline cancer in lung biopsies with good accuracy. This network could be further developed into a decision support tool for pathologists in their routine diagnostic process.

Funding by: VINNOVA—Sweden's innovation agency


Her family genes, DDR2 and PI3KCA mutations may be early events in pre-neoplastic lesions and in atypical bronchial type epithelium

L. Carvalho*, A.F. Ladeirinha, A. Alarcão, M.R. Silva, T. Ferreira, A. Rodrigues, C. Vilasboas, A. Figueiredo, F. Barata, V. Sousa

*Institute of Anatomical and Molecular Pathology, Faculty of Medicine of the University of Coimbra, Portugal; CIMAGO - Research Center for Environment, Genetics and Oncobiology, Faculty of Medicine, University of Coimbra, Portugal; University Hospital Anatomical Pathology Coimbra, Portugal

Background & objectives: Early diagnosis will decrease lung cancer incidence through molecular changes identification in pre-neoplastic and in pre-invasive lesions.

NGS was applied to either adenocarcinoma and atypical bronchial type epithelium and to cases of AIS and MIA.

Methods: Three pulmonary adenocarcinomas and concomitant atypical bronchial type epithelium were manually/independently microdissected from FFPE blocks; adenocarcinoma in situ (1 case) and minimally invasive adenocarcinoma (2 cases) were also selected.

Routine immunohistochemical panel was applied for precise diagnosis. NGS (colon/lung panel) was performed in a PGM platform and ALK, ROS1 and MET FISH analysis was also performed on 4-μm-thick.

Results: Adenocarcinomas expressed CK7 /TTF1; two also expressed Vim and p63 to a lesser extent. AIS and MIA cells expressed CK7 and TTF1.

Identified somatic mutations concerned KRAS, FGFR2 and DDR2 in two adenocarcinomas and respective ABTE; PI3KCA, EGFR and MET represented another duet while ERBB2 raised up also.

ALK and ROS1 rearrangements or MET amplifications were not found by FISH.

The AIS case exhibited ERBB2/4, EGFR and SMAD4 somatic mutations while the two MIA cases were sequencing apparently wild-type for the applied panel.

Conclusion: Mutational status in ABTE identical to concomitant adenocarcinomas provides concern about early genetic events present in lung adenocarcinoma carcinogenesis deserving further studies to be emphasized as pre-neoplastic lesion to lead early clinical guidance for high-risk patients identification in bronchial – pulmonary carcinoma in screening.


Nucleolin expression in pleural mesothelioma – a potential therapeutic target

L. Carvalho*, A.C. Gregório, N.A. Fonseca, V. Sousa, A. Alarcão, A.F. Ladeirinha, M.R. Silva, T. Ferreira, A. Rodrigues, C. Vilasboas, J.N. Moreira

*Institute of Anatomical and Molecular Pathology, Faculty of Medicine of the University of Coimbra, Portugal; CIMAGO - Research Center for Environment, Genetics and Oncobiology, Faculty of Medicine, University of Coimbra, Portugal; University Hospital Anatomical Pathology Coimbra, Portugal

Background & objectives: Treatment of pleural mesothelioma includes surgery/chemotherapy often palliative. Cisplatin/pemetrexed constitute current standard but curative therapy remains unclear. Nucleolin involvement in tumorigenesis/angiogenesis might be related to metastisation raising nucleolin expression determination in pleural mesothelioma necessary.

Methods: Biopsies/lobectomy samples of 21 mesotheliomas without any preoperative chemotherapy or radiotherapy treatment and classified after calretinin/CK5.6/WT1/vimentin/podoplanin/Ber-Ep4/TTF1 had nucleolin expression validated through positive cells percentage/field: 0% = 0 (negative); 1-10% = +1 (low positive); 11-49% = +2 (positive) and ³50% = +3 (highly positive).

Results: Histopathological characteristics and nucleolin scoring correlated biphasic mesothelioma +3 – eight cases; epithelioid mesothelioma +2-five cases and +3-four cases and sarcomatoid mesothelioma +1 – +3 – three cases, all with consistent fusiform cellular stroma. Positive and strong staining of the nucleus was observed in all the cases, albeit it was also possible to see some cells that did not express the protein. Cytoplasmic staining accompanied by membrane staining was observed in 3 of the 20 mesothelioma samples analysed.

Conclusion: Nucleolin value as both prognostic factor and possible therapeutic target in several types of cancer has been extensively studied. Also subject of interest for targeted delivery systems in the context of oncological diseases reveals to be a promising mesothelioma therapeutic target.


NUT carcinoma: a tertiary single institute experience with a case of EWSR1 rearrangement

H. Cho*, M.J. Song, K. Cho, J.S. Song

*Department of Pathology, University of Ulsan, College of Medicine, Asan Medical Center, Seoul, Republic of Korea

Background & objectives: NUT carcinoma is a rare and aggressive tumour, defined by chromosomal rearrangement of NUT (nuclear protein in testis, i.e. NUTM1) gene. Herein we describe clinical and histological characteristics of seven cases in a single institute experience.

Methods: Seven cases of NUT carcinomas were identified from June 2014 to February 2020. All cases were confirmed by diffuse nuclear NUT immunoreactivity and/or NUT rearrangement by FISH. Clinico-pathologic parameters were reviewed. Next-generation sequencing (NGS) was performed in three cases.

Results: Primary sites included lung, frontal and ethmoid sinuses, larynx, and axillary area. Five patients had multiple metastases at the time of diagnosis. The tumours showed small round cell morphology with/without abrupt keratinization. The tumour were immuno-positive for cytokeratin (ck, 6/6, 100 %) and p40 (4/5, 80%). One showed EWSR1 rearrangement by FISH which initially diagnosed as Ewing sarcoma. Another one showed mutations of NF1 and APC genes by NGS.

Conclusion: NUT carcinoma is a diagnostic challenging disease and has been misdiagnosed as poorly differentiated carcinoma, squamous cell carcinoma, Ewing sarcoma, and desmoplastic small round cell tumour. The recognition of this entity is important to prevent misdiagnosis and to suggest the proper treatment. In addition, the name of NUT rearranged tumour is more favourable due to CK -negative NUT positive tumour.


A unique case of extrasomatic angiomatoid fibrous histiocytoma arising from the pulmonary hilum

T. Costa Pereira*, E. Rios, S. Guimarães

*Centro Hospitalar Universitário de São João, Portugal

Background & objectives: Angiomatoid fibrous histiocytoma (AFH) is an uncommon soft tissue tumour that typically occurs in the extremities. Fewer than 10 cases having been reported in the pulmonary region. Herein we report the first case of AFH arising from the pulmonary hilum.

Methods: A 32-year-old male presenting with recurrent haemoptysis was found to have a hypervascular mass (53mm) in the right pulmonary hilum, involving the pulmonary vein, the interlobar artery and the middle lobe bronchi on CT scan. The routine staging including PET scanning, disclosed no evidence of disease elsewhere. After several inconclusive biopsies, a right pneumonectomy was performed due to massive haemoptysis.

Results: The tumour comprised a multinodular proliferation of bland spindle to histiocytoid cells with dense lymphoplasmacytic infiltrate and angiomatoid features, which had invaded into bronchial and vessel walls (exhibiting an impressive endovascular growth) and two hilar lymph nodes. There was no significant atypia, mitosis or necrosis. Immunohistochemical profile (CD99, CD68, EMA diffusely positive; desmin focally positive with dendritic-like pattern) and detection of EWSR1(22q12) gene rearrangements confirmed the diagnosis of AFH.

Conclusion: AFH can occur at unusual sites, including the pulmonary hilum. Diagnosis can be extremely difficult, especially in limited biopsies or when involving lymph nodes. Once completely resected, pulmonary AFP discloses a benign clinical behaviour, even if nodal metastases occur.


Pitfalls in ROS1 immunohistochemistry in non-small cell lung carcinoma: equivocal ROS1 immunostaining can be associated with alternative mutation status

M. Cotter*, J. Gorman, N. Nadeem, C. O’Brien, S. Finn, A. Fabre

*Department of Histopathology, St. Vincent’s University Hospital, Elm Park, Dublin, Ireland

Background & objectives: ROS-1 gene rearrangement is an important predictive biomarker for targeted tyrosine kinase inhibition. The College of American Pathologists supports the use of ROS-1 immunohistochemistry as a screening tool followed by confirmation of rearrangement by sequencing in all positive cases.

Methods: We retrospectively examined the correlation between immunohistochemistry (IHC) using the Ventana SP384 ROS-1 antibody, on routine diagnostic tissue sections, and rearrangement status by next generation sequencing (Lung panel Oncomine Focus Assay, Ion Torrent). Clinicopathological details including age at diagnosis, tumour morphology and rearrangement /molecular status were collated.

Results: The cohort included 39females and 25males, age range 36-88 (average 66). 64 were tested: 2 lung resections, 52 biopsies(40 lung/pleura, 5 bone/soft tissue, 5 lymph node, 2 liver) and 10 cytology specimens. 55/64(86%) were adenocarcinomas. ROS-1 expression was detected in 33%(21/64). 3/21 showed strong positive staining with 1 confirmed CD74-ROS1 fusion, 1 KRAS codon 12 mutation and 1 negative for fusions/mutations. Equivocal expression was present in 28%(n=18) without subsequent ROS-1 rearrangement, but with KRAS(39%, n=7), EGFR(17%, n=3), and HER2(6%, n=1) mutations, giving a false positive rate of 17%. 3 showed no fusions/mutations and 4 were insufficient/not sequenced. ROS-1 expression was also noted in reactive type II pneumocytes and mesothelial cells.

Conclusion: ROS-1 immunoreactivity should be interpreted with caution as tumours lacking ROS-1 rearrangement may still express ROS-1 by IHC and harbour alternative mutations (i.e. KRAS in 39%). Reporting of IHC staining should be done in conjunction with mutational status if possible.


Bronchiolar adenoma: a report of four cases with morphological, immunophaenotypic and genomic analysis

X. Wang, J. Huang, J. Da*

*National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital & Shenzhen Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, China

Background & objectives: To improve the capacity of differential diagnosis and avoid confusion of recognition caused by various morphological changes of bronchiolar adenoma(BA).

Methods: Various patterns and trilineage cell of four cases were reviewed morphologically and immunohistochemically (IHC), and meantime, performed genomic analysis by Next-generation sequencing (NGS, Illumina Hiseq).

Results: Different intensity/pattern of immunophenotype was demonstrated at trilineage cell, including basel cells(BCs), ciliated cells(CCs), mucous cells(MCs). #1 was composed of bilayers of BCs with both MCs and CCs, the MCs was predominant. IHC expressed strong for TTF-1, NapsinA, p40, and Braf-specific antibodies with Braf mutation(p.V600E). #2 was similar to #1. However, luminal cells consisted of MCs, negative for NapsinA without Braf mutation. #3, “distal-type BA” contained predominant CCs and cuboidal cells without MCs, papillary/glandular architecture. Bilayered architecture were equivocal, and possible to overlie BCs layer by luminal cells. IHC showed strong for TTF-1, NapsinA, EGFR; weak p40; with EGFR mutation (L858R, E709K). #4 appeared to BA mixed with malignant component.

Conclusion: Variour morphology, immunophenotype and gene mutation appeared in our four cases. This suggests that BA may arise from different stem cells or differentiate for different lineage cells. It is possible that BA occured at respiratory bronchiole lacks completely bi-layered architecture.


Alveolar proteinosis: The Cruces University Hospital experience in the last 20 years

S. Gamba Torrez*, S. Fernández Solé, A. Marcos Muñoz, G. Garcia De Casasola Rodriguez, D. Parron Collar, I. Diaz De Lezcano Manrique De Lara, A. Gartzia Rivero, Y. Aberasturi Plata, M.I. Imaz Murga, M. Saiz Camin

*Department of Pathology, Cruces University Hospital, Barakaldo, Spain

Background & objectives: To describe the clinic-pathological features of patients with alveolar proteinosis (AP) diagnosed in our Institution during the last 20 years.

Methods: Archive retrieval and retrospective study of the histological sections of cases diagnosed of alveolar proteinosis and review of clinical records (including follow-up) of patients in the period 1998-2019.

Results: Four cases with the diagnosis of alveolar proteinosis (3 adults - 2 males, 1 female) were found in the retrospective review. The average age was 37 (range 27-44). A fourth case was diagnosed in a 2-year-old girl and was confirmed as hereditary. Diagnostic biopsies were obtained by thoracotomy. The 3 adult cases were smokers, one of them having had professional contact with aluminium, and another being cocaine user. Crazy paving pattern was detected in the two patients in whose CT scan was performed. Histological sections showed in all cases the typical pasty eosinophilic material fulfilling the alveolar spaces. Three cases were treated with whole-lung lavage with good initial response.

Conclusion: Alveolar proteinosis consists in the accumulation of surfactant in alveolar macrophages and alveoli and results in severe gas exchange impairment. It is considered a rare disease. 90-95% of adult cases have an autoimmune origin and the rest are secondary. Less than 1% of the cases are congenital. The diagnosis requires lung biopsy which shows the typical histology of diffuse alveolar fulfilment of proteinaceous eosinophilic material.


Driver mutations and PD-L1 expression frequency in Galician non-small cell lung cancer patients

S.M. García Acuña*, M. Sanchez Ares, J.R. Antúnez, J.M. Cameselle Teijeiro, M.F. Fraga Rodriguez, I. Abdulkader Nallib

*Clinical University Hospital of Santiago de Compostela, University of Santiago de Compostela, Spain

Background & objectives: Driver mutations and PD-L1 expression in lung cancer are potential therapeutic targets. We investigate the prevalence of these molecular alterations in a group of Galician non-small cell lung cancer (NSCLC) patients and compare it with reported in the literature.

Methods: Paraffin-embedded tissue samples from 103 NSCLC patients from our files were reviewed. Immunohistochemical study for PD-L1 expression, mutational screening for EGFR, KRAS, NRAS and BRAF by PCR and analyses for ALK and ROS1 rearrangements by fluorescence in situ hybridization were performed.

Results: PD-L1 expression levels were as follows: ≥50% in 14.5% cases; 1%-49% in 27.2% and <1% in 58.3%. The overall frequency of mutations were: EGFR 10.7%; ALK 2.9%; ROS1 0.97%; KRAS 22.3% and BRAF 1.94%. There were no tumours with NRAS mutations.

Conclusion: Compared to the literature, frequencies of driver mutations and PD-L1 expression level ≥50% are consistent, but frequencies of 1-49% levels are lower and those of <1% higher than expected, probably due to interobserver variability and tumour heterogeneity.


Matrix metalloproteinases and their inhibitors in foci of pulmonary fibrosis in workers of a plutonium production facility

D. Gogoleva*, E. Kazachkov, G. Sychugov, T. Azizova, V. Revina, A. Sychugov

*Chelyabinsk Bureau of Pathology, Russia

Background & objectives: Morphogenesis of radiation-related pulmonary fibrosis (PF) has not been sufficiently investigated. Current studies are focused mainly on matrix metalloproteinases (MMP) and their inhibitors (TIMP). The present study was aimed to investigate the role of MMP-TIMP in pathogenesis of radiation-related PF.

Methods: We investigated immunomorphological characteristics of PF based on autopsy material taken from 101 Mayak nuclear workers. 32 samples of plutonium-induced PF (PuPF); 34 samples of non-plutonium PF (non-PuPF) collected from workers with a clinical diagnosis of chronic obstructive pulmonary disease; 35 samples of normal tissue free from pulmonary pathology. The sections were incubated with antibodies against MMP-2, MMP-9, TIMP-1, TIMP-2.

Results: MMP and TIMP expression was examined in macrophages and stromal fibroblastic components. MMP-2 density was markedly lower in PuPF specimens (0.37:0.15;1.14) compared to non-PuPF specimens (1.41:0.53;3.40) and normal lung tissues (0.84:0.48;1.76), p=0.0012. On the contrary, MMP-9 was hyper-expressed in PuPF and non-PuPF (5.02:4.08;7.75 and 4.17:3.30;6.55, respectively) compared to normal lung tissues (0.87:0.53;1.47), p<0.0001. TIMP-1 was downregulated in PuPF specimens, while TIMP-2 was markedly upregulated (p=0.0365 and p=0.0021, respectively).

MMP hyper-expression and proteinase-anti-proteinase disbalance in PF foci in individuals who had been occupationally internally exposed to alpha radiation over prolonged periods might promote disruption of the extracellular matrix and basal membranes and the lung connective tissue remodelling.

Conclusion: The MMP disbalance is likely to contribute to the pulmonary neoplastic process. This study provides findings on expression of components of metalloproteinase-anti-proteinase system of the pulmonary tissue and its role as a potential histological marker for diagnostics and prognosis purposes.


A potential panel of four-long non-coding RNA signature to improve survival prediction of small cell lung cancer

J. Kim*

*Chonnam National University Hwasun, Republic of Korea

Background & objectives: The prognostic significance of long non-coding RNAs (lncRNAs) in small cell lung cancer (SCLC) remains unclear and needs to be investigated.

Methods: We obtained and analysed lncRNA expression profiles in two cohorts of 69 SCLC patients by repurposing the publicly available RNA sequencing and microarray datasets from the Gene Expression Omnibus (GEO) database. Functional enrichment analysis of lncRNAs-related protein-coding genes (PCGs) includes DAVID bioinformatic tool, GSEA, and cytoscape enrichement map.

Results: In the discovery series of 48 patients, we identified a set of four lncRNAs that was significantly associated with patients' overall survival (OS) using univariate Cox regression analysis. The four-lncRNA signature classified patients of the discovery series into the high-risk group and low-risk group with significantly different survival outcome (p < 0.0002). The four-lncRNA signature was further tested in the independent dataset. Moreover, the prognostic value of the four-lncRNA signature is independent of conventional clinical factors. Functional analysis suggested that four-lncRNA signature may be involved with SCLC through exerting their regulatory roles in known cancer-related pathways, DNA repair, glycosylation and metabolism.

Conclusion: Our study not only highlighted the potential role for lncRNAs to improve clinical prognosis prediction in patients with SCLC but also provided alternative biomarkers and therapeutic targets for SCLC patients.


A lipofuscin-detecting immunohistochemical marker of senescence relates with low proliferation and poor prognosis in non-small-cell-lung cancer

M. Kouroupi*, K. Balaska, M.I. Koukourakis, A. Giatromanolaki

*General Hospital of Alexandroupolis, Pathology Department, Greece

Background & objectives: Cancer cell senescence is associated with a permanent cell cycle arrest. Senescent cells exert potent bystander effects that may regulate clinical tumour behaviour. Senescence role in defining local and metastatic aggressiveness at a clinical level remains obscure.

Methods: A sensitive histochemical/immunohistochemical method (SenTraGor™/STG) has been developed to detect senescent cells, based on the synthesis of a hapten-linked Sudan Black B analogue that selectively binds to lipofuscins and labels accumulating dysfunctional lysosomes. In our study, we validated the STG method in 98 paraffin embedded surgically resected non-small-cell-lung carcinomas. Proliferation index MIB1 (PI), p16 and p21 was in parallel assessed.

Results: Strong expression of SGR in 10-100% in 36/98 cases (36.7%), but no association with stage, histology or differentiation was noted. Linear regression analysis showed a significant inverse association between SGR and PI (p=0.007, r=0.28). SGR was related with p21, but not with p16 expression. Kaplan-Meier survival curves showed a direct association of high STG expression with poor postoperative prognosis and independent of stage. MIB1 was not related with prognosis.

Conclusion: STG provides a reliable methodology to detect lipofuscin accumulation in cancer cells, in paraffin-embedded tissues, opening a new field for translational studies focusing on senescence. High presence of SGR+ senescent cells relates with poor prognosis in NSCLC.


PD-L1 assays and their performance in Nordic immunohistochemical quality control (NordiQC)\

H.L. Kristoffersen*, R. Røge, S. Nielsen

*NordiQC, Denmark

Background & objectives: Immunohistochemistry for PD-L1 expression is applied in different cancer types to identify patients eligible for treatment with immune checkpoint inhibitors. NordiQC provides proficiency testing for immunohistochemical (IHC) demonstration of PD-L1 expression with primary focus on non-small cell lung carcinomas (NSCLC).

Methods: NSCLC samples characterized for PD-L1 with critical and relevant expression levels, using the companion diagnostic (CDx) assay 22C3, Agilent, as reference standard method, were used for the assessments. In each assessment, tissue micro arrays comprising 6–12 NSCLCs, were constructed. Sections were circulated to the participants performing IHC for PD-L1 by their routine method. Results were evaluated by experienced assessors.

Results: Between 2017-19, six PD-L1 assessments were performed. The number of participants have increased from 68 in C1 to 182 in C6 with a total number of 881 submitted protocols in the first six runs. The pass-rate varied from 50% in C1 to 91% in C3, with an average of 81%. 37% of the participants used CDx assays performed accordingly to vendor guidelines provided an average pass-rate of 92%, compared to 67% for laboratory developed (LD) assays, used by 44%. 19% modified the protocol settings for CDx assays, providing a pass-rate of 88%. 75% of insufficient results were characterized by false negative staining reactions changing PD-L1 status of the included carcinomas.

Conclusion: Results obtained in all six assessments showed performance differences between LD and CDx assays. Within LD assays, meticulous calibration and validation is required. Participation in proficiency testing has improved the overall pass-rate for PD-L1 in NSCLC in NordiQC.


BAP1 loss in malignant mesothelioma: distribution according the histological subtype

J. Martin Lopez*, P. Martin Acosta, C. Salas Antón

*Department of Pathology, Hospital Universitario Puerta de Hierro, Spain

Background & objectives: Malignant mesothelioma (MM) constitutes a diagnostic challenge overall in small biopsies and cytology. Recently, BAP1 Loss, MTAP and EZH2 IHC and NF2 and CDKN2A/p16 FISH has immerged as useful tools and could help in problematic cases of mesothelial proliferations.

Methods: We evaluate the distribution of histological subtype of MM and the immunohistochemical expression of BAP1 in complete sections of 42 cases diagnosed between 2005 and 2018 in our institution (Hospital Universitario Puerta de Hierro).

Results: A cohort of 42 patients was recruited, 31 males (73,8%) and 11 females (26,2%) from 35 to 79 age. The specimens were small biopsies in 4 cases (9,6%), surgical biopsies in 30 cases (71,4%), surgical resection in 6 cases (14,2%) and metastatic tissue in 2 cases (4,8%). All cases were initially evaluated with at least two mesothelial and two carcinoma markers. Regarding to the histologic subtype were 30 cases of epithelioid mesothelioma (71,4%), 3 sarcomatoid (7,1%) and 9 biphasic (21,5%). Loss of BAP1 protein expression was observed in 21 of 42 MM cases (50%). According the histologic type, 66.6% of biphasic, 46.6% of epithelioid, and 33.3% of sarcomatoid MM were BAP1-deficient.

Conclusion: In our series we usually obtained the diagnosis of MM in surgical biopsies. BAP1 immunohistochemistry could help in mesothelial proliferations. According our data we observed BAP1 loss more frequently in biphasic and epithelioid subtypes.


Retrospective analysis of lung transplant explants: incidence of incidental malignancies in our institution

J. Martin Lopez*, R. Laporta Hernández, C. Salas Antón

*Department of Pathology, Hospital Universitario Puerta de Hierro, Spain

Background & objectives: The incidental discovered of unexpected neoplasms in lung explants has importance due to the immunosuppressive therapy that patients received and, in some cases determine the overall survival. Few series evaluate this incidence, and usually describe it as 0,7% and 2%.

Methods: We evaluated the rate of incidental malignancies detected in explanted lungs at our institution in a period of 24 years. A literature review was performed to evaluate the prevalence and the risk factors mainly involved.

Results: 6 of 639 consecutive explanted lungs have unexpected cancer (0,9%). All male smokers. There were 4 cases of adenocarcinoma (66,6%), 1 squamous cell carcinoma (16,6%) and 1 case of metastatic papillary thyroid cancer (16,6%). Of them, 3 cases were in the setting of idiopathic pulmonary fibrosis (IPF) (50%), 2 cases in the setting of chronic obstructive pulmonary disease (COPD) (33,3%) and one case due to bronchiectasis (16,6%). 1 of 6 neoplasms was detected at an advanced stage in the setting of IPF and recurrence occurred 5 months after transplantation.

Conclusion: Incidence of unexpected cancer in our lung explants was 0,9 % and more frequently due to adenocarcinoma in the setting of IPF similar with the data published in the literature.

IPF is an indepent risk factor associated with lung cancer and regarding to the literature, IPF seem to be risk factor for undetected neoplasms in lung explants. Efforts to improve screening in these populations could be considered.


Peribronchiola metaplasia (lambertosis) what is the pathological significance?

O. Matsubara*, Y. Jin

*National Defense Medical College, Japan

Background & objectives: Peribronchiolar metaplasia (PBM) is a nonspecific reaction to injury, resulting in the extension of bronchiolar epithelium through the canals of Lambert into alveoli. The genesis remains unknown. We aim to investigate the character of this lesion.

Methods: We reviewed the resected operation materials of 70 patients with the primary lung cancer and 13 patients of metastatic lung cancer (MLC). Comprehensive histological review and immunihistochemical staining for TTF-1, p40, ALK and Ki-67. We checked the lesions of PBM, atypical adenomatous hyperplasia (AAH), lung fibrosis, tumourlet and smoker’s bronchiolitis.

Results: We observed PBM was in 20(28.6%) in 70 cases; 14(28%) in adenocarcinoma (AC), 2(15.4%) in 13 squamous cell carcinomas.

(SCC), in 3(60%) in 5 large cell neuroendocrine carcinoma (LCNEC), and in one (7.7%) in MLC 13 cases. AAH wass in 12(17.1%) in 70 cases; 11(22%) in AC, 0 in SCC, one (20%) in LCNEC and in one (7.7%) in MLC cases. Lung fibrosis was in 9(12.9%) in 70 cases; in 5(10%) in AC, 4(30.7%) in SCC, in 0 in LCNEC and in one (7.7%) in MLC cases. Tumourlet was in (11.4%) in 70 cases; in 7(14%) in AC, in 0 in SCC, in one (20%) in LCNEC.

Conclusion: PBM is a very common finding in the lungs of the primary lung cancer (28.6%) vs. in the lung of MLC (7.7%). Our results suggest also PBM may be related to the pathology of AAH and tumourlet in lung adenocarcinoma.


ROS1 (SP384) screening in non-small cell lung cancer: experience in real conditions

L.O. Moscoso Miranda*, J. Hernandez Losa, L. Salazar Huayna, J. Castellvi Vives, S. Ramón y Cajal, I. Sansano Valero

*Vall d´Hebron University Hospital, Spain

Background & objectives: ROS1 is a mandatory biomarker in metastatic lung adenocarcinoma. Immunohistochemistry (IHC) is accepted as a screening method. Our study aims to show our experience with Ventana ROS1 clone antibody SP384 and to propose a diagnostic algorithm based on ROS1 H-score.

Methods: We have reviewed all the ROS1 tested in our centre since the introduction of anti-ROS1 antibody (SP384 clone) and re-evaluated 2 old positive cases. We have calculated an H-score 0-300 using chocolate as a visual aid for ensuring internal reproducibility. We have calculated the best cut-off value with a ROC curve test using STATA software.

Results: We have performed 170 ROS1 IHC and found some expression in 41. We could perform in situ hybridization in a total of 33 cases, identifying positivity by FISH in 7 cases (21,2%). The H-score range was 10-300 (mean 117 and median 95). The best cutoff to identify ROS1 rearrangements was ≥ 200 with a sensitivity of 100% and a specificity of 100%. The total percentage of ROS1 positive rearrangements in the new set of samples was 2,9%.

Conclusion: In our experience ROS1 SP384 clone is a useful screening tool and performing FISH/NGS only in those cases with an H-score ≥200 would be a cost-effective approach. We plan to enlarge the sample and test the reproducibility of the H-score method in a multicenter study.


PD-L1 expression in non-small cell lung cancers, data of the molecular pathology lab of National Institute of Oncology in Hungary between May 2018 and February 2020

K. Németh*, E. Tóth, L. Fülöp

*National Institute of Oncology, Hungary

Background & objectives: Immune checkpoint inhibitor therapy have improved the survival of non-small cell lung cancer (NSCLC) patients. Programmed death ligand-1 (PD-L1) tumour proportion score is correlating with response and survival. Our goal was to examine the PD-L1 expression rates of NSCLC cases.

Methods: PD-L1 immunohistochemistry (IHC) was carried out on 1687 NSCLC patients’ formalin fixed paraffin embedded material. In vitro diagnostic DAKO PD-L1 IHC 22C3 pharmDx assay performed on DAKO Autostainer Link 48. The evaluation of the PD-L1 immunhistochemistry was done according to the guidelines. PD-L1 tumour proportion score (TPS), the histologic subtype and the mutational status of the tumours were compared.

Results: From the 1275 adenocarcinoma, 496 (38,9%) was negative, 368 (28,9%) showed 1-49% TPS and 411 (32,2%) were more or equal to 50%. Of the 412 squamous cell carcinomas TPS distribution was equal in each group, 34,7%, 34% and 31,3% respectively.

42,1 % of the adenocarcinomas were KRAS mutant, 12,5 % were EGFR mutant. KRAS mutant tumours showed higher fraction of TPS>50%, than KRAS wild type tumours. EGFR mutant tumours generally showed low PD-L1 expression.

Conclusion: Our results show strong correlation with the literature datas. Mutation types has effect on PD-L1 expression.


Mortality for lung cancer among women in the state of Ceara, Brazil

D. Nunes Oliveira*, S. André de Souza Júnior, P. Vitória Pereira Motoyama, V. Holanda Ferreira, J. Carneiro Melo, A. Rolim Campos, R.L. Freitas de Almeida, N. A Roger Marie Piton, J. Benoit Sabourin

*University of Fortaleza, Brazil

Background & objectives: Lung cancer in women has shown an increase in its incidence and mortality worldwide. The objective of this study was describe the epidemiological profile and the temporal and spatial trend of lung cancer mortality of the female population.

Methods: This is an ecologic study. Data was collected from the Mortality Information System - Brazilian Ministry of Health. Cases of lung cancer were selected in women recorded in Ceará, Brazilian state, between 2000 and 2015.

Results: A predominantly upward growth pattern was observed in the analysed period. Of a total of 4883 deaths for lung cancer in women ,2729 (55,9%) were in the age groups of 60-69 and 70-79 years of age; mainly among brown and white women, with little or no education. The eastern region of the state had a higher relative risk of lung cancer mortality. Results consistent with the global trend, showing an increase in rates, especially in advanced age groups.

Conclusion: The survey sought to contribute to a better understanding of the mortality of lung cancer; targeted preventive interventions are needed to combat smoking and occupational risk factors associated with the incidence of lung cancer.


Histological pattern and trend of malignant pleuropulmonary tumours: a ten-year experience review

O. Ogunbiyi*, W. Ogunsanya, I. Nwanji, U. Ezenkwa, D. Mashor

*Department of Pathology and Forensic Medicine, Lagos State University College of Medicine (LASUCOM), Ikeja, Lagos, Nigeria

Background & objectives: In both sexes combined, lung cancer is the most commonly diagnosed cancer and the leading cause of cancer death globally. Diagnosis is increasingly being made on small tissue samples and cytological specimens. We present here our institutional experience regarding this.

Methods: We reviewed histo- and cytomorphological data of lung and pleural tumour specimens received over a 10-year period (2010-2019). Data was sourced from the surgical pathology records and population-based cancer registry. Diagnostic materials were mainly trucut biopsies of the pleura and lung. Appropriate statistics was applied to evaluate tumour subtype proportions and associate clinicopathological factors.

Results: There were 230 cases consisting of 166 (73%) biopsies and 64 (27%) cytology specimens from 103 (44.8%) males and 127 (55.2%) females giving a female-to-male ratio of 1.2:1. The mean age was 58±16 years. Primary tumours constituted 167 (72.6%) of the cases whilst 63 (27.4%) were metastatic tumours. Of the primary tumours, adenocarcinoma was the predominant histologic subtype (52.7%), with small cell carcinoma and mesotheliomas occurring in 3.6%% and 2.4% of patients, respectively. The number of cases diagnosed rose by 37% in the past 5 years (2014-218). Diagnosis of primary versus metastatic tumours was significantly associated with gender (p=0.003), females having more metastatic tumours, likely a reflection of gender incidence.

Conclusion: Our finding suggests a rising incidence of diagnosed pleuropulmonary tumours in this population. This reflects both the increased effort by surgeons to obtain tissue for diagnosis as well as a real projected increase in cancer burden being witnessed in developing countries including lung cancers. As in previous studies, adenocarcinomas predominate in the histomorphology. There is now a need to interrogate adenocarcinomas for smoking association using available genetic markers.


Synchronous squamous cell carcinoma and malignant pleural mesothelioma: a rare case report

D. Ozyigit Buyuktalanci*, A. Veral, D. Nart, S. Tasbakan, A.G. Ergonul

*Ege University Faculty of Medicine, Department of Pathology, Izmir, Turkey

Background & objectives: The co-existence of malignant pleural mesothelioma (MPM) and lung carcinoma is extremely rare. Herein, we report an uncommon case of co-presence of squamous cell carcinoma (SHC) and MPM.

Methods: A 75-year-old man presented with hemoptizia and dyspnea. He had a history of smoking but no exposure to asbestos. Chest computed tomography (CT) revealed the presence of a right lung mass combined with right pleural effusion and multiple pleural nodules. Positron emission tomography/CT showed increased uptake in the right upper lung mass (maximum standard uptake value:21.16). Then a right thoracentesis was performed.

Results: The smear of the pleural fluid revealed atypical cells with anisokaryosis and hyperchromasia forming papillary arrangements. The tumour cells were immunoreactive to mesothelial markers (calretinin, HBME-1, WT-1, CK5/6 were positive; BAP1 and desmin were negative) and the patient was diagnosed with MPM. The patient underwent bronchoscopy and there was no endobronchial lesion. Bronchial washing and brushing were diagnosed as benign cytology. The lung mass,24x20 mm in dimension, was evaluated on frozen section during surgery. Histologically, islands of large polygonal malignant cells containing keratin and intercellular bridges were seen and it was diagnosed SHC. Formalin-fixed paraffin-embedded sections of the same specimens showed immunoreactivity for p40 and CK5/6. At the same surgery, pleura biopsy was performed and epithelioid type MPM was confirmed.

Conclusion: We presented an extremely rare case of collision of MPM and pulmonary SHC. Possible coexistence of MPM and lung carcinoma should be considered in patients with pleural nodules and effusion.


Primary pulmonary classical Hodgkin lymphoma with underlying interstitial lung disease: a case report and review of the literature

M.B. Pimentão*, J. Fraga, L. Carvalho

*Centro Hospitalar e Universitário de Coimbra, Portugal

Background & objectives: A primary pulmonary lymphoma consists of clonal lymphoid proliferation in the lung parenchyma or bronchus, without extrapulmonary disease in the next 3 months. Classical Hodgkin Lymphoma (CHL) is an indolent clonal lymphoproliferative neoplasia originated in germinative follicular centre.

Methods: We present a case of an 81 years old man, with hypertension, chronic obstructive pulmonary disease, revealed left lower lung lobe nodule in a Chest X-ray. CT scan demonstrated multiple mediastinal lymph nodes, a spiculated solid lesion, with 3x2cm, hypercaptant on PET scan and diffuse thickening of pleural leaflets on the same lobe. Pleural thickening was considered as inflammatory changes.

Results: It was performed an wedge resection of a semilunar pulmonary fragment weighing 17.6g and measuring 5x3,5x2,5cm. Grossly it was observed an area of 3,2x2,1x1,5cm, heterogeneous, where whitish areas were mixed by black reticulation. Remaining scarce whitish and spongy lung tissue with subpleural cavitation. Histologically revealing a Classic Hodgkin's Lymphoma - nodular sclerosis variant. It was composed by several distinct populations, with predominance of two: Reed-Stenberg neoplastic cells and bulky Hodgkin cells. Both populations expressed CD20, PAX5, CD30, CD15, MUM1 and EBER test was positive. Involving the neoplasia, BALT hyperplasia with lymphoid follicles and activated germinative centres, were present. This case also presented an Usual interstitial pneumonia and an Epstein-Barr virus infection.

Conclusion: The misunderstood spectrum of interstitial preceding tissue where pulmonary Classical Hodgkin Lymphoma – nodular sclerosis, might be more frequent than what is commonly observed, suggesting that pulmonary inflammation and time remodelling can be similar to EBV lymph node infection.


Comparative histopathological and digital scoring between microfluidic and standard immunohistochemistry for p40 and TTF1 on non-small cell lung carcinoma

M. Procopio*, M. Houmani, S. Brajkovic, A. Leblond, D.G. Dupouy, A. Soltermann

*Lunaphore Technologies SA, Switzerland

Background & objectives: Microfluidic-based immunohistochemistry (IHC) for the non-small cell lung carcinoma (NSCLC) differential diagnostic markers p40 (squamous cell carcinoma) and TTF1 (adenocarcinoma) was previously optimized.

Here, we aim to prove its non-inferiority with respect to standard assay for NSCLC differential diagnosis.

Methods: Comparative scoring of p40 and TTF1 immunoreactivity between the two techniques was assessed on a NSCLC tumour microarray (TMA) cohort (160 resected patients, two cores each). The QuPath software was trained by manual annotation for cancer cell identification and employed for digital quantification of p40 and TTF1 signal intensity, reported as average score of tumour cells’ nuclei per TMA core.

Results: The microfluidic-based IHC for p40 and TTF1 was previously optimized to achieve an analytical performance comparable to standard staining, while shortened to 26 minutes - including the antigen retrieval step.

The concordance rate between the microfluidic (LabSat®) and gold-standard (BenchMark® ULTRA) IHC staining technique was 100% for both p40 (n=194 cores, 126 patients) and TTF1 (n=175 cores, 119 patients) when visually scored as positive or negative as routinely done in the histopathological practice.

Furthermore, comparison of digitally quantified staining intensity on the same NSCLC TMA for p40 and TTF1 resulted in a very strong positive correlation for the two techniques with a Pearson coefficient of 0.94 and 0.95, respectively.

Conclusion: Overall, the microfluidic-based IHC assay represents an equivalent approach to the standard chromogenic staining, allowing for NSCLC classification into squamous cell carcinoma or adenocarcinoma by automated, rapid and accurate detection of p40 and TTF1, respectively.

Funding: KTI 25736.1 PFLS-LS


Two unexpected and overlapped endobronchial benign lesions

C. Rivero Colmenarez*, S. Palomo Cousido, J. Castelao Naval, J. Fernandez Frances, D. Luján Rodriguez

*Guadalajara University Hospital, Spain

Background & objectives: Benign neoplasms comprise less than 10% of tumours involving the bronchi. Primary Pulmonary Leiomyoma (PL) is very rare and usually solitary. In the other hand, solitary squamous cell papillomas (SSP) are exceedingly rare and account <1% of all lung neoplasms.

Methods: We present a 57-year old man presented with a several month history of cough. The bronchoscopy image showed an obstructive upper left bronchial lobe mass. Histology revealed an squamous epithelial exophytic-papillary tumour with acanthosis and a submucosal proliferation of monomorphic cells arranged in whorls and fascicles with spindly nuclei. No necrosis or mitotic activity.

Results: PL are uncommon both in adults and children, constituting 2% of benign lung tumours, symptoms are due to partial/complete airway obstruction, can simulate asthma or be complicated (bronchiectasis and recurrent infections), besides that, endobronchial growth can cause cough, wheeze, haemoptysis. There are four types of respiratory papillomas: recurrent respiratory papillomatosis, solitary squamous papilloma, solitary glandular papilloma and mixed papilloma. They are 3 times more common in men that are usually in their 6th decade of life, presented with obstructive symptoms and up to 25% could be asymptomatic. CT scan reveals endobronchial plaques, nodules or thickening. They are usually central and endobronchial. The size range is 0,7 to 9 cm.

Conclusion: PL are the rarest benign neoplasms of the respiratory tract and treatment should be conservative but as SSP`s local recurrence-transformation into squamous cell carcinoma is uncertain, surgical and more aggressive manage is recommended. We present this overlapped benign endobronchial lesion case due to their low incidence, the fact that SSP could be misdiagnosed as a carcinoma and the need of diagnostic/manage algorithms to avoid unnecessary thoracotomies and wedge resections.


Proposition of a morphological score to predict the STK11 variation status in lung adenocarcinomas without genotyping

L. Claude, N. Piton, A. Lamy, F. Marguet, J. Sabourin*

*Rouen University Hospital, France

Background & objectives: STK11 is the third mutated gene in lung adenocarcinomas (LUAC). STK11-mutated LUAC are resistant or poor responders to immunotherapy. Our objective was to build a morphologically predictive score that could assess STK11 mutation status before genotyping.

Methods: LUAC surgical specimens resected during a 2-year period were included. Twenty-eight morphological criteria were assessed. Among them, 2 criteria, i.e. presence of mucinous contingent and presence of lymphoid nodule at low magnification, seemed more frequent in STK11-mutated LUAC. A morphological score was built attributing one point to the presence of these 2 criteria: minimum 0 and maximum 2.

Results: Forty-eight STK11-mutated and 235 STK11-wild type LUAC were morphologically evaluated. With a threshold defined at 1, our morphological score showed a sensitivity of 94%, a specificity of 34%, a negative predictive value of 95% and a positive predictive value of 29%. Of note, lymphoid nodules are usually associated with a better response to immunotherapy. In STK11-mutated LUAC, immune response seemed to be impaired, explaining this paradoxical finding.

Conclusion: In a recent study using deep learning-image analysis, STK11 mutational status was predicted using only morphological criteria, which were not determined.

Our good results, using a 2-criteria morphological score, warrant further validation in a larger muticentre series of LUAC.


Cryobiopsy in lung transplant patients: diagnostic yield and long-term mortality

L.E. Salazar Huayna*, K. Loor, O. Moscoso Miranda, J. De Gracia, M. Culebras, C. Berastegui, A. Alvarez, I. Sansano Valero

*Vall d´Hebron University Hospital, Spain

Background & objectives: Transbronchial biopsy (TB) is a routine procedure to assess rejection in lung transplant patients. Cryobiopsies are a kind of TB that provides more tissue with few complications. We report the diagnostic yield and long-term mortality in our series.

Methods: We have reviewed the 429 cryobiopsies’ diagnosis and follow-up of 256 patients transplanted at Vall d’Hebron University Hospital between April 2011 and April 2016.

Results: Of the 429 cryobiopsies analysed we could reach a multidisciplinary diagnosis in 421. All-cause 5-year mortality was 25.8% (n=66). Three patients died within 30 days (1.2%). Only one of these cases (0.2%) was directly related to the procedure (pneumothorax). Nine patients died within 90 days (3.5%) and 29 patients died within 360 days (11.3%).

Conclusion: Safety of cryobiopses has been recently questioned in terms of long-term mortality in patients with difuse parenchymal disease. We can say that this observation is not true for transplanted patients. Our series has a high diagnostic yield and a 5 years mortality rate that is halve the observed in lung transplanted patients demonstrating that cryobiopsies are safe and can potentially improve patients’ outcomes.


Pluripotency factors in mesothelioma

F. Seiwerth, F. Sedlic, A. Sepac, S. Sikiric, L. Batelja Vuletic, S. Seiwerth*

*UZSM, Croatia

Background & objectives: Pluripotency factors and PI3K-AKT pathway regulate tumour differentiation status and could be responsible for local and distal spreading of the tumour, resistance to therapy and other unfavourable characteristics of malignancy. We tested their expression in mesothelioma and mesothelium.

Methods: We tested samples of 66 patients diagnosed with Epitheloid Malignant Pleural Mesothelioma. Of them male (93%), with median age of 62 (27- 82) at the time of diagnosis. Pathohistological samples have been analysed and subclassified into 6 subtypes: solid (23 cases, 34.8%) , tubulopapillary (18, 27.7%), pleomorphic (12, 18.1%), trabecular (8, 12%), micropapillary (3, 4.5%) and microcystic (2 cases, 3%).

Results: Kaplan-Meier survival curves showed median overall survival of 15 months (95% CI 10.2- 19.7), but without statistical significance between various histological subtypes (chi square 9.176, p=0.102). Immunohistochemical staining was performed to determine expression of pluripotency factors Sox2, Oct-4, Akt and Nanog as well as the Pi3k-bcl 2 signalling pathway in mesothelioma histological subtypes. Sox 2 expression was high in all MPM samples, while the expression of oct-4 was negative regardless of subtype. Other proteins had moderate to high expression. Correlation between transcription factors expression and overall survival time was calculated. Nanog (Pearson corr. 0.282, p=0.022) and Sox-2 (0.281, p=0.023) suggesting correlation significance.

Conclusion: Human mesothelioma exhibits enhanced expression of NANOG, SOX2 and AKT proteins and reduced expression of BCL2 representing the basis for further pluripotency studies.

Funding by: HRZZ Grant


Recurrent respiratory papillomatosis with extensive lung involvement in a young woman with juvenile-onset form

M. Souto Moura*, A. Tavares, S. Saleiro, L.P. Afonso, M. Jácome, A.L. Cunha

*Department of Pathology, IPO-Porto, Portugal

Background & objectives: Recurrent respiratory papillomatosis(RRP), human papilloma virus(HPV–mostly 6 and 11) related, is characterized primarily by laryngeal papillomatous lesions rarely affecting the nasopharynx, tracheobronchial tree(2-5%) and pulmonary parenchyma(1-3.3%). Juvenile-onset form (JORRP) develops before 20-years-of-age (mean 3-5.5 years) mainly due to vertical transmission.

Methods: We report the case of a 28-years-old female, non-HPV vaccinated, non-smoker, with JORRP since 3-years-of-age, submitted to dozens of larynx papillary lesions endoscopic CO2-laser excisions. Lastly, tracheal lesions were discovered and CT-scan revealed several bilateral (mostly inferior lobes) lung cavitated lesions(4-24mm) and a 12mm inferior right lung lobe solid lesion, consistent with RRP imaging findings. The solid lesion was biopsied.

Results: All the larynx lesions previously removed were histologically compatible with squamous cell papillomas with HPV infection cytopathic signs and some with areas of mild dysplasia. The current lung biopsy revealed intra-alveolar nests of squamous epithelium (consistent with RRP lung involvement) with focal necrotic and degenerative aspects that were suspicious but not sufficient for a malignancy diagnosis. HPV genotyping of one of the most recent larynx lesions and of the lung biopsy revealed infection with HPV11.

In this case, histopathological findings were consistent with RRP with lung involvement and the scarcity of material prevented the conclusion of malignant transformation.

Conclusion: JORRP is generally more aggressive than adult form with multiple papillomas, high recurrence rate and need of several surgeries, increasing downward/lung dissemination possibility. HPV11, age-of-onset and multiple recurrences represent risk factors for aggressiveness (our case) and malignant transformation (<1% of cases).


The role of pulmonary collapse in radiation-induced lung injury

S. Timofeev*, Y. Kirillov, I. Chernov, Y. Malysheva, O. Rozenberg

*Tyumen State Medical University, Russia

Background & objectives: Radiation therapy is used for the treatment of cancer but it has a severe effect on other organs. The goal of this study is to find out the morphological characteristics of pulmonary collapse after a single dose of X-ray exposure.

Methods: For this study, two groups of Wistar male rats, aged between 8-9 weeks, were used. The main group of rats received a single dose of X-Ray. In both groups, surfactant phospholipids were checked. At various time points, each of the rats were sacrificed and lung samples were analysed through light and electron microscopy.

Results: On days 1 - 3, changes in the lung tissue could only be observed by electron microscopy in the subpleural areas. We discovered signs of endothelial and type II alveolocyte’s damage, such as oedema, disturbances in the location of the organelles, osmiophilic bodies and destruction of the surfactant. At the end of the first week, partial atelectasis were seen by light microscopy. Moreover, during this time, zones of pulmonary collapse expanded from the subpleural area to the lung’s root. Between 2 and 7 weeks, there was a noticeable change in the lung and constriction of the bronchus was widespread. After 7 weeks, a ‘honeycomb’ pattern and pulmonary fibrosis was observed.

Conclusion: This study showed that after a single dose of X-ray exposure, atelectasis starts from the distal, subpleural areas and moves down. There are different causes of pulmonary collapse, including surfactant damage, hypoxia, the destruction of the epithelium cells and bronchial constriction.


The oxidative stress and endothelial dysfunction in radiation-induced lung injury

Y. Malysheva, Y. Kirillov, I. Chernov, S. Timofeev*, V. Kukushkin

*Tyumen State Medical University, Russia

Background & objectives: Irradiation of organs of the thoracic cage to treat malignancies causes frequent complications that manifest as radiation pneumonitis and fibrosis. The purpose of this study is to evaluate oxidative stress and endothelial dysfunction in radiation induced lung injury(RILI).

Methods: Two groups or the Wistar-male-rats were used. One group had been irradiated by a single dose 12Gy of X-ray on the right lung. Non-irradiated group had been instilled with a saline solution into trachea. Animals from both groups were sacrificed on the different days after the beginning of the experiment and their lungs specimen had been analysed.

Results: Signs of the endothelial dysfunction were discovered by electron microscopy in the lung’s capillaries such as spasm, endothelial dystrophy and neoangiogenesis in the first hours after the start. IHC showed the reduction of the endothelial function markers (CD31, CD34, CD117, VEGFR1, VEGFR2, e-NOS) but their activity had been recovered in a few weeks. The local maximum of the residual fluorescence identified on the spectrum of the specimens is conditioned by toxicity of– OH radicals, superoxide anion radicals – O2 and some quantity of – CH3 radicals in the tissues. Superoxide anion radicals reduced the amount of nitrogen oxides converting them into peroxynitrites (ONOOH).

Conclusion: The results show that signs of oxidative stress and endothelial dysfunction could be detected within the first 24 hours after the initiation of the experiment by electron microscopy. Raman-fluorescent shows peaks by the 3rd day and the activity decreases by the 50th day of the experiment. Free radicals play the key role in oxydative stress and mediate the extent and intensity of the endothelial dysfunction.


The use of multigene next generation sequencing to improve accuracy of lung cancer staging in multiple tumours

S. Toomey*, P. Bennett, D. Moore

*United Kingdom

Background & objectives: In patients with multiple lung carcinomas, pathological staging depends upon whether these are considered independent. Molecular profiling has value in making this distinction. We reviewed a case series in which multigene sequencing was performed and assessed the impact on staging.

Methods: We searched the case database of the Sarah Cannon Molecular Diagnostics laboratory to identify patients with multiple synchronous lung carcinomas which had undergone NGS. The histopathology reports from these cases were reviewed alongside the tumour mutational profiles to determine evidence of clonality and whether the staging of their lung carcinoma was affected following the results of NGS.

Results: The patients included in the study were all diagnosed with lung adenocarcinoma, and the tumour nodules showed similar histologic types. Seven patients were identified and for 5 of these cases the tumours showed differing somatic mutations indicating that these are likely to be independent primaries. In the remaining 2 patients, there were overlapping variants in the detected mutation profile, indicating that these are highly likely to represent separate nodules of the same tumour. TNM stage was altered in 2 cases overall as a result of the molecular profiling.

Conclusion: The integration of multigene molecular profiling alongside conventional histopathological reporting can improve the accuracy of TNM staging in multiple pulmonary tumours.


Clinical and economic impact of the current testing scenario for ALK rearrangements in Spain compared to a hypothetical no-testing scenario

H.E. Torres Rivas*, L. Cabezón, E. Nadal, D. Bautista, A.L. Ortega, F. Rojo, L. Ruiz de Alda, F. García, P. Vieitez, D. Carcedo

*Hospital Universitario Central de Asturias, Spain

Background & objectives: Biomarkers play an essential role in diagnosis, treatment, and management of lung cancer. The aim of this study is to assess, for the first time, the clinical and economic impact of current ALK testing scenario in Spain.

Methods: Estimate the cost-health outcomes of NSCLC patient comparing current testing scenario vs no-testing scenario. A panel of expert established the assignment of the target treatment (true and false positive-negative). 3-states Markov model was developed, where progression free-survival and overall-survival curves were parameterized using exponential extrapolations to model transition of patients among health states. Only medical direct costs were included (€2019).

Results: A target population of 7,628 NSCLC (non-squamous and never-smoker squamous) patients was estimated. Over a lifetime horizon, the current ALK testing scenario produced additional 4,291 life years and 3,120 quality adjusted life years (QALYs), compared with no-testing scenario. More analyses such as the incremental cost-effectiveness ratio (ICER) (9.855 €/QALY), effect of increasing the testing rate and sensitivity analyses, to confirmed the robustness of the base-case results, were carried out.

Conclusion: The resulting ICER shows that performing molecular testing to the appropriate population of NSCLC patients is a cost-effective strategy in Spain.

Funding by: ROCHE Farma


Analysis of frequency of EGFR and ALK mutations, and PD-L1 expression in patients with lung adenocarcinoma in Bosnia and Herzegovina

D. Udovicic-Gagula*

*Clinical Center University of Sarajevo, Bosnia and Herzegovina

Background & objectives: Molecular testing of lung adenocarcinomas has become a standard and required part of pathological report. We analysed the EGFR, ALK-mutation status, and PD-L1 expression in a representative cohort of patients with lung adenocarcinomas in Bosnia & Herzegovina and correlate with clinical data.

Methods: Newly diagnosed patients within 12 months with histological proven primary lung adenocarcinomas were included. Mutational analyses for EGFR mutations were performed on a Cobas z 480 analyser. ALK immunohistochemistry was performed using the D5F3 clone on Ventana Ultra Benchmark instrument, and PD-L expression data obtained with Antibody DAKO 22C3.

Results: Of the 158 patients, 61,4 % (97) were male. Median years was 61, range from 29 to 84 years. Most of the patients were current or former smokers (95%). EGFR mutations were found in 12% of the patients, and of these mutations, exon 19 deletion was the most common (45.1 %). ALK mutation were present in 3,8% of the patients. The EGFR was more often detected in women and non-smoking, while ALK mutation had no gender predilection. Analysis of PD-L1 expression showed that 21% had a PD-L1 tumour proportion score (TPS) < 1%, 33 % had a PD-L1 TPS of 1–49% and 46% had a PD-L1 TPS of ≥50%.

Conclusion: The detected mutation rates demonstrated expected prevalence of EGFR-mutations and ALK-gene rearrangement, in comparison with the rates in other European countries, while PD-L1 expression was some higher than previously reported, but it maybe be ascribe to high percentage of smokers.


Histopathologic features of the pulmonary sarcomatoid carcinoma

H. Ürer*, N. Fener, N. Unver

*University of Health Sciences, Turkey

Background & objectives: Pulmonary sarcomatoid carcinoma are poorly differentiated cancers. The current classification includes pleomorphic carcinoma (PC), spindle cell carcinoma (SCC), giant cell carcinoma (GCC), carcinosarcoma (CS) and pulmonary blastoma. Our goal is to determine the histological features of sarcomatoid carcinomas.

Methods: Sarcomatoid carcinoma cases reported in our pathology laboratory over the last decade have been scanned. Histopathological features of the cases were evaluated.

Results: A total of 70 cases were detected. In cases, 53 PC, 3 SCC, 3 GCC, 9 CS, 2 pulmonary blastomas were found. In CS, carcinoma component was 6 squamous cell carcinoma, 2 adenocarcinoma, 1 nonsmall cell carcinoma. Sarcoma component was 5 chondrosarcoma, 2 osteosarcoma, 1 synovial sarcoma. Lymphovascular invasion was detected in 48 cases. Nodal involvement was found in 17 cases at N1 level and 7 at N2 level.

Conclusion: Pulmonary sarcomatoid carcinomas are a heterogeneous tumour group containing different histological components. This tumour group has its own spesific histologic profile.


The decline of surgical lung biopsy in interstitial lung disease: a tertiary centre experience 2009-2019

S. Waise*, N. Singh, E. Shaw, L. Veryard, S. Jogai

*University Hospital Southampton, United Kingdom

Background & objectives: High-resolution computerised tomography (HRCT) is increasingly used as a non-invasive diagnostic tool in interstitial lung disease (ILD). The aim of this analysis was to examine the impact of HRCT on annual rates of histological ILD diagnosis over the last decade.

Methods: A search of the laboratory information management system was performed to identify cases diagnosed between 2009 and 2019 at Southampton General Hospital. Paediatric cases were excluded. The remaining reports were examined, and only those in which ILD was the primary or favoured diagnosis were included in the final count.

Results: A total of 503 cases were identified, 462 of which were in adults. Of these, 202 had a diagnosis appropriate for inclusion in analysis. Over the 10-year period, the number of annual diagnoses was variable (range 7-32). However, the overall trend was a decrease in the annual rate of ILD cases diagnosed at histology, down 50% in 2019 from 2009. In contrast, the number of annual referrals to the ILD team has increased approximately fourfold.

Conclusion: The use of HRCT by specialist thoracic radiologists, together with specialist chest physicians, has obviated the need for diagnosis on surgical biopsy: the majority of cases can be diagnosed on the basis of clinical and imaging features. However, there is a subset of cases with overlapping features where histology plays a pivotal role in determining the diagnosis and accordingly, management.


Pulmonary carcinoid tumours with elevated proliferation rates – should there be a grade 3 subgroup of neuroendocrine tumours (NET)?

J. Wright*, E. Lim, A. Rice, C. Brambilla, J.L. Robertus, A.G. Nicholson

*Department of Histopathology, Royal Brompton & Harefield NHS Foundation Trust, United Kingdom

Background & objectives: The current WHO classification of pulmonary neuroendocrine tumours (NETs) includes low-grade typical carcinoid (TC) tumours to intermediate-grade atypical carcinoid tumours (AC) to high-grade large cell neuroendocrine tumours (LCNEC) and small cell carcinomas (SCC).

Methods: We wished to review of an emerging group of tumours that have histologic features of carcinoids but mitotic counts exceeding 10 per 2 mm2 which by the current WHO 2015 criteria are classified as LCNEC. We performed a retrospective analysis of 50 neuroendocrine

tumour cases diagnosed at our institutions between 2005 – 2020.Detailed microscopic review was undertaken.

Results: Of the 50 cases reviewed,14 cases (28%) showed histological features of carcinoids but mitotic counts exceeding 10 per 2 mm2.Three cases showed combined AC and LCNEC morphology and one case showed combined AC and SCC morphology. In all included cases, the highest mitotic count was >10 per 2mm2.The median highest mitotic count was 19 (IQR 15.5 -31.0).In eight out of fourteen cases, the Ki67 proliferation index was >20 (IQR 13.7 – 40).

Conclusion: A

subgroup of NETs exists with morphology closer to carcinoid than non-small cell carcinoma, but with a raised proliferation rate 10 per 2 mm2.

These may be better classified in alignment with carcinoids than LCNEC.


Plastic bronchitis: expanding clinical associations and identifying specific histologic characteristics compared to other intraluminal casts

J. Wright*, M. Kokosi, E. Renzoni, S. Desai, A. Devaraj, P.L. Molyneaux, V. Kouranos, F. Chua, G. Peter, A.U. Wells, A. Rice, A.G. Nicholson

*Department of Histopathology, Royal Brompton & Harefield NHS Foundation Trust, United Kingdom

Background & objectives: Plastic bronchitis is a rare condition in which airway casts develop in the tracheobronchial tree causing airway obstruction. Casts may be expectorated or lead to asphyxiation. Patients with casts can clinically be divided into those with allergic bronchopulmonary aspergillosis (ABPA) and plastic bronchitis (PB).

Methods: The PB group may be subdivided into post-operative cases including those following congenital, paediatric, adult or transplant surgery. We wished to determine whether there is a “primary” PB subgroup and, if so, did it have specific histopathological features. We performed a retrospective analysis of 30 consecutive-cases diagnosed at our institutions between 1999-2019.Clinical history was reviewed to classify the cases. Relevant histological features were semi-quantitatively scored.

Results: The 30 cases reviewed were classified as follows: APBA (12), postoperative (6) and “primary” PB (12).M<F (11:19). Median age:38 (2 months – 82 years). Casts from the “primary” PB group contained significantly more lymphocytes compared to neutrophils (p=0.0037) and increased lymphocytes compared to the ABPA group casts (p=0.0292). All “Primary” PB casts comprised loose fibrillary material which was only seen in 1/6 post-operative cases (p=0.0007) and none of the ABPA cases which all comprised thick mucin (p=0.0001).

Conclusion: Five cases showed clinical evidence suggestive of abnormalities in lipid metabolism. We have identified a group of “primary” plastic bronchitis casts with distinct microscopic features, in some cases associated with abnormal lipid metabolism.


Upregulation of phagocytosis in senescence cells in UIP and COPD to supplement their metabolic need, and their interaction with immune cells

D. Xhemalaj*, L. Brcic, S. Eidenhammer, W. Timens, P. Kapisyzi, H. Popper

*University Hospital of Lung Diseases "Shefqet Ndroqi", Tirana, Albania

Background & objectives: Senescence cells (SeC) were demonstrated in usual interstitial pneumonia/idiopathic pulmonary fibrosis (UIP/IPF), driving the fibrotic process. They upregulate LDH and Glut1 to keep with hypoxia. How senescent cell interfere with the immune system has not been studied.

Methods: Tissues from patients with UIP of different aetiologies and COPD were investigated. Sections were incubated with antibodies for phagocytosis activation markers tartrate-resistant acid phosphatase, Rab7, and LAMP1. The tissues were evaluated for CD47, an inhibitor of phagocytosis. SeC/p16+/p21+ were evaluated for the inflammatory mediators TNF·, iNOS, and VEGF. To elucidate the interaction of SeC with the immune system.

Results: Macrophages and lymphocytes were analysed using antibodies for CD4, CD8, CD20, CD68, CD206.Senescent cells are found in remodeling areas in UIP and COPD. They express positivity for p16 and p21, upregulate autophagy, and express phagocytosis markers.

They are negative for CD47. The cells secrete inflammatory molecules, and thus drive the repair process inducing proliferations of myofibroblasts, finally resulting in fibrosis.

Conclusion: Senescent cells secure their metabolic needs by phagocytosis, and to keep with hypoxia activate alternative metabolic pathways by LDH and GLUT1 upregulation, either for anaerobic glycolysis, or using the Warburg effect of glycolysis in hypoxia.

They also influence the polarization of macrophages, whereas the distribution of lymphocyte subtypes are not influenced.


Mature cystic teratoma of the lung: an exceptional disease

F. Zerd*, E. Vuhahula

*MUHAS, Tanzania

Background & objectives: Teratomas are tumours composed of tissues derived from more than one germ cell line. Intrathoracic teratomas are usually seen in the mediastinum; they rarely occur in the lung as intrapulmonary teratomas. The criteria for pulmonary origin are the exclusion of a gonadal site or other extragonadal primary sites and the exclusive origin of the tumour from the lung. This case aims to demonstrate the clinicopathological description of a teratoma mimicking a lung mass querying aspergilloma.

Methods: A retrospective review was conducted. The case was obtained from the Pathology archives at the Muhimbili National Hospital, Dar es Salaam, Tanzania. Age, gender, the clinical presentation was recorded. Histopathological diagnosis was confirmed by gross examination and hematoxylin and eosin sections, characteristics of mature cystic teratoma was seen.

Results: A 32-years-old woman presented with long standing cough for over 13 years and episodes of hemoptysis and chest pain, no weight loss. She has been treated with anti TB with no improvement. CT showed left sided upper heterogeneous mass with areas of calcification. A 7x8x6cm specimen was received at histopathology showed cysts and calcifications grossly, and microscopy revealed a tumour with mature tissue from all the 3 germ cell layers.

Conclusion: The diagnosis has to rely on the radiologic imaging, which demonstrates calcification, cavitations, and peripheral translucent areas and confirm by histopathology. Patients present with chest pain, cough, hemoptysis and trichoptysis. Surgical removal is the curative treatment of this condition.

PS-12 Autopsy Pathology


Incidental lung hamartomas

C. Amalinei*, L. Riscanu, T. Iov, C. Amalinei, A. Grigoraș

*"Grigore T. Popa" University of Medicine and Pharmacy, Institute of Legal Medicine, Romania

Background & objectives: Lung hamartomas or pulmonary chondromas or pulmonary chondromatous hamartomas are considered rare benign tumours. We report two cases of lung hamartomas incidentally discovered in the necroptic examination, in a man of 58 years and a woman of 61 years old.

Methods: Necroptic examination has been associated to collection of tissue specimens for microscopy. Paraffin-embedding, followed by routine hematoxylin-eosin (HE) and Masson’s trichrome stainings have been performed.

Results: One case revealed, in a bronchial wall, a circumscribed incidental lesion, of approximately 10 mm and another had a parenchymal nodule, of approximately 13 mm diameter. They consisted in mature, disordered hyaline cartilage, containing areas of ossification, in one case, along with variable amounts of adipose tissue, smooth muscle, and entrapped clefts of respiratory epithelium, corresponding to the features of lung hamartomas. No cellular atypia has been observed. Lung hamartomas may occur as a component of Carney triad, a rare non-hereditary condition with young women prevalence that also includes gastrointestinal stromal tumours and extra-adrenal paragangliomas, which has been excluded in our cases.

Conclusion: Despite their rarity, lung hamartomas should be considered in the differential diagnosis of lung cancers or benign tumours, such as lipoma, leiomyoma, myxoma, and fibroadenoma. They may be incidentally discovered during necropsy and microscopic examination may certify the diagnosis.


Developmental hepatic lesions in forensic pathology

C. Amalinei*, A. Grigoraș, L. Riscanu, C. Teodorescu, T. Iov

*"Grigore T. Popa" University of Medicine and Pharmacy, Institute of Legal Medicine, Romania

Background & objectives: Developmental hepatic lesions are attributed to abnormal growth of the biliary tree. The aim of our study is to evaluate the necroptic findings of developmental hepatic lesions from our files.

Methods: The reports from autopsies performed in the last 5 years in our Department, have been reviewed and 7 cases of developmental hepatic lesions, with age distribution between 50 to 73 years old, 4 male and 3 female, have been selected. These cases have been investigated by routine paraffin-embedding, followed by haematoxylin-eosin (HE) and Masson’s trichrome stainings.

Results: The microscopic exam revealed multiple large cysts associated with renal cysts, in 3 cases, diagnosed as polycystic liver disease, small or dilated bile ducts surrounded by fibrous stroma, characteristic features for incidental biliary hamartoma (“von Meyenburg complex”), in 2 cases, and incidental simple solitary hepatic cysts, in other 2 cases. Differential diagnoses included pseudocysts, infection pathology, neoplastic diseases, and traumatic-related lesions.

Conclusion: Developmental hepatic lesions are relatively rare, occur as simple cysts, biliary hamartomas, polycystic liver disease, as reported in our files, along with Caroli disease and ciliated hepatic foregut duplication cyst, reported in literature, their differential diagnosis being important in forensics.


Case report: post-mortem diagnosis of eosinophilic myocarditis

Z. Argyropoulou*, C. Santos, C. Gomes, R. Manso

*National Institute of Legal Medicine and Forensic Sciences of Portugal, Portugal

Background & objectives: Eosinophilic myocarditis is a form of myocardial inflammation, characterized by eosinophilic infiltration of the myocardium and often accompanied by eosinophilia. We present a case of a cardiorespiratory arrest with histologic evidence of eosinophilic infiltrate of the myocardium during the autopsy.

Methods: A medico-legal autopsy, toxicological laboratory exams and a histological diagnosis were conducted.

Results: A 48-year old, female patient with no relevant background history presented cardiorespiratory arrest, with previous complaints of interscapular pain. Laboratory findings revealed elevated levels of d-dimer, troponin I, reactive protein C and creatine kinase and no peripheral eosinophilia. Imagiological testing and coronary angiography, excluded aortic dissection, pulmonary embolism as well as, an acute coronary syndrome. Organ support was suspended because of the presented instability and an unknown reversible cause. Macroscopic appreciation, during autopsy revealed a slight heterogenic coloration of the myocardium, and no other apparent causes of death. The histology revealed interstitial haemorrhage of the heart and an eosinophilic infiltrate of the septal subendocardic myocardium.

Conclusion: The clinical presentation of Eosinophilic myocarditis varies from paucisymptomatic to acute fulminant myocarditis. It is often fatal with high in-hospital mortality, in particular when it presents as fulminant form. Eosinophilic myocarditis is probably under-recognized and frequently discovered on post-mortem examination. We present a case of EM discovered on post-mortem examination were no associated systemic disorder was identified, as such considered an idiopathic/undefined EM.


Correlation of digital and invasive post-mortem examinations

K. Brougham*, K. Manoharan

*St Helens and Knowsley NHS Trust, United Kingdom

Background & objectives: Invasive autopsy is the gold standard for determining cause of death (COD). Digital autopsies have been used in the Sefton Coroner’s jurisdiction since August 2018. We compared proposed digital COD with invasive COD and compared assessment of coronary artery disease.

Methods: All autopsies that had both digital and invasive examination at Whiston Hospital between August 2018 and January 2019 (6 month period) were included. Reports were interrogated for major and minor discrepancies along with coronary artery disease assessment. The data was compiled in Microsoft Excel and analysed in both Microsoft Excel and IBM SPSS.

Results: A total of 303 autopsies were digitally scanned of which 134 proceeded to invasive post-mortem examination. A total of 51 minor discrepancies were identified, which would not affected cause of death not were otherwise significant. There were 6 major discrepancies identified that would have altered cause of death. 57% had similar estimations of coronary artery disease. Only 64% of cases had no discrepancies between the radiological and invasive examinations.

Conclusion: Our cohort population is significantly different from those of previous studies. Often due to lack of clinical information there are many unanswered questions remaining after DA, so invasive PM is required. The use of digital autopsies has to be refined based on appropriate case selection since some COD cannot be detected or are likely to be missed on radiological examination.


Acute fatty liver disease of pregnancy: a difficult diagnosis in a case of maternal death

L. Chinezu*, H. Jung

*George Emil Palade University of Medicine, Pharmacy, Science and Technology of Targu Mures, Romania

Background & objectives: Acute fatty liver disease of pregnancy (AFLP) is a rare condition and an obstetric emergency with high rates of severe maternal morbidity and mortality. We present a case of a 29-years old female, who died shortly after the delivery.

Methods: She presented in emergency department with abdominal pain, nausea, and vomiting five days after her first delivery. She died shortly after due to liver failure. To establish the cause of death, a full autopsy was performed at the Institute of Forensic Medicine of Targu Mures, Romania.

Results: On autopsy examination, massive oedema of the lungs and fatty appearance of the liver were seen. At microscopy, the lungs exhibited acute extensive oedema and pulmonary capillary congestion. The liver presented microvesicular fatty infiltration of the hepatocytes visible only in the central and mid zonal parts of the lobule. The fat droplets surrounded centrally located nuclei with a foamy appearance of the cytoplasm. The fatty infiltration spared a thin rim of cells around the portal tracts.

Conclusion: The histological aspect of the liver was consistent with AFLP. The differential diagnosis with hemolysis, elevated liver enzymes and low platelets syndrome (HELLP) is difficult, since there are some clinical, biochemical and pathological overlapping. Some of the authors consider these entities as different stages of the same disease.


CMV pneumonia mimicking lung tumour - an autopsy case report

D. Crisan*, R. Ghica, C. Albu, C. Lazar, B. Pop

*Dept. of Pathology, “Iuliu Hatieganu” University of Medicine and Pharmacy” Cluj-Napoca, Dept. of Pathology, Emergency Clinical County Hospital Cluj-Napoca, Romania

Background & objectives: Viral infections may cause acute exacerbations in patients with usual interstitial pneumonia. Cytomegalovirus (CMV) pneumonia ranges from mild interstitial infiltrates to diffuse alveolar damage and can sometimes mimic a lung tumour on macroscopy and Hodgkin’s disease on microscopic examination.

Methods: We present a case of a 46-year-old woman, previously diagnosed with usual interstitial pneumonia (UIP), suffering progressive worsening of respiratory function, brought by ambulance into the emergency department. Chest CT scan showed ground glass appearance and multiple nodules in her lungs, suggestive of acute respiratory distress syndrome. The patient died several hours after admission.

Results: Autopsy showed multiple confluent firm tumour-like gray-white nodules in the middle and lower lobes of the right lung, encasing hilar structures, with yellow-gray areas of necrosis and associated pleural and pericardial retraction. Dark red areas of haemorrhage were also seen. Frozen section examination during autopsy raised suspicions of malignancy, but permanent sections showed no malignant cells. Pneumocytes were large and displayed a single basophilic intranuclear inclusion, surrounded by a clear halo, resembling Reed-Sternberg (RS) cells. We also found the honeycombing pattern characteristic of UIP and interstitial lymphocytic infiltrate, focal hyaline membranes, areas of hemorrhagic necrosis with central neutrophilic response and histiocytes within the airspaces.

Conclusion: On immunohistochemistry, the RS-like cells were pan-cytokeratin positive, TTF-1 and LCA negative and stained for anti-CMV antibody. CD15 was positive in some RS-like infected cells, but CD30 was negative.This case illustrates the diagnostic pitfalls in CMV pneumonia, mimicking a tumour, both macroscopically and histologically, and highlights the challenges pathologists face in autopsy examination.


Video documentation in autopsy practice

D. Dzhenkov*, G. Stoyanov, L. Petkova

*Medical University Varna, Bulgaria

Background & objectives: Video documentation devices for personal use have become widely available in the last decade, many of whom are able to record the point of view of the pathologist and serve as aids in protocol finalization and educational tools for students.

Methods: Five separate wearable devices for video documentation were used in the standard autopsy practice of a single pathologist – two sport cameras, a pair of camera glasses and two pairs of smart glasses – Cloud-I II and Google glass – XE V2. The five devices were compared for their individual pros and cons and feasibility in autopsy and educational practice.

Results: A total of 50 co