Oral Free Paper Sessions

OFP-01 | Oral Free Paper Session Digestive Diseases Pathology - GI


Gastric polyps in familial adenomatous polyposis (FAP) Portuguese patients - the first Western cohort with Asian features

D. Baptista*, M. Fernandes, F. Sousa, M. Garrido, L. Pinho, R. Morais, M. Baptista, F. Carneiro, I. Gullo

* Pathology Department, Centro Hospitalar Universitário de São João (CHUSJ), Portugal

Background & objectives: Chronic atrophic gastritis may contribute to the phenotype of gastric polyps in familial adenomatous polyposis (FAP). As the prevalence of Helicobacter-pylori infection in Portugal is up to 90%, we aim to characterize gastric polyps in a series of Portuguese patients.

Methods: Fifty-six FAP patients followed up at our hospital in High-Risk Consultation of Digestive Tumours, from 1992 to 2021 were retrospectively selected. Thirty-two patients were males (57.1%), and the medium age was 52 (range: 26-87). Clinico-pathological features, with particular emphasis on periodic upper endoscopic examinations, were studied. IBM SPSS (Release 27.0) was used for statistical analysis.

Results: Our series encompassed 95 gastric polyps, including 53 (55.8%) fundic gland polyps (FGPs) without dysplasia (n=34) or with dysplasia (n=19) and 42 (44.2%) intestinal-type adenomas. Half of FAP patients (n=28, 50.0%) developed endoscopically visible gastric polyps, including FGPs in 12 patients (21.4%) and adenomas with or without FGPs in 16 patients (28.6%). Foveolar-type adenomas and pyloric gland adenomas were not identified in this series. Intestinal-type adenomas occurred predominantly in the distal stomach (62.5%, p=0.031), were larger than 7mm in 9/16 cases (56.3%, p=0.03), and were more frequently associated with duodenal adenomas (87.5%, p<0.001). Chronic atrophic gastritis and intestinal metaplasia was observed in the background mucosa in most cases (75.0%, p=0.009).

Conclusion: To our knowledge, this is the first Western series showing high prevalence of intestinal-type adenomas in FAP patients, comparable to Asian studies. Chronic atrophic gastritis/intestinal metaplasia are likely responsible for this difference, with risk of neoplastic transformation and management implications. Endoscopists should have a high degree of suspicion in FAP patients and low threshold to biopsy/excision of gastric polyps, particularly in patients with chronic atrophic gastritis/intestinal metaplasia, in those with distal gastric polyps with worrisome features (namely >7mm) and/or duodenal adenomas.


Road-mapping for gastric intestinal metaplasia

E.K. Çagdas*, İ. Adanır, C. Ersöz, Ş. Ersöz, B. Savaş, A. Ensari

*Ankara University School of Med., Turkey

Background & objectives: Uneven distribution of gastric intestinal metaplasia (GIM) may require mapping biopsies rather than random biopsies which may fail to reflect the extent of the lesions. We, hereby, evaluated the value of mapping in determining the extent and severity of IM.

Methods: Random biopsies (RB) obtained according to updated Sydney protocol and mapping biopsies (MB) taken from 6 different sites of corpus and antrum during surveillance were evaluated for the severity and extent of histologic parameters of gastritis, mainly focusing on GIM with atrophy in a cohort of 202 patients. Statistical analysis was performed using Wilcoxon test on SPSS version 22.

Results: Mean age of 202 patients (104 females, 86 males) was 61.78±11,5 years. Average time interval between RB and MB was 18 months. There were 98 cases with isolated antral IM and 75 cases with isolated corpus IM in RB while MB revealed IM in the corpus in 13 of 98 cases (13.26%) and antral IM in 23 of 75 (30.66%) cases. IM at both sites was observed in 17 (8.9%) RB and 31 (16.4%) MB which yielded a significant increase by an increment of 1.84 (p<0.05). Severity of antral IM significantly increased in MB in 28% of cases (p<0.05) while no such difference was found for corpus IM.

Conclusion: The results of the present study suggest that mapping with multiple biopsies improves the detection rate of IM both in the corpus and antrum, providing information for the extent and severity of the lesions. This approach also seems to be useful in cases with isolated antral IM in monitoring severity of IM as already demonstrated by MB taken from the antrum in our study. However, large prospective, randomized, multicentre studies comparing different follow-up strategies are necessary for a better roadmap.


Myths and facts: reflux oesophagitis vs eosinophilic oesophagitis

E.K. Çagdas*, N. Aras, C. Ersöz, Ş. Ersöz, Z. Kuloglu, A. Kansu Tanca, B. Savaş, A. Ensari

*Ankara University School of Med., Turkey

Background & objectives: Basal cell hyperplasia and papillomatosis are non- discriminatory for eosinophilic oesophagitis (EoO) or reflux oesophagitis (RO). The aim here is to clarify the true incidence of these features in RO and EoO using morphometry to highlight their diagnostic significance.

Methods: A total of 543 RO and 59 EoO cases were re-evaluated for basal cell hyperplasia and papillomatosis using an ocular grid and basal cell thickness (BCT) and papillary height (PH) were measured on H&E stained sections of biopsies with the most severe histology. Data were evaluated using one sample t test on SPSS version 22. A p<0.05 was considered significant.

Results: EoO group (41 females, 17 males) had a significantly younger mean age of 15.25±13.9 (p<0.001) compared to RO (297 females, 246 males) with a mean age of 37.65±23.6. Papillomatosis was significantly more common in RO (85%) compared to EoO (55%) which showed basal cell hyperplasia (12.7% vs 100%) significantly more frequently. Morphometrically, BCT was 284 (25-250) and 153 (25-250) microns, PH was 209.75 (25-975) and 118.75 (0-325) in RO and EoO, respectively. The ratio of PH/epithelial thickness was significantly (p<0.05) higher (57%) in RO than EoO (39%). Although BCT was higher in RO the ratio was lower compared to EoO due to epithelial thickness which was much higher in RO.

Conclusion: Morphometric analysis allowed more accurate interpretation of papillomatosis and basal cell hyperplasia measured as BCT and PH with respect to epithelial thickness. Indeed, proportioning yielded better results in distinguishing RO from EoO. The results also showed that basal cell hyperplasia proved to be a constant feature of EoO besides eosinophilia. Papillomatosis on the other hand is less discriminatory as it can be present in both conditions, though more common in RO.


Clinical relevance of tumour response patterns in rectal cancer

S. Kus Ozturk*, C. Graham Martínez, K. Sheahan, D. Winter, S. Aherne, E. Ryan, C.J. van de Velde, C.A. Marijnen, G.A. Hospers, R.S. van der Post, I. Nagtegaal

*Radboud university medical centre, The Netherlands

Background & objectives: Tumour regression grading (TRG) and tumour downstaging are standard methods for examination of pathological response after neoadjuvant chemoradiotherapy (CRT). However, different response patterns can be observed. We investigate these response patterns and their clinical relevance in rectal cancer.

Methods: The study included a test and a validation cohort consisting of post-CRT rectal cancer patients with adenocarcinoma n.o.s and response to the therapy. TRG was established according to both CAP and Dworak. Response patterns were scored based on the three-step flowchart by two independent observers and correlated with pathological features and outcome.

Results: The test and validation cohorts included 236 and 103 patients respectively. In both cohorts, the predominant response pattern was fragmentation (74% vs 66%) and the interobserver agreement was excellent (k=0.85). The fragmented pattern presented with a significantly higher pathological stage (TNM III/IV: 31% vs 20%; p<0.001), less tumour regression (p=0.001), a tendency towards less downstaging (downstaging: 45% vs 65%; p=0.06). The shrinkage pattern presented better overall survival (OS) (p=0.049) in the test cohort and longer recurrence-free survival (RFS) (HR 2.85, 95% CI 1.09-7.49, p=0.033) in the validation cohort. In the regression analysis of combined cohorts, pathological stage and Dworak TRG were independent prognostic factors of survival.

Conclusion: The heterogeneous nature of tumour response following CRT is reflected in different response patterns, which can be scored reproducibly. Fragmentation and shrinkage are the main response patterns in rectal cancer with a predominancy of the fragmented pattern. This pattern is associated with deeper invasion and positive lymph nodes. While not independently associated with outcome, knowledge of response pattern can guide future treatment decisions, in particular concerning local treatment.


Consultation rate and pathologist diagnostic rate in 6,020 oesophageal biopsy specimens

R. Cheng, A. Naqvi, C. Finley, M. Bonert*

*Pathology, McMaster University, Canada

Background & objectives: Institution level data analyses can supplement case reviews for quality assessment. The objective of this project was to characterize the pathology in all institutional oesophageal biopsies and assess consultation rate and variation of the pathologist diagnostic rate (PDR).

Methods: All in house oesophageal biopsy specimens (EBS) accession 2011-2020 were extracted, diagnostically categorized with a hierarchical string-matching algorithm (HSMA) and tabulated by pathologist with documented (informal and/or formal) consultations. HSMA categorizations were audited by reviewing 200 EBS reports. PDR was compared using funnel plots with 95%(p<0.05) and 99.9%(p<0.001) confidence intervals (CI) centred on the group median diagnostic rate.

Results: The cohort contained 6,020 EBS and the HSMA was 99% (198/200) accurate. These were 524(8.7%) malignant/tumour, 34(0.6%) suspicious (SUSP), 72(1.2%) high-grade dysplasia (HGD), 78(1.3%) low-grade dysplasia (LGD), 65(1.1%) indefinite for dysplasia (IFD). The malignant/tumour were 339(5.6%) adenocarcinoma (ADN), 162(2.7%) squamous carcinoma(SCC), and 23(0.4%) other tumour(OTH). The consultation rates were 22.7%, 15.4%, 34.8%, 61.8%, 56.9%, 25.6%, 24.6% for ADN/SCC/OTH/SUSP/HGD/LGD/IFD respectively, and 5.6% (339/6,020) for all EBS. Thirteen pathologists interpreted >150 EBS each (158-626) and together saw 5,243. The number of 95%/99.9% CI outliers were 3/1, 2/0, 1/0, 4/2, 4/4, 1/0, 5/4 for ADN/SCC/OTH/SUSP/HGD/LGD/IFD respectively. Intestinal metaplasia (IM) (818), eosinophilic esophagitis (EE)(293) and gastroesophageal reflux (GERD)(897) had 9/4, 3/1 and 12/7 outliers respectively.

Conclusion: The funnel plots demonstrated moderate to poor PDR similarity on malignant, pre-malignant and benign diagnoses. Reviews are most frequent at the benign-malignant interface. The relative rarity, and PDR variation support the recommendation for reviewing all HGD. Concordance for benign diagnoses (with the exception of EE) likely can be improved significantly. Observational data can be useful for quality assessment and for helping guide quality improvement efforts.


Endoscopic biopsies in the diagnosis of colorectal carcinoma – is it quantity or quality?

K. Teo*, M. Elgoweini

*University Hospital Crosshouse, United Kingdom

Background & objectives: Endoscopic biopsy is required not only for definitive diagnosis of colorectal carcinoma (CRC), but also for additional molecular tests. Our aim was to evaluate the relationship between the number of biopsies taken and diagnostic accuracy in our hospital.

Methods: A search was performed on the departmental database for colonic biopsies obtained for suspected CRC between March and October 2021. Data was then retrieved from both endoscopy and pathology reports.

Results: In total 135 cases were identified and 68% of them had ≥6 biopsies taken. Initial biopsies were reported as invasive malignancy in 101(74.8%) and the remaining 34(29.4%) reported as suspicious of malignancy (19), dysplasia (12) and normal/other(3). Of the 101 cases with confirmed CRC, 73.7% had ≥6 biopsies and 26.3% had <6 biopsies. Of the 34 cases without a definite diagnosis, 55.9% had ≥6 biopsies. Repeat procedure was required for diagnostic purposes in 10 cases, 4 of which already had ≥6 initial biopsies taken. For molecular testing, estimated tumour content was provided in 57% of confirmed cases. Cases with <6 or ≥6 biopsies had comparable tumour content of 37% and 38% respectively.

Conclusion: In the majority of cases, the number of biopsies taken for suspected CRC at our hospital is in line with ESGE recommendations. Our findings suggest that the quality of targeted biopsies is also relevant, as a definite diagnosis was achieved in more than quarter of cases with less than 6 initial biopsies. Furthermore, more than half of initial unconfirmed cases met the recommended number of 6. In addition, the number of biopsies taken does not appear to affect tumour content.


Epithelial-mesenchymal transition and α-SMA expression in the tumour microenvironment: role in tumour stroma composition and association with prognosis in colorectal cancer

R. Souza da Silva*, F.F.N. Gomes, J.P. Andrade, L.E.d.M. Barbosa, D.S.G. Silva

*Universidade Federal da Paraíba, Brazil

Background & objectives: The epithelial-mesenchymal transition is characterized by increased expression of mesenchymal markers and plays an essential role in promoting tumour invasion and metastasis. We analysed the association between overall survival and α-SMA expression in colorectal cancer (CRC).

Methods: The TSR in colorectal adenocarcinoma was categorized into 2 groups: ≤50%- low stroma and >50%- high stroma. The immunohistochemical expression of α-SMA was observed in cancer cells (CCs) and cancer-associated fibroblasts (CAFs) present in different tumour areas: estimative stromal (ES), invasion front and desmoplastic stroma. α-SMa immunostaining >10% was defined as positive. The association between α-SMA expression and overall survival was evaluated.

Results: A total of 158 cases were analysed, with 54% of the tumours presenting stroma high. Positive immunostaining of α-SMA was detected, in CCs and CAFs, in 80.3% of cases. Stroma-high tumours showed increased expression of α-SMA. Cancer cells with expression of α-SMA were observed in 59.6% of cases. CAFs expressing α-SMA were found in 87.2% of the cases, higher positivity was observed predominantly in the ES and invasion front areas: 88.9% and 86.7% of cases, respectively. The positive immunostaining observed in CCs and CAFs present in the area of stromal estimation showed correspondence with death outcome. The high expression of α-SMA, in CAFs present in the invasion front and in the desmoplastic stroma, was associated with lower overall survival.

Conclusion: The α-SMA is a mesenchymal biomarker related to the epithelial-mesenchymal transition and is highly expressed in different tumour areas of the CRC, which are associated with a worse prognosis. Cancer cells and CAFs show high expression of α-SMA. The immunoexpression of this biomarker showed correspondence with the death outcome. In CRC, high expression of α-SMA in CAFs is associated with shorter overall survival.


Digital spatial profiling and proteomics identifies differences in biological phenotypes of tumour deposits and lymph node metastases in colorectal cancer

N. Brouwer*, L. Webbink, S. van Lent-van Vliet, P. Jansen, R. Geene, I. Nijman, F. Simmer, D. Tauriello, M. Vermeulen, I. Nagtegaal

*Department of Pathology, Radboud University Medical Centre, The Netherlands

Background & objectives: Both lymph node metastases (LNM) and tumour deposits (TD) are currently included in nodal staging of colorectal cancer (CRC). However, knowledge regarding the biological background of these biomarkers is lacking, which is essential in understanding their role in CRC spread.

Methods: Spatial profiling was performed on TD and LNM from 10 CRC patients using 1,800 RNA targets, and segmentation for tumour and tumour microenvironment (TME). From 10 other CRC patients, one TD and LNM were included for filter aided shotgun proteomics, identifying 5,578 differentially expressed proteins. Differences in RNA and protein expression were visualized using heatmaps, volcano plots, and enrichment analyses.

Results: Digital spatial profiling showed distinct transcriptome profiles between TD and LNM. The most significant results were found for the TME where TD showed a more tumour supportive environment (e.g., overexpression of SFRP2, COMP, THBS1, COL11A1, FN1) compared to LNM. Enrichment analyses showed enrichment of focal adhesion, proteoglycans in cancer, and ECM-receptor interaction in the TD (p<0.05), using the KEGG pathways, and the hallmark of epithelial mesenchymal transition (p<0.05), using the Molecular Signatures Databases (MSigDB). The proteomics analyses of 10 other CRC patients validated the transcriptome results from the digital spatial profiling, with largely overlapping expression profiles as well as similar enriched pathways.

Conclusion: This study shows that TD have a distinct and more invasive phenotype compared to LNM on both the RNA and protein level. The most pronounced differences were found in the TME, which was more pro-tumorigenic in TD. Furthermore, the hallmark of epithelial mesenchymal transition was enriched in TD compared to LNM. These results show that TD are biologically distinct from LNM and give insight into the heterogeneity of different modes of locoregional spread in CRC.


Extraappendiceal goblet cell carcinoid like amphicrine tumours of GI tract: a long known, not routinely used entity, associated with aggressive behaviour

Z.B. Erdem*, H.E. Pasaoglu, E. Yarikkaya, M. Cin, T.B. Özcan, H. Dincer, H. Bektas, N. Dursun Kepkep

*Basaksehir Cam and Sakura City Hospital, Turkey

Background & objectives: Amphicrine tumours are a rare group of tumours in which single tumour cells coexist both epithelial and neuroendocrine differentiation. This entity is included in the 5th WHO classification of appendiceal tumours, but it is not included in other organ classifications.

Methods: Total of 24 gastrointestinal (GI) carcinomas showing amphicrine features reported between January,2016 and February,2022 were retrieved from pathology files. Cases that didn’t express at least two neuroendocrine markers and whose appendix weren’t examined for the primary tumour presence (histopathologically or radiologically) were excluded from the study. Twenty non-amphicrine GI-carcinomas reported at the same time interval were randomly selected to compare tumour stages.

Results: Of the 24 patients 13 were female and 11 were male; mean age was 59 (22 to 72 years). Tumour localizations were as follows: 8 gastric-antrum, 2 gastric-cardia, 2 ampullary-duodenum, 10 ascending colon, 2 transvers colon. On microscopic examination of tumours the most prominent feature that attracted our attention was that majority of the cases (20 out of 24) formed at least focal crypt-like structures formed by signet ring-like cells in either myxoid(mostly) or desmoplastic stroma. Remaining 4 cases showed conventional intestinal carcinoma morphology with amphicrine immunophenotype. Another significant finding was the presence of multiple lymph node metastases and advanced stage disease in all of the cases, except for four cases.

Conclusion: Despite the fact that this entity is not included in the current WHO classification of GI organs other than appendix, they usually present in advanced stages and exhibit aggressive behaviour. In most, if not all, amphicrine tumours have characteristic morphologic clues (crypt-like structures formed by signet-ring-like cells) and clinical features(83% of the lower-GI cases on ascending colon). These clues can be defined clearly, and the terminology should be used in daily practice in order to assure an adequate therapy in a timely manner.


Mismatch repair protein and microsatellite instability status in gastric cancer: a comparative study between endoscopic biopsies and surgical specimens

I. Gullo*, D. Sousa Marques, J. Ricardo Silva, L. Mascarenhas-Lemos, X. Wen, C. Nascimento, A. Costa, A. Faria, C. Gouveia, L. Pinho, P. Patrícia, M. Cravo, F. Carneiro

*Department of Pathology, Centro Hospitalar Universitário de São João; Faculty of Medicine of University of Porto (FMUP); i3S - Instituto de Investigação e Inovação em Saúde da Universidade do Porto, Portugal

Background & objectives: Evaluation of mismatch repair protein (MMR) and microsatellite instability (MSI) status is of utmost importance for the management of gastric cancer (GC) patients. Aim: to assess if MMR/MSI-status in surgical specimens (SSs) may be predicted accurately in endoscopic biopsies (EBs).

Methods: One-hundred GC cases with EBs and respective SSs were selected retrospectively from two institutions between 2004 and 2015. Both EBs and SSs were evaluated for MMR-status by immunohistochemistry (IHC) and classified as MMR-proficient (MMRp) or deficient (MMRd). Cases were also classified for MSI-status by IdyllaTM and Bethesda panel (five mononucleotide markers) as microsatellite stable (MSS) or unstable (MSI-high).

Results: Sixty-three patients were submitted to surgery alone, while 37 patients underwent neoadjuvant chemotherapy. In SSs, 64/100 cases (64%) were MMRp and 36/100 (36%) were MMRd by IHC; 72/100 (72%) were MSS and 28/100 (28%) were MSI-high by IdyllaTM. In SSs, no cases classified as MMRp were MSI-high, but 21/100 cases (21%) were reported as MMRd by IHC and MSS by IdyllaTM. MSI-status, evaluated by IdyllaTM, was concordant to the gold-standard Bethesda panel in 64/65 SSs (98.5%). When comparing EBs and SSs, only 5/100 cases (5%) were discordant by IHC (k=0.889%, sensitivity=88.9%, specificity=98.4%), while 14/100 cases (14%) were discordant by IdyllaTM (k=0.620, sensitivity=60.1%, specificity=95.8%).

Conclusion: High concordance rate was found when comparing EBs and SSs for MMR-status by IHC, suggesting that we can rely on the immunohistochemical evaluation of EBs when assessing GC cases before surgery/neoadjuvant chemotherapy. The few discordant cases (n=5) may be explained by insufficient tumour sampling to account for GC heterogeneity (4/5 had ≤2 EBs), and/or neoadjuvant chemotherapy (used in 3/5 patients). Although IdyllaTM reliably evaluated MSI-status in SSs, as compared to gold-standard, it doesn’t seem to accurately identify MSI-status in EBs.

Funding: Bolsas de Investigação Luz Saúde, Portugal


Which is which? Autoimmune ( H. pylori ) gastritis

N. Aras*, E.K. Çagdas, S. Kurt, B. Savas, A. Ensari

*Ankara University Department of Pathology, Turkey

Background & objectives: Pyloric metaplasia(PM) and neuroendocrine-cell hyperplasia(NCH) are common features of autoimmune gastritis(AIG), but are also seen in HP-gastritis leading to diagnostic difficulty. Co-ocurrence of two complicates their distinction. We aimed to evaluate features useful in the differential diagnosis when overlaps occur.

Methods: Total of 123 cases of gastritis comprising 77 AIG cases(group1) with normal/reactive antrum, 30 cases of HP-gastritis with concurrent atrophy and metaplasia in the corpus and antrum(group 2), and 16 cases with AIG-like changes in the corpus, and atrophy and/or IM in the antrum(group 3) were reevaluated for updated Sydney parameters, PM and NCH. Chi-square test was used for statistics.

Results: Though all cases in group1 and 3 had inflamed corpus, severe chronic inflammation was significantly (p=0.03) higher in group 1(71.4%) compared to group3(37%) while activity of group3(43.7%) was significantly higher (p<0.001) than group1(25,8%). Atrophy was significantly (p=0.026) higher in group2(86.6%) than group3 (50%) similar to IM (100% and 93,75 in groups 2 and 3, %, respectively). NCH was present in 80,5% of all cases and was significantly more common (p<0.001) in group3(100%) than group2(23.3%). PM, on the other hand, was seen in %77 of all cases and its frequency was significantly higher(p=0.002) in group1 (%85.7) compared to groups 2 and 3 (56.6%, 75%, respectively).

Conclusion: Inflammation, both active and chronic, not differing between HP-gastritis with(group3) or without(group2) features of AIG, suggest that overlaps do indeed exist. As expected, IM and atrophy were more severe in HP-gastritis compared to HP-gastritis with features of AIG in contrast to NCH and PM which were more predominantly observed in AIG or HP-gastritis with such features. These, taken together, support the idea of incorporating PM and/or NCH into the Sydney system of gastritis for accurate diagnosis, treatment of overlapping cases.


Systemic neutrophil-to-lymphocyte ratio in colorectal cancer: differences in systemic cytokine profile and immune effector cell populations

D. Bhattacharjee*, M. Cummings, N. Orsi, M. Cullen, S. Richards, R. Prasad, D. Jayne, P. Quirke, J. Pine

*Pathology and Data Analytics, Leeds Institute of Medical Research, School of Medicine, University of Leeds, United Kingdom

Background & objectives: A raised pre-operative neutrophil-to-lymphocyte ratio (NLR) is correlated with poorer outcomes in colorectal cancer (CRC). We assessed the plasma cytokine profiles and immune effector cell populations of NLR<5 and NLR>5 groups in operable CRC to establish a mechanism for this.

Methods: Patients undergoing elective bowel resection for CRC were prospectively recruited. Preoperative plasma from 47 patients underwent cytokine analysis by multiplex fluid-phase immunoassay. A second group of 33 patients was recruited for flow cytometric evaluation of full blood count for immune cell population assessment.

Results: The NLR>/=5 group showed significant depression of some T -cell subgroups, activated natural killer cells and eosinophils and significant elevation of memory B cells and neutrophils. Significant depression of the cytokines IL-2, IL-1β, IL-7, IL-13, basic FGF, IFN-γ and MIP-1α occurred in the NLR>/=5 group

Conclusion: The down regulation of IFN-γ, IL-2 and IL7, involved in immunoregulation in the NLR>5 group suggests the failure of a coherent, T-cell driven, Th1-polarised response to cancer. The pan-T cell lymphocytopaenia seen in the NLR>5 group demonstrates an inappropriate T-cell driven immune response to tumour. The increased neutrophilia seen in the NLR≥5 group may relate to tumour microenvironmental factors that make them pro-tumourigenic.

Funding: Mr Pine was supported by a grant from Leeds Cares. Dr Cummings was supported by a Wellbeing of Women project grant. Dr Orsi is supported by an NIHR clinical lectureship. Professor Quirke is supported by programme grants from Yorkshire Cancer Research (YCR) L386 and by an NIHR Senior Investigator award.


An innovative artificial intelligence based application for the differentiation of invasive adenocarcinoma from pseudoinvasion in colorectal polyps

L. Chen*, Z. Yang, B. Wang, D. Driman, C. Ling, Q. Zhang

*Department of Laboratory Medicine and Molecular Diagnostics, Sunnybrook Health Sciences Centre, University of Toronto, Canada

Background & objectives: Dysplastic glands misplaced in the submucosa (pseudoinvasion) of colorectal polyps sometimes cannot be differentiated from invasive adenocarcinoma, even by expert pathologists. We are developing an innovative application to differentiate invasive adenocarcinoma from pseudoinvasion on whole-slide images (WSI).

Methods: Under low power (x2) magnification, we trained the algorithm to identify areas of interest (dysplastic glands) on WSI. Using our images and NCT-CRC-HE-100K datasets, we developed a combined non-patch and patch-based algorithm to identify 12 morphological categories in differentiating pseudoinvasion from invasive adenocarcinoma (true invasion). We used two models to aggregate the per-patch/area classification results into a final classification.

Results: Slides from 130 colon polyps (70 pseudoinvasion and 60 true invasion) were digitized. Low power detection successfully identified all areas of interest. The convolutional neural network (CNN) model achieved an overall accuracy of 98% in recognizing and classifying each area/patch into categories. Based on the 9 categories currently completed, the linear model and the 3-layer CNN model show accuracy rates of 83% and 88% respectively in the final classification (true versus pseudoinvasion).

Conclusion: Our AI based application mimics how pathologists work and has achieved reliable results with 9 of 12 categories used. We are confident that with the other 3 categories added into the algorithm, reliability will be increased. The algorithm will be validated using another 60 cases and diagnostic accuracy will be compared with expert GI pathologists blinded to the results. The application will be available on our website for pathologists to access worldwide (http://ai4path.ca/).

Funding: Ontario Molecular Pathology Research Network CPTRG Pathology Image Analysis Fund


MMR-deficient crypts detection by immunohistochemistry in normal colonic mucosa of patients with MMR deficient (dMMR)/MicroSatellite Instable (MSI) colorectal cancer: a helpful tool for the diagnosis of Lynch syndrome

M. Svrcek*, S. Breton, I. Sourrouille, N. Basset, E. Guillerm, I. Brocheriou, D. Enea, P. Bourgoin, A. Duval, T. Andre, M. Muleris, F. Coulet

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

Background & objectives: The diagnosis of Lynch syndrome (LS) is challenging. We investigated whether the detection of MMR-deficient non-neoplastic intestinal crypts could be helpful in patients with inconclusive germline mutation studies of MMR genes (Lynch-like syndrome; LLS) or variants of uncertain significance (VUS).

Methods: We evaluated the expression of MMR proteins in non-neoplastic mucosa of colorectal cancers from patients with LS (n=15), including 7 with multiple CRCs (mRCCs), LLS (n=7) and VUS (n=7). Ten immunohistochemistry (IHC) slides were performed on 1 or 2 blocks of both adjacent (adj-muc) and distant (dist-muc) mucosa. A crypt ratio (number of dMMR crypts/total number of crypts) was determined.

Results: dMMR crypts were identified in 12/15 patients with LS (80%). dMMR crypts tended to be more frequently observed in dist-muc [11/15 (73%) vs 8/14 (57%), p=0.157], and in mRCC [7/7 (100%) vs 5/8 (63%), p=0.244]. The ratio of dMMR crypts was significantly higher in patients with mCRC [mCRC-ratio = 0.00337 vs non-mCRC-ratio = 0.0005 (p=0.003)]. A minimum number of n=8 IHC slides analysed in adj-muc and n=6 slides in dist-muc identified all patients with dMMR crypts and 66.7% and 91.7% respectively of all patients with LS. dMMR crypts were identified in 1/7 patients with LLS (14%) and in 4/7 patients with VUS (57%).

Conclusion: The detection of dMMR crypts, with a minimum number of 6 IHC slides in dist-muc, could be an integral part of the decision-making algorithm for the diagnosis of LS in patients with LLS or VUS.


Tumour budding is an independent prognostic factor in stage III colon cancer patients: a post-hoc analysis of the IDEA-France phase III trial (PRODIGE-GERCOR)

M. Svrcek*, D. Basile, C. Broudin, J. Emile, A. Falcoz, F. Pagès, L. Mineur, J. Bennouna, C. Louvet, P. Artru, S. Fratte, F. Ghiringhelli, T. Andre, V. Derangère, D. Vernerey, J. Taieb

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

Background & objectives: Tumour budding (Bd) is an emerging prognostic biomarker in colon cancer (CC). We explored further the significance of Bd for risk stratification by evaluating survival of stage III CC patients included in the IDEA-France phase III trial.

Methods: Bd was assessed on scanned HE-stained slides and scored by central review by the Bd criteria of the 2016 International Tumour Budding Consensus Conference (ITBCC2016) and classified as Bd1 (0-4 buds/0.785 mm2), Bd2 (5-9 buds), and Bd3 (≥10 buds) categories. Disease-free survival (DFS) and overall survival (OS) were analysed by log-rank test. Clinicopathologic features and Immunoscore® were correlated with Bd.

Results: Samples of 1048 CC patients were analysed. Overall, Bd1, Bd2, and Bd3 were observed in 39%, 28%, and 33% of CC, respectively. Bd2 and Bd3 were associated with vascular (P = .002) and perineural invasions (P =.0009). The 3-year DFS and the 5-year OS rates for Bd (1 versus 2-3) was of 79.4% versus 67.2% (P=.001) and 89.2% versus 80.8% (P=.001), respectively. This was confirmed after adjustment for relevant clinicopathological features for DFS (HR, 1.41; 95% CI, 1.12 to 1.77; P =.003) and OS (HR, 1.65; 95% CI 1.22 to 2.22; P = .001). When combined with pTN stage and Immunoscore subgroups, Bd significantly improved disease prognostication.

Conclusion: Bd demonstrated its independent prognostic value for DFS and OS. Given these findings, Bd per the ITBCC 2016 should be mandatory in every pathology report in stage III CC patients. Bd and Immunoscore could play a complementary role in personalized healthcare in this setting.

Funding: We thank the GERCOR team, the PRODIGE investigators and the National Cancer Institute (INCa) for funding the trial.

OFP-02 | Joint Oral Free Paper Session Gynaecological Pathology / Cytopathology


Consensus based recommendations for the diagnosis of Serous Tubal Intraepithelial Carcinoma; an international Delphi study

J. Bogaerts*, M. van Bommel, M. Steenbeek, R. Hermens, J. de Hullu, J. van der Laak, M. Simons

*Radboud University Medical Centre, The Netherlands

Background & objectives: Diagnosis of Serous Tubal Intraepithelial Carcinoma (STIC), a precursor lesion to high-grade serous carcinoma, has moderate reproducibility. We aim to inventory criteria for STIC diagnosis among gynaecopathologists and formulate consensus based recommendations.

Methods: We invited 70 gynaecopathologists to a 3 round Delphi study. The first round consisted of open ended questions concerning their diagnostic process. The answers of round 1 were used to formulate 64 statements. In the subsequent rounds, participants were asked to rate their level of agreement with these statements, using a 9-point Likert-scale, ranging from fully disagree to fully agree.

Results: Gynaecopathologists participating in this study (n=34, 49%) scored 64 statements, subdivided in topics: tissue handling, morphological criteria, immunohistochemical staining and reporting recommendations. Consensus was reached for 27/64 (42%) statements, such as: each fallopian tube has to have the fimbriated end fully embedded; nuclear pleomorphism, nuclear enlargement, high nuclear to cytoplasmatic ratio and nuclear hyperchromasia are morphological criteria that need to be present for diagnosing STIC; P53 and Ki67 staining only have to be performed in case a STIC is considered based on morphology; WT1, CyclinE, STMN1 and p16 have no added value in diagnosing STIC.

Conclusion: We describe current practices concerning STIC diagnostics among 34 gynaecopathologists and present a list of 27 recommendations based on consensus vote. Consistent and reproducible STIC diagnostics is important, as it holds prognostic value for individual patients. Moreover, it is a prerequisite to safely offer alternative risk reducing surgical interventions to women who are at an increased risk of ovarian carcinoma within the protection of a clinical trial. The recommendations from this study contribute to further standardization of the diagnostic process.

Funding: Funded by the Dutch Cancer Society (KWF Kankerbestrijding)


Endocervical endometrioid adenocarcinoma: clinicopathologic characterisation of a rare human papillomavirus-independent tumour type

A. Hodgson*, S. Stolnicu, C. Mateoiu, T. Kiyokawa, A. Felix, H. Trihia, L. De Brot, G. Karpathiou, R. Soslow, C. Parra-Herran, W.G. McCluggage, K.J. Park

*Toronto General Hospital, Canada

Background & objectives: Endocervical endometrioid adenocarcinoma (EEA) is a rare human papillomavirus (HPV)-independent tumour type included in the 2020 World Health Organization Classification of cervical adenocarcinoma. Due to its apparent rarity, this entity has been poorly characterised in the literature to date.

Methods: EEAs that fulfilled the following strict criteria were collected from multiple institutions: presence of confirmatory endometrioid features; surgically treated; exclusion of endometrial or ovarian origin; negative high risk HPV by in situ hybridization or polymerase chain reaction. Demographic, pathologic and follow up information, if available, was recorded for each case.

Results: There were 14 patients with a median age of 56 years (range 30-74 years). Endometriosis and areas of mesonephric(-like) differentiation were seen each in 2 cases (one tumour had both). 4/14 tumours demonstrated evident squamous differentiation/metaplasia. The median tumour size was 3.6 cm (4 tumours 2-3.9 cm and 6 tumours ≥ 4 cm, range 0.8 to 8.6 cm). All were at least stage IB1 (FIGO 2018). Initial nodal involvement was seen in 2/13 patients and recurrence (14 months, liver; 60 months, paraaortic nodes) in 2/11 patients (follow up median 46 months, range 2-192 months). Oestrogen receptor expression was at least focal in 13/14 tumours while all showed non-diffuse p16 expression.

Conclusion: This endeavour represents the largest reported series of EEA. Diligent sampling, comprehensive microscopic examination and ancillary studies for detection of HPV are essential to establish the correct diagnosis. Additional studies are needed to determine the molecular pathogenesis and optimal management for these neoplasms and to compare their clinicopathologic behaviour with other endocervical adenocarcinoma types.


QPOLE; a rapid, simple and cheap approach for POLE assessment in endometrial cancer by multiplex qPCR

A. van den Heerik*, N. Ter Haar, L. Vermij, J. Jobsen, M. Brinkhuis, S. Roothaan, A. Leon-Castillo, G. Ørtoft, E. Hogdall, C. Hogdall, T. van Wezel, L. Lutgens, E. van der Steen-Banasik, J.N. McAlpine, C. Creutzberg, V. Smit, B. Gilks, N. Horeweg, T. Bosse

*Radiation Oncology, Leiden University Medical Center, The Netherlands

Background & objectives: Detection of pathogenic POLE-mutations in endometrial cancer is of prognostic and therapeutic importance. Currently, POLE-status is determined by DNA-sequencing, which is time-consuming, not widely available and expensive. We validated a rapid, low-cost quantitative polymerase-chain-reaction (qPCR) assay for pathogenic POLE-mutations; QPOLE.

Methods: Primer and fluorescence-labelled 5’-nuclease probe-sequences of pathogenic POLE-mutations within the POLE exonuclease domain were designed. Two multiplex mixes, QPOLE-frequent for the most occurring mutations (P286R, V411L, A456P, S459F) and QPOLE-rare for the rare variants (M295R, F367S, D368Y, L424I, P436R, M444K) were developed using DNA extracted from formalin-fixed paraffin-embedded tumour tissues from our extensive EC tumour tissue repository.

Results: Cut offs for POLE-wild type, -mutant and failed results were predefined based on 50 POLE-wild types and 7 POLE-mutant cases. For cases with values in the equivocal range between wild type and mutant, additional DNA-sequencing (i.e. Next Generation Sequencing (NGS)) is recommended. In our testing set of 227 cases (71 POLE-mutated, 156 POLE-wild type), 20 samples (8.8%) fell in the equivocal range (10 POLE-mutant, 10 POLE-wildtype). Sensitivity and specificity for the combined QPOLE assays were 93.4% (95%CI 90.1-96.7%) and 100%. QPOLE combined with NGS for equivocal cases yields a final sensitivity of 94.4% (95%CI 91.6-97.2%), while maintaining a 100% specificity.

Conclusion: With this qPCR assay, QPOLE, we have developed a simple, faster, reliable and sensitive alternative for targeted NGS of all pathogenic somatic variants in the exonuclease domain of the POLE-gene. The simplicity of the design enables assessment of POLE-status within 4 hours, and will make universal low-cost POLE-testing available for all EC patients. An interlaboratory validation study to determine the assay’s practical feasibility is ongoing; results are available at the time of the conference.


Molecular characterisation of endometrial carcinoma and prognostic risk group classification

M.E. Brizzi*, Á. López-Janeiro, V. Heredia Soto, A. Berjón, L. Yébenes, A. Peláez-García, M. Mendiola, D. Hardisson

*Department of Pathology, Hospital Universitario La Paz, Spain

Background & objectives: Molecular classification of endometrial carcinoma (EC) has been recently incorporated into prognostic risk classification of endometrial carcinoma and should be integrated into conventional pathologic diagnosis.

Methods: We included EC cases diagnosed in our Department between September 2019 and March 2022. Clinicopathological features were retrieved from the electronic medical records of the patients. Molecular classification was performed by the TCGA surrogate testing immunohistochemical markers (p53, MLH1, PMS2, MSH2 and MSH6) and somatic mutation analysis of POLE. Prognostic risk group stratification was established according to ESGO-ESTRO-ESP 2021 guidelines.

Results: We found 92 patients with EC, 80 of which underwent surgery. Most cases were low-grade early stage endometrioid carcinomas (85%). According to the molecular classification, 25% EC showed mismatch repair deficiency (MMRd), 15% were p53abn (all high-grade, mostly at advanced stages), 6.5% showed a pathogenic POLE mutation, and 50% were of no specific molecular profile (NSMP). Three cases displayed more than one alteration (“multiple classifiers”). According to clinicopathological features, 38 cases were low-risk, 12 intermediate, 9 intermediate-high, 18 high-risk, and 2 advanced metastatic. After integrating molecular classification, a risk-group shift occurred in 3 patients (one p53abn from low to intermediate risk, and 2 POLEmut from high-intermediate to low-risk).

Conclusion: There is good correlation between molecular classification and histological subtypes: all p53abn tumours were high-grade carcinomas (43% presented at advanced stage), whereas POLEmut cases were mostly low-grade endometrioid carcinomas. Inclusion of this surrogate marker approach to the molecular-based classification is prognostically informative in low-, intermediate-, and high-risk endometrial carcinoma. Changes in prognostic risk group classification occur mainly in p53abn and POLEmut cases. According to current guidelines, molecular classification is encouraged in all endometrial carcinomas, especially high-grade tumours.


Comparison of tumour infiltrating lymphocytes between endometrial carcinoma primary tumours and matched metastases

D. Martins*, E. Sustatxa, M. Pires, L. Azevedo, C. Jerónimo, C. Bartosch

*Pathology Department , Centro Hospitalar São João, Porto; Medical Faculty, University of Porto, Portugal

Background & objectives: Tumour infiltrating lymphocytes (TILs) in endometrial carcinoma can stimulate anti-tumour immune response and have been shown to impact survival. In some tumour models decreased TILs in metastases was shown, but in metastatic/recurrent endometrial carcinoma limited data is available.

Methods: We studied a cohort of 39 primary endometrial endometrioid carcinomas and matched metastases, diagnosed between 2000 and 2013, at two Portuguese tertiary centers. Patient’s clinical files and histological slides were retrospectively reviewed. The number of TILs per square millimeters was automatically determine using QuPath in 6 (primary) and 3 (metastasis) random fields for each case.

Results: The number of TILs in metastases had a significant, but weak, correlation with the number of TILs in primary tumours (rs=0.40, p=0.01). Metastases had a median of 238 TILs/mm2 (range:4,1871), with no difference between distant versus regional metastases, nor between lymph-node versus other sites (peritoneum, lung, bone). Primary tumours had a median of 262 TILs/mm2 (range:0,1013). Twenty metastases showed a decrease in the number of TILS compared to matched primary tumours, and 18 showed an increase. Variations in TILs numbers do not correlate with metastases site, grade or mitotic count. Even though not statistically significant, patients with higher number of TILs in metastasis had a better overall survival (HR:0.43, p=0.11).

Conclusion: The immune microenvironment in endometrial carcinoma metastases seems to be variable, either with an increase or decrease in TILs compared to primary tumours. Further studies with larger series are needed to confirm our findings and determine the factors underlying this variation.


DNA methylation analysis of endometrial carcinomas identifies molecularly and clinically distinct subgroups

J. Li1, F. Kommoss1, A. Lum, D. Chiu, V. Yuan, S. Leung, D.G. Huntsman, J.N. McAlpine, A. von Deimling2, B. Tessier-Cloutier*

*Memorial Sloan Kettering Cancer Center, USA

Background & objectives: DNA methylation analysis is an emerging approach with great potential to further our understanding of oncogenic mechanisms and assisting with tumour classification. In this study we set to characterize a group of endometrial carcinomas using their methylation profile.

Methods: We collected a series of molecularly profiled (using ProMisE) and clinically annotated endometrial carcinomas. Methylation analysis was done using the Illumina Infinium EPIC (850k) BeadChip with subsequent unsupervised clustering of the differentially methylated regions. Statistical analyses including two-tailed T-Tests, Chi-Squared tests, and Kaplan Meier survival analysis were done across various clinical and molecular variables.

Results: Our cohort (n=50) included 10 POLE mutated (POLEmut), 10 MMR-deficient (MMRd), 10 p53 abnormal (p53abn), and 20 with no specific molecular profile (NSMP). The unsupervised clustering analysis identified two major methylation groups (A and B), each with two subgroups (A1/2 and B1/2). Clusters A and B differed significantly in terms of molecular subtypes (p<0.001): cluster A only included NSMP and p53abn cases, while cluster B had high representation of MMR-deficient and POLEmut tumours. Deep myometrial invasion (stage IB), lower BMI, and beta-catenin expression were associated with cluster B (p=0.040, 0.009, and 0.037 respectively). Survival analysis of sub-cluster B2 showed significantly better overall survival compared to the others three subgroups (p=0.040).

Conclusion: Endometrial carcinoma is a complex disease with a spectrum of different genomic and epigenetic alteration profiles. Our findings support that methylation analysis has the potential to predict molecular and clinical variables. Further analysis of the impact of specific differentially methylated regions on clinicopathologic variables is ongoing.

1 Both authors contributed equally 2 Co-senior authors


Integrated clinicopathological and molecular analysis of endometrial carcinoma: prognostic impact of the new ESGO-ESTRO-ESP endometrial cancer risk classification

A. De Leo*, A.G. Corradini, F. Rosini, J. Lenzi, M. Grillini, M. Ruscelli, M. Errani, T. Maloberti, S. Coluccelli, G. Tallini, D. Santini, C. Ceccarelli, D. de Biase

*Pathology Unit, University of Bologna Medical Center, Italy

Background & objectives: The ESGO/ESTRO/ESP committee has been recently proposed a new risk stratification system for endometrial cancer (EC) patients incorporating both clinicopathological and molecular characteristics. The study aims to compare the ESGO/ESTRO/ESP risk classification system with the previous 2016 risk classification.

Methods: The cohort included 187 consecutive patients with endometrial carcinoma. Immunohistochemistry (IHC) and Next-Generation Sequencing (NGS) were used to assign TCGA molecular EC subgroups: POLE mutant (POLE), mismatch repair deficient (MMRd), p53 mutant (p53abn), and no specific molecular profile (NSMP).

Results: TCGA class assignment of EC cohort: 7% POLE group, 31% MMRd group, 23.5% p53abn group, 38.5% NSMP group. In the 2020 risk classification system, 39.1% of patients were allocated to low risk compared with 22.6% in the 2016 risk classification system, mainly due to reclassification of patients previously classified especially as high-intermediate risk. The recent 2020 guidelines revealed a total of 61 patients (32,6%) with a change in risk group in relation to the 2016 classification system: the shift was due to p53abn, POLE alterations and lymph vascular invasion. The application of the 2020 risk stratification system shows Kaplan-Meier curves with a more significant difference between the groups throughout survival.

Conclusion: In our cohort, the application of the new 2020 risk classification integrating clinicopathological and molecular parameters provided a more accurate identification of low-risk and high-risk patients, potentially allowing a more specific selection for post-operative adjuvant therapy. Integrated molecular classification is a promising tool for a better therapeutic management of patients.


CCND1 amplification and cyclin D1 overexpression correlate with adverse outcome in vulvar squamous cell carcinoma

N. Carreras-Dieguez, C. Manzotti, M. del Pino, I. Trias, I. Ribera-Cortada, S. Diaz-Mercedes, L. Marimon, N. Vega, M.T. Rodrigo, J. Ordi, N. Rakislova*

*Hospital Clínic Barcelona, Spain

Background & objectives: There is scarce evidence regarding the role of CCND1 amplification and protein overexpression in vulvar squamous cell carcinomas (VSCC). We aimed to 1) correlate cyclin D1 DNA amplification with protein overexpression and 2) correlate cyclin D1 overexpression with clinical prognosis.

Methods: Whole exome sequencing and cyclin D1 immunohistochemistry (IHC) were performed in 65 VSCC. Copy number alterations were predicted for the tumour-control pairs using ControlfreeC. The prognostic significance of cyclin D1 IHC overexpression and its correlation with clinical-pathological features was further assessed in a subset of 90 VSCC. Cyclin D1 was considered overexpressed when >50% of the tumour cells stained positive.

Results: A total of 18/65 (27.6%) VSCC showed amplifications in CCND1 and 26/65 (40.0%) showed strong cyclin D1 overexpression. A strong positive correlation between CCND1 amplification and cyclin D1 overexpression was found (p<0.001). Both CCND1 gains and cyclin D1 overexpression strongly correlated with worse disease-free survival (p<0.001 for each). Only cyclin D1 IHC overexpression had significant association with worse disease-specific survival (p<0.01).

In the cohort of 90 VSCC, tumours overexpressing cyclin D1 showed worse disease-specific and disease-free survival than cyclin D1-negative tumours (p<0.001 for each). Cyclin D1 overexpression correlated also with HPV-negative status (p<0.001), presence of p53 abnormalities (p<0.001) and advanced FIGO stage [III-IV vs. I-II] (p=0.02).

Conclusion: Our findings indicate that CCND1 is amplified and overexpressed in a significant proportion of VSCC, mostly in HPV-independent tumours. Cyclin D1 overexpression is strongly associated with adverse patient outcome (worse disease-specific and disease-free survival) and could be a useful tool for prognostic stratification. Innovative targeted therapies could be explored based on these findings.

Funding: Project “PI20/00368; Caracterización genómica de los carcinomas de vulva independientes de virus del papiloma humano y de sus precursores”, funded by Instituto de Salud Carlos III and co-funded by the European Union (ERDF) “A way to make Europe”. ISGlobal receives support from the Spanish Ministry of Science and Innovation through the “Centro de Excelencia Severo Ochoa 2019-2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program.


Worse disease-free survival for patients with vulvar squamous cell carcinoma arising on HSIL-like or VAAD/DEVIL lesions

N. Rakislova*, N. Carreras-Dieguez, C. Pumarola, M. del Pino, C. Manzotti, A. Saco, I. Trias, I. Ribera-Cortada, M.T. Rodrigo, S. Diaz-Mercedes, R. López del Campo, L. Marimon, J. Ordi

*Hospital Clínic Barcelona, Spain

Background & objectives: A number of new precursors of Human Papillomavirus (HPV)-independent vulvar squamous cell carcinomas (VSCC) have been recently defined: vulvar acanthosis with altered differentiation (VAAD), differentiated exophytic intraepithelial lesions (DEVIL) and high-grade squamous intraepithelial (HSIL)-like lesions.

Methods: We assessed whether patients with HPV-independent VSCC had any clinico-pathological differences depending on the associated adjacent precursor. The study comprised 157 patients with HPV-independent VSCC surgically treated between 1975 and 2021. The skin adjacent to the tumour was histologically reviewed. The median follow-up was 69 months. Correlations with outcome were analysed using multivariable Cox regression and log-rank analysis.

Results: 50 patients (31.8%) showed differentiated VIN (dVIN), 49 patients (31.2%) showed adjacent inflammatory dermatosis, 25 (15.9%) had HSIL-like lesions, 9 (5.7%) showed VAAD or DEVIL and in 24 women (15.3%) no adjacent skin lesion was identified. Patients with HSIL-like and VAAD/DEVIL lesions showed impaired disease-free survival (higher rates of relapse and/or persistence of disease) compared with other groups (p<0.001). In addition, despite no statistically significant, the tumour size of VSCC associated to HSIL-like lesions was smaller than of other groups (p=0.17). No differences in overall or disease-specific survival were identified among the six groups (p=0.15). None of the women with VAAD or DEVIL died of disease.

Conclusion: Our findings indicate higher risk of relapse or persistence of disease in patients with VSCC arising on HSIL-like or VAAD/DEVIL lesions. Closer surveillance after surgical resection of VSCC may be indicated for these cases.

Funding: Project “PI20/00368; Caracterización genómica de los carcinomas de vulva independientes de virus del papiloma humano y de sus precursores”, funded by Instituto de Salud Carlos III and co-funded by the European Union (ERDF) “A way to make Europe”. ISGlobal receives support from the Spanish Ministry of Science and Innovation through the “Centro de Excelencia Severo Ochoa 2019-2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program.


Reproducibility of low-volume lymph node metastasis assessment in endometrial cancer

M. Shahi*, D. Sadeghian, A. Yilmaz Altay, P. Biagio, M. Ghioni, G. Mehmet, D. Fanni, J. Bennett, E. Unti, J. Cheville, A. Larish

*Mayo Clinic, Rochester, MN, USA

Background & objectives: Lymph node (LN) assessment is critical for staging of endometrial cancer (EC). Low volume metastasis (LVM), upstages patients, but early data suggests

some of these patients can be spared adjuvant treatment. So, accurate pathologic determination of LVM is critical.

Methods: Whole slide images (WSI) (3 H&E levels, 40 μm apart and 1 CK AE1/AE3 slide) of 11 pelvic sentinel lymph nodes (SLN) involved by LVM were reviewed by 9 pathologists from multiple institutions. Various parameters were assessed: number of foci, size of the largest focus, clusters versus scattered cell, site of involvement in the LN and extra-nodal extension.

Results: An international consortium of 9 pathologists were participated in this study. 9 out of 11 cases, ≥8 pathologists had complete agreement determining the type of involvement (MM vs ITC). In the remaining 2 cases, there was an even split in the designation of the involvement type. These two cases showed multiple scattered tumour foci with dispersed cells, making the accurate measurement and determination of the involvement type challenging. The largest focus measurements showed some disagreement in both micro-metastasis and ITC cases, with slightly higher variability in the ITC sub-group. Agreement in #foci/plane, pattern, and site of involvement were high.

Conclusion: Our study indicates an excellent interobserver agreement for the separation of ITCs and MM amongst an international group of gynaecologic pathologists using WSI. Problematic cases arise when tumour foci extend along a distance within a lymph node. Additional studies are needed to determine the impact of these findings and to formulate recommendations to address.


Evaluation of the implementation and diagnostic accuracy of the Paris classification for reporting urinary cytology in voided urine specimens: a cyto-histological correlation study in a high-volume cancer centre

J. Lobo*, C. Lobo, L. Leça, Â. Rodrigues, R. Henrique, P. Monteiro

*IPO Porto, Portugal

Background & objectives: The Paris classification was introduced for reporting urinary cytology, highlighting the need to focus on accurately identifying high-grade urothelial carcinoma (HGUC), and aiming to improve criteria, terminology and ultimately improving patient management.

Methods: To assess the overall implementation and diagnostic performance of the Paris classification for reporting urinary cytology. All urinary cytology reports from July 2018-December 2019 were collected (n=1240). Only voided urine samples were included (n=1180), of which 9.9% had histological confirmation. Risk of malignancy (ROM) was calculated. Diagnostic performance of urinary cytology was assessed, including sensitivity, specificity, PPV, NPV and accuracy.

Results: The median age of the study population was 69 years, and 71.2% were male. The Paris system categories were widely used (in 99.7% of reports). The distribution of categories was: 0.3% unsatisfactory, 90.5% negative for HGUC, 5.6% atypical urothelial cells (AUC), 1.6% suspicious for HGUC, 1.9% HGUC and 0.1% other malignancies. No diagnosis of low-grade urothelial neoplasia was given. ROM was 21.4% for negative for HGUC, 66.7% for AUC, 91.7% for suspicious for HGUC and 100% for HGUC. When using suspicious for HGUC as a cut-off, the diagnostic performance of urinary cytology in identifying HGUC was 46% sensitivity, 98% specificity, 96% PPV, 68% NPV and 74% accuracy.

Conclusion: Specificity of urinary cytology is very high. ROM for each category was in accordance with literature, except for AUC where ROM was slightly higher (66.7%). Sensitivity of voided urine specimens is known to be lower than that of instrumented specimens, explaining the lower sensitivity in our study. Study population characteristics (high-volume cancer centre with many patients treated with intra-vesical therapies) may explain part of our results. This pilot study motivated an inter-observer variability study among three Cytopathologists, which is ongoing.


Application of a standardised terminology and nomenclature for respiratory cytology: experience from a large tertiary respiratory cancer centre

D. O'Connor*, A. Fabre, M. Mc Nally, D. Gibbons

*St Vincent's University Hospital, Ireland

Background & objectives: International cytopathology coding systems have a valuable role in facilitating report standardisation. A new international respiratory cytopathology coding system is currently being developed. Our institution has been coding all respiratory cytology specimens in a similar manner for over 10 years.

Methods: Specimens are coded as non-diagnostic (C1), benign (C2), atypical, favour reactive (C3), suspicious for malignancy (C4) or malignant (C5).

We calculated rates of diagnostic categories on our cohort over a two-year period by evaluating all endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA), bronchial washing, bronchial brushing, bronchial lavage and sputum specimens performed at our institution using the laboratory information system.

Results: We assessed all aforementioned respiratory cytological specimens received at our institution in the years 2020 and 2021. In total, 1433 cytological specimens fulfilled the inclusion criteria and were analysed. Of these, 15.8 % (n=226) were coded as malignant (C5) and 1.47% (n=21) were coded as suspicious for malignancy (C4).

The calculated rates of diagnostic categories, bases on cytological findings, were as follows:

1. EBUS-TBNA (n=415): C1=8.7%, C2=54.7%, C3=2.4%, C4=1.4%, C5=32.8%.

2. Bronchial brushings (n=96): C1=1.0%, C2=49.0%, C3=8.3%, C4=3.1%, C5=38.5%.

3. Bronchial washings (n=486): C1=3.1%, C2=75.7%, C3=8.4%, C4=2.3%, C5=10.5%.

4. Bronchial lavages (n=429): C1=1.9%, C2=97.0%, C3=0.5%, C4=0.2%, C5=0.5%.

5. Sputum (n=7): C1=14.3%, C2=85.7%, C3=0.0%, C4=0.0%, C5=0.0%.

Conclusion: Coding systems are widely utilised in other areas of cytopathology, such as in thyroid and salivary gland specimens. The consistency and standardisation of reporting achieved from such systems provides greater clarity for the treating physician and facilitates clear communication of essential information.

We have been using a classification system analogous to the proposed new respiratory cytology classification system for approximately 10 years. This data provides indicative rates of cytological outcomes in a tertiary referral lung oncology centre.


A retrospective report-based review of 4,155 consecutive patients undergoing minimally invasive thoracic lymph node sampling

M. Bonert*, S. Sayeda, H. Begum, J. Agzarian, J. Cutz, C. Finley, A. Naqvi

*Pathology, McMaster University, Canada

Background & objectives: Endoscopic bronchial ultrasound (EBUS) and endoscopic ultrasound (EUS) are minimally invasive strategies to assess thoracic lymph nodes. This project examined the pathology of patients from a regional thoracic surgery centre that underwent EBUS/EUS and (if applicable) lung cancer resection.

Methods: All EBUS/EUS specimens accessioned 2011-2020 were retrieved and linked via anonymized patient identifier to lung cancer resections with synoptic reports. Cases were automatically classified and grouped into mutually exclusive categories (malignant(MAL), suspicious(SUSP), insufficient(INSUF), benign(BEN)), based on the most recent specimens using a previously validated program. Data was stratified by location (station(ST)7, ST4R, ST4L, all other N2 ST(STN2others), all N1 ST(STN1all)).

Results: The cohort contained 4,155 patients with 10,922 EBUS/EUS specimens. Patients had 1 to 13 specimens and median number of specimens per patient was three. Patients with station MAL/number of patients(% MAL) was: ST7 750/3,407(22%), ST4R 816/2,844(29%), ST4L 293/1,504(20%), STN2others 283/668(42%), STN1all 381/1,264(30%). Overall, N2 MAL status/number of patients(%) was 1,399/3,992(35%) and multiple N2 stations/STN2others were involved in 604 of 1,399(43%) patients. 829(20%) patients went for lung cancer resection; 47 had two surgeries and two had three surgeries. Nodal stage by most recent surgical specimen was 20 (patients) pNX, 563 pN0, 175 pN1 and 71 pN2. 469 of 4,155 patients (11%) had granulomatous inflammation in 838 EBUS/EUS specimens.

Conclusion: EBUS/EUS is used for lung cancer staging, sarcoidosis and other indications. Patients with N2 positivity frequently have multiple positive stations. The low pN2 rate on resection in our environment confirms EBUS/EUS is effective at selecting lung cancer patients for surgery. The assessment of pathologic categorizations can provide insights in thoracic surgery and tumour biology.


The Paris System for reporting urinary cytology: 5 years of institutional experience in a tertiary hospital

M. Pinho*, R. Moiteiro da Cruz, J. Boavida, R. Luís

*Department of Pathology - Hospital de Santa Maria, CHULN; Faculdade de Medicina da Universidade de Lisboa, Portugal

Background & objectives: The Paris System for Reporting Urinary Cytology (TPS) standardizes the triage criteria for high-grade urothelial carcinoma (HGUC) in urine specimens. Our aim was to assess the TPS accuracy through cytohistological correlation, stratified by both cytological category and histological grade.

Methods: The reports of urinary tract histological diagnoses and pertaining retrospective cytological specimens, examined in 2016-2021, were retrieved from our Department software database; non-satisfactory/non-diagnostic samples were unaccounted for. The TPS categories were divided into negative and positive tiers (suspicious and HGUC combined); the risk of malignancy (ROM) and performance parameters were then calculated, following histological correlation.

Results: A total of 1261 cytological and 638 histological samples were assessed. Each biopsy/surgical diagnosis correlated with an average of 2 urinary (voided/instrumented) specimens (ranging from 1 to 12), sampled either simultaneously or up to 52 months beforehand. The ROM ranged from 17.4% in negative samples to 71.5% in positive categories; the performance analysis revealed a sensitivity of 46.7%, a specificity of 91.8%, a positive predictive value of 78.4% and a negative predictive value of 72.7%. Atypical urothelial cells were independently analysed and revealed mixed results.

Conclusion: We present a heterogeneous series, encompassing the learning curve following TPS implementation and clustering both “de novo” diagnoses and urothelial neoplasm follow-ups. The large volume of these samples impacts its diagnostic yield and hence a low sensitivity was expected, contrasting with a high specificity that translates the atypia of even scarce shed tumour cells. On the other hand, although pathologists are not blinded to clinical information and previous diagnosis, histological false negatives due to undersampling must not be overlooked.


Fine needle aspiration biopsy of orbital masses: a review of 36 cases

H. Domanski*, J. Köster

*Department of Genetics and Pathology, Division of Laboratory Medicine Region Skåne, Lund, Sweden

Background & objectives: Fine needle aspiration biopsy (FNA) is a useful tool to triage orbital lesions in cases where a diagnosis cannot be made by clinical and imaging findings alone. FNAB can provide crucial information before a surgical and oncologic procedure is undertaken.

Methods: Over a period of 5 years, 2017-2021, 36 patients underwent FNAB of orbital masses. Twenty-nine lesions were sampled by FNAB using a transcutaneous or transconjunctival approach and 7 patients underwent CT guided FNAB with the assistance of a cytopathologist. Thirteen patients had a prior history of malignancies. Flow cytometry was performed in 16 cases and immunocytochemical examination in one.

Results: Diagnostic material was obtained in 28 lesions. Malignant haematolymphoid lesions represented most of the malignancies: 6 primary and 4 recurrences of non-Hodgkins lymphomas and acute myeloid leukaemia. There were 8 metastatic malignancies: 3 breast, 1 lung, 1 adrenal and 2 neuroendocrine carcinomas, and 1 metastasis of glioblastoma. One patient with history of pleomorphic adenoma in the orbit was diagnosed with carcinoma ex pleomorphic adenoma. In 2 patients FNAB disclosed primary salivary duct carcinoma and recurrence of basal cell carcinoma respectively. The remaining cases included 3 cases of lymphoid hyperplasia, 3 cases of inflammatory pseudotumour and 1 haemangioma. With exception for minor hematomas in some patients, no severe complications were observed.

Conclusion: FNAB may be considered a useful technique in the diagnostic approach to orbital masses according to strictly defined indications. This technique allows a confident diagnosis with a low risk of complications and is particularly useful in the evaluation of malignant, unresectable and retrobulbar orbital lesions.

OFP-03 | Oral Free Paper Session Digestive Diseases Pathology - Liver/Pancreas


Histopathological tumour response scoring in resected pancreatic cancer following neoadjuvant therapy (ISGPP-1): an international interobserver study

B. Janssen*, S. van Roessel, S. van Dieren, O. de Boer, M. Besselink, J. Verheij, H. Wang, C. Verbeke, A. Farina, for the International Study Group of Pancreatic Pathologists (ISGPP)

*Amsterdam UMC, The Netherlands

Background & objectives: This study investigated whether gastrointestinal/pancreatic pathologists can reliably identify neoadjuvant treatment effects in pancreatic tumours based on histomorphology. Moreover, it aimed to determine the interobserver agreement for current (internationally used) tumour response scoring (TRS) systems.

Methods: Overall, 23 gastrointestinal/pancreatic pathologists reviewed whole H&E-slides of neoadjuvantly treated or treatment-naive resection specimens of pancreatic cancer. The accuracy in identifying treatment effect was investigated in 60 patients (30 treatment-naïve, 30 after NAT). Interobserver agreement for the College of American Pathologists (CAP) and MD Anderson Cancer Center (MDACC) TRS systems was assessed in 50 patients using intraclass correlation coefficients (ICC).

Results: The sensitivity and specificity for identifying NAT effect were 76.2% and 49.0%, respectively. The histological features: reduced cancer cell density, mucin pools, and cell degeneration were most frequently stated to allow distinction between treated and treatment-naïve cases. In 50 patients after NAT, the ICC values for both TRS systems were ‘moderate’; 0.66 CAP and 0.71 MDACC. The ICC values of <0.50, ≥0.50 and <0.75, ≥0.75 and <0.90, and >0.90 indicate poor, moderate, good, and excellent reliability, respectively. None of the cases scored as a complete response by at least one pathologist had 100% concordance.

Conclusion: Identification of the effect of NAT in resected pancreatic cancer proved unreliable. The interobserver agreement for the current TRS systems was suboptimal. These findings support the recently published ISGPP recommendations to score residual tumour burden rather than tumour regression following NAT, which will require a new TRS system.


Two step- progression of non-functioning pancreatic neuroendocrine tumours (PanNET)

I. Marinoni*, A. Di Domenico, P. Kirchner, K. Bräutigam, R. Maire, C. Thirlwell, C. Kim-Fuchs, A. Perren

*Institute of Pathology, Switzerland

Background & objectives: PanNETs with mutations in ATRX, DAXX and MEN1 (ADM) originate from α-cells. Alpha-like PanNETs, small and indolent, progress into intermediate (ADM), larger and with high relapse risk. We dissected epigenetic changes and activation of related pathways, occurring during such progression.

Methods: We combined DNA methylation profiles of 155 and RNA sequencing of 45 PanNET samples. DNA methylation analysis was performed using the ChAMP pipeline. Consensus clustering was performed following the ConsensusClusterPlus (v1.54.0) pipeline. Spearman correlation between expression values and correspondent beta or M values of methylation was used to identify genes which changes in both DNA methylation and expression.

Results: Combining RNAseq and DNAme data, we delineated three groups of PanNET originating from alpha cells: alpha-like PanNET, intermediate-ADM and intermediate-ADM3. We compared DNA methylation and transcriptome profiles of the three groups. We found that alpha-like PanNETs develop first into intermediate-ADM and then into intermediated-ADM3 following a two steps progression. Notably, alpha-like tumours develop into intermediate-ADM upon DAXX/ATRX mutations and changes in DNA methylation mainly at heterochromatin regions increasing Chromosomal Instability. Interestingly, only genes regulating cell proliferation were found differently expressed between alpha-like and intermediate-ADM tumours. Intermediate-ADM develop into intermediated-ADM3 acquiring changes in DNA methylation at regulatory regions, resulting in an altered expression of genes involved in cell differentiation and metabolism.

Conclusion: We described pathways of progression dependent on DAXX and ATRX and subsequent epigenetic changes, characterized by increased cellular dedifferentiation and metabolic adaptation. Additionally, our study confirmed the high relevance of DNA methylation in stratifying and classifying PanNETs with different molecular and clinical characteristics.


Mutational profile of hepatocellular carcinomas with microvascular invasion and microscopic portal vein invasion. Implications for tumour progression and recurrence

S. Chillotti*, T. Maloberti, D. de Biase, G. Gerinario, M. Cescon, M. Ravaioli, A. D'Errico, F. Vasuri

*Department of Pathology, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Italy

Background & objectives: The aims of the present study are to confirm the prognostic role of microscopic portal vein invasion (MPVI) in resected hepatocellular carcinoma (HCC), and to correlate MPVI with the mutational status to create risk categories based on NGS data.

Methods: Out of 400 retrospective resected HCCs we selected all cases with a diagnosis of microvascular invasion (MVI). Then we reviewed the histopathology, subclassifying each case according to the presence of MVI vs MPVI. Survival data for each patient was then obtained. We then performed NGS analysis with a custom panel on a perspective cohort of recent resected HCCs.

Results: Kaplan-Mayer survival analysis was performed on the retrospective cohort which showed a statistically significant better Overall Survival (OS) for HCCs without MPVI (p=0.007). NGS analysis found that TERT(65%) TP53(26%), and CTNNB1(22%) were the most frequently observed mutations. NGS results and MVI were studied using a Chi-Square test and found that TP53-mut and MPVI were positively correlated (p=0.039), while TERT-promoter mutation correlates with the presence of any MVI (p=0.038). Although MPVI and CTNNB1-mut correlation was not significant (p=0.076), none of the 5 CTNNB1-mut cases had MPVI. TP53-mut HCCs were characterized by a higher histological grade (p=0.005) and a solid/macrotrabecular architecture (p=0.017).

Conclusion: Our retrospective data confirmed the literature on resected HCCs, showing a worse OS in patients exhibiting MPVI vs MVI. The perspective phase of our study is still in progress, but our preliminary data on NGS results suggest a progression from TERT-mutated HCCs, which develop MVI, to TP53 mutation, which can predict MPVI, besides a more aggressive behaviour.


Acinar cystic transformation of the pancreas: clinical and molecular analysis for unravelling its heterogeneous nature

P. Mattiolo*, O. Basturk, A. Mafficini, O. Kerem, R.T. Lawlor, S. Hong, L.A. Brosens, V. Adsay, A. Scarpa, C. Luchini

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

Background & objectives: Acinar cystic transformation (ACT) of the pancreas is a poorly understood and rare entity among pancreatic cystic lesions. This study aims at clarifying their real nature.

Methods: The study cohort includes 25 pancreatic ACT, representing the largest series of ACT in the literature. Here we provide their clinicopathological characterization along with molecular profiling by next-generation sequencing (NGS).

Results: ACT were more common in female patients, frequently in body-tail region. At follow-up, all patients were alive and free of disease. Histologically, all cysts were lined by typical acinar epithelium, sometimes intermingled with columnar or ductal-like epithelium. Cell atypia, necrosis, mitoses, and invasive carcinoma were absent. Three cases showed a patchy distribution, and two cases were associated to ductuloinsular complexes with centroacinar microcysts. Two ACT showed peculiar histologic features: one showed a distinctive microcystic pattern, and another harboured foci of low-grade dysplasia in the areas lined by ductal-like epithelium. NGS detected the presence of two pathogenic / likely-pathogenic mutations in two different cases: KRAS, c.34G>C, p.G12R, and SMO, c.1685G>A, p.R562Q.

Conclusion: Overall considered, our findings indicate that ACT is as a heterogeneous entity. It seems to encompass lesions with different possible pathogenesis, which includes the evolution from a centroacinar microcyst, and malformative, obstructive, or neoplastic origins. The potential presence of driver mutations call for a careful management of ACT patients, taking into account also surgical resection and active imaging surveillance / life-long follow-up.


Spatially resolved transcriptomic and proteomic analysis of pancreatic cancer reveals distinct profiles which correlate with site of recurrence

A.S. Wenning*, B. Gloor, P. Aeschbacher, A. Perren, E. Karamitopoulou-Diamantis

*Department of Visceral Surgery, University Hospital Bern, Switzerland

Background & objectives: Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal neoplasm. The majority of patients with localized disease will eventually develop tumour recurrence after complete surgical resection. Here we investigate tumour- and immune cell determinants of PDAC recurrence.

Methods: PDACs (n=284) were classified according to recurrence site as liver (n=93/33%), lung (n=49/17%), local (n=31/11%), peritoneal recurrences (n=38/13%) and no-recurrence (n=73/26%). Four regions of interest per tumour were selected. Spatial compartments were identified with fluorescent imaging followed by transcriptomic (pancytokeratin+tumour cell compartment) and proteomic (CD45+leukocyte compartment) analysis for immune pathway associated targets by using a digital spatial profiling platform.

Results: Median overall-survival for the PDAC-groups was 16months (liver), 27months (lung), 26months (local), 12.5months (peritoneal) and 64months (no-recurrence). The tumour cell-compartment of 60% of the non-recurrent PDACs, as well as 45%, 40% and 31% of the lung, local and peritoneal recurrences respectively, showed significant upregulation of pathways involved in T cell activation, immune cell adhesion/migration, antigen presentation and cytokine signalling, as compared to only 20% of liver recurrences. CD66b (granulocyte-marker) and STING (mediator of immunosuppressive microenvironment and feature of the basal-like subtype) were strongly up-regulated in the liver leukocyte-compartment. The non-recurrent leukocyte-compartment revealed significant up-regulation of CD3, CD4, CD8, CD20, GZMB, HLA-DR, checkpoint molecules and beta-2-microglobulin compared to all recurrent tumours.

Conclusion: We found distinct spatial and microenvironmental profiles on gene and protein level in each recurrence group, which differed from each other as well as from the no-recurrence group, underlining the heterogeneity of PDAC. Tumours with liver recurrence display poor prognosis associated with unfavourable immune signalling and features of the basal-like molecular subtype.


A comparative analysis of CPA1, BCL10 and chymotrypsin for the distinction of pancreatic acinar cell carcinomas

R. Uhlig, S. Minner, A. Luebke, S. Weidemann, P. Lebok, N. Gorbokon, G. Sauter, F. Jacobsen, T.S. Clauditz*

*University Medical Center Hamburg, Germany

Background & objectives: Pancreatic acinar cell carcinoma (PACC) is a rare tumour of the pancreas with an intermediate prognosis as compared to pancreatic neuroendocrine tumours (PNE) and pancreatic ductal adenocarcinoma (PDAC) from which it may be difficult to distinguish by morphology alone.

Methods: To study was the efficiency of immunohistochemical markers, 18 PACCs, 531 PDACs, 64 PNEs, 117 extrapancreatic neuroendocrine neoplasms (EPNN), 826 colorectal carcinomas (CRC) and 252 gastric carcinomas (GC) were analysed with antibodies for CPA1 (MSVA-601M), bcl10 (Santa Cruz sc5273), and chymotrypsin (Biorad 2100-0657) in a tissue microarray format.

Results: CPA1 was positive in 18 of 18 (100%) of PACCs, 0 of 49 (0%) of PNEs, 0 of 88 (0%) of EPNNs, 10 of 404 (2.5%) of CRCs, and 0 of 178 (0%) of GCs. Chymotrypsin was positive in 16 (87,5%) PACCs, 1 (2%) PNEs, 2 (2.3%) EPNNs, 10 (2.5%) CRCs, and 1 (0.6%) GCs. Bcl10 was positive in 18 (100%) PACCs, 2 (4.1%) PNEs, 5 (1%) EPNNs, 109 (27%) CRCs, and 18 (10%) GCs. These data resulted in a sensitivity and specificity of 100%/99.2% for CPA1, 100%/88.4% for bcl10, and 94.4%/98.6% for chymotrypsin.

Conclusion: CPA1 and chymotrypsin are both highly specific and sensitive for ACC while bcl10 is sensitive but has markedly lower specificity. Because all “false positive” cases identified by CPA1 were CRCs that only showed a positive staining in goblet cells and an identical staining pattern was observed in all these cases for chymotrypsin and bcl10, a pancreatic origin of the mucus in these goblet cells is concluded.


Targeted next-generation sequencing of endoscopic ultrasound-guided through-the-needle-biopsies from pancreatic cystic lesions

C. Rift*, L.C. Melchior, B. Kovacevic, P. Klausen, A. Toxværd, H. Grossjohann, J. Karstensen, L. Brink, H. Hassan, E. Kalaitzakis, J. Storkholm, D. Scheie, C.P. Hansen, E. Lund, P. Vilmann, J.P. Hasselby

*Department of Pathology Rigshospitalet, Denmark

Background & objectives: Endoscopic sampling of pancreatic cystic lesions (PCLs) with through-the-needle-biopsies (TTNBs) has been introduced in clinical management. The aim of this study was to evaluate the feasibility and diagnostic accuracy of additional next-generation sequencing (NGS) of TTNBs as a diagnostic tool.

Methods: We prospectively included patients with PCLs > 15 mm in cross-section for endoscopic ultrasound and TTNB-sampling. The TTNBs were microscopically evaluated and classified according to the WHO classification, using up to 12 slides from each TTNB with a 3 μm thickness. Secondly, additional 10 slides of each TTNB were analysed by NGS using a 51 gene customized hotspot panel.

Results: We included 101 patients of which 95 had sufficient tissue for microscopic evaluation. The TTNBs were evaluated according to the WHO classification for pancreatic neoplasms including appropriate immunohistochemical staining. Subsequently, 91 patients had available tissue for NGS. We identified genetic aberrations in 49 patients, mostly alterations in KRAS and GNAS, diagnostic for intraductal papillary mucinous neoplasm (IPMN). Sensitivity and specificity of the NGS analysis were calculated with TTNB histology as the gold standard. A sensitivity and specificity of 83.7 % (70.3-92.7 %) and 81.8 % (48.2-97.7 %), respectively, were demonstrated for a mucinous cyst diagnosis, and 87.2 % (74.2-95.2 %) and 84.6 % (54.5-98.1 %), respectively, for an IPMN diagnosis.

Conclusion: We have demonstrated that TTNBs can be used for both microscopic evaluation, immunohistochemical classification, and additional targeted NGS in the majority of patients in a large prospective cohort. TTNBs offer the possibility of assessment of an intact piece of the cyst wall with correlation to genetic aberrations. NGS has high sensitivity and specificity for the diagnosis of mucinous cysts including IPMNs. We propose TTNBs as a potential alternative to cyst fluid cytology in the diagnostic management of patients with PCLs.

Funding: The study received funding from Rigshospitalets Research Foundation, The Novo Nordisk Foundation, The Danish Cancer Society, The Danish Cancer Research Foundation, Neye Fonden, Holms Mindelegat, Harboefonden, Agnete Løvgreens legat, Carl & Ellen Hertz legat, Direktør Jakob Madsen og Hustru Olga Madsens fond, Frimodt Heineke fonden, Torben & Alice Frimodts fond, Else og Mogens Wedell – Wedellsborgs fond, Fabrikant Ejnar Willumsens Mindelegat, Aase & Ejnar Danielsens fond, AP Møllers fond, and Anita & Tage Therkelsens Fond.


EUS FNA vs. EUS FNB of pancreatic lesions: a comparative study

T. Pasupati Meenakshi*, A. Narayanan, D. Shalini

*Gribbles Pathology M Sdn Bhd, Malaysia

Background & objectives: Endoscopic ultrasound (EUS)-guided fine-needle biopsy (FNB) is replacing EUSFNA in the diagnosis and management of pancreatic lesions in Malaysia. The aim is to document and compare the diagnostic precision, accuracy and IHC studies of EUS-FNB and EUSFNA of pancreatic lesions.

Methods: This is a cross sectional, comparative study of EUS FNA and EUS FNB samples of pancreas received in Gribbles Pathology in the year 2021, with clinical, CT scan and endoscopy correlation. A total number of 341 EUS FNA and 68 EUS FNB samples of pancreatic lesions were documented for cytological and histological evaluation, respectively, along with IHC analysis.

Results: A wide range of pancreatic lesions, mostly primary adenocarcinomas, along with a small number of neuroendocrine tumours were documented. Spindle cell tumours, mucinous cystic neoplasms, serous papillary lesions, lymphomas, and cases of chronic pancreatitis were also seen. Metastatic lesions from the lung and kidney were also noted. Correlation with clinical picture, tumour markers and CT scan findings were undertaken in all cases. EUS FNA cytology had shown fruitful results in majority of the cases and cell block with good yield helped in IHC outcome. However, EUS FNB was found to be easier, advantageous, precise, and had an additional advantage of more material for IHC and further molecular studies.

Conclusion: EUSFNB is emerging as the most safe, precise and reliable diagnostic procedure replacing the conventional EUSFNA and cell block of pancreatic lesions in Malaysia. The precision of the procedure and accuracy of the results of EUSFNB samples have considerably increased in the year 2021. This initial, first-time comparative study carried out in Gribbles Pathology, Malaysia is to document the increasing and accurate diagnostic precision of EUSFNB in the management of pancreatic lesions.


Expression and prognostic significance of HMGA2 in pancreatic ductal adenocarcinoma and ampullary adenocarcinoma

D. Oflas*, F. Canaz, L. Demir, İ. Özer, E. Çolak

*Eskisehir Osmangazi University, Turkey

Background & objectives: High mobility group protein 2 (HMGA2) is a structural transcriptional protein involved in tumourigenesis, and epithelial-mesenchymal transition (EMT). We evaluated the expression and clinical prognostic value of HMGA2 in pancreatic ductal adenocarcinoma (PDAC), and ampullary adenocarcinoma (AAC).

Methods: HMGA2 expression was immunohistochemically assessed in normal pancreatic tissue (n=57), chronic pancreatitis (n=86), low-grade PanIN (n=80), high-grade PanIN (n=30), PDAC (n=57) and AAC (n=30). Immunochemical staining of EMT markers (E-cadherin and vimentin) were applied in PDAC and AAC. The relationship between HMGA2 expression and clinicopathological characteristics in the PDAC, AAC, and the neoplasia cohort (n=87) including these groups was evaluated.

Results: HMGA2 expression was not observed in normal pancreatic tissue, chronic pancreatitis, and low-grade PanIN. High expression was detected in high-grade PanIN and PDAC (P<0.001). Between HMGA2 expression and age, gender, tumour location, size, differentiation, lymphovascular invasion, perineural invasion, pT, and pN status in the PDAC, AAC, and the neoplasia cohort significant relationship was not found (P>0.05). A significant correlation was observed between loss of E-cadherin expression and vimentin positivity and HMGA2 expression (P<0.05). HMGA2 expression increased the risk of disease-related death and decreased overall survival in AAC and the neoplasia cohort (P=0.002, P=0.016, respectively). HMGA2 was not related to overall survival and risk of death in PDAC (P>0.05).

Conclusion: Our results suggest that HMGA2 is an effective immunohistochemical marker in detecting benign and malignant lesions in the pancreas, as well as a potential new prognostic marker and therapeutic target in periampullary tumours, especially AAC. The statistically significant correlation between HMGA2 protein expression, loss of E-cadherin expression, and vimentin positivity support the role of HMGA2 in EMT.

Funding: This study was supported by Eskişehir Osmangazi University Scientific Research Projects Coordination Unit Commission within the scope of project numbered 202111D02.


Detection of perineural invasion in pancreatic adenocarcinoma using artificial intelligence

S. Borsekofsky*, R. Hagege, D. Hershkovitz

*Pathology Department of Tel Aviv Sourasky Medical Center, Israel

Background & objectives: Perineural invasion (PNI) refers to invasion of cancerous cells around/in nerve. PNI is associated with a worse prognosis in pancreatic ductal adenocarcinoma (PDAC) with therapeutic target potential. We aimed to build an algorithm to identify PNI in PDAC more reliably/efficiently.

Methods: Training the algorithm involved manual segmentation of nerve and tumour using 260 slide images from 6 scanned PDAC cases. Analytical validation used 168 additional images. Clinical validation had the algorithm applied to 59 cases of previously diagnosed PDAC, presenting images of areas tumour and nerve were in closest proximity. A pathologist then determined presence or absence of PNI per case.

Results: In the analytical validation, the algorithm showed sensitivity of 86%, 55% and specificity of 78% and 83% for the detection of nerve and tumour, respectively. After incorporation of the tumour-nerve distance into the algorithm, PNI was identified in an additional 18 previously misclassified cases increasing the rate of detection from 52.5 to 81.4%. Interestingly, this required an average of only 24 seconds per case.

Conclusion: This algorithm was shown to be a very useful and time efficient tool to assist pathologists to more accurately and reliably identify PNI in PDAC. Of interest, training the algorithm to imitate pathologist thought processes (measuring the distance between tumour and nerve) allows development of a robust algorithm even based on a small cohort.


Pre-invasive lesions of pancreatobiliary cancer: immunohistochemical signatures defining biological behaviour

R. Vesce*, A. Yavas, L.J. Häberle, I. Esposito

*Institute of Pathology, University Hospital Düsseldorf, Germany

Background & objectives: We investigated the immunohistochemical expression profile of pre-invasive lesions of pancreatic ductal adenocarcinoma (PDAC) and cholangiocarcinoma (CCA) to detect staining patterns predicting risk of tumour-progression at early stages.

Methods: We analysed 33 PanIN, 28 gastric (gIPMN) and 20 intestinal (iIPMN) IPMN, 18 BilIN and their associated invasive cancers, when present (PDAC, n=23; CCA, n=6). We investigated the expression of markers associated with subtyping (MUC1, MUC2, MUC5AC, MUC6, CDX2), tumour biology (p16, p53, GATA6, Smad4, ki67), along with recently identified markers (TFF3, MUCL3).

Results: PanINs, gIPMNs and BilINs displayed similar phenotypes with MUC stains; however, MUCL3 was increased in gIPMNs(p=0.03) and BilINs(p=0.01) compared to PanINs. TFF3 was increased in BilINs, compared to PanINs(p=0.03). BilINs revealed focal MUC2(17%) and CDX2(38%) expressions. iIPMNs showed higher expression of MUC2,CDX2,TFF3, and higher Ki67-index than PanINs and gIPMNs(p<0.001). MUC1 was overexpressed in high-grade PanIN(p=0.02) and PDAC(p<0.001) compared to low-grade PanIN and in CCA compared to BilIN(p=0.001). MUC5AC was decreased in IPMNs and BilINs with associated invasive tumours compared to cases without invasive tumours(p=0.03 and p=0.03, respectively). MUC6 was decreased in PDAC compared to PanIN and gIPMN(p<0.001). Altered expression of GATA6,p53,p16 and SMAD4 was observed in high-grade lesions and/or cancers.

Conclusion: iIPMNs display peculiar marker expression and higher proliferation rates compared to gIPMNs and PanINs, which are very similar to each other apart from the expression of MUCL3. BilINs and PanINs also show overall similar immunophenotypes. However, BilINs often present aberrant marker expression, different TFF3 and MUCL3 expression and higher proliferation rate. Altered expression of differentiation markers, such as MUC1, MUC5AC, MUC6 and GATA6 and of tumour-suppressors is associated with high-grade precursors and invasive cancers.

Funding: Deutsche Forschungsgemeinschaft, project number ES 285/8-1

OFP-04 | Joint Oral Free Paper Session Soft Tissue and Bone Pathology / Infectious Diseases Pathology


Whole-exome sequencing of chordoma with special emphasis on chromatin regulatory genes and recurrences

S.R. Ullmann*, A. Roessner, J. Schreier, D. Schanze, C. Lohmann, M. Röpke, D. Jechorek, S. Franke

*OvGU Magdeburg Department of Pathology, Germany

Background & objectives: More insight into molecular genetics of chordoma is desired for defining new therapeutic strategies. We investigated 9 primary chordomas including one case with four recurrences. Special emphasis was put on chromatin regulatory genes and differences between recurrences and primary tumours.

Methods: DNA was extracted from formalin- fixed, paraffin-embedded tissue samples of sacrococcygeal chordomas from patients between 39 and 78 years of age. After thorough quality checking, the DNA was analysed by whole- exome sequencing with a NextSeq Illumina sequencer. In addition to histopathological tumour examination the expression of brachyury p53, Ki-67,SMARCB and H3K36me3 was investigated by immunohistochemical techniques.

Results: Consistent with our immunohistochemical findings and current literature our study revealed a pattern of typical molecular alterations including brachyury, p53, APC, BRCA, CDKN, and PI3K-signaling, in both recurrences and primary tumours. Interestingly our study found an increase in quantity of mutations over time in the recurrences, most of all in chromatin regulatory genes but also in rarer Genes like LYST and HYDIN. In all cases histone modifiers (KMT, KDM) and CRC-family members especially coding for the SWI/SNF- and ISWI-complexes (SMARC, ARID, PBRM) had a high number and variety of mutations.

The number of SNVs and InDels did not show significant differences between the primary and recurrent tumours.

Conclusion: The increasing number of alterations in chromatin-regulatory genes in recurrences compared to primary tumours could point to a possible importance of these alterations in recurrence development and be the basis for new targeted therapy strategies of chordoma. The progress in tumour mutation burden as well as copy number variations in recurrences were more limited compared to the genetic evolution of carcinomas with long time recurrences. Further studies focusing on chromatin regulators could elucidate the molecular pathogenesis of chordoma.


Extraskeletal myxoid chondrosarcoma: a morphological, immunohistochemical and molecular analysis of 31 cases

A. Llombart-Bosch*, F. Giner, i. Machado, S. Navarro, A. Ferrandez Izquierdo

*Dep Pathology Univ. Valencia, Spain

Background & objectives: Extraskeletal myxoid chondrosarcoma (EMC) is a rare malignant tumour. Their spectrum is wide but a diagnosis based on histology can be difficult. NR4A3 gene rearrangement is the hallmark in these tumours but still lack specific immunohistochemical profile for this neoplasm.

Methods: We collected 31 cases diagnosed as EMC between 1999 and 2018 from two institutions. Corresponding clinical data were recorded. We performed a histopathological and molecular study with a wide immunohistochemical panel.

Results: The mean age of our patients was 50 years with a range between 22 and 86 years. We found a slight predominance for males. The majority of EMCs showed a typical architectural pattern. EMCs expressed (focal or positive) the following markers: FLI-1 (100%), CDK4 (100%), TRK-A (96.8%), STAT-6 (90.3%), CD99 (90.3%), CD117 (83.9%), HNK-1 (80.6%), SATB2 (67.7%) and S-100 (58.1%). Neuroendocrine markers chromogranin, synaptophysin and INSM1 showed intense and focal expression in 22.6%, 22.6% and 38.7% of cases respectively. The EWSR1-NR4A3 rearrangement was found in 19 cases and 7 patients presented the TAF-15-NR4A3 fusion. The TAF15-NRA43 rearrangement correlated with a non-typical histology (more cellular with solid pattern).

Conclusion: EMCs express some immunohistochemical markers which are used in diagnosis for other neoplasms that can also be positive for EMC and can cause extra difficulty for differential diagnosis in EMCs with non-typical histology and where molecular rearrangement is not informative. Some immunohistochemical markers such as CD99, CDK4, FLI-1, SATB2 and STAT6 may be considered positive in EMCs suggesting the possibility of incorporating these markers in the differential diagnosis with other entities.

Funding: Supported by the IVO Cancer Fundation.Valencia Spain


A single-institution experience with 11 cases of extraskeletal myxoid chondrosarcoma: rare fusions, unusual morphology and the utility of INSM1 immunohistochemistry

N. Klubickova*, J. Lenz, M. Michal, M. Michal

*Bioptical Laboratory Ltd., Czech Republic

Background & objectives: Extraskeletal myxoid chondrosarcoma (EMC) is a rare soft tissue sarcoma of uncertain differentiation, most commonly driven by fusion of EWSR1 and NR4A3 genes. A recent report identified INSM1 as a useful immunohistochemical marker, being positive in 90% of EMC cases.

Methods: 11 cases of EMC from our institutional files where molecular genetic results were available were included in the study. Immunohistochemistry for INSM1 was performed in 10 cases. Targeted RNA sequencing (RNA-seq) using customized Archer FusionPlex Kits or FISH with EWSR1 and NR4A3 break-apart probes were carried out in 5 and 6 cases, respectively.

Results: 8/10 cases were positive for INSM1. However, only 4 cases showed strong expression in more than 50% of tumour cells. Using FISH or targeted RNA-seq, the classic EWSR1::NR4A3 fusion was detected in 7/11 cases. Less common TAF15::NR4A3 and distinctly rare TCF12::NR4A3 fusions were detected in 1 case each. In 1 case, FISH was positive for NR4A3 but negative for EWSR1 rearrangement. 10 cases displayed typical morphology i.e., anastomosing cords of round/oval tumour cells situated in abundant myxoid stroma, while the case with the TCF12::NR4A3 fusion showed a highly unusual morphology consisting of a solid cellular proliferation of bland monomorphic ovoid/spindled cells with only small foci of myxoid stroma.

Conclusion: Our analysis confirms that most cases of EMC are positive for INSM1. However, the diagnostic utility of this marker is limited by the fact that only 50% of cases show a strong INSM1 expression in most tumour cells. Furthermore, our study highlights that EMC with alternative gene partners may present with unusual morphology. As illustrated by the case with the TCF12::NR4A3 fusion, some of these cases can be confidently diagnosed only with the use of high throughput sequencing methods.

Funding: This study was supported by study grant SVV 260539 from the Ministry of Education, Czech Republic (NK).


Differential Cyclin-E1 expression in CIC-rearranged sarcoma and Ewing sarcoma

B. Karabulut*, O. Kandemir, S. Yenidunya, K. Kösemehmetoglu, F. Ardıc Yukruk

*Ankara Oncology Training and Research Hospital, Department of Pathology, Turkey

Background & objectives: CIC-rearranged sarcomas(CRS) - one of EWSR1-negative undifferentiated round cell sarcoma(URS)- show more aggressive behaviour than Ewing Sarcoma(ES). As CCNE1 expression is associated with tumour growth in CIC::DUX4-rearranged CRS, we aimed to demonstrate the value of CyclinE1 expression in CRS.

Methods: CyclinE1 immunohistochemistry (Abcam, 1/50, EDTA) and break-apart FISH for EWSR1 and CIC gene rearrangements (ZytoLight, Zytovision) were performed on 3-mm tissue microarrays composed of 40 small round cell tumours. CyclinE1 expression was evaluated as low and high similar to the previous study by Wei et al (J Orthop Res. 2020;38(9):1952-1964).

Results: By morphology and FISH, 5 cases were CRS, and 22 cases were ES, while 13 cases were regarded as URS. Among all three diagnostic groups, Cyclin E1 expression was higher in CRS (4/5,80%) and URS (8/13,62%) groups compared to ES (1/22,5%; p<0.001). Higher mean age at diagnosis, presence of atypical histological findings (such as nuclear aberrations and myxoid stroma), lack of CD99 expression, and presence of metastasis at diagnosis were significantly associated with high CyclinE1 expression. The sensitivity and specificity of the high expression of CyclinE1 in detecting EWSR1(-) cases were 66.7% and 95.5%, respectively. However, the correlation between CyclinE1 expression level and survival was not statistically significant.

Conclusion: CyclinE1 expression is significantly lower in ES compared to CRS and URS suggesting that it can be used as an adjunct in the diagnostic immune panel of small round cell sarcomas.


PDL1 expression correlates with the density of tumour-infiltrating lymphocytes (TILs) in high-grade osteosarcoma and is an independent marker irrespective of disease progression and metastasis - an ambispective cross sectional study

S. Salman*, A. Barwad, A.R. Mridha, S. Bakhshi, V.K. Iyer

*AIIMS, New Delhi, India

Background & objectives: Osteosarcoma is the most common primary malignant bone tumour in children and young adults. Not much work has been done to explore immunotherapy targeting PD1-PDL1 axis in osteosarcoma. This study shows correlation between PDL1 expression and TILs in high-grade osteosarcoma.

Methods: PDL1 expression by immunohistochemistry and TILs were evaluated in 40 resection specimens of high-grade osteosarcoma. A score of 0-3 was given for PDL1 expression. TILs were calculated under 400X (in 10 fields) and a score of 0-3 was given based on intensity. Clinical data was collected from record section.

Results: The mean age of presentation was 16 years with male to female ratio 1.6:1. The most common bone involved was femur followed by tibia. The most common site of metastasis was lung. PDL1 was positive in 82.5% cases (33/40). 17.5% (7/40) did not show PDL1 expression. 15% (6/40) showed a score of +1, 27.5% a score of +2 and 40% (16/40) a score of +3. It was observed that PDL1 expression correlated with TILs scoring (p=0.029). However, PDL1 expression and TILs did not correlate with any parameter like age; sex; histologic type; tumour size, site and necrosis; metastasis; progression and relapse; duration of chemotherapy; radiotherapy; follow up and survival data.

Conclusion: Our research shows that PDL1 is an independent marker in high-grade osteosarcoma and cannot be taken as a basis for judging progression of the disease. However, since in our study it correlated directly with the TILs density, it can be regarded as a potential therapeutic target for tumour immunotherapy.


Osteoblastoma-like osteosarcoma: a case series and literature review

S. Sevim*, E. Gedik, S. Yuksel, M.O. Karaca, H.Y. Yildiz, G. Kaygusuz

*Ankara University Medical School, Pathology Department, Turkey

Background & objectives: Osteosarcoma (OS) is the most common primary bone tumour in young adults and children. Osteoblastoma-like osteosarcoma (OBLOS) is a rare subtype that accounts approximately 1% of all OSs. In this study, we present three cases of OBLOS with clinicopathological correlation.

Methods: Gross materials were fixed with 10% buffered formalin. After decalcification process, hematoxylin and eosin (H&E) sections of 4-5 microns were obtained. The cases (n=3) were analysed according to their histopathological, radiological and clinical features. In one case (talus localized lesion), beta-catenin was performed immunohistochemically.

Results: Two of the three cases were male and one female, and the mean age was 43.33 years. The tumours were located in the iliac bone, 4th finger of the hand, and talus. The cross-sectional surface of the tumours was solid and heterogeneous, and haemorrhagic areas were also observed. The tumours had infiltrative borders and were composed of oval-spindle shaped cells with vesicular nuclei and eosinophilic cytoplasm. Osteoclastic giant cells and mitoses were observed. Nuclear beta-catenin staining was not observed in the talus localized lesion. Two patients had a history of neoadjuvant therapy and, the other patient died due to lung metastasis.

Conclusion: Two histological features are helpful in differential diagnosis of osteoblastoma, OBLOS. Permeation around host bone and loss of peripheral maturation of the lesion favours OBLOS, whereas osteoblastoma is well-defined and there is peripheral maturation. These lesions may be radiolucent/radiodense, and OBLOS can contain features of osteoblastoma and OS. Some studies revealed that COX2, FOS, beta-catenin immunoprofile differs between osteoblastoma-OBLOS. We consider that excisional biopsy is important to support this distinction, especially if radiological findings cannot rule out or support malignancy.


Prognostic value of vessels encapsulating tumour clusters (VETC) in sarcoma

S.L. Renne*, M. Tassan-Mangina, I. Santori, L. Ruspi, F. Sicoli, P. Colombo, M. Cammelli, V. Quagliuolo, L. Terracciano, L. Di Tommaso, F. Cananzi

*Humanitas Clinical and Research Center – IRCCS, Humanitas University, Department of Biomedical Sciences, Rozzano, Italy

Background & objectives: How sarcoma metastasize is unknown. VETC has been described as an epithelial-to-mesenchymal-transition independent process of metastasis: endothelium covers neoplastic clusters allowing tumour dissemination. Our aims are to assess the presence of VETC in sarcoma, and to model its prognostic role.

Methods: The study was retrospective. We selected 54 cases of sarcomas (6_DDLPS, 10_GIST, 6_LMS, 9_MLPS, 8_MPNST, 10_SFT, 5_UPS); of them 31 were metastatic (M1 group), 23 were not (M0 group, defined as least 5 years of negative follow-up). VETC was assessed with CD31 immunohistochemistry and defined as a continuous endothelial lining around tumour clusters. We used probabilistic modelling for the analysis.

Results: Within each histology, the two groups (M0 & M1) were substantially homogeneous: most (89%CI) of posterior probability(PP) difference –i.e. the contrast CPP– included 0 for sex, age, size, and grade. VETC in SFT was basically only expressed in M1, with almost all the CPP mass above the 0. Also, in UPS and GIST, VETC was more probable to be in metastatic diseases with 79% and 78% respectively of the CPP mass above 0. VETC was prognostic of metastasis free survival in SFT and UPS with a coefficient of 2.42(CI_0.73–4.65) and 1.94(CI_0.16–3.67); only UPS reached median survival of 65 months(mo)(standard deviation, SD:74_mo) for VETC- Vs 11 mo (SD:14_mo) for VETC+.

Conclusion: VETC was present in all the investigated histotypes but two (MLPS, MPNST). VETC was prognostic of disease free survival in UPS and SFT. These findings warrants confirmations on a larger series. Moreover, in some carcinomas VETC has been shown to be predictive of tyrosine-kinase-inhibitors (TKI) response; our results prompt us to to verify if this is also true for SFT, where TKI are often used in clinical practice.


Immunohistochemical expression of H3.3G34W in 67 giant cell tumours of bone and its diagnostic mimics: a single institutional study at a tertiary cancer referral centre in India

B. Rekhi*, V. Dave, S. Keltle, O. Shetty, A. Puri

*Department of Surgical Pathology/Tata Memorial Hospital, Parel, Mumbai, India

Background & objectives: Despite its classic histopathological features, sometimes there is a challenge in differentiating giant cell tumour of bone(GCTB) from its various mimics. Lately, Histone 3.3G34W has been identified as a useful immunohistochemical marker.

To evaluate H3.3G34W immunohistochemical staining in 67 GCTBs.

Methods: Immunohistochemical staining for H3.3G34W (monoclonal, RM263, 1:100 dilution) was graded in terms of staining intensity(1+to 3+) and the percentage of tumour cells showing crisp nuclear staining. Seventy-one(65.7 %) GCTBs occurred in patients 15-66 years old (average=32, median=9), in femur (26,36.6%), proximal tibia (11,15.5%), distal radius (9,12.6%), pelvis, including sacrum (8,11.2%) and other bones (17,23.9% ), including a single multicentric case.

Results: Out of 67 GCTBs, wherein H3F3G34W immunostaining worked, 55(82.1%) cases showed positive staining in the mononuclear cells, including tumours with fibrous histiocytoma-like areas, sparing the osteoclast-like giant cells. The average percentage of tumour cells showing positive immunostaining was 69%, with 3+ staining intensity in 42/55(76.4%) cases and 2+ in 13(23.6%) GCTBs. All 4/4(100%) malignant GCTBs showed positive staining, including the mononuclear and pleomorphic/sarcomatous cells. Three(4.3%) cases developed metastasis(axillary nodes, mediastinum and lung). All 3/3(100%) metastatic GCTBs showed positive immunostaining in the metastatic lesions. Out of seven post-denosumab treated GCTBs, four showed no residual giant cells and lacked H3.3G34W immunostaining. None of the other 37 “giant cell-rich” lesions displayed H3.3G34W immunostaining.

Conclusion: The diagnostic sensitivity of H3.3G34W for GCTB was 82.1% and specificity was 100%. The present study, constituting one of the first reports from our country, further validates the value of H3.3G34W in differentiating GCTB, including metastatic and malignant type from its diagnostic mimics. Its utility in identifying residual tumour cells in post-denosumab treated GCTBs is worth exploring.

Funding: Intramural grant, Tata memorial Hospital, Parel, Mumbai


COVID-19 placentitis in pregnant women may be complicated by foetal death

P. Babál*, L. Krivošíková, I. Deckov, M. Ondrejka, P. Martanovic, P. Janega

*Institute of Pathological Anatomy, Comenius University, Bratislava, Slovakia

Background & objectives: Dominant clinical manifestations of the SARS-CoV-2 infection are in the lungs, however, pathological consequences of COVID-19 in different organs have been documented in multiple studies. Only limited data are available about the outcome of this infection in pregnant women.

Methods: Two cases of intrauterine death of the foetus in mothers after overcoming COVID-19 with mild symptoms were studied in detail. Placentas hebd. 38 and 35, respectively, were nodular, hard-elastic, with whitish to dark pink areas affecting 70 – 80%. Autopsy of the babies was performed. No pathological abnormalities were grossly identified in either case.

Results: Microscopic evaluation of the placental tissue revealed broad intervillous and perivillous deposits of fibrin with marked histiocytic infiltration, scant lymphocytes and focally with neutrophils, especially in areas of forming infarctions. Inflammation of villi was not prominent. Immunohistochemistry and in-situ hybridization proved the ongoing presence of syncytiotrophoblast infection by the SARS-CoV-2 virus. This finding was also confirmed by the +PCR test from placental tissue samples. Histological evaluation of tissues of the foetuses showed only mild interstitial lymphocytic infiltration in lungs and sporadic immunohistochemical and in-situ hybridization positivity of SARS-CoV-2 in individual cells in lungs and kidneys, which was also confirmed by the +PCR test in lung tissue samples.

Conclusion: The two presented cases documented the development of placentitis caused by SARS-CoV-2 infection after uncomplicated COVID-19. This process is associated with massive deposition of intervillous fibrin and placental infarctions with consequent ischemia and death of the foetus. Although the vertical transfer of the infection was documented, the SARS-CoV-2 virus infection did not contribute to the foetal demise in the presented cases. This rare complication of pregnancy appears to be independent of the seriousness of COVID-19 clinical course in the mother.

Funding: Supported by APVV grant PP-COVID-20-051.


Histological and molecular features of placental and foetal tissues in pregnancies with SARS-CoV-2- positive mothers during second and third trimester: the Bergamo experience in vertical transmission

D. Morotti*, L. Patane, M. Cadamuro, V. Poletti de Chaurand, M. Arosio, L. Goisis, G. Massazza, E. Rigoli, P. Tebaldi, A. Gianatti

*Department of Pathology, Papa Giovanni XXIII Hospital, Italy

Background & objectives: Intrauterine transplacental transmission of SARS-CoV-2 to the foetus is rare but possible in infected pregnant women. According to WHO 2021 classification criteria, both stillborn and liveborn neonates appear to have the possibility to acquire transplacental COVID-19 infection prior to delivery.

Methods: During the first pandemic wave, two Covid-19 positive women delivered two positive babies at third trimester. In September 2021 a case of second trimester twin stillbirth have found positives to SarS-Cov-2 from an asymptomatic infected mother. All specimens collected were analysed by real time PCR, immunohistochemistry and in situ hybridization RNA in order to detect and to localize SARS-CoV-2.

Results: All placentas showed unusual pathology abnormalities that include chronic histiocytic intervillositis with presence of CD68+ macrophages, syncytiotrophoblast necrosis and positivity of the syncytiotrophoblast for SARS-CoV-2 antigen or RNA. Notably, Hofbauer cells do not resulted infected by viral particles as outlined by double staining for CD163+(marker for Hofbauer cells) and ISH for SARS-CoV-2 (spike protein) RNA. Foetal autopsies didn’t show any malformations. The lung of the first foetus only showed interstitial pneumonia features with vascular congestion and neutrophilic infiltrate. Viral genome sequencing identified the lineage B.1.1 in two women who delivered in March 2020, while in the third positive woman, who aborted during the third pandemic wave, was detected B.1.36 lineage.

Conclusion: We gave evidence that SARS-CoV-2 vertical transmission mother-foetus is possible also in second trimester of pregnancy and in asymptomatic pregnant women and can lead to severe morbidity. We found a specific histologic pattern in all infected placentas and our studies ruled out the pathogenic involvement of Hofbauer cells. Furthermore, as far as we know, it does not appear that different genetic variants of the virus affect its possible transplacental vertical transmission.


Diagnostic value of ITS sequencing in mucormycosis – a retrospective single-centre study

M. Zacharias*, A. Thüringer, K. Kashofer, R. Krause, G. Gorkiewicz

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

Background & objectives: Mucormycosis is a fast-progressing disease with a high mortality rate. Rapid and accurate diagnosis is of utmost importance, but microbiological culture may lack sensitivity. Molecular techniques are therefore increasingly recommended; however, real-world data about their value are still sparse.

Methods: Between 2015 and 2022, we performed internal transcribed spacer (ITS) sequencing in 491 tissue and body fluid samples from 432 patients. Clinical, pathological, and microbiological data were extracted from medical records and correlated with sequencing results.

Results: Taxa from the order Mucorales could be detected in 11/432 patients. 7 of these 11 patients were male, and the median age was 54 years (range 5-74 years). All 11 patients had severe comorbidities (9 with hematological cancers and 2 with other cancers; 1 had additionally COVID-19). Specimen sites included lung (6x), upper respiratory tract (2x), myocardium (2x), and soft tissue (1x). The most commonly detected Mucor species was Rhizopus microsporus (5x), followed by Lichtheimia corymbifera (2x), Rhizopus oryzae (1x), Actinomucor elegans (1x), Rhizomucor pusillus (1x), and Rhizomucor miehei (1x). Microbiological culture was performed in 7 cases, always yielding negative results.

Conclusion: The rising incidence of mucormycosis in patients with COVID-19 has raised awareness of this rare but lethal fungal infection. Due to the fast-progressing clinical course of mucormycosis, early diagnosis is key. Our study demonstrates that ITS sequencing is a reliable tool in diagnosing this devastating disease with a much higher sensitivity than microbiological culture. Thus, especially when combined with histopathology, ITS sequencing enables a rapid and accurate diagnosis of mucormycosis, with important implications for clinical practice.


Comprehensive analysis of bronchoalveolar lavage in severe COVID-19 patients

G. Olteanu*, E. Baldasso, F. Lunardi, F. Fortarezza, F. Pezzuto, A. Kilitci, L. Moracci, V. Tauro, L. Vedovelli, C. Del Vecchio, A. Boscolo, P. Navalesi, F. Calabrese

*Spitalul Clinic de Boli Infecțioase și Pneumoftiziologie Dr.Victor Babeș Timișoara, Romania

Background & objectives: A few and inconclusive data are reported about the broad analysis of bronchoalveolar lavage (BAL), a demonstrated safe procedure in COVID-19 patients. We aim to report the comprehensive cytological, microbiological, and molecular analysis of BAL from critically ill COVID-19 patients.

Methods: BAL fluid was obtained from 12 COVID-19 patients admitted in the intensive care unit in the second wave (February 2021 - May 2021). BAL from lung transplant recipients (n=12), and lung donors (n=12), represented the ‘fragile’ and ‘healthy’ control groups, respectively. Cytological analysis, microbiological investigation (culture and molecular determination), and wide cytokine expression analysis using Real-Time PCR were performed.

Results: SARS-CoV-2 (mainly A variant) was positive only in severe COVID-19 patients. Microbiological analysis showed bacterial coinfections with K. pneumoniae, P. aeruginosa, and S. aureus in COVID-19 and transplant patients. Fungal coinfections were found only in COVID-19 patients (C. albicans and A. fumigatus). Molecular analysis identified the viral coexistence of Epstein Barr virus, Cytomegalovirus, and Herpes simplex virus type-1 in both COVID-19 and lung transplant groups; lung donors were negative for all microorganisms. The molecular cytokine profile in the COVID-19 group was remarkable for a low expression of interferon-gamma (IFN-γ), and significantly overexpression of interleukins (IL) IL-1β, and IL-9.

Conclusion: The full comprehensive analysis of BAL is crucial to specifically detect frequent coinfections in patients with severe COVID19 pneumonia. IL-1β, and IL-9 overexpression could represent a possible therapeutic target. Although confirmation is required on larger case series, the obtained results underline a complex and overall different inflammatory profile in COVID-19 BAL in comparison to blood or upper airways as reported in the literature.

OFP-05 | Joint Oral Free Paper Session Uropathology / Nephropathology


Outcomes according to consensus molecular subtypes of urothelial carcinoma after neoadjuvant chemotherapy in the GETUG-AFU V05 VESPER trial

Y. Allory*, C. S. Groeneveld, V. Harter, C. Krucker, V. Dixon, A. de Reynies, S. Culine, C. Pfister, F. Radvanyi

*Institut Curie, Pathology department, Saint-Cloud, France

Background & objectives: The molecular subtypes of urothelial carcinoma may impact the outcomes after neoadjuvant chemotherapy in muscle invasive bladder cancer. Our aim was to assess the 3-year progression free survival according to the consensus molecular subtypes within the prospective Vesper clinical trial.

Methods: 493 patients received dd-MVAC or GC after randomization in the VESPER trial (NCT01812369). This ancillary study was restricted to neoadjuvant treated patients. To take into account intra-tumoural heterogeneity, we performed 3’ mRNA sequencing of distinct tumour areas when morphology and/or multiplexed GATA3 Cytokeratin 5/6 TUBB2a immunostaining highlighted distinct pattern. Consensus molecular subtype was determined for each area from transcriptomic profile.

Results: Out of 296 cases, 97 with heterogeneous immunostaining were selected for multiple sampling. For 251 cases, one single subtype was detected per tumour (i.e. pure): 37 luminal papillary, 60 luminal unstable, 17 luminal non specified, 53 stroma-rich, 81 basal/squamous and 3 neuroendocrine-like. 45 cases were mixed with 2 or more subtypes (27 with basal/squamous component). Pathological response was not different between pure subtypes but was decreased for mixed cases (OR adjusted for randomization arm: 0.43, 95% CI 0.19-0.96, p=0.040). Compared with luminal and stroma rich, the 3yr PFS was decreased for basal/squamous, either pure or admixed with another subtype (HR adjusted for randomization arm: 2.16, 95% CI 1.46-3.20, p<1e-3).

Conclusion: In the VESPER trial, the basal/squamous molecular subtype (pure or mixed) is associated with a decreased 3 yr PFS after neoadjuvant chemotherapy. Further studies will investigate the molecular basis associated with this adverse feature and explore new systemic treatments for the basal/squamous subtype.

Funding: French governmental grant (Institut National du Cancer)


Clinicopathologic and molecular spectrum of testicular sex-cord stromal tumours not amenable to specific histopathologic subclassification

S. Siegmund*, H. Tsai, I. Tran, Y. Yang, V. Vasudevaraja, M. Snuderl, K. Cornejo, M. Idrees, K. Al-Obaidy, K. Collins, J. Gordetsky, S. Wobker, K. Trpkov, A. Yilmaz, G. Quiroga-Garza, W. Anderson, M. Hirsch, C. Magi-Galluzzi, A. Acosta

*Department of Pathology, Brigham and Women's Hospital; Department of Pathology, Harvard Medical School, Boston, MA, USA

Background & objectives: Testicular neoplasms of sex-cord or stromal derivation that cannot be definitively classified into a specific tumour subtype are designated "unclassified sex-cord stromal tumours" (USCSTs). Given the rarity of USCSTs, their clinicopathologic and molecular features remain largely unexplored.

Methods: This study evaluated a multi-institutional series of testicular USCSTs to better define the spectrum of tumours comprised in this diagnostic category. Twenty-six USCSTs from 25 patients diagnosed between 1996 and 2021 were evaluated by review of histology slides, review of clinicopathologic data and massively parallel DNA sequencing. Further evaluation by comparative methylation profiling was conducted on a subset of tumours.

Results: Cytomorphologic patterns included monophasic spindled (5/26), monophasic epithelioid (10/26), and biphasic or mixed (11/23). Histopathologic features suggestive of malignancy (size >5 cm, invasive growth, necrosis or prominent mitotic activity) were seen in 11 cases. DNA sequencing was successful in 19 tumours. Two molecular patterns emerged, including recurrent whole-chromosome gains of 3, 6, 7, 8, 9, 12, 14, 15, 17 and 20. (5/23), and pathogenic mutations in the WNT signalling pathway (8/19). A subset of WNT-altered tumours showed methylation profiles consistent with Sertoli cell tumours, NOS. Follow-up for fifteen cases included 9 patients alive without disease (median 9 mo), 4 alive with disease (median 7 mo), and 2 dead of disease.

Conclusion: The results of this study demonstrate that USCSTs comprise tumours with variable clinicopathologic features and molecular alterations. Two major trends emerged in the cytologic and molecular analysis that facilitated re-classification of ~40% tumours into distinct sex cord stromal tumour entities, including a significant proportion driven by mutations of WNT pathway genes (CTNNB1, APC), some of which could be reclassified as Sertoli Cell tumours, NOS. The remaining unclassified tumours were enriched for aggressive pathological features and malignant clinical behaviour.


Defining lineage plasticity in androgen indifferent prostate cancer

S. Logotheti*, H. Vundavilli, M. Estecio, R. Soundararajan, P. Shepherd, J. Dong, A. Hoang, S. Guo, N. Navone, V. Tzelepi, C. Logothetis, Y. Lu, W. Wang, A. Aparicio

*Department of Genitourinary Medical Oncology, UT MD Anderson Cancer Center, USA

Background & objectives: Combined alterations in TP53 with RB1 and/or PTEN characterize aggressive variant prostate cancers (AVPC) and are associated with androgen indifference and lineage plasticity. A measurable definition of lineage plasticity is needed to enable the development of ‘plasticity-inhibiting’ therapies.

Methods: MDA PCa-177-B (AR-negative, basal expression profile) and MDA PCa-189-1 (AR-positive, luminal expression profile) are AVPC PDX models derived from one patient’s prostate tumour, before and after chemotherapy respectively. They share a p.Y163N Tp53 mutation, suggesting a common clonal origin. We performed scRNAseq, DNA methylation profiling and H3K27me3, H3K27ac and H3K4me3 ChIPseq to identify a shared candidate signature of lineage plasticity.

Results: scRNAseq analysis revealed clusters with a “high-plasticity cell state” (HPCS), in each PDX, characterized by a diversity of lineage identities, similar to that associated with aggressive features and progression in lung cancer mouse models (Marjanovic et al., 2020). ChIPseq of histone markers (H3K27ac, H3K27me3, H3K4me3) and DNA methylation profiling showed shared chromatin profiles at enhancer and super-enhancer regions linked to lineage plasticity events. Taken together, these results suggest that lineage plasticity events drive the evolution of lethal PCa and identified a cell plasticity signature for AVPC.

Conclusion: We identified shared HPCS clusters and chromatin profiles in phenotypically distinct PDX, which may serve as candidate signatures of lineage plasticity. Ongoing analyses are determining the association between the chromatin profiles and the HPCS clusters to arrive at clinically applicable markers. Future studies will determine the effect of drugs presumed to target plasticity on these candidate signatures. These markers will be tested as prognostic indicators and may help identify tumours destined to follow the aggressive variant pathway of PCa progression.

Funding: MD Anderson prostate Cancer Moonshot platform; MD Anderson ATGC Core NCI Grant (CA016672) MD Anderson CPRIT SINGLE CORE Facilities Grant (RP180684)


RNA-seq profiling of upper tract urothelial carcinoma: relevance of the bladder cancer consensus molecular classification, molecular heterogeneity, and differential immune signatures

J. Fontugne*, E. Xylinas, C. Krucker, V. Dixon, C. S. Groeneveld, H. Pinar, G. Califano, M. Bucau, J. Verine, F. Desgrandchamps, J. Hermieu, F. Radvanyi, Y. Allory, A. Masson-Lecomte

*Institut Curie, Pathology department, Saint-Cloud, France

Background & objectives: Extensive analyses of transcriptomic data in large datasets of muscle-invasive bladder cancer (MIBC) have resulted in a consensus molecular classification. Our objective was to determine the relevance of the consensus classification in ≥pT1 upper tract urothelial carcinoma (UTUC).

Methods: We constituted a novel cohort of ≥pT1 UTUC patients with clinico-pathological data. We evaluated GATA3-CK5/6-TUBB2B in multiplex, CK20, p16, MMR proteins and PD-L1 expression by immunohistochemistry (IHC). Heterogeneity was assessed morphologically and/or with subtype marker IHC expression. FGFR3 mutational status was determined using pyrosequencing. Gene expression was profiled using 3’ RNA-seq for each tumour, including multiple samples in heterogeneous cases.

Results: In our cohort of 66 patients with ≥pT1 UTUC, the majority were men (77.3%) with pT1 (35.4%) or pT3 (46.2%) stage disease. FGFR3 mutations and MSI-H status were identified in 41.5% and 4.7% of patients, respectively. The consensus classifier was robustly applicable to UTUC samples and reflected intrinsic subtypes, determined by unsupervised clustering. The proportion of LumP samples (68.4% in ≥pT1, 57.2% in ≥pT2 UTUC) was significantly higher than in MIBC. Ten patients (15.2%) harboured areas of distinct consensus classes. Consensus classes were associated with FGFR3 mutational status, pT stage, morphology and subtype IHC. The majority of LumP tumours were characterized by low immune infiltration and low PD-L1 IHC expression.

Conclusion: We characterized a novel cohort of 66 patients with ≥pT1 UTUC based on morphology, immunohistochemistry, FGFR3 mutation status and transcriptomics. The consensus classification of MIBC efficiently classified UTUC samples, and highlighted intratumoural molecular heterogeneity. In contrast to MIBCs, the majority of ≥pT1 UTUC patients harboured a LumP tumour, a class mostly characterized by low immune infiltration, low PD-L1 expression and a high proportion of FGFR3 mutations. These findings may suggest differential response to novel therapies between UTUC and MIBC patients.


Spatial interplay between TIM3+, PD-L1+, PD-1+ and CLTA-4+ immune/ tumour cells using 18+1 BLEACH&STAIN mfIHC in more than 5 000 tissue samples

N.F. Debatin, E. Bady, J.H. Müller, T. Mandelkow, M.C.J. Lurati, R. Simon, C. Hube-Magg, N.C. Blessin*

*University Medical Center Hamburg, Germany

Background & objectives: A combination of different immune-checkpoint-inhibitors (ICIs) have shown remarkable success in several tumour entities. However, the likelihood of positive response to ICIs is poor in most tumour entities. Little is known about the spatial orchestration between immune checkpoint+ cells.

Methods: To study the spatial interplay between TIM3, PD-L1, PD-1, and CTLA-4 expression on lymphocyte-, macrophage subsets, dendritic cells, in relation to panCK+ malignant cells, and other structural tumour compartments, a 18 marker BLEACH&STAIN multiplex fluorescence immunohistochemistry approach was used to analyse >5000 carcinoma samples from 40 different carcinoma entities. A deep learning-based framework for image analysis was used.

Results: TIM3, PD-1, PD-L1, and CTLA-4 expression was successfully quantified on tumour cells (panCK+), cytotoxic T-cells (CD3+CD8+), T-helper cells (CD3+CD4+), regulatory T-cells (CD3+CD4+FOXP3+), M1 and M2 macrophages (CD68+CD163+/ CD68+ CD163-) and dendritic cells (CD11c+). TIM3 as well as CTLA-4 expression on CD3+CD8+ cytotoxic T-cells and CD3+CD4+FOXP3+ regulatory T-cells showed a spatially more diverse expression pattern – particularly in bladder cancer – compared to PD-1 expression on all analysed T-cells subsets that was consistently accompanied by PD-L1 expression on immune and tumour cells (p<0.001). A high density of immune checkpoint positive T-cells, macrophages and dendritic cells was linked to low pT stage (p≤0.014 each).

Conclusion: BLEACH&STAIN facilitates deep profiling of 18 biomarkers in more than 40 different carcinoma entities and revealed complex changes in the spatial orchestration of a wide range of immune cell subsets that were driven by the expression profile and composition of TIM3, PD-L1, PD-1 and CTLA-4.


Successful deployment of an AI solution for prostate biopsies diagnosis in clinical practice

O. Sukmanov*, A. Yosepovich, S. Ish – Shalom, S. Ikher, N. Ziv-Sokolovski, N. Temper, R. Ziv, I. Krasnitsky, I. Yazbin, G. Mallel, M. Grinwald, C. Linhart, M. Vecsler

*Kaplan Medical Center, Israel

Background & objectives: This study aimed to prospectively evaluate the performance and clinical utility of the AI-based Galen Second Read Prostate workflow solution on detection of prostate adenocarcinoma in real world clinical routine use.

Methods: A prospective, single-centre observational diagnostic study including digitized histopathology slides of all consecutive prostate core needle biopsies (CNBs), TRUS and MRI-targeted, was performed. Slides were blindly processed by the AI solution, while, in parallel, pathologists reviewed the cases. Alerts were triggered in case of discrepancies between the AI results and initial pathologist’s diagnosis, prompting a second review by the pathologist.

Results: Five senior pathologists participated in the study and reported on 109 prostate CNBs comprising 2,684 H&E slides, 60% of which were MRI-targeted biopsies that had up to 66 H&E slides/case. Analysis was performed at block level, 109 cases comprised 986 blocks, 190 (19.3%) were reported as adenocarcinoma, 2 (0.2%) as ASAP and 794 (80.5%) were benign. The AI solution demonstrated extremely high performance for detection of cancer with AUC = 0.994 (95% CI: 0.991-0.997), sensitivity of 96.9%, specificity of 94.96% and NPV of 99.21%. Moreover, following the second review by the pathologists, five alerted cases were revised from benign to cancer, leading to 4.6% decrease in diagnostic error rate.

Conclusion: This prospective study reports the successful deployment of the Galen Prostate diagnostic support solution, in routine clinical practice. The AI solution enabled 100% Quality Control on prostate biopsies and increased diagnostic accuracy and patient safety, decreasing diagnostic errors by 4.6% and preventing missed cancers. Thus, AI solutions could be used as significant aiding tools for pathologists in clinical decision-making in routine pathology practice.


The new entities LOT and EVT among oncocytic tumours of the kidney: a retrospective mono-institutional experience with re-analysis of 16 cases

A. Bressan*, P. Colombo, M. Valeri, M. Cieri, G. Elefante, S. Pancetti, V. Belsito, L. Terracciano, R. Hurle, M. Lazzeri, P. Casale

*Department of Pathology, IRCCS Humanitas Clinical and Research Hospital, Rozzano, Italy

Background & objectives: Low-grade oncocytic and eosinophilic vacuolated tumours (LOT and EVT) have been proposed as distinct entities with low malignant potential in the spectrum of renal oncocytic neoplasms. We report 16 further cases of these rare and controversial categories with clinico-pathological description.

Methods: Oncocytomas (RO), Hybrid Tumours (HT), and Chromophobe carcinomas (ChRCC) diagnosed in our Institution from 2015 to 2021 were retrospectively reviewed. On all selected cases, we performed immunohistochemical analysis for CD117 and CK7 to identify LOTs and EVTs. Histological features, phenotype, molecular profile, and clinical data were recorded.

Results: From 431 tumours, 7 LOTs and 9 EVTs were identified. Male/female ratio was 1:1,3 and 2:1, with median age of 67 and 58 yrs, and median size of 2,7 and 4,2 cm, respectively. LOTs overlapped RO/ChRCC with eosinophilic cytoplasm and perinuclear halos; EVTs showed intracytoplasmic vacuoles and atypical nuclei. LOTs were positive for CK7, CKpan, CD63, AMACR, CD15 (but CD117 negative overlapping ChRCC). EVT were positive for CD117, AMACR, CKpan, and CD10 (but CK7 negative/focally+, opposed to LOT/ChRCC). Interestingly, EVTs CKpan immunoreactivity often reflected a biphasic cellularity (6/9 cases). Rearranged genes in mTOR pathway were occasionally found in both tumours. Both LOTs and EVTs behaved indolently (follow-up 9-72 months).

Conclusion: Here, we described a further group of LOTs and EVTs from a retrospective cohort analysis. Our data confirm LOT and EVT as emerging entities with peculiar histological features, a specific immune-profile, and indolent behaviour, which should be identified among “pink” tumours of the kidney. In the future, these tumours deserve further clinico-pathological studies for promoting the awareness and improving classification of these new categories that will be described in the 2022 Genitourinary WHO classification.


A novel pT1 substaging system for high-grade urothelial bladder carcinoma: a prospective mono-institutional confirmatory progression risk analysis

M. Valeri*, R. Contieri, V. Fasulo, M. Cieri, G. Elefante, C. De Carlo, A. Bressan, C. Saitta, A. Gobbo, P.P. Avolio, R. Hurle, M. Lazzeri, G. Taverna, L. Terracciano, P. Colombo

*Department of Pathology, IRCCS Humanitas Clinical and Research Hospital, Rozzano, Italy

Background & objectives: The Rete Oncologica Lombarda (ROL) system for substaging pT1 high-grade (HG) urothelial carcinoma (UC) showed high predictive value for progression after transurethral resections (TUR) in retrospective studies. We aimed to validate ROL system on a prospective large mono-institutional series.

Methods: From 2013 to 2020, we adopted ROL for all patients with pT1HGUC on TUR and collected clinico-pathological data. We employed a cut-off of 1 mm (or diameter of an objective 20x high-power field) to stratify tumours in ROL1 and ROL2, corresponding to one invasive focus or multiple foci extending together for <1 mm and for >1 mm, respectively.

Results: A total of 229 confirmed pT1HGUC were analysed. Mean age was 73yrs, with male predominance (74.7%); 70 tumours showed multifocality (30.57%), 33 divergent differentiation (14.4%). Associated CIS and vascular invasion occurred in 14% and 9% of cases. ROL was feasible in all but one case (99.6%): 94 cases were ROL1 (41%) and 134 ROL2 (59%). At a median follow up of 23 months (IQR 12.33-38.5), 59 patients had recurrence (25.76%) and 37 progression (16%). ROL predicted progression in univariate (OR= 3.58, 95% CI 1.50-8.56; p=0.004) and multivariate (OR= 2.95, 95% CI 1.11-7.87; p=0.03) Cox regression analysis. At Kaplan-Meier estimates, ROL showed correlation with progression (p<0.01), but not with recurrence (p>0.05).

Conclusion: Our results confirmed the strong predictive role of ROL system for progression in pT1HGUC on a large prospective series. The management of pT1HGUC patients is still a challenging issue in urological practice, and depth and amount of lamina propria tumour invasion is a key prognostic variable. We foster the application of ROL system for substaging T1HGUC, a simple and feasible method alternative to pT1a/b that might identify high-risk patients and drive urological decision-making.


Computational analysis of nuclear features as a grading tool for urothelial carcinoma

I. Fahoum*, D. Rattner, A. Zubkov, A. Greenberg, O. Greenberg, R. Naamneh, V. Zemser-Werner, S. Tsuriel, R. Hagege, D. Hershkovitz

*Tel-Aviv Sourasky Medical Centre, Tel-Aviv, Israel

Background & objectives: Tumour grade determines prognosis in urothelial carcinoma. The two-tiered classification of low and high grade is based on nuclear morphological features. The purpose of this study is to assess the value of computer-based image analysis tool for urothelial carcinoma grading.

Methods: 400 images of urothelial tumours were graded by five pathologists and one uropathologist using a scale of 1 (lowest grade) to 5 (highest grade). A computer algorithm was used to segment the nuclei and to provide 40 morphometric parameters for each nucleus, which were used to establish the grading algorithm. Grading algorithm was compared to pathologists' agreement.

Results: In the training cohort 10 different nuclear parameters showed >85% agreement with the uropathologist's score. All 10 parameters showed >85% agreement with the uropathologist's score in the independent validation cohort. Three parameters showed 94.5% agreement in the validation cohort. The agreement of the pathologists with the uropathologist ranged from 88.5% to 97.5%. Unexpectedly, the parameter that was most associated with grade was the 10th percentile of the nuclear circumference, and high grade was surprisingly associated with lower 10th percentile nuclei, caused by the presence of more inflammatory cells in the high-grade tumours.

Conclusion: Quantitative nuclear features could be applied to quantitate urothelial carcinoma grade. AI assisted grading systems could explore new nuclear parameters with better correlation to grade than those currently used.


Tumour microenvironment immune markers associated with pathologic response to neoadjuvant pembrolizumab in muscle-invasive bladder cancer (Pure-01 trial: an open label, single arm, Phase II study)

L. Cattaneo*, L. Carpenito, C. Napolitano, V. Lagano, I. Bersani, P. Giannatempo, A. Necchi, N. Nicolai, P. Biagio, M. Colecchia, G. Pruneri, R. Mortarini, A. Anichini

*IRCCS Fondazione Istituto Tumouri, Italy

Background & objectives: PURE 01 trial enrolled 143 patients who received 3 cycles of pembrolizumab (IO) every 3 weeks before radical cystectomy. We compared tumour-microenvironment immune markers expression in pre-IO TURB specimens in complete/major and non-responders to identify features associated with pathologic response.

Methods: Immunohistochemistry expression of CD3, CD8, CD68-KP1, CD163, CD20, PD1, PD-L1, MHC-I, HLA-DR and Beta2-microglobulin on tumour cells and tumour-microenvironment was evaluated in 18 complete responders (CR: ypT0), 6 major responders (MR: ypTa/ypT1) and 19 non-responders (NR: ypT2/ypT3, N0 or N+) with a semiquantitative count of the positive cells percentage (0, 0%; 1, <25%; 2 , 25-50%; 3, 51-74%; 4, 75-100%).

Results: Seven markers (CD8, stromal or tumour PD-L1, HLA-DR on tumour cells and B2M on tumour cells) were significantly more expressed (p value range: 0.001 to 0.048) on TURB lesions from responders (CR + MR) compared to non responders (N0 or N+). In addition to such markers, patients achieving a CR, compared to non responders, showed significantly higher expression of CD68 and CD163, of PD-1 on lymphocytes, as well as of tumour MHC-I molecules. Comparison of lesions from patients with CR vs MR revealed significant differences for CD3, CD163, PD-1 and MHC-I, all these markers being more frequently expressed on the former group compared to the latter.

Conclusion: The tumour immune microenvironment of pre-therapy TURB lesions of patients achieving a complete or major pathologic response after neoadjuvant pembrolizumab shows significant enrichment for T cells and myeloid cells, for stromal or tumour PD-L1 as well as increased MHC-I expression on tumour cells compared to lesions from non responders.

Funding: Grant support. Ministry of Health, Lombardy and Tuscany regions, Bando Ricerca Finalizzata, grant number NET-2016-02361632


CD163+ M2 macrophage tissue infiltration and urinary soluble CD163 in IgA nephropathy

V. Agrawal*, S. Singh, S. Kamthan, N. Prasad, V. Agarwal


Background & objectives: Macrophages play an important role in renal inflammation and fibrosis. We evaluated M2 macrophage (CD163+) infiltration in IgAN and correlated it with other parameters of monocyte activation including monocyte-derived circulating microparticles (MMPs), urinary soluble CD163 (sCD163), KIM-1 and MCP-1.

Methods: Twenty-one IgAN, classified with Oxford MEST-C score, two age and sex-matched healthy and four Lupus Nephritis (LN) disease controls were included. Plasma MMPs (AnnexinV+/CD14+), quantified by flow cytometry were estimated as % of total MPs. CD163 immunohistochemistry (clone-EP324) was performed on renal biopsies and quantified in glomeruli and the tubulo-interstitium. Urinary sCD163, KIM-1 and MCP-1 levels were estimated by ELISA.

Results: The mean age in IgAN was 34±10 years; fourteen were males; mean s. creatinine 3.1±1.8mg/dl and 24-hour proteinuria 2.5±0.8gm/day. Mean circulating MPs levels in IgAN, LN and healthy were 8.1x105/μl, 6.7x103/μl and 2.4x105/μl, respectively. MMPs in IgAN constituted 54% of total MPs. Mean urinary sCD163, KIM-1 and MCP-1 in IgAN, LN and healthy controls were 11.4ng/ml, 27ng/ml and 0.18ng/ml; 2.5ng/ml, 1.84ng/ml and 0.37ng/ml; 2.3ng/ml, 7.05ng/ml and 0.13ng/ml respectively. CD163+ macrophages in IgAN were 4.8±5.1/glomeruli and correlated significantly with presence of endocapillary hypercellularity (E1) and crescents (C2). The mean number of CD163+ cells in tubulo-interstitium were 69±35/hpf. Urinary sCD163 levels correlated significantly with number of CD163+ cells in glomeruli.

Conclusion: We found monocyte activation and M2 (CD163+) macrophage tissue infiltration in IgAN. M2 macrophage tissue infiltration and urinary sCD163 levels correlate with proliferative glomerular changes suggesting its role in the early active stage of renal disease. Urinary sCD163 may act as non-invasive biomarker in assessing active proliferative lesions in IgAN.

Funding: SERB-DST Grant (CRG/2018/003042)


Podocyte injury – aristolochic acid nephropathy in mice

D. Miljkovic*, J. Grujic-Milanovic, I. Capo, M. Popovic, T. Kravic-Stevovic, J. Popovic, D. Lalosevic

*Department of Histology and Embryology, Faculty of Medicine, University of Novi Sad; Center for Medical and Pharmaceutical Research and Quality Control (CEMPhIC), Faculty of Medicine, University of Novi Sad, Serbia

Background & objectives: Aristolochic acid nephropathy is a chronic tubulointerstitial renal disease in which important symptoms can be proteinuria and albuminuria. In this study, we examined glomerular morphometric features and protein excretions in NMRI mice treated with aristolochic acid I.

Methods: Experimental animals were treated intraperitoneally with 10 mg/kg aristolochic acid I for seven consecutive days, vehicle control received 2.5% polyethylene glycol 400, and the control received saline only. The experiment lasted 60 days, with several different euthanasia time points for light and transmission electron microscopy glomerular injury assessment. Nestin and WT1 were used as immunohistochemical markers for identifying podocytes.

Results: For every euthanasia time point, mean mesangial score in glomeruli between aristolochic acid treated mice and control groups showed no significant difference. Furthermore, glomeruli of aristolochic acid treated mice had a decreased number of WT1 positive podocytes, lower cytoplasmic nestin expression and area fraction than mice that received 2.5% polyethylene glycol 400 and saline. In addition, ultrastructural changes of podocytes in the aristolochic acid treated group, observed under a transmission electron microscope, indicate foot process effacement, karyopyknosis, and thickening of the glomerular basement membrane with electron-dense deposits. Significant albuminuria occurred in experimental animals from later experiment phases compared with control groups.

Conclusion: Our findings suggest that exposure to aristolochic acid I induce glomerular damage by reducing the number of podocytes and affecting the normal functioning of the glomerular filtration barrier, thus serving as valuable data in further research related to the treatment of aristolochic acid nephropathy.


Deep learning-based histopathologic segmentation of peritubular capillaries in kidney transplant biopsies

D. van Midden*, M. Hermsen, E. Steenbergen, L. Hilbrands, J. van der Laak

*Radboud University Medical Center, The Netherlands

Background & objectives: Peritubular capillaritis scoring is an important feature for diagnosing antibody-mediated rejection (ABMR). This task suffers from interobserver variability and might benefit from automation. As a first step towards automatic peritubular capillaritis quantification, we developed a peritubular capillary (PTC) segmentation algorithm.

Methods: Kidney transplant biopsies (n=54) were 1) stained with periodic-acid Schiff (PAS), 2) scanned into whole-slide images (PAS WSI), 3) re-stained using CD34-antibody, and 4) scanned again (CD34 WSI). Guided by the CD34 WSI, a pathologist manually annotated approximately 19.000 PTCs on the PAS WSI. The dataset was used to train (n=40) and test (n=14) a deep learning (DL)-based network.

Results: We developed a U-net DL network architecture, with an Efficientnetb2 backbone and a pre-trained encoder using ImageNet. The network was trained using 12,000 patches (512 x 512 pixels) per epoch. Various techniques were applied to prevent overfitting and to improve the model’s generalization. Training the network on a resolution of 0.5 μm/pixel using a non-PTC/PTC ratio of 3:1 yielded an F1 score of 0.74, with a precision and recall of 0.78 and 0.70, respectively. We observed reduced performance on cases with prominent interstitial alterations, as PTCs become less recognizable, while certain pathologies mimic PTCs (e.g. atrophic tubules, matrix deposition).

Conclusion: This study presents a DL-based algorithm for the segmentation of PTCs in PAS-stained kidney transplant biopsies. This is a first step towards a more accurate, reproducible scoring of peritubular capillaritis using DL. The results highlight the applicability of DL for clinical use to guide pathologist in routine diagnostics. Next steps will include incorporation of this algorithm in the development of a fully automated Banff classification algorithm, as part of our DIAGGRAFT project, funded by the Dutch Kidney Foundation.


Training a deep learning model for quantification of fibrosis in non-neoplastic kidney biopsies - a feasibility study

N. Mola, E. Hodneland, H. Weishaupt, S. Leh*

*Haukeland University Hospital, Norway

Background & objectives: Interstitial fibrosis is a key prognostic marker of kidney disease. Accurate quantification is therefore important. The study aim is to develop a deep learning algorithm for quantification of interstitial fibrosis that can be used on haematoxylin-eosin (HE) stained kidney sections.

Methods: To create annotated training data, tissue sections were first stained with HE and then - after destaining - with sirius red. Masks of the sirius-red stained fibrosis areas were created using conventional image analysis. A deep learning algorithm was trained with these masks to measure fibrosis in the identical HE stained slide. The model performance was validated with the F-statistics.

Results: The advantage of this approach is that time and resource consuming manual annotations of the fibrosis areas are avoided but supervised learning still can be performed. A deep neural network based on U-Net was used for image segmentation. HE and the mask images were divided into tiles (512 x 512 pixels). Feasibility of the method was tested in a pilot study with 10 representative renal biopsies with varying degrees of interstitial fibrosis. The deep learning model was able to learn from the masks and found the fibrotic areas in the HE stained digital sections with an associated F-score of 0.76 in the validation data set.

Conclusion: The proposed method is feasible and can provide a rapid and reproducible quantitative result for interstitial fibrosis in HE stained kidney biopsies. We will train and fine-tune the deep learning model with more data and expect to see even better performance. The model will then be tested for robustness in an independent cohort.

Funding: Western Norway Regional Health Authority


Relationship between immunosuppressive treatment, morphology, and gene expression in T-cell-mediated rejection of the transplanted kidney

D. Dobi*, S. Chandran, J.R. Greenland, F. Vincenti, Z. Laszik

*Semmelweis University, Hungary

Background & objectives: Belatacept preserves renal function better than calcineurin inhibitor (CNI)-based immunosuppression. However, higher frequency of first-year T-cell-mediated rejection (TCMR) in belatacept-treated patients hampered the adoption of costimulation blockade. We set out to study the patomechanism of TCMR in this patient group.

Methods: Formalin-fixed paraffin-embedded renal biopsy samples were analysed from 92 patients stratified by histopathologic diagnosis (TCMR, borderline changes, or normal) and immunosuppression regimen (belatacept, CNI). We applied gene expression analysis and whole slide inflammatory cell quantification to assess the impact of belatacept on intragraft immune signature.

Results: Ninety-one percent of genes overexpressed in TCMR showed significant correlation with whole-section inflammatory load. There were 27 genes that had a positive association with belatacept treatment. These were mostly related to myeloid cells and innate immunity. Genes negatively associated with costimulation blockade (n=14) could be linked to B-cell differentiation and proliferation.

Conclusion: We concluded that expression levels of genes characteristic of TCMR are strongly interconnected with quantitative changes of the biopsy inflammatory load. Our results might suggest differential involvement of the innate immune system, and an altered B-cell engagement during TCMR in belatacept-treated patients relative to CNI-treated referents.

OFP-06 | Joint Oral Free Paper Session Pulmonary Pathology / Thymic and Mediastinal Pathology


Multi-case learning model for predicting EGFR and KRAS gene mutation in non-small cell lung cancer

Y. Ding, C. Wu, Y. Zhao, J. Yao, Y. Liu*

*The Fourth Hospital, China

Background & objectives: To develop an artificial intelligence learning model for predicting EGFR and KRAS gene mutation in non-small cell lung cancer (NSCLC) by integrating the information of pathological images.

Methods: 934 NSCLC biopsy whole slice images (WSIs) were collected. EGFR and KRAS gene mutation were detected by next-generation sequencing (NGS). The WSIs were divided into training set, validation set and test set, and a transformer-based multi-instance learning (T-MIL) model was developed to predict EGFR and KRAS gene mutation. Moreover, T-MIL model was compared with the other models.

Results: The area under the cure (AUC) was 0.711 by using T-MIL model to predict EGFR gene mutation, and the sensitivity, specificity, the positive predictive value (PPV), and the negative predictive value (NPV) were 81.6%, 55.6%, 61.7%, 77.5%, respectively. For predicting KRAS gene mutation, T-MIL model AUC value was 0.601, and the sensitivity, specificity, PPV, and NPV were 56.2%, 65.1%, 13.2%, 94.0%, respectively. Compared with other learning models, including attention-based multiple instance learning (A-MIL) and RNN architecture for bag representation generation in MIL (RNN-MIL), T-MIL model demonstrated better performance. For EGFR gene mutation, the AUC value were 0.485, 0.6767, 0.711 respectively, and 0.5753, 0.5593, 0.601 respectively for KRAS gene mutation.

Conclusion: We developed a T-MIL learning model for predicting NSCLC gene mutation, and demonstrated well performance. Its performance in predicting EGFR mutation was better than KRAS(AUC 0.711 vs 0.601). Our research proved that the performance of T-MIL learning model through pathological images was better than A-MIL and RNN-MIL model in predicting key driver gene mutation.


Three cancer associated fibroblasts subtypes are associated with histological features, immune environment and prognosis in resected non-small cell lung cancer (NSCLC)

E. Guenzi*, K. El Husseini, A. Guyard, A. Mailleux, P. Mordant, A. Couvelard, B. Crestani, A. Cazes, G. Zalcman, N. Pote

*Department of Pathology, Hôpital Bichat, Assistance Publique - Hôpitaux de Paris, France

Background & objectives: Three cancer-associated fibroblasts (CAFs) subtypes have been recently identified in breast cancer, characterized by the differential expression of FAP and ANTXR1. We aimed to assess, in NSCLC, the association of these CAF subtypes with histological features, immune environment and prognosis.

Methods: Expression of FAP and ANTXR1 was assessed by immunohistochemistry (H-score) on tissue micro-array built from a retrospective series of 186 NSCLC surgical samples. Three CAF subtypes were defined by the differential expression of FAP and ANTXR1 (FAPLow; FAPHigh/ANTXR1Low; FAPHigh/ANTXR1High) and correlated with histological features, immune environment (assessed by CD8, CD4, and FOXP3 expression) and prognosis.

Results: 82 adenocarcinomas (ADC) and 104 squamous cell carcinomas (SCC) were included. We found a predominance of FAPHigh/ANTXR1High CAFs in SCC and FAPLow CAFs in ADC (p<0.001). In SCC, FAPhigh/ANTXR1low CAFs were associated with a higher CD8/CD4+CD8 ratio (p=0.02), but there was no correlation of CAFs subtypes and the immune environment in ADC. In ADC, a higher proportion of FAPHigh/ANTXR1High CAFs was detected in poorly differentiated tumours (p<0.001). Finally, in ADC, tumours with FAPHigh/ANTXR1High CAFs were associated with a poorer disease-free survival in patients that did not have adjuvant chemotherapy (p<0.05). In SCC, no association of CAFs subtypes with disease free survival was found.

Conclusion: These three CAF subtypes are differentially expressed between ADC and SCC, and are associated with the immune environment in SCC and with tumour differentiation and disease free survival in ADC. These preliminary results suggest that FAP and ANTXR1 could be used as prognostic biomarkers in ADC.


Some aspects of lung fibrosis in COVID-19

V. Zinserling*, N. Semenova, D. Baram

*V.A. Almazov Research Center, Russia

Background & objectives: Lung fibrosis is considered as one of the most significant lesions in late stages of acute COVID-19 infection and post Covid syndrome. Many questions related to its pathogenesis and sequels remain unclear.

Methods: We studied lungs in 16 lethal cases with known genotype of SARSCOVi2, nearby routine methods the slices were stained according Mallory, IHC included sera against macrophages CD68+, CD163+ and collagens of 1 and 3 types. In 14 cases the slices, stained for CD68+ cells were scanned and then counted with the help of morphometric program and related to square.

Results: We found that fibrosis was practically equally expressed in all cases nevertheless exact duration of the disease, which was difficult to evaluate in several cases. Genotype of the virus. Collagen was of both types 1 and 3, with the prevalence of the latter. The number of CD68+ macrophages varied from 27 till 93/ mm2. Notable that both, collagen 1 and 3 were detected not only typical fibres but also in cytoplasm of macrophages. In these regions accumulation of CD163+ macrophages was noted. In one case cirrhotic changes of the lung developed in previously healthy man in less than a year after first disease (finished clinical recovery) during the second episode.

Conclusion: Thus, many issues of lung fibrosis of clinical importance have to be additionally studied.


Evaluation of acquired resistance to sotorasib in patients with KRAS p.G12C-mutated non-small cell lung cancer: biomarker analysis using plasma from the CodeBreaK 100 trial

A. Addeo*, J. Wolf, B.T. Li, V. Velcheti, G.K. Dy, S.S. Ramalingam, A. Hindoyan, A. Anderson, A. Ang, F. Skoulidis, J. Delord

*University Hospital of Geneva, Switzerland

Background & objectives: The CodeBreaK 100 trial supported approval of sotorasib, a specific, irreversible KRASG12C inhibitor, for adults with KRAS p.G12C-mutated locally advanced or metastatic non-small cell lung cancer (NSCLC) previously treated with systemic therapy. We consider acquired resistance to sotorasib.

Methods: In the Phase 1/2 CodeBreaK 100 trial, patients with advanced KRAS p.G12C-mutated NSCLC received sotorasib monotherapy 960 mg once daily. The primary endpoint was objective response rate (ORR). An exploratory endpoint examined genomic alterations, absent at baseline but present at disease progression. Plasma samples collected at baseline and progression were analysed with the 23-gene Resolution Bioscience ctDx Lung™ assay.

Results: The ORR in 174 sotorasib-treated patients was 41%. Median progression-free survival and median overall survival were 6.3 and 12.5 months, respectively (median 22.5-months follow-up). Of 67 patients with sequenced plasma samples, 19 (28%) exhibited at least one newly acquired genomic alteration; 7 (10%) had more than one mutation. Variants were detected across multiple genes and pathways. The receptor tyrosine kinase (RTK) genes were the most prevalent putative mechanism of resistance, with alterations apparent in 16 (24%) patients, including 6 (9%) with EGFR gene mutations. PI3K/AKT/mTOR, secondary RAS, and ERK/MAPK mutations were evident in 3 (4%), 2 (3%), and 1 (1%) patient, respectively. Six (9%) patients had undetectable tumour shedding.

Conclusion: Genomic alterations observed in KRAS p.G12C-mutated NSCLC patients treated with sotorasib suggest acquired resistance can arise via a range of mechanisms; however, new RTK alterations were frequently apparent at disease progression. This highlights a potential benefit of combining sotorasib with upstream inhibitors of RTK, such as SHP2 or EGFR inhibitors. Overall, patterns of acquired resistance suggested by DNA analysis of plasma samples at baseline and disease progression support the development of KRASG12C inhibitor combination therapies.

Funding: Amgen Inc.


PD-L1 22C3 Lab Developed Test (LDT) for the Ventana’s BenchMark platform is clinically effective in NSCLC and can be used safely instead of the FDA approved DAKO platform: nation-wide experience and real-life validation (2016-2022)

T. Neuman*, G. Vainer

*Hadassah-Hebrew university medical centre, Jerusalem, Israel

Background & objectives: PD-L1 companion diagnostic by Dako (22C3 clone), for immunotherapy patient stratification, is a common requirement. Our group described a 22C3-based LDT for the Ventana platform, and showed its reliability and reproducibility (2016). However, real-time data about the reliability is lacking.

Methods: Ventana's BenchMark immunohistochemistry (IHC) platform is widely used around the world. Between July 2016 and January 2022, 1444 non-small cell lung cancer (NSCLC) patients were evaluated at the Hadassah Medical Center for PD-L1 by immunohistochemistry. All patients were evaluated by using the clone 22C3 Ventana's BenchMark immunohistochemistry (IHC) platform as a Lab Developed Test (LDT).

Results: The overall PD-L1 tumour proportion score (TPS) of ≥50%, 49-1%, and <1% of the keynote010 trial and our cohort is 28.48%, 37.89%, 33.63%, and 28.39%, 33.85%, 37.80%, respectively. Tumours with a PD-L1 TPS of ≥50% were not associated with patient gender, ethnicity, or biopsy type.

Conclusion: Our PD-L1 22C3 Lab Develpoed Test (LDT) for the Ventana's BenchMark platform and the Keynote 010 display similar scoring distribution (strongly positive cases versus weakly or negative results) in NSCLC. Our cohort represent a nation-wide, real-time, heterogenic group, outside of clinical trial setup.

This support the notion that our PD-L1 LDT is clinically effective in NSCLC and can be used safely instead of the FDA approved DAKO platform for all of the indications based on the 22C3 clone.


Pulmonary asbestos fibre burden, fibre types and their effects on mortality in patients with malignant pleural mesothelioma

H. Wolff*, S. Laaksonen, E. Kettunen, E. Sutinen, I. Ilonen, T. Vehmas, T. Tormakangas, J. Rasanen, M. Myllarniemi

*Finnish Institute of Occupational Health, Helsinki, Finland

Background & objectives: Malignant pleural mesothelioma (MPM) is associated with a dismal prognosis and is strongly related to occupational asbestos exposure. Our aim was to survey retrospectively the asbestos fibre types and concentrations and their effect on the mortality of MPM patients.

Methods: We used a national dataset of MPM patients. For this study, we included patients where an evaluation of the pulmonary asbestos fibre and type had been made using electron microscopy at the Finnish Institute of Occupational Health (FIOH).

Results: A total of 590 patients were included. The median asbestos concentration within dry lung tissue was 3.20 million fibres/gram (range: 0 - 1700). The most prevalent asbestos fibre types detected in lung issue were crocidolite and anthophyllite, respectively. In multivariable survival analyses, overall asbestos fibre concentration increased the Hazard ratio (HR) for mortality. Interestingly if the survival time was under 7 months the HR decreased with asbestos fibre concentration. The age of these patients was high, and they were probably not involved in follow up programs for the asbestos exposed possibly resulting in a delayed diagnosis. No effect of fibre type for the HR of mortality could be established.

Conclusion: We found that the total asbestos fibre concentration increased mortality over follow up time in general except for an initial phase. The most common fibre types were anthophyllite and crocidolite, the usage of crocidolite has been relatively small. However crocidolite has been used for asbestos spraying explaining its prevalence in the lungs. Anthophyllite was recognized to be the sole fibre sizable population of patients with isolated anthophyllite exposure supporting its role in the pathogenesis of MPM.

Funding: Several grants from the Helsinki University Hospital, the Finnish Cancer Foundation, the Finnish Work Environment Fund, and the Foundation of Finnish Anti-Tuberculosis Association have funded this study.


Do different ALK positivity rates affect treatment response and prognosis in non small cell carcinoma of the lung?

E. Yumuk*, A.A. Ağalar, D. Gürel, M. Arslan, M. Keser, Ç. Ulukuş, İ. Öztop

*Department of Pathology, Dokuz Eylül University Faculty of Medicine, Turkey

Background & objectives: Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase and therapeutic target in non-small cell lung cancer (NSCLC). We compared the therapeutic efficiency of targeted therapy between ALK-positive cases near the threshold value (≥15%) and other ALK-positive cases.

Methods: Our study included 73 patients with ALK-positive adenocarcinoma or NSCLC, 29 of whom were treated with ALK inhibitors and were followed up and treated at our centre. The percentage of ALK-positive tumour cells and the predominant signal pattern (break-apart or single red) were obtained from pathology reports, and their relationship to prognosis was statistically evaluated.

Results: The median age was 64.4±0.8(41-91). 52 were male (71.2%). The percentage of ALK rearrangements was 15-20% in 29, %15-25 in 47 cases. 51.4% of the cases died. There was no statistical significance between the percentage and signal pattern groups and prognosis. However, the single-red signal dominant group had a lower mortality rate than the break-apart dominant group (33.3%vs58.3%, p=0.056). The treatment response in the 15-20% ALK-positivity group was lower than in the ≥21% group (%14.3vs%35.7, p=0.314).<gwofw> The treatment response in the single red dominant group was higher than the break apart dominant group (62.5%vs26.3%, p=0.09). 15-25% group showed progression in 5.9 months, while the ≥26% group showed in 8.3 months.</gwofw>

Conclusion: The mortality rate was lower and the treatment response rate was higher in the single red dominant group. Treatment response was lower in the group with 15-20% compared to the ≥21%group. The time to progression was 2.4 months shorter in the group with 15-25% compared to the ≥26%group. In conclusion, "borderline ALK-positive tumours" and cases with predominant break apart signal may have a worse prognosis. Nonetheless, these findings must be validated by larger-scale research with a greater number of cases.


Pathologic assessment of resected stage III non-small cell lung cancer after neoadjuvant chemotherapy: identification of new prognostic factors

F. Lunardi*, S. Tzorakoleftheraki, L. Vedovelli, F. Fortarezza, F. Pezzuto, A. Ferro, M. Schiavon, D. Gregori, F. Rea, G. Pasello, F. Calabrese

*University of Padova, Italy

Background & objectives: Non-small cell lung cancer (NSCLC) patients undergoing neoadjuvant chemotherapy followed by surgery represent an ideal clinical setting to discover prognostic/predictive factors. The aim of the study was to identify clinical/pathological features useful for a better patient stratification.

Methods: Fifty-four stage III NSCLC patients were included between 2013 and 2021. Main clinical/laboratory data at the time of the diagnosis were recorded. All the morphological parameters of the surgical samples were evaluated, including the tumour bed and the new WHO grading for adenocarcinomas. Computer-assisted morphometrical quantification of fibrosis and inflammation extension was performed. Survival analyses were done by Kaplan-Meier curves.

Results: Longer disease-free survival (DFS) was found in patients with higher blood lymphocytes count (p=0.005) and higher fibrosis extension (p=0.05). Overall survival (OS) was related to gender (p=0.02), histotype (p=0.03) and pleural infiltration (p=0.05). When considering only adenocarcinomas, DFS was longer in patients with numerous blood lymphocytes (p=0.0006), lower WHO grades (p=0.01), lower proliferative index (p=0.01), less necrosis (p=0.004) and higher fibrosis extension (p=0.04). OS was related only to stage (p=0.02). A combined score that included lymphocytes, vascular infiltration, proliferative index, necrosis, fibrosis and inflammation, resulted more useful in stratifying patients for DFS(p=0.008). In adenocarcinomas the combined score seems to show a better performance when also WHO grading was included (p<0.0001).

Conclusion: Different morphological aspects resulted crucial for the patient prognostic stratification, especially for DFS. The precise computer-assisted quantification of stromal components can overcome observer bias and inaccuracy, and the combination of different parameters will result in a more effective prognostic stratification of the stage III NSCLC patients.


Asbestos body number in the lung of malignant pleural mesothelioma resected by extrapleural pneumonectomy

K. Okabe*, K. Hara, H. Miyamoto, N. Furukawa, T. Kimura

*Bell Land General Hospital, Japan

Background & objectives: Malignant pleural mesothelioma (MPM) is still dreadful disease, and has been recognized as related to asbestos inhalation. The aim of this study is to analyse the asbestos body number in the lung of MPM patients who underwent extrapleural pneumonectomy (EPP).

Methods: Sixty consecutive MPM patients who underwent EPP from 2006 to 2019 were reviewed. Asbestos body quantification involved the digestion of 1-4 grams of lung tissue in bleach employing a modified Smith and Naylor method (Smith MJ, Naylor B. Am J Clin Pathol 1972; 58:250-254). In addition, age, sex, affected side, MPM type, cause of asbestos exposure, and prognosis were investigated.

Results: The median age at EPP was 62 years old. 49 males and 11 females were operated. Right side was 30, and left side was 30. Epithelioid was 40, biphasic was 15, sarcomatoid was 2, and special variant was 3. 5-year survival and median survival of 30 epithelioid patients after 2011 were 36% and 58 months. Many patients had occupational asbestos exposure. 9 patients were judged as environmental asbestos exposure. The median asbestos body number in one gram of dried lung was 7,189. The asbestos body numbers in one gram of dried lung of 13 patients (22%) were less than 1,000, and those of 35 patients (58%) were more than 5,000.

Conclusion: Strong relationship between inhaled asbestos in the lung and MPM was re-confirmed. The representative occupations were construction worker, asbestos factory worker, plumber, and railroad car maker. Although the dried lung of MPM patients were investigated, the asbestos body numbers of 22% were less than 1,000/g, and those of only 58% were more than 5,000/g.


Prevalence of TTF-1 negative lung adenocarcinoma on lung core biopsy with EGFR, ALK and PD-L1 status

S. Sayeda*, A. Naqvi, H. Begum, C. Finley, C. Coschi, R. Juergens, M. Bonert

*Pathology, McMaster University, Canada

Background & objectives: TTF-1 negative (TTF1neg) lung adenocarcinoma is relatively infrequent, typically CK7+ HepPar1+ and has SMARCA4 gene mutations. The objective was to retrospectively examine biomarkers/immunohistochemical characteristics of local TTF1neg lung adenocarcinomas. Locally, the TTF-1 immunostain clone in use is 8G7G3/1 (Dako).

Methods: All in-house lung core biopsies (LCBs) from Jan 2011-Dec 2020 were retrieved and analysed using a hierarchical free text string matching algorithm (HFTSMA) to establish the diagnosis, and a logical text parsing tool (LTPT) to retrieve results for immunostains, PD-L1, EGFR, and ALK status. A full review/audit was done by pathologists on cases selected by the HFTSMA and LTPT.

Results: Of 5,867 LCBs, 3,142 had immunostains (IHC) from 4,973 patients. The HFTSMA classified 5,725 (98%) LCBs. LTPT identified 85% of LCBs based on TTF-1 status. TTF-1 was done but not reported in 11%. 748 of 1,640 LCBs with adenocarcinoma were TTF-1 positive (TTF1+). 73 cases were TTF1neg, CK7+ and negative for non-lung markers. 50 of these 73 were deemed primary lung, of which 0/29 were EGFR+ and 0/28 were ALK+. PD-L1 was positive in 2/17 (11%), low positive 3/17 (18%) and negative in 12/17 (71%) cases. Only 1/28 cases was positive for Napsin A. HepPar1 was done in only four cases; three matched the SMARCA4 deficient profile TTF1neg CK7+ HepPar1+.

Conclusion: Using the HFTSMA and LTPT, additional TTF1neg lung adenocarcinomas were identified, and these were uniformly negative for EGFR and ALK. In the cohort, 6% (50/ (748+50)) are TTF1neg lung adenocarcinomas. PD-L1 appears to be frequently negative compared to TTF1+ adenocarcinomas. IHC reporting is uneven in our environment. The possible utility of HepPar1 positivity in identifying TTF1neg LCBs appears to be underutilized in our environment. Napsin A negativity appears to be a common finding in TTF1neg lung adenocarcinomas.


Histologic features, nuclear grading, BAP1 and PD-L1 expression in malignant pleural mesothelioma: analysis of a mono-institutional series

H.G. Terzioglu*, S. Onder

*Hacettepe University, Turkey

Background & objectives: The most common primary malignant tumour of the pleura is malignant pleural mesothelioma (MPM) which has a poor outcome. This study aims to identify any relation between histological features, nuclear grading, and expression status of BAP1 and PD-L1 in MPM.

Methods: 52 tumour samples diagnosed as MPM between 2001-2022 were re-evaluated and nuclear grading was performed. Whole sections were analysed for BAP1 and PD-L1 (73-10 clone) expression by using IHC assays. Presence or absence of the nuclear expression of BAP1 was noted. Any pattern of staining was accepted in PD-L1 the 73-10 clone, and cut-offs were set as ≥1%, ≥50%, ≥80%.

Results: Male/female ratio was 27:25 and age range was 33-83 (mean:60). Forty-nine cases were epithelioid and 3 were biphasic. Among epithelioid MPMs, 37/49 were low grade(LG). BAP1 expression was lost in 69%(67% of epithelioid, 100% of biphasic) of cases. There was no statistically significant correlation between BAP1 loss and nuclear grade or overall tumour grade. PD-L1 was negative in 43%(95% epithelioid, 84% LG, 65% BAP1-lost); ≥1% positive in 40%(95% epithelioid, 74% LG, 65% BAP1-lost); ≥50% positive in 11%(75% epithelioid,33% LG, 100% BAP1 lost) and ≥80% positive in 6%(100% epithelioid, 67% LG, 67% BAP1-lost) of the cases. Rhabdoid features were seen in 5/49 cases all of which were BAP1-lost, and PD-L1 positive.

Conclusion: MPM is an aggressive tumour. BAP1 is the most common somatic mutation, and its loss of expression remains to be common regardless of tumour grade or PD-L1 status. In epithelioid MPMs, expression of PD-L1 seems to be associated with certain histologic features such as rhabdoid morphology, but not with tumour grade or BAP1 expression. Immune checkpoint inhibitors were shown to be effective for some aggressive tumour types, and it may be promising for a subset of MPM patients as well.


Evaluation of predictive markers for the patterns of metastatic disease in patients with pulmonary adenocarcinoma

J. Wolf*, P. Clahsen, E. Andrinopoulou, D. Mustafa, M. Kros, J. von der Thüsen

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

Background & objectives: The majority of patients diagnosed with pulmonary adenocarcinoma present at an advanced stage of the disease or will develop metastases during follow-up. Finding predictive biomarkers for the development of metastatic disease during diagnostic workup is important to guide therapeutic strategies.

Methods: Histologic growth pattern and a diagnostic genetic panel customized for lung cancer alterations, were evaluated in biopsy and resection specimens of 319 patients presenting with metastases from pulmonary adenocarcinoma. A subset of patients with early-stage lung adenocarcinoma who developed metastasis during follow up was compared to a group who did not develop metastatic disease.

Results: Primary lung tumours presenting with a dominant solid growth pattern and absence of driver mutations correlate with brain metastasis and are found predominantly in early brain metastasis. EGFR mutations are found in early metastasis of brain and non-brain origin irrespectively of the growth pattern. MET mutations were seen in early non-brain metastasis. We observed a major change of the dominant growth pattern between the primary lung tumour and the corresponding metastasis. Both, early and late brain metastasis show more often a papillary dominant growth pattern whereas the acinar dominant pattern is found in early brain and non-brain metastasis.

Conclusion: Routine histopathology and genetic biomarkers of primary pulmonary adenocarcinoma specimens predict to some extent the development of metastasis. These parameters are by themselves insufficiently specific to reliably predict metastatic behaviour. This is, however, a first step towards the development of a predictive algorithm on which therapeutic strategies can be based. We are currently investigating additional parameters such as tumour microenvironment as well as additional histological and clinical parameters to improve the specificity of our predictive model.


Expression of phagocytosis markers and phagocytosis inhibitors in Usual Interstitial Pneumonia (UIP)

M. Neururer*, L. Brcic, S. Eidenhammer, H. Popper

*Medizinische Universität Graz, Austria

Background & objectives: Fibrosis in UIP is facilitated by senescence cells, which secrete inflammatory cytokines. Phagocytosis counteracts the inflammation by removing cellular debris. We want to identify cells expressing phagocytosis-associated molecules and cells protecting themselves by expressing CD47 within the peripheral lung.

Methods: We performed immunohistochemistry on 44 cases of UIP with different aetiology using antibodies against LAMP1, Rab7, TRAP (all phagocytosis-associated), and CD47 (phagocytosis-protecting).

Results: Phagocytosis-associated molecules were expressed in all macrophages, but also by a considerable proportion of regenerating cells within the remodelled areas. Myofibroblasts, endothelia, and bronchial/bronchiolar epithelial cells were all negative. Expression of LAMP1 and Rab7 were sometimes focally seen in type II pneumocytes within normally structured lung. CD47 was expressed by macrophages, but also by few regenerating epithelial cells within the remodelled peripheral lung, much less compared to the expression of phagocytosis markers. As we suspected these to be senescent cells, we performed double immunohistochemical staining for p16 (senescence marker) and CD47.

Conclusion: Our results indicate that senescence cells have probably activated a mechanism, protecting them from being attacked and phagocytosed by leukocytes. Thus, they maintain inflammation and proliferation of myofibroblasts.


Large-scale human tissue analysis identifies Uroplakin 3B as a useful diagnostic marker for mesothelioma and normal mesothelial cells

V. Reiswich, D. Atug, C. Fraune, R. Uhlig, V. Chirico, C. Völkel, T.S. Clauditz, F. Büscheck, A. Marx, F. Jacobsen*

*University Medical Center Hamburg, Germany

Background & objectives: Uroplakin 3B (Upk3b) stabilizes the urothelial cell layer lining the bladder. Based on RNA expression studies Upk3b is expressed in a only limited number of normal tissues and tumour entities. This study assessed the diagnostic utility of Upk3b immunohistochemistry.

Methods: A set of tissue microarrays containing 8071 samples from 125 different tumour types and subtypes and 608 samples of 76 different normal tissue types was analysed by immunohistochemistry.

Results: Normal cell expression of Upk3b was largely limited to mesothelial cells, umbrella cells of the urothelium, and amnion cells. Upk3b was detectable in 13 (10,4%) of 125 tumour entities. The rate of Upk3b positivity was highest in malignant epitheloid mesotheliomas (81,5%), followed by various categories of urothelial tumours (14,6-45,7%) including Brenner tumours of the ovary (10,8%), four further subtypes of ovarian cancers (1,1-10,4%) and adenocarcinoma of the ampulla of Vater (2,7%). Within urothelial tumours, Upk3b positivity decreased from 45,7% in pTaG2 (low grade) to 34,2% in pTaG3 (high grade), and 14,6% in pT2-4 cancers (p<0,0001). Within pT2-4 cancers, Upk3b staining was unrelated to pT, pN, and patient prognosis.

Conclusion: Upk3b immunohistochemistry is a useful diagnostic tool for the distinction of mesotheliomas from other thoracic tumours and the visualization of normal mesothelial and umbrella cells.


The relationship of VSIR(VISTA) and PD-L1 expressions with histologicalsubtypes in mesothelioma

M. Dicleli*

*Dicle University, Faculty of Medicine, Department of Medical Pathology, Turkey

Background & objectives: Mesothelioma is a tumour with low response to treatment and poor prognosis. This has led to the search for new treatment methods such as antibodies that block immunocontrol points. We evaluated the immune checkpoint markers PD-L1 and VISTA in mesotheliomas.

Methods: VISTA and PD-L1 expression analysed in 45 epithelioid,10 sarcomatoid, 9 biphasic mesotheliomas 3 well-differentiated mesothelial tumours (WDPMT) and 2 atypical mesothelial proliferation cases (AMP). Positive staining for VISTA in tumour cells was defined as the presence of any cytoplasmic and/or membranous staining, and for PD-L1, the presence of any membranous staining.

Results: Positive VISTA staining was seen %91 of cases, more frequently in epithelioid (%98) and biphasic (89%) compared to sarcomatoid (60%) mesotheliomas. Benign mesothelium, WDPMT, AMP, testicular and peritoneal mesotheliomas were all positive with VISTA. PD-L1 was expressed more frequently in sarcomatoid (80%) mesotheliomas compared to epithelioid (29%) and biphasic (33%) mesotheliomas. All PD-L1 positive cases were pleural mesothelioma except 2 epithelioid peritoneal mesothelioma. While 1/23 low-grade and 10/17 high-grade pleural epithelioid mesotheliomas were positive with PD-L1. A significant correlation was found between VISTA(p=0.00) expression with epithelioid mesotheliomas, while PD-L1 expression with sarcomatoid (p=0.01) and high-grade epithelioid mesotheliomas (p=0.00). An inverse negative relationship between VISTA and PD-L1 scores was observed.

Conclusion: VISTA expression was observed more frequently in sarcomatoid mesotheliomas compared to epithelioid mesotheliomas, while PD-L1 expression was mostly observed in sarcomatoid mesothelioma. It is known in the literature that PD-L1 expression is associated with a bad prognosis and VISTA expression is associated with a good prognosis. We also observed higher PD-L1 expression in high-grade epithelioid mesothelioma than in low-grade. We wanted to emphasize the importance of the expression of immuc check point inhibitors in mesothelioma subtypes in target-directed drug selection.

OFP-07 | Oral Free Paper Session Dermatopathology


Mutations detected by next generation sequencing in primary and metastatic melanoma samples and correlation with clinico-histopathological parameters

J.B.S. Liu, K. Zwaenepoel, K. De Winne, A. Pouliakis, P. Pauwels, V. Siozopoulou*

*Department of Pathology, Antwerp University Hospital, Edegem, Belgium

Background & objectives: Melanoma accounts for the vast majority of death related to skin cancer. Its incidence increased during the last few decades. The underlying pathogenetic mechanisms have to be further elucidated, but several oncogenic mutation have been reported to be involved.

Methods: This is a retrospective study on 220 melanoma samples (primary or metastatic) from equal patient number. A NGS-DNA Oncomine Focus Assay was performed in all cases. The results are correlated with different clinico-pathological parameters, among which metastatic status and response to therapy. We present the preliminary results on 114 patients; the study is ongoing. The final results follow upon presentation.

Results: The median age at the time of diagnosis was 62,3 years. Less than half of the patients underwent a sentinel node (SN) procedure and 60,87% of the SNs showed metastatic deposits. Distant metastasis was seen in 46,5% of the cases. The metastatic locations ranged from one up to six. Among the mutations, BRAF and NRAS comprised the majority. Patients with a BRAF but no TERTp mutation had more metastatic locations than those with both a BRAF and TERTp mutation (p=0.044). However, the latter had higher rates of brain metastasis than those with BRAF but no TERTp(p=0.037). Patients with NRAS and TERTp mutations displayed a higher Breslow thickness than NRAS alone(p=0.045).

Conclusion: We present the preliminary results of our retrospective study on 114 melanoma samples. We correlated their molecular status with different clinico-pathological parameters. BRAF mutated melanomas seem to can give rise to multifocal metastatic disease, even in the absence of TERTp mutation. However, TERTp mutations are responsible for the more aggressive, brain metastasis. TERTp in combination with NRAS mutation correlates with higher Breslow thickness of the primary tumours. The study is ongoing and final results are about to be completed.


The utility of PRAME in the diagnostic approach of cutaneous melanocytic lesions

K. De Corte, K. Zwaenepoel, A. Pouliakis, S. Koljenovic, V. Siozopoulou*

*Department of Pathology, Antwerp University Hospital, Edegem, Belgium

Background & objectives: The PReferentially expressed Antigen in Melanoma(PRAME) has been extensively researched for its expression in cutaneous melanomas. Yet, little is known about its expression in non-malignant melanocytic lesions. Here we investigate PRAME expression in a large series of cutaneous melanocytic lesions.

Methods: We performed a retrospective study on the immunohistochemical expression of PRAME in 296 melanocytic lesions. These were classified according to a modified MAPTH-Dx classification in group 1:benign, group 2:moderate atypical, group 3:severe atypical and group 4:malignant. PRAME expression was analysed based on the percentage of cells with expression (< 1%, 1-25%, 26-50%, 51-75%, 76-100%) and on intensity (none/light, moderate, intense).

Results: PRAME percentage of 76-100% was seen in 2%, 4%, 11 % and 52% for groups 1 to 4 respectively (p<0.05). Strong intensity was seen in 10%, 9%, 27% and 62% for groups 1 to 4 respectively (p<0.05). The AUC for percentage of PRAME expression in discriminating between groups 1 and 4 was 85.09%, between groups 2 and 4 84.90% and between groups 3 and 4 74.61%. The AUC was 64.07% for discrimination between groups 2 and 3, 51.02% between groups 1 and 2 and 63.57% between groups 1 and 3. Comparison of the AUC for percentage, intensity and their combination had no significant difference (p>0.05 in all cases).

Conclusion: These results suggest that immunohistochemical analysis for PRAME expression is a useful adjunct for distinguish melanoma from non-malignant cutaneous melanocytic lesions. In the non-malignant category it may play a role in discriminating moderate from severe atypia. The significance of these findings need to be further determined in a larger cohort including the follow up clinical data.


PRAME immunoexpression in 275 cutaneous melanocytic lesions: a single institutional experience

G. Cazzato*, A. Colagrande, D. Di Nanni, G. Ingravallo, E. Maiorano, L. Resta

*University of Bari "Aldo Moro", Italy

Background & objectives: In recent years PReferentially expressed Antigen in MElanoma (PRAME) has been used in the histopathological diagnosis of melanocytic lesions. We performed a single-centre study to evaluate the data on the usefulness of PRAME could also be confirmed by our group.

Methods: From 1 December 2021 to 29/03/2022 we collected 275 cases of melanocytic lesions that were immunostained with PRAME. We categorized PRAME tumour cells percentage positivity and intensity of immunostaining in a cumulative score obtained by adding the quartile of positive tumour cells (0,1 +, 2 +, 3 +, 4 +) to PRAME expression intensity in tumour cells.

Results: Of these 275 lesions, 136 were benign, 12 were of uncertain potential for malignancy, and 127 were malignant. The immunoexpression of PRAME was totally negative in 125/136 benign lesions with only a few positive melanocytes, with intensity 1+ in the remaining 11 cases (8.1%). Of the 127 cases of melanoma, PRAME was strongly positive in 104/127 cases with intensity 4+ and 3+. In 17 cases PRAME was positive in percentage 2+ and with intensity ranging from 2+ to 3+. In 6 cases of desmoplastic melanoma, PRAME was 1+ positive or completely negative. Of the 12 cases of spitzoid lesions, PRAME was much more heterogeneous and irregularly distributed throughout the lesion.

Conclusion: These data are perfectly in agreement with the current literature, they demonstrate that the reliability of PRAME is quite high, but its use cannot disregard the morphological information and the execution of other ancillary immunohistochemical stains such as Melan-A, HMB-45, MiTF and SOX-10.


Cutaneous histopathological findings in systemic amyloidosis

B. Yaman, C.A. Gomez Gonzalez*, A. Acar, T. Akalın, B. Sarsık Kumbaracı, A. Çeltik, S. Şen

*Department of Pathology, Medical Faculty, Ege University, Izmir, Turkey

Background & objectives: Amyloidosis comprehends a group of diseases characterized by the deposition of amyloid fibres within tissues and organs. Skin biopsy is a simple and safe procedure with a high yield and might be used to support the diagnosis of systemic amyloidosis.

Methods: We analysed 59 skin biopsies from 47 patients with systemic amyloidosis (SA) (26 males, 21 females), including 18 amyloid light chain (AL); 19 serum amyloid A (AA), and 10 non-AL/non-AA amyloid types. We evaluated the distribution of amyloid deposits within the tissue.

Results: For each group of systemic amyloidosis, AL, AA, and non-AL/non-AA type, secondary cutaneous amyloidosis was confirmed in 15 (83,3%), 6 (31,5%), and 6 (60%) cases respectively. Amyloid deposits within the skin biopsies in AL type were in the papillary dermis, interstitial infiltration, and hair follicle shaft involvement. In AA-type skin biopsies showed amyloid infiltration at the sweat glands and hair follicle shaft involvement. In non-AL/ Non-AA type the amyloid deposits described an interstitial infiltration and deep small blood vessel wall deposition. In a subgroup of biopsies(N=13), C4d staining was evaluated for identifying amyloid deposition being positive in 84,6%(n=11) of cases.

Conclusion: The diagnosis of SA sampling abdominal fat tissue has variable rates of sensitivity. Skin biopsy is a simple procedure providing valuable findings for amyloidosis. Descriptions of skin involvement and histopathological findings in SA are scant noticing skin compromise in 40% of cases. We detected amyloid deposits at different layers and components of the skin, increasing the probability of detection of SA, supporting this procedure as a diagnostic tool, especially for AL-type amyloidosis where positive findings are frequently detected allowing an early diagnosis for this condition.


Rare histopathological findings in erythema migrans: a 5 year retrospective study from Freiburg Dermatopathology Unit and review of the literature

C. Faria*, A. Pina, K. Deml, K. Wallerius, S. Kraft, S. Hörster, W. Weyers

*Pathology Unit, Hospitais da Universidade de Coimbra, Centro Hospitalar e Universitário de Coimbra, Portugal

Background & objectives: Erythema migrans represents the skin lesion of early manifestation of Lyme disease typically as an erythematous, circular, annular plaque with central clearing, generally greater than 5cm. Some cases can be defiant, leading to a histopathological evaluation, occasionally with uncommon findings.

Methods: We describe 88 cases with PCR test positivity for Borrelia burgdorferi and diagnosis of erythema migrans with rare histopathological findings diagnosed from 2016 to 2021. Our series had a female predilection, representing 71% of total patients and an age distribution between 21 and 82 years old. There was correlation with the clinical information in 59% of the cases.

Results: Erythema migrans is characteristically described as a superficial and deep perivascular inflammatory infiltrate, predominantly composed by lymphocytes with occasional plasma cells and eosinophils. Besides these features, our study presents cases with rare findings. There were interface changes like vacuolar and lichenoid infiltrate in 29% of the cases. Granulomatous features were present in 50,6% of this casuistic, with granuloma formation and a predominantly interstitial histiocytic infiltrate. There was also observed a pseudolymphomatous pattern with superficial, deep and severe lymphocytic infiltrate in 17% of the cases. And 3,4% of these patients presented with histopathological findings similar to mycosis fungoides with epidermotropism and papillary dermal fibroplasia.

Conclusion: The diagnosis of erythema migrans is usually clinical and serologic testing is not recommended due to low sensitivity; but some difficult clinical cases benefit from a biopsy evaluation. However, the findings can be too subtle and unusual, suggesting other entities like granuloma annulare, lupus erythematosus, drug eruptions, and mycosis fungoides, all of which require a distinct therapeutic approach. Although those differential diagnosis can often be excluded with additional histopathologic findings, close clinicopathologic correlation is necessary for a definite diagnosis.


Cutaneous lupus erythematosus: a 5-year casuistic study from a Portuguese dermatopathology department

C. Faria*, A. Alves, C. Courelas, O. Tellechea, J.C. Cardoso

*Pathology Unit, Hospitais da Universidade de Coimbra, Centro Hospitalar e Universitário de Coimbra, Portugal

Background & objectives: Cutaneous lupus erythematosus (LE) occurs in the context of systemic LE or restricted to the skin, and it can be subclassified into acute, subacute or chronic. While there are differences between them, some histopathological hallmarks are present in most subtypes.

Methods: Retrospective study of 54 patients with histopathological diagnosis of cutaneous lupus erythematosus from 2016 to 2021. There was a female predilection representing 70,4% and our study had a broad age distribution, the youngest case with 8 years-old and the oldest one with 88 years-old, with almost 6% of cases being diagnosed at the paediatric age.

Results: Cutaneous lupus erythematosus diagnosed in the setting of systemic lupus erythematosus represents 13% of the patients while the cases limited to the skin represent 87%. Subacute cutaneous lupus erythematosus accounts for 23,4% and chronic cutaneous LE 76,6%. No cases of acute lupus erythematosus were described.

In our study, discoid LE (DLE) is the most common subtype of chronic cutaneous LE (69,4%), followed by tumid LE (19,4%). Bullous LE represents 5,6%, while hypertrophic / verrucous LE and lupus panniculitis account for 2,8% each.

Four cases (8,4%) were diagnosed in the context of scarring lymphocytic alopecia.

Conclusion: The diagnosis of cutaneous LE requires clinical and histopathological correlation. However, the overlap with other connective tissue diseases warrants integration of serologic findings, as a systemic autoimmune disorder may be considered in the differential diagnosis.

Our study also reveals that the majority of cases were responsive to treatment, presenting only hyperpigmented residual lesions. Nevertheless, 11% had persistent lesions, especially patients with other associated autoimmune diseases like Sjogren syndrome, psoriasis and alopecia, or in the context of HIV infection.


Correlation between line-field confocal optical coherence tomography and histopathology. Preliminary results

R. Caltabiano*, G. Broggi, F. Lacarrubba, A.E. Verzì, G. Micali

*Department of Medical and Surgical Sciences and Advanced Technologies "G.F. Ingrassia", Anatomic Pathology, University of Catania, Italy

Background & objectives: Line-field confocal optical coherence tomography (LC-OCT) is a new, non-invasive technique that provides in-vivo, high-resolution images in both vertical and horizontal sections. Our study evaluated LC-OCT imaging in some inflammatory disorders and to correlate the resulting features with histopathology.

Methods: The retrospective study included patients with histopathological confirmed diagnosis of plaque psoriasis, atopic eczema and lichen planus, who were imaged with LC-OCT before the biopsy. LC-OCT was performed with the commercially available LC-OCT device.

Results: A total of 15 adult patients with histopathologically proven plaque psoriasis (N: 5), atopic eczema (N: 5), and lichen planus (N: 5) were included. In all cases, LC-OCT allowed the in-vivo recognition of the main microscopic features of the examined inflammatory skin disease, with a strong correlation with histopathology.

Conclusion: Although future studies on larger series of patients are necessary, LC-OCT, based on these preliminary findings, may represent a promising tool in inflammatory skin disorders with potential applications including enhanced diagnosis, biopsy guidance, follow-up and treatment monitoring.


The challenging differential diagnosis of ALK-positive cutaneous lesions, a case series

L. Keulen*, J. Liu, K. Dewinne, P. Pauwels, V. Siozopoulou

*Department of Pathology, Antwerp University Hospital, Edegem, Belgium

Background & objectives: ALK-positive cutaneous lesions comprise epithelioid fibrous histiocytoma (eFH), ALK-positive non-Langerhans cell histiocytosis (non-LCH) and Spitz lesions. These are rare entities, sometimes with overlapping histologic features, hence their differential diagnosis can be challenging.

Methods: We collected a series of 14 patients with ALK-positivity on immunohistochemistry (IHC). Two case were diagnosed as non-LCH, 4 as eFH and 8 as Spitz lesions. IHC with the same ALK clone (5A4) was implemented in all cases. A NGS-RNA Oncomine Focus Assay was performed in the 6 non-Spitz lesions. We compared the morphological, immunohistochemical and molecular findings.

Results: The median age for the non-LCH was 16,5 years(all females), for the eFH 40 years(females=males) and for the Spitz lesions 20 years(75% females).

The entities shared common morphologic features: circumscribed or ill-defined lesion, spindle or epithelioid cell morphology, fascicular, storiform or nodular growth pattern.

On IHC, all tumours demonstrated diffuse and strong ALK expression. The histiocytic tumours were at least partially positive for CD68. S100 was focally expressed in some eFH, whereas SOX 10 and/or melan-A were confined to the Spitz lesions. No other IHC stainings aided in the differential diagnosis.

Molecular analysis in the non-Spitz lesions showed a SQSTM-ALK fusion in 2 eFHs and a KIF5B-ALK in a non-LCH.

Conclusion: We present a series of ALK-positive non-LCH, eFH and Spitz lesions. These three entities show overlapping morphological features. However, Spitz lesions are characterized by SOX10 and/or Melan-A positivity, which is not seen in eFHs and non-LCH. Still, for the histiocytic entities, IHC is not able to narrow down the differential diagnosis. Additional molecular testing may help, since specific ALK fusion partners have been demonstrated in certain entities.


Melanoma in situ with regression

O. Gokoz, D. Ateş Özdemir, H.G. Terzioglu*

*Hacettepe University, Turkey

Background & objectives: Regression in melanoma in situ (MIS) has been reported but not well studied. Defining the clinical and histopathological features of cases with regression can help work-up of patients and be a step to determine the value of regression in prognosis.

Methods: Ffifty-six cases with a diagnosis of MIS in 2014-2022 were retrieved from the archives and retrospectively examined for the type of MIS, presence of melanophages, fibrosis, vascular proliferation and/or vertically arranged vessels, presence and degree of inflammation, elastosis, presence of folliculotropism, immunohistochemical stainings and surgical margins. Demographic features, clinical history and follow-up findings were recorded.

Results: There were 40 patients with regression, 19 males and 21 females with an average age of 61.8 and 51.3 respectively. Head and neck were the most frequent localization (45%). The mostly seen MIS type was lentigo maligna (62.5%), MIS developing on dysplastic nevus being the second (20%). Melanophages together with inflammation were present in 71.4% of cases. Folliculotropism was seen in 55%, vascular proliferation in 42.5%, and fibrosis in 25% of cases. Immunohistochemistry with melan A and/or HMB45 was studied for the detection of dermal invasion in 40%. Sentinel lymph node sampling was done in 2 patients with negative results. Invasive melanoma was detected in 22.5% of patients.

Conclusion: Evaluation of regression in different types of MIS is valuable in the sense that upstaging can be conceivable in some clinical settings since it can be a sign of invasive melanoma. Step sectioning and immunohistochemical studies are important tools to detect invasion. Folliculotropism and vascular proliferation along with inflammation and melanophagocytosis are frequently seen in cases with regression. Regression in a dysplastic nevus is a known phenomenon and the severity of structural and cytologic atypia derive the diagnosis of MIS.


Progressive disease in sentinel-negative melanoma patients: biological differences and importance of sentinel lymph node biopsy

A. Conrad, U. Maccio*, M. Reinehr, D. Holzmann, J. Mangana, M. Wanner, M. Huellner, R. Barnhill, C. Lugassy, N. Lindenblatt, D. Mihic-Probst

*University Hospital of Zurich, Switzerland

Background & objectives: Among the most important prognostic factors in melanoma is the sentinel lymph node (SLN) status.

Methods: Using our electronic database we identified 109 of 890 SLN-negative patients with progressive disease (PD). These patients were characterized for melanoma type, molecular type, sequence and extent of metastatic spread.

Results: A total of 61 of 109 SLN-negative patients had PD in the SLN-basin indicating false-negative SLN (group-1). 48 of 109 patients had PD at distant sites and were therefore impossible to be identified using SLN biopsy (group-2). Despite distant spread these patients had significantly more single organ metastasis (p<0.001) and significantly longer disease-free-survival (p=0.001) compared to group-1. Additionally, to significant differences on a molecular basis between the two groups (p=0.01), all lentigo maligna and spindle-cell-melanomas belonged to group-2 and all, except one lentigo maligna melanoma, had single visceral metastasis.

Conclusion: Two different biological groups among SLN-negative patients with PD were demonstrated. Extravascular-migratory-metastasis, rather than hematogenous spread, might be responsible for the observed PD with single organ involvement.


PRAME expression in dysplastic nevi

L. Innocenti*, R. Scarpitta, V. Ortenzi, A.G. Bonadio, B. Loggini, C. Scatena

*University of Pisa, Italy

Background & objectives: PRAME is a melanoma-associated antigen whose expression is well documented in cutaneous and ocular melanoma. Our study aimed to investigate the expression of PRAME in a series of low- and high-grade dysplastic nevi in which little is still known.

Methods: Immunohistochemistry for PRAME was carried out on formalin-fixed paraffin-embedded samples applying the specific antibody (clone EPR20330, Rabbit Monoclonal, Abcam) on automated system (Ventana Benchmark Ultra), according to the manufacturer's instructions. Samples were scored blindly according to both the percentage of positive cells and the intensity of expression (H-score). The study was approved by the local Ethical Committee.

Results: The study included a total of 250 melanocytic tumours of which: 50 high grade and 50 low grade dysplastic nevi; 50 nevi with architectural disorder and minimal cytological atypia; 50 melanomas and 50 common nevi as controls. The histopathological diagnosis was reviewed collegially by four expert dermatopathologists according to the 2018 WHO classification of skin tumours. Written consent was obtained from each participant. Statistical analysis was performed using ANOVA analysis and Kruskal-Wallis test. As expected, PRAME immunoreactivity was markedly different between melanomas (diffuse and strong staining) and common nevi (weak and focal). Instead, among dysplastic nevi PRAME protein displayed different staining patterns in the epidermal and dermal portions.

Conclusion: This work represents the first large study on PRAME expression in dysplastic nevi in a real-world setting. Our results suggest that immunohistochemical analysis for PRAME expression may aid in the differential diagnosis between low grade and high grade dysplastic nevi as well as between high grade dysplastic nevus and melanoma, in addition to and as a complement to the WHO morphological criteria used in routine practice.


Somatostatin receptor type 2a (SSTR2a) immunohistochemical expression pattern in Merkel cell carcinoma- a pilot study

A. Sykaras*, C. Vourlakou, C. Kouvidou

*Department of Pathology, Evangelismos General Hospital, Athens, Greece

Background & objectives: Merkel Cell Carcinoma (MCC) is an aggressive, high-grade cutaneous neuroendocrine carcinoma. Recent data suggest that MCC patients may benefit from therapies targeting somatostatin receptors. Our aim was to evaluate the expression pattern of somatostatin receptor type 2a (SSTR2a) in MCC.

Methods: SSTR2a immunohistochemistry was performed in ten MCC specimens (five primary and five metastatic to lymph nodes). SSTR2a expression was evaluated by using a scoring system proposed from Volante and colleagues. Specifically, score 0 describes no staining and score 1+ cytoplasmic staining whereas membrane expression corresponds to score 2+ (staining in <50% of tumour cells) or score 3+ (>50% of cells).

Results: All primary MCC were SSTR2a positive with membrane staining in 4/5 cases and focal cytoplasmic staining (score 1+) in one case. Membrane SSTR2a staining was scored as 2+ in two cases and 3+ in the other two. Score 2+ cases displayed heterogeneous-focal membrane staining of moderate intensity with partial or circumferential pattern whereas score 3+ cases were characterized by a circumferential, strong and diffuse membrane expression of SSTR2a. In contrast with primary MCC, 3/5 metastatic MCC were SSTR2a negative (score 0) with endothelial and follicular dendritic cells serving as internal positive controls. The other two metastatic MCC displayed focal cytoplasmic SSTR2a staining (score 1+) of weak to moderate intensity.

Conclusion: SSTR2a is expressed in 6/10 MCC cases, with membrane staining that is considered as clinically important in 4/10. Although preliminary, our data are in agreement with the few imaging and immunohistochemical studies that previously assessed the SSTR2a expression in MCC. Given that SSTR2a is typically expressed in well-differentiated neuroendocrine tumours (NETs), the positive SSTR2a staining in MCC is a surprising finding. SSTR2a may represent a potential target for imaging and therapies with somatostatin analogues, in a similar fashion with NETs.

OFP-08 | Joint Oral Free Paper Session Paediatric and Perinatal Pathology / Autopsy Pathology


Digital pathology approach for prognostisation of neuroblastoma

N. Tari, T. Werber, T. Micsik*

*Semmelweis University, Hungary

Background & objectives: Neuroblastoma (NB) diagnosis needs precise percentages of differentiating/proliferating cells and mitosis-karyorrhexis index (MKI), which are hard to assess manually and objectively. Our aim was to test the benefit of using digital pathological evaluation to harbour these features, especially for prognostisation.

Methods: Our retrospective study was performed on 41 NB-cases from Semmelweis University, Budapest, Hungary. Hematoxylin-Eosin and Ki-67 immunostained slides were digitalized by Pannoramic 1000 Scanner (3DHistech, Budapest, Hungary). MKI, tumour differentiation, Ki-67 proliferation index were digitally calculated with Quant Center algorithms of the same vendor. Statistical correlations, ROC analysis, t-tests and graphic visualisation were performed with Microsoft Excel and XLSTAT programs.

Results: Our pilot study incorporated 5 undifferentiated, 28 poorly differentiated and 7 differentiating NBs. Average age at diagnosis was 25,54±38,39 months, with slightly more males (23:18). 5-year overall survival was 80%, disease-free survival was 65%. Manual and digital MKI values correlated well (r=0,78, p<0,05) and delivered prognostic value. Manual and digital Ki-67 values correlated well (r1=0,63, r2=0,56, r3=0,91, p1,2,3<0,05), and proved to be prognostic in different aspects. Tumour cells’ biometric data visualisation by violin-plots showed differences among differentiation classes, offering promising ways for digital classification. Combining the various digitally assessed features into the Neuroblastoma Digital Pathology Index we reached a classification accuracy of 94% in non-high-risk and 88% in high-risk NB-patients.

Conclusion: Our epidemiological findings were similar to literature data. Digital MKI counting is accessible and delivers prognostic data. Ki-67 proliferation index’s own prognostic value falls behind some classic prognostic factors such as MKI, though it adds valuable tools for NB prognostisation through complex formulas, especially by automatic digital evaluation on hot-spots or whole slides. Our Neuroblastoma Digital Pathology Index’s classification accuracy is promising, just like the visual representation of tumour cells with violin-plots which might show differentiation related distribution patterns.


Wilms’ tumour 1 expression in eutopic and ectopic decidua: a correlation with the pregnancy status

J. Pacheco*, F. Rosa, E. Dvindenko, R. Barros, A. Costa Braga

*Centro Hospitalar Universitário de São João, Portugal

Background & objectives: Wilms’ tumour 1 gene (WT1) is reported to be overexpressed in decidual cells during pregnancy, however, case series are lacking. Our aim is to evaluate WT1 expression in eutopic and ectopic decidua and correlate it with pregnancy status.

Methods: We evaluated 99 cases (94 tissue microarrays) of decidual tissue (72 eutopic; 27 ectopic). WT1 nuclear immunoexpression was independently evaluated by four pathologists. Percentage and intensity of positive cells were estimated using a semi-automated open-source software. A staining score was obtained by multiplying the percentage and intensity of positive cells. Clinical data was reviewed and the results were statistically analysed.

Results: We evaluated 99 cases of decidual tissue in total, 36,4% associated with pregnancy (23 eutopic; 13 ectopic) and 63,6% non-pregnancy associated (49 eutopic; 14 ectopic). Nuclear expression of WT1 was observed in 93,9% cases (97,2% in pregnant women; 92,1% in non-pregnant women), with no statistical differences (p=0,550). The percentage and nuclear intensity of cells were higher in non-pregnancy related decidua, mainly in eutopic location (p<0,001 and p=0,003, respectively). Accordingly, the WT1 score was also higher in non-pregnancy related eutopic decidua [moderate (14,3%); strong (77,6%); p=0.034]. The WT1 percentage of positive decidual cells was associated, independently of the pregnancy status, with the eutopic location (p=0,024).

Conclusion: In our study of a case series we did not verify a higher imunoexpression of WT1 associated with pregnancy status. We verify a higher percentage of WT1-positive decidual cells in eutopic tissues, especially in non-pregnancy related cases.


Chronic histiocytic intervillositis and related clinical findings: a multicentric, retrospective analyses of 34 cases in a 17 year period

M. de Brito Pereira*, S. Carralas Antunes, E. Vitorino, R. Ilgenfritz, J. Tavares

*Pathology Department, Hospital CUF Descobertas, Lisbon, Portugal

Background & objectives: Chronic histiocytic intervillositis (CHI) of unknown aetiology is a rare placental disorder defined by the presence of an histiocytic infiltrate in the intervillous compartment. According to literature, it is related to poor foetal outcome, maternal autoimmune diseases (AD) and recurrence.

Methods: A retrospective review was conducted in all placentas and products of conception (over 21000 specimens) received in three major hospitals in Lisbon metropolitan area, between June 2003 and February 2022, to detect cases with CHI as a major finding; cases with other major placental findings were excluded. Medical records were analysed regarding the associated clinical and obstetric data.

Results: A total of 34 cases were selected, which corresponded to 32 women with mean age of 37 years (+/- 5 years). Half (17 cases) of pregnancies resulted in viable births, 9% (3 cases) in intrauterine deaths and 41% (14 cases) in spontaneous abortions. In 41% of cases there was intrauterine growth restriction, 3 cases (9%) had a single umbilical artery and 5 (15%) were associated with foetal malformations.

Only 25 women had an available medical record, AD were present in 3 (1 a previous diagnosis, 2 revealed after placental evaluation). Recorded recurrency was seen in 2 women and 3 other had a history of repeated aborditions without known histologic assessment.

Conclusion: Our incidence of CHI is similar to the literature; regarding the prevalence of maternal AD, our study reveals a lower incidence than usually observed. The question remains if an undiagnosed AD is present at the time of gestation or if CHI can precede the full setting of an AD and, if so, how long it takes between both events. We propose follow-up of these patients in a 5 years period, with special focus on CHI recurrency and AD incidence/prevalence.


Left ventricular non-compaction in foetal autopsy: a report of 6 cases

A. Nadal*, C. Fuster, N. Masoller, Ò. Rosiñol, J. Camacho, O. Gómez

*Hospital Clínic, Spain

Background & objectives: Left ventricular non-compaction (LVNC) results from arrest of normal process of ventricular compaction, resulting in a ratio between non-compacted and compacted myocardial layers >1. It is a rare condition of uncertain aetiology although likely genetic with dominant pattern of inheritance.

Methods: Cases with LVNC were retrieved from pathology files. Ultrasonographic and genetic data when available were retrieved from clinical charts. Histology when available was reviewed.

Results: LVNC was identified in 6 cases (three termination of pregnancies and three intrauterine foetal deaths) out of 3000 foetal autopsies. Gestational ages ranged from 13 to 25 weeks. Four out of five cases had ultrasound abnormalities with two foetal hydrops and one additional subcutaneous oedema. Cardiac abnormalities were identified in two cases. Three out of four cases with available genetic analysis had genetic alterations: X monosomy, 8q23 deletion and heterozygous compound LDB3 mutations. Healthy carriers of LDB3 mutations were identified among the relatives of this case. Two of three sibs that shared the heterozygous compound LDB3 mutations had LVNC, one being a live-born girl.

Conclusion: LVNC incidence was higher in this foetal autopsy series than it is reported in the paediatric population. Noncompacted cardiomyopathy must be considered in the differential diagnosis in cases of foetal hydrops and dilated cardiomyopathy. It must also be ruled out when other structural cardiac defects are found. The inheritance pattern found in LDB3 mutations, consistent with a recessive pattern, was not previously described.


Umbilical cord compromise versus other clinical conditions predisposing to placental foetal vascular malperfusion

J. Stanek*

*Cincinnati Children's Hospital, USA

Background & objectives: Foetal vascular malperfusion (FVM) can be due to cord compromise, hypercoagulability, foetal cardiac dysfunction, or hypoxia. Although the cord compromise is most common, the relative importance of other factors is unclear which is the aim or this retrospective analysis.

Methods: 580 placentas were examined: Group 1: 52 placentas with clinical cord compromise/anatomical abnormalities, Group 2: 204 placentas with maternal/ foetal conditions predisposing to FVM; Group 3: 286 placentas with coexisting at least one variable of Group 1 and one variable of Group 2 predisposing to FVM, Group 4: 38 placentas with no clinical conditions or cord factors predisposing to FVM.

Results: Average gestational age was the shortest in Group 4, followed by Groups 1, 2 and 3. Groups 1 and 4 featured more cases with poor prenatal care, less frequent cesarean sections, more frequent macerated stillbirths, less frequent neonatal stay in intensive care unit, atherosis of spiral arterioles, retroplacental hematomas, and luminal vascular abnormalities of chorionic villi. Clusters of sclerotic/stromal vascular karyorrhectic/mineralized chorionic villi, large vessel foetal vascular malperfusion, and low grade distal foetal vascular malperfusion were statistically significantly more common in Groups 1 and 3. There were no statistically significant differences in inflammatory and hypoxic lesions or patterns (acute or chronic) or lesions of shallow placental implantation among the groups.

Conclusion: Cases with isolated clinical umbilical cord compromise were associated with the most unfavourable clinical outcome as umbilical cord complications and pathology strike unexpectedly and are notorious for unpredictability, causing stillbirths not associated with other maternal or foetal diseases. This finding paralleled the histological segmental FVM most common in Groups 1 and 3, both with the umbilical cord aetiology. The clinical risks alone for FVM alone, without the umbilical cord factors (Group 2), were not associated with increased rate of FVM.


Placental recent/on-going foetal vascular malperfusion with endothelial fragmentation is diagnostically equivalent to established distal villous lesions of foetal vascular malperfusion

J. Stanek*

*Cincinnati Children's Hospital, USA

Background & objectives: CD34 immunostaining increases sensitivity of placental diagnosis of foetal vascular malperfusion (FVM). This comparative retrospective study was performed to find out whether recent distal FVM lesions diagnosed with CD34 immunostaining are diagnostically equivalent to remote FVM lesions diagnosed with hematoxylin-eosin

Methods: Clinical and placental phenotypes of 562 placentas from ≥20weeks high-risk pregnancies were analysed: Group 1 - 158 placentas with remote distal villous FVM (by H&E only), Group 2 - 142 placentas showing clustered endothelial fragmentation by CD34 immunostaining, 98 of them also with H&E distal FVM lesions (on-going, temporal heterogeneity), Group 3 - 262 placentas without distal villous FVM.

Results: Foetal congenital malformations were seen in most cases of each group (58.5% of all cases). Using Bonferroni correction, there were no statistically significant differences in clinical or placental phenotypes between Group 1 and Group 2, or among the 3 groups (p>0.002). However, in Group 1, gestational age was the shortest, postnatal mortality most frequent, placental weight the smallest, intra villous haemorrhage, erythroblasts in foetal blood, hypertrophic decidual arteriopathy, and foetal vascular thrombi most common, and in Group 2, placental infarction, post-uterine pattern of chronic placental injury and excessive extra villous trophoblasts of chorionic disc were most common (p<0.05).

Conclusion: In this cohort of foetuses/neonates dominated by congenital malformations, distal villous FVM was the most common pattern of placental injury. The absence of statistically significant differences in clinical or placental phenotypes among all 3 groups indicates that distal villous FVM diagnosed by CD34 and that diagnosed by H&E are diagnostically/prognostically equivalent. CD34 immunostaining is therefore a powerful tool in diagnosis of distal villous FVM.


Cardiac arrest with successful cardiopulmonary resuscitation induces histologic changes that correlate with survival time and lead to misdiagnosis in sudden arrhythmic death syndrome

J. Coelho Lima*, J. Westaby, M. Sheppard

*Department of Pathology, University of Cambridge, United Kingdom

Background & objectives: Sudden arrhythmic death syndrome (SADS) is defined as sudden cardiac death (SCD) with a morphologically normal heart. Cardiac arrest with cardiopulmonary resuscitation (CPR) may induce cardiac histologic changes. We aimed to assess whether such changes could confound SADS diagnosis.

Methods: Retrospective observational study analysing all consecutive cases of sudden death prospectively referred to a UK national cardiac pathology centre between January 2017 and November 2021. Cases showing hypoperfusion features due to cardiac arrest followed by CPR were identified after review of clinical information and examination by two expert cardiac pathologists. Data is presented as percentage or median.

Results: Out of 2,568 SCD cases, 126 (4.9%) were identified with hypoperfusion changes. Macroscopically, the commonest finding was left ventricular focal or diffuse subendocardial haemorrhage (13.5%). Microscopically, haemorrhage and contraction band necrosis (n=50, 37.7%), subendocardial acute infarction (n=44, 34.1%), interstitial mixed inflammatory cell infiltrates (n=31, 24.9%), granulation tissue (n=9, 7.1%) and subendocardial fibrosis (n=1, 0.7%) were observed. These changes correlated to duration of survival following resuscitation, with subendocardial infarction and granulation tissue being observed later at 2 and 9.5 days, respectively (p<0.001). In a subcohort of 41 cases, autopsy pathologists misinterpreted such changes as ischaemic myocardial infarction (n=7; 17%), myocarditis (n=5; 12.1%), or other pathologies (n=2; 4.8%) in 14 SADS cases.

Conclusion: We provide a comprehensive characterisation of hypoperfusion-related changes in the heart following successful CPR with survival, which are time related. These features can lead to diagnostic confusion among pathologists but knowledge of history of resuscitation with survival should help with general and expert pathology assessment and improve SADS diagnostic yield, prompting genetic screening of decedents’ relatives.

Funding: Cardiac Risk in the Young Grant numbers: 12840-14


Post-mortem pulmonary findings in a large series of COVID-19 cases at the University of Texas Medical Branch (2020-2022): insights from an ancient and still relevant procedure

J.P. Olano*, I. Marin, J. Estrada

*University of Texas Medical Branch, USA

Background & objectives: COVID-19 is an infection due to SARS-CoV-2 and was declared by WHO a pandemic on March 11, 2020. This project addresses the spectrum of pulmonary pathology in 294 autopsy cases performed at a single tertiary care institution.

Methods: A total of 294 autopsies were performed in our service between April 2020-2022. All cases were diagnosed by nucleic acid amplification (real-time RT-PCR) using post-mortem nasal swabs. Demographics, clinical history, gross and histologic findings were collected prospectively. Histologic sections were routinely stained with H&E, Masson’s Trichrome and MOVAT pentachrome. Other special stains were performed as needed (PAS-D, MSB, etc).

Results: The average age was 60 years (range: 28-94). Clinical spectrum ranged from asymptomatic infections (cause of death unrelated to COVID-19) to lethal outcome. Clinical course was 2-120 days with an average of 19. The main risk factors included systemic hypertension, morbid obesity, diabetes mellitus, coronary artery disease, congestive heart failure, emphysema, cirrhosis and chronic renal disease. The main complications were hepatic encephalopathy, thrombotic microangiopathy, acute kidney injury (AKI) and bacterial bronchopneumonia. Virtually all lungs showed markedly increase weight and consolidation. Histologic findings included interstitial pneumonitis, oedema, hyaline membranes, acute fibrinous organizing pneumonia, type 2 pneumocyte hyperplasia, reactive/atypical type 2 pneumocytes, fibroblastic foci in alveolar spaces, fibrosis, squamous and bronchiolar metaplasia.

Conclusion: Virtually all patients had at least one risk factor, the most common being systemic hypertension. Numerous patients had two or more risk factors. The spectrum of histologic findings in the lung is broad with overlapping features, and spatial and temporal heterogeneity as opposed to conventional diffuse alveolar damage. The most common complication is hepatic encephalopathy due to the presence of liver cirrhosis in this population, followed by thrombotic microangiopathy and AKI. Other complications were related to hypercoagulability or bleeding diathesis.

Funding: George Dock Professorship. University of Texas Medical Branch


Patterns of infectious disease identified in clinical autopsy. The role of autopsy as an ancillary study in determining the cause of death

M. Khaba*, M. Dikotope, N. Makhado

*Sefako Makgatho Health Sciences University, South Africa

Background & objectives: Infectious diseases are one of the most prevalent diseases in clinical autopsies. Despite its value, the use of autopsies has decreased and infectious diseases are often undiagnosed or misdiagnosed due to the lack of pathological understanding.

Methods: This was an 8 year retrospective study of autopsy cases with final diagnosis of infectious diseases.

Results: The study consisted of 52 autopsies which comprised of 32 female (61.53%) and 20 males (38.46%) with mean age of 28 years. HIV/AIDS was the commonest comorbidity (17/52; 32.69%). Bacterial pneumonia (80.76%) was one of the most common infectious diseases, followed by septic shock (19.23%), candidiasis (14%), bacterial meningitis (9.61%), tuberculosis (7,69%), cryptococcosis (7.69%), cytomegalovirus infection (5.26%) and herpes simplex virus infection and mucormycosis each 1.92%. Antemortem diagnosis was incorrect in 63.46% (33/52) of the cases.

Conclusion: The use of autopsy for years has been the gold standard for establishing the causes of death and have played a valuable role in the diagnosis and understanding of diseases. Autopsies have also informed public health strategies in the fight against these communicable diseases.


The impact of the COVID-19 pandemic on autopsy practice

M. Carter*, S. Brennan, L. Clarke, K. O'Hare

*Tallaght University Hospital, Ireland

Background & objectives: Tallaght University Hospital autopsies from the two years before and after the onset of the COVID-19 pandemic were analysed. We hypothesised that the pandemic would cause fewer autopsies, increased use of ancillary testing and prolonged time from death to autopsy.

Methods: All autopsies conducted between mid-March 2018 and mid-March 2022 were analysed for basic demographic information, location of death (community, emergency department or hospital inpatient) and the use of ancillary studies such as toxicology, histology, microbiology and subspecialty examination. The data from the two years prior to the pandemic (Study Period 1) and following two years (Study Period 2) were compared.

Results: Fewer post-mortem examinations were conducted in Study Period 2 (n = 238) compared to Study Period 1 (n = 418). An institutional change in autopsy practice accounts for much of this decrease, however the data for emergency department deaths only also showed a significantly fewer autopsies between Study Period 1 and 2 (n = 90 v n = 52). Average time from death to autopsy significantly increased (2.3 days v 3.8 days, p-value <0.01). The use of toxicology, histology, neuropathology and non-SARS-CoV-2 microbiology did not change significantly. The use of post-mortem microbiology has increased primarily due to screening for SARS-CoV-2 with 95.4% of decedents undergoing nasopharyngeal swabbing since mid-March 2020.

Conclusion: Since the beginning of the pandemic our institution has found a reduction in autopsy examinations, an increased interval between death and autopsy and an increased use of microbiological ancillary testing. The demographic characteristics of our autopsy cases, and rates of use of non-microbiology ancillary testing have not changed significantly.


Analysis of the causes of death in cases of COVID-19 based on maternal mortality cases during the pandemic

A. Sapargaliyeva*, D. Mirzakhmetova, I. Yefimenko

*Pathology Bureau of Almaty City; Al-Farabi Kazakh National University (Department of Fundamental Medicine), Kazakhstan

Background & objectives: Officially COVID-19 led to a sharp increase in maternal mortality in the Republic of Kazakhstan. There were 156 cases of maternal death in 2020 and 200 cases in 2021.

Methods: We conducted a retrospective analysis of 78 cases of maternal death for the period of 2020-2021 with a clinical diagnosis of COVID-19 to determine a cause of death. All women (32-39 weeks of pregnancy) were admitted to the hospital with positive PCR results for COVID-19. The death of women occurred within 3 to 45 days after hospitalization.

Results: In 67 cases out of 78 morphological signs of COVID-19 were identified. However, the morphological spectrum of lung damage was quite diverse, including acute tracheobronchitis, pulmonary oedema, and massive hyaline membranes (in 8 cases). In 31 cases, there were signs of bacterial and fungal infection with COVID-19. In 28 cases, the cause of maternal death was obstetric pathology, but squamous metaplasia of the epithelium of the trachea and large bronchi and signs of exudate organization in the alveoli were detected.

Conclusion: Based on our analysis of COVID-19, massive hyaline membranes were the cause of death, but in a small number of cases. The death of patients with COVID-19 in most cases was associated with a bacterial or fungal infection. Squamous metaplasia of the epithelium of the trachea and large bronchi and giant cell transformation of alveolar macrophages indicate a viral infection.

OFP-09 | Joint Oral Free Paper Session Endocrine Pathology / Head & Neck Pathology


Cadherin-16 (CDH16) immunohistochemistry: a novel diagnostic tool for renal cell carcinoma and papillary carcinomas of the thyroid

M. Lennartz*, S. Minner, N. Gorbokon, A. Menz, T. Krech, D. Höflmayer, R. Simon, G. Sauter, T.S. Clauditz, A. Hinsch

*University Medical Center Hamburg, Germany

Background & objectives: Cadherin-16 (CDH16), also termed kidney specific cadherin (ksp-cadherin), is a membrane-associated glycoprotein with a role in the embryonal development of tubuli in kidney and thyroid. Downregulation of CDH16 RNA was found in papillary carcinomas of the thyroid.

Methods: A set of tissue microarrays containing 14,978 samples from 149 different tumour types and subtypes as well as 608 samples of 76 different normal tissue types was analysed by immunohistochemistry to determine the expression of CDH16 in cancer and to assess the diagnostic utility of immunohistochemical CDH16 analysis.

Results: Among normal tissues, a membranous CDH16 immunostaining predominated in thyroid, kidney, cauda epididymis, and in mesonephric remnants. In the thyroid, CDH16 staining was seen in all normal samples, 83% of follicular adenomas, 58% of follicular carcinomas, but in only 9% of papillary carcinomas (p<0.0001). CDH16 positivity was particularly frequent in nephrogenic adenomas (100%), oncocytomas (98%), chromophobe (97%), clear cell (85%), and papillary (76%) renal cell carcinomas (RCCs), clear cell (56%), mucinous (36%), and endometroid (16%) carcinomas as well as carcinosarcomas (18%) of the ovary, adenocarcinomas of the cervix uteri (40%), serous (33%), clear cell (33%), and endometroid carcinomas (18%) of the endometrium and in various subtypes of neuroendocrine neoplasms (4-26%).

Conclusion: Given the massive loss of CDH16 expression in >90% of papillary carcinomas of the thyroid, CDH16 is a highly useful diagnostic marker for these tumours. CDH16 immunohistochemistry is also useful for the identification of nephrogenic adenomas and the distinction of renal cell carcinomas from other neoplasms.


Pediatric thyroid nodules: a multi-institutional study from India based on the applicability of the Bethesda System with analysis of the risk of malignancy (ROM) and comparison with adult thyroid nodules

C. Rana*, N. Nigam, S. Agarwal, P. Mishra, A. Bychkov

*King George's Medical University, India

Background & objectives: This study analyses the paediatric thyroid nodules based on the Bethesda system and compares the risk of malignancy (ROM) with adults (>18 yrs) across various Bethesda categories. It also compares young adults (19-21 yrs) with the other two age groups.

Methods: This is a retrospective multi-institutional, where archival thyroid cytology and histology data were retrieved. The cases were segregated into paediatric (<18 years), young adult (19-21 years), and adults (>18 years) age groups. The Bethesda distribution across the different age groups and diagnosis on follow-up resection were collated. The ROM in the various categories was compared across the different age groups.

Results: A total of 5,958 FNA for thyroid swelling were performed over a period of 5 years. The paediatric patients constituted 3.3% (n=199) of all the cases. Follow-up histology was available in 2276 patients. The malignancy was significantly higher in patients <18b yrs as compared to adults (18 % vs. 12.7% p-value 0.02). Interestingly, surgical resection rates were also higher in paediatric groups in almost all the categories except benign. Similar to the paediatric age group, the young adult patients also underwent a significantly higher number of resections as compared to adults. The risk of malignancy was comparable between paediatric and young adults age groups.

Conclusion: There was no significant difference in the distribution of Bethesda categories between the adult and paediatric age groups. When resected, paediatric patients are more likely to harbour malignancies than adults with thyroid nodules as the overall risk of malignancy is higher in children as compared to adults. Importantly, the young adult group (19-21 yrs) may behave in a similar manner to paediatric suggesting a reconsideration of the upper age limit, however, more studies are required to further validate this finding.


Diagnostic utility of menin immunohistochemistry in multiple endocrine neoplasia type 1 syndrome patients

A.S. Kok, A.V. Verschuur*, F.H. Morsink, M.F. van den Broek, J.A. Offerhaus, G.D. Valk, M.R. Vriens, B.P. Nesselrooij, W.M. Hackeng, L.A. Brosens

*Department of Pathology, University Medical Center Utrecht, Utrecht University, The Netherlands

Background & objectives: The diagnosis of multiple endocrine neoplasia type 1 (MEN1) syndrome is confirmed with a germline mutation in the MEN1 gene. As 5-25% of patients suspected of MEN1 remain without definitive genetic diagnosis we investigate the added value of menin immunohistochemistry.

Methods: From 16 MEN1 syndrome patients 31 parathyroid adenomas were collected. As control group, 61 parathyroid adenomas were collected from 32 non MEN1 syndrome patients, these included sporadic (n=30), multiple endocrine neoplasia type 2A (n=1) and hyperparathyroidism-jaw tumour (n=1) patients. Menin immunohistochemistry was performed and its use for identification of MEN1 syndrome related tumours was assessed.

Results: 14 out of 16 MEN1 syndrome patients and 3 out of 32 non MEN1 patients showed nuclear menin loss on immunohistochemistry. On average 1,9 tumours were resected per patient. Using a cut-off of at least one tumour showing menin loss on immunohistochemistry per patient, the sensitivity and specificity in diagnosing MEN1 syndrome was 87,5% and 90,1% respectively. Using a cut off of 2 tumours showing menin loss on immunohistochemistry, the specificity raised to 100%. Menin immunostaining on two cases with a germline variance of unknown significance in the MEN1 gene illustrates the additional value of menin immunohistochemistry in MEN1 diagnosis.

Conclusion: Menin immunohistochemistry is useful in the recognition of MEN1 syndrome and in the genetic analysis of patients with inconclusive MEN1 germline testing.


BRAF mutation and AXL (hyper)expression as markers of high risk for persistent/recurrent papillary thyroid cancer (PTC)

M. Martini*, C. Pizzimenti, A. Ieni, A. Campennì, L. Giovanella, G. Fadda

*Università degli Studi di Messina, Italy

Background & objectives: Thyroidectomy followed with 131-radioiodine therapy (RIT) is the main treatment for differentiated thyroid cancer (DTC) patients with an excellent response rate. The understanding of the PTC molecular mechanisms may be useful to identify patients with higher risk of persistent disease.

Methods: We analysed 42 low (n=32) or intermediate (n=10) risk PTC patients subjected to total-thyroidectomy and RIT with ablative or adjuvant purpose. The response to treatments were evaluated 6-12 months after RIT. The mutational status of BRAF, RAS, TERT, PIK3 and RET, the expression of PD-L1 (as CPS score) and AXL gene and CD4/CD8 ratio were analysed on surgical pathology specimens.

Results: Thirty patients had an excellent response (ER), 6 an indeterminate/incomplete bio-chemical response (BIndR/ BIR) and 6 a structural incomplete response (SIR). A significant correlation was found between BRAF mutation and high expression of AXL gene (p= 0.001) and between these parameters and persistent/recurrent disease, respectively (p=0.02 and p=0.03). BRAF mutation and high expression of AXL gene were also correlated to PD-L1 expression (p=0.004 and p=0.002). Moreover, PTC patients with persistent disease showed significantly higher PD-L1 and AXL expression and lower level of CD4/CD8 ratio (p=0.021, p=0.032 and p= 0.015, respectively). No significant association was found about RAS, TERT, PIK3 and RET alterations and persistent/recurrent disease, PD-L1 and AXL expression.

Conclusion: This data suggests that BRAF mutation and AXL (hyper)expression are correlated with increased expression of PD-L1 and CD8 in PTC patients with persistent disease with respect to those without persistent/recurrent disease after initial treatments. Accordingly, they should be considered as potential biomarkers of aggressive behaviour of PTC also suggesting other possible therapeutical targets.


Medullary thyroid carcinoma: conventional and emerging prognostic factors

M. A. Bani*, M. Faron, C. Kanaan, P. Khneisser, L. Lacroix, L. Lamartina, J. Hadoux, D. Hartl, V. Suciu, X. Sastre-Garau, E. Baudin, J. Scoazec, A. Al Ghuzlan

*Department of Medical Biology and Pathology, Morphological pathology laboratory, Gustave Roussy Cancer Campus, Villejuif, France / Paris-Saclay university, Gustave Roussy Cancer Centre, Inserm US23, CNRS UMS3655, AMMICa, Villejuif, France

Background & objectives: Prognosis of patients with medullary thyroid carcinoma (MTC) remains difficult to establish. The objective of our study was to evaluate the prognostic value of the patho-molecular characteristics in MTC.

Methods: Tumour tissues from primary MTC patients diagnosed at Gustave Roussy between 2003 and 2020 were reviewed to assess conventional prognostic factors and immunophenotype. Mutational status of RET was determined using New Generation Sequencing. Outcomes included overall survival (OS), biological (B-DFS) and morphological disease free survival (M-DFS). Univariate and multivariate statistical studies were conducted.

Results: 207 patients were included: 56% were females and the median age was 54 years. In the univariate study for OS: age (>50 years), size (>4cm), extrathyroidal extension, positive surgical margins, absence of encapsulation (AE), necrosis, high mitotic index (MI) (≥5 mitosis/2mm2), lymphovascular invasion, atypical mitosis were significantly associated with poor outcome. In the multivariate study, only AE(p=0.034), necrosis(p=0.001) and MI(p<0.001) remained statistically significant. Using the IMTCGS grading system, high grade tumours (MI≥5 and/or Ki67≥5 and/or necrosis) were associated to a decreased OS, B-DFS and M-DFS. Somatic RET_M918T mutation was found in 31/127(37%). There were no prognostic significant difference between high grade (15/50) and low grade (16/77) RET mutated MTC.

Conclusion: In patients with MTC, AE, necrosis and high MI were associated with poor OS, thus validating the prognostic value of IMTCGS in an independent cohort. Ten of the high grade and seven of the low grade RET_M918T mutated cases experienced relapse, suggesting that this biological trait may represent an independent biological prognostic factor from IMTCGS. Analysis of a larger panel is necessary to confirm these data.


Differential expression profiles of immunoregulatory genes in poorly differentiated and anaplastic thyroid carcinomas progressive from papillary carcinoma

G. Orlando*, J. Metovic, F. Maletta, C. Tampieri, F. Napoli, V. Zambelli, M. Volante, M. Papotti

*University of Turin, Italy

Background & objectives: Few data are available on the immunoregulatory mechanisms of thyroid cancer, with special reference to aggressive forms. We aimed at identifying profiles of expression of immune-related genes in anaplastic (AC) and poorly-differentiated (PDC) carcinomas with associated papillary carcinoma (PC) components.

Methods: Expression levels of over 700 genes involved in immune to tumour response mechanisms were investigated by using the nCounter® PanCancer Immune Profiling Panel in 9 PDC and 12 AC, all having a PC associated component. Moreover, in the 12 AC cases the matched PC component was analysed in parallel.

Results: Over 200 genes were differentially expressed in PDC as compared to AC samples, affecting all main pathways covered by the panel. All pathways were down-regulated in PDC. Pairwise analysis of AC and matched PC components showed a stable pattern of expression for most genes, with a few showing a high statistically significant differential expression (p<0.001 in t test analysis). Among those, 5 (MAP3K1, PRKCD, CYFIP2, BLNK and EPCAM) were down- and 6 (RIPK2, ITGB1, CCL3L1, ITGA5, PLAUR and TICAM2) were up-regulated in AC. Interestingly, for 9 of these 11 genes the pattern of deregulation between PC and AC components was consistent in all samples.

Conclusion: PDC and AC possess specific and different expression profiles of immunoregulatory genes even in the presence of a similar histological pattern of progression. Unexpectedly, progression of PC to AC is not associated with a wide deregulation of immunoregulatory mechanisms. However, a subset of genes is consistently impaired during AC progression, whose role as biomarkers and functional mechanisms need to be assessed and validated in future studies.

Funding: This work was supported by the Associazione Italiana per la Ricerca sul Cancro (Milan, Grant IG 20110 to M.P.).


High-grade medullary thyroid carcinoma, morphological features and prognostic value of the international grading system

C. Ariño-Palao*, R. Meléndez Gispert, H. Pian-Arias, T. Caniego-Casas, J. Molina Cerrillo, T. Alonso Gordoa, I. Ruz Caracuel

*Ramón y Cajal University Hospital, Spain

Background & objectives: Medullary thyroid carcinoma (MTC) is a rare malignant tumour without a widely accepted grading scheme. In 2021, a multicentric study developed the International Medullary Thyroid Carcinoma Grading System. We seek to employ this system and analyse its prognostic value.

Methods: We reviewed slides from a single-centre cohort of 28 MTC since 2002. Tumours were assigned high-grade based on the presence of at least one of three parameters: mitotic index ≥5 mitosis/2mm2, Ki67 proliferative index ≥5%, and tumour necrosis. We compiled additional pathological features and clinical information to perform a survival analysis.

Results: We identified 7 high-grade carcinomas (25%) and 21 low-grade carcinomas (75%). Two high-grade carcinomas showed all three parameters, whereas one presented both Ki67 proliferative index and tumour necrosis. Two expressed only Ki67 proliferative index ≥5% and the remaining two had tumour necrosis. High-grade carcinomas frequently showed an infiltrative pattern, spindle cell morphology, lymphovascular invasion, lymph node metastasis at diagnosis, AJCC stage III or IV, extrathyroidal extension, and affected surgical margins. Statistical analysis demonstrated decreased overall survival (median 41.57 months vs 64.80 months; LR= 17.412; p<0.001) and decreased disease-free survival (median 6.57 months vs 46.87 months; LR= 10.276; p=0.001) in high-grade carcinomas.

Conclusion: We confirm the prognostic value of the International Medullary Thyroid Carcinoma Grading System in an independent cohort. High-grade carcinomas associated significant decreased overall survival and disease-free survival, in addition to other pathological parameters of worse prognosis. Therefore, we advocate for a widespread use of this system to help clinicians on patient risk assessment.

Funding: Instituto Ramón y Cajal de Investigación Sanitaria - Intramural 2021 (projects grant 2021/0453).


Apocrine differentiation in salivary duct carcinoma: immunohistochemical evaluation for GATA3, p62 (Sequestosome1) and FABP7 in 106 cases

K. Kusafuka*, K. Ueda, H. Inagaki, J. Itakura, Y. Otsuki, I. Ito, N. Kuroda, T. Daa, K. Suzuki, H. Iwai, K. Yamanegi, Y. Imamura, M. Hamada, M. Yasuda, S. Baba, E. Nakatani, M. Suzuki

*Shizuoka General Hospital, Japan

Background & objectives: The up-regulation of p62(sequestosome1) and brain fatty acid binding protein(FABP7) was reported in apocrine carcinoma of the breast(ACB). Salivary duct carcinoma(SDC) is frequently seen as carcinomatous component of carcinoma ex pleomorphic adenoma(CXPA). We aim to elucidate their expressions in SDC.

Methods: We selected SDC cases from a pathology file of 11 institutions during 2000-2020, immunostained them for p62, FABP7 and GATA3, adding to androgen receptor(AR), and gross cystic disease fluid protein(GCDFP)-15. In the assessment for p62, nuclear signals were estimated as N type, whereas cytoplasmic signals were estimated as CY type. We examined with statistical analysis using R version 3.6.2 software.

Results: One hundred six cases were selected as SDC, including 59 cases of CXPA (20 cases of intracapsular type, 8 cases of minimally invasive type and 31 cases of widely invasive type). Eighty one percent of SDC was positive for p62 (N type, 59%; CY type, 10%; N+CY type, 23%). Twenty-nine percent and 90% of SDC were positive for GATA3 and FABP7, respectively. Fifty-two percent and 45% of SDC were positive for AR and GCDFP-15, respectively. The expression status of p62 CY type or (-) was related to worse outcome, whereas the GATA3(+) cases were related to better outcome. Atypical pleomorphic adenoma expressed p62(N type only) in the atypical luminal cells.

Conclusion: SDC should be called “apocrine carcinoma of the salivary glands”, due to the positivity for p62, FABP7 and GATA3, like ACB. Adding to GCDFP-15 and AR, the combinations of p62, FABP7 and GATA3 were considered to be new diagnostic markers for SDC. On the other hands, in the cases of CXPA, the luminal cells of PA may acquire the apocrine differentiation during the malignant transformation to SDC.


The prognostic role of cortactin overexpression in oral squamous cell carcinoma of tongue: large cohort study

B. Toközlü*, D. Sapkota, E.M. Vallenari, O. Schreurs, T. Soland

*Gazi University, Turkey

Background & objectives: Among oral cancer patients, those with squamous cell carcinoma of the oral (anterior 2/3rd) tongue are more likely to have higher mortality rates. Cortactin protein has been linked with progression and nodal involvement in the head and neck carcinoma.

Methods: One hundred twenty-five HPV negative, formalin fixed paraffin embedded specimens were used to show cortacin expression by constructing tissue microarrays using tissue cores of 2 mm diameter from both tumours invading front and the corresponding superficial/central areas.

Followed by an analysis of tumour cell invasion in 3D-organotypic co-culture models after siRNA mediated silencing of CTTN gene.

Results: Cortactin expression was higher at the invading core than at the corresponding central cores. High overall cortactin expression was positively associated with tumour size and distant metastases. High overall cortactin expression was associated with reduced 5-year overall survival. siRNA-mediated knockdown of cortactin resulted in reduced proliferative and invasive abilities of cell lines in 3D-organotypic co-culture models.

Conclusion: We showed that the expression of cortactin was significantly higher at the invading cores than at the corresponding central cores. The results of current study also demonstrated that the overexpression of cortactin was associated with a significantly reduced overall survival. Increased expression of cortactin was similarly associated with tumour size and distant metastasis. These findings were further confirmed by analysing the invasive and proliferative abilities of cells in 3D-organotypics. Our findings suggest a prognostic value for cortactin in oral cancer.

Funding: The author Burcu Toközlü was supported by a grant from TUBITAK 2021 during her stay in Oslo, and the project was supported by North Norway Regional Health Authority (Helse Nord project no. SFP1276-16).


Diagnostic utility of NR4A3 and NR4A2 immunohistochemistry in salivary gland pathology: a single-institution experience with 108 cases of acinic cell carcinoma

N. Klubickova*, A. Skalova, O. Koshyk, E. Mosaieby

*Bioptical Laboratory Ltd., Czech Republic

Background & objectives: Acinic cell carcinoma (AciCC) is a common salivary gland malignancy, typically composed of neoplastic acinic cells with zymogen granules. The vast majority of cases are driven by a t(4;9)(q13;q31) leading to enhancer hijacking and upregulation of the NR4A3 gene.

Methods: Seventy-one cases of classic low-grade AciCC, as well as 37 cases with high-grade transformation (HGT), were retrieved from institutional files and included in the analysis (n=108). Immunohistochemistry for DOG1, SOX10 and NR4A3 was performed in all cases; all NR4A3-negative and 10 NR4A3-positive cases were subsequently stained for NR4A2. FISH analysis of NR4A3 gene rearrangement was carried out in 1 case.

Results: The patients’ age ranged from 17 to 86 years (mean=56). Patients with HGT were almost two decades older than patients with low-grade AciCC (mean=67 vs. mean=50). 67% of patients were female. One hundred cases were primary tumours, most commonly occurring in the parotid gland, 6 cases represented recurrences, and 2 lung/pleural metastases were sampled. Immunohistochemical staining for DOG1 and SOX10 was positive in 94% and 97% cases, respectively. NR4A3 was at least focally positive in 104/108 (96%) cases. NR4A3 rearrangement was confirmed by FISH in 1 analysed case. Out of the 4 NR4A3-negative cases, 2 displayed strong nuclear immunopositivity with the NR4A2 antibody, while all 10 NR4A3-positive cases were negative.

Conclusion: Our analysis confirms that majority of AciCC, including cases with HGT, are immunopositive for NR4A3, and suggests that NR4A3 immunohistochemistry is a powerful tool in the differential diagnosis of salivary gland tumours. However, its utility is limited in older and/or sub-optimally fixed samples which often display weaker and focal positivity. Our study also indicates that in minority of cases, AciCC might be negative for NR4A3 immunostaining because the pathogenic genetic event in these cases is rather the overexpression of NR4A2.

Funding: This study was supported by study grant SVV 260539 from the Ministry of Education, Czech Republic (NK).


Molecular-genetic profile of sinonasal tumours: molecularly heterogeneous but histologically distinctive low-grade and high-grade tumours

M. Banecková*, A. Agaimy, M. Hyrcza, N. Ptáková, P. Martínek, J. Laco, N.J. Rupp, M.F. van den Hout, O. Koshyk, M. Michal, A. Skalova

*Department of Pathology, Charles University, Faculty of Medicine in Plzen, and Bioptic Laboratory, Ltd, Plzen, Czech Republic

Background & objectives: Sinonasal adenocarcinomas (SNAC) are classified as salivary and non-salivary adenocarcinomas. Non-salivary adenocarcinomas encompass a spectrum of high-grade intestinal-type adenocarcinomas (ITAC) and low-grade lesions potentially originating from seromucinous structures non-intestinal type adenocarcinomas (non-ITAC). The molecular genetic background is pleomorphic and inconsistent.

Methods: Retrospectively, 12 cases of sinonasal lesions with a detected gene mutation or gene fusion were selected from the registry of tumours. The cohort included seven cases of low-grade lesions or benign tumours and 5 cases of high-grade carcinomas. These tumours were compared morphologically and immunohistochemically.

Results: Low-grade lesions represented classic forms of seromucinous hamartoma / respiratory epithelial adenomatoid hamartoma or non-ITAC. High-grade tumours were basaloid, keratin positive, solid to papillary. The architecture was mostly monotonous.

In the low-grade malignant/benign subgroup there were detected 6 gene mutations (2x BRAF 2x RET, 1x KRAS, and 1x PDGFRA – this case showed an MYB::NFIB gene fusion).

In a group of high-grade carcinomas, ETV6::NTRK3 and EWSR1::COLCA1/2 were found in 2 cases. One case of epithelial-myoepithelial sinonasal carcinoma harboured an HRAS mutation. One case of sinonasal undifferentiated carcinoma developed two gene mutations in IDH2 and ASXL1 genes. One case of high-grade carcinoma showed dual CREBBP and BRIP1 gene mutation.

Conclusion: The sinonasal tract encompasses a wide spectrum of high-grade or low-grade lesions with pleomorphic molecular-genetic backgrounds and only a few of them are truly defined by gene fusion/mutation. There is a need to perform a further investigation to state the correlation between morphology and molecular genetics to better understand pathogenesis.

Funding: This study was supported by study grant SVV 260539 from the Ministry of Education, the Czech Republic.


Update to seromucinous hamartomas and respiratory epithelial adenomatoid hamartomas with dysplastic features and malignant transformation

M. Banecková*, M. Hyrcza, J. Laco, T. Vaněček, P. Martínek, P. Grossmann, S. Ihrler, N.J. Rupp, F. Petersson, O. Koshyk, D.V. Spagnolo, J. Sanjiv, M. Schlageter, A. Skalova, M. Michal

*Department of Pathology, Charles University, Faculty of Medicine in Plzen, and Bioptic Laboratory, Ltd, Plzen, Czech Republic

Background & objectives: Sinonasal hamartomas (SH) and respiratory epithelial adenomatoid hamartoma (REAH) are rare and underestimated lesions. The term hamartoma is used for an indolent lesion without neoplastic potential, however, SH/REAH does not always behave accordingly.

Methods: We have investigated twenty cases of “dysplastic” polypoid lesions diagnosed either as REAH and SH, adenoid cystic carcinoma (AdCC) arising in SH or REAH, and low-grade non-intestinal-type tubulopapillary adenocarcinoma (LG non-ITAC) arising in SH, low-grade adenocarcinoma ex REAH, and solitary fibrous tumour with SH. All cases were evaluated morphologically and immunohistochemically and 17 cases were tested by molecular-genetic methods.

Results: Dysplastic features of SH/REAH contained irregular cystic glands with atypical epithelial lining with occasional loss of stratification and often intraluminal snouts. The cells had nuclear membrane irregularities and coarse chromatin. The stroma was densely fibrotic almost of desmoplastic character. Immunohistochemical expression of p63 was patchy or negative in SH but preserved in AdCC. Interestingly, expressions of both S100 and SOX10 markers were observed in the seromucinous component but were lost in the AdCC component. We have detected 4 cases with gene fusions (MYB::NFIB and MYBL1::NFIB) and 5 cases with developed gene mutation (BRAF, RET, and PDGFRA).

Conclusion: Our findings not only strongly support that SH/REAH are genuine tumours, but also suggest that may represent a precursor lesion for the development of malignancies. SH/REAH may develop dysplastic features with the potential for risky behaviour. To the best of our knowledge, this is the largest molecular-genetic study of REAH/SH with the occurrence of glandular atypia in them and with a subset of cases with a direct transition into AdCC or LG non-ITAC.

Funding: This study was supported by study grant SVV 22639 from the Ministry of Education, the Czech Republic.


Multiparametric, in situ study of the microenvironment (MTE) of head and neck squamous cell carcinomas (HNSCC): impact of human papillomavirus (HPV). Macrophagic Infiltration Study

J. Martin*, M. Durand, C. Lepine, Y. Harrar, J. Bastian, C. Badoual

*laboratoire Inserm U970, PARCC, Université Paris Cité, France

Background & objectives: Our study aims to explore the composition of HPV-driven Head & Neck Squamous cell carcinoma (HNSCC) tumour microenvironment (TME), in particular the macrophagic infiltration.

Methods: A multiplex immunofluorescence macrophage signature was applied to 127 patients with HPV-driven HNSCC, using the OPAL® technique developed by AKOYA BioSciences®.

This technique allowed us to apply a macrophage labelling of 7 markers in a sequential manner. Macrophage polarization (CD68, CD163), expression of immune checkpoint inhibitors (PD-L1, PD-L2), tumour cells (CK) and negative regulation pathway of phagocytosis (SIRPa, CD47).

Results: A mapping of the distribution of macrophages between stroma and tumour could be established: unconventional macrophages called "M2" would be more abundant in the stroma, conventional macrophages called "M1" appear to be evenly distributed between tissues and stroma.

The expression of PD-L1 by M1 and M2 macrophages does not seem to be associated with the level of expression of HPV E6 and E7 mRNA in CISH. However, there is a statistical trend in favour of a correlation between the population of intratumoural M1 macrophages expressing SIRPa and the level of E6 and E7 expression.

Conclusion: Thus, E6 and E7 oncoproteins appear to influence SIRPa expression on macrophages M1 in HPV-driven OPSCC. There is a need to confirm the prognostic value of SIRPa expression and to further identify interactions with different lymphocyte populations.


Differential expansion of innate lymphoid cells and their role in oral squamous cell carcinoma

S.A. Syed*, M.A. Qureshi, S. Khan, R. Kumar

*Dow University of Health Sciences, Karachi, Pakistan

Background & objectives: Oral cancers are the commonest reported cancers in Pakistani males and 2nd commonest in females. We investigated infiltration and potential role(s) of innate lymphoid cells (ILCs) in an accelerated murine model of oral carcinogenesis and in well-defined human cancers.

Methods: We established an accelerated murine model of oral carcinogenesis and characterized tissue infiltration of ILCs in harvested cancer tissues using flowcytometry. We also investigated ILCs infiltration in well-defined human oral cancer tissues. Moreover, we inhibited ILCs using α-Th1 antibody to investigate potential role of ILCs in progression of oral cancers.

Results: 84% of the mice treated with 9,10-dimethyl-1,2-benzanthracene (DMBA) carcinogenic regime developed moderately differentiated squamous cell carcinoma and showed differential expansion of ILCs in various cancers. Moreover, well defined human oral squamous cell carcinoma samples also exhibited increased infiltration of ILCs (along with increased expression of selected/relevant cytokines). Tumour progression did not significantly differ upon inhibition of ILCs using α-Th1 antibody. However, ILCs expansion was different amongst the antibody treated and untreated groups.

Conclusion: We present novel data on differential expansion of ILCs in oral cancers which demands further exploration to exploit these newly discovered cells to devise novel diagnostic, therapeutic and prognostic strategies to prevent/treat oral cancers.


High-grade transformation in salivary gland tumours: a rare and under-recognized phenomenon

N. Batra*, A. Patil, N. Mittal, S. Rane, K. Kante, M. Bal

*Tata Memorial Hospital, India

Background & objectives: High-grade transformation (HGT) in salivary gland tumours (SGT) is an extremely rare phenomenon associated with aggressive clinical course. Herein, we aimed to study the clinicopathological spectrum of SGT with HGT diagnosed at our institute.

Methods: Clinical data and pathologic material of all cases (2014-2022) was reviewed and diagnosis confirmed as per the WHO 2017. HGT was defined by presence of unequivocal areas of high-grade carcinoma coexisting with conventional low-grade salivary gland carcinoma. Clinical and treatment details were recorded from the electronic medical records. Pathologic features of HGT and conventional areas were recorded and compared.

Results: 18 cases were identified. The median age was 53 years; male-to-female ratio was 2:3. The sites included:submandibular (n=5), parotid (n=4), palate (n=4), tongue (n=2), and 1 case each in nasopharynx, nasal cavity, floor of mouth, buccal mucosa, and maxilla. The histologic types included: adenoid cystic carcinoma (ACC, n=12), epithelial-myoepithelial carcinoma (n=4), polymorphous adenocarcinoma (n=1), acinic cell carcinoma (n=1). HGT areas displayed high-grade cytology, necrosis, brisk mitoses, and higher Ki-67 in comparison to their low-grade counterparts. Poorly differentiated adenocarcinoma was the most common HGT histology. Extra-parenchymal spread, margin involvement, lymphovascular and perineural invasion were identified in 50%, 25%, 30%, and 50%, respectively.

Conclusion: HGT of salivary neoplasms is a rare and frequently under-recognized occurrence that portends a poor prognosis. ACC is the most frequent underlying histology. Accurate diagnosis is essential as aggressive treatment is warranted.


OFP-10 | Oral Free Paper Session Breast Pathology


Mucoepidermoid carcinoma of the breast: multidimensional profiling reveals novel biomarkers and genetic drivers

M. Ivanova*, K. Venetis, E. Sajjadi, S. Andaloro, C. Rossi, M. Lucioni, U. Malapelle, F. Pagni, M. Barberis, E. Guerini Rocco, G. Viale, N. Fusco

*IEO, European Institute of Oncology, Italy

Background & objectives: Breast mucoepidermoid carcinomas (MEC) are rare salivary gland-type triple-negative breast cancers (TNBC), often considered low-risk malignancies. Their biology is poorly understood, so they pose diagnostic and clinical challenges. We sought to characterize the molecular landscape of breast MECs.

Methods: Thirteen breast MEC were histologically confirmed and subjected to tumour-infiltrating lymphocytes (TILs) profiling and PD-L1 (combined positive score, CPS), EGFR, and amphiregulin (AREG) immunohistochemistry. The MAML2 and EWSR1 rearrangements (recurrent in salivary MECs) were investigated by fluorescent in situ hybridization. Eight cases with enough tissue material were subjected to next-generation sequencing (NGS) of 161 cancer-related genes.

Results: Most cases were of low histological grade with Ki67<30% (n=10/13, 77%). TILs were found in 2 (17%) cases, while PD-L1 CPS ranged from 0 to 20 (median 12.5). All cases with available material showed EGFR overexpression and were AREG-positive (n=9/9, 100%). No MAML2 and/or EWSR1 rearrangements were detected. Pathogenic mutations in PIK3CA were highly recurrent (n=4/8; 50%), though only 1 (12.5%) case harboured a TP53 mutation. Additional somatic mutations affecting cancer-related genes found in MECs included CDK2, NF1/2, AKT1, SMARCB1, MYC, KRAS, CDK4, NOTCH1, and FGFR3/4. Taken together, complex patterns of genetic alterations were observed in PI3K/AKT/mTOR and cell cycle regulation pathways.

Conclusion: Here, we present the broadest collection of breast MECs comprehensively profiled for their molecular alterations. We demonstrate that these tumours lack the hallmark TP53 mutations often found in high-grade TNBC as well as MAML2 or EWSR1 rearrangements. The low TILs and PD-L1 levels suggest an immunoediting deficiency and that MECs may unlikely respond to immunotherapy combination strategies. Finally, the EGFR-AREG axis activation, and genomic alterations in PI3K/AKT/mTOR and cell cycle regulation pathways warrant caution in considering MECs as low-grade TNBCs.


Association of metastatic pattern in breast cancer with tumour type and patient specific factors: a nationwide autopsy study using artificial intelligence

F. Kazemzadeh*, A. Snoek, Q. Voorham, N. Hugen, M.G.H. van Oijen, I. Nagtegaal

*Department of Pathology, Radboud University Medical Center, Nijmegen, The Netherlands; Department of Medical Oncology, Amsterdam University Medical Center, University of Amsterdam, The Netherlands, Cancer Center Amsterdam, Therapy Program, The Netherlands

Background & objectives: Heterogeneity of metastatic pattern of breast cancer hampers treatment decisions. Large scale studies into underlying biological and patient specific factors are necessary to progress for future treatment of metastatic diseases.

Methods: Pathological records of 4831 patients diagnosed with breast cancer who underwent autopsy between 1974 and 2010 were retrieved from Dutch nationwide pathology databank (PALGA). Natural language processing (NLP) methods were applied to extract data from autopsy reports. Named entity recognition was based on SNOMED codes that are utilized in PALGA.

Results: The accuracy of data retrieval with NLP was above 0.9 and recall was 0.94 for majority of cases. Our model outperformed manual extraction of value of interest. We identified 2622 (54.2%) of patients with metastatic disease. Invasive ductal carcinoma, and mucinous carcinoma more frequently metastasized to lung and liver, whereas for invasive lobular carcinoma and mixed type this was to bone and liver respectively. There was no statistically significant association between lateralization and metastatic patterns, except for kidney (right= 5.0%, left= 8.9%, bilateral= 13.3%, p= 0.007). In a subgroup of patients, we found that (ER+/HER2+) patients, were more likely to metastasize to liver and bone, compared to (ER-/HER2+) patients.

Conclusion: This is the first large-scale study that demonstrates artificial intelligence methods are efficient for extracting information from Dutch pathology reports. We show differences in frequencies and combinations of metastatic sites between histological subtypes. The patterns and frequencies identified in this autopsy study may reflect the underlying biology of metastatic breast cancer and potentially influence the future follow-ups and patient-tailored treatment strategies depending on other clinical correlations.


Automated prognosis marker assessment in 2,004 breast cancers using an artificial intelligence-based framework for BLEACH&STAIN mfIHC

T. Mandelkow, E. Bady, J.H. Müller, N.F. Debatin, M.C.J. Lurati, C. Hube-Magg, N.C. Blessin, G. Sauter*

*University Medical Center Hamburg, Germany

Background & objectives: Prognostic markers in routine clinical practice of breast cancer are currently assessed using multi-gene panels. However, the fluctuating tumour purity can reduce the predictive value of such tests. Immunohistochemistry holds the potential for a better risk assessment.

Methods: To enable automated prognosis marker detection (i.e. HER2, GATA3, progesterone- [PR], oestrogen- [ER], and androgen receptor [AR], TOP2A, Ki-67, TROP2), we have developed and validated a framework for automated breast cancer identification, which comprises three different artificial intelligence analysis steps and an algorithm for cell-distance analysis of 11+1 marker BLEACH&STAIN multiplex fluorescence immunohistochemistry (mfIHC) staining in 2’004 breast cancers.

Results: The optimal distance between Myosin+ basal cells and benign panCK+ cells was identified as 25 μm and used to exclude benign glands from the analysis combined with several deep learning-based algorithms. Our framework discriminated normal glands from malignant glands with an AUC of 0.96. The accuracy of the approach was also validated by well-characterized biological findings, such as the identification of 13% HER2+, 73% PR+/ER+, and 14 triple negative cases. Furthermore, the automated assessment of GATA3, PR, ER, TOP2A-LI, Ki-67-LI and TROP2 was significantly liked to the tumour grade (p<0.001each). Furthermore, a high expression level of HER2, GATA3, PR, and ER was associated with a prolonged overall survival (p≥0.002 each).

Conclusion: A deep learning-based framework for automated breast cancer identification using BLEACH&STAIN multiplex fluorescence IHC facilitates automated prognosis marker quantification in breast cancer.


Upgrade rate and predictive factors for benign breast intraductal papilloma on core biopsy in Vancouver, Canada

T. Salisbury*, S. Koonmee, L. Ali, O. Ondic, R. Bhan, K. Pivovarcikova, A. Gurung, R. Alaghehbandan

*Department of Pathology, Faculty of Medicine, University of British Columbia, Royal Columbian Hospital, Vancouver, BC, Canada

Background & objectives: The management of benign intraductal papillomas (IDPs) diagnosed on core biopsy is controversial. We aim to determine the upgrade rate of IDPs diagnosed on core biopsy in subsequent surgical excision specimens and to identify associated clinical, pathologic, and radiologic factors.

Methods: This is a retrospective population-based study of all breast papillary lesions diagnosed on core biopsy from 2017-2019 in Fraser Health Authority in Greater Vancouver, Canada. Patient demographics, histopathologic, and radiologic findings were analysed. Upgrade was defined as atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS), encapsulated papillary carcinoma (EPC), solid papillary carcinoma (SPC), and invasive carcinoma on surgical excision.

Results: A total of 129 patients with benign IDPs diagnosed on core biopsy were included. The overall upgrade rate to atypia or malignancy was 9.3% (12/129) on final excision. This included 7 with ADH, 7 with DCIS, EPC or SPC, and 1 with invasive carcinoma. Predictors of upgrade included older age (55.6 vs 66.1 years, p < 0.0001) and larger lesion size (11.1 vs 15.1 mm, p = 0.001). Older age (≥ 55 years) (OR [95%CI] 5.3 [1.04-27.08]) was an independent predictor of upgrade. In our study, location (central vs peripheral) and BI-RADS radiologic category were not associated with predicting upgrade.

Conclusion: Our findings support surgical excision of IDPs diagnosed on core biopsy in women aged 55 years or older with large lesions, while a conservative approach (close clinical follow-up) may be warranted for younger women with smaller lesions.


Evaluation of dual colour-dual in-situ hybridization (D-DISH) for HER2/neu testing in breast cancer

S. Desai*, A. Rathi, P. Shah, A. Sahay, A. Patil, T. Shet

*Tata Memorial Centre, Homi Bhabha National Institute, India

Background & objectives: To standardize and validate the HER2 Dual ISH DNA Probe Cocktail (D-DISH) assay by VENTANA for HER2/neu testing in breast cancer using FISH as the gold standard and to assess the interobserver variability in interpreting D-DISH.

Methods: HER2/neu IHC, FISH, and D-DISH assay by HER2 Dual ISH DNA Probe Cocktail Assay (Ventana Medical Systems, Inc., Tucson) were performed on 120 breast carcinoma cases. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were calculated against D-DISH results for four pathologists (FISH-gold standard). The absolute agreement between FISH and D-DISH ratios, average signals was assessed.

Results: A concordance of 98.3% was observed between FISH and D-DISH assays. D-DISH showed a sensitivity of 95.92%, specificity of 100%, PPV of 100%, and NPV of 97.26%, with respect to FISH. Cohen's kappa statistic was 0.96 demonstrating perfect agreement. Intraclass correlation coefficient (ICC) values of 0.97 (HER2), 0.92 (CEP17), and 0.97 (HER2/CEP17) were observed. Interobserver variability showed an almost perfect agreement (kappa values 0.98-1.0) and ICC values of 0.96-0.98, 0.91-0.95, and 0.96-0.98 in measuring the HER2 signals, CEP17 signals, and HER2/CEP17 ratio, by D-DISH respectively. Interobserver variability between the four pathologists showed perfect agreement for genomic heterogeneity and group categorization, and moderate agreement for polysomy by D-DISH.

Conclusion: Our study successfully validated the D-DISH test using the updated version of HER2 Dual ISH DNA Probe Cocktail Assay (Ventana Medical Systems, Inc., Tucson, AZ) as a substitute for FISH assay in accurately predicting the HER2 gene status with significant interobserver reproducibility. We conclude that this D-DISH test may be introduced in rouine diagnostic services as a reflex test for detecting HER2 gene status.


Image-based identification of HER2 status in H&E-stained breast cancer slides

O. Greenberg, A. Zubkov, L. Trejo, D. Nataf, I. Hayun, A. Avinoam, I. Gazy, N. Paz-Yaacov, D. Hershkovitz*

*Pathology Department, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel

Background & objectives: Determination of HER2 status is critical for prognosis and guiding treatment decisions in breast cancer patients. Here we present a method to detect HER2 expression directly from routinely prepared diagnostic H&E slide images, using an image-based deep learning approach.

Methods: A HER2-classifier was generated using 251 H&E-stained slide images and their relevant IHC and FISH status from the pathology department at Sourasky Medical Center. Convolutional neural network (CNN) analysis was performed on on-the-fly augmented images. Multiple instance learning (MIL) algorithms and ranking training schemes were applied to create the categorical HER2-classifier (positive/negative), powered by Imagene-AI.

Results: The HER2-classifier was evaluated on a validation set including H&E images only of 104 retrospective cases. The model performance values were 87.5% sensitivity, 85.9% specificity, 86.14% accuracy and 0.89 AUC. To further evaluate the AI-solution additional 245 cases were analysed. In this set, a high proportion (n=9/20; 45%) of false callings was observed in samples with HER2 IHC=3. The IHC slides of samples with score 3 in the set were re-evaluated by two pathologists. While three of the 9 false-negative cases (33%) status was changed to 2, by at least one pathologist, there were no changes in the true-positive group (n=9).

Conclusion: Implementation of Image-based solution to routine pathology workflow can support fast, cost-effective and standardized method for biomarker detection. We evaluated the use of an AI-model to analyse HER2 status compared to conventional IHC and FISH methods. Analysis of 349 cases resulted in 85.3% accuracy. IHC, manually analysed by pathologists, is a subjective method with both intra- and inter-observer discordance's reported. An AI-solution can support the routine workflow flagging cases where re-evaluation can support the pathologist analysis of difficult cases.


Application of molecular imaging as potential prognostic biomarker for triple-negative breast cancer (TNBC)

C. Villa*, P. Rainone, M. Cadamuro, S. Valtorta, S. Todde, R.M. Moresco, M. Lavitrano

*Department of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy

Background & objectives: The administration of the antihypertensive syrosingopine in combination with metformin provided a synergistic effect against different tumours, including breast cancer. We aimed to investigate the response to metformin and/or syrosingopine, evaluating the [18F]FDG and [18F]FLT as potential early prognostic biomarkers.

Methods: Balb/c female mice were inoculated subcutaneously with murine TNBC cells (4T1) and divided into six treatment groups: vehicle, cisplatinum, metformin, syrosingopine or cisplatinum plus metformin and metformin plus syrosingopine. The response to treatment was monitored by caliper measuring, for tumour volume and PET studies. Molecular biomarkers of glucose metabolism and tumour invasiveness were analysed by means of immunohistochemistry and q-RT-PCR.

Results: A significant tumour growth inhibition (%TGI) has been observed only after metformin plus syrosingopine administration, confirming a synergistic effect after ten days of treatment. PET analyses revealed a significant reduction of [18F]FLT tumour uptake in cisplatinum plus metformin (*p<0.05) and metformin plus syrosingopine (*p<0.001) treated groups, whereas [18F]FDG uptake increased in all experimental conditions. Molecular analyses performed by both immunohistochemistry and q-RT-PCR demonstrated a significant decrease of the lactate transporter MCT4 levels in mice treated with cisplatinum and metformin plus syrosingopine (*p<0.05) as well as low levels of the EMT biomarker Snail only in the group treated with the combination of metformin plus syrosingopine (*p<0.05).

Conclusion: Our data suggested that the combined administration of metformin plus syrosingopine is able to modulate the glucose metabolism and inhibit the tumour invasiveness and growth of cancer cells. Moreover, the use of [18F]FLT radiotracer may represent a potential biomarker for the early response to treatment of TNBC.


Challenges of breast NEN: NEN G3 and genetic analysis

Y. Liu*, M. Zhao

*The Fourth Hospital, China

Background & objectives: The Breast NEN(Br-NEN)was graded according to Nottingham standard, resulting in confusion between Br-NENG3 andNETG3. It was unclear whether therapeutic targets associated with gastroenteropancreatic NENare applicable in Br-NEN. This study aims to illustrate these issues.

Methods: The 50 cases were re-diagnosed according to the 5thWHO classification criteria. The gastroenteropancreatic NEN characteristic immunohistological staining (SSTR1-5, DLL3, ISL-1, INSM-1) were performed, and DNA was isolated from the primary tumour for exome sequencing. In this study, NET G3 was set as Nottingham grade 3, excluding small cell carcinoma and large cell neuroendocrine carcinoma.

Results: 1)Compared with G3 and NEC,the MYC pathway was enriched in G3, and the TP53 pathway was more common in the latter (P=0.036).

2)The G3 had more ZNF703 and FGFR1 genetic mutations than NEC, and was more likely to exhibit the genetic characteristics of Luminal B.

3)The SSTR2 and INSM1 positive expression was more common in the G1-2, P=0.006 vs 0.012, and showed decreasing trend in NEC compared with G3. No positive expression of DLL3 was found in G3except NEC.

4)The metastasis occurred in 3 cases (6% G3,11% NEC, 4% G1-2) and the breast cancer-related deaths occurred in 4 cases(12% G3,22% NEC). G3 showed poor prognosis than G1-2, but better than NEC.

Conclusion: 1)The poorly differentiated group has more frequent genetic alterations and poor prognosis.Br-NET G3 show genetic alterations and a better prognosis distinct from Br-NEC. Whether the Br-NET G3 concept is different from the Br-NEN G3 needs to be explored by more data.

2)The expression pattern of SSTR1-5, DLL3, ISl-1 and INSM-1 in Br-NEN was similar to that in gastroenteropancreatic NEN, so based on these biomarkers, it may be necessary to explore the potential therapeutic significance in Br-NEN.


Prediction of the efficacy of neoadjuvant therapy for breast cancer with immunologic and genome characteristics

Y. Liu*, M. Han

*The Fourth Hospital, China

Background & objectives: The effect of different immune cells and genomic alterations in tumour microenvironment before neoadjuvant therapy (NAT) in breast cancer patients on efficacy is currently uncertain. This study aimed to explore its impact on efficacy of NAT in breast cancer patients.

Methods: 81 cases of breast invasive cancer diagnosed by core needle biopsy before NAT were selected. Multiple Immunohistochemical/Immunofluorescence (mIHC/IF) detected CD3, CD8, PD-L1 and PD-1. Next generation sequencing (NGS) technology analysed the genome and detect somatic cell variation. Logistic regression analysis was used to draw a nomogram to predict the pCR rate of breast cancer patients.

Results: Patients were divided into two groups by NAT efficacy: pCR and non-pCR. The mIHC/IF results showed expression level of stromaCD8+cells, parenchyma and stroma PD-L1+cells in pCR was higher, with statistically significant differences (P < 0.05).NGS testing results showed the highest mutation rate was TP53(81%), followed by ERBB2(49%), PIK3CA(47%), CDK12(46%) and LRP1B (8%), et al. The mutation types include missense mutation, copy number amplification, synonymous mutation, et al. Only the difference in LRP1B mutation rate between two groups was statistically significant (P=0.011).Cox regression analysis showed expression level of stromal CD8+ cells, stromal and parenchymal PD-L1+ cells and LRP1B mutation rate influenced pCR (P < 0.05). The calibration chart(AUC:0.78) showed that the nomogram performs well and has high pCR predictionability.

Conclusion: There is a difference in immune microenvironment and gene expression profiles between pCR and non pCR patients. And the expression levels of CD8 + cells in tumour stroma, PD-L1+ cells in tumour parenchyma and tumour stroma are high, and LRP1B gene is prone to mutationin pCR patients. The nomogram of predicting pCR rate has good consistency, which can provide a certain predictive value for breast cancer patients with neoadjuvant therapy.


Histological assessment of large fresh breast surgical specimens with ultra-fast slide-free confocal microscopy: a feasibility study

M. Mathieu*, M. Ragazzi, M. Ferchiou, P. Van Diest, O. Casiraghi, M. Nap, A. Ben Lakhdar, N. Labaied, A. Conversano, M. Abbaci

*Gustave Roussy Cancer Campus, France

Background & objectives: A new generation of ultra-fast confocal microscope (UFCM) with a large field of view allows ex-vivo analysis of surgical specimens. We present the standardization of UFCM protocol for breast lumpectomy and the original online training program in breast UFCM images.

Methods: Fresh lumpectomies from 55 patients with breast conservative surgery were cut in two, stained with acridine and imaged with Histolog-Scanner-field of view of 20 cm² (Samantree Medical). The images were colored in purple to simulate frozen sections and annotated by three pathologists expert in breast and confocal pathology. The images were used for a training program followed by two pathologists.

Results: All surgical specimens were successfully imaged. Among these 55 cases, 49 were carcinomas (31 invasive no special type, 11 invasive lobular, 7 ductal carcinoma in situ) and in 6 cases no tumour was present at definitive histological examination. The UFCM protocol was completed in 8-10mn. The global architecture of the tissue was evaluated at low magnification and the cellular details at zoom (x40). The 30μm of axial resolution resulted hypercellularity compared to final histology. Training program was composed of 135 reading sheets including tumoural and non-tumoural features. Pathologists who completed the training program performed a diagnosis on the 88 self-assessment sheets getting 100% accuracy.

Conclusion: UFCM allowed to quickly image a 20 cm² area of an entire section of fresh breast lumpectomy. The training of pathologists in reading UFCM images was successful, probably since the images resemble traditional frozen sections. However a training is mandatory because the magnification of the images is lower than with microscope and the optical section is 30 μm thick. The pilot study points the feasibility of UFCM for fast and extensive intraoperative margin assessment during breast surgery.

Funding: SamanTree medical, European Union’s Horizon 2020 research and innovation program under grant agreement No 823284


PIK3CA mutations in endocrine-resistant breast cancer

C. Schagerholm*, S. Robertson, B. Holm, H. Awier, H. Toosi, E. Sifakis, J. Hartman

*Department of Oncology-Pathology, Karolinska Institutet, Sweden

Background & objectives: The majority of breast cancer patients' tumours express oestrogen receptor alpha (ER) but despite endocrine therapy, one-third progress or relapse. Studies investigate the phosphoinositide-3-kinase pathway, introducing therapies targeting PIK3CA. This study aims to examine the mutational prevalence in endocrine-resistant tumours.

Methods: In a retrospectively collected cohort of verified endocrine-resistant breast tumours diagnosed in 2008-2012, primary ER+ and human epidermal growth factor receptor 2 (HER2) negative breast cancers with an ER+/HER2- relapse during ongoing endocrine therapy were included. Targeted gene panel sequencing was performed on formalin-fixed paraffin-embedded tumour tissue and paired analysis was carried out between the relapse and primary tumours.

Results: The overall preliminary analysis showed PIK3CA mutations in 48.9% of all patients (n/N=23/47), of which 65.2% (n=15) had mutations in the 11 known hotspot regions. 65.2% of the mutations were similar between the primary and relapse tumours, 73.3% of these were found in hotspots, whilst 34.8% seem to develop or disappear during ongoing endocrine therapy. In primary tumours of patients that eventually relapsed during endocrine therapy, 60.9% had mutations in hotspots and 30.4% outside of these regions. In their relapse tumours, 56.5% had hotspot mutations and 26.1% elsewhere.

Conclusion: Our study of this unique cohort suggests that endocrine-resistant breast cancers are associated with high PIK3CA mutation frequencies, with significant proportions occurring outside of the known hotspot regions; analysis is ongoing for prognostic value combined with clinical data. Similar mutational profiles of primary and relapse tumours suggest invariability through treatment and may thus aid in utilizing diagnostic tools for therapeutic response prediction. Moreover, similar and alternating mutations of the tumours can be further investigated for incorporation of future mutation-targeted treatments.

Funding: This trial is a non-interventional study set up as a research collaboration between Karolinska Institutet and Novartis Sweden AB.


Breast carcinoma with apocrine differentiation: less indolent than expected?

C. Rossi*, S. Fraticelli, E. Boveri, G. Di Giulio, M. Fanizza, B. Francesco, A. Sgarella, A. Ferrari, A. Della Valle, E. Ferraris, A. Lasagna, G. Rizzo, E. Bonzano, M. Paulli, M. Lucioni

*Unit of Anatomic Pathology, Department of Molecular Medicine, Fondazione IRCCS Policlinico San Matteo, University of Pavia, Italy

Background & objectives: Breast carcinoma with apocrine differentiation is a rare (1%) breast cancer histotype characterized by apocrine morphology in >90% of cells, that typically shows positivity for androgen receptor (AR) and it’s either triple-negative or HER2-positive, non luminal.

Methods: At our Institution, 37 patients were diagnosed with breast carcinoma with apocrine differentiation between January 2010 and December 2020. Data about patient’s age, and tumour histological grade, ER, PR, Ki-67, AR and HER2 status and TNM staging was obtained from pathological report. Of note, no patient had undergone neoadjuvant therapy.

Results: Of our 37 cases, 25 were classified as high grade, and 11 as low or intermediate grade according to the Nottingham Histologic Score. The high grade group comprised slightly younger patients (median age: 69 vs 71,5) and showed a statistically significant prevalence of HER2-amplified tumours (14/25 vs 1/12, p=0.0106) and a higher Ki-67 average score (28% vs 13% in the low and intermediate grade tumours); no difference was observed in loco-regional stage of the tumour (average primary tumour dimension: 22,7 mm vs 18,1 mm; cases with positive lymph nodes: 11/25 vs 6/12).

Conclusion: Despite being regarded as an histotype with a more indolent course, our data shows that even lower grade apocrine carcinoma has a high propensity for loco-regional spread, and should be managed accordingly; the net difference in prevalence of HER2 amplification between low and high grade tumours suggests that different, unknown molecular events underlie disease progression in the two groups, and warrants further studies to define the biology of this histotype.


Claudin-1 expression in triple-negative breast cancers (TNBCs) and its clinical significance

A. Ouban*

*Alfaisal University College of Med, Saudi Arabia

Background & objectives: TNBC is an aggressive disease, lacking therapeutic and prognostic markers. Claudin-1, a biomarker with a prognostic value in several tumours, reportedly has conflicting results in TNBCs. The authors shed some light on claudin-1 expression in TNBC and its value.

Methods: We analysed the expression of claudin-1, a tight-junction protein that has a promising prognostic value in several cancers, by immunohistochemistry, in TNBC cases from the King Khaled university hospital. This expression was cross-checked against clinical-pathological criteria of TNBC patients, and against beta-Catenin expression in the same patients' sample.

Results: Claudin-1 was significantly expressed in the majority of TNBC cases. This expression was significantly correlated with lymph node metastases, tumour invasion, higher tumour nuclear grade, higher clinical and TNM staging, adverse survival outcome and failure to achieve remission following neoadjuvant chemotherapy (NAC). TNBCs' claudin-1 expression was also correlated with grade-2 abnormal expression of beta-Catenin (AEB) in the same patients' sample.

Conclusion: Most cases of TNBCs in our institution expressed the claudin-1. This expression is strongly linked to parameters of poor prognosis. The above may shed some light on the possible role of claudin-1 in TNBC; and may present an opportunity for the use of this biomarker in the management of TNBC patients.


Filamin-A expression in triple negative breast cancer (TNBC) and its clinical significance

A. Ouban*

*Alfaisal University College of Med, Saudi Arabia

Background & objectives: TNBC presents a clinical dilemma with early recurrence, metastases and poor survival outcome. Filamin-A is recently discovered protein which plays a dual role as oncogene and tumour-suppressor gene in many cancers. This study analyzes the expression of Filamin-A in TNBCs.

Methods: This study analysed the expression of Filamin-A, using immunohistochemistry, in a tissue microarray of 50 cases of triple-negative, invasive ductal breast carcinomas. Filamin-A expression was cross-checked against clinic-pathological attributes of the TNBC cases including age, grade, clinical stage and TNM staging. Filamin-A expression within the cell was analysed. The TNBC cases were further categorized by the intensity of Filamin-A expression.

Results: A significant majority of this study's TNBCs exhibited positive expression of Filamin-A. All Filamin-A positive TNBC cases expressed the protein in the cytoplasm of the tumour cells. Filamin-A expression was significantly correlated with TNBC grade, clinical stage, and TNM staging. A significant majority of TNBC cases had the highest intensity of Filamin-A expression (35/45, IRS=12). This study is the first to analyse expression of Filamin-A only in TNBC cases. When compared with other studies which analysed this expression in breast cancer sets (mixed sets); it was noted that the number of Filamin-A positive cases were much higher in this study's TNBC set.

Conclusion: Given the versatile role of Filamin-A, with its numerous interactions with cytoskeleton components of tumour cells and with signalling proteins, in addition to its role in modulating sensitivity to the chemotherapeutic agent Docetaxel, make the results of this study particularly important in TNBC patients, who have few management options. This study provides evidence of the clinical significance of Filamin-A in TNBCs, and proposes additional studies to pinpoint the exact function of this protein in this subset of breast cancer patients.


Nullisomy of chromosome 17 in invasive breast cancer: characterisation of a rare and puzzling genomic event

A. Valent, A. Fitouri, F. Delalande, N. Joyon, P. Michenet, M. Lacroix-Triki*

*Department of Pathology, Gustave Roussy, France

Background & objectives: In situ hybridization (ISH) is systematically performed for HER2 2+ invasive breast cancer. Nullisomy is a rare genomic event that may mislead the pathologist in ISH interpretation. We aimed at describe the pathological and molecular features of three nullisomy cases

Methods: Three cases of cen17 nullisomy breast cancer (BC) were retrieved from our archives. Clinical, pathological and treatment data were collected for each case. HER2 immunohistochemistry and HER2/cen17 fluorescent ISH were scored following the last ASCO/CAP guidelines. DNA was extracted from formalin-fixed paraffin-embedded samples and subjected to SNP array (OncoscanTM). A Foundation One liquid CDx analysis was available for one patient.

Results: All patients presented with metastatic BC, two of them from the outset. All tumours were high grade, oestrogen receptor positive. HER2 score was 2+ (n=2), or 3+ heterogeneous (n=1). All cases showed no signal in tumour cells with cen17 probe, whilst a signal was observed in normal cells. Two cases were HER2 amplified (HER2=7.9 and 9). All cases displayed a complex genomic profile with homozygous loss of the chromosome 17 centromeric region, associated to multiple quantitative chromosomic alterations. All cases showed chromosome 8q gain involving CCNE2 and MYC genes. Liquid biopsy sequencing of the lobular case additionally identified ESR1, PIK3CA, CDH1, DNMT3A, FAM123B, PTPN11 and TP53 mutations.

Conclusion: Nullisomy of chromosome 17 centromeric region is a poorly known genomic event that may prove puzzling for the pathologist while reading HER2 ISH in invasive BC. Our study shows that this exceedingly rare alteration is associated to BC showing a strikingly aggressive clinical course, and displaying a consistent complex genomic profile.

OFP-11 | Joint Oral Free Paper Session IT & Other Topics (Electron Microscopy / Cardiovascular Pathology)


Epidermolysis bullosa: an electron microscopy study of 6 cases

A. Cohn*, C. Salavastru, M. Gherghiceanu

*Emergency University Hospital, Bucharest, Romania

Background & objectives: Epidermolysis bullosa (EB) is a group of rare inherited disorders characterized by skin fragility and blistering after minor trauma. This study presents the ultrastructural features of six cases of EB, highlighting the diagnostic role of transmission electron microscopy (EM).

Methods: Skin biopsies from six infants were processed for both light microscopy (LM) and EM. Five of six patients were younger than 1 month. Clinical presentation included skin fragility and blisters in all cases. Oral mucosa involvement was reported in one case. LM revealed a completely detached epidermis in all cases of dystrophic EB.

Results: EB cases were classified based on the split level as EB simplex (EBS), junctional EB (JEB), and dystrophic EB. One case showed intraepidermal cleavage, intrakeratinocyte splitting, and tonofilament clumps suggesting generalized severe EBS.

EM findings of JEB were found in one case. These included multiple microsplits through the lamina lucida and a reduced number of anchoring filaments into the lamina densa.

Ultrastructural features of both EBS and JEB were found in one case, revealing intra-lamina lucida splits, suprabasal cleavage, and intracytoplasmic fractures.

All three cases of dystrophic EB showed a split below the lamina densa, with absent or markedly reduced anchoring fibrils between the lamina densa and collagen bundles.

Conclusion: Even though TEM tends to be replaced by immunolabeling and genetic testing, ultrastructural studies are still relevant in the diagnosis of EB. Not only can EM establish the major type of EB by identifying the level of the split in the skin, but also it can provide critical prognostic information in several subtypes. Moreover, EM can detect subtle changes in the dermo-epidermal junction and was proven to be superior in diagnosing EBS in absence of evident blisters.


Clinico-pathologic correlation in cardiac amyloidosis: is there a substrate for the echocardiographic “relative apical sparing” sign?

M. De Gaspari*, G. Sinigiani, S. Rizzo, M. Perazzolo Marra, M. Della Barbera, D. Mele, C. Basso, A. Cipriani

*Cardiovascular Pathology Unit, Department of Cardiac, Thoracic, Vascular Sciences and Public Health, University of Padua - Azienda Ospedaliera, Padova, Italy

Background & objectives: Myocardial longitudinal strain (LS) and strain rate by speckle-tracking echocardiography play a prognostic role in cardiac amyloidosis (CA) and relative apical sparing of LS (RELAPS) is useful for early diagnosis. Our aim was to assess regional differences in amyloid burden.

Methods: Retrospective study of whole hearts from autopsy with histological evidence and immunoelectron microscopy typing of CA. Amyloid burden was assessed quantitatively by histomorphometry on sodium sulphate-Alcian Blue stained transmural slides at different levels from base to apex. The parameters of myocardial LS by echocardiography performed before death were compared to the amyloid burden and basal-to-apex distribution.

Results: Of the 29 hearts examined, amyloid typing identified 19 cases of immunoglobulin light chains (AL, 65.5%) and 10 transthyretin (ATTR, 34.5%). A prevalent interstitial deposition was found in 20 (69%) and vascular in 9 (31%). Among the latter, all but one (88.9%) were AL. A homogeneous distribution of amyloid was demonstrated, with a median of 25.38, 26.70 and 18.8 (P=NS) at the basal, mid and apical sections, respectively. In 11 patients, echocardiography during the same hospitalization showed a significant LS basal-to-apex gradient. A correlation was found between total histological amyloid burden and the reduction of LS at echocardiography, although the RELAPS didn’t match to a basal-to-apex gradient of amyloid.

Conclusion: This clinico-pathological study demonstrates that amyloid is evenly distributed in the ventricular myocardium both in AL and ATTR. While there is a correlation between total amyloid burden and reduction of LS, the typical basal-to-apex gradient in LS at echocardiography does not appear to be explained by a gradient of amyloid burden in whole hearts. Our findings thus suggest that RELAPS is an epiphenomenon of complex interactions among amyloid infiltration, myocardial structure and consequent adaptation.


Acute myocarditis in COVID-19 era: pre-pandemic and pandemic periods compared

M. Riefolo*, M. Sabatino, C. Baldovini, C. Marcelli, L. Potena, O. Leone

*Pathology Unit, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Italy

Background & objectives: Acute Myocarditis (AM) has been much discussed as one of the most frequent cardiac complications of COVID-19. Despite serious clinical suspicion, however, there is no substantial EMB histological series.

Methods: At the S.Orsola Bologna Centre, cardiologic referral Centre for Emilia-Romagna region, we retrospectively analysed and compared two groups of patients who underwent EMB for clinical suspicion of myocarditis in the 5 years pre-pandemic (2015-2019, Group 1) and in the initial 15 months of the pandemic (April 2020-June 2021, Group 2), before the vaccine became generally available in our region.

Results: In group 1, of 65 patients who underwent EMB, 31 (47.7%) had a histological diagnosis of AM, with histotype: lymphocytic in 24 cases, giant cell in 1, toxic in 1, mixed in 1. Extension of inflammation was focal in 19 and multifocal/diffuse in 12. Month ratio in suspected cases was 1.1; in histological confirmed cases 0.5.

In group 2, of 23 patients 13 had EMB positive for AM (56.5%), with histotype: lymphocytic in 8 cases, giant cell in 1, eosinophilic in 2, mixed in 2. Extension of inflammation: was focal in 7 and multifocal/diffuse in 6. Month ratio in suspected cases was 1.5, in histological confirmed AM, 0.9.

Conclusion: In this study we analysed the frequency, clinical presentation and histological parameters of suspected AM before and during the pandemic. During the pandemic there was an increase in number of cases, in terms of both clinical suspicion and of histologic confirmation at EMB. The cases were generally more severe and showed a different range of histotypes, but there was no real correlation with SARS-CoV-2.


From conventional congenital cardiac surgery to molecular cardiac surgery: between darkness and light. New paradigms for investigations and treatments during pregnancy

A. Capuani*

*Private Organization Carrara, Italy

Background & objectives: We advocate new biological models and protocols for investigation and treatment of severe CHD on the light of nowadays spectacular progresses of the Molecular Biology with the Next Generation Sequency (NGS), Microarray Technologies and CRISPR-Cass9 Technique.

Methods: We reconsidered the embryogenesis and the morphology of the most severe forms of CHD, from TF to Transpositions and Univentricular Hearts, considering the diagnostic potentiality of maternal liquid biopsies in pregnancy and with emphasis on the CHD embryogenetic patterns. (Capuani et al. Ann Thorac Surg 1995, J Cardiothorac Surg 2014, Virchow’s Archives 2015, 2016, 2020).

Results: We found a common morphological denominator encompassing all pathological cardiac settings: the Trabecula Septomarginalis (Leonardo’s Cord) sequential counterclockwise malrotation. Several genes are involved in the process with over and under expression and network interactions. Microarray and NGS analysis applied early in pregnancy and to each single step of the malrotation may lead to a very early diagnosis and possible treatments. We present our research protocol.

Conclusion: Each malformed cardiac phenotype has a specific molecular profile. The TSM malrotation protocol encompass all cardiac pathological phenotypes and is proposed as a model for investigation and treatment of CHD early in pregnancy by NGS, Microarray and CRISPR-Cass9 technique, what we refer as Molecular Cardiac Surgery.


Reporting guidelines for pathology AI research – a review

C. Mcgenity*, D. Treanor

*Leeds Teaching Hospitals NHS Trust, United Kingdom

Background & objectives: An explosion of interest into applications of artificial intelligence (AI) is transforming pathology research. Complete reporting of research is essential for avoiding research waste and benefitting patients. The objective of this work was identifying reporting guidelines for pathology AI research.

Methods: The Equator Network library of 499 reporting guidelines and extensions was systematically searched to identify those applicable to pathology AI research. Inclusion and exclusion criteria were used and guidance was screened for utility at different stages of research and for a range of study types. Items were compiled to create a summary for easy identification of useful guidance and templates.

Results: 70 reporting guidelines and extensions applicable to stages of pathology AI research were identified. These were categorised into 5 groups: Literature & Research Priorities, Discovery, Clinical Trial, Implementation and Post-Implementation & Guidelines. A summary resource was developed for pathology AI researchers, with links to guidelines for these 5 groups, to assist in complete reporting of research. Guidelines currently in development and those useful at multiple stages of research were also highlighted. Our group recently published a study demonstrating that essential information is underreported in pathology AI studies, making replication difficult. Therefore, this summary will be shared publicly to highlight the availability of reporting guidance to the pathology AI research community.

Conclusion: Replication and research waste are recognised to be problematic in AI research. Reporting guidelines can be used as templates to ensure the essential information needed to replicate research is included within journal articles and abstracts. Reporting guidelines are available and useful for many study types, but greater awareness is needed to encourage researchers to utilise them and for journals to adopt them. This review and summary resource highlights guidance to pathology AI researchers, aiming to improve completeness of reporting.

Funding: Dr McGenity is funded by Leeds Hospitals Charity and the National Institute for Health Research (NIHR). Prof. Treanor is funded by National Pathology Imaging Co-operative (NPIC). NPIC (project no. 104687) is supported by a £50m investment from the Data to Early Diagnosis and Precision Medicine strand of the Government’s Industrial Strategy Challenge Fund, managed and delivered by UK Research and Innovation (UKRI).


A holistic evaluation of three-dimensional biomarker expression and genetic alterations in non-small cell lung cancer using tissue clearing technology

L. Wang*, Y. Hsieh, Y. Hung, Y. Chen, Y. Lin, Y. Lin, T. Chou

*Taipei Veterans General Hospital, Taiwan

Background & objectives: The common practice of assessing programmed death-ligand 1 (PD-L1) expression based on a single section may not be representative. To overcome this problem, we developed a novel protocol that can make formalin-fixed, paraffin-embedded tissue translucent, allowing three-dimensional (3D) imaging.

Methods: Our protocol can process tissues up to 150-μm in thickness, allowing anti-PD-L1 staining of the entire tissue and producing high resolution 3D images. After 3D imaging process, the thick sections were recovered for epidermal growth factor receptor (EGFR) mutation analysis. We further developed artificial intelligence-assisted models to calculate the tumour proportion score (TPS) of the entire 3D tissue.

Results: Artificial intelligence-assisted PD-L1 quantitation of these images revealed a marked variation of PD-L1 expression in 3D. In 5 of 33 needle-biopsy-sized specimens (15.2%), the TPS varied by greater than 10% at different depth levels. In 14 cases (42.4%), the TPS at different depth levels fell into different categories (<1%, 1–49%, or ≥50%), which can potentially influence treatment decisions. The EGFR mutation analysis performed using pre- and post-processing tissue yielded identical results in all the tested cases, including 4 cases with EGFR L858R mutation, 6 cases with EGFR exon 19 deletion, 1 case with EGFR exon 20 insertion, 1 case with EGFR G719X mutation, and 8 cases without detectable EGFR mutation.

Conclusion: Our novel method has the potential to increase the accuracy of tumour PD-L1 expression assessment and enable precise deployment of cancer immunotherapy. In addition to application in PD-L1 expression assessment in non-small cell lung cancer, 3D tissue imaging can also potentially apply to the evaluation of biomarkers in other cancer types. Importantly, our technology permits recovery of the processed tissue for subsequent mutation analysis, enabling holistic evaluation of the protein-level expression and genetic alterations in small specimens.

Funding: Industry-academia collaboration grant from JelloX Biotech Inc. (grant number R-19005)


CoNIC Challenge: large scale assessment of automated methods for identification and counting of Colon Nuclei

S. Graham*, Q.D. Vu, M. Jahanifar, D. Snead, S. Raza, F. Minhas, N. Rajpoot

*University of Warwick, United Kingdom

Background & objectives: Identification of nuclei in histology images, such as those from epithelial and inflammatory cells, enables large-scale profiling of the tumour microenvironment. To help drive forward innovation for automatic nuclear recognition, we organised the Colon Nuclei Identification and Counting (CoNIC) Challenge.

Methods: We created the largest dataset for nuclear recognition in computational pathology, containing around 550K labelled nuclei, and invited researchers to develop algorithms on the data, aimed at solving 2 tasks: 1) nuclear segmentation & classification and 2) prediction of cellular composition. Participants submitted model code to the challenge, which enabled the test data to remain completely unseen.

Results: In total, we had 323 and 50 submissions to the preliminary and full test phases, respectively. Submissions were ranked by multi-class panoptic quality (mPQ+) and multi-class coefficient of determination (R2) for tasks 1 and 2, respectively. The top segmentation and classification submission achieved an mPQ+ of 0.501 and the top cellular composition prediction submission achieved an R2 of 0.764. Best models were able to successfully identify under-represented classes, such as neutrophils, eosinophils and plasma cells, helping them to achieve competitive mPQ+ and R2 scores.

Conclusion: Nuclear morphology and co-localisation of different subtypes have shown to be important indicators of cancer prognosis and diagnosis. However, manual assessment is not feasible as each tissue sample typically contains thousands of nuclei. We organised the CoNIC Challenge to encourage the development of automatic approaches for nuclear recognition in computational pathology. We hope that the widespread participation will motivate researchers to further develop methods on the provided data and accelerate the development of downstream cell-based models for clinical applications.

Funding: Simon Graham, Mostafa Jahanifar, David Snead, Shan Raza, Fayyaz Minhas and Nasir Rajpoot are part of the PathLAKE digital pathology consortium, which is funded from the Data to Early Diagnosis and Precision Medicine strand of the governments Industrial Strategy Challenge Fund, managed and delivered by UK Research and Innovation (UKRI).


Quantification of metabolic heterogeneity across multiple imaging modalities

E. Smeets*, M. Trajkovic-Arsic, D. Geijs, M. van Zanten, L. Brosens, B. Feuerecker, M. Gotthardt, J. Siveke, R. Braren, F. Ciompi, E. Aarntzen

*Department of Medical Imaging, Radboud UMC, The Netherlands

Background & objectives: Radiomics based on clinical imaging are increasingly applied to classify tumours, but often lack sufficient biological rationale, hampering clinical implementation. We developed an [18F]FDG-PET/CT signature in pancreatic cancer based on MCT4-expression, quantified by texture analyses of whole tumour cross-sections.

Methods: We developed a cross-modal image analyses pipeline using a cohort of pancreatic cancer patients (n=29) of which tumour cross-sections and PET-scans were available. We computed density maps of MCT4-expression on whole-slide images to extract texture features. Using k-means, we defined two subgroups with distinct MCT4-expression patterns. From corresponding [18F]FDG-PET scans, texture features were selected that associate with the pre-defined subgroups.

Results: Clustering techniques, based on k-means, umap and heatmap analyses, revealed two distinct MCT4-expression patterns. MCT4-expression pattern A was dominated by a higher MCT4-expression level and more local variation. MCT4-expression pattern B was characterized by less MCT4-expression. Using MCT4-expression patterns as label, we investigated which [18F]FDG-PET derived texture features associate with these tumour characteristics. MCT4-expression pattern A was linked to a specific [18F]FDG-PET signature, characterized by higher tracer uptake values and second order features correlated to local variation of tracer uptake on the corresponding clinical scans. The MCT4-based [18F]FDG-PET signatures were applied to an additional cohort (n=71) pancreatic cancer patients who received palliative systemic treatment and showed prognostic value.

Conclusion: The presented cross-modal image analyses pipeline allows to build PET-scan signatures based on a biological rationale using quantitative immunohistochemistry. As use-case we focussed on tumour glycolysis as hallmark of cancer, measured by MCT4-expression patterns on resected whole tumour sections and by [18F]FDG-PET scans. We show that a subgroup of pancreatic cancer patients with high and heterogenous MCT4-expression can accurately be identified in vivo using [18F]FDG PET-derived texture features. This MCT4-based [18F]FDG-PET-signature associated with worse prognosis.


Artificial intelligence as a potential tool for pathologists to evaluate lymphocyte infiltration in melanoma

A. Romano Martínez Martínez, D. Jiménez-Sánchez, J.I. Echeveste, S. Martín Algarra, M.D. Lozano Escario, C.E. de Andrea*

*Clínica Universidad de Navarra, Spain

Background & objectives: Melanoma is one of the most frequent types of skin cancer, as well as one of the most aggressive, commonly known for having very poor survival outcomes.

Methods: Although lymphocyte presence in a tumour has been described as important, there is no standardization of the criteria pertaining to its analysis; it is highly subjective and dependent on the individual looking at the sample. Therefore, an annotation-free artificial intelligence technique, called NaroNet, was developed to identify melanoma cells and tumour infiltrating lymphocytes objectively.

Results: It was then applied to samples in order to explore the differences between biopsies at their baseline and at progression. As NaroNet’s parameters determine the scope of tissue pattern learning, they were optimized to identify both cell types. Patch analysis approach (70x70 pixels) was used on 22-paired samples to detect cells of different morphologies and determine their distribution pattern. Each patch could hold one large melanoma cell and up to nine lymphocytes, which allowed the algorithm to discover interactions within their environment. NaroNet predicted baseline from progression with a 95.45% accuracy using phenotype abundances. T-distributed stochastic neighbor embedding of each phenotype showed high confidence when identifying cell types.

Conclusion: Biopsies at progression had little to no brisk infiltration of the tumour, which was significantly different (p=0.03) from baseline samples. NaroNet can be trained to detect cell types without manual annotations, and used to identify important patterns of cell interactions in melanoma that could differentiate samples at baseline and progression. With further development, the algorithm could be optimized to become a useful tool for pathologists.


Artificial intelligence-guided spatial transcriptomics in high grade serous carcinoma: toward image-analysis based precision oncology

A. Laury*, S. Zheng, O. Youssef, J. Tang, O. Carpen

*University of Helsinki, Research Program in Systems Oncology, Finland

Background & objectives: H&E images of high-grade serous ovarian carcinoma (HGSC) may contain prognostic information detectable only by artificial intelligence (AI). We hypothesise that AI can pinpoint regions with prognostic value, and the biology of these regions can be revealed with spatial transcriptomics.

Methods: The cohort included 55 stage III-IV patients with distinct platinum-free intervals (PFI) (<6 vs >18 months). A deep learning neural network tool identified tumour regions most indicative of outcome. These high-confidence (HC) and background regions were probed with 10x Visium for FFPE spatial transcriptomics technology. Output was visualized with 10x Loupe browser and analysed using the R package Seurat.

Results: The neural network was first trained to identify tumour tissue, then to classify the tumour into short or long PFI group. Using a HC mask, regions indicative of outcome were identified. These HC regions were then used to train the final neural network. Testing a combined inference pipeline to classify an independent tumour set showed high sensitivity (73%) and specificity (91%). UMAP visualization of the spatial transcriptomics demonstrated that while data from the same patient are close to each other, HC and background regions are mostly distinct within the cluster for each patient. Transcriptomics profiles from HC regions predicted PFI group status significantly better than background regions.

Conclusion: Artificial intelligence-based image-analysis (AI-IA) of HGSC tissue can identify morphologic patterns invisible for human eye and guide selection of biologically meaningful regions for spatial transcriptomics. When combined, these novel technologies identified several signalling pathways and transcripts separating HGSC tumours with short vs. long PFI. In conclusion, AI-IA together with spatial transcriptomics offers a promising toolkit to identify biological features associated with cancer behaviour, making the AI-based diagnosis more interpretable and clinically relevant.

Funding: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 965193 for DECIDER.


Development of trial quality assurance program for digital pathology of the Korean Society of Pathologists

Y. Chong*, J.M. Bae, D. Kang, K.I. Kim, H.S. Han

*The Catholic University of Korea, Republic of Korea

Background & objectives: Digital pathology (DP) can fundamentally change the way of working in pathology. Since the Korean Society of Pathologists (KSP) published the consensus recommendation paper for DP application recently, the need for quality assurance program (QAP) for DP has been raised.

Methods: To provide standard baseline reference for internal and external QAP for DP, the Committee of Quality Assurance of KSP developed a checklist for DP QAP and started a trial QAP in 2021. After several revisions, the checklist was finalized. Five leading institutes participated the trial QAP in the first year and we gathered feedback from these institutes afterwards.

Results: The newly developed checklists of QAP for DP contains a total of 39 items (212 score) to check, 8 items for quality control of DP systems, 3 items for DP personnel, 9 items for hardware and software requirement for DP systems, 15 items for validation, operation, and management of DP systems, and 4 items for data security and personal information protection. Full text in both Korean and English is attached as appendices. Most participant institutes in the trial QAP replied that continuous education on unfamiliar terminology of new technology and more practical experience is demanding.

Conclusion: QAP for DP is essential for the safe implementation of DP in pathologic practice. Each laboratory should prepare institutional QAP according to this checklist and consecutive revision of the checklist with the feedback from trial QAP for DP needs to follow.

Funding: This study was supported by the Korean Society of Pathologists Study Group/Committee Supporting Research Grant (2020).


Detecting premalignant lesions in the Fallopian tube, using a deep-learning model. A pilot study

J. Bogaerts*, J. Linmans, M. van Bommel, M. Steenbeek, J. Bulten, J. de Hullu, M. Simons, J. van der Laak

*Radboud University Medical Centre, The Netherlands

Background & objectives: Serous Tubal Intraepithelial Carcinoma (STIC) is a precursor lesion to High Grade Serous Carcinoma (HGSC). Interobserver variability in STIC diagnosis is high. We aim to develop an Artificial Intelligence model that can detect STIC in digitalized whole slide images.

Methods: We collected, digitalized and annotated 91 cases of STIC/STIL and 75 control cases. Diagnosis was confirmed using p53 and Ki-67 immunohistochemical stains, when available. The cases were split into 71 training cases and 20 test cases. A two-step deep-learning algorithm was trained: first all epithelium was detected, and next aberrant epithelium was distinguished from normal epithelium.

Results: The two-step model approach reached an area under the receiver operating curve of 0.946 on slide level. Visual inspection confirms adequate detection of the aberrant epithelium, in concordance with morphology and immunohistochemistry.

Conclusion: We present a deep-learning algorithm that can successfully detect STIC. Adequate STIC diagnosis is important to better understand the oncogenesis of HGSC, holds prognostic implications for individual patients, and is a prerequisite to safely offer alternative risk reducing surgeries, such as salpingectomy with delayed oophorectomy, currently studied in prospective international trials. We believe an AI model has the potential to aid the pathologist in this challenging diagnosis. Expanding the dataset is expected to aid further development of this model.

Funding: Supported by the Dutch Cancer Society.


Eyeballing and hot-spot counting of ki67 may misguide therapy in invasive breast carcinoma, NST and the quick fix is automated counting

S. Sevim*, E. Dicle Serbes, G. Ozdogan, S. Dizbay Sak

*Ankara University Medical School, Pathology Department, Turkey

Background & objectives: Ki67 evaluation is essential in invasive breast carcinomas. This can be a very laborious process for pathologists. In this study, we aimed to compare Ki67 scores by a.)eyeballing, b.)manual-counting (MC), and c.)using an artificial intelligence based automated counting program (AIACP).

Methods: For 54 cases, three regions of interests (ROIs) (1 hot-spot and 2 reflecting the first impression), each of 0.2 mm2, were selected on Ki67 (SP6) stained whole digital sections. These ROIs were counted manually and by AIACP (3D-HISTECH, Panoramic P250 Flash3, VPS3.0.2., Quant Center). Blinded to these countings, three independent observers determined Ki67 scores for the same cases, by eyeballing.


Conclusion: In this study, our findings of high agreement between AIACP and time-consuming MC shows that a standardized automated Ki67 scoring tool is very beneficial. It should also be stressed that, although interclass agreement between eyeballing and other methods seems acceptable, when Ki67 scores are grouped categorically based on International Ki67 in Breast Cancer Working Group 2021 Consensus, concordance was only moderate. Eyeballing and hotspot-only counting should not be used to determine Ki67 scores, which are critical in determining therapy options.


AI versus microscope in primary diagnosis of breast biopsies: multi-site clinical reader study

A. Vincent-Salomon, A. Nudelman, J. Cyrta, M. Maklakovski, A. Albrecht Shach, G. Sebag, G. Mallel, I. Krasnitsky, T. Feinberg, C. Linhart, M. Vecsler, J. Sandbank*

*Maccabi Healthcare Services, Israel

Background & objectives: This study aimed to clinically validate the use of an AI-based solution by pathologists for reviewing and reporting breast core needle biopsies as compared with the gold standard practice, review on the microscope.

Methods: A two-arm prospective reader study comparing the performance of pathologists using an AI-based solution with pathologists using a microscope was performed at two sites (different staining and scanners). Both arms were compared to ground truth (GT) established by consensus of two breast pathologists. Rates of major discrepancies between each arm and GT, as determined by an adjudicating pathologist, were compared.

Results: Eight pathologists participated in the study and reported on 385 cases (442 HES and 330 H&E slides), each case being reported twice, once in each study arm. Pathologists first reviewed only H&E/HES slides, if requested and available, they were provided with IHCs, while the AI results were on H&E/HES only. The major discrepancy rates of the microscope arm and of the AI arm against GT were 4.42% and 3.12%, respectively, demonstrating 29.4% reduction in major discrepancies. Pathologists with AI demonstrated very high accuracy for the detection of invasive carcinoma with sensitivity and specificity of 100% for both, as well as for DCIS/ADH with sensitivity of 92.4% and specificity of 97.8%.

Conclusion: This multi-site reader study reports diagnostic accuracy improvements by pathologists performing diagnosis and reporting with the support of a first read AI solution for breast biopsies. The AI solution performed accurately and generalized well for different staining platforms and different scanners. Thus, AI solutions could be used as significant aiding tools for pathologists in clinical decision-making in routine pathology practice, enhancing the quality and reproducibility of diagnosis.


Digital score of Ki67 in prostate cancer is associated with high-grade disease and presence of metastasis

Q.D. Vu*, L. Mendes, C. D. Brawley, S. Raza, E. Grist, A. Ali, S.S. Vidal, M. Parry, S. Lall, N. B. Atako, M. Richmond, A. Haran, L. Zakka, N. W. Clarke, M. K. B. Parmar, N. D. James, L. C. Brown, D. Berney, G. Attard, N. Rajpoot

*University of Warwick, United Kingdom

Background & objectives: Ki67 proliferation-index (PI) has been identified as a valuable prognostic marker in prostate cancer. However, manual scoring is laborious and highly subjective. We explore the association of our digital Ki67 score with high-grade prostate cancer disease and presence of metastasis.

Methods: Diagnostic paraffin-embedded needle core biopsies were stained for Ki67 using MIB-1 antibody (DAKO, Carpinteria, CA, USA). Manual scoring used the unweighted global assessment method. Digital scores were calculated using our deep learning method. Scores were correlated with Gleason score and metastatic status.

Results: Samples from 54 patients randomised to STAMPEDE arm A (2006-2015) were assessed. There were 29 M0, 9 M1 low burden and 6 M1 high burden. Gleason score was centrally assigned with 72% classified as GG5. The correlation between manual and digital scores was calculated to be 0.7562 (p=3.78e-11). The manually assigned Ki67 score was associated with high-grade disease (GG5 and 4) (p = 0.031) and the presence of extra-pelvic metastases (p<0.001), whereas our digital Ki67 score (DigiKiPI) also showed a statistically significant association with high-grade disease and presence of metastasis (p = 0.047 and p = 0.001, respectively) with the added benefits of being objective and reproducible.

Conclusion: Ki67 PI is a robust prognostic tool in clinically advanced prostate cancer that can refine patient prognostication. However, it has not been broadly used in clinical routine due to lack of standardisation and high intra and inter observer variations. Automated deep learning based scoring offers a promising tool for better way to objectively and reproducibly quantify Ki67 PI while reducing manual labour. We find that our DigiKiPI score is significantly associated with high-grade disease and the presence of extra-pelvic metastasis.

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


The Nrf2-ARE pathway: a potential novel therapeutic target in papillary renal cancer patients

S. Angori*, A. Banaei-Esfahani, K. Mühlbauer, A. Kahraman, V. Pietiäinen, S. Potdar, O. Kallionemi, P. Schraml, H. Moch

*University Hospital Zurich, Switzerland

Background & objectives: Nrf2-ARE signalling, a key sensor of oxidative stress in humans, is involved in papillary renal cell carcinoma (pRCC). We characterised Nrf2-ARE pathway over-activation in pRCCs and screen pathway inhibitors to identify targeted treatment for pRCC patients.

Methods: Comprehensive characterisation of 60 formalin-fixed, paraffin-embedded pRCCs by copy number analysis and Whole Exome Sequencing allowed us to identify pRCC groups based on their genetic background. Protein expression of NQO1, the downstream target of the Nrf2-ARE pathway, was analysed in pRCCs by immunohistochemistry and activity assay. Newly established patient-derived cell models that resemble pRCC tumours were applied for drug profiling.

Results: Immunohistochemical tissue microarray analysis of 119 pRCC correlated Nrf2-ARE pathway over-activation with worse patient outcome and higher tumour grade and stage. Secondly, based on the STRING network, we identified 15 members of the Nrf2-ARE pathway of which 4 genes NFE2L2, Keap1, CUL3 and Bach1 had mutations in 12% of all samples. The investigation of 9 matched pRCC samples and patient-derived tumour cell cultures demonstrated increased NQO1 mRNA and protein expression and activity in 56% of tumours. Finally, drug screening with 18 Nrf2-ARE pathway inhibitors using 6 pRCC PDCs with deregulated Nrf2-ARE pathway activation showed Brusatol and Convallatoxin, two Nrf2 inhibitors, had the most potent responses in our PDCs.

Conclusion: pRCC is not a single disease but consists of at least two main subtypes with distinct molecular backgrounds and patient outcomes. We first characterised a subset of aggressive pRCCs with aberrant activation of the Nrf2-ARE pathway. Moreover, we showed that pharmacological inhibition of Nrf2 represents a promising therapeutic target for this tumour subtype. We anticipate that this will open up new possibilities for the clinical management of these patients.


Improving the quality of cfDNA testing – results from two years of EQA

J. Fairley*, M.H. Cheetham, S.J. Patton, Z.C. Deans

*GenQA, United Kingdom

Background & objectives: Testing of cfDNA for biomarkers is now a recognised part of clinical practice in lung cancer. EQA plays an important role in assessing and improving standards. The results from two global EQAs testing cfDNA in lung cancer are presented.

Methods: Artificial plasma samples with cfDNA containing prescribed variants at defined allelic frequencies were distributed. Laboratories tested the samples according to their usual cfDNA protocols and reported the results in the context of specific clinical cases. Reports were peer-assessed, and feedback provided to laboratories in the form of individual reports.

Results: EQAs were provided in 2020 and 2021 with the number of laboratories submitting results increasing from 259 to 292. Both EQAs included assessment of EGFR testing and 2021 also included KRAS. The genotyping error rate decreased from 22% in 2020 to 11% in 2021. Common issues observed included the use of inappropriate methods for cfDNA testing and a lack of awareness available treatment for tumours with KRAS mutations, over-interpretation of the absence of a variant and failing to provide sufficient details of the test methodology and limitations.

Conclusion: The increase in the number of participants reflects the recognition for the need for EQA for cfDNA testing. These EQAs have shown a large variation of methodologies being used and variability in genotyping accuracy and interpretation of results being reported. This has the potential to adversely impact on patient care highlighting the clear need for education to improve testing methods and the clear reporting of the result. EQA is a key mechanism to deliver this knowledge.


Common pitfalls in interpreting fusion testing results highlighted by EQA

J. Fairley*, Z.C. Deans

*GenQA, United Kingdom

Background & objectives: Testing of tumours for fusion transcripts by molecular methods is becoming increasingly widespread. GenQA has adapted to the change in testing strategies by including assessment of fusions by genomic testing into existing EQAs and the introduction of an NTRK EQA.

Methods: Fusion -testing by molecular methods is assessed in the lung, thyroid and renal cancer, sarcoma and NTRK EQAs. FFPE tissue is provided to laboratories to test for fusions according to their usual procedures and report the results in the context of the clinical case provided. Returns are assessed by expert assessors and laboratories provided with individual reports of their results.

Results: There has been a significant increase in the number of laboratories performing routine fusion transcript testing. Common reporting errors across the EQAs were identified which resulted in the incorrect interpretation of the clinical relevance of detected transcripts. Laboratories reported the presence of multiple transcripts, described transcripts incorrectly and incorrectly predicted the productive nature of transcript.

Conclusion: The introduction of molecular testing for fusion transcript has the benefit that multiple targets can be examined using the same assay compared to techniques such as FISH and IHC. EQAs for the detection of fusion transcripts in FFPE tissue from solid tumours have identified issues with both interpretation and reporting of results. This could impact on patient care and therefore there is a need for continual assessment of this testing.


The Geneva HRD test: clinical validation on 469 samples from the PAOLA-1 trial

Y. Christinat*, L. Ho, S. Clément, C. Genestie, J. Sehouli, S. Cinieri, A. Gonzalez Martin, U. Denison, K. Fujiwara, I. Vergote, G. Tognon, S. Hietanen, E. Pujade-Lauraine, I. Ray-Coquard, T. McKee

*Hôpitaux universitaires de Genève, Switzerland

Background & objectives: The efficiency of the Myriad HRD test to guide use of PARP inhibitors has been demonstrated in several phase III trials. However, its high failure rate and limited accessibility establish a need for a clinically validated laboratory developed test.

Methods: The algorithm behind the Geneva HRD test was developed on 457 high grade serous ovarian samples and 112 triple negative breast cancer samples from the TCGA. As part of the ENGOT HRD European Initiative the algorithm, applied on OncoScan(TM) CNV Assay data, was compared to Myriad with respect to the PFS on 85+384 samples from the PAOLA-1/ENGOT-ov25 phase 3 trial.

Results: The analysis of the TCGA cohort revealed that a normalization of the number of large-scale state transitions by the number of whole genome doubling events allows a better separation and classification of HR-deficient samples than the tripartite score used by Myriad or the genomic LOH score used by Foundation Medicine. On the PAOLA-1 samples, the Geneva test yielded a similar hazard ratio as the Myriad test with respect to the addition of Olaparib or placebo to the Bevacizumab maintenance treatment (HR=0.32 vs 0.31). Compared to Myriad, the test yielded a lower technical failure rate (2% vs 11%) and a positive and negative predictive value of 90% and 85%.

Conclusion: The proposed test is a viable alternative to the Myriad myChoice HRD test and can be easily deployed in a clinical laboratory. The performance is similar to the commercial test in terms of hazard ratio but the lower failure rate of the Geneva HRD test allows a 10% increase (375 vs 340) in the number of patients that will receive a conclusive laboratory result.

Funding: AstraZeneca/Merck


Clinical utility of tumour-only targeted panel sequencing in childhood tumours

Y. Mok*, C.H. Kuick, J.Y. Goh, N. Chia, M.S. Nwe, K.T. Chang

*National University Health System, Singapore

Background & objectives: Broad based genomic profiling is increasingly part of the standard workup for paediatric cancers. In this study, we evaluate the clinical utility of tumour-only sequencing in a cohort of 94 paediatric tumours using a targeted next generation sequencing panel.

Methods: 94 cases of paediatric solid tumours spanning multiple subtypes were recruited, including relapsed and refractory childhood cancers. Nucleic acid extracted from formalin-fixed paraffin embedded tissue was tested with Illumina Ampliseq Childhood Cancer Panel, which includes single nucleotide variants, copy number variants and gene fusions involving >130 genes. All clinically significant variants were reviewed at a multi-disciplinary tumour board.

Results: 94 cases of paediatric solid tumours from 87 individuals were sequenced, including bone and soft tissue tumours (n=30), central nervous system (CNS) tumours (n=17), lymphomas (n=9), sympathetic nervous system tumours (n=8), renal tumours (n=8), liver tumours (n= 5) and other tumour types.

Clinically relevant variants were identified in 57 cases (60.6%). Variants were of diagnostic significance in 54 cases (57.5%), therapeutic significance in 34 cases (36.2%), and prognostic significance in 11 cases (11.7%). A potential germline alteration was detected in 9 cases (9.3%). Based on the sequencing findings, 3 cases had a change in diagnosis: pancreatic Ewing sarcoma, myeloid sarcoma with KMT2A-MLLT3 gene fusion and metastatic colitis-associated colorectal adenocarcinoma (poorly-differentiated).

Conclusion: Paediatric tumour types that benefitted most from tumour-only sequencing were soft tissue tumours (67% of cases with clinically relevant variants) and CNS tumours (65% of cases with clinically relevant variants). Whilst changes in histological diagnoses were rare, molecular findings were extremely useful in assisting diagnosis of rare or poorly-differentiated entities, as well as tumours occurring in uncharacteristic locations.

Funding: VIVA-KKH Brain and Solid Tumour Programme


The activation of Jagged1 signalling by chemotherapeutic agents counteracts the Oxaliplatin/5Fluorouracil- mediated anti-cancer effects: a novel mechanism of drug resistance in colon cancer

M. Pelullo*, S. Zema, M. Decarolis, A. Villari, A. Montalti, I. Screpanti, D. Bellavia

*Center for Life Nano-&Neuro-Science (CL2NS@Sapienza), Istituto Italiano di Tecnologia, Rome, Italy

Background & objectives: Colorectal cancer (CRC) is a leading cause of mortality worldwide, characterized by metastasis and resistance to therapy. Recently, we demonstrated that Kras mutation drives the activation of Jag1-ICD oncogene, via-ERK1/2. Herein, we explore the new intrinsic drug-resistance mechanisms, Jag1-ICD-mediated.

Methods: Human CRC cell lines were treated with different chemotherapeutic compounds (e.g. OXP, 5FU and GSIs), alone or in combination, and subjected to in-vitro assays, to evaluate proliferation, metastasis and chemoresistance. CRC resistant cells were obtained by chronical treatment with low doses of OXP/5FU. The resistant cells were analysed by colony-formation assays and by qRT-PCR to assess growth and gene-reprogramming ability.

Results: Herein, we evaluate the effects of OXP, 5FU and GSIs alone or in combination, on Jagged1 processing in CRC cell lines. We demonstrate that the anticancer drugs, OXP and 5FU, lead directly to a massive Jag1-ICD activation that results in the selection of a drug-resistant subpopulation. The chemoresistance mechanism is induced by a forced Jag1-ICD accumulation that protects cells from apoptosis, under the activation of Jag1-ICD-dependent pro-survival targets. In addition, GSIs induce the proliferation of Jag1-ICD positive CRC cells, functioning as tumour-promoting agents. Finally, the Jagged1 abrogation in OXP- or 5FU-resistant subpopulations is enough to restore the sensitivity to chemotherapy, confirming that drug resistance is Jag1-ICD-dependent.

Conclusion: Overall, our data show that Jagged1 processing is directly activated by the most potent chemotherapeutic agents (OXP/5FU) or by GSIs compounds. Moreover, we unveil a new role for Jag1-ICD oncogene which controls both apoptosis and proliferation, in CRC cells upon chemotherapeutic treatments. Therefore, we demonstrate the existence of a new mechanism of intrinsic drug-resistance, where Jag1-ICD functions as pivotal nuclear effector. Finally, we suggest Jagged1 as molecular predictive biomarker for the chemotherapy-outcome in CRC patients bearing Krasmut and over-expressing Jagged1.


Ultra-fast gene fusion assessment as a reflex testing in daily clinical practice for advanced non-small cell lung cancer patients

V. Hofman, S. Heeke, C. Bontoux*, L. Chalabreysse, M. Barritault, P. Bringuier, T. Fenouil, N. Benzerdjeb, H. Begueret, J. Merlio, C. Caumont, N. Piton, J. Sabourin, S. Evrard, C. Syrykh, E. Long-Mira, S. Lassalle, M. Ilié, P. Hofman

*Laboratoire de pathologie Clinique et experimentale, CHU de Nice, Université Côte d’Azur, Nice, France

Background & objectives: There is an urgent need to improve the broad molecular profiling of advanced non-squamous non-small cell lung carcinoma (NS-NSCLC) patients, notably for a rapid assessment of multiple genomic alterations.

Methods: We compared two ultra-fast gene fusion assessment assays, using a next generation sequencing (Genexus, Oncomine™ Precision Assay, Thermo-Fisher) or an RT-PCR (Idylla™, GeneFusion Assay, Biocartis) approaches, set up as a reflex testing at diagnosis.

Results: 250 NS-NSCLC patients (68 ALK, 26 ROS1, 15 RET, 6 NTRK, 11 MET positive and 125 wild type patients) from 8 centers were included. 83% of patients were stage IIIB-IV.

The sensitivity (98%) and specificity (99%) of the two approaches were analogous, when compared to gold standard methods, accredited according to the ISO15189 norm in the Laboratory of Clinical and Experimental Pathology (Nice, France).

Conclusion: Ultra-fast gene fusion evaluation using NGS or RT-PCR approaches should be developed as a reflex testing for NS-NSCLC at diagnosis in order to treat these patients according to the international recommendations and guidelines.


Nuclear markers for the diagnosis of histiocytosis

I. Ungureanu*, S. Héritier, F. Cohen-Aubart, Z. Hélias-Rodzewicz, J. Donadieu, J. Haroche, J. Emile

*Department of Pathology, Ambroise-Paré Hospital, France

Background & objectives: Newly described immunomarkers could bring potential benefit in the diagnosis and treatment of histiocytosis. Our objective was to analyse recently described nuclear markers and to expand the immunohistochemical panel in the diagnosis of histiocytosis.

Methods: Biopsy samples diagnosed with Erdheim-Chester Disease (ECD), Langerhans cell histiocytosis (LCH), Rosai Dorfman Disease (RDD), malignant histiocytosis, and cutaneous histiocytosis were retrieved from the files of our Pathology Department. Haematoxylin & eosin-stained slides were reviewed. Immunohistochemistry was performed with the following antibodies: PU.1(EPR3158Y, Abcam), OCT.2(EPR12482-106, Abcam), phosphoERK(Erk 1/2, Cell Signalling). Molecular biology was performed using PCR or Next-Generation Sequencing.

Results: phosphoERK was performed in 567 biopsy samples of histiocytosis. It was positive in 91%, 86%, 73%, 67%, 70% and 83% of LCH (n=118), ECD (n=198), mixed (n=22), RDD (n=119), cutaneous (n=86) and malignant histiocytosis (n=24), respectively. All the types of histiocytosis were positive for PU.1 (5 LCH, 5 ECD, 7 RDD, 4 ALK+ and 8 C group). Among the 17 cases referred as malignant histiocytosis, all the 9 confirmed cases were positive, contrasting with the 8 excluded cases that were negative. OCT.2 was positive in 42/80 cases of RDD. All 9 mixed RDD-ECD or RDD-LCH were OCT.2 positive. 18/38 cases of non-RDD had at least a low positivity for OCT.2.

Conclusion: PU.1 is a marker indicating a histiocytic origin and it is useful to exclude a tumour rich in reactive histiocytes mimicking histiocytosis. OCT.2 can be used to confirm Rosai-Dorfman Disease. phosphoERK is a nuclear and cytoplasmic marker highlighting the activation of MAPkinase pathway and it can be used to initiate targeted therapy in histiocytosis when molecular biology is not available.


Clonality analysis of Richter’s transformation in CLL treated with targeted therapy

I. Xie*, I. Rashedi, Y. Amemiya, D. Spaner, A. Seth, Z. Ghorab, R. Goswami

*Department of Laboratory Medicine and Pathobiology, University of Toronto, Canada

Background & objectives: Richter’s syndrome (RS) occurs in 1-10% of patients with chronic/small lymphocytic leukaemia (CLL/SLL). Clonal transformation has a poorer prognosis than de novo transformation, and benefits from more aggressive chemotherapy. We sought to characterize RS clonality in a Canadian case series.

Methods: DNA was extracted from FFPE lymph node, bone marrow, or banked cells. Targeted NGS of heavy and light chain regions was performed using the Oncomine BCR Pan-Clonality Assay (ThermoFisher). IGH, IGK, and IGL rearrangements and clonal frequencies were assessed through the Ion Reporter Oncomine BCR workflow and MiXCR. Slides and IHC were reviewed to confirm the diagnosis.

Results: We identified nine patients with 20 pre- and post-transformation samples. The median age at diagnosis was 52 (range 44-84), with median 9 years (range 1.9-15) between the diagnosis of CLL vs RS. Most cases transformed to DLBCL (n=7), and the remainder to classical Hodgkin lymphoma (n=2). 7/9 patients had received therapy prior to transformation, including ibrutinib, acalabrutinib, or venetoclax. Analysis of the heavy chain and light chain loci revealed a clonal relationship in all nine cases, including one case where the secondary DLBCL was CD5-negative. Interestingly, several cases exhibited multiple productive IGH rearrangements (2/9) or light chain rearrangements (7/9) shared between the pre- and post-transformation cells.

Conclusion: Targeted sequencing of a case series of CLL patients with RS revealed a clonal relationship in all nine cases. Multiple productive Ig rearrangements were identified in a surprising number of cases, consistent with a growing number of NGS studies supporting greater diversity in CLL clonality than previously appreciated.


Delineating the spatial compartmentalization of human follicular B cell metabolic dynamics

M.M. Perra*, A. Noto, K. Ioannidou, M. Foglierini-Perez, C. Fenwick, L. de Leval, C. Petrovas, G. Pantaleo

*Unil-CHUV, Switzerland

Background & objectives: Despite the well-established role of germinal centers (GC) for the generation of protective B cell response, an insight on the in-situ human GC immune reactions remains unclear. We aimed to evaluate the energetic and metabolic profile of follicular B cells.

Methods: Metabolic profile of primary tonsillar B cells were characterized by applying complementary methodologies. Phenotyping and 2-NBDG uptake of relevant B cell subsets were assessed by multiparametric flow-cytometry. Ex vivo metabolic function (Seahorse) and transcriptomic signatures (bulk NGS) were investigated on cryopreserved TNMCs (tonsillar mononuclear cells). Multiplex tissue imaging was applied for the in situ B cell metabolic profiling.

Results: Seahorse analysis revealed that OXPHOS supported 70% and glycolysis 30% of total ATP produced in GCBCs. Coherently, an increased 2-NBDG uptake was observed in GCBCs. GSEA of sorted B-cell subsets showed significant enrichment of OXPHOS and mTORC1 pathways in GCBCs, with several glycolysis-related genes being significantly expressed. In-vitro treatment with UK5099 (MPC inhibitor), negatively affected the SRC, MRC, and Krebs capacity in GCBCs as compared to untreated controls. Tissue imaging of FFPE-tonsillar sections revealed a polarization of GLUT1, MCT4, HIF1a and HIF1b in Light Zone, while Opa1 was expressed within Dark Zone. Gene expression of MCT1 (lactate transporter) was increased in GCBCs, while imaging showed its distribution across the GC.

Conclusion: Despite an intense dependence of GCBCs on OXPHOS, glycolysis is a significant energy supporter too. Tissue in situ analysis suggests the coupling of massive division in DZ with OXPHOS and local exchange of lactate, while glycolysis is more frequent among B cells in LZ. Moreover, the lactate transporters’ expression and the impaired Krebs cycle capacity induced by MPC-blockade point to a significant role of glucose-derived carbon atoms and monocarboxylates in sustaining the oxidative machinery.


Comparison of the accuracy of cytomorphology, flow cytometry immunophenotyping and immunohistochemistry in determining diagnostic and prognostic blast percentage groups in bone marrow in myelodysplastic syndrome and acute myeloid leukaemia cases

E. Hacihasanoglu*

*Yeditepe University, Turkey

Background & objectives: Bone marrow (BM) blast percentage (BP) is important in diagnosis, classification and prognosis of myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). We aimed to compare accuracy of cytomorphology (CM), flow cytometry immunophenotyping (FCI), immunohistochemistry (IHC) in determining BP groups.

Methods: BM biopsy and aspiration samples from MDS and AML patients and patients diagnosed with AML underwent bone marrow transplantation (BMT) between 9/2019 and 6/2021 were analysed. CM was evaluated. CD34 positive BP was determined by FCI and IHC. Cases were divided into four groups according to BP: <5%, ≥5%-<10%, ≥10%-<20%, ≥20%. Three methods were compared with the Pearson-r correlation.

Results: A total of 68 BM materials from 55 patients were analysed. Thirty-nine of the cases were MDS (7 MDS-EB), 2 of them were AML, and 14 of them were AML patients who underwent BMT. Pearson-r correlations for absolute values in CM-FCI, CM-IHC, and FCI-IHC comparisons were 0.8865, 0.8787, 0.9670, respectively, indicating a good correlation. When CM-FCI was compared, 86.7% of the cases were in the same blast range. In the comparison of CM-IHC, 79.4% of the cases were in the same blast range. Comparison of IHC-FCI showed 88.2% correlation in blast intervals.

Conclusion: BM blast rate determination is important in MDS classification and MDS-AML differentiation. Although CM is the gold standard, reproducibility is low even with high-quality smears. In our study, we observed good correlations between CM, FCI and IHC. Correlation between FCI and IHC, which have high reproducibility and low interobserver variability, was even higher. However, this study is limited to cases showing CD34 expression. Although FCI and IHC methods have high accuracy with CM, using the three methods together is recommended.


Prevalence and impact of co-infections in patients with lymphoma and HIV

C. Padrão*, A.M. Gonçalves Pereira, F. Pereira, J. Tinoco, M.G. Gasparinho

*Hospital Prof. Dr. Fernando Fonseca, Portugal

Background & objectives: HIV infection is associated with the development of lymphomas and some other co-infections (eg., HBV and HCV). The aim of our study was to review lymphomas arising in HIV setting and linked co-infections, in a patient’s cohort from our hospital.

Methods: We selected patients diagnosed with HIV and lymphoma between 2010-2020 in our hospital, from our electronic medical database. Demographic data, date of diagnosis, date of death/last contact, lymphomas types and location, serologies for hepatitis B and C, cytomegalovirus, toxoplasma and Epstein-Barr virus (6 months prior until 3 months after lymphoma diagnosis) were collected. Descriptive statistical analysis was performed.

Results: We selected 52 patients (71.2% males). HIV-1 was predominant (90.4% of the cases). Average age of HIV diagnosis was 42.38 years and of lymphoma diagnosis was 47.79 years; mean interval between diagnoses was 64 months (36.5% diagnosed within 1-year).

Laboratory data demonstrated high prevalence of chronic hepatitis C (9.6%) and B (28,8%) and low prevalence of recent EBV (1.9) or CMV (3.8) infection.

Diffuse large B cell lymphoma (40.4%), Hodgkin’s lymphoma (23.1%) and plasmablastic lymphoma (11.5%) were the most frequent types. Lymph node involvement (59.6%) was predominant, followed by bone marrow (7.9%) and gastric involvement (5.8%). Mortality rate was 59.6%. Median survival was 11.71 months after lymphoma diagnosis.

Conclusion: Our patients’ cohort follows the general patterns of gender, HIV type distribution and main types of lymphomas diagnosed in HIV-infection setting usually described in literature. We have a higher frequency of plasmablastic lymphoma though, not readily explained by a similar rise of frequency of EBV infection – not all patients were tested for EBV, what may account for the lower frequency observed. We hope to further investigate this point in future studies.

OFP-13 | Joint Oral Free Paper Session Neuropathology / Ophthalmic Pathology


Immunohistochemistry against RB1 is useful for the distinction between giant cell glioblastoma and pleomorphic xanthoastrocytoma

V. Barresi*, S. Michele, C. Ciaparrone, S. Pedron, A. Mafficini, A. Scarpa

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

Background & objectives: Giant cell glioblastoma (GC-GBM) displays frequent RB1 alterations, aside from TP53 mutations. Herein, we aimed to assess the value of RB1 immunohistochemistry in the differential diagnosis between GC-GBM and pleomorphic xanthoastrocytoma (PXA), which harbours better prognosis and frequent BRAF mutations.

Methods: In 34 GC-GBMs and 8 PXA (5 grade 2 and 3 grade 3), we analysed: i) mutations and copy number variations of 409 genes using NGS; ii) RB1, P53 and BRAF p.V600E immunostainings. Cases were classified P53 positive when showing at least 10% stained tumour cells.

Results: GC-GBMs were RB1-altered in 15 cases (including 4 with RB1 homozygous deletion, 5 with RB1 heterozygous deletion coupled with mutation of the other allele and 6 with RB1 mutations), TP53-mutated in 27 (including 6 with truncating mutations) and BRAF-mutated in none. At immunohistochemistry, all 15 GC-GBMs with RB1-alterations and 11 RB1-unaltered cases had RB1 loss, 21 were P53 positive and all were BRAF negative. GC-GBMs were RB1-/P53+ in 16 cases, RB1-/P53- in 10, RB1+/P53+ in 5, RB1+/P53- in 3.

PXA were BRAF-mutated in 6 cases, TP53-mutated in 3 and RB1-altered in none. All six cases with BRAF mutation were BRAF p.V600E positive; no PXA was RB1 negative or P53 positive.

Conclusion: An immunohistochemical profile consisting in RB1 loss associated with, or alternative to, P53 positivity had the same specificity (100%), but higher sensitivity (91% vs 61%) for GC-GBM than P53 positivity alone. The addition of RB1 staining in an immunohistochemical algorithm including P53 and BRAF p.V600E may be helpful to differentiate GC-GBM from PXA.


Myoepithelial neoplasia of central nervous system: a series of 4 cases

S. Yeni Yildirim, K. Kosemehmetoglu*, F. Soylemezoglu

*Hacettepe University, Department of Pathology, Turkey

Background & objectives: Myoepithelial neoplasms, composed of myoepithelioma and myoepithelial carcinoma, have not been documented in the central nervous system. Here we present clinicopathological characteristics of 4 neoplasms with myoepithelial differentiation involving the brain and spinal cord.

Methods: The clinicopathological findings of 4 cases were evaluated: 1) 16-year-old female with a 6-cm tumour in the right frontoparietal region.2) 24-year-old female with a 6-cm tumour in T12-L2. 3) 8-year-old female with a 3,5-cm tumour in T11-L1. 4) 20-year-old female with a 3-cm tumour in C4-T2. Spinal tumours were located in intradural-extramedullary space, and the cranial tumour was dural-based.

Results: All patients were female with a mean age of 17(8-24). Histopathologically; 3 tumours were characterized by nests/cords and sheets composed of malignant epithelioid/rhabdoid cells in myxocollagenous background at least focally, thus categorized as myoepithelial carcinoma. Case 2 was reminiscent of myoepithelioma/mixed tumour of soft tissue; however, showed a malignant behaviour. We found loss of INI1 expression in 3 of 4 cases; S100+EMA coexpression, and positivity for at least one keratin marker in all cases. SMA was positive in 3 of 4 cases. Break-apart FISH revealed EWSR1 rearrangements in 2 of 3 cases. Two cases (cases 2 and 3) died, one of whom (case 2) with multiple recurrences and lung metastasis.

Conclusion: Myoepithelial neoplasms occur in the central nervous system of children and young adult females and should be considered in the differential diagnosis of INI1 deficient tumours, after exclusion of AT/RT, poorly-differentiated chordoma, and epithelioid sarcoma. Multiple myoepithelial marker expression and INI1 loss along with EWSR rearrangements favour the diagnosis of myoepithelial neoplasm.


Solitary fibrous tumour of central nervous system: a series of 23 cases

O. Sular, B. Babaoglu, G. Yazici, K. Kosemehmetoglu*, F. Soylemezoglu

*Hacettepe University, Department of Pathology, Turkey

Background & objectives: Meningeal solitary fibrous tumour is characterized by fibroblastic proliferation with hemangiopericytomatous pattern and NAB2::STAT6 alterations. Here, we present clinicopathological features of our series with a special emphasis on the predictability of biological behaviour with the current grading schemes.

Methods: The clinical, radiological, and pathological findings of 23 cases were evaluated. Immunohistochemically, all cases were STAT6 positive. Age, sex, tumour size, location, hypercellularity, mitotic activity, presence of necrosis, hemorrhage, moderate to high-grade nuclear pleomorphism, recurrence, death, and time for recurrence were noted. Tumours were graded according to WHO2016 and WHO2021 classifications. Recurrence-free and overall survival statistics were applied.

Results: The mean age was 37 (14-51) with M:F ratio of 12:11. Tumours had intracranial (n=19) and spinal (n=4) locations; sizes ranged from 2 to 7 cm with a mean of 4,2 cm. Tumours were classified by WHO2021 as grade 1 (n = 12), 2 (n = 6), or 3 (n = 5), and by WHO2016 as grade 1 (n =4), grade 2 (n=8), or grade 3 (n=11). Necrosis was present in 7 (30%). Recurrence occurred in 10 (44%) patients within a mean of 5.6 years, 7 of which were dead during follow-up. On univariate analysis, neither of the above-mentioned parameters nor grading schemes were associated with recurrence-free or overall survival.

Conclusion: According to our data, morphological and clinical parameters, as well as WHO grading schemes, failed to stratify this family of tumours accurately.


Response to regorafenib of recurrent glioblastoma. A clinical and NGS study

M. Martini*, Q.G. D'Alessandris, T. Cenci, V. Fiorentino, L. Lauretti, M. Balducci, R. Pallini

*Università degli Studi di Messina, Italy

Background & objectives: Predictive factors for response to regorafenib in recurrent glioblastoma, IDH-wildtype, are scarcely recognized. The objective of this study was to identify molecular predictive factors for response to regorafenib using a clinically available platform.

Methods: We analysed a prospective cohort of 30 patients harbouring recurrent glioblastoma, IDH-wildtype, and treated with regorafenib. Next-generation sequencing (NGS) analysis was performed on DNA extracted from paraffin-embedded tissues using a clinically available platform. Moreover, MGMT methylation and EGFRvIII expression analyses were performed.

Results: Six-month progression-free survival (PFS) was 30% and median overall survival (OS) was 7.5 months. NGS analysis revealed a mutation of EGFR pathway in 18% of cases and a mutation in the mitogen-activated protein-kinase (MAPK) pathway in 18% of cases. In the remaining cases, no mutations were detected. MAPK pathway mutated patients had a poor response to regorafenib treatment, with a significantly shorter PFS and a nonsignificantly shorter OS compared to EGFR-mutated patients (for PFS, p=0.0061; for OS, p=0.1076). Multivariate analysis confirmed that MAPK pathway mutations independently predicted a shorter PFS after regorafenib treatment (p=0.0188). The negative prognostic role of MAPK alteration was reinforced when we combined EGFR-mutated with EGFRvIII-positive cases.

Conclusion: Recurrent glioblastoma tumours with an alteration in MAPK pathway could belong to the mesenchymal subtype and respond poorly to regorafenib treatment, while EGFR-altered cases have a better response to regorafenib. We thus provide a molecular selection criteria easy to implement in the clinical practice.


Digital image analysis is a powerful tool to demonstrate the prognostic impact of proliferation assessed with Ki67 and PHH3 in meningeal solitary fibrous tumours

H. Nihous, C. Bouvier*, B. Lemarié, D. Figarella Branger, N. Macagno

*Department of Pathology, Timone Hospital, France

Background & objectives: The risk of recurrence and metastasis of solitary fibrous tumours is greatly dependent of proliferation. We aimed to determine the prognostic significance of Ki67 and PHH3 evaluated by digital image analysis in a cohort of 86 meningeal solitary fibrous tumours.

Methods: We compared eye-balling estimation, manual counting, and standardized digital image analysis using QuPath software to evaluate cellularity and proliferation based on Ki67 and PHH3, with correlation with survival in a retrospective cohort of 86 meningeal solitary fibrous tumours. The concordance between the methods was calculated and the processing time was compared.

Results: Evaluation of proliferation by digital image analysis with Ki67≥0.05 or PHH3≥0.0006 cut-off was significantly associated with worse PFS and OS in univariate and multivariate analyses, with a significantly shorter processing time for digital image analysis. Manual counting and digital image analysis were highly correlated, with a concordance correlation coefficient of 0.973 (95% CI: 0.061-0.981) for Ki67 and 0,810 (95% CI: 0.725-0.867) for PHH3. Eye-balling estimation showed less correlation with other methods.

Conclusion: Digital image analysis is a fast and accurate method for evaluating proliferation markers. Using this method, Ki67 ≥0.05 and PHH3≥0.0006 have a negative impact on PFS and OS in univariate and multivariate analyses.


Morphological analysis with morphometry of meningioma calcifications

A. Denysenko*, R. Moskalenko

*Sumy State University, Ukraine

Background & objectives: Meningiomas are the most common non-glial tumours of the central nervous system. Differential diagnosis of meningiomas is a challenging problem due to their location. The work aims to study the morphology, structure, and phase analysis of meningiomas' psammoma bodies (PB).

Methods: The study group included 30 patients with calcified meningiomas. We use histological techniques, transmission and scanning electron microscopy with microanalysis. We captured all photos with the digital visualisation system on Zeiss Primo Star microscope with digital camera ZEISS Axiocam ERc 5s and software package “Zen 2.0”. We performed the statistical analysis of the results using GraphPad Prism 7.04.

Results: All meningioma samples contained PB in the fibrous tissue of the tumour. According to the histological examination and SEM results, PB had a layered structure. They were often in the form of fragments and fragments that preserved the original structure. The number of PB in meningioma tissue varied from one to hundreds of units. In our study, the size of the PB ranged from 20.01 to 197.02 μm. We can generally divide PB by dimensional characteristics (larger formation diameter) into three groups: large (more than 100 μm), medium (70-100 μm) and small (less than 69 μm).

Conclusion: PB is a promising diagnostic marker for the prognosis of dura mater tumours, which doctors and scientists can use in radiological and histological methods. According to morphometry results, we can divide PB into three groups by size - large, medium and small.


Thalamic diffuse midline glioma, a series of cases from a single cancer centre

S. Al Sharie, D. Abu Laban, S. Ghnaimat, M. Al- Hussaini*

*King Hussien Cancer Center, Jordan

Background & objectives: Diffuse midline glioma (DMG) of thalamus is rare, as thalamic tumours, in general, represent 1% of all intracranial neoplasms. It is associated with aggressive behaviours and poor prognosis. Recently, DMGs are characterized by the presence of an H3K27M gene mutation.

Methods: Eighteen thalamic DMGs are reviewed, including the age, gender, and laterality. Immunostains for GFAP, H3 K27M, and H3 K27me, IDH1 (R132H), P53. The outcome of the cases is reported.

Results: There were 11 males. The average age is 34 years (median 27.5). All cases were positive for GFAP. Eight cases were mutant for H3 K27M, all with loss of H3 K27me staining. In addition, a single case showed loss of H3 K27me immunostain in the presence of non-mutant H3K27M. There were 8 P53-mutant cases. All cases were IDH1 (R132H) wildtype. Of interest, there were 3 cases diagnosed as pilocytic astrocytoma, including a case that was H3 K27M mutant. Of those with available survival data, 10 were dead, including 4 cases of mutant H3 K27M and a single case of wildtype H3 K27M, but with loss of H3 K27me.

Conclusion: Around 50% of the cases are consistent with the diagnosis of DMG where H3 K27M is altered. However, there remains a number of cases with dismal outcomes that were non-H3 K27M altered. These should be further tested for potential other mutations.


Histone H3 trimethylated in lysine 27 (H3K27me3) immunohistochemical loss predicts shorter progression-free survival in intracranial meningiomas treated with radiosurgery

P.C. Rizzo*, S. Ammendola, M. Longhi, E. Zivelonghi, S. Pedron, G. Pinna, F. Sala, A. Nicolato, A. Scarpa, V. Barresi

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

Background & objectives: Loss of H3K27me3 has been recently associated with an increased risk of recurrence in meningiomas. In the current study, we aim to investigate whether H327 trimethylation status can predict the response of meningiomas to stereotactic radiosurgery.

Methods: H3K27me3 expression was evaluated by immunohistochemistry in 39 surgically resected, treatment-naive intra-cranial meningiomas, treated with stereotactic radiosurgery (SRS) for a residue or at recurrence. The immunohistochemical results were correlated with tumour recurrence and recurrence-free survival (RFS) after SRS.

Results: Seven meningiomas had H3K27me3 loss, 27 retained H3K27me3 expression and five has inconclusive immunostaining. The immunohistochemical loss of H3K27me3 was significantly associated with tumour recurrence (P= 0.0143) and with shorter RFS (P=0.0036) after SRS.

Conclusion: Our findings suggest that the absence of H3K27me3 in neoplastic cells may concur to a weaker response to stereotactic radiosurgery in meningiomas.


Concerted deposition of vascular basal membrane proteins laminin beta 1 and 2 supports neo-angiogenesis and contributes to vascular permeability

S. Bannykh*, K. Bannykh

*Cedars-Sinai Medical Center, USA

Background & objectives: Expression profiling studies of glioblastomas (GBM) showed striking over-expression of laminin beta 1 (LAMB1) isoform with concomitant loss of LAMB2. Inhibition of LAMB1 expression by nanoconjugates suppresses tumour growth in animal model of glioblastoma through unclear mechanism.

Methods: We use immunohistochemistry and in situ hybridization to identify location and cell of origin for LAMB1/2 production in human brain.

Results: In quiescent state brain the microvasculature but does not express LAMB1, but expresses LAMB2 at the sites of blood brain and CSF brain barrier. Remodelling of microvasculature in subacute stroke involves initial deposition of LAMB1 by proliferating endothelium, followed by an encasement by LAMB2. Low grade diffuse astrocytomas show no LAMB1 expression. Microvascular hyperplasia in GBM show striking deposition of LAMB1, while LAMB2 coverage trails behind. Bevacizumab induces a loss of LAMB1 and normalizes LAMB2 coverage.

Conclusion: Distribution of LAMB2 correlates with sites controlling fluid permeability. Expression of LAMB1 corresponds to endothelial cells and coincides with angiogenesis. Expression of LAMB2 lags behind LAMB1 and its deficiency in the microvascular wall correlates with increased permeability.


Molecular genetics and immunostaining of relapsing inoperable meningiomas located at the base of skull (BS) or in frontal areas, before radiotherapy

M. Alshemeili*, M. Barritault, D. Poncet, E. Jouanneau, D. Meyronet

*Hospice Civil de Lyon, France

Background & objectives: Our experience in screening for targetable molecular alterations of untreatable meningiomas led us to hypothesise that these mutations were less frequent than expected considering available studies.

Our goal to provide prospective clinic-pathological/ molecular data to support future pharmacological targeted therapies.

Methods: From 2018 to 2022, the molecular status of p-AKT and SHH activating molecular alterations was prospectively determined using NGS in the first biopsy of all relapsing patients after surgery with pathologically diagnosed meningioma located at BS or in frontal areas, before radiotherapy prescribed in our center’s multidisciplinary meeting. Further evaluation of IHC prescreening using GAB1 and OTX2 was also conducted.

Results: -61 patients were included: 61.66% (N=36) in BS with median age 53 while 80.33% (N=47) were female. 62% (N=38) were grade 1, 35% (N=21) were grade 2 and 3% (N=2) grade 3.

-2 (3%) meningiomas displayed a SMO mutation and 4 (6%) meningiomas a PIK3CA mutation. Five (83%) meningiomas with SMO or PIK3CA mutations were grade I.

-Among the 8 cases (16%) that express GAB1 with a H-score >120, 2 were associated with SMO and 1 with PIK3CA mutation. OTX2 expression was contributive.

- All AKT mutated meningiomas (N=9,100%) , displayed a GAB1 H-score < 120, a score considered as negative. Conversely GAB1 was expressed in SHH activated meningiomas.

Conclusion: -In real life clinical situation we showed that SHH and mTOR activating mutations are rarer than previously described in exhaustive “molecular landscape” studies. These new data should be taken in account for future therapeutic trial designs.

-GAB1 could be a useful marker for immunohistochemical pre-screening of cases amenable to sequencing for hedgehog pathway or mTOR pathway genes sequencing while OTX2 is not a beneficial marker.

-The WHO grading system has no role in predicting tumour molecular alterations of meningiomas.


miR-196b-5p and miR-107 expression differentiates ocular sebaceous carcinoma from squamous cell carcinoma of the conjunctiva

R.M. Verdijk*, R.O. de Keizer, A. Vriends, G.J. Hötte, D..A. Paridaens, E.A. Wiemer

*Erasmus MC University Medical Center Rotterdam, The Netherlands

Background & objectives: Ocular Sebaceous Carcinoma (OSC) can mimic Squamous Cell Carcinoma of the Conjunctiva (SCCC). Aim of this study was to find microRNA biomarkers to distinguish OSC and SCCC from normal tissue and from each other.

Methods: Clinical OSC and SCCC case files and corresponding histopathological slides were collected and reviewed. Microdissected formalin-fixed paraffin-embedded tumour and control tissue were subjected to semi-high throughput microRNA profiling.

Results: MicroRNA expression distinguishes OSC and SCCC from corresponding control tissues. Selected differentially expressed miRNAs were validated using single RT-PCR assays. A comparison between OSC (n=14) and SCCC (n=18) revealed 38 differentially expressed microRNAs (p<0.05). Differentially expressed miRNAs were selected for validation in the discovery cohort and an independent validation cohort (OSC, n=11; SCCC, n=12). At least two miRNAs miR-196b-5p (p ≤ 0.05) and miR-107 (p ≤ 0.001) displayed a statistically significant differential expression between OSC and SCCC with miR-196b-5p upregulated in SCCC and miR-107 upregulated in OSC. ROC analyses indicated that the combined miR-196b-5p and miR-107 expression levels predicted OSC with 90.0% sensitivity and 83.3% specificity.

Conclusion: Our findings indicate that deregulated miRNAs, identified by comparing tumour tissue with corresponding control tissue, may play a role in the tumourigenic processes in OSC and SCCC. We provide evidence that miR-196b-5p and miR-107 can differentiate OSC from SCCC. Combined testing of miR-196b-5p and miR-107, may be of additional use in routine diagnostics to discriminate OSC from SCCC in conjunctival tumour lesions.

Funding: This research was funded by SWOO-Flierenga, grant number SSWOO2018S12


Audit of outcomes for metastatic uveal melanoma patients in Ireland

C. Hegarty*, K. Kulakova, N. Walsh, C. Hui, S. Kennedy

* Research Foundation, Royal Victoria Eye and Ear Hospital, Dublin, Ireland

Background & objectives: Uveal Melanoma (UM) accounts for approximately 85% of ocular melanomas. The published rate of metastasis is approximately 50%, 5 years following diagnosis. This retrospective analysis investigated the percentage of patients who metastasised and their outcome over a 21 year period.

Methods: A detail review of pathology files, cancer registry files and death certs were accessioned, and results were tabulated on excel. UM patients who received enucleation were selected and further analysis was used to determine who presented with metastasis. Prior to 2011 patients were treated by enucleation. From 2011 on patients were treated by either plaque rt, proton beam or enucleation.

Results: From 194 identified metastatic cases, 93 were female and 101 male. Patient age ranged from 11-90 (average age: 62) at initial diagnosis. 10 patients were aged 20-40, 61 aged 40-60, 112 aged 60-80 and 17 aged 80-100. Youngest patient in this cohort was 11 and metastasised and died shortly after diagnosis. Primary tumour location: choroid and ciliary body. Average time for metastasis was 44 months. Sites of metastasis included liver, lung, and skin. Metastasis rates were as follows: 116 patients metastasised 0-24 months after diagnosis, 57 patients 24-48 months and 51 exhibited late metastasis ranging from 60-216 months after initial diagnosis. Patients died on average 1.5 years following metastatic diagnosis.

Conclusion: From the analysis in our institution the incidence of metastatic disease corresponds with the European published figures. Our cohort includes a number of patients with a metastatic UM diagnosis prior to the age of 35. We found that 47.2% of patients treated for UM metastasised on average 43 months following diagnosis and the average time to death following metastasis was 1.5 years. Genetic analysis is currently underway to examine the contribution of specific genes role in early and late metastasis.

CP-02| Computational Pathology Symposium: Abstract presentations and Best Abstract Award


Swarm learning for decentralized deep learning in gastric cancer histopathology

O.L. Saldanha*, H.I. Grabsch, H.S. Muti, R. Langer, B. Dislich, M. Kohlruß, G. Keller, J.N. Kather

*UniKlinik Aachen, Germany

Background & objectives: A limitation for computational pathology is the difficulty of data exchange. Swarm learning (SL) is a protocol for decentralized training of deep learning models. We evaluate SL for the prediction of microsatellite instability (MSI) from gastric cancer histopathology images.

Methods: We collected tissue samples from four cohorts of patients with gastric cancer from four countries (Switzerland, Germany, the UK and the USA). Each dataset was stored in a physically separate computer. We trained a deep learning-based classifier to detect microsatellite instability using SL from digitized haematoxylin and eosin-stained resection slides without annotating tumour containing regions.

Results: We evaluated the patient-level performance for the prediction of MSI status in the TCGA cohort (N=334 patients). We found that local models achieved AUROCs of 0.7016 (+/- 0.0087), 0.5600 (+/- 0.0238) and 0.6638 (+/- 0.0170) when trained on local datasets. Merging the three training cohorts on a central server (merged model) improved the prediction of AUROC to 0.7508 (+/- 0.0074). This was compared to the performance of SL-trained models, and we assessed the performance of a weighted Swarm Learning model (w-chkpt) for MSI mutation prediction. In this task, w-chkpt achieved an AUROC of 0.7469 (+/- 0.0214), which was not significantly different from the merged model (p=0.7806).

Conclusion: Computational pathology problems in gastric cancer requires large datasets. Preferably, such data should be derived from different centres so as to avoid bias. However, the collection of such datasets faces practical, ethical and legal obstacles. These obstacles can be overcome using SL. In the future, this could be an alternative for sharing patient-related data across sites.


BLEACH&STAIN, a novel multiplex fluorescence immunohistochemistry framework that facilitates a fast high throughput analysis of >15 biomarkers in more than 3000 human carcinomas

E. Bady, K. Möller, N.F. Debatin, T. Mandelkow, C. Hube-Magg, N.C. Blessin*

*University Medical Center Hamburg, Germany

Background & objectives: Multiplex fluorescence immunohistochemistry (mfIHC) approaches were yet either limited to 6 markers or limited to a small (1.5cmx1.5cm) tissue size that hampers translational studies on large tissue microarray (TMA) cohorts.

Methods: To assess more markers in a large patient cohort, we have developed a BLEACH&STAIN mfIHC approach that enables the analysis of ≥15 biomarkers in 3098 tumour samples from 44 different carcinoma entities within one week and without costly instrumentalization. An artificial intelligence-based framework –incorporating three different deep learning systems– for automated marker quantification was used to interoperate the BLEACH&STAIN data.

Results: This approach was used to study the relationship between PD-L1 expression on multiple different cell types and the relationship with various leucocyte subtypes (PD-L1,PD-1,CTLA-4,panCK,CD68,CD163,CD11c,iNOS,CD3,CD8,CD4,FOXP3,CD20,Ki67,CD31). Comparing the automated and deep learning-based BLEACH&STAIN PD-L1 analysis framework with conventional brightfield PD-L1 data revealed a high concordance in tumour cells (p<0.0001) as well as immune cells (p<0.0001) and an accuracy of our approach ranging from 90% to 95.2%. Unsupervised clustering showed that a major proportion of the three PD-L1 phenotypes (i.e., PD-L1+ tumour and immune cells [G1], PD-L1+ immune cells [G2], PD-L1 negative [G3]) were either inflamed (G1.1, G2.1, G3.1) or non-inflamed (G1.2, G2.2, G3.2) and showed distinct spatial orchestration patterns.

Conclusion: BLEACH&STAIN mfIHC in combination with a deep learning-based framework for automated PD-L1 assessment on tumour and immune cells enabled a rapid and comprehensive assessment of 15 biomarkers across more than 3000 tumour entities that is quick and easy to establish in all laboratories. In breast cancer, the PD-L1 relative expression on tumour cells showed a significantly higher predictive performance for overall survival compared to the commonly used PD-L1 tumour proportion score.


Pathologists’ first perspectives on barriers and facilitators of computational pathology implementation in histopathology

J. Swillens*, I. Nagtegaal, S. Engels, A. Lugli, R. Hermens, J. van der Laak

*Radboudumc, The Netherlands

Background & objectives: Computational pathology algorithms detect, segment or classify cancer in whole slide images in histopathology. Currently, challenges have to be overcome before they can be used. We aim to explore international perspectives on the role of computational pathology in clinical practice.

Methods: We will focus on opinions and first experiences regarding barriers and facilitators, which will inform establishment of validation studies, implementation trajectories and communication activities to generate widespread stakeholder acceptance. We conducted an international explorative eSurvey study and semi-structured interviews with pathologists and pathology residents. We used an implementation framework to classify potential influencing factors.

Results: Results of the eSurvey showed remarkable variation in opinions regarding attitude, understandability and validation of computational pathology. Results of the interviews showed that barriers focused on the quality of available evidence, while most facilitators concerned strengths of using computational pathology. A lack of consensus was present for multiple barriers and facilitators, such as the determination of sufficient validation using computational pathology, the preferred function of computational pathology within the digital workflow and the appropriate timing of computational pathology introduction in pathology education.

Conclusion: The diversity in opinions illustrates variety in barriers and facilitators in computational pathology implementation. A next step would be to quantitatively determine important influencing factors among all relevant stakeholders. Simultaneously, prospective validation studies may be developed and initiated, to collect evidence on the most effective way of implementation. This will further propel the use of computational pathology into clinical practice.

Funding: This study received funding from the Dutch Cancer Society (grant number 2017-10602). This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 945358. This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation program and EFPIA.


The prognostic value of deep learning based mitotic count for breast cancer molecular subtypes

M. Balkenhol*, C. Mercan, L. Tessier, D. Tellez, A. Niendorf, A. Wegscheider, P. Bult, F. Ciompi, J. van der Laak

*Radboudumc, The Netherlands

Background & objectives: Breast cancer grading was introduced decades ago and its prognostic value has not yet been studied in the context of contemporary molecular classification. This study uses automatic mitotic count to evaluate its prognostic value for different breast cancer molecular subtypes.

Methods: A previously developed artificial intelligence (AI) algorithm detected mitoses and assessed mitotic counts in H&E-stained whole-slide images from a multicentre cohort of 846 breast cancer patients. Stratified analyses based on hormonal receptor (HR) and HER2 status were performed to study potential different prognostic mitotic cut off values. Multivariable Cox regression survival models were used to study its independent prognostic value.

Results: We found that the mitotic count, assessed by AI was prognostic in univariate Cox analysis for HR positive / HER2 negative breast cancers, applying the widely used Nottingham cut-offs, both for recurrence free and overall survival. Prognostic value could be optimized applying a cut-off of 10 mitoses per 2 mm2 (recurrence free survival hazard ratio = 2.05 (1.14-3.68; p=0.02); overall survival hazard ratio = 1.84 (1.09-3.11; p=0.02) in multivariable analysis). However, for HER2-positive tumours, no mitotic cut off was found to be prognostic.

Conclusion: This study shows that automatic mitotic count yields different prognostic information for specific subtypes of breast cancer, suggesting the need for a molecular subtype specific grading assessment in clinical practice. In addition, it showed the potential of AI to automate part of the pathologists’ workflow, as well as the feasibility of applying modern AI technologies to re-assess widely used histopathological features by evaluating large numbers of cases in a systematic, accurate and reproducible manner.


Pathologist validation of a machine learned biomarker for risk stratification in colon cancer

V. L'Imperio, E. Wulczyn, M. Plass, H. Muller, N. Tamini, L. Gianotti, N. Zucchini, R. Reihs, G.S. Corrado, L.H. Peng, P. Cameron Chen, M. Lavitrano, Y. Liu, D.F. Steiner, K. Zatloukal, F. Pagni*

*Department of Medicine and Surgery, Pathology, ASST Monza, San Gerardo Hospital, University of Milan-Bicocca, Monza, Italy

Background & objectives: Identifying new prognostic features in colon cancer may refine histopathology review. While prognostic artificial intelligence (AI) systems have demonstrated significant risk stratification in several cancer types, studies have not yet shown that the machine learned features are interpretable by pathologists.

Methods: This retrospective study utilized de-identified, archived colorectal cancer cases from 2013 to 2015 from University of Milano-Bicocca (UNIMIB). Histologic slides from 258 consecutive colon adenocarcinoma cases were reviewed at UNIMIB by two institutional pathologists. The pathologists conducted semiquantitative scoring for Tumor Adipose Feature (TAF), which was previously identified via a prognostic deep-learning model developed using an independent colorectal cancer cohort.

Results: 258 colon adenocarcinoma histopathology cases from 258 patients (median age 67 years; interquartile range 65-81; 47% female) with stage II (n=122) or stage III (n=139) cancer were included. TAF was identified in 120 cases (widespread n=63; multifocal n=31; unifocal n=26). For OS analysis adjusting for tumour stage, TAF was independently prognostic: Hazard Ratio (HR)=1.55 (95%CI 1.07-2.25; p=0.02) for TAF as a binary feature (presence vs. absence); and HR=1.87 (95%CI 1.23-2.85; p<0.005) for the highest TAF category (widespread) when evaluating semiquantitative scoring. Inter-pathologist agreement for widespread TAF vs. lower categories (absent/unifocal/multifocal) was 90%, corresponding to kappa at this threshold of 0.69 (95%CI 0.58-0.80).

Conclusion: Pathologists were able to learn and reproducibly score for TAF providing significant risk stratification on this independent dataset. While additional work is warranted to understand the biological significance of this feature and to establish broadly reproducible TAF scoring, this work represents an important milestone as the first validation of human expert learning from machine learning in pathology. This validation demonstrates that a computationally identified histologic feature can represent a human-identifiable, prognostic biomarker with the potential for integration into pathology practice.


Predicting genetic variation from quantitative tissue phenotypes using explainable machine learning

J. Connelly*, J. Luft, C.J. Anderson, P. Bankhead, F. Connor, P. Flicek, N. López-Bigas, C.A. Semple, D.T. Odom, S.J. Aitken, M.S. Taylor

*MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, United Kingdom

Background & objectives: Most human cancer genomes exhibit multiple mutational signatures, reflecting the complex milieu of damage and repair occurring during carcinogenesis. We used a robustly controlled, highly powered in vivo experiment to investigate genotype-phenotype correlates.

Methods: Inbred mice were exposed to a single dose of diethylnitrosamine shortly after birth. Resultant liver tumours were isolated and submitted for WGS, total RNAseq, and histopathology. This cohort was used to discover lesion segregation, which drives cancer genome evolution. We used deep learning to segment nuclei in these images, computed quantitative morphometric features, and modelled these using machine learning.

Results: We find that supervised learning of quantitative nuclear morphology robustly predicts (i) germline variation between ancestrally divergent mouse strains, (ii) germline heterozygosity within strain, and (iii) somatic mutations in driver oncogenes. We apply a game-theoretic approach to uncover morphometric features which explain the inference, identifying nuclear geometry as key to inferring driver gene mutations, and nuclear histochemical staining most relevant for germline variation. Interestingly, we often find that statistical measures of variance in morphology are more relevant than measures of central tendency, and that the relationships are frequently non-linear and best modelled using tree ensembles. We further uncover pervasive batch effects and describe an approach to address these.

Conclusion: We defined quantitative relationships between histological phenotype and both germline and somatic genetic variation in tumour tissue using explainable machine learning. This approach has the potential to influence how clinical grade molecular inference models are optimised for generalisability in the future and allow histopathologists to gain intuition into the predictions made by deep models.

Funding: This work was supported by: the MRC Human Genetics Unit core funding programme grants (MC_UU_00007/11 and MC_UU_00007/16), MRC Toxicology Unit core funding (RG94521), Cancer Research UK Cambridge Institute core funding (20412), European Molecular Biology Laboratory, ERDF/Spanish Ministry of Science, Innovation and Universities-Spanish State Research Agency/DamReMap Project (RTI2018-094095-B-I00), and the Wellcome Trust (WT202878/B/16/Z). Edinburgh Genomics is partly supported through core grants from NERC (R8/H10/56), MRC (MR/K001744/1) and BBSRC (BB/J004243/1). J.C. is supported by a Wellcome Trust PhD Training Fellowship for Clinicians (WT223088/Z/21/Z) as part of the Edinburgh Clinical Academic Track (ECAT) programme. S.J.A. received a Wellcome Trust PhD Training Fellowship for Clinicians (WT106563/Z/14/Z) and National Institute for Health Research (NIHR) Clinical Lectureship.


Successful deployment of an AI solution for primary diagnosis of prostate biopsies in clinical practice

M. Aslam*, A. Bansal, M. Atkinson, K. Sawalem, M. Mehdi, H. Abdelsalam, A. Heath, P. Huey, R. Nicholson, J. Theunissen, M. Grinwald, M. Vecsler, C. Linhart

*Betsi Cadwaladr University Health Board, United Kingdom

Background & objectives: This project aimed to validate, clinically deploy and integrate an AI decision support solution for prostate biopsies into the digital pathology workflow as a first read for primary diagnosis.

Methods: The project included a technical validation and integration phase of the AI solution into the lab workflow prior to the deployment. Seven pathologists underwent training and used the solution for prospective primary diagnosis of consecutive prostate core needle biopsies, reporting on 334 cases (1197 H&E slides). AI-assisted diagnoses were compared to the ground truth (GT = concordance of two pathologists).

Results: The AI solution demonstrated high performance when pre-classifying slides with highest likelihood to be benign or malignant, with NPV = 98.8% (331 / 335) and PPV = 99.8% (399 / 400), respectively. 32% of slides have been classified as undetermined by AI. In 4 out of 7 discrepancies that were compared subsequently to the GT, the AI classification was correct. User experience survey, as reported by pathologists, showed high satisfaction marks for the AI solution. Pathologists felt more confident to review and report both benign and cancerous slides using the AI system and prefer to continue working with the system compared to a microscope.

Conclusion: We report here successful implementation of a multi feature AI solution that automatically imparts clinically relevant diagnostic parameters regarding prostate cancer and other pathologic features. The solution demonstrated its ability to accurately triage cancerous prostate cases and improve diagnostic quality. Thus, Galen Prostate AI solution could be used as significant aiding tool for pathologists in clinical decision-making in routine pathology practice.

Funding: SBRI Centre of Excellence

MD-01| Molecular Diagnostics Pathology Symposium: Selected Abstracts


Comparison of whole genome with broad gene panel sequencing to identify actionable targets for cancer treatment

D. Leunissen*, L. Kester, E. Driehuis, A. zur Hausen, P. Van Diest, W. de Leng, E. Speel

*Maastricht UMC+, The Netherlands

Background & objectives: DNA mutation analysis by broad panel NGS and WGS is currently used to guide cancer treatment. WGS can detect all genetic alterations, however, its implementation in daily clinic holds practical considerations. We evaluated the potential of WGS alternatives in diagnostics.

Methods: Publicly available WGS data of lung (n=86), colon (n=118), melanoma (n=63) and ovarian (n=42) cancers was used to identify clinically relevant variants using variant interpretation software (VarSome Clinical). We compared reported variants between the whole genome and targeted panels and their clinical relevance in Dutch routine care or clinical trials.

Results: For each tumour type unique single nucleotide variants were identified (on average 1834 (likely) pathogenic variants (LPV) per tumour type). Structural variants were not included in this study. After applying in silico filters for commercially available cancer hotspot panel (CHP), Foundation Medicine (FMI) and TSO500 panels (50, 324 and 523 genes, respectively), of the LPV detected by WGS, an average of 12.4%, 6.6% and 3.5% was predicted to be detected by TSO500, FMI and CHP, respectively. Of the detected variants, all that were deemed clinically relevant were detected by the broad TSO500 and FMI gene panels while 15% would be missed using a smaller CHP gene panel.

Conclusion: Of the clinically actionable LPV detected by WGS in four tumour types, 100% is assumed to be identified by broad gene panels NGS (TSO500, FMI) and 85% by CHP. We conclude that in current clinical practice, the added value of WGS compared to broad gene panels is limited for clinically actionable single nucleotide variant detection in the tumour types analysed.


Validation of TruSight TM Oncology Comprehensive (EU) assay

V. Sementchenko*, M. Harris, N. Haseley, A. Yazdanparast, P. Wenz, J. Dockter, T. Pawlowski

*Illumina, Inc., USA

Background & objectives: TruSightTM Oncology Comprehensive (TSO Comp) is a CE-marked comprehensive genomic profiling (CGP) assay designed to interrogate solid tumours for relevant single nucleotide variants, multi-nucleotide variants, insertions, deletions and gene amplifications from DNA, and gene fusions and splice variants from RNA.

Methods: It is an enrichment-based next-generation sequencing assay that targets 517 genes for detection of small DNA variants, 2 genes for detection of gene amplifications, 23 genes for detection of gene fusions and 2 genes for detection of splice variants. TSO Comp performance was evaluated in various analytical studies, including limit of detection/blank, accuracy, precision, utilizing FFPE-derived DNA and/or RNA samples.

Results: Performance of the assay was assessed for multiple variant classes: small variants and gene amplifications (amps) from DNA, TMB and MSI genomic signatures in DNA, as well as RNA fusions and splice variants.

Limit of Detection was as low as 1.6% variant allele frequency for small DNA variants, 2-fold change for gene amps, 10 supporting reads for fusions, and 19 supporting reads for splice variants. Specificity was 99.9999% for small DNA variants, 99.9% for fusion variants and 100% for gene amps and MSI. Positive Percent Agreement (PPA) with whole exome sequencing (WES) for small DNA variants was 84.7% (382/451) for WES somatic and 99.8% (33,163/33,224) for WES germline variants.

Conclusion: DNA small variant Negative Percent Agreement was 99.999% (70,000,481/70,000,907). PPA for gene amps was 92.3% (337/365), for MSI status 93% (40/43). PPA for RNA fusions and for RNA splice variants was 80.5% (70/87). Qualitative precision analysis across multiple operators, instruments, reagent lots, and days showed high concordance (>90% positive percent call).

This CGP assay helps maximize the ability to find actionable biomarkers and help inform therapy decisions according to clinical guidelines that have the potential to improve patient management.


Evaluation of MET amplification in lung cancer via Idylla™ GeneFusion cartridges

J. Siemanowski*, V. Welter, A. Ehteschami, A. Quaas, S. Merkelbach-Bruse

*University hospital Cologne, Germany

Background & objectives: MET amplification in lung cancer is known as resistance mechanism to epidermal growth factor receptor tyrosine–kinase inhibitors. This retrospective study used Idylla™ GeneFusion assay (Biocartis) cartridges to develop a delta Cq cut-off for discrimination of non-amplified versus MET amplified samples.

Methods: A cohort of 70 samples including 31 non-amplified and 39 MET amplified samples was analysed. MET amplification status was previously detected by fluorescence in situ hybridization (FISH). One to five 10 μm slices with a tumour cell content (TC) between 10% and 90% of the same FFPE tumour tissues were taken for analysis with the IdyllaTM GeneFusion Assay.

Results: For initial data analysis a threshold of -3.0 (Delta housekeeping gene-MET) was used for discrimination between non-amplified and MET amplified samples. Hereby 27 out of 31 non-amplified samples (specificity= 87%) and 26 out of 39 MET amplified samples (sensitivity=67%) were detected. After a threshold adjustment from -3.0 to -2.0 the specificity was lowered to 74% (23/31 non-amplified samples) but sensitivity increased to 84% (33/39 MET amplified samples). No further optimization was reached by implementing a % TC cut-off since false positives and false negatives were distributed at different TC content. All top-level amplifications (copy number gain (CNG) > 10) were true positive.

Conclusion: This study showed that the IdyllaTM GeneFusion Assay might be a promising screening tool for top level MET amplification assessment in lung cancer samples. Nevertheless, even after threshold adjustment both specificity and sensitivity within different levels of MET amplification remained lower than 90%. Therefore, this test with our proposed cut-off is not suited to identify MET amplification at lower thresholds. This is in line with other published PCR-based approaches using for example next generation sequencing.


Shallow whole genome sequencing accurately detects homologous recombination deficiency in ovarian cancer

J. Dagher*, N. Scamuffa, V. Vocat-Mottier, K. Lefort, L. de Leval, B. Bisig, E. Missiaglia

*Institute of Pathology, CHUV, Switzerland

Background & objectives: Homologous Recombination Deficiency (HRD) predicts benefit from PARP inhibitors. MyChoiceCDx test (Myriad) is approved to assess HRD, based on BRCA1/2 mutations and genomic instability (GIS) score. We assessed HRD by shallow whole-genome sequencing (sWGS) using Large-scale Genomic Alteration (LGA) score.

Methods: Fifteen high-grade serous ovarian carcinomas underwent sWGS on a NextSeq550 (Illumina), using FFPE-extracted DNA (tumour content ≥25%). All samples had known tumour BRCA1/2 status (9 mutated, 6 wild-type), while 9 also had Myriad GIS score available. Raw reads were aligned to hg19 human genome assembly and analysed using shallowHRD software to compute LGA score, which was considered positive if ≥20.

Results: We successfully performed sWGS on all samples, generating an average of 22 million (M) reads per sample (11-42M) and achieving a mean coverage of 0.8X (0.3X-1.6X). LGA computation was still robust by artificially down-sampling to 5M reads (0.2X coverage). LGA score was positive in 8/9 BRCA-mutated cases, and negative in all wild-type cases (median 26 (11-40) vs. 6.5 (0-14), Wilcoxon rank test P=0.004). Correlation between LGA and GIS scores was statistically significant (Spearman rank rho 0.78; P=0.014), with only 1/9 cases showing discordant results. This was a BRCA wild-type sample with positive GIS score but negative LGA score, presumably due to low tumour cell content (25%) and low DNA quality.

Conclusion: In our series of 15 high-grade serous ovarian carcinomas, determination of LGA score by sWGS – using a predefined positivity cut-off ≥20 – was highly concordant with both BRCA1/2 mutational status and Myriad GIS score. Despite a limited number of samples analysed so far, this preliminary cohort shows promising results, supporting further work to refine the cut-off value(s) and validate this tool as a predictive molecular biomarker for PARP inhibitor therapy, for patients with ovarian cancer or potentially other malignancies.


Microsatellite instability in intestinal T-cell lymphomas

V. Rattina, D. Vallois, J. Bouilly, E. Lingre, V. Vocat Mottier, L. Veloza Cabrera, K. Lefort, B. Bisig, L. de Leval, E. Missiaglia*

*Institute of Pathology, University Hospital Lausanne (CHUV), Switzerland

Background & objectives: Microsatellite instability (MSI) is a consequence of defective DNA mismatch repair (MMR), leading to a hypermutant phenotype, with a high tumour mutational burden (TMB). We explored the potential impact of MSI in the oncogenesis of primary intestinal T-cell lymphomas (ITCLs).

Methods: Whole Exome Sequencing (WES) was performed on 54 ITCLs and matched non-tumour DNA: 34 MEITLs (Monomorphic Epitheliotropic Intestinal T-cell Lymphomas) and 20 EATLs (Enteropathy-Associated T-cell Lymphomas). Mutation signatures were extracted from somatic variants and compared with COSMIC reference signatures (MuSiCa package). MSI status was predicted from instability scores (MANTIS software), and assessed by PCR (5 mononucleotide markers) on 43 samples.

Results: We observed an overall median TMB of 1.8 non-synonymous somatic mutations per Mb, with a higher median in EATLs relative to MEITLs (2.3 vs 1.7/Mb). Nonetheless, the highest TMBs were detected in three MEITLs (maximum: 15/Mb). Accordingly, these three cases showed signatures associated with MMR deficiency, and high instability scores as measured by MANTIS. Their MSI status was confirmed by standard PCR analyses, while none of the other MEITLs or EATLs showed instability. Altogether, our analyses estimated a prevalence of MSI of 11% (3/28) in the MEITL subtype. Notably, none of the MSI samples presented somatic mutations within MMR pathway genes (MLH1, PMS2, MSH2, MSH6), suggesting alternative mutagenic mechanisms.

Conclusion: Taken together, these data reveal a relatively high TMB in ITCLs when compared to other peripheral T-cell lymphomas, and an MSI status in a subset of MEITLs. This suggests a role of MMR deficiency in the oncogenesis of a proportion of ITCLs, with potential clinical implications. Alternative mutagenic mechanisms, possibly involving the intestinal environment, seem to play a role in the tumorigenesis of the majority of ITCLs.


Extensive spatial characterisation of the tumour-microenvironment in a large clinical non-small cell lung cancer cohort and correlation with clinical and pathological parameters

S. Ruane*, S. Schallenberg, C. Böhm, G. Dernbach, M. Gottschalk, N. Aldoj, X. Liang, R. Büttner, A. Quaas, S. Merkelbach-Bruse, K. Hekmat, M. Heldwein, G. Schlachtenberger, D. Horst, M. Dragomir, J. Rückert, N. Frost, F. Leiss, M. Alber, F. Klauschen

*Aignostics, Germany

Background & objectives: We present an AI-driven image analysis workflow combining histomorphological and multiplex immunofluorescence data for automated, cell-level characterization of the tumour microenvironment (TME) that can facilitate biomarker identification. The workflow is utilized for the exploration of a clinical NSCLC cohort.

Methods: 340 clinical NSCLC cases from an FFPE tissue microarray were stained with a 12-plex immunofluorescence panel (IF) and subsequently with hematoxylin and eosin (H&E). All stains were scanned and co-registered with single cell accuracy. Deep learning models were developed to detect tumour regions from H&E and cell subtypes from IF and H&E. The resulting readouts were correlated with clinical parameters.

Results: Deep learning models were trained to quantify the presence of tumour cells expressing PD-L1 and lymphocyte cell subtypes. For the task of separating PD-L1+ carcinoma, PD-L1- carcinoma and other cells a balanced accuracy (BA) of 92% was reached. The task of separating FoxP3+ T-cells, CD8+ T-cells and B-cells respectively from other cells was performed with a BA of > 90%. The models were trained and evaluated using over 13,000 IF-informed manual pathologist annotations. The evaluation was performed on a held out TMA section with separate cases. The correlation of PD-L1 expression and various tumour infiltrating lymphocyte subpopulation levels with patient prognosis and other clinical parameters was computed.

Conclusion: The presented workflow allows for scalable characterization of the NSCLC TME by (1) staining the same section with H&E and multiplex IF images and registering resulting scans and (2) applying deep learning for robust cell subtype detection from both modalities at once. First results show that cells involved in the immune response within the TME can be precisely quantified and correlated with clinical parameters.


PS-01 | Poster Session Breast Pathology


Insulinoma-associated protein 1 (INSM1) expression in breast carcinomas with neuroendocrine morphologies: application and future perspectives

T. Kawasaki*, T. Tashima, Y. Nakamura, A. Fujimoto, Y. Usami, A. Fujita, H. Imai, Y. Baba, M. Nakahira, S. Ryozawa, K. Okamoto, H. Kagamu, T. Inozume, C. Muramatsu, M. Saitoh, K. Taniyama, H. Nagai, T. Kondo, A. Enomoto, K. Kaira

*Department of Pathology, Saitama Medical University International Medical Center, Japan

Background & objectives: Insulinoma-associated protein 1 (INSM1) is a zinc-finger transcription factor initially isolated from a human insulinoma subtraction library. INSM1 was recently demonstrated to be a better diagnostic and prognostic indicator for small cell lung carcinoma than the traditional neuroendocrine (NE) markers.

Methods: Herein, for the first time, we present eight cases with NE phenotype mammary neoplasms in which the NE nature of the tumours was confirmed solely by INSM1. Patients were 35–64 (mean: 48.9) year-old women with breast tumours showing characteristic NE morphologies, i.e. solid growth of polygonal, short-spindle or plasmacytoid cells with fine-granular cytoplasm and nuclei, and a well-developed vascular network.

Results: On immunohistochemical examinations, these malignancies showed diffuse nuclear expressions of INSM1 (mouse monoclonal, clone A-8: sc-271408, dilution 1:100; Santa Cruz Biotechnology, Inc., Dallas, TX), whereas chromogranin A [three sources: 1) mouse monoclonal, clone LK2H10; Roche Diagnostics, Mannheim, Germany, 2) rabbit polyclonal, dilution 1:500; Dako, Copenhagen, Denmark, and 3) rabbit polyclonal, 412751; Nichirei Bioscience Inc., Tokyo, Japan] and synaptophysin [two sources: 1) rabbit polyclonal, dilution 1:50; Dako, and 2) mouse monoclonal, clone 27G12: 413831; Nichirei] staining did not correspond to distinct NE features in the neoplastic cytoplasm. Finally, we diagnosed these cancers of luminal-like immuno-subtype as 4 neuroendocrine neoplasms (NENs), three hypercellular mucinous carcinomas, and one neuroendocrine ductal carcinoma in situ.

Conclusion: Based on the establishment of INSM1, a promising NE marker with high sensitivity and specificity, accompanied by our current immunohistochemical results, the frequency of detecting NE differentiation in systemic neoplasms, including mammary NENs as well as carcinomas with NE differentiation such as type B mucoid carcinoma and solid papillary carcinoma, is anticipated to increase. Our observations might contribute to the development of novel treatments including molecular-targeted therapies for these tumour entities.

Funding: Tomonori Kawasaki is supported by Grants-in-Aid for Scientific Research (No. 21K06910 and No. 20K08131) from the Japanese Ministry of Education, Culture, Sports, Science and Technology and the National Hospital Organization (NHO) Grant (H29-NHO-01).


PRAME expression in invasive breast carcinoma

L. Korša*, M. Abramovic, T. Pikivaca, L. Kovačević, M. Milošević, M. Prutki, Z. Marušić

*Clinical Department of Pathology and Cytology, University Hospital Centre Zagreb, Croatia

Background & objectives: PRAME (PReferentially expressed Antigen in MElanoma) is a carcinoma testis antigen expressed in numerous tumour types. The aim of this study was to assess PRAME expression in different surrogate subtypes of breast carcinoma and its correlation with other prognostic factors.

Methods: A total of 25 Luminal A like, 31 Luminal B like, 15 triple negative (TN) and 18 HER2 positive breast carcinomas were assessed for PRAME expression by immunohistochemistry (IHC) using the EPR20330 (ab219650; Abcam) monoclonal antibody. Expression of PRAME was quantified as positive (nuclear and/or cytoplasmic staining) or negative, and also as a percentage of tumour cells expressing PRAME.

Results: A significantly higher expression of PRAME was detected in HER2 positive carcinomas and TN breast carcinomas compared to ER positive (luminal like) subtype of breast carcinomas. PRAME expression was detected in 53% (8/15) TN carcinomas and 72% (13/18) HER2 positive carcinomas, as opposed to luminal A and B like breast carcinomas, where it was expressed in 32% (8/25) and 26% (8/31) of cases, respectively. Percentage of PRAME positive tumour cells showed positive correlation with tumour size, Ki67 proliferation index, HER2 status, nuclear grade and presence of metastasis, and negative correlation with ER status.

Conclusion: Previous studies on PRAME in breast carcinoma were mainly based on RT-PCR detection, with immunohistochemical studies limited to polyclonal antibody results. Our study showed that HER2 positive and TN breast carcinomas more commonly express PRAME than ER positive carcinomas and that PRAME expression shows positive correlation with certain prognostic factors. The importance of PRAME expression in breast carcinoma lies in its potential use as an immunotherapeutic target, particularly in patients with limited therapeutic options (e.g. in TN carcinomas).


Alpha-methylacyl-CoA Racemase (AMACR/P504S) over-expression occurs in the early proliferative lesions of the breast irrespective of apocrine differentiation

Z. Gatalica, P. Stafford, S. Vranic*

*Qatar University, Qatar

Background & objectives: Alpha-methylacyl-CoA racemase (AMACR/P504S) is a mitochondrial and peroxisomal enzyme involved in the branched-chain fatty acid and bile acid metabolism. We explored AMACR expression in a large cohort of patients undergoing breast biopsies to investigate its role in cancer progression.

Methods: The first, exploratory cohort of all cancer types (Caris Life Sciences) was investigated for AMACR mRNA expression followed by the second cohort of 150 patients’ breast biopsies (77 with invasive carcinomas) studied for the discrete lesions’ expression of AMACR using an automated IHC. The lesions were considered positive if AMACR was detected in ≥10% of the cells.

Results: AMACR mRNA expression was detected in all cancer types and in breast carcinoma, its median value was substantially and consistently lower than in prostate carcinomas. However, AMACR protein expression was detected not only in apocrine carcinomas, as recently described, but also in normal breast epithelium (6/77 samples, 8%), and with increased frequency in proliferative epithelial lesions and carcinomas: 23% of UDH, 73% of ADH and in-situ carcinomas, 60% of invasive carcinomas, including lymph node/distant metastases. LCIS and invasive lobular carcinomas expressed AMACR in 50% of cases, respectively. Apocrine lesions showed strong, nearly uniform overexpression of AMACR (100% of metaplasias, hyperplasias, and in situ carcinomas and 88% of invasive apocrine carcinomas).

Conclusion: AMACR expression in the breast is a common, early pathogenic event in the development of breast carcinoma and not exclusive to the apocrine morphology. It points to altered lipid metabolism as one of the hallmarks of breast carcinogenesis, similar to several other malignancies, notably prostate carcinoma. It may hence represent a potential target for early cancer intervention and management.


Evaluation of NLRP3 immunohistochemical levels in breast cancer

M. Lambropoulou*, V. Papadatou, A. Karatza, S. Tologkos, T. Deftereou, V. Lampropoulou, T. Alexiadis, I. Olbasalis, C. Alexiadi, T. ChatzIneophytou, G. Tripsianis, O. Pagonopoulou

*Histology-Embryology Lab., Medical Department, Democritus University of Thrace, Alexadroupolis, Greece

Background & objectives: NLRP3 belongs to a complex of proteins triggering proteolytic degradation via caspase-1. This molecular pathway is involved in breast oncogenesis. The purpose of our research was the investigation of NLRP3 expression in breast cancer and its association with clinicohistopathological factors.

Methods: Formalin-fixed, paraffin-embedded breast tissues from 43 patients were studied. 31 of them diagnosed with breast cancer (study group) and the other 12 (control group) diagnosed with fibroadenoma. NLRP3’s expression was investigated immunohistochemically using a monoclonal anti-NLRP3 antibody. NLRP3 expression was statistically associated with various clinical and histological parameters using the SPSS program.

Results: Our results showed statistically significantly higher expression of NLRP3 in well- differentiated carcinomas G3 (p<0.005), a tendency for higher NLRP3 expression in carcinomas with severe Ki-67 expression (p< 0.01) and also with the presence of lymph node metastases (p<0.022). In contrast, no statistically significant correlation was observed between NLRP3 expression and patient age (p=0.662), expression of ER (p=0.236) and PR (p=0.244), HER2 gene expression (p=0.342) and the cancer type (p=0.871).

Conclusion: Based in our results, we conclude that NLRP3 could be a potential breast cancer biomarker as its high immunohistochemical expression is directly linked to advanced breast cancer. However, this is just a preliminary study and a more extended research needs to be conducted in order to establish these outcomes.


Encapsulated papillary carcinoma. Our experience from 2005

A. Córdoba*, R. Beloqui, I. Fernandez, C. Cerezo, A. Pasco, M.R. Mercado, I. Amat

*Complejo Hospitalario de Navarra, Spain

Background & objectives: The diagnosis of Encapsulated Papillary Carcinoma (EPC) of the breast is challenging. These lesions are staged as lesions in situ, and it is not possible to confirm EPC diagnosis until the capsule has been examined to rule out invasive growth.

Methods: All the cases treated at our institution since 2005 are reviewed. To analyse the involvement of sentinel lymph nodes, as the indication for their study is controversial. We have collected 54 patients who were treated surgically for EPC. Selective SLN biopsy was performed in 39 (72%) of them.

Results: We have observed that the mean size of EPC was 12 millimetres. The Nottingham grade was 1 in 46 cases (85.1%), 2 in 6 cases (11.1%) and 3 in 2 cases (3.7%). EPC was associated with invasive carcinoma in 17 cases (31.4%).

Of the 39 patients in whom SN was performed, only three cases (5.5%) showed invasion, whereas one was ITC, and 2 micrometastases. In these three patients, the EPC showed invasion between 3 and 8 mm.


-SLN involvement is very low in our series of EPC (5.5%).

-The frequences of EPC without infiltration and invasive carcinoma are 0% and 11%, respectively.

-The presence of invasive carcinoma is only confirmed in the surgical specimen.

-Therefore, we suggest assessing the indication for SLN in those cases where invasive carcinoma is confirmed.


Applicability of One Step Nucleic Acid in cytokeratin 19 negative tumours, based on a series of cases

A. Córdoba*, M. Bronte, I. Fernandez, I. Amat, R. Beloqui, A. Larrea

*Complejo Hospitalario de Navarra, Spain

Background & objectives: The One Step Nucleic Acid (OSNA)is a molecular technique for the study of sentinel lymph node (SLN) in breast carcinomas. There is no general agreement on the lowest limit of detection of CK19 expression. A cut-off of 30% is proposed.

Methods: We analysed the expression of CK19 in BC diagnosed in our institution since May 2018 and selected those cases with negative or partial expression of CK19. A total of 1189 cases of BC biopsied in our hospital were reviewed, taking the cut-off value of 30%. In the selected cases, tumour type, grade and phenotype were included.

Results: 23 cases out of 1189 biopsies (1.93%) showed negative or partial CK19 expression. 19 cases (82.6%) showed less than 30% expression and four cases showed focal positivity >than 30%. 21 cases (91.3%) were invasive carcinoma-NOS with 15 (65.2%) being grade 1 and 2, one metaplastic carcinoma and one solid papillary carcinoma. The most frequent phenotype was luminal A (52.1%). In all cases with minimal and/or moderate expression (<30%), SLN study by traditional histological examination was recommended. Axillary lymph node metastasis was observed in 6 cases, in which no CK19 expression was verified by immunohistochemistry (IHC). In the 4 cases studied by OSNA, micrometastases was found in 1, CK19 negative.

Conclusion: The assessment of CK19 in tumour cells can be challenging in cases with partial positivity.

The majority of CK19-negative cases in our series are low-grade tumours, in contrast to previous evidence describing this finding in more aggressive tumours (intermediate-high grade and/or triple negative tumours).

A cut-off point of 30% in the assessment of CK19 IHC expression is considered an appropriate value in tumours with partial positivity to perform the OSNA technique, as evidenced in our series.


Gross examination of post neoadjuvant chemotherapy (NACT) breast resections in resource constraint setting – how much is enough?

P. Shah*, S. Desai, T. Shet

*Tata Memorial Hospital, India

Background & objectives: Post-NACT breast carcinoma gross examination poses a challenge when primary tumour is not localized with a radio-opaque wire/clip – a situation common in resource-constrained settings. We undertook a blinded study on two grossing approaches demonstrating how much sampling is adequate.

Methods: Fifty breast carcinoma cases were grossed prospectively by a single pathologist (n=50). Tumour bed was localized by clinico-radiological and visual correlation only. Tumour bed was submitted entirely in grids of multiple slices (Method 1) and one slice was marked as the one with maximum tumour bed area (Method 2). Kappa values were calculated to evaluate agreement between the two methods.

Results: Twenty patients underwent breast conservation surgery; whereas 30 patients underwent mastectomy. Mean of 8 blocks per case were prepared for a single slice (Method 2); whereas 26 blocks were prepared from sampling of entire tumour bed (Method 1). Pathological complete response (pCR) by both methods was calculated to analyse non-pCR cases missed by Method 2. Method 2 documented 23 cases with pCR of which 21 were picked up in Method 1.The two cases missed by Method 2 had minimal residual disease with <2 mm residual tumour (RCB I).The concordance of the two methods was 91.3%. Kappa value for Method 2 was 0.919 thus demonstrating very good correlation with Method 1.

Conclusion: Submitting slice of largest visible tumour bed captures 90% of pCR and RCB accurately. In resource-constrained settings, this would be a viable substitute option for primary tumour not localized prior to NACT by radio-opaque wire/clip. The average cost of 1 Hematoxylin and Eosin stained slide is INR.100 (equivalent to approximately 1$). The method 2 was shown to curtail the expenditure to 33%. RCB class could also be assigned using this technique.


Clinicopathological features and response to neoadjuvant chemotherapy in triple positive breast cancer

A. Dhaoui*, Y. Houcine, K. Hamza, S. Kammoun, G. Sahraoui, S. Ayadi, R. Chargui, M. Driss

*Forces de Securité Interieure, Tunisia

Background & objectives: Breast cancers with positive expression of hormone receptor and overexpression of HER2/neu, are called “triple positive breast cancers TPBC”.Data is relatively limited on TPBC. We aim to characterize the clinical and pathological features associated with TPBC and study their responses to neoadjuvant chemotherapy.

Methods: Clinicopathologic data associated with TPBC from 2015 to 2021 were retrieved from our pathology database. A variety of clinicopathologic parameters, including patient age, tumour Nottingham grade, HER2 IHC status, tumour necrosis, and frequency of pathologically-determined complete response to neoadjuvant treatment (PCR).

Results: Of 123 consecutive TPBC, all cases were invasive ductal carcinomas. The mean age was 52 years with extremities of 26 and 102 years. TPBCs were more often of Nottingham grade II in 78 cases (63%). TPBC were node-positive in 63 cases (52%) and were less often IHC 3+ in 80 cases (65%). Tumoural necrosis was observed in 78% (n=96). Among the patients that underwent neoadjuvant chemotherapy, PCR was achieved in 3 of 20 (15%) TPBC.

Conclusion: In our study, TPBC are associated with Nottingham grade and higher rate of node-positivity. Notably, these tumours show lower rate of complete pathologic response to neoadjuvant chemotherapy.


MMP-2 expression in BC and its association with clinicopathological features and lymph nodes status

V. Kometova*, L. Mikhaleva, V. Rodionov, I. Kolyadina, M. Dardyk, M. Rodionova

*FSBI "V.I.Kulakov NMRC for OGP", Russia

Background & objectives: Matrix metalloproteinase-2(MMP-2) plays a role in the invasion and metastasis of cancer through the destruction of the basal membrane and extracellular matrix. The study aimed to investigate MMP-2 expression levels in breast cancer(BC) and its relationships with lymph nodes status.

Methods: We tested the expression of MMP-2 in 358 BC specimens with immunohistochemistry (EPR1184, Abcam, USA). All cases were divided into two groups according to intensity of stained tumour cells (negative and mild vs strong cytoplasmic expression). MMP-2 positivity was detected in most cases (262, 72.2%). Nevertheless, strong positive expression (>50% tumour cells,3+) was only in every fifth case (78, 22%).

Results: Statistical significant association was found between MMP-2 overexpression(3+) and tumour size(pT2-3)(HR=34.05,p<0.001); Grade3(HR=73.08,p<0.001); ER(0-2 score, Allred)(HR=15.91,p<0.001); PgR(0-2 score, Allred)(HR=13.83,p<0.001); Ki-67≥50%(HR=68.58,p<0.001). There is loss of MMP-2 expression in most luminal A BC(93.8%). There was no statistically significant correlation of MMP-2 expression with patient’s age, number of tumour nodes, its localization, histological type,HER2/neu expression. A significant relationship was found between MMP-2 expression and regional metastasis(р<0,001). In the group with lymph nodes BC metastases the number of cases with MMP-2 overexpression was higher (52 cases,66,7%) than in the group without lymph nodes metastases(26 cases,28.6%). There was negative MMP-2 expression(-) in 86 cases(38.1%) in the group without lymph nodes metastases vs the group with lymph nodes BC metastases (10cases,7,6%)( р<0,001).

Conclusion: Identification of specific biomarkers is very important for prediction of regional metastases. We demonstrated that MMP-2 expression is associated with clinicopathological parameters of BC and its lymph nodes status. Loss of MMP-2 expression is statistically significantly reduced risk of lymph nodes metastases (p<0.001). Loss of MMP-2 expression was predominant in BC with pT1, Grade I-II, ER-positive, PgR-positive, Ki-67-low parameters. These results suggest that MMP-2 plays a role in the biology of BC.

Funding: НИОКТР АААА-А18-118053190016-7


Sentinel lymph node localization with Magtrace. Our experience so far

E. Panopoulou, E. Koniaris*, A. Stylianaki, G. Zografos, G. Kafiri

*Pathology Department, General hospital of Athens "Hippokratio", Greece

Background & objectives: Magtrace lymphatic tracer is a liquid non-radioactive tracer, specifically designed for sentinel lymph node biopsy, assisting surgeons to locate the first draining axillary lymph nodes. Sentinel lymph nodes (SL) are the first nodes most likely to be infiltrated by metastasis.

Methods: The cohort of our study consisted of 22 female patients aged 33-66, surgically treated (WLE or mastectomy) for breast cancer. All patients gave a written informed consent to participate in the experimental study with this lymphatic tracer. In one patient the technique was performed bilaterally. In all cases 1-6 SL were received in each patient, measuring 0,5-2 cm.

Results: Patients received the tracer 24 hours to 30 minutes prior to surgery. Macroscopically 1-6 SL were isolated and examined on frozen sections with H&E. SLN were totally enclosed and studied. Microscopically, increased presence of pigment laden macrophage aggregates was noted, found mainly in a central location in the node. Although the amount of the macrophage aggregates were striking, did not interfere with the correct diagnosis and was mainly found in one or two SL, indicating the first one or two SL. At present, common practice involves dual technique, Blue de Methylene or Patent blue in combination with radioactive technetium 99 and this technique doesn't seem to be inferior.

Conclusion: Magtrace, even though a relatively new SLN tracer method, is a standardized technique with a steep learning and teaching curve, as 2-3 applications are sufficient. The main advantages of this tracer are its safety, convenience, the non-stigmatizing technique for the patient, the significant lower allergy rates, the radiation free setting (reducing nuclear medicine dependency) and the longer half lifetime reaching up to one month. Overall, Magtrace has highly reproducible and accurate results and produces less discomfort for the patient.


FOXA1 and lymph nodes status in breast cancer

V. Kometova*, V. Rodionov, I. Kolyadina, M. Rodionova, M. Dardyk, L. Mikhaleva

*FSBI "V.I.Kulakov NMRC for OGP", Russia

Background & objectives: Forkhead box protein A1(FOXA1) promotes the transcription of a gene that induces luminal cell differentiation and suppresses the processes of epithelial-mesenchymal transition in breast cancer(BC). In our study, we purposed to investigate the relationship between FOXA1 and lymph node BC metastasis.

Methods: We tested the expression of FOXA1 in 358 BC specimens with immunohistochemistry (SP88,Abcam,USA). FOXA1-scoring was done according to the Allred system. Scores of 3-8 are considered positive. Tumours with score 0-4 were considered as FOXA1-low, whereas tumours with score 5-8 were considered as FOXA1-high. FOXA1-negative BC was detected in 89 cases (25%). FOXA1-high expression was detected in 131 cases (36.6%).

Results: FOXA1-expression significantly correlates with clinicopathological and immunohistochemical parameters, including those that have a predictive value in the process of lymph node BC metastasis, such as the patient's age(p=0.004), tumour node size(p=0.028), Grade(p=0.001), ER(p<0.001) and PgR-expression(p=0.003), Ki-67(p<0.001). FOXA1-positive tumours were predominant in both comparing groups (with/without lymph nodes metastases-71.2% and 75.7%, respectively) (р=0.354). However, a significant relationship was found between the level of FOXA1 expression and lymph node BC metastasis(p=0.010). The intensity of FOXA1 expression was higher in the group without lymph node BC metastasis 94 cases(41.6%) compared to the group with lymph node BC metastasis 37 cases(28.2%). No significant difference was obtained in the two compared groups for FOXA1-low expression(p=0.081).

Conclusion: FOXA1 was a significant independent prognostic factor for the lymph nodes BC metastases. The intensity of the marker expression has statistical significance (p=0.01). With moderate FOXA1 expression, risk of lymph node metastasis decreases compared with weak staining of tumour cells. FOXA1-low expression reversely correlates with BC metastasis and progression and may serve as a prognostic biomarker for predicting lymph node metastasis.

Funding: НИОКТР АААА-А18-118053190016-7


SLC7A5 expression in breast cancer and its association with molecular subtypes

V. Kometova*, I. Kolyadina, L. Mikhaleva, M. Rodionova, M. Dardyk, V. Rodionov

*FSBI "V.I.Kulakov NMRC for OGP", Russia

Background & objectives: Solute carrier family7 member5(SLC7A5), known as LAT1, is a transporter factor delivering amino acids to cancer cells.SLC7A5 is an important prognostic marker of the breast cancer(BC) aggressive behaviour. This study aimed to investigate correlation between SLC7A5 expression and molecular subtypes.

Methods: We tested the expression of SLC7A5 in 358 BC specimens by immunohistochemistry (PA5-34215, ThermoFisher, USA). Full membranous staining in >10% tumour cells was considered as positive. Moderate/strong SLC7A5-positive expression was detected in 199 cases (55.6%). Loss of SLC7A5 expression was detected only in 56 cases(15.6%). Strong membranous staining in >50% tumour cells was determined in a quarter of cases(26.5%).

Results: SLC7A5 expression was increased in BC with HER2/neu overexpression, regardless of hormonal receptor status. Strong SLC7A5 expression was detected in 14 cases(66.7%) of luminal B HER2-positive subtype and in 12 cases (80%) of non-luminal HER2-positive subtype. There was no SLC7A5-negative BC cases with HER2/neu overexpression. In TN breast cancer and in luminal B subtypes, the transport protein was expressed more than in half cases, with approximately the same frequency in all three subtypes – 65%-66.7%. In luminal A subtype, cases with no SLC7A5 expression were 1.5 times more often (34 cases, 21%) than in luminal B HER2-negative BC (18 cases, 14.5%)(p<0.001).

Conclusion: We demonstrated that SLC7A5-expression is associated with molecular BC subtypes (p<0.001). SLC7A5-negative BC positively correlates with luminal-A subtype. The largest number of cases with strong SLC7A5 expression in >50% cancer cells was detected in cases of non-luminal BC subtype with HER2/neu overexpression. These results suggest that SLC7A5 plays a role in the biology of BC.

Funding: НИОКТР АААА-А18-118053190016-7


Comparison of HercepTest™ mAb pharmDx (Dako Omnis) (GE001) with Ventana PATHWAY anti-HER-2/neu (4B5) in breast cancer

J. Rüschoff*, M. Friedrich, I. Nagelmeier, M. Kirchner, L. Andresen, K. Salomon, B. Portier, S. Sredni, H.U. Schildhaus, B. Jasani, M. Grzelinski, G. Viale

*Targos - A DLS company, Germany

Background & objectives: For HER2 assessment in Breast Cancer, immunohistochemistry (IHC) staining is the method of choice. Here, we compare the clinical performance of the new CE-IVD-marked HercepTest™ mAb pharmDx (Dako Omnis) (GE001) with Ventana PATHWAY anti-HER-2/neu (4B5).

Methods: In total, 119 pre-selected breast cancer samples covering the entire range of HER2 IHC expression scores were tested by HercepTest (mAb), PATHWAY 4B5, and fluorescent in situ hybridization (FISH). Three pathologists independently evaluated HER2 IHC according to 2018 American Society of Clinical Oncology/College of American Pathologists guidelines. Sensitivity and specificity of both IHC assays were assessed based on FISH data.

Results: There was a high concordance between results from the HercepTest (mAb) and PATHWAY 4B5 assays for HER2-negative (IHC 0, 1+, 2+ and FISH negative) and HER2-positive (IHC 3+, 2+ and FISH positive) breast carcinomas (98.2%). Regarding individual IHC scores, complete agreement was achieved in 69.7% (83/119) of cases and all but one of the discordant cases were due to higher HER2-status scoring using the HercepTest (mAb). Thus, more tumours were scored as IHC 2+ by HercepTest (mAb) (27 versus 15) as evidenced by their lower FISH positivity rate (48.1% versus 80%). However, two amplified tumours identified as IHC 2+ by HercepTest (mAb) were missed by PATHWAY 4B5 (IHC 1+).

Conclusion: The HercepTest (mAb) detects HER2 expression with higher sensitivity in tumours with gene amplification (ISH group 1) and increased gene counts (ISH group 4) as well as in HER2-low tumours (HER2 IHC 2+ / FISH negative or IHC 1+). These findings could be critical for the identification of patients eligible to new HER2-targeting treatment options. Future studies will demonstrate whether this new assay has the capacity to provide better patient stratification, leading to better patient response rates and clinical outcomes.

Funding: This study was funded by Agilent Technologies, Inc.


Phosphohistone H3 versus mitotic count in breast cancer grading: a single institution study

N. Mansouri*, F. Gargouri, M. Ben Thayer, K. Tlili, R. Aouadi, I. Msakni, B. Laabidi

*Department of Pathology, Military Hospital of Tunis, Tunisia

Background & objectives: Mitotic index is an important prognosis marker in invasive breast carcinoma (BC). The aim of this study was to evaluate the utility of Phosphohistone H3 in grading of BC in comparison with mitotic count on hematoxylin and eosin-stained slides.

Methods: A retrospective study on a series of 40 BCs diagnosed from March 2020 to August 2021 at the Pathology Department of the Military Hospital of Tunis was performed. We compared grade variability according to the mitotic count on H&E-stained slides and on PHH3 stained slides.

Results: Although, mean average count was higher by IHC method, good correlation was observed (R²=0.799). Using PHH3 IHC, three cases of grade I tumours were upgraded in to grade II and six cases of grade II were upgraded in to grade III. None of the tumours were downgraded.

Conclusion: In summary, we have performed the first study to explore the utility of PHH3 in breast cancer grading in Tunisia. Similar to some other previous studies, we found PHH3 a robust sensitive and practical marker for mitotic count in breast carcinoma. Especially it is helpful to identify the most proliferating area.


Immune checkpoint genes expression in breast carcinoma: correlation with clinicopathological features and patients’ survival

Y.G. Montoyo Pujol*, M. García-Escolano, T. Martin-Bayon, S. Delgado-García, H. Ballester, J.J. Ponce, E. Castellón-Molla, J.M. Sempere-Ortells, F.I. Aranda López, G. Peiró

*University General Hospital and Alicante Institute for Health and Biomedical Research –ISABIAL-, Spain

Background & objectives: Immune checkpoints deregulation can lead to tumour progression and invasion. However, very little data is available on breast carcinoma (BC). Therefore, we analysed CTLA-4, PDCD1, CD274, PDCD1LG2, and CD276 expression in BC and their association with clinicopathological factors and survival.

Methods: We included 275 non-consecutive BC. mRNA expression was analysed by qRT-PCR using TaqMan® primers and probes. PUM1 and β-actin were used as reference genes, and healthy breast tissue served as a calibrator. The 2-ΔΔCT calculated relative changes in expression. Results were correlated with clinicopathological factors and prognosis. Significant differences were calculated with χ2 and log-rank test.

Results: Overexpression of at least one immune checkpoint was found in 95.2% of samples. CTLA-4 was overexpressed mostly in Triple-Negative/Basal-like phenotypes (p=0.046), whereas PDCD1 and CD276 were more frequently found in Luminal tumours (all p<0.023). CD274 and PDCD1LG2 showed no correlation with immunophenotype. High CTLA-4, PDCD1, CD274 and PDCD1LG2 expression was associated with a middle/high proliferation rate (all p<0.039), and presence of TILs (all p<0.011), with opposite results for CD276 (p=0.048, and 0.026, respectively). CTLA-4 overexpression correlated with better disease-free survival -DFS- (p=0.037), and specifically among HER2-enriched subtype (DFS p=0.033, and overall survival p=0.011).

Conclusion: Our data support that CTLA-4, PDCD1, CD274, and PDCD1LG2 are potential biomarkers of BC aggressiveness. Paradoxically, CTLA-4 stratified a subgroup of HER2-enriched patients with a better outcome.

Supported by Grants ISABIAL (NR-180202, UGP-18-231, UGP-19-291, UGP-20-090)


The malignancy of benignancy. The malignant consequences of benign breast tumours. Is the onus on pathologists? Post-pregnancy associated infarcted fibroadenoma, a rapidly growing benign phyllodes tumour, a benign phyllodes tumour in an adolescent girl

S. Di Palma*, A. Gamage, P. Partlett, F. Pakzad, E. Rakha, E. Clayton

*Royal Surrey County Hospital, United Kingdom

Background & objectives: Fibro-epithelial lesions of the breast can be difficult to assess, especially when dealing with core biopsies. In uncommon clinical settings such as pregnancy, young girls or a size that would require mastectomy, they are even more difficult to assess.

Methods: Here we report on three cases of fibroepithelial lesions, where caring clinicians were concerned about malignancy despite benign pathology reports. We discuss the difficulties of making clinicians accept a benign pathology diagnosis despite the worrying clinical presentation. Literature for similar cases confirmed that these are rare but well-known conditions usually described in single or very small series case reports.

Results: In one case, the core biopsy was reported as B2, fibroadenoma with necrosis. Despite a benign report, diagnostic surgical excision was performed. Following MDT, it was revealed that the patient was post-partum and lactating. Patient 2 had a B3 core biopsy for a spindle cells tumour, occupying most of the breast. Excision biopsy was unwelcome because the size would have required a mastectomy, not consistent with a B3 report. Additional biopsies revealing an epithelial component consistent with phyllodes tumour were required for diagnosis. Patient 3 was a 17-year-old girl with a 70mm lump in her breast. Clinical concern led to surgical resection without prior core biopsy revealing benign phyllodes tumour.

Conclusion: Benign fibroepithelial tumours simulating malignancy on clinical presentation are well known to pathologists but still create uncertainty in clinicians. Although rare, in one year we have encountered and correctly identified three such cases. Our experience, where clinicians are not prepared to accept a diagnosis of benign/uncertain tumour on core biopsies, suggests that more education of these tumour subtypes is essential if clinicians are to accept our diagnosis and organize patient management accordingly.


Breast interstitium or "Hartveit’s Labyrinth": morphology and relationship to disease processes

S. Gordin*, B. Belovarac, A. Serrano, L. Chiriboga, B. Zeck, F. Darvishian, D. Nimeh, U. Ozerdem, S. Badve, R. Wells, N. Theise

*NYU Langone Medical Center, New York, NY, USA

Background & objectives: The breast stroma contains interstitial spaces (IS) supported by collagen bundles and intermittently lined by CD34+/vimentin+ interstitial lining cells (ILC) identified previously by Hartveit (Histopathology, 1990) as pre-lymphatic "labyrinth." Current study investigates morphology and role of interstitium in disease processes.

Methods: Normal breast tissue with skin and breast tissue with PASH were stained for HA with peroxidase labelled HABP (hyaluronic acid binding protein) to investigate continuity between intralobular/interlobular and skin IS. IHC multiplex co-staining for CD34 and vimentin was used to demonstrate spatial relationship of the interstitial HA with ILC (Discovery Ultra, Ventana) and IHC for Galectin/Vimentin and p63/Vimentin in PASH.

Results: HABP staining showed HA throughout the IS of the normal breast sections and demonstrated continuity of IS between intralobular, interlobular, perivascular and perineural interstitium. In samples with PASH, HABP showed expansion of IS associated with thickened collagen bundles. IHC for p16/Vimentin showed significantly increased staining in PASH comparing to normal breast tissue and Galectin/Vimentin demonstrated positive Galectin staining along cell membrane of some fibroblasts in PASH.

Conclusion: Breast IS form a continuous space permeating all stromal compartments: periductal, interlobular, nipple, and dermis. Additionally, IS are continuous between breast stroma and perivascular adventitia and perineurium, connections to a body-wide system of IS. Our data support that PASH is a keloid-type expansion and remodelling of the mammary IS, which further suggests that mammary interstitium may play roles in fibrocystic disease, gynecomastia and malignant states such as invasive lobular carcinoma and could provide a route to spread via lymphatics/perineural routes.


The effect of neoadjuvant chemotherapy on histologic grade and hormone receptor status in HER2/Neu-negative Luminal-B tumours of the breast

O.C. Eren*, S.E. Horoz, N. Arı, G. Menetlioglu, H. Kaya

*Marmara University, Turkey

Background & objectives: With stratification of tumours, neoadjuvant chemotherapy (NACT) has become more common in Her2/Neu-negative Luminal-B group. Here we investigate the effect of NACT on histologic grade/hormone receptor (HR) status and tumour representation adequacy in pre-NACT core biopsies.

Methods: Her2/Neu-negative luminal-B cases between 2017 and 2020 with available core biopsy and post-NACT resection slides were retrieved. Hospital records, original sign-out reports and glass slides were retrospectively reviewed for demographic data, pre-/post-NACT histologic grade and HR status of tumours, as well as number of pre-NACT core biopsy pieces available for tumoral representation.

Results: 45 cases were identified, with a mean age of 49.8. In the pre-NACT core biopsy samples, average number of pieces and percentage of tumoral tissue in the pieces were 3.82 and 52% respectively. 40% (n=18) of cases were attributed a histologic grade different from what had been originally assigned to them with the core biopsy, while 4.4% of cases (n=2) showed complete regression in post-NACT resection specimens. From core biopsy to resection, in terms of oestrogen receptor expression change, 8.9% (n=4) and 8.9% (n=4) of cases marked a decrease and increase, respectively. For progesterone receptor status, 33.3% (n=15) of cases demonstrated a decrease, while 11.1% (n=5) showed an increase.

Conclusion: Changes in histologic grade, increased/decreased ER or decreased PR status have been described in literature as potential effects of NACT. Yet 11.1% (n=5) of our cases showed significant increase in PR status, which can better be explained by intratumoral heterogeneity and inadequate pre-NACT representation of tumoral tissue. In fact these cases had significantly lower percentage (30%) of tumoral tissue representation in the pre-NACT core biopsy specimen.


Morphological and crystalchemical features of breast cancer microcalcificates

O. Kolomiiets*, A. Piddubnyi, S. Danilchenko, R. Moskalenko

*Department of Pathology Sumy State University, Sumy, Ukraine

Background & objectives: The aim of the study is to study the main morphological and crystal chemical properties of microcalcifications of breast cancer.

Methods: In our study, we examined 30 specimens of breast carcer by histology (hematoxylin-eosin staining), histochemistry, scanning electron microscopy with EDS and TEM.

Results: Histological examination of breast cancer samples revealed the presence of microcalcifications in the form of dark blue deposits of round and irregular shape, different sizes. A positive reaction to von Koss staining indicates the presence of calcium phosphate compounds in their composition. We confirmed the presence of round calcifications using SEM. SEM with X-ray microanalysis confirmed that the biomineral part of the samples of the group consists mainly of hydroxyapatites. In 6 cases, the presence of hydroxyapatite is combined with oxalates. However, oxalates and apatites had different localizations: apatites were associated with tumour, and oxalates were in intact adjacent tissue.

Conclusion: We found the possibility of the simultaneous presence of microcalcifications of hydroxyapatite and oxalate nature in samples of invasive breast cancer. Different spatial localization of biominerals indicates different mechanisms and conditions for the formation of microcalcifications.


HER-2 ISH: do we need to count?

S. Costache*, A. Baltan, A. Jurkiewicz, M. Porter, K. Billingham, H. Haynes, A. Chefani, S. Wedden, M. Sajin, C. D'Arrigo

*Poundbury Cancer Institute, Dorchester, UK, Carol Davila University of Medicine and Pharmacy Bucharest, Romania

Background & objectives: Assessment of Her2 amplification requires counting 20-60 cells. This is time-consuming and may be unnecessary since morphological assessment can provide accurate diagnosis in a proportion of cases. We define parameters for selection of cases that can be diagnosed morphologically.

Methods: Using both glass slides and DP, we studied 200 consecutive breast core biopsies for which DDISH was requested following an indeterminate (2+) IHC result with 4B5. We assessed whole slides for presence and frequency of amplification clusters and polysomy of Ch17 by eyeballing. We separately performed formal counting and compared the time to reach a diagnosis.

Results: We identified a number of morphological groups, including tumours with large amplification clusters (regardless of the number of Ch17 signals), tumours with sparse Her-2 signals (that are clearly below a ratio of 2 per Ch17) and tumours with high prevalence of borderline/non-amplified features. We evaluated concordance between morphological diagnosis and formal counting. Concordance was high in tumours with amplification clusters and in tumours with low gene copy numbers and/or low Ch17 signals. Concordance was poor in tumours with intermediate features. Time required for morphological assessment (30-60s) was 5-6 times faster than formal counting. For these assessments, DP was comparable to glass slides.

Conclusion: Some cases will still require the formal counting approach but we demonstrate that morphological assessment can be as accurate as formal counting in selected cases. For this to become established, training will need to be provided in order to ensure concordance amongst pathologists. This training can be delivered by DP. Digital programmes of Proficiency Testing would ensure continuing diagnostic alignment. This approach would save reporting time and therefore Pathologists and Healthcare resources could be used more efficiently.


Pathological features of CT-guided bone lesion biopsy specimens in breast cancer patients

M. Abramovic*, L. Korša, L. Grbanović, M. Čavka, I. Švagelj, M. Prutki, Z. Marušić

*Clinical Department of Pathology and Cytology, University Hospital Centre Zagreb, Croatia

Background & objectives: Current breast cancer guidelines recommend biopsy of metastatic lesions at presentation or first recurrence. Objective is to provide a cross-sectional pathological study of CT-guided bone biopsy specimens in breast cancer patients with suspicious bone lesions.

Methods: A cross-sectional study on 56 consecutive female breast cancer patients who underwent CT-guided biopsy of suspicious bone lesion was performed. Quantity of the specimen was defined as optimal (>5% cancer cellularity), suboptimal and low (non-diagnostic). Immunophenotype of the primary tumour and matching metastasis was compared, where available. Diagnostic accuracy of CT-guided bone biopsy was determined.

Results: A total of 58 bone lesions were biopsied in 56 patients and 44 (75.9%) breast cancer metastases were found. CT-guided biopsy enabled optimal quantity of specimens in most cases (56.9%), followed by suboptimal (32.8%) and low (10.3%). In 10% of positive cases, the number of tumour cells was too low for further immunohistochemical analysis. In 36 paired cases (primary vs metastatic), a shift in immunophenotype was observed in 13 cases (36.1%), most commonly from PR-positive to PR-negative (10/13, 76.9%). Sensitivity of CT-guided biopsy for detecting bone metastases was 93.6% and specificity was 100%.

Conclusion: CT-guided bone biopsy is a method with high sensitivity and specificity for detecting breast cancer metastasis that can be used when soft tissue metastatic lesions are not obtainable. The proportion of positive cases with sufficient tumour quantity for further immunohistochemical analysis is high. Comparison of primary tumour and metastasis immunophenotype revealed discordance in more than one third of patients, with potential therapeutic implications.


Luminal breast cancer subtypes and associated prognostic factors: a population-based study from Osijek, Croatia

D. Damjanović*, J. Rajc, K. Marjanović, R. Gudelj, M. Bakula, R. Alaghehbandan

*University Hospital Osijek, Croatia

Background & objectives: Breast cancer is a heterogeneous disease, with a spectrum of biological features and differences in clinical outcome. The aim of this study was to examine the prognostic factors of luminal breast cancer subtypes among Croatian breast cancer population.

Methods: This is a large retrospective population-based study including 1127 primary breast cancer cases, during a six year period (2016–2020) in Osijek, Croatia. The clinico-pathologic and immunohistochemical (IHC) and in situ hybridization (ISH) data were extracted from pathology reports to examine the luminal subtypes A and B. The cross-tabulated statistics of the observed characteristics were performed between the two subtypes.

Results: Luminal cancers comprised 86.9% (980/1127) of the total cohort, including 724 (64.2%) cases of luminal B and 256 (22.7%) luminal A. Age profile of Luminal A and B cancers were similar (63.5 vs 62.3 years). Mean tumour size was higher in luminal B than luminal A cancers (19.6 vs 22.2 mm, p=0.03). Luminal B cancers were significantly associated with adverse prognostic features than luminal A cancer, including high histologic grade (14% grade III luminal B vs 3.5% luminal A, p<0.0001), vascular invasion (18% luminal B vs 7.5% luminal A, p<0.0001), and nodal metastasis (21.2% luminal B vs 15.1% luminal A, p=0.006).

Conclusion: Luminal B is the most frequent subtype of breast cancer in Croatian patients. They were associated with adverse clinico-histologic parameters such as higher grade, vascular invasion, and nodal metastasis. Our findings suggest that, despite lack of molecular studies in routine practice, IHC/ISH-based typing are sufficient for prognostic and therapeutic stratifications in breast cancers.


Ki67 proliferative index in patients with early breast cancer and its association with clinicopathological factors

D. Damjanović*, J. Rajc, E. Matuzalem Marinović, B. Dumenčić, R. Gudelj, M. Bakula, R. Alaghehbandan

*University Hospital Osijek, Croatia

Background & objectives: Standard cut-off for distinguishing low and high Ki67 index as a prognostic marker in early breast cancer do not exist. The aim of this study was to determine the role of Ki67 index and its association with other prognostic parameters.

Methods: A large population-based retrospective study including 957 primary early breast cancers (pT2 or less) was conducted during a 5-year period (2016–2020) in Osijek, Croatia. The clinico-pathologic data were extracted from pathology reports. Multiple logistic regression analysis was used to evaluate the associations between Ki67 and other clinicopathological factors.

Results: The median Ki67 was 30% (range 2–98%), with 251 (26.2%) patients had a Ki67 <20%. Triple-negative breast cancers showed highest ki67 index (mean 72.7 ± 23.9%) followed by HER2-positive (mean 60.2 ± 21.3%) and luminal B cancers (mean 39.8 ± 19.9%) (p<0.0001). Metaplastic and medullary breast cancers significantly showed higher Ki67 index compared to ductal carcinoma, NOS. Using a multivariable logistic regression with Ki67 (<20% vs. ≥20%) as binary dependent variable, younger age, positive nodal status, higher grading, negative HR status, and positive HER2 status were shown to be significantly associated with a higher proliferative index (Ki67 ≥20%).

Conclusion: Ki67 index is a valuable biomarker of breast cancer as higher ki67 correlates with more aggressive tumour biology. However, definition of low and high proliferation index itself is challenging. It is essential to interpret Ki67 indices carefully with regard to the own institutional values and other clinicopathological factors.


To determine the accuracy of axillary staging using ultrasound-guided fine-needle aspiration cytology in breast cancer patients: a single institutional experience

I. Aloglu*, M. Doğan, S. Aydın Aksu, F. Aker

*Department of Pathology, Haydarpaşa Numune Training and Research Hospital, Turkey

Background & objectives: Ultrasound-guided fine-needle aspiration cytology (US-FNAC) of axillary lymphadenopathy is a helpful tool in identifying candidates for axillary dissection and neoadjuvant chemotherapy for breast cancer patients. This study investigated the diagnostic value of evaluating the axillary lymph nodes with US-FNAC.

Methods: Between 2011 and 2021, 284 cases who underwent the axillary US-FNAC procedure during the diagnosis of primary breast cancer and then underwent axillary / sentinel surgery were collected retrospectively. Cytological diagnoses and corresponding histological diagnoses were documented. Final histopathology was taken as gold standard. The diagnostic parameters, including sensitivity, specificity, positive (PPV), and negative predictive values (NPV), were calculated.

Results: Hundred-fifty-two of 284 cases were evaluated as malignant cytology in FNAC. There were no malignant histopathological findings in 4 of them. Conversely, 132 cases were evaluated as benign cytology in FNAC. While 41 of them had malignant tumour metastases histopathologically, 91 of them did not have any malignancy findings. Overall sensitivity of lymph node FNAC was 78.3% and specificity was 95.7%. Positive predictive value (PPV) was 97.3% and negative predictive value (NPV) was 68.9%.

Conclusion: Axillary FNAC is a diagnostic method that helps surgeons decide whether to perform sentinel node biopsies. This technique is favourable due to its minimally invasive nature. In addition, we suggest that FNAC can prevent unnecessary axillary dissection due to its high positive predictive value (PPV). The reason for the lower negative predictive value may be hypocellularity and micrometastasis. So FNAC has adequate sensitivity and high specificity in diagnosing axillary lymph node metastasis.


Predictive factors of lymph node metastasis in breast cancer patients: a Croatian population-based study

Z. Radičević*, J. Rajc, D. Damjanović, R. Gudelj, M. Bakula, R. Alaghehbandan

*University Hospital Osijek, Croatia

Background & objectives: Breast cancers are heterogeneous groups of tumours, and its subtypes correlates with therapeutic response and overall survival. In this study, we assessed whether the subtype can predict the presence of nodal metastasis in a large cohort of breast cancer patients.

Methods: A large population-based retrospective study including 1127 primary breast cancers was conducted during a 5-year period (2016–2020) in Osijek, Croatia. The clinico-pathologic data were extracted from pathology reports. The Pearson chi-square test was used to determine whether progesterone receptor (PR) status had an impact on the incidence of lymph node positivity in oestrogen receptor (ER) positive patients.

Results: Lymph node metastasis was significantly associated with HER2-positive and luminal B breast cancers, higher histologic grade, as well as higher Ki67 proliferation index (p<0.01). Independent predictors of nodal positivity included tumour size and vascular invasion (p<0.0001). There were significant differences between subtypes and nodal positivity, with luminal A cancers (23.3%) had the lowest and Her2-positive (50%) the highest rates (p=0.001). There was no difference in lymph node positivity between PR+ and PR- tumours amongst all subtypes (p=.1).

Conclusion: Tumour size and vascular invasion are independent predictors of nodal positivity in this study. HER2-positive breast cancers, higher histologic grade and Ki67 proliferation index are significantly associated with lymph node metastasis. These findings may play a role in guiding regional management considerations.


GATA3 expression in human tumours: a tissue microarray study on 13,204 tumours

V. Reiswich, M. Lennartz, F. Büscheck, K. Möller, C. Bernreuther, G. Sauter, R. Simon, S. Minner*, E. Burandt, A. Marx, T. Krech, A. Hinsch, F. Jacobsen

*University Medical Center Hamburg, Germany

Background & objectives: GATA3 is a transcription factor involved in epithelial cell differentiation. Positive GATA3 immunostaining is used as a diagnostic marker for breast and urothelial cancer but can also occur in other neoplasms.

Methods: A set of tissue microarrays containing 16,611 samples from 133 different tumour types and subtypes and 608 samples of 76 different normal tissue types were analysed by immunohistochemistry to comprehensively evaluate GATA3 expression in normal and tumour tissues.

Results: GATA3 positivity occurred in 70 different tumour types including 24 (18%) with ≥1 strongly positive tumour. Highest positivity rates occurred in non-invasive papillary urothelial carcinoma (92-99%), lobular (98%) and NST (92%) carcinoma of the breast, cutaneous basal cell carcinoma (97%), invasive urothelial carcinoma (73%), T-cell lymphoma (23%), adenocarcinoma of the salivary gland (16%), and cutaneous squamous cell carcinoma (16%). In breast cancer, low GATA3 staining was linked to advanced pT stage (p=0.03), high BRE grade (p<0.0001), HER2 overexpression (p=0.0085), oestrogen and progesterone receptor negativity (p<0.0001 each) and reduced survival (p=0.03). In urothelial neoplasms, low GATA3 was linked to poor grade within pTa tumours (p=0.01) and with advanced stage (p<0.0001).

Conclusion: GATA3 positivity can be seen in various tumour entities. Particularly high frequency and levels of GATA3 expression occur in breast and urothelial carcinoma. A reduced level of GATA3 reflects cancer progression and poor patient prognosis in these tumour entities.


Analysis of intraoperative frozen sections of sentinel lymph nodes - a 10-year long series in a tertiary Portuguese hospital

J. Boavida*, R. Moiteiro da Cruz, L. Correia

*Department of Pathology - Hospital de Santa Maria, CHULN; Faculdade de Medicina da Universidade de Lisboa, Portugal

Background & objectives: Intraoperative frozen sections of sentinel lymph node (IFSSLN) allowed better management of breast cancer (BC) particularly in the decision to perform axillary lymphadenectomy (AL). This study reviews results of IFSSLN, their definitive evaluation and correlation with AL status when performed.

Methods: Through our department’s information system, we compiled every case of BC where IFSSLN was performed along with data pertaining diagnosis and AL status, from 2010 to 2020; 929 out of 1056 cases were selected. Only cases with a diagnosis of invasive disease made in surgical specimens were chosen and AL was solely considered when referred as such by the surgeon.

Results: Median age of diagnosis was 59,4 years (range 27-95 years), with a female predominance (n=923). 695 were IFSSNL-negative with 88,9%(n=618) being true-negatives; the remaining showed signs of disease in definitive evaluation (mostly isolated tumour cells[ITC] and 2 cases with metastasis). AL was performed in 7 false-negative cases which revealed metastasis in 2 cases and micrometastasis in 1; 42 false-negatives had nodal evaluation not by AL (either satellite lymph node or axillary inspection), showing metastatic disease in 6 of these (4 metastasis, 1 micrometastasis and 1 ITC). There were no false-positive cases. Two cases were deferred. Invasive breast carcinoma of no special type was the most common diagnosis (n=725).

Conclusion: IFSSNL continues to be a useful assessment method in BC staging, showing in this retrospective study highly favourable statistical values when compared with posterior definitive evaluation (75,1% sensibility; 100% specificity; 100% positive predictive value; 88,9% negative predictive value); this results are vastly supported by our use of immunohistochemical stains during the procedure. 34,5% of true-positive cases benefited from this evaluation, since AL revealed the presence of metastatic disease; furthermore, it spared 571 patients of AL-associated complications.


Determination of inter-observer agreement in the immunohistochemical interpretation of PD-L1 clones 22C3 and SP142 in triple-negative breast cancer (TNBC)

R. Abreu, R. Peixoto, M. Corassa, W. Nunes, T. Neotti, T. Rodrigues, C. Toledo, T. Domingos, D. Carraro, H. Gobbi, M. de Brot, R. de Paula*

*Ac Camargo Cancer Center, Brazil

Background & objectives: Advanced triple-negative breast cancer can be treated with immune checkpoint inhibitors, and the expression of PD-L1 is one of the biomarkers used to select patients with clinical benefit. This study analyses the interobserver agreement in the immunohistochemical report of PD-L1.

Methods: 168 cases of TNBC were previously tested and diagnosed between 1987-2016 in 3 institutions. Medical records were reviewed for clinicopathological data collection. The study was performed on 2 TMAs, using two clones. The samples were analysed by 2 breast pathology subspecialists and 2 general pathologists without training. The Kappa value was calculated to assess interobserver agreement, with p < 0.001.

Results: For clone 22C3, (κ) among the 4 evaluators was 0.387, with κ = 0.625 among subspecialist examiners and κ = 0.264 among general pathologists, with 73% concordant cases. Of the discordant cases, a subspecialist evaluator considered 1.7% samples as positive, while the others classified them as negative. For clone SP142, (κ) among the 4 evaluators was κ = 0.331, with κ = 0.420 among subspecialists and κ = 0.285 among general pathologists, with 109 (64.8%) cases in agreement. Of the discordant cases, in 10.1% one evaluator was inconsistent and interpreted the sample as positive, with the discordant interpretation given by subspecialists in 14 cases and by general pathologists in 3.

Conclusion: We demonstrated different rates of interobserver variability between 4 evaluators and different PD-L1 clones, with a minimum overall level of agreement. However, in comparison with general pathologists, the agreement rate between subspecialist evaluators was higher, reaching a moderate level for clone 22C3 and pointing to the possibility of improvement with tutorials. It is noteworthy that even among trained examiners, the agreement rate did not reach optimal levels, indicating the existence of challenges in the analysis of PD-L1 expression.

PS-02 | Poster Session Head & Neck Pathology


DNA methylation analysis in oropharyngeal squamous cell carcinoma – unraveling novel prognostic biomarkers in a clinico-pathological and molecular genetic study of 51 cases

J. Laco*, N. Birknerová, H. Kovaříková, I. Baranová, A. Přikrylová, H. Vošmiková, B. Gajdošová, M. Hodek, M. Vošmik, M. Chmelařová, A. Ryška

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

Background & objectives: Hypermethylation of tumour suppressor genes leads eventually to malignant transformation. The aim of our study was to determine DNA methylation status of selected tumour suppressor genes in oropharyngeal squamous cell carcinoma (OPSCC) and to find correlation with clinico-pathological characteristics.

Methods: A total of 101 samples were analysed in the study (31 primary tumours with 31 corresponding metastases, 20 non-metastasizing primary tumours, and 19 control samples). In every patient, classical clinico-pathological parameters were recorded. For methylation analysis, methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) was performed for a set of 25 tumour suppressor genes (Probe mix ME002-C1).

Results: The study sample comprised 37 males and 14 females, aged 45–80 years (median 58 years). A total of 80% of tumours were HPV-positive. During the follow-up period (range 3–180 months; median 82 months), 10% of tumours recurred and 10% of patients died due to tumour. We observed significantly higher methylation of WT1, PAX6, and CADM1 genes in primary tumours compared to controls (p < 0.05). WT1 and CADM1 genes were significantly hypermethylated in HPV-positive OPSCC compared to HPV-negative OPSCC (p < 0.01). Kaplan-Meier survival curve showed that patients with higher methylation levels of PAX5 gene had impaired overal survival compared to patients with PAX5-unmethylated tumours (p = 0.04).

Conclusion: In summary, significant correlation was observed between methylation status of selected tumour suppressor genes and clinico-pathological parameters in our OPSCC study sample. Our promising results unravel novel potential biomarkers which, if confirmed by further studies, could be used as prognostic markers in the sense of tailored therapy and treatment individualization of patients with OPSCC.

Funding: This study was supported by the Ministry of Health Czech Republic conceptual development of research organization (UHHK, 00179906), by the Specific University Research Program (SVV 260396) from Charles University, Faculty of Medicine in Hradec Kralove, by the program PROGRES Q40/11, and by the project BBMRI-CZ LM2018125.


Predictive gene expression model for detection of SDHx mutation in carotid paragangliomas

V. Pavlov*, A. Kobelyatskaya, M. Fedorova, D. Kalinin, A. Golovyuk, G. Krasnov, A. Kudryavtseva, A. Snezhkina

*Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia

Background & objectives: Mutations in SDHx genes occur in more than 40% of carotid paragangliomas (CPGLs). SDHB and SDHD variants are associated with the high risk of metastasis and multifocality, respectively. Identification of SDHx mutation is important for management of patients with CPGLs.

Methods: Whole-transcriptome sequencing on an Illumina platform and bioinformatics analysis were performed for 71 CPGLs. Based on gene expression data (CPMs), a fully connected neural network (FCNN) was constructed using Keras, Tensorflow, and KerasR libraries. The predictive model was trained and tested on a studied cohort, as well as additionally tested with RNA-Seq data for pheochromocytomas (PCCs) from TCGA.

Results: We created a two-step predictive gene expression model for identification of deleterious variants in SDHx genes. At first step, the model defines variants in SDHx genes overall based on the expression of four genes (CEP104, ERP29, EYA3, and NFRKB) and a lncRNA (STXBP5-AS1). The first gene set has the following metrics: sensitivity–0.85/specificity–1/accuracy–0.92/AUC–0.92 for the CPGL cohort and sensitivity–0.52/specificity–0.69/accuracy–0.61/AUC–0.61 for the PCC cohort. In the second round, the model predicts SDHB and SDHD mutations using data on CEP104, ERP29, and EYA3 gene expression and had the following metrics: sensitivity–1/specificity–1/accuracy–1/AUC–1 for CPGLs, and sensitivity–1/specificity–0.7/accuracy–0.77/AUC–0.85 for PCCs. This work was financially supported by the grant from the Russian Science Foundation (no.21-14-00353).

Conclusion: Clinical genetic testing of patients with CPGLs requires target sequencing of four genes (SDHA, SDHB, SDHC, and SDHD) or whole-exome sequencing, which is often long and expensive. Immunohistochemistry of SDHB subunit and measurement of succinate-to-fumarate ratio were recently proposed as alternative methods for primary prediction of SDHx mutations. However, these approaches do not detect the specific mutated gene. This model allows identifying not only SDHx-mutated tumours but also detect mutated genes (SDHB and SDHD) that are essential for tumour management.

Funding: This work was financially supported by a grant from the Russian Science Foundation (no. 21-14-00353) and performed using the equipment of the EIMB RAS “Genome” center (http://www.eimb.ru/rus/ckp/ccu_genome_c.php).


Molecular pathways associated with SDHx mutations in vagal paragangliomas

V. Pavlov*, M. Fedorova, E. Pudova, A. Kobelyatskaya, D. Kalinin, A. Golovyuk, G. Krasnov, A. Snezhkina, A. Kudryavtseva

*Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia

Background & objectives: Vagal paraganglioma (VPGL) is a rare neuroendocrine tumour occurring along the vagus nerve. Approximately half of head and neck paragangliomas are associated with mutations in SDHx genes. However, molecular changes associated with these mutations have not been fully understood.

Methods: Illumina whole-transcriptome libraries were prepared for 33 VPGLs with known SDHx status and subsequently sequenced on a NextSeq 500 at 76bp, single-end mode. Raw sequencing data were subjected to the standard bioinformatics analysis. Analysis of differential gene expression and pathways enrichment were performed using the RTrans pipeline and KEGG and GO databases.

Results: Using top-40 differently expressed genes between SDHx-mutated and non-SDHx-mutated tumours, pathway enrichment analysis was performed. Based on KEGG database, four pathways enriched with upregulated genes and seven pathways enriched with downregulated genes were found (FDR ≤ 0.05). Using GO database, we revealed 75 pathways enriched by upregulated genes (FDR ≤ 0.05) and no significant changes in pathways enriched by downregulated genes. According to both databases, SDHx mutations are associated with expression changes in genes related to cell adhesion, extracellular matrix, PI3K-Akt, and VEGF signalling pathways. This work was performed using the equipment of EIMB RAS “Genome” centre (http://www.eimb.ru/ru1/ckp/ccu_genome_c.php).

Conclusion: Changes in biological pathways associated with SDHx mutations in VPGLs were firstly detected. We showed that well-known tumour-associated PI3K-Akt pathway can potentially be activated in SDHx-mutated tumours. Upregulation of genes involved in the VEGF signalling pathway in SDHx-mutated VPGLs is possibly associated with pseudohypoxic state that occurs as a result of succinate accumulation and HIF stabilization.

Funding: This work was financially supported by a grant from Russian Science Foundation no.19-15-00419.


The significance of H3K9Me3 and H3K18Ac in salivary gland neoplasms

E. Phattarataratip*, A. Lam-Ubol

*Chulalongkorn University, Thailand

Background & objectives: Histone modifications had been reported in different cancers with varying prognostic implications. The objectives of this study were to examine the degree of acetylation and methylation of histone H3 in salivary gland neoplasms and their associations with prognostically-relevant pathologic characteristics.

Methods: The expression of H3K18Ac and H3K9Me3 in 70 specimens of salivary gland neoplasms, consisting of 30 mucoepidermoid carcinoma (MEC), 20 adenoid cystic carcinoma (ACC) and 20 pleomorphic adenoma (PA), were investigated immunohistochemically. The immunohistochemical scoring was calculated in each case, based on both the staining intensity and the percentage of positive tumour cells.

Results: H3K18Ac and H3K9Me3 were variably expressed in the majority of MEC, ACC and PA cases. Their expression appeared significantly correlated within this group of neoplasms. The increased H3K9Me3 in MEC was positively correlated with small nest invasion at tumour front and advanced grade pathologically. In addition, the solid subtype of ACC showed significant up-regulation of both H3K18Ac and H3K9Me3, compared with cribriform/tubular subtypes.

Conclusion: Salivary gland neoplasms differentially acquire distinct pattern of histone modification. Hyperacetylation and methylation of histone H3 could be underpinning the prognostically worsen solid type of ACC, and the trimethylation of H3K9 may be involved in aggressive pathologic characteristics of MEC.

Funding: This study was supported by Faculty Research Grant (DRF 64008), Faculty of Dentistry, Chulalongkorn University.


H3K9Ac expression in salivary gland tumours: correlation with histopathologic characteristics

A. Lam-Ubol*, E. Phattarataratip

*Srinakharinwirot University, Thailand

Background & objectives: Acetylation of histone protein plays an important role in regulation of gene expression. Dysregulation of histone H3K9 acetylation was reported in various cancers and presented prognostic and therapeutic values. We aimed to examine H3K9Ac expression among common salivary gland tumours.

Methods: Archived paraffin-embedded tissue of 30 mucoepidermoid carcinomas, 20 adenoid cystic carcinomas and 20 pleomorphic adenomas were included in the study. H3K9Ac expression was evaluated by immunohistochemical staining. Expression of H3K9Ac were determined semi-quantitatively based on both staining intensity and percentage of positive cells. The immunohistochemical scores were then evaluated according to various histopathologic features.

Results: Mucoepidermoid carcinoma demonstrated significantly increased H3K9Ac expression compared with pleomorphic adenoma. Moreover, the solid subtype of adenoid cystic carcinoma showed significantly higher H3K9Ac expression than the cribriform/tubular subtypes.

Conclusion: High levels of H3K9Ac appear to be associated with malignant salivary gland neoplasm and a more aggressive subtype of adenoid cystic carcinoma.


Predictive modeling for the diagnosis of oral and laryngeal premalignant and malignant lesions using expression of p53 and ki-67

J. Jeong*, K. Cho, H.J. Lee, Y.S. Lee, J.S. Song

*Department of Pathology, Asan Medical Center, Republic of Korea

Background & objectives: Oral and laryngeal epithelial lesions are diagnosed based on histologic criteria of WHO classification which may appear interobserver variability. Integrated diagnostic approach based on immunohistochemistry (IHC) is required that can help the interpretation of ambiguous histological findings of epithelial lesions.

Methods: The oral cavity and larynx tissues of 118 cases from 108 patients were examined by IHC for p53 and Ki-67. Logistic regression analysis and decision tree algorithm were employed to develop the scoring system and predictive model for differentiating the epithelial lesions. The comparison between TP53 mutation and expression patterns of p53 was conducted by Next-generation sequencing (NGS) and IHC.

Results: The diffuse expression type (pattern HI) and null type (pattern LS) for p53, and pattern HI for Ki-67 were significantly associated with high-grade dysplasia (HGD) or squamous cell carcinoma (SqCC). With accuracy and the area under a receiver operating characteristic curve (AUC) of 85.3% and 85.4%, the scoring system based on p53 and Ki-67 classified epithelial lesions into two types: non-dysplasia or low-grade dysplasia, and HGD or SqCC. The decision tree model using p53 and Ki-67 classified epithelial lesions into non-dysplasia, dysplasia, and SqCC with accuracy and AUC of 64.7% and 80%. The patterns HI and LS for p53 were confirmed to be correlated with missense and nonsense/frameshift mutations, respectively.

Conclusion: The scoring system using p53 and Ki-67 may aid in the differentiation of epithelial lesions, especially when their morphologic features are ambiguous.


Comparison of PD-L1 immunohistochemical assays in head and neck carcinoma

J. Jeong*, U. Jo, K. Cho, J.S. Song

*Department of Pathology, Asan Medical Center, Republic of Korea

Background & objectives: Programmed death ligand 1 (PD-L1) expression is predictive biomarker for immune checkpoint inhibitor in head and neck squamous cell carcinoma. We compared the variable immunohistochemical (IHC) assays using the tumour proportion score (TPS) and the combined positive score (CPS).

Methods: In total 56 cases of head and neck carcinoma (HNC), PD-L1 expression was evaluated for formalin fixed paraffin embedded (FFPE) blocks from biopsy and surgical resection specimens using 4 IHC assays including 22C3 pharmDx on the Dako Link 48 platform and on Ventana Benchmark Ultra platform, SP263, and SP142 by TPS and CPS using cut-offs of >1% and >50%.

Results: Overall PD-L1 positivity rates were 83.9% (47/56) with a cut-off ≥1% and 21.4% (12/56) with a cut-off ≥50%. The average of TPS scores were 18.03 ±24.83 with 22C3 on Ventana assay, 16.56±25.84 with 22C3 pharmDx, 16.76± 25.16 with SP263 assay, and 3.72±11.48 with SP142 assay, resulting in the lowest expression rate in SP142 assay. A statistically significant correlation (p=0.012) was analysed between location and PD-L1 expression using a cut-off ≥50%. When comparing different assays, 22C3 pharmDx and SP263 assay using TPS score showed good correlation with Spearman correlation coefficients calculated as 0.892 (p <0.001). However, less agreements were observed among 22C3 pharmDx, SP263 assay, and SP142 assay.

Conclusion: Overall PD-L1 positivity rate with a cut-off ≥1% was 83.9%. PD-L1 expression was statistically significant correlation with the location (P = 0.012). The correlation of PD-L1 expression between paired the biopsy and resection specimen is evaluating in this ongoing research.


PD-L1 expression and its clinicopathological significance in odontogenic carcinomas

K. Oh*, J. Kim, S. Cho, J. Lee, H. Yoon, S. Hong

*Seoul National University, Republic of Korea

Background & objectives: Programmed cell death-ligand 1 (PD-L1) expression has been investigated in various malignancies and is currently used for immunotherapy selection in patients with several cancers. This study aims to primarily identify PD-L1 expression and determine its clinicopathological significance in odontogenic carcinomas.

Methods: PD-L1 (clone E1L3N) immunohistochemistry was performed in 20 odontogenic carcinomas after validated compared to the 22C3 pharmDx assay. The percentage of tumour cells with membranous staining at any intensity (TPS) ≥ 1% was defined as PD-L1-positive. Associations between PD-L1 expression and clinicopathological factors were statistically analysed.

Results: PD-L1 was positively expressed in 85.7% (6/7) of ameloblastic carcinomas, 37.5% (3/8) of primary intraosseous carcinomas, 33.3% (1/3) of clear cell odontogenic carcinomas, and 50% (1/2) ghost cell odontogenic carcinomas. Positive PD-L1 expression was associated with larger tumour size (P = 0.031), whereas no correlation was observed with age, sex, and tumour location (P > 0.05). Most cases (3/4; 75.0%) of odontogenic carcinomas with metastasis showed high PD-L1 expression (TPS ≥ 50%).

Conclusion: These results suggest the possible utility of immune checkpoint inhibitors for the treatment of patients with advanced odontogenic carcinomas, which warrants further clinical investigation.


Assessment of TP53 and CDKN2A status can be a predictive marker of malignant transformation of sinonasal inverted papilloma

S. Kwon*, E.S. Kim, J.H. Paik, J. Chung, J.H. Wee, S. Cho, T. Won, J. Kim, C.S. Rhee, H. Kim

*Seoul National University Bundang Hospital, Republic of Korea

Background & objectives: Sinonasal inverted papilloma (IP) has the potential to transform into squamous cell carcinoma (SCC), but there are no diagnostic methods to predict it. We investigated genetic mutations involved in progression of IP-SCC and explored biomarkers that can predict malignant transformation.

Methods: 14 patients diagnosed with SCC arising in IP and six patients diagnosed with IP without malignant transformation were included. DNA was extracted from IP, IP with dysplasia, and SCC, respectively, and whole exome sequencing and immunohistochemistry (IHC) was performed.

Results: Major oncogenic mutations were observed with high frequency in the stepwise progression from IP to SCC. TP53 was the most frequently mutated gene (39%), followed by CDKN2A (27%), TTN (27%), ARID1A (21%), and PIK3CA (15%). Mutations in TP53 and/or CDKN2A were observed in 3 out of 6 IPs with malignant transformation, whereas none of the mutations were observed in IPs without malignant transformation. As a result of TP53 and CDKN2A IHC, three of six IPs with malignant transformation showed a diffuse strong or null pattern in p53, and one showed a total loss of p16 which is distinct pattern from pure IPs.

Conclusion: Our result suggests that assessment of TP53 and CDKN2A status can be a predictive marker of malignant transformation of IP and assessment of p53 and p16 status using IHC can be a surrogate marker for TP53 and CDKN2A status.


Ossifying fibroma of the jaws: a clinicopathological case series study

M. Triki*, S. Makni, S. Graja, O. Boudawara, N. Gouiaa, W. Ghribi, T. Boudawara, R. Kallel

*Department of Pathology, Habib Bourguiba University Hospital, Sfax, Tunisia

Background & objectives: Juvenile trabecular ossifying fibroma (JTOF) and juvenile psammomatoid ossifying fibroma (JPOF) are two rare histological variants of ossifying fibroma (OF). Theses variants are 3 distinct clinicopathological entities. Our aim is to assess clinico-pathologic features of a case series of OF.

Methods: Eleven consecutive cases of OF diagnosed in the department of pathology of Habib Bourguiba Hospital, were collected from 2011 to 2021. The clinical data and microscopic features of these cases were reviewed and analysed with the most recent diagnostic criteria for OF.

Results: Patients mean age at diagnosis was 35 years with a sex-ratio of 0,2. Eight cases were found in the mandible and three in the maxilla. Bone swelling or expansion was the most frequent clinical presentation (90%). Microscopically there was 8 cases of classic OF comprised of globulous woven or lamellar bone with rare or without osteoblastic rimming mixed with fibrous tissue and three cases of JPOF comprised predominantly of psammoma bodies mixed with a highly cellular fibrous tissue. No case of JTOF was recorded. No cellular atypia or mitosis were identified. Recurrences were recorded in two women with JPOF, aged 16 and 22 years respectively. These recurrences were surgically removed.

Conclusion: OFs occur more frequently in female patients and in those in the second to fourth decades of life. The most commonly affected site is the mandible. Most OF can be treated by conservative and complete surgical excision. Recurrences are infrequent in classic OF but more common in Juvenile ossifying fibroma. Therefore, a complete excision with long term supervision is needed.


Verification and validation of the anti-PD-L1 antibody, clone 22C3 on a laboratory developed test

S. Brennan*, J. O'Neill, S. Kennedy

*St. Vincent's University Hospital, Dublin, Ireland

Background & objectives: PD-L1 IHC 22C3 pharmDx assay kit processed via the Dako Autostainer Link-48 platform is the licensed immunohistochemical assay and companion diagnostic test(CDT) for the assessment and thus administration of Pembrolizumab. However, laboratories may not have access to this specified CDT.

Methods: 47 whole tumour slides off head and neck squamous cell carcinomas were stained with the PD-L1 IHC 22C3 pharmDx assay kit processed via the Dako Autostainer Link 48 (CDT) and the Dako Omnis platform, referred to as a laboratory developed test (LDT). A combined positive score (CPS) were provided by 2 pathologists, with discordant cases provided with an agreed score.

Results: PD-L1 IHC 22C3 pharmDx assay kit processed via CDT and LDT showed identical staining in terms of intensity and pattern of staining. On review of concordance between the CDT and LDT, implementing a CPS cut-off of ≥ 1 showed the following: overall percentage agreement (OPA) 94%; positive percentage agreement (PPA) 100%; negative percentage agreement (NPA) 88%. A CPS cut-off of ≥ 20 showed the following: OPA 96%; PPA 95%; NPA 100%. With a CPS cut-off of ≥ 1, the intra-examiner concordance between the platforms was 89%, and the inter-examiner concordance between examiners on each platform was 92%(CDT) and 85%(LDT).

Conclusion: Firstly, the high level of concordance between scoring Pathologists illustrates with adequate training there is a reduction in the variable of scoring interpretation by examiners, which may have a therapeutic implication. Secondly, we open for discussion the deconstruction of the current practice of a compulsory CDT for a particular PD-L1 immunohistochemical assay in head and neck cancers. The implementation of LDTs as an alternative to the CDT are a novel and readily available method to surmount limitations posed.


The accuracy of head and neck core needle biopsies and fine needle aspirates at a large teaching hospital

P. Hankinson*, R. Gorania, A. Khurram

*The University of Sheffield, United Kingdom

Background & objectives: Fine needle aspirate and core needle biopsies are common techniques for diagnosis of head and neck diseases. Diagnosis can be challenging due to limited tissue for examination. Here we record the accuracy of these techniques at Sheffield Teaching Hospitals.

Methods: We accessed records of patients receiving core needle biopsy (CNB) or fine needle aspirate (FNA) at a head and neck site from 2015 to 2020. Cases without definitive diagnosis on subsequent excision were excluded. Both specific diagnosis and benign/malignant category was compared between CNB/FNA and the excision specimen. Accuracy of FNA and CNB was compared with a Fischer’s exact test.

Results: A total of 79 cases were identified in which the CNB/FNA diagnosis could be compared with a definitive diagnosis made on an excision specimen. Of these, 63 cases were CNB and 16 were FNA. CNB showed 95% agreement between the benign and malignant category and 87% in specific diagnosis. FNA showed 94% agreement between the benign and malignant category and 69% in specific diagnosis. The most common diagnoses on CNB/FNA were pleomorphic adenoma and metastatic squamous cell carcinoma. A Fisher’s exact test did not show a statistically significant difference in the accuracy of CNB and FNA, this may be due to the limited sample size of the FNA group.

Conclusion: CNB and FNA are both non-invasive techniques in comparison to a conventional biopsy and are often utilised in the head and neck region. The accuracy of these techniques presented here is similar to those previously reported in the literature, approximately 90% for CNB and 70% for FNA. However, where previous studies found CNB to be more accurate than FNA this difference was not observed in this cohort. This may be due to the limited sample size of the FNA group.


Transcription factors profile in sinonasal neuroendocrine neoplasms (snNENs) and olfactory neuroblastoma (ONB)

F. Pettenon*, C. Facco, A.M. Chiaravalli, M. Turri-Zanoni, P. Castelnuovo, S. La Rosa, M. Cerati, F. Sessa, S. Uccella

*Università degli Studi dell’Insubria, Department of Anatomical Pathology, Varese, Italy

Background & objectives: snNENs are rare and mostly include neuroendocrine carcinomas (NECs). ONB is a unique sinonasal neoplasm, expressing neuroendocrine markers. We aimed to investigate transcription factors expression profile in snNENs and ONBs and to explore their role in distinguishing these two entities.

Methods: GATA3, SATB2 and CDX2 expression were investigated in a series of 26 ONBs and 7 snNENs diagnosed and treated in our Institution. ONBs were graded according to Hyams’ system (2 grade 1, 13 grade 2, 8 grade 3 and 3 grade 4) and epithelial NENs were reclassified into 5 NECs, 1 mixed neuroendocrine/non-neuroendocrine neoplasms (MiNENs) and 1 amphicrine carcinoma.

Results: Hyams’ grade 1-3 ONBs stained diffusely and intensely for SATB2. Grade 4 ONBs and NECs were globally negative, including the NEC component of the MiNEN. The intestinal-type adenocarcinoma (ITAC) component of the MiNEN, like the amphicrine carcinoma, was intensely positive. GATA3 was heterogeneously expressed in Hyams’ grade 1-3 ONBs, whereas grade 4 ONBs, all NENs and the amphicrine carcinoma were completely negative. CDX2 was only expressed in the amphicrine carcinoma and, as expected, in the ITAC component of the MiNEN.

Conclusion: Our study expands the spectrum of SATB2 and GATA3-positive neoplasms and identifies, for the first time, SATB2 and GATA3 expression as features of Hyams grade 1-3 ONBs, suggesting that Hyams grade 4 ONBs are not only clinically but also biologically different from low graded ONBs. These results are useful in diagnostic daily practice, as highlight that the joudicious employment of transcription factors may help in correctly diagnosing rare but clinically relevant neoplasms.


Cervical lymph node extemporaneous assessment by confocal microscopy during surgery: an alternative to frozen section examination?

O. Casiraghi*, A. Villard, A. Aupérin, I. Breuskin, S. Asmandar, M. Abbaci

*Gustave Roussy Cancer Campus, France

Background & objectives: To evaluate the value of the Ultra-fast Confocal Microscopy (UFCM) imaging of cervical lymph nodes in patients with squamous cell carcinoma, compared to conventional histology. This technique would be considered interesting if it is more accurate than frozen section examination.

Methods: We carried out an ex vivo study on lymph nodes from patients N0. The two parts of fresh lymph nodes were marked with acridine, then imaged with the Histolog Scanner (SamanTree Medical, Switzerland) during one minute. In post processing, all acquired and anonymized UFCM images were read independently by two pathologists and the “UFCM diagnoses” were compared to conventional histology.

Results: We included 11/44 patients, i.e. 64 lymph nodes. 8/64 lymph nodes were metastatic (N+) on conventional histology. On Ultra-fast Confocal Microscopy images, one pathologist (PT1) was in accordance with conventional histology in 93.75% (95% CI= 84.8-98.3%) and the other (PT2), in 95.3% (IC95=86.9%-99%). PT1 considered as negative one patient, who was finally N+. The errors were as follows: the metastatic area has escaped the screening of the Histolog Scanner image (n=3), and a metastasic area has been analysed as «suspect », but not identified as carcinomatous (n=1)

Conclusion: Inclusions have now reached 44 patients and 206 lymph nodes, allowing a robust statistical analysis, which is in progress. At this stage, the analysis by Ultra-fast Confocal Microscopy seems to be a very promising technique. It could eventually replace the frozen section examination, because its diagnostic reliability seems at least equivalent. Furthermore, its speed processing (5 minutes) constitutes a major asset.



Laminin is a useful marker in the differentiation between actinic cheilitis and invasive squamous cell carcinoma in oral biopsies: new insights

K. Zacharouli, M. Strataki*, P.G. Doukas, D.P. Vageli, V. Tsangari, S.G. Doukas, G. Kabanos, V. Kakanis, R. Papamichali, S. Tzika, G.K. Koukoulis, M. Ioannou

*Department of Pathology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Greece

Background & objectives: Oral cancer can be life-threatening if not diagnosed early. Precancerous lesions, such as actinic cheilitis, can transform into oral cancer. Laminin is a fundamental component of basement membrane (BM), its degradation can contribute to mucosal malignant transformation.

Methods: Formalin-fixed and paraffin-embedded biopsies from 46 patients with oral lesions (37 males and 9 females, mean age = 67 years) were histologically analysed by hematoxylin and eosin staining to diagnose the entity of actinic cheilitis and to classify epithelial dysplasia and invasive cancer. Immunohistochemical (IHC) analysis was performed to evaluate laminin expression in biopsies.

Results: Histological analysis revealed 34 patients with actinic cheilitis and 12 patients with squamous cell carcinoma (SCC) of the lip. Three patients with actinic cheilitis had concomitant in situ carcinoma. IHC analysis for laminin revealed intense and continuous staining of the BM in all cases of actinic cheilitis with low dysplasia, while loss of laminin expression was observed in invasive SCC cases. Interestingly, intracellular expression of laminin in parabasal layers of the epithelium was noted in cases of actinic cheilitis with high-grade dysplasia/in situ carcinoma.

Conclusion: Laminin expression, by IHC analysis, could be useful in the differential diagnosis between actinic cheilitis and invasive squamous cell carcinoma, as well as actinic cheilitis with low and high-grade dysplasia. Findings from this study provide new insights into the mechanisms involved in the process of progression of actinic cheilitis into SCC of the lip, encouraging in vitro and in vivo studies that may document the mechanistic role of laminin in this process.


Exploration of the transcriptomic landscape of HPV-positive and HPV-negative oropharyngeal squamous cell carcinoma upon development of cisplatin resistance

H. Crane*, K. Hunter, S.F. El-Khamisy

*University of Sheffield, United Kingdom

Background & objectives: Oropharyngeal Squamous Cell Carcinoma (OPSCC) has seen a dramatic increase over the past few decades. OPSCC is frequently treated with a chemoradiotherapy regime including cisplatin, but resistance can develop in some patients which may be difficult to treat.

Methods: To develop a cisplatin resistant cell line model, a HPV-positive (UDSCC2) and a HPV-negative (UMSCC89) cell line were treated over several months with increasing cisplatin concentrations. Single cell clones were selected and assessed using clonogenic survival assays. Following RNA-Sequencing the reads were quantified using Salmon and differential expression analysis was conducted using the package DESeq2 in R.

Results: Clonogenic survival assays revealed the selected clones were more resistant to cisplatin compared to the parental cells. One HPV-positive and one HPV-negative resistant clone were selected for RNA-Sequencing alongside their parental counterparts. Differential expression analysis between parental and resistant cells revealed there were 1234 differentially expressed genes in the HPV-positive group and 1521 differentially expressed genes in the HPV-negative group. 243 of these genes were seen to be differentially expressed in both the HPV-positive and HPV-negative resistant clones. Using gene pathway analysis, multiple pathways were seen to be involved upon development of cisplatin resistance, including epithelial to mesenchymal transition and apoptotic signalling.

Conclusion: This study provides an insight into the transcriptomic landscape of HPV-positive and HPV-negative OPSCC upon development of cisplatin resistance. Following validation, expression of selected markers will be assessed in tumour samples to explore their potential as a prognostic biomarker.

Funding: Funded by a Cancer Research UK (CRUK) and the Pathological Society Joint Predoctoral Research Bursary and Wellcome Trust Clinical PhD Fellowship (4Ward North Programme)


"Molecular" resection margins in squamous cell carcinoma of the oral cavity – report of the first results of the multidisciplinary view

P. Hurník*, J. Stembirek, Z. Chyra, T. Sevcikova, J. Reznarova, B. Putnova, Z. Cermakova, T. Blazek, V. Zidlik, O. Res, J. Stransky, M. Buchtova

*Department of Clinical and Molecular Pathology, University Hospital Ostrava and Faculty of Medicine, University of Ostrava, Department of histology and embryology, Faculty of Medicine, Masaryk University Brno, Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Brno, Czech Republic

Background & objectives: The therapy of squamous cell carcinoma of the oral cavity has significantly intensified in the last decade. Here, we focused on sensitive mutation analysis of resection margins that could improve the prediction of relapse and/or sensitivity to specific drugs.

Methods: DNA was isolated from 26 patients (tumour, peripheral blood and margins in total 3 samples/patient). We performed Illumina sequencing using a panel of 88 cancer genes. Only non-synonymous variants in tumour margins that were reported in the ClinVar database as “pathogenic”, “likely pathogenic” or “uncertain significance”, and were simultaneously not present in the peripheral blood, were selected for further analysis.

Results: In total, we found 21 mutated genes, among them mainly tumour suppressor genes involved in DNA repair. We detected mutations in DNA isolated from 22/26 tumours, and in 5/26 tumour margins. Gene TP53 was the most commonly mutated gene followed by BRCA1/2 and CDKN2A. The median tumour load was 2 pathogenic mutation per patient on average (range 0-9). This parameter did not correlate with the presence of histological markers like perineural invasion probably due to small cohort size. Similarly, we did not observe association of mutations in resection margins and probability of disease relapse.

Conclusion: The spectrum of the tumour mutations is similar as in other studies with the exception of mutations in the BRCA genes, which were not found frequently mutated in OSCC (12-19%). The data in the literature suggests BRCA mutation in OSCC examined by imunohistochemistry technique ranges from 44% (139patients) to 63% (60patients). Variants identified in our dataset are often introducing stop codons leading to truncated and non-functional proteins. The potential application of BRCA(PARP) inhibitors for OSCC needs to be elucidated.


Keratinising pleomorphic adenoma of parotid salivary glands: analysis of three cases from practice

M. Myroshnychenko*, I. Brodetskyi, V. Malanchuk, O. Dyadyk, Y. Kalashnyk-Vakulenko

*Kharkiv National Medical University, Ukraine

Background & objectives: Pleomorphic adenoma (РА) of parotid salivary glands (PSG) with squamous metaplasia, keratin cysts formation (keratinizing PA) is not common and causes difficulties in diagnosis. The objective was to analyse three cases from practice of keratinizing PA of parotid salivary glands.

Methods: Surgical material from two women, one man with keratinizing PA was studied. The mean patients’ age was 36.3±2.1 years. In two cases, primary surgical treatment was performed, in one case – secondary, due to relapse. During examination it was noted in PSG a painless nodule of dense consistency in diameter from 1.5 to 4.5 cm. Histological, histochemical methods were used.

Results: Macroscopically in three cases the nodes on the cut were of whitish-pinkish colour with cyst formation. Microscopically, the tumour was characterized by the predominance of the parenchymal (epithelial) component over the mesenchymal (stromal) one. The epithelial component was represented by epithelial, myoepithelial cells. Epithelial cells were of basaloid, spindle-cell, squamous, clear-cell type. They formed nests, strands with numerous foci of squamous metaplasia and keratinous cysts lined by squamous epithelium and containing eosinophilic keratin substance. The stroma was represented by connective tissue, vessels and myxoid, chondroid, osteoid, mucoid zones. In two cases, the tumour was surrounded by a distinct fibrous capsule with tumour invasion; in one case the capsule was absent.

Conclusion: Morphological diagnosis of keratinizing PA causes certain difficulties for pathologists and requires a differential diagnosis with mucoepidermoid carcinoma, necrotizing sialometaplasia, squamous cell carcinoma. In the studied cases, morphological examination revealed a keratinizing PA of PSG with a predominance of the epithelial component over the mesenchymal one. The presence the relapse in one case in anamnesis; tumour invasion into the capsule in two cases, absence the capsule in one case indicate that keratinizing PA is prone to recurrence.


Frequency of SDHx variants in middle ear paragangliomas

V. Pavlov*, M. Fedorova, E. Pudova, D. Kalinin, A. Golovyuk, G. Krasnov, A. Kobelyatskaya, A. Snezhkina, A. Kudryavtseva

*Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia

Background & objectives: Middle ear paragangliomas (MEPGLs) are rare neuroendocrine tumours occurring on the medial promontory wall of the middle ear. MEPGLs are highly hereditable, being susceptible to mutations in driver genes, such as SDHx coding for succinate dehydrogenase subunits.

Methods: Total of 28 MEPGLs were subjected to genetic testing on the presence of SDHx variants using Sanger sequencing on an ABI PRISM 3500xL (Thermo Fisher Scientific) based on designed specific primers for all exons of target genes.

Results: Four pathogenic mutations (4/28, 14%) were found among studied MEPGLs: missense and nonsense variants in the SDHB gene, [NM_003000.3:c.689G>A (rs587782604) and NM_003000.3:c.79C>T (rs74315369)], respectively, as well as two missense mutations in the SDHD gene, [NM_003002.4:c.305A>G (rs104894302) and NM_003002.4:c.274G>С (rs80338845)]. This work was performed using the equipment of EIMB RAS “Genome” centre (http://www.eimb.ru/ru1/ckp/ccu_genome_c.php).

Conclusion: According to the literature on head and neck paragangliomas, SDHB variants are most frequent (33%), followed by SDHD variants (21%). This research showed low and close frequencies (7%) for variants both in SDHB and SDHD genes for Russian patients with MEPGLs. These results are similar to those for patients with MEPGL in other populations. Thus, development of MEPGLs may be linked with prevalence of mutations in other PGL susceptibility or cancer-associated genes.

The study was funded by grant МК-5956.2021.1.4.


Sinonasal inverted papillomas with multiples recurrences: revision of our experience and HPV status

L. López Vilaró*, M.C. Campos Marmol, J.R. Gras Cabrerizo, M. Casasayas Plass, S. Bagué Rosell, J. Szafranska

*Department of Pathology. Hospital de la Santa Creu i de Sant Pau, Spain

Background & objectives: Inverted Papilloma (IP) is a rare sinonasal tumour with local destructive potential and risk of malignant transformation. The aim of this study is to compare clinicopathologic features of a multirecurrent IP group and a none-multirecurrent IP group and its HPV status.

Methods: We reviewed clinical and pathological data of patients diagnosed of IP during the period of 1978-2020 in our institution and selected the recurrent cases. The IP group with more than one recurrence, treated by diverse surgical procedures, was compared with another group of IP with one recurrence. We performed p16 IHQ (Dako) study and HPV Genomic PCR.

Results: The study included 152 patients from which 22 had recurrent IP.13 patients had multirecurrent IP,9 were males with a median age of 59, 11tumours were localized in ethmoid-sphenoid sinuses. Four patients were smokers,2 had allergy and 2 had toxic exposures. Three IPs in the multirecurrent group showed low-grade dysplasia and one SCC.p16 was positive in 8cases, in 2 PCR showed presence of HPV, one determination negative and 10 not valuable. Nine patients presented with none-multirecurrent IP,8 were males with a median age of 56, 6 were localized in ethmoid-sphenoid sinuses. Five patients were smoker and 1 had toxic exposure. They had not dysplasia or SCC associated.p16 was positive in 3cases but PCR were not valuable.

Conclusion: IP is a benign sinonasal tumour with malignant potential. In our series, 1/22 had squamous cell carcinoma transformation and 13/22 had more than one recurrences. The aetiology is yet unknown and probably multifactorial with a main role of HPV infection. Our findings suggest that the HPV status may be associated with a higher risk of recurrences and dysplastic transformation, but further investigation is needed.

PS-03 | Poster Session IT in Pathology / Computational Pathology Symposium


MarrowQuant 2.0: clinical application of a user-friendly digital hematopathology tool for human bone marrow trephine biopsies

R. Sarkis*, O. Burri, C. Royer-Chardon, S. Blum, F. Schyrr, M. Costanza, S. Sherix, C. Bárcena, B. Bisig, V. Nardi, R. Sarro, O. Spertini, S. Blum, M. Weigert, A. Seitz, B. Deplancke, L. de Leval, O. Naveiras

*Laboratory of Regenerative Hematopoiesis, Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne (EPFL) & Department of Biomedical Sciences, University of Lausanne (UNIL), Switzerland

Background & objectives: Bone marrow(BM) assessment is a multiparametric evaluation of which cellularity constitutes one parameter. In diagnostic practice, the assessment is based on a semi-quantitative estimation which is time-consuming. In this study, we validated MarrowQuant2.0, within QuPath software, a digital hemopathology tool.

Methods: MarrowQuant2.0 quantifies four compartments within the BM (hematopoietic cells, adipocytes (using Stardist), interstitiumμvasculature, and bones) and measures the cellularity of human BM trephine biopsies. We calculated the cellularity in a series of retrospective biopsies (training set n=36; experimental set n=157H&E). Using intraclass coefficient of correlation(ICC), specificity and sensitivity tests, we measured the agreement between MarrowQuant 2.0’s quantification and clinical reference.

Results: Our algorithm was capable of accurate, rapid, and robust segmentation (average accuracy 0.86, n=36). There was an excellent agreement between MarrowQuant 2.0 and the clinical reference (ICC=0.978(95% CI0.955-0.989), R2=0.93). MarrowQuant 2.0 performed in a comparable way as to the clinical reference when used on a set of BM trephine biopsies from clinical routine diagnosis(n=42). We found reciprocity between the hematopoietic and adipocytic compartments in the context of an extreme case of BM remodelling, except for cases with stromal expansion. MarrowQuant2.0 can also leverage an adipocyte-based StarDist model, a deep-learning-based segmentation algorithm, implemented as an extension in QuPath, offering an accurate segmentation of individual adipocytes and a size-based classification.

Conclusion: Our tool may represent a useful adjunct for experimental and clinical hematopathology. We will use MarrowQuant 2.0. to link output with clinical parameters using dimension reduction and clustering methods to visualize and explore a potential prognostic value in myeloid malignancies.

Funding: PHRT-Personalized and Health-Related Technologies


Convolutional neural network-based algorithm for the detection and quantification of the components of the histologic grading of breast ductal adenocarcinoma - the first results

G. Olteanu*, D. Kumar, M. Köteles, I. Mihai

*Spitalul Clinic de Boli Infecțioase și Pneumoftiziologie Dr.Victor Babeș Timișoara, Romania

Background & objectives: The current grading system for ductal adenocarcinoma NOS of the breast (DAC-NOS), is prone to low reproducibility, subjectivity, and is overall time-consuming. Here, we trained a convolutional neural network-based algorithm (CNN-bA) to detect and quantify the components for grading DAC-NOS.

Methods: 100 whole slide images (WSI) of diagnostic slides and 10 training slides (TS) with DAC-NOS were selected from the TCGA-BRCA dataset and subsequently uploaded to a WSI management server (Aiforia Technologies Oy, Helsinki, Finland). Briefly, the CNN-bA (Aiforia version 4.8, Aiforia Create, Aiforia Technologies Oy) was trained on the 10 TS to detect and quantify the components for grading DAC-NOS.

Results: After a successful model was established, the model was used to detect and quantify the histological components of DAC-NOS: tumour tissue (TT), tubule and gland formation (T + GF), solid tumour (ST) aspect, nuclear pleomorphism (NP) and mitotic count (MC). Next the results were exported and interpreted. Here, we report the first results of this pilot program. Successful training for TT, T, and GF or ST aspects was achieved after 3 iterations of the model. Unsuccessful training for NP and MC was reported and this represents the limitations of the results, and the baseline for future improvements that need to be addressed for the subsequent improved artificial intelligence (AI) model.

Conclusion: AI in digital pathology represents the successful evolution of diagnostic pathology. While at times difficult, this transition is successfully implemented in in vitro diagnostics (IVD) platforms. Complex diagnostic algorithms used by pathologists like grading DAC-NOS are laborious to translate into an AI model. We report preliminary data (successful and unsuccessful data points) for a CNN-bA AI model that detects and quantifies components of the histologic grading of DAC-NOS with the aim of constructing the framework for a future IVD model.

Funding: Aiforia Aiforwarding Program


Refining pre-analytic deficiency reporting and capture in the anatomical pathology laboratory: a quality improvement initiative

T. Truong*, P. Roopchand, T. Jordan, Z. Ghorab, E. Slodkowska, M. Downes

*Sunnybrook Health Science Centre, Canada

Background & objectives: Pre-analytic, notably pre-laboratory, deficiencies are estimated to account for ~70% of laboratory errors. Herein, we reviewed pre-analytic deficiency (PAD) data at our institution to develop strategies for improved PAD documentation, allowing for identification of PADs and improvement of quality performance.

Methods: We retrospectively reviewed 12 months of data at an academic, tertiary referral centre, which was captured from three sources: Laboratory information system (LIS), corporate risk management reporting system and manual paper logs. We also interviewed accessioning staff to determine barriers to data recording.

Results: 237 PAD were recorded in one year. Of these, 72% (n=171) were pre-laboratory and 28% (n=66) were in laboratory. Specimen procurement accounted for 79% (n=134) of all pre-laboratory PAD followed by deficient requisitions, 12% (n= 21). Failure to adhere to specimen handling protocols accounted for 37.5% (n=25) of in-laboratory PAD with accessioning errors contributing to 23% (n= 15). Barriers to incident documentation were: time required, multiple systems being used and free-hand data entry under 92 separate headings with no protocol to determine categories of incidents. Therefore, the following consensus categories were created: specimen collection, requisition, packaging, transportation, reception, accessioning, specimen preparation and missing histology alerts.

Conclusion: To streamline PAD documentation, manual logs were eliminated, all reporting was moved to LIS under the above eight categories and accessioning staff was trained on data entry under the new categories. Mandatory deficiency check has been implemented in LIS for the accessioning bench, before the specimen is further processed. Deficiency entry protocol for the grossing bench and histology lab are also currently being optimized for LIS integration in the next phase of the project.


NLP in diagnostic texts from nephropathology

M. Legnar, C. Weis*

*University Medical Centre, Germany

Background & objectives: Nephropathology is a sub-discipline with complex diagnostic patterns and terms. In addition, reporting in a structured manner is a special feature.

Against this background, we investigate whether predicting the final diagnosis based on the written description is possible.

Methods: For his work, 1,185 unlabelled nephropathological reports were included. (i) First, the diagnosis sections were clustered unsupervised to <20 diagnosis groups. Therefore, bag of word-based and embedding-based text-vectorization methods were used. (ii) Second, different natural language processing (NLP) methods for classification were trained to predict based on the descriptive report section the diagnosis group.

Results: Regarding text clustering (i), the silhouette-score and the classification performance of a support vector machine were used to measure clustering accuracy. For both, embedding-based approaches (best is a Bidirectional Encoder Representations from Transformer (BERT)) performed slightly better compared to bag of word-based (best is latent Dirichlet allocation) approaches. Analysing the clusters for keywords shows that some clusters can be mapped to diagnostic groups. For example, there is a cluster for IgA-nephropathy or one for diseases with glomerular necrosis.

Again, the BERT-based approach worked best regarding diagnosis prediction based on the histological description (ii). Notably, there are classification quality differences between the diagnostic clusters. Only some groups are almost perfectly predicted.

Conclusion: For nephropathological reports, the morphological description alone enables retrieving the correct diagnosis for some entities. For other entities, this associative approach does not work well. This is in accordance with a previous study on glomerular change patterns, where some diagnoses are associated with one pattern, and for others, there is a complex pattern combination.

Mapping every diagnosis cluster to a diagnosis group (or a chapter in a textbook) is still under investigation while writing this abstract.


A multi-feature AI solution for diagnosis support in gastric biopsies: a multi-site clinical study

J. Sandbank*, J. Calvo, A. Nudelman, T. Garcia, E. Lanteri, J. Reyre, I. Laouar, B. Terris, M. Montagne, C. Rancati, M. Maklakovski, A. Albrecht Shach, A. Arad, G. Sebag, R. Mikulinsky, T. Amit, I. Gross, M. Grinwald, C. Linhart, M. Vecsler

*Institute of Pathology, Maccabi Healthcare Services, Israel

Background & objectives: This study aimed to clinically validate the performance of a multi-feature AI-based solution on the detection of gastric carcinoma, high-grade dysplasia and high-grade lymphoma, and Helicobacter pylori against rigorous ground truth (GT) established by multiple blinded pathologists in gastric biopsies.

Methods: The Galen™ Gastric algorithm was examined in a prospective stand-alone performance study using retrospectively collected histopathology slides from two sites. We compared GT diagnosis of adult gastric biopsies with the algorithmic results on H&E. GT was reached by concordance between two pathologists (original report and a new blinded diagnosis by pathologist reviewing slides/WSIs). Discrepancies were adjudicated by an expert pathologist.

Results: The AI algorithm demonstrated very high accuracy for the detection of gastric adenocarcinoma, high-grade dysplasia and high- grade lymphoma, with AUC of 0.986. Analysing 544 cases (82 positive), demonstrated sensitivity of 96.34%, specificity of 88.74%, and NPV of 99.27%. Additionally, the algorithm achieved an AUC of 0.966 for the detection of H. pylori in analysis of 525 cases (112 positives), with sensitivity of 91.07%, specificity of 90.56%, and NPV of 97.40% %. We will further report on additional pathologies, e.g., low-grade lymphoma, low-grade dysplasia, and Adenoma.

Conclusion: This study reports the successful clinical validation of the Galen™ Gastric multi-feature AI solution in the accurate detection of a broad range of pathological features, including gastric adenocarcinoma, H. pylori, neuroendocrine neoplasms and more, offering an important tool for computer-aided diagnosis in routine pathology practice, supporting pathologists in their diagnostic work.


Computer aided iron quantification on liver biopsy whole-slide images

D. Panzeri*, R. Scodellaro, G. Chirico, C. Lancellotti, L. Di Tommaso, L. Sironi

*Department of Physics "G.Occhialini", University of Milano-Bicocca, Italy

Background & objectives: Iron overload disorders diagnosis currently relies on blood tests and genotyping. However, liver biopsies remain an invaluable tool for prognostic purpose. We developed a pipeline to quantify iron deposits in whole-slide images (WSI) of liver biopsy marked with Perls stain.

Methods: The study is based on 10 WSI of liver biopsies with different amount of iron. WSI were analysed on different scales to quantitatively and objectively reproduce the standard clinical procedure followed by pathologists. The cells stained by Perls stain (PS) were segmented and quantified by exploiting two methods: Optical Density (OD) colour thresholding and a custom spectral phasor approach.

Results: Our pipeline is able to quantitatively retrieve: the percentage liver biopsy covered by PS, the mean intensity of PS stain, the density and dimension of PS granules, PS heterogeneity. Different background properties of WSI were buffered using a colour correction procedure. Moreover, heatmap overlays are generated to address statistically significant tissue patches (i.e., more densely stained, higher/lower intensity areas, PS deposits dimensions). Data generated by the pipeline had < 5% of error from pathologist’s evaluation.

Conclusion: Our pipeline warrants a rapid and objective method for the evaluation of hepatic iron. Moreover, it can also be tuned and applied to different staining protocols (for example PicroSirius Red, used for the quantification of liver fibrosis). Finally, the estimated parameters are coupled to heatmap overlays in order to obtain intuitive graphical representations of parameter differences in the WSI.


AI-aided assessment of HER2 status in primary and metastatic breast carcinoma

C. Palm*, C. Connolly, R. Masser, B. Padberg Sgier, E. Diamantis Karamitopoulou, B. Bode, M. Tinguely Kovarik

*Pathologie Institut Enge, Switzerland

Background & objectives: Re-evaluation of HER2 receptor status is recommended following metastatic transformation of invasive breast carcinoma to assess for receptor status conversion. We examine the performance of artificial intelligence in the determination of HER2 status in metastatic compared to primary breast carcinoma.

Methods: 60 slides of matched primary and metastatic invasive breast carcinoma were selected from our digitalised cohort of 476 cases (Roche Ventana DP200 Scanner). HER2 IHC scoring was performed by 3 pathologists, and followed with ISH when IHC score ≥ +1. Results were compared to those from uPath HER2 4B5 and Dual ISH algorithms for IHC and ISH respectively.

Results: Overall, there was moderate agreement between pathologist and AI results (Cohen’s κ 0.43, 95CI 0.25-0.61), with higher concordance on metastatic (κ 0.48, 95CI 0.24-0.73) versus primary cases (κ 0.36, 95CI 0.07-0.64). Breakdown analyses revealed the lowest concordance with ISH on metastatic lesions (κ 0.23, 95CI 0.04-0.49) where unedited AI overestimated the number of positive and equivocal cases. Inter-observer-variability of HER2 IHC scoring among pathologists was similar for primary (Fleiss’ κ 0.77, 95CI 0.68-0.86) and metastatic lesions (κ 0.73, 95CI 0.58-0.89). Conversion of HER2 status between primary and metastatic lesions was observed in one case, confirmed by pathologists and AI on IHC and ISH.

Conclusion: Moderate concordance was observed between AI and pathologists in the assessment of HER2 status on primary and metastatic breast carcinoma. Concordance between AI and pathologists was notably higher with IHC compared to ISH. Examination of larger cohort with a diverse range of metastatic sites, combined with increased operator input at the time of digital analysis, may help to determine the feasibility of an automated digitalised HER2 workup for metastatic breast carcinoma.


Evaluation of Ki67 by image-analysis-enhanced quantitative digital pathology

M.J. Krämer*, B.V. Sinn, P. Jank, A. Grass, A. Litmeyer, M. Untch, D. Gerber, A. Schneeweiss, K. Saeger, M. Gleitsmann, J. Furlanetto, S. von Gerlach, B. Felder, A. Ramaswamy, S. Loibl, C. Denkert, W.D. Schmitt

*Institute of Pathology, Philipps-University Marburg, Germany

Background & objectives: Assessment of prognosis of breast cancer by Ki67 immunohistochemistry is an important element of personalized treatment strategies. A precise assessment is crucial for clinically relevant therapy decisions. We aimed to evaluate a Ki67 quantification tool using whole slide images (WSI).

Methods: 61 Ki67 stained, pre- (preTx) and intra-therapeutic core biopsies, as well as corresponding surgical residual disease tissue from neoadjuvant GBG trials, were digitalized. Manual Ki67 scoring was performed by five individual pathologists on WSI (multi-observer), while semi-automated Ki67 scoring was done using VMscope’s Scan Connect (multi-tumour-area). Scan Connect analysed up to four 600x600dpi areas on WSI, followed by pathologist supervision.

Results: The Pearson correlation between manual (man.) and computational (aut.) Ki67 assessment was r=0.781, with no significant differences in overall scoring or global precision (p=0.333, p=0.070). Semi-automated multi-area analysis on preTx tissues had a significantly lower variability than manual assessment (n=29, p<0.001), while showing no differences on intra-therapeutic samples (n=29, p=0.885). Using predefined cut offs, we observed that in Ki67 low and intermediate (int.) groups standard deviations (sd) were lower, while being higher for Ki67 high group, irrespectively of the assessment method (man. & aut.: low sd=±3.4 & ±2.0; int. sd=±4.3 & ±3.9; high sd=±18.2 & ±12.7). In comparison with full-automated assessment, the supervised semi-automated assessment improved the precision significantly (p=0.008).

Conclusion: We found strong correlation between manual (multi-observer) and supervised semi-automated (multi-tumour-area) Ki67 assessment. In comparison to inter-observer variance in manual Ki67 assessment, improved precision was seen in semi-automated assessment considering intra-tissue variance. VMscope’s ScanConnect software could be a useful tool in pathological cancer diagnostic. As a next step, results should be validated in a larger cohort and survival data could be included to examine prognostic differences between the two methods.

Funding: This project is partly funded by joined BMBF/DLR project “CognoScan” (13GW0207).


Leveraging deep learning-based mitosis detection models for supporting automated breast cancer grading

K. Korski*, K. Badowski, X. Li, Y. Nie, S. Abbasi-Sureshjani

*F. Hoffmann-La Roche AG, Switzerland

Background & objectives: The mitotic figure count within 10 HPF in a mitosis-dense region is the only mitosis-based metric used for tumour grading in breast cancer. We present an innovative spatial statistical analysis of mitoses driven by an automatic mitotic figure detection method.

Methods: Mitotic figure candidates are detected using a detector neural network and are then filtered by a classifier neural network with customised architecture. The density and spatial distribution of detected mitotic figures and their relationship to tumour grade are investigated, using G-function that characterises probability distribution of nearest neighbour distances and Getis-Ord Gi statistics that detects local hotspots.

Results: A mitotic figure classification model with 83.06% validation accuracy, was applied on 131 test slides including 18, 47 and 66 slides with tumour grade I, II and III, respectively. Mitotic density showed 0.3987 Pearson (0.4660 Spearman) correlation with tumour grade. The area between observed and theoretic G-function suggested that nearest neighbours of mitotic figures were closer when tumour grade is higher. Group comparison p-values for tumour grade I vs II, II vs III, were 0.0069, 0.019 respectively. The hotspot ratio suggested more hotspots present in higher tumour grade. Group comparison p-values for tumour grade I vs II, II vs III, were 0.015, 0.0036 respectively.

Conclusion: Automatic mitosis detection models enable identifying regions of highest mitotic activity and quantification of the tumour proliferation and aggressiveness based both on the presence of mitotic figures and their relation to their neighbourhood. Our spatial analysis identifies metrics that quantify the mitotic figure spatial distribution and have statistical power to differentiate between low, intermediate, and higher grade tumours. Future work will focus on assessing whether these spatial metrics provide additional information over the current standard and ideally better prognostic value.


Computer-aided algorithm and 3D imaging technology are sensitive methods in the diagnosis of HER2 expression-low breast cancers

Y. Lee*, Y. Hsieh, S. Chang, Y. Chen, Y. Lin, Y. Lin

*National Taiwan University Hospital, Taiwan

Background & objectives: The developed antibody-drug conjugates (ADC) show promising results especially in HER2-low expression breast cancer defined as immunohistochemically 1+ or 2+ with no gene amplification. More sensitive methods can be helpful in HER2-low samples diagnosis compared with traditional light microscopy examination.

Methods: Two approaches were used to determine HER2 expression. The computer-aided algorithm on digital pathological slides can determine HER2 expression levels. The 3D (three-dimensional) imaging approach used 100-μm thickness slide labelled by HER2 antibody with fluorescence and acquiring image under confocal microscopy with optical clearing method. Both methods can be successfully applied to retrospective and forward clinical studies.

Results: In computer-aided approach, we developed a workflow including tumour recognition and HER2-positive cell counting based on 70 WSIs (Whole slide imaging) training dataset. Using 68-ROIs (Region of interest) cropped from 15-WSIs as validation, this method reached 86.7% accuracy and 94.34% sensitivity. 2 ROIs originally categorized as HER2-negative were reclassified as HER2-low by this method.

In 3D imaging approach, HER2 fluorescent stained slides from the same 15 validation cohort were acquired under confocal microscopy. One of four HER2-negative specimens originally categorized by IHC report was reclassified as HER2-low. Using 3D image, 2 of 15 specimens showed heterogeneous HER2 expression in different depth of their thick slides.

Conclusion: This study demonstrated both computer-aided algorithm and 3D imaging technology were able to identify more HER2-low samples than light microscopy. These sensitive methods can detect cases with very low HER2 expression which are considered as HER2-negative by traditional light microscopy. For patients with very low HER2 expression identified by these sensitive methods, more studies are needed to see their clinical response to HER2 antibody-drug conjugates.


Differential diagnosis of Crohn’s disease and Ulcerative Colitis with deep learning based on hyperspectral infrared images

T. Arto, F. Großerüschkamp*, T.M. Müller, A. Mosig, M.F. Neurath, S. Zundler, K. Gerwert

*Ruhr University Bochum, Center for Protein Diagnostics (PRODI), Biospectroscopy, Germany

Background & objectives: Differential diagnosis of inflammatory bowel disease (IBD) can be challenging but is important for treatment decisions and follow-up strategies. We aimed to establish label-free quantum cascade laser (QCL)-based infrared imaging combined with deep learning as a tool for differential diagnosis.

Methods: Infrared imaging was used to analyse IBD and non-IBD cases. It is based on the interaction of electromagnetic waves with molecules within the tissue creating specific molecular fingerprints. A two-step deep learning approach, based on a modified U-Net (CompSegNet), was applied. The first instance differentiates IBD from non-IBD. The second instance differentiates between Crohn’s disease (CD) and Ulcerative Colitis (UC).

Results: The cross-sectional sample set consisted of formalin-fixed, paraffin-embedded (FFPE) tissue sections from biopsies of CD (n=102), UC (n=52), and control cases (n=70). These cases were equally separated in the sub-cohort train (n=99), test (n=66), and validation (n=59). With the first instance CompSegNet, we achieved a validation area under receiver operating characteristic (AUROC) of 0.99 (sensitivity 95%, specificity 91%) in distinguishing between non-IBD and IBD. The subsequent second instance differentiation between CD and UC provided a AUROC of 0.89 (sensitivity 93%, specificity 83%).

Conclusion: Our approach provides an objective and label-free tissue diagnosis in IBD distinguishing between CD and UC. With an increased number of patients and longer training phases, we expect a more accurate and robust classification. The combination of spatial and biochemical information encoded in the infrared images allows to track changes on the molecular level. Overall, this approach has the potential to become a widely applicable diagnostic tool for IBD and maintains intact tissue for further molecular analysis.

Funding: This work was founded by a synergy award of the Kenneth Rainin Foundation.


Generalisation of deep learning for breast cancer metastasis detection

S. Jarkman*, M. Karlberg, M. Pocevičiūtė, A. Bodén, P. Bándi, G. Litjens, C. Lundström, D. Treanor, J. van der Laak

*Department of Clinical Pathology, and Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden and Center for Medical Image Science and Visualization (CMIV), Linköping University, Sweden

Background & objectives: For clinical adoption of AI, models must be robust when applied to new settings. Objective of this study was to test the generalization potential of a pretrained deep learning (DL) model to unseen data and to a new diagnostic domain.

Methods: Whole slide images from Linköping, Sweden, with exhaustively annotated tumour regions, and publicly available CAMELYON data were used. Previously developed DL for breast cancer metastases detection in sentinel lymph nodes, developed using CAMELYON data, was used as baseline. The model was tested on sentinel nodes and lymph nodes from axillary dissections (n =51 and n=17, respectively; both Linköping).

Results: Base model showed decreased performance on Linköping data (AUC 0.929; 95%CI 0.800-0.998 and FROC 0.744; 95%CI 0.566-0.912), compared to the performance on CAMELYON data. A large FROC decrease was found for the base model applied to axillary nodes. The model was retrained, using both CAMELYON and Linköping WSI, resulting in increased performance for both sentinel nodes and axillary nodes (both AUC p<0.05). Pathologist qualitative evaluation of the outputs of the retrained model showed no missed positive slides and in 21 of 24 positive slides slide-level diagnoses matched the clinical ground truth. False positives and false negatives were observed. One previously undetected micrometastasis was identified as a result of using DL.

Conclusion: The study highlights the generalization challenge (even when using DL trained on multi-centre data), both as a result of applying DL in a new diagnostic setting for the initial indication, but even more remarkably, a slight change in indication impacted the model´s performance even more. Retraining the model, including data from target application, could mitigate the problem. Further studies are required to explore strategies to overcome the generalization challenge and evaluate what model performance is needed for different clinical applications.


The use of digital pathology and artificial intelligence in the assessment of multiple myeloma

M. McCabe, K. Sheehan*, S. Glavey

*Royal College of Surgeons, Ireland

Background & objectives: Plasma cell quantification in bone marrow trephines is an essential part of the diagnosis of multiple myeloma. Artificial intelligence (AI) is ideally suited for classification and quantification tasks but has not yet been employed in analysing the full myeloma microenvironment.

Methods: Twenty-two trephines from patients with myeloma were retrieved from the archives and whole slide imaging performed at 40x (Objective Imaging). Using a deep learning convolutional neural network (HALO-AI, Indica), the algorithm was trained to segregate and quantify bone marrow tissue and cellular phenotypes. In addition, spatial analysis was performed to assess the relationship of plasma cells within the marrow microenvironment.

Results: The trained classifier showed excellent segregation of marrow tissue elements as well as quantification of the different cell phenotypes on a Haematoxylin & Eosin stain. The mean number of plasma cells across the cases was 60,300 and mean density 1,637/mm2, occupying an average of 67% of the cell constituents. It was also noted that eosinophil numbers were increased (mean 4,900/trephine; 7% of all cells). Analysis of other marrow constituents demonstrated an inverse correlation between increasing plasma cell numbers and other cell types. Spatial analysis revealed a higher plasma cell density/mm2 with increasing distance from bone, although at a distance of 200uM, overall plasma cell numbers were reduced.

Conclusion: In this study, we have successfully applied AI to the classification of tissue types in multiple myeloma bone marrow biopsies. Using cellular phenotyping, it was possible to quantify plasma cells without immunohistochemistry, as well as other cell types in the marrow microenvironment in a more reproducible manner. This is a useful diagnostic adjunct for Pathologists and enables us to further study the relationship between the pattern of disease burden and overall prognostic indicators for patients with this disease.


Optimization of automated tissue classification in histopathological images: use of a deep transfer learning approach on a pancreatic cancer cohort

L. Haeberle*, R. Kronberg, M. Pfaus, H. Xu, K. Krings, M. Schlensog, T. Rau, A. Pandyra, K. Lang, I. Esposito, P. Lang

*Institute of Pathology, Heinrich Heine University & University Hospital Duesseldorf, Germany

Background & objectives: Neuronal networks (NNs) can assist with the analysis of digitalized histological slides. However, training of NNs can be hampered when training samples contain not one, but several tissue types. Our aim was overcome this problem by using deep transfer learning.

Methods: Image tiles were extracted from tissue microarrays with samples from 223 pancreatic cancer patients. Tiles contained pancreatic cancer, healthy pancreas, lymph nodes, but also confounders such as adiopse tissue. To purify the training data, we performed a data clean-up step using two communicating NNs (communicators). Subsequently, data was used to train NNs, which were then validated using an independent dataset.

Results: By feeding pre-existing datasets containing confounders such as adipose tissue as well as our own training datasets into two communicating NNs (communicators), we received a selection of unequivocal tissue tiles for NN training. A ResNet-18-based NN re-trained with these data achieved a higher weighted accuracy over all tissue classes (94%) than after training with raw data (90%). Additionally, we tested 72 NNs using an independent dataset created from H&E-stained whole-slide images. NNs were able to distinguish between pancreatic cancer, healthy pancreas, lymph nodes and adipose tissue following training with purified data. Performances varied and depended on various factors, such as the learning rate and the optimizers used.

Conclusion: Automated classification of histological tissue types in a pancreatic cancer cohort can be optimized by using communicator-driven data pre-processing. In the future, we aim to explore whether a similar approach can also be used to optimize other classification tasks, e.g., the distinction between pancreatic ductal adenocarcinoma and cholangiocellular carcinoma on digitalized H&E slides.


Inter-rater agreement of pathologists on determining cell-level PD-L1 status in non-small cell lung cancer

L. van Eekelen*, E. Munari, I. Girolami, A. Eccher, J. van der Laak, K. Grünberg, M. Looijen-Salamon, S. Vos, F. Ciompi

*Radboud University Medical Center, The Netherlands

Background & objectives: Artificial intelligence (AI) based quantification of cell-level PD-L1 status enables spatial analysis and allows reliable and reproducible assessment of the tumour proportion score. In this study, we assess the cell-level inter-pathologist agreement as human benchmark for AI development and validation.

Methods: Three pathologists manually annotated the centres of all nuclei within 53 regions of interest in 12 whole-slide images (40X magnification) of NSCLC cases and classified them as PD-L1 negative/positive tumour cells, PD-L1 positive immune cells or other cells. Agreement was quantified using F1 score analysis, with agreement defined as annotations less than 10 um apart and of the same class.

Results: An average of 9044 nuclei (1550 negative, 2367 positive tumour cells, 1244 positive immune cells, 3881 other cells) were manually annotated by the three pathologists. The mean F1 score over pairs of pathologists at dataset level was 0.59 (range 0.54-0.65). When split across classes, the mean per-pair F1 scores stay approximately the same, indicating the readers perform similarly regardless of cell type. Besides human variability in manual point annotations with respect to the centre of nuclei, lack of context contributed to disagreement: readers who reported they solely examined the ROIs tended to disagree more with readers that reported they also looked outside the ROIs for additional (morphological/density) information.

Conclusion: Agreement on determining the PD-L1 status of individual cells is only moderate, suggesting a role for AI. By quantifying the inter-rater agreement of pathologists, we have created a human benchmark which may serve as an upper bound (and could be combined via majority vote) for the validation of AI at cell level, something not done previously. Cell-level AI-based assessment of PD-L1 may supersede slide level scoring, adding significant information on the heterogeneity and spatial distribution over the tumour.

Funding: VIDI (F. Ciompi)


Nuclei detection with YOLOv5 in PD-L1 stained non-small cell lung cancer whole-slide images

L. van Eekelen*, E. Munari, L. Dulce Meesters, G. Silva de Souza, M. Demirel-Andishmand, D. Zegers, M. Looijen-Salamon, S. Vos, F. Ciompi

*Radboud University Medical Center, The Netherlands

Background & objectives: Nuclei detection in histopathology images is an important prerequisite step of downstream research and clinical analyses, such as counting cells and spatial interactions. In this study, we developed an AI-based nuclei detector using the YOLOv5 framework in whole-slide NSCLC cases.

Methods: Our dataset consisted of 42 PD-L1 stained cases (30 training, 12 test). Four trained (non-expert) readers manually annotated all nuclei (both positive/negative) within regions of interest (ROIs) viewed at 40X magnification. We trained a YOLOv5(s) network on annotations of one reader. Performance was measured using F1 score analysis; hits were defined as being less than 10 um away from annotations.

Results: We evaluate YOLOv5 on the test set by pairing it against all four readers separately. There, YOLOv5 performs excellently, falling within the interrater variability of the four readers: the mean F1 score over algorithm-reader pairs is 0.84 (range 0.76-0.92) while the mean F1 score over pairs of readers is 0.82 (range 0.76-0.86). When we determine the cell count (number of annotations/predictions) per ROI in the test set, agreement of algorithm-reader pairs and reader pairs is equally well aligned: 0.93 (range 0.90-0.97) versus 0.94 (range 0.92-0.96). Visual inspection indicates YOLOv5 performs equally well on PD-L1 positive and negative cells.

Conclusion: We have trained a nuclei detector that performs within the interrater variability of four human readers. In future work, we could extend this detector to additional tissues and immunohistochemistry stainings. Moreover, this detector could be used as a AI-assisted manual point annotation tool: while human readers perform the (context-driven) task of delineating homogeneous regions (e.g. clusters of PD-L1 positive stained cells), the detector performs the (local, yet laborious) task of identifying individual nuclei within these regions, providing labelled point annotations.

Funding: VIDI (F. Ciompi)


Automated annotation of digital H&E/SOX10 dual stains generates high-performing convolutional neural network for calculating tumour burden in H&E-stained cutaneous melanoma

P.S. Nielsen*, J.B. Georgsen, M.S. Vinding, L.R. Østergaard, T. Steiniche

*Dept. of Pathology, Aarhus University Hospital and Dept. of Clinical Medicine, Aarhus University, Denmark

Background & objectives: Deep learning for analysis of H&E stains requires a large annotated training set; a labor-intensive task that often involves highly skilled pathologists. We aim to develop and evaluate computer-assisted annotation based on digital dual stains of the same tissue section.

Methods: H&E stains of primary (n=48) and metastatic (n=48) melanoma were digitized, re-stained with SOX10, and re-scanned. Images were aligned, and automated annotations of SOX10 stains based on thresholding and a trained convolutional neural network (CNN) were thus directly transferred to H&E stains of the training set (n=37). Training of the final CNN for calculating tumour burden included 1,221,367 annotated nuclei.

Results: For primary melanomas, nuclei-annotation precision was 99.7% (95%CI=99.4%;99.9%) for tumour cells and 99.2% (95%CI=97.7%;99.7%) for normal cells. With a mean difference of 7.9% (95%CI=6.1%;9.7%), precision for normal cells was markedly reduced for metastases compared with primary melanomas (p<0.001). Associated false-positive annotations were predominantly related to SOX10-negative tumour cells. Correspondingly, mean SOX10 intensity (red chromaticity) was 0.37 (95%CI=0.35;0.39) for primary melanomas and subcutaneous metastases but 0.32 (95%CI=0.30;0.34) for lymph-node and organ metastases (p=0.002). Accuracy of trained CNN for calculating tumour burden in primary and subcutaneous lesions was 92.6% (95%CI=83.6%;96.8%). Compared with stereological counting, mean difference in tumour burden was 5.8% (95%CI=-1.2%;12.9%, p=0.10) for CNN and 16% (95%CI=3.7%;28.3%, p=0.02) for routine eyeballing.

Conclusion: With this annotation technique, a large annotated H&E training set with high quality was created within a reasonable timeframe for primary melanomas and subcutaneous metastases. For these lesion types, the training set generated a high-performing CNN for calculating tumour burden, which was superior to routine eyeballing. Yet, due to low or missing tumour-cell SOX10 positivity, advantages were limited in lymph-node and organ metastases. To include other cancer types or objects of interest, immunohistochemistry of the technique may easily be modified.

Funding: Health Research Foundation of Central Denmark Region


Collaborative web platform: lab organisation, research and digital pathology practice

V. Sousa*, R. Almeida, V. Almeida, M. Reis Silva, R. Jesus, L. Bastião Silva, D. Gonzalez, T. Adão, J. Carias, C. Costa, L. Carvalho

*Institute of Anatomical and Molecular Pathology, Faculty of Medicine of the University of Coimbra; CIMAGO – Research Center for Environment, Genetics and Oncobiology, Faculty of Medicine, University of Coimbra; University Hospital Anatomical Pathology Coimbra, Portugal

Background & objectives: Digital pathology has become increasingly important in Pathology laboratory practice. The combination of whole-slide-based imaging techniques and machine learning has been greatly applied to develop visual analytics tools, with great contribute to a more accurate and time-effective practice.

Methods: A consortium between BMDSoftware, Computer Graphics Center and Institute of Anatomical Pathology and Molecular Pathology - Faculty of Medicine/University of Coimbra, is developing a collaborative Web platform for digital pathology. It is a three years project that began in January of 2020 and joins the efforts of pathologists, computer science researchers and software developers.

Results: The result is a cloud-based platform known as iPATH. It allows the visualization of whole-slide images, easy navigation through the images, annotations, delimitation of areas of interest, and a wide range of measurements. It aims the informatization of laboratory routine, tracking of all steps, from the reception of samples to the final pathologic report, including the management of response time. The platform allows the management of the datasets annotation process for production but also for supporting the development and integration of artificial intelligence tools. Currently, Helicobacter pylori identification and quantification instrument to apply to gastric biopsies and mitoses identification in different neoplasms are the challenges being considered as case-study.

Conclusion: Digital pathology is already a reality in many Pathology labs across the world, perhaps driven by Covid-19 pandemic crisis, as it allows remote work. We believe that digital pathology is just the beginning and a platform for the creation of decision aid tools through artificial intelligence and deep learning technologies, that will improve the speed and quality of diagnosis processes.

iPATH is a multidisciplinary project aiming to create useful solutions for pathologists and Pathology labs.


Upconversion nanoparticles as labels for histopathological tissue evaluation

T. Nilsson*, K. Krawczyk, M. Mickert, S. Andersson-Engels

*Lumito, Sweden

Background & objectives: H&E staining and DAB-labelling are the gold standard in pathology, suffer from narrow dynamic range, difficulties in quantification and limited possibilities regarding multiplexing. We present an upconversion-nanoparticle (UCNP)-based technique that allows to overcome problems associated with commonly used labelling techniques.

Methods: Formalin-fixed paraffin-embedded breast cancer cell line and human breast cancer tissue were sectioned and labelled. Upconversion imaging of the human tissue sections was conducted in our prototype device and compared with a standard DAB-based IHC. The combination of UCNP and haematoxylin counterstaining on the same slide was investigated.

Results: Images obtained with our novel device demonstrate that our UCNP bioconjugates are excellent labels for the detection of cancer markers in tissue sections. Brightfield images prove that UCNPs do not interfere with the standard tissue evaluation by a pathologist. Additionally, brightfield and luminescent images can be merged to provide a better understanding of tissue morphology.

Conclusion: The emerging field of UCNP-based labelling techniques provides new possibilities for more accurate diagnosis. Staining solutions and a novel device developed by us keep the advantage of H&E staining and combine it, in one image, with the UCNP luminescent data. The high-contrast images of the UCNP labelling – generated by our scanning device – set the foundation for generating ground truth for machine learning algorithms.


Computer-assisted diagnosis of early-stage lung adenocarcinoma using deep learning

T. Trandafir*, J. Wolf, F. Akram, Y. Li, A. Dingemans, A. Stubbs, J. von der Thüsen

*Department of Pathology and Clinical Bioinformatics, Erasmus MC, Rotterdam, The Netherlands

Background & objectives: Early-stage lung adenocarcinoma growth patterns strongly associate with disease progression. Tumour biopsies are subtyped regarding microenvironment alterations and growth patterns: lepidic, acinar, papillary, micropapillary, and solid. We developed a deep learning (DL) pipeline to sub-classify adenocarcinomas to improve pre-operative assessments.

Methods: We developed a multi-class DL classification model for the prognostically relevant patterns. A retrospective cohort of 129 whole-slide images of needle-biopsy sections of stage I and II lung adenocarcinomas stained with haematoxylin and eosin and their corresponding annotations for the regions of interest were used to train and validate our DL classification models.

Results: In preliminary experiments, we designed a three-class DL model to classify normal tissue, tumour area of combined growth patterns, and tumour microenvironment. Compared to the ground truth, we reported a high overall accuracy of 0.86. Next, we designed a nine-class DL model to individually classify all patterns, yielding an overall accuracy of 0.77 and a Dice similarity coefficient of 0.66. We believe the results are influenced by class imbalance, introduced by dominant normal tissue, stroma, lepidic and solid patterns. Furthermore, high intra-class variability and inter-class similarity might have also influenced the results. For example, acinar patterns cover heterogeneous morphologies ranging from glandular to cribriform patterns, that may resemble lepidic patterns.

Conclusion: Our results indicate the potential aggressiveness of early-stage lung adenocarcinomas from small biopsy samples by sub-classifying growth patterns using DL. As we have a limited cohort, further training of the model on enriched datasets is required. This analysis can guide the extent of the surgical approach to maximise the preservation of healthy adjacent tissue and increase the patient’s quality of life. However, biopsies might misclassify the dominant growth pattern due to sampling error.


A novel machine learning pipeline to analyse unstained liver biopsies and automatically quantify tumour-related structures

R. Scodellaro*, D. Panzeri, E. Pagani, L. D'Alfonso, M. Bouzin, M. Collini, G. Chirico, D. Inverso, L. Sironi

*Department of Physics "G.Occhialini", University of Milano-Bicocca, Italy

Background & objectives: Tumour diagnosis is usually performed through the visual inspection of stained biopsies. Here, we propose a pipeline to support the pathologists’ clinical routine: it avoids staining procedures and provides novel quantitative insights to improve the diagnosis accuracy.

Methods: Images of unstained liver biopsies, acquired by a whole-slide scanner, are virtually H&E-stained through a convolutional neural network (CNN). Relevant biological structures (e.g. dead hepatocytes, cell nuclei, collagen) and tissue dis-architecture (e.g. steatosis) are retrieved by exploiting semantic segmentation and texture analysis procedures, coupling the phasor approach with clustering and semi-supervised machine learning techniques.

Results: The pipeline accuracy has been evaluated on 20 liver murine biopsies (10 affected by hepatocarcinoma and 10 healthy controls) by comparing the algorithm output with the pathologist’s quantification. In each biopsy, the amount of dead hepatocytes, cell nuclei and the extension of steatosis-affected regions have been automatically provided, with a mean accuracy > 95%. Moreover, the CNN performance in the virtual staining procedure has been evaluated for H&E samples. 2000 real and virtually stained tissue patches have been pixelwise compared, resulting in a colour content discrepancy < 5%.

Conclusion: These preliminary results demonstrate the capability of the proposed pipeline to extract quantitative and objective information, expanding the tumour feature dictionary, assisting pathologists with a more accurate diagnosis and overcoming the limitations due to inter-observer variability. Moreover, time and resources consumptions due to staining procedures are avoided by the H&E virtual colouring provided by the CNN in the images pre-processing steps.


Segmentation of anthracosis – a needed first step in digital analysis of lung tissue

P. Zens*, E. Baumann, A. Janowczyk, M. Kreutzfeldt, D. Merkler, S. Berezowska

*Institute of Pathology, University of Bern, Switzerland

Background & objectives: Anthracosis, the black granular pigment in lung tissue, is often wrongfully detected as nuclei by image analysis algorithms, leading to high false positive rates in immunohistochemical slides. Here, deep learning models for preemptive removal of anthracosis were evaluated.

Methods: From n=8 CD8 stained whole slide images, 128 tiles (256x256 px at 0.2431 μm/px) were manually selected and annotated to reflect expected lung tissue heterogeneity. Tiles were used in 4-fold validation to comparatively evaluate a traditional U-Net vs. Xception-based U-Net model (31e6 vs 2e6 parameters). For image augmentation, we focused on colour augmentation. Model performance was assessed by Dice score.

Results: The traditional U-Net outperformed the Xception-based model (0.78 ± 0.22 vs. 0.74 ± 0.22), with its unaugmented version performing the best (0.85 ± 0.16). Qualitative assessment showed that the models were more precisely segmenting individual granules versus the coarse annotations provided in the ground truth, suggesting superior performance over those suggested by quantitative metrics. Faint anthracotic pigments on darker background (condensate macrophages) and intensively stained CD8-postitve lymphocytes were common sources of error, with the dark combined hematoxylin and DAB at the nuclear membranes being detected as false positive anthracosis.

Conclusion: We show that simple U-Net-based models are powerful tools for localization of anthracosis. These models should likely be included in image analysis pipelines to help eliminate biologically irrelevant artifacts, thus improving specificity of downstream analyses. Augmentation methods did not appear to improve the model in identifying potentially relevant morphological features, suggesting that colour is insufficiently discriminatory in many instances. Next steps will include the refinement of our anthracosis model, including more targeted augmentation methods, and combination with nuclei segmentation.

Funding: MD-PhD scholarship 5088-06-2020 of Cancer Research Switzerland


Deep learning neural networks for real-time discrimination between osteosarcoma and fracture callus on conventional histological (H&E) sections

S. Stavropoulos, K. Gourdoupis, S. Georgopoulos, V. Plagianakos, D. Papachristou*

*University of Patras, School of Medicine, Unit of Bone and Soft Tissue, Patras, Greece

Background & objectives: Histopathological discrimination between osteosarcomas and fractures can be extremely challenging. Herein, we aimed at developing a novel Machine Learning architecture (Convolutional Neural Network) for real-time discrimination between osteosarcoma and fracture callus based on conventional H&E sections under brightfield microscopy.

Methods: We collected 2136 H&E images from 1154 osteosarcomas and 982 fractures from our archives (n=925) and the web. With the data collected, we trained a Convolutional Neural Network (CNN) based on the mobilenet version 3 architecture. Training set included 2021 images (1086 osteosarcomas, 935 fractures), and test set 115 images (68 osteosarcomas, 47 fractures) from new/unknown to the system cases.

Results: We created a web application that allows microscopes to directly connect to either desktop/laptop computers or mobile phones, in order to facilitate easy access to the Neural Network classifier.

The proposed system is simple to use and delivers real-time high accuracy classification regarding the separation of osteosarcoma from fracture, with sensitivity 91.3% and specificity 93.75%. Four (4) osteosarcomas were falsely classified as fractures; among them 2 were postchemotherapy osteosarcoma cases with 100% necrosis. Two (2) fractures were falsely classified as osteosarcomas.

Conclusion: CNN trained on conventional H&E sections can significantly decrease the pathologist’s workload and serve as an additional tool towards accurate diagnosis of several pathologies in everyday-routine practice. Since this tool is based on conventional H&E images and not on WSI, it can be easily used by pathologists from remote areas and small hospitals for the diagnosis of osteosarcomas, but also for virtually every pathologic condition, after specific training.


Novel approach for adaptive colour normalisation based on tissue thickness and biological tissue type variability

J. Jain*, P. Perugupalli, R. Gupta, D. Dodle, S. Krishna, V. Rao

*Pramana, Cambridge, MA, USA/India

Background & objectives: Colour variation in H&E and IHC slides poses a huge challenge for computational pathology algorithms. We present a novel method for colour normalisation based on tissue thickness and biological tissue variability to achieve significantly improved performance over existing methods.

Methods: 30 H&E slides of endometrium tissue were scanned using Pramana WSI scanner (inline analysis of tissue thickness and colour) and data was collected for colour and tissue thickness variation using colour distribution map and tissue thickness graphs. Colour normalisation was done based on tissue thickness, biological tissue variability and colour differences between thick and thin tissue areas in the slide.

Results: Visual evaluation by histopathologist revealed that our method yielded better results in comparison to existing methods. Our method does selective normalisation in the AOI as per the requirement after consideration of vectors for tissue thickness, biological tissue variability and colour distribution of the target AOIs instead of adopting the generic approach uniformly across the slide like existing methods. Reference pairing based on close matching of the colour distribution range of different colour in the target AOI to the reference AOI has improved the performance when biological tissue variability vector was also considered. Selective application of this approach based on tissue type and thickness reduces the over and under colour saturation.

Conclusion: Inline analysis of tissue thickness variation and colour hue distribution for all the AOIs in a WSI can help in achieving better colour normalisation when biological tissue variability is taken into consideration. Target to Reference AOI pairing done on the basis of biological tissue type and colour distribution graph allows for selective colour normalisation in the target AOI if needed. Both tissue type and thickness variation across the AOIs determine the need of selective colour normalisation for optimal results.


Digital image analyses applied to HSIL cervical biopsies

A. Alves*, V. Almeida, R. Almeida, M. Reis Silva, V. Sousa, L. Carvalho

*Centro Hospitalar e Universitário, Portugal

Background & objectives: Pathology has been integrating technology into its workflow and there are several algorithms we can runout to generate reliable information.

We aimed to determine if nuclear features of cervical HSIL differ among patients infected with distinct HPV genotypes.

Methods: We randomly selected 57 HSIL (CIN II and CIN III) cervical biopsies with previous HPV genotypification and scanned one representative slide of each. We then used a built-in algorithm on Aperio ImageScope software [] to estimate the average nuclear size and the nuclear chromasia of cervical epithelium with HSIL and used statistical software to compare the data.

Results: Our sample comprised 18 cases of HPV16, 26 cases of HPV-HR and 13 cases of HPV16 and HPV-HR coinfection.

The presence of HPV16 genotype, alone or together with HPV-HR, was associated with CIN III (chi-squared = 5,28; p = 0,02).

When comparing the HPV16 group to coinfected 16+HR, we found that HPV16 alone had significantly larger nuclei than the coinfection group (t-test = 2,3; p = 0,03). However, these groups showed no difference regarding the nuclear chromasia.

Conclusion: Digital image analysis applied to pathology seems a promisor field since it can provide us with essential information about our specimen that human eye simply can´t measure. In our study, the stronger association was between HPV 16 and CIN III but the clinical value of that is uncertain and may need a larger study. The same applies to the fact that HPV16 specimens have larger nuclei than coinfected 16+HR.


Using a digital platform to organize and manage pathology residents curricula in Portugal

F. Ramalhosa*, F. Pereira, C. Alves-Vale, D. Argyropoulou, C. Dahlstedt-Ferreira, C. Padrão, D. João, A. Lozada, G. Gerardo, J. Vaz da Silva, A.M. Gonçalves Pereira, R. Coelho, J. Gancho Figueiredo, R. Veiga

*Serviço de Anatomia Patológica, Centro Hospitalar e Universitário de Coimbra, Portugal

Background & objectives: In Portugal, for yearly and national board exams (NBE),a formal curriculum must be written down. In order to organize the registry of routine cases signed out during residency, a free internet platform dedicated to curricula organization and management (XERPA-MD) has been working together with Portuguese residents.

Methods: Members of the residents' committee of the Portuguese Society of Pathologists (NiSPAP) and an independent pathologist (coordinator of Anatomic Pathology for XERPA-MD) met regularly over a period of 12 months. A database, with neoplastic and non-neoplastic entities, was built according to the most recent editions of the WHO Classification of Tumours books, as well as other well-known bibliographic references in the field of surgical pathology.

Results: The database follows the Portuguese College of Pathologists curricular guidelines. After three rounds of work (data input, data correction, and data review), a total of 5500 entities were organized. Users are now able to access the database online and to register all their curricular activities in a structured and systematic way. All inserted records can be reviewed, organized, managed, and exported automatically, saving several hours of work.

Conclusion: Pathology residency is a critical period in the lives of all pathologists worldwide, with lots of study, routine cases and diverse scientific activities to be performed. The formal curriculum must include, among several other extensive registries, a thorough and organized list of all routine cases signed out during residency. Quite understandably, this becomes a daunting, exhaustive and stressful task, to be done every year, until the NBE. Automatization of curricula creation and management greatly decreases the time spent by Portuguese Pathologists writing down and organizing data.


AI(H): deep learning model for standing and grading autoimmune hepatitis from histology

C. Ercan*, K. Kordy, A. Knuuttila, X. Zhou, D. Kumar, P. Mesenbrink, S. Eppenberger-Castori, L.M. Terracciano, M.C. Pedrosa

*Institute of Pathology and Medical Genetics, University Hospital Basel, University of Basel, Switzerland

Background & objectives: Autoimmune Hepatitis(AIH) is one of the most challenging diagnoses in liver pathology. We aim to develop a deep learning model, Artificial Intelligence for Hepatitis[AI(H)] that evaluates liver biopsies, to provide granular, quantifiable, and rapid analysis of histological features of AIH.

Methods: One hundred twenty-five pretreatment liver biopsies with AIH diagnosis from the biobank of the University Hospital Basel were selected and split into training (80%) and test (20%) datasets and utilised to train several convolutional neural network models in the Aiforia platform. Manuel annotations of target regions were created by a hepato-pathologist, and used to train and test AI models.

Results: The liver microstructure detection model was trained to segment liver tissue into portal or, lobular areas and central vein compartments, while the necroinflammation model was trained for focal necrosis, interface hepatitis, or confluent necrosis. The immune cell classification model can detect, classify, and quantify lymphocytes, plasma cells, macrophages, eosinophils, and neutrophils. The bile duct model was trained for detecting the damaged bile duct.

The four AI models are accurate and efficient in diagnosis of various morphological components of AIH biopsies. When evaluated on a separate test set(ratios of correct predictions) of 92.9%, 97.1%, 84.5%, and 99.5% on liver microstructures, necroinflammation features, immune cell classification and bile duct damage detection, respectively.

Conclusion: AI(H) is a novel diagnostic tool for AIH histology. It demonstrates comparable results to manual hepato-pathologist assessment for several specific diagnostic tasks on AIH biopsies and classifies cell/tissue types a much shorter time. AI(H) is an intelligent, fast, accurate, and efficient diagnostic tool. Further planned development of AI(H) will allow for more functionalities such as portal and lobular necroinflammation, specific inflammatory cells, fibrosis, and bile duct damage.

Funding: Study was partially sponsored by Novartis.​


An assisting deep learning tool for accurate detection of colorectal cancer lymph node metastasis

A. Khan*, N. Brouwer, F. Müller, H. Dawson, J. Thiran, I. Nagtegaal, A. Blank, A. Perren, A. Lugli, I. Zlobec

*Institute of Pathology, University of Bern, Switzerland

Background & objectives: Histopathological evaluation of lymph node metastasis (LNM) for colorectal cancer (CRC) patients can be laborious and time-consuming. We propose an assisting deep learning method for CRC-LNM detection by leveraging transfer learning with an ensemble model on hematoxylin and eosin slides.

Methods: The proposed deep learning method consists of an LN segmentation (UNet) and an ensemble (Xception, Vision Transformer) metastasis detection models. LN segmentation model was trained on one hundred annotated CRC slides. An ensemble metastasis detection model was trained first on the public breast cancer dataset, PatchCamelyon and then fine-tuned on CRC data.

Results: The proposed method was validated on an internal and external CRC cohort by analysing AUC, sensitivity, and specificity on a whole slide level. The method achieved an AUC of 98.1% in the internal validation cohort (2803 slides) with a sensitivity of 99.5% and specificity of 96.7%. Around 0.5% of positive slides were incorrectly classified as negative due to small isolated tumour cell clusters (<100um). About 3.3% of negative slides were falsely detected as positive, mainly due to tissue folds and active germinal centres. In the external validation cohort (1033 slides), the method correctly classified 100% of all slides.

Conclusion: The deep learning method developed in this study showed excellent performance, making it suitable as an assisting tool for CRC-LNM screening. Future studies should include other histological subtypes (mucinous adenocarcinomas, signet ring cell carcinomas), investigate the application for other solid tumour types next to breast and CRC, as well as improve sensitivity for more challenging cases (isolated tumour cell clusters). The specificity could be improved by detecting and excluding tissue folds and germinal centres before applying the metastasis detection model.

Funding: Rising Tide Foundation for Clinical Research (CCR-18-295800) and Swiss Cancer Research Foundation (KFS-4427-02-2018)


The creation of a virtual pathology department: a novel solution to a modern crisis

R. Chetty*, J. Fitzgerald, M. Colleluori, D. O'Shea

*DECIPHEX, Ireland

Background & objectives: There are several well-known global challenges confronting Pathology that translate into poor TATs, lack of expertise/access and ultimately poor patient care. Our aim was to alleviate these pressure points to improve patient management.

Methods: A multitude of challenges are currently faced in Pathology resourcing. To this end, we developed an AI-empowered digital pathology system using a proprietary, customizable, scanner agnostic, cloud-based, LIS integratable, state-of-the-art pathology PACS. We then recruited and validated a team of recognized sub-specialists to constitute the world’s first CQC/CLIA accredited virtual pathology department.

Results: Across our pathologist network we currently have 56 pathologists in funnel across 12 subspecialities. The team is geographically distributed across Europe, UK, Canada and the US. We have fully validated our pathologists according to RCPath/CAP guidelines and have shown over 97% concordance for glass versus digital cases. Only minor discordances occurred, and a discordance remediation process was put in place whereby blind review of the discordant case was performed again and diagnostic accuracy recorded. We are currently producing successful turnaround times of <2 days in pilot trials. Pathologist efficiency has improved with the use of measuring and mitoses counting tools, including Ki-67 quantification.

Conclusion: Overall Client hospital satisfaction is highly evident with TAT decreased, no mailing of slides required, MDT ready reports not being re-typed, and going into LIS directly, saving both time and overheads. Pathologist satisfaction, quality control and efficiency are evaluated for this disruptive workflow, with net promoter score systems and tools employed to ensure optimal pathologist experience and high-quality reporting achieved.

PS-04 | Poster Session Nephropathology


Morphometric analysis of lysosomes in the renal tubule in monoclonal gammopathy using transmission electron microscopy: ‘mottled appearance’ and beyond

M. Jung*, H. Lee, K.C. Moon

*Department of Pathology, Yonsei University College of Medicine, Republic of Korea

Background & objectives: Lysosomal ‘mottled appearance’, or uneven electron-dense contents related to monoclonal gammopathy (MG), has been described in light chain proximal tubulopathy (LCPT). We aimed to determine the ultrastructural characteristics of lysosomal mottled appearance in kidney biopsies and its association with LCPT.

Methods: Seventy-seven biopsies were grouped into LCPT (n = 5), MG conditions other than LCPT (n = 43), and non-MG conditions (n = 29). The mottled lysosomes in the renal tubules were evaluated using transmission electron microscopy and morphometric analysis.

Results: Mottled lysosomes were more prevalent (% of present cases) and frequent (no. of mottled lysosomes/20,000x ultramicroscopic field) in the LCPT group (100% and 8.20 ± 4.15/field) than in the MG (41.9% and 1.13 ± 2.05/field) and non-MG (37.9% and 0.80 ± 1.44/field) groups. In morphometric analysis of all mottled lysosomes (n = 520) detected from the 34 biopsies (5 LCPT, 18 MG, and 11 non-MG), we found that mottled lysosomes were larger, more irregular, and more electron-dense for the LCPT group than for the MG and non-MG groups.

Conclusion: Mottled lysosomes can be present in disorders other than LCPT or even without MG. The morphological characteristics of mottled lysosomes could provide objective guidance for the diagnosis of LCPT.


Gene expression characteristics of T-cell-mediated alloimmune response in HIV-infected kidney transplant recipients

D. Dobi*, A. Zarinsefat, Y. Kelly, P. Stock, Z. Laszik

*Semmelweis University, Hungary

Background & objectives: The graft survival of HIV-infected renal transplant recipients are comparable to that of non-HIV infected referents. However, the frequency of first-year T-cell-mediated rejection (TCMR) in the former group is higher. We assessed the molecular characteristics of TCMR in HIV-infected individuals.

Methods: We studied formalin-fixed paraffin-embedded (FFPE) renal biopsy samples from 68 transplant recipients (34 HIV+, 34 HIV-). The diagnostic groups were normal, borderline changes, and TCMR. We applied gene expression analysis on the FFPE material by using a panel of 760 targets that included immune-response-related and HIV genes. We performed differential gene expression (DE) analysis and pathway analysis (PA) (reactome database).

Results: DE analysis revealed multiple genes with significantly increased expression in the diagnostic groups of HIV+ borderline changes and HIV+ TCMR relative to their HIV- counterparts. PA of these genes showed gene enrichment in the following pathways: toll-like receptor cascades, MYD88:MAL cascade, cytosolic sensors of pathogen-associated DNA, and NLR-signalling among others. HIV genes were not found to be present in the biopsy material of HIV-infected patients.

Conclusion: Upregulation of the innate immune pathways in the biopsies of HIV+ patients with borderline changes and TCMR may indicate the enhanced involvement of natural immunity during T-cell-mediated alloimmune response in this patient group. This can potentially stem from immune dysregulation caused by HIV infection.


Chief renal medullary osmolytes NaCl and urea differentially modulate tubular cellcytokine expression and monocyte recruitment

J. Schmitz*, N. Brauns, A.M. Hüsing, M. Flechsig, T. Lepletier Glomb, H. Haller, J.H. Bräsen, S. von Vietinghoff

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

Background & objectives: Extreme electrolyte concentrations characterize the renal medulla. Renal immune cells are sentinels against ascending bacteria but also promote detrimental inflammation. Here, we investigated how the renal main osmolytes, NaCl and urea, regulate tubular cell cytokine expression and monocyte chemotaxis.

Methods: Normal kidneys, transplant surveillance and minimal change biopsies were stained for macrophages, monocytes and cytokines using immunofluorescence and RNA in situ hybridization. Tissue cytokine concentrations were measured with ELISA. Clinical data, immunosuppressant and diuretic medication were extracted from the records. Human renal tubular cells (HK2) were exposed to NaCl, urea or mannitol. Gene expression was assessed by gene array analysis.

Results: In the healthy human kidney, more monocytes were detected in medulla than cortex. The monocyte gradient was attenuated in patients with medullary NaCl depletion by loop diuretic therapy and in nephrotic syndrome. Renal tubular epithelial cell gene expression responded similarly to NaCl and tonicity control mannitol, but not urea. NaCl significantly upregulated chemotactic cytokines, e.g., CCL2 and CSF1. This induction was inhibited by ROS scavenger n-acetylcysteine. In contrast urea, the main medullary osmolyte in catabolism, dampened tubular epithelial cytokine expression. NaCl-, but not urea stimulated tubular epithelium or cytokine combinations promoted human classical monocyte migration. Consistently, gene array data revealed renal medullary chemokine and monocyte marker decrease in catabolic mice.

Conclusion: Our results depict two different renal medullary scenarios depending on whether NaCl or urea is the main osmolyte: The energy intense state with tubular cell cytokine production and recruited myeloid cells in the presence of elevated NaCl may aid antibacterial host response. Less cytokine production and stable myeloid cell populations in presence of elevated urea concentrations may benefit organisms with limited energy supply which may also limit detrimental inflammation. This could be a basis for additional management strategies of patients.

Funding: NB was supported by a Hannover Biomedical Research School stipend, JS and JHB by Jackstädt Stiftung and German Ministry for Education and Research (BMBF 13GW0399B), and SvV by grants from MHH HilF2019 and MHH Transplant Centre and Deutsche Forschungsgemeinschaft (450775971 and EXC2151 – 390873048).


Adverse renal effects of immune checkpoint inhibitors: presentation of 12 patients undergoing renal biopsy

K. Palamaris, A. Stofas*, D. Alexandris, I. Giatras, A. Patereli, H. Theodoropoulou, A. Koutsovasili, C. Kaitatzoglou, E. Psimenou, K. Stylianou, A. Gerakis, N. Alevizopoulos, S. Theocharis, E. Kastritis, H. Gakiopoulou

*1st Department of Pathology, School of Medicine, National and Kapodistrian University of Athens, Greece

Background & objectives: The immunomodulatory activity of immune checkpoint inhibitors (ICIs) often elicits a broad spectrum of immune-related adverse effects (IRAEs), in multiple tissues, including kidney. We present a case series of renal IRAEs with various clinical and histological manifestations.

Methods: Twelve patients, bearing a wide range of solid malignancies, received either PDL-1, or a combination of PDL-1 and CTLA-4 inhibitors. Following ICIs administration, clinical signs indicative of renal toxicity included acute kidney injury (AKI), proteinuria, nephrotic syndrome and/or haematuria. All patients underwent renal biopsy, which was processed for light microscopy, immunofluorescence, and when tissue sufficed, electron microscopy.

Results: The most frequent clinical presentation was AKI and the most frequent pathologic alteration was tubulointerstitial nephritis (TIN) encountered in six cases. In one of these cases, TIN was accompanied by IgA glomerulonephritis. Two patients, presenting with nephrotic syndrome, exhibited a secondary “lupus-like” membranous glomerulopathy. In one of the latter patients, there was TIN as well. Among the remaining three patients with AKI, two displayed acute tubular injury as the most prominent finding, while in the third, a combination of membranoproliferative glomerulonephritis and thrombotic microangiopathy was identified. The last patient developed nephrotic syndrome and a secondary renal amyloidosis was detected, on a rheumatoid arthritis background presenting after the initiation of ICIs.

Conclusion: Our findings harmonize with bibliographical data that identify TIN as the most frequent histological lesion related to ICIs administration. The preferential involvement of tubulointerstitial tissue could be associated with the higher expression levels of PD-1 on tubular epithelial cells, compared to glomeruli. On the other hand, both secondary “lupus-like” membranous glomerulopathy and secondary amyloidosis upon rheumatoid arthritis, are postulated to emerge as a consequence of a systemic immune system reconstruction, induced by immune-checkpoints inhibition.


Compensatory and regenerative potential in kidneys of newborns from mothers with complicated pregnancy by preeclampsia and iron deficiency anaemia

M. Myroshnychenko*, N. Kapustnyk, A. Shapkin, T. Moiseienko, I. Torianyk, V. Ivannik, T. Chastii, I. Mozhaiev

*Kharkiv National Medical University, Ukraine

Background & objectives: Preeclampsia (PE) and iron deficiency anaemia (IDA) are pregnancy complications that have a negative effect on women health, their offspring. The objective was to reveal the compensatory and regenerative potential in newborns kidneys that developed under maternal PE, IDA conditions.

Methods: The study material was the tissue of kidneys of newborns from mothers with physiological pregnancy (n=28) (group (G) 1); complicated pregnancy by PE of varying degrees of severity (n=78) (G 2), IDA of varying degrees of severity (n=85) (G 3). Histological, immunohistochemical, morphometrical, statistical methods were used.

Results: In newborns kidneys of G 2-3 it was revealed a deficiency of nephrons with the presence of alterative changes in them, hypertrophy of glomeruli with hyperplasia of capillary loops mainly in G 3. Proliferative activity of nephrons structural elements increased in G 2 (Ki-67 proliferative index (PI) – (21.3±2.1)%), G 3 (Ki-67 PI – (38.9±2.7)%) compared with G 1 (Ki-67 PI – (12.5±1.9)%), however, was more pronounced in G 3. In G 2 and especially G 3, there was compensatory angiogenesis activation, as evidenced by an increase in the number of vessels in stroma in these groups (G 2 – 8.3±1.2, G 3 – 11.4±2.1) compared to G 1 (5.6±0.9).

Conclusion: In kidneys of newborns from mothers whose pregnancy was complicated by PE and IDA, compensatory and regenerative processes characterized by hypertrophy of glomeruli with hyperplasia of capillary loops, activation of proliferative potential of nephrons cells, angiogenesis activation. The latter were more pronounced in kidneys of newborns that developed under maternal IDA conditions. The data obtained by the authors indicate a more pronounced damaging effect on the newborns kidneys of maternal PE compared to IDA.


Renal amyloid deposition limited to glomeruli in caveolin-1 knockout mice

G. Terinte-Balcan*, D. Marta, M. Gherghiceanu

*"Victor Babes" National Institute of Pathology, Romania

Background & objectives: Caveolae are invaginations of the plasma membrane involved in many different disease processes, such as cardiovascular diseases, infections, diabetes, drug sensitivity and cancer. The objective of this study was to assess the renal pathological abnormalities in cav-1 knockout ageing mice.

Methods: Renal tissue from 6 and 18 month-old cav-1 knockout mice was processed for paraffin and plastic embedding in order to be examined using light microscopy (LM) and electron microscopy (EM). Slides with paraffin embedded tissue were processed for immunofluorescence microscopy (IF) and examined using the following markers: IgA, IgM, IgG, C3, C1q, fibrin, kappa and lambda light chains.

Results: LM showed normal structure of kidney from 6 month-old mice and large amorphous deposits located only in the glomeruli from 18 month-old mice. There was no endocapillary hypercellularity, fibrinoid necrosis or crescents in the glomeruli. The tubulo-interstitial and vascular compartments of the kidney had a normal structure. IF was negative in the glomeruli and there were no extraglomerular deposits on any marker examined. EM showed non-branching, randomly arranged fibrils, with a diameter between and 7 and 12 nm located only in the glomeruli.

Based on the findings observed by LM, IF and EM, a diagnosis of amyloidosis limited to the glomeruli in the kidneys of aged mice was made.

Conclusion: There are several experiments in animal models and HEK cells that demonstrate a connection between cav-1 levels and amyloid precursor proteins. In humans, variations in the CAV1 gene have been reported in association with metabolic disorders and cardiovascular disease.

This study represents the first report of amyloid deposition in the kidneys of cav-1 knockout aged mice, further strengthening the evidence that cav-1 and caveolae are implicated in disease pathophysiology.


Clinical and pathohistological characteristics of COL4A3 c.2881+1G>A variant causing Alport spectrum disorders in Croatian population

M. Horaček*, P. Šenjug, S. Kuzmac, M. Šenjug Perica, D. Klarić, F. Paić, T. Nikuševa Martić, D. Galešić Ljubanović

*Institute of Pathology, School of Medicine, University of Zagreb, Croatia

Background & objectives: Alport syndrome (AS) and thin basement membrane nephropathy (TBMN) are kidney disorders caused by mutations in COL4A3, COL4A4, or COL4A5 genes that encode polypeptide chains of collagen IV, the major structural component of basement membranes.

Methods: We identified 13 patients from 12 unrelated families with a pathohistological diagnosis of AS or TBMN who tested positive for a heterozygous variant COL4A3 c.2881+1G>A on conducted next-generation sequencing (NGS). Subsequently, their family members were recruited for genetic counselling, urinalysis, and blood sampling for targeted NGS. A correlation of clinical and pathohistological data and genealogy study was also performed.

Results: Overall, 34 patients (58.8% male) were found positive for heterozygous, disease-causing variant COL4A3 c.2881+1G>A. Haematuria was present in 33 patients (97.1%), while 19 (55.9%) had proteinuria. Follow-up data showed that four more patients developed proteinuria (23 total; 67.6%) and 6 (17.6%) developed chronic kidney disease, started dialysis or underwent kidney transplantation by the median age of 51 years. There were 6 (17.6%) patients with hearing loss (3 confirmed with audiogram) and 4 (11.8%) with ocular lesions. Among 13 patients who underwent kidney biopsy, 12 had glomeruli available for electron microscopy. Five patients had classic AS morphology and 7 had TBMN (3 of them with focal lamellation).

Conclusion: The suspected founder variant COL4A3 c.2881+1G>A is disease-causing. There is variability among these patients not only in clinical presentation but also in pathohistological findings. Interestingly five out 12 heterozygous patients had classic AS morphology on kidney biopsy. It is essential to conduct a detailed analysis of each collagen IV variant to optimize the affected patients' prognostic and therapeutic approach.


Ursolic acid prevents the dysregulation in the expression of histone methylation-related epigenetic enzymes in diabetic kidney

S. Manea*, A. Lazar, M.L. Vlad, A. Manea

*Institute of Cellular Biology and Pathology “Nicolae Simionescu” of the Romanian Academy, Romania

Background & objectives: Histone methyltransferases (KMTs)/demethylases (KDMs) play a major role in the pathology of diabetic kidney disease (DKD). We aimed at investigating the potential role of ursolic acid (UA), a pentacyclic triterpenoid, to modulate the expression of archetypal KMTs/KDMs in diabetic kidney.

Methods: Non-diabetic and streptozotocin-induced diabetic C57BL/6J mice were randomized to receive via intraperitoneal injection 1 mg/kg UA, or its vehicle for 4 weeks. Human endothelial cells (EA.hy926) were exposed to normal (5 mM) or high (25 mM) concentrations of glucose in the absence/presence of UA (5 μM). Hematoxylin-eosin staining, fluorescence microscopy, real-time PCR and Western blot were employed.

Results: No significant changes in blood glucose levels and body weights were detected following UA administration to diabetic mice as compared with vehicle-treated diabetic animals. Glomerular hypertrophy and enhanced accumulation of extracellular matrix proteins were detected in diabetic kidney. The mRNA and protein levels of KMT (DOT1L, SETD7, EHMT1, EHMT2, EZH1, EZH2) and KDM (KDM1A, KDM2A, KDM3A, KDM4A, KDM5A, KDM5B) subtypes were found significantly elevated in the kidney of diabetic mice as compared with non-diabetic animals. Treatment of diabetic mice with UA suppressed the up-regulation of KMTs and KDMs. High glucose-induced increased expression of the archetypal KMT and KDM subtypes was significantly reduced by UA in cultured endothelial cells.

Conclusion: Selective triterpenic acids are generally acknowledged as potential medicines for the treatment of a wide range of human pathologies. In this study, we provide evidence that ursolic acid prevents the alterations in gene and protein expression levels of archetypal KMTs and KDMs in the kidney of diabetic mice. Ursolic acid or its pharmacologically active chemical derivates may become important therapeutic tools to prevent epigenetic instability and the ensuing gene expression and phenotypic alterations in DKD.

Funding: Work supported by UEFISCDI (PN-III-P4-ID-PCE-2020-1898, PN-III-P1-1.1-TE-2021-0180, PN-III-P2-2.1-PED-2019-2497, PN-III-P2-2.1-PED-2019-2512).


A multihierarchical terminology for non-neoplastic kidney biopsies: kidney biopsy codes for pathologists

A. Dendooven*, H. Weishaupt, K. Amann, H. Hopfer, R. Cornet, L. Gesualdo, S. Leh

*UZ Gent, Belgium

Background & objectives: A kidney biopsy is often the only option to correctly diagnose a kidney disease and to gain information about prognosis and possible treatment. However, an international standardized system for coding morphological findings and diagnoses has been missing.

Methods: An expert workshop defined the principles for the Kidney Biopsy Codes for Pathologists (KBC) system. Based on experience, literature review, and 60 nephropathology reports, a terminology with synonyms and parent-child relationships was established. Then, a project-internal review process and a second workshop were carried out. Several visualisations of the system have been developed using R/Shiny.

Results: The aim of the Kidney Biopsy Codes for Pathologists project (KBC, https://kibico.org/) is to establish a comprehensive coding system for non-neoplastic kidney biopsies. KBC currently consists of 576 active concepts, of which 168 belong to a compact and 408 to a detailed set of terms. The KBC structure is multi-hierarchical with a pattern of injury (276 concepts) and a disease concept axis (266 concepts) as well as attributes (43 concepts) including qualifiers for certainty (3 concepts). Concepts are further grouped according to kidney compartments. For each concept, a preferred term and synonyms have been defined.

Conclusion: A comprehensive coding system for non-neoplastic kidney diseases is established. In order to provide governance and to promote use within existing frameworks, the KBC team aims to collaborate with SNOMED international to make a subset in SNOMED CT. Finally, an international review process will be conducted.


Association of amyloid deposits with C4d immunohistochemical staining in kidney allografts on the onset of AA amyloidosis recurrence

S. Şen, C.A. Gomez Gonzalez*, A. Çeltik, G. Aşcı, B. Sarsık Kumbaracı, H. Töz

*Department of Pathology, Medical Faculty, Ege University, Izmir, Turkey

Background & objectives: Serum amyloid A protein-related AA amyloidosis can lead to ESRD,therefore renal transplantation.Surveillance of allografts with C4d immunohistochemical staining assesses humoral rejection.Immune complex-mediated diseases and amyloidosis describe positive staining.We analysed the C4d immunohistochemical staining pattern in allograft biopsies with recurrent amyloidosis.

Methods: This retrospective analysis included allograft biopsies performed in our centre that were previously positive for amyloid with congo red and C4d evaluation. Amyloidosis scoring and description of C4d immunohistochemical pattern staining (glomerular, vascular, interstitial and/or capillary peritubular) were evaluated, and C4d staining was correlated with congo red staining.

Results: 41 biopsies belonging to 22 patients were analysed. Among the indication of biopsy were graft dysfunction (n=9; 40,9%), non-nephrotic proteinuria (n=5; 22,7%) and nephrotic proteinuria (n=8; 36,3%). During the investigation 12 graft cases had preserved function, 6 cases had graft dysfunction and 4 patients died. Amyloid deposits were frequently founded in arteriolar vessel walls (N=12; 54,5%) and combined glomerular/vascular staining (n=6;45,4%). There was one biopsy with C4d insufficient staining, in 5 cases the amyloid deposits were small and the evaluation of C4d and congo red at the same time was necessary. In the other cases, C4d staining highlighted amyloid deposits within blood vessels and glomeruli.

Conclusion: Amyloid deposits in recurrent AA amyloidosis in kidney allografts show a distribution over blood vessel walls. This nonspecific finding at the walls of the arterioles is seen as well in arteriolar hyalinization, in contrast, glomeruli staining with arteriolar staining seems to be more specific for amyloidosis. C4d can be used as a marker for amyloid using mass spectrophotometry identifying C4d protein as part of the AA amyloid component.

PS-05 | Poster Session Ophthalmic Pathology


Conjunctival Melanoma in Ireland – a sixty year review

L. Coady*, C. Hegarty, K. Kulakova, S. Kennedy

*St. Vincent's University Hospital, Ireland

Background & objectives: Conjunctival melanoma is a rare ocular neoplasm with an unpredictable pathological course. Reported incidences vary from 0.1–0.9/1,000,000. Lack of population studies coupled with the rarity of the tumour has resulted in poor understanding of risk-factors and limited therapeutic options.

Methods: A retrospective review of all cases of conjunctival melanoma accessioned in the largest eye unit in Ireland over a 60 year period (1961 – 2021) was performed. The age, sex, eye laterality, size of tumour, development of metastasis and/or recurrence was determined. Genome sequencing was performed on a select number of samples from the cohort and any mutations found recorded.

Results: 72 cases of conjunctival melanoma were diagnosed since 1961. There was a female preponderance (n = 42, 58.3%). Median age of diagnosis was 77 (Range:32-91). Tumour size varied from 5–56mm in maximum dimension. Thirty-five (48.6%) have died. Time of death ranged from 1 month to 12 years post diagnosis.

28 (40%) developed metastases (brain, lung, liver, kidney, bowel, thyroid, prostate, parotid, lymph nodes), 20 (28.6%) developed recurrences. The left eye was more commonly affected (n=36, 50%).

14 of 70 specimens dating back to 1996 were sent for analysis with either Sequenom or Oncomine platforms. Mutations were detected in 12 patients. These included PIK3R1, M326I, PIK3CA, MET, N3755, BRAF and NRAS.

Conclusion: Conjunctival melanoma is a rare neoplasm with only 72 cases diagnosed in Ireland in the last 60 years. Recurrence and metastases are common.

Options for treatment of conjunctival melanoma include excision, cryotherapy, corneal epitheliectomy, radiotherapy and topical mitomycin C. Adjuvant therapy is limited, with conjunctival melanomas showing intermediate sensitivity to immunotherapy.

Extended follow up of patients will allow identification of risk factors for the disease, while further genetic sequencing will enable identification of potential therapeutic targets.


Uveal melanoma: ten years of experience and behaviour in the Principality of Asturias. A retrospective analysis

M. Berríos-Hernández*, M.d.l.P. González-Gutiérrez, H.E. Torres-Rivas, N. González-Ortega, L.M. Fernández Fernández, C. Fuente-Díaz, M. García-Martínez, V. Blanco-Lorenzo, B. Vivanco-Allende, C. González del Rey-Rodríguez, A. Encinas-Muñiz

*Hospital Universitario Central de Asturias, Spain

Background & objectives: Uveal melanoma (UM) is the most common primary intraocular neoplasm in adults, arising from melanocytes located in the iris (4%), ciliary body (6%), or choroid (90%). It represents 2 cases per million per year in Spain and Italy (Southern Europe).

Methods: We performed a descriptive and retrospective analysis for ten years (2011-2021) on patients diagnosed of UM from different public medical institutions from the Health Service of the Principality of Asturias (SESPA). The information was obtained through the Hospital Registry of Tumours in Asturias.

Results: Twenty-five patients were included in this study with a median age of 62-year-old. Predominant histologic subtype was epithelioid melanoma (28%), followed by spindle cells (24%) and mixed (24%), subtype was not available in 6 patients (24%). Most of them presented as local stage (64%) and underwent radical surgery (88%). Most cases affected the choroid (80%) and a smaller group the ciliary body (20%). We found no cases with iris involvement. A small percentage (8%) required radiotherapy and immunotherapy respectively. Four patients died with a mean of 4,43 years after diagnostic, three of them with metastatic disease. The other patients are being followed-up at the referral hospital.

Conclusion: In recent years, numerous advances have been made in genetics and behaviour of UM. However, despite these efforts, survival has not been improved and once metastatic disease progresses, the prognosis is poor and therapeutic options are very limited. The heterogeneity of the molecular pathways involved in this pathology has hindered the development of a specific drug for advanced disease. Therefore, more studies are needed to achieve this.


Retinoblastoma in Ireland; the next generation sequencing molecular profiles of selected enucleation cases with a special focus on non-RB1 mutations and a comprehensive review of the retinoblastoma caseload in Ireland over the past 20 years

J. Maguire*, A. Greene, M. Capra, L. Bradley, A. Green, C. Hartnett, S. Kennedy

*St Vincent’s University Hospital, Ireland

Background & objectives: Increasingly, retinoblastoma has been linked to somatic gene abnormalities beyond RB1, especially BCOR, a BCL-6 co-repressor gene. Our aim is to assess the molecular profile of selected retinoblastoma specimens and to review 20 years of retinoblastoma in the Irish population.

Methods: Using the hospital database of the national ophthalmic centre, all enucleation specimens for suspected retinoblastoma performed in Ireland over the past 20 years (2001 – 2020) were analysed under various headings, including age at surgery, sex, laterality, diagnosis, and histopathological features including tumour differentiation, extent and stage. 6 selected cases were sent for somatic molecular analysis using next generation sequencing.

Results: 65 enucleations were performed on 63 children. 61 specimens showed retinoblastoma. On average 3.2 surgeries were performed per year. 41% were female, 59% male. The average age at surgery was 2.72 years. 8% of the patients had undergone neoadjuvant therapy prior to the enucleation procedure. 75% of the tumours were moderately to poorly differentiated, 61% had optic nerve invasion and 5% had a positive optic nerve margin. 5 of the cases referred for molecular profiling had a somatic RB1 variant. The remaining case, a 3 year old patient with a left sided, high grade, poorly differentiated retinoblastoma was found to have a mutation in BCOR, with no associated RB1 mutation.

Conclusion: Our review shows that the rate of performance of enucleations for retinoblastoma in the Irish population over the past 20 years has remained stable. We identified a non-RB1 BCOR mutated retinoblastoma, associated with a high pathological grade. This supports the literature in highlighting BCOR as the most common non-RB1 gene to be mutated in retinoblastoma, and the importance of molecular profiling to identify non-RB1 somatic mutations and potentially higher grade, more aggressive tumour types to improve patient treatment.


Histopathologic findings of lens capsule and persistent hyperplastic primary vitreous of Korean juvenile cataract patients

Y.M. Kim*, C. Cho, J.Y. Kim, W.K. Kim, H.W. Kim, Y. Chun, W.S. Kim

*Haeundae Paik Hospital, Republic of Korea

Background & objectives: Persistent hyperplastic primary vitreous (PHPV) is a rare congenital anomaly in which the regression of the hyaloid vessel fails and primary vitreous persists after birth. The purpose of study was to determine the histologic features of congenital cataract and PHPV.

Methods: Histopathologic examination of a total of 142 lens capsules from a retrospective cohort study of 106 consecutive unilateral and bilateral Korean juvenile cataract patients was conducted. Retrolenticular membranes of the 12 PHPV patients were reviewed for histology and immunohistochemistry with antibodies of CD31, CD34, Von Willebrand factor, Cytokeratin, Vimentin and TGF-β1 were performed.

Results: 1) The frequency of PHPV in unilateral and bilateral Korean juvenile cataract patients was 11.3% (12/106). 2) Characteristic histologic features of PHPV, hypercellular membrane tissue consisting of either vascular structures and/or mesenchymal cells, were found in 75% (9/12) of cases at an age younger than 123 months. However, the hyaloid arteries and endothelium-lined blood vessels in the retrolenticular membranes were found only in 3 cases. 3) Six cases, including one case of bilateral PHPV, showed only mesenchymal cells. Three cases of clinically diagnosed PHPV did not show fibrovascular membranes by histology. 4) Endothelial cells of the vessels but not mesenchymal cells expressed TGF-β1 by immunohistochemistry.

Conclusion: 1) Not all cases of PHPV have vascular structures in the retrolenticular membrane tissues. 2) Mesenchymal cells with or without vascular structures are found in most of cases (80%), which suggests that the vascular mesenchymal transformation could be one of the possible mechanisms in the process of vascular regression of PHPV. 3) Mesenchymal transition of remnant foetal vascular structures in PHPV is independent of the patient’s age at the time of operation, or the size of retrolenticular membranes.

Funding: This work was supported by a grant from Research year of Inje University in 20150684.

PS-06 | Poster Session Paediatric and Perinatal Pathology


Paediatric soft tissue malignant tumours: a 16 year experience with literature review

A. Bchir, N. Abdessayed*, T. Tlili, Y. Fejji, B. Sriha, M. Mokni

*Department of pathology, Farhat Hached University Hospital, Sousse, Tunisia

Background & objectives: Paediatric soft tissue tumours are rare heterogeneous group. Although most tumours are benign, developing an appropriate diagnosis requires knowledge of clinical and radiologic characteristics. The objective of our study is highlight the clinico-pathologic characteristic of paediatric soft tissue malignant tumours.

Methods: A total of 25 cases of soft tissue malignant tumours diagnosed in children under 16 years at the Department of Pathology of Farhat Hached University Hospital in Sousse, over a period of 16 years (from 2005 to 2020). A review of clinical, paraclinical, pathological and evolutionary data was performed in all cases.

Results: These were 9 female and 16 male, with an average age of 9 years. The average diagnosis time was 7 months. The main clinical presentation was abdominal mass. The main tumour size was 7,15 cm. 7 tumours were in upper limbs and 18 were in lower limbs. MRI was an essential exam for the diagnosis. There were 8 Ewing sarcoma/PNET (32%), 5 rhabdomyosarcoma (20%), 3 fibrosarcoma (12%), 2 dermatofibrosarcoma protuberans (8%), one inflammatory fibroblastic sarcoma (4%), one low grade fibromyxoid sarcoma (4%), one kaposi sarcoma (4%) and one myxoid liposarcoma (4%). Treatment is a combination of chemotherapy, surgery, and radiation. 5 patients were dead and 20 are still alive.

Conclusion: Evaluation of paediatric soft-tissue tumours can be challenging. To formulate a differential diagnosis and, ultimately, diagnose a presenting lesion, the clinician should have an organised and systematic approach to the evaluation. Biopsy is a relevant and indolent exam for the diagnosis. Sarcomas represent the most common paediatric soft tissue cancers. To optimize their management and survival, patients should be treated at specialised centres to provide appropriate therapy and follow-up.


The role of preeclamptic stem villi obliterative angiopathy in the prognosis of newborn status

O. Voronova*, L. Mikhaleva

*PHI Clinical Hospital RZhD-Medicine, Rostov-on-Don, Russia

Background & objectives: The prevention of ischemic changes in the placenta, where obliterative angiopathy of the villus vessels plays a leading role, requires the development of an objective prognosis tool. 70 placentas from women with preeclampsia of varying severity, 20 from healthy women.

Methods: Morphometry was carried out using the Leica Application Suite module, Leica DM4000B. Micromorphometric indicators were determined: the area of the stem villi, the area of the lumen of the arterioles; diameters and areas of arterioles, including their wall thickness; measurements were made in 20 stem villi, the arteriole obliteration degree was calculated according to the coefficient Ксо = Sат\Sа.

Results: Foetal distress manifestation in preeclampsia depends on the severity of obliterative angiopathy. The normal significance of the obliteration coefficient for arterioles1-2 the order of stem villi was 1.18-1.34, for the 3rd -1.19-1.37. With an increase in the obliteration coefficient of arterioles of the 1st and 2nd order of villi more than 1.34, and of the 3rd order more than 1.37, the frequency of chronic intrauterine hypoxia increased to 61.0%and 59.4%, respectively. The increase of this indicator in the combination of preeclampsia with extragenital pathology over 1.89 for villi of 1-2orders and 1.92 of 3rd was accompanied by the addition of an increase in intrauterine growth restriction to 31.3% and 29.6%, respective.

Conclusion: The coefficient of arteriole obliteration is directly proportional to the incidence of chronic intrauterine hypoxia. When the placental villi arteriole obliteration coefficient exceeds critical value, the frequency of chronic intrauterine hypoxia and intrauterine growth restriction occurring in combination increases significantly. The significance of obliterative angiopathy in preeclamptic placentas in the functional state of the foetus and newborn was confirmed, and a prognostic model was created to calculate the probability of its occurrence.


Immunohistochemical characteristics of the vessels of the supporting villi of the placenta during preeclampsia

O. Voronova*, L. Mikhaleva

*PHI Clinical Hospital RZhD-Medicine, Rostov-on-Don, Russia

Background & objectives: Morphological changes in the vessels in preeclampsia are not sufficiently reflected in the literature, especially for the stem villi and their branches. There are no clear morphological criteria for assessing the severity of hypoxia of a newborn.

Methods: Studied 70 placentas from women with preeclampsia of varying severity, 20 placentas from healthy women. A macro- and microscopic examination of the placenta was carried out according to the generally accepted method. Immunohistochemical markers were used: CD34 (clone QBEnd\10), VEGF-A, eNOS. The level of expression of immunohistochemical markers was assessed in points: 0-no reaction, 1-weak reaction, 2-moderate reaction, 3-pronounced reaction.

Results: Immunohistochemical markers detailed the cells producing these factors in the vessels and stroma of supporting villi. Using the CD34 marker, hyperplasia and desquamation of endotheliocytes were revealed with a pronounced formation of reticulate structures in arterioles (placental endotheliosis) and its "palisade" location in venules. A decrease in the expression of eNOS and VEGF-A was clearly accompanied by the presence of rheological disorders in the vascular bed, there was a progression of collagenization of the supporting villi stroma with the formation of perivascular sleeves and obliteration of the lumen of their vessels with signs of reduction of the paravascular bed.

Conclusion: Morphological changes in the supporting villi are most pronounced in pregnancy complicated by preeclampsia. The lack of conditions for the implementation of compensatory reactions at the tissue level in the presence of preeclampsia exacerbates the severity of placental insufficiency, the development of obliterative angiopathy of the vessels of the stem villi, a decrease in blood flow in the capillaries of the terminal villi, which significantly worsens the prognosis for foetal development, increases the risk of adverse perinatal outcomes.


Foetal autopsy: causes of foetal death

L. Simic*, J. Jevtic, M. Jovanovic, M. Ninkovic, J. Sopta

*University of Belgrade, Faculty of Medicine, Institute of Pathology, Belgrade, Serbia

Background & objectives: Perinatal mortality remains globally unacceptably high with up to three million stillbirth every year. Intrauterine foetal death and stillbirth are crucial part of perinatal mortality. Autopsy, correlated with clinical data, remains the “gold standard” for identifying causes of foetal death.

Methods: A cross-sectional study, during the 2018 year, was performed by reviewing autopsy reports from the Institute of Pathology, Faculty of Medicine University of Belgrade. It was 1171 autopsies generally, among them 146 foetal. Based on autopsy findings, we identify the most common causes in perinatal death. Data were analysed using methods of descriptive statistics.

Results: The clinical diagnoses were: anomalies 45, intrauterine death 42, chromosomal aberrations 18, placental and umbilical lesions 10, spontaneous abortion 2, tumour 3 and multiple causes 26. Autopsy confirmed anomalies are the most frequent primary disease and cause of death (56%), where CNS anomalies were leading lesions (41%). Based on patohistology general asphyxia is the following cause of foetal death in 47%. Asphyxia was associated mostly with placental lesions (37%) as a result of vascular placental changes, followed by inflammation in women older than 35 years. We found the highest association of chromosomal aberrations in the same mother’s ages. Foetal anomalies were mostly found in women younger than 25 years.

Conclusion: Anomalies and asphyxia are the most frequent cause of foetal death between 26-35 gestation weeks. Foetal asphyxia as a result of placental ischemic lesions, umbilical cord pathology is mostly seen in mothers older than 35 years. In the same age group were mothers of foetuses with chromosomal aberrations. Foetal anomalies are mostly found in mothers younger than 25 years.


The association of mir-204 and mir-483 5p expression with clinicopathological features of Wilms tumour: could provide foresight?

A. Orgen Calli, G. Issin, İ. Yılmaz, D. Ince, I. Guzelis*, E. Tural, R.E. Cecen, H.N. Olgun, D. Kabakci, E. Ozer

*Izmir Katip Celebi University, Turkey

Background & objectives: Wilms tumour(WT) is the most common neoplasm of the kidneys in childhood. With the improvement of up-to-date treatment protocols, an increase in survival rates in WT was observed. However, metastases or local relapses are still observed in 15% of patients.

Methods: The evaluation of genetic and epigenetic features such as miRNA analyses might allow us to comment on the behaviour of the tumour and the treatment response. For this purpose, expression levels of mir-204 and mir-483-5p were evaluated in tumoral and normal tissue by qRT-PCR. The relationship of miRNA expression levels with clinicopathological, histological features, and survival was also investigated.

Results: This study recruited 24 WT cases who had paraffin blocks available for the study. Anaplasia (focal and diffuse) was identified in six patients(12%) based on the distribution of anaplastic changes. The result of the study indicated that the relative expression levels of mir-204 in WT tissues were significantly lower than that in adjacent normal tissues(mean value0.11). In contrast, tumour tissue had higher miR-483-5p expression than corresponding normal tissues. A statistically significant association between miR-204 expression level with age and the presence of anaplasia was observed in this study. Significantly high levels of miR-483-5p expression were found in cases who underwent preoperative CT compared to those who did not receive CT.

Conclusion: According to the literature data, decreased expression of miR-204 is associated with a poor prognosis. Our findings also suggest that poor prognostic data are accompanied by a downregulation of miR-204. In our study mir-483-5p expression level was found to be higher in patients who underwent preoperative CT compared to patients who did not receive CT. Of particular interest is the finding that the mir-483-5p can be a promising biomarker in the early detection of CT response in WT.


Histopathological assessment of placenta in maternal SARS-CoV-2 infection

R.A. Balan*, I.D. Caruntu, I. Pavaleanu, T. Butureanu, M. Pavaleanu, D. Popovici, S.E. Giusca, L. Lozneanu, T.A. Balan, C. Amalinei

*"Grigore T. Popa" University of Medicine and Pharmacy, Romania

Background & objectives: The placenta represents an important witness of pregnancy and its related complications in SARS-CoV-2-infected mothers. We aim to evaluate specific histopathological placental changes associated with maternal SARS-Cov-2 infection and their correlation with pregnancy dynamics.

Methods: The study includes 36 pregnant women, with pregnancy age ranging from 11 to 42 weeks, admitted between September 2020 and February 2022 with positive nasopharyngeal RT-PCR for SARS-CoV-2. All placenta specimens were macroscopically and histologically examined, the slides being conventionally stained with Hematoxylin and Eosin and immunohistochemically stained for CD8, CD68, and CD20.

Results: Of the 36 women, 34 (94.44%) had singleton pregnancies, one (2.77%) had twin pregnancy, and one (2.77%) had quadruplets. 28 (77.77%) pregnancies were term deliveries, with one (2.77%) placenta accreta and one (2.77%) marginal placenta praevia, two (5.55%) were miscarriages, and 4 (11.11%) were foetus exitus. The histopathological assessment revealed chorioamnionitis in 9 (25%) cases, foetal vascular malperfusion in 15 (41.66%) cases, villitis and chronic intervillositis in 8 (22.22%) and 4 (11.11%) cases, chronic deciduitis in 4 (11.11%) cases, hematomas in 7 (19.44%) cases, infarction in 11 (30.55%) cases. All placentas showed an association of these abnormalities, the local inflammatory response being underlined by positive immunoexpression of studied markers.

Conclusion: SARS-CoV-2 infection in pregnant women is associated with an increased frequency of placental histopathological abnormalities, especially foetal vascular malperfusion, various inflammatory lesions, infarction, and hematomas. In complicated pregnancies of patients with SARS-CoV-2 infection, histopathological evaluation of the placenta, supported by a specific panel of antibodies, may help elucidate the pathological mechanisms that occur at the foetal-maternal interface associated with COVID-19 infection, providing additional data in the obstetrical management of this pathology.

PS-07 | Poster Session Digestive Diseases Pathology - GI


Podoplanin expression in neoplastic cells and cancer-associated fibroblasts in colorectal cancer predicts unfavourable clinicopathological features

R. Souza da Silva*, E. Queiroga, C. Osório, K. Cunha, E. Dias

*Universidade Federal da Paraíba, Brazil

Background & objectives: The pattern of activity of the cellular elements that compose the tumour microenvironment influences tumour behaviour. To analyse the activity of cancer cells (CCs) and cancer-associated fibroblasts (CAFs) in colorectal cancer (CRC), based on the expression of podoplanin stratified by tumour-stroma ratio.

Methods: We performed immunohistochemistry for podoplanin on tissue microarrays from 357 cases of colorectal adenocarcinoma (CRA). The tumour-stroma ratio (TSR) was evaluated: stromal percentage ≤50%- stroma low; >50%-stroma-high. The expression of podoplanin was evaluated in different areas: TSR, most invasive, centre of tumour, tumour budding, and desmoplastic stroma. The association between the markers and clinicopathological parameters, including TSR, was evaluated.

Results: Immunostaining of podoplanin was detected in CCs and CAFs, with positivity of 36.8% and 70%, respectively. Higher positivity of podoplanin in CCs was observed predominantly at TSR area: 64.3% of cases. Status podoplanin CAFs+ was higher in the desmoplasic region (71.6%). Stroma-high tumours showed increased expression of podoplanin in CCs and CAFs in comparison with stroma-low tumours. The status of podoplanin in CCs was observed in association with women (p=0.042), angiolymphatic involvement (p=0.021; p=0.047) and distant metastasis (p=0.014).

Conclusion: In the CCR microenvironment, CAFs and CCs express podoplanin. Our research found an increase in podoplanin expression in high stromal tumours, known to be more aggressive than low stromal tumours, and an association with angioinvasion and distant metastasis. The expression of this marker, in CRA stratified by tumour-stroma ratio, contributes to aggressive behaviour. It may represent a patient stratification tool, in the prediction of possible outcomes.


IgG4 as a biomarker for inflammatory bowel disease

D. Raduta*, S. Zurac, M. Busca, R. Ardeleanu, A. Vilaia, A. Dinculescu, M. Filip, L. Cristina, C. Popp

*Colentina Clinical Hospital, Romania

Background & objectives: Inflammatory bowel disease (IBD) is a non-specific inflammatory condition affecting the gastrointestinal tract. The pathogenesis of IBD is insufficiently understood, but a key role is attributed to immune dysregulation. Our objective was to approach the IgG4 contribution to mucosal injury.

Methods: We conducted a case-control study comprising 12 patients with IBD with different stages of disease activity (remission, mild, moderate, severe), and we focused our research on immunohistochemical identification and quantification of IgG4+ plasma cells found in lamina propria. The infiltration of IgG4+ plasma cells in patients with IBD was compared with plasma cells level of 12 healthy control individuals.

Results: Patients with IBD had higher intestinal mucosal IgG4 counts than normal colonic mucosa (over 10 times more plasma cells IgG4+ in IBD per 10 HPF). Cases of ulcerative colitis (UC) showed higher numbers of IgG4+ plasma cells in lamina propria, compared to patients with Crohn disease (CD) for the same stage of activity. Mucosal infiltration of IgG4+ plasmocytes in active disease was higher comparative to remission stage of disease, as it follows: 5 IgG4+ per 10 HPF when CD is remitted and up to 25 IgG4+ in severely active CD, in contrast to 13 IgG4+ in remitted UC which goes up to 33-41 IgG4+ in moderately to severely active UC.

Conclusion: Our study led to the following conclusions:

The potential pathogenic involvement of B cell lineage in the pathogenesis of IBD deserves further research and in-depth studies, as it may contribute to personalized therapy.


MMR proteins and PD-L1 status: impact on gastric adenocarcinoma’s prognosis

F. Sassi*, R. Jouini, F. Khanchel, I. Helal, R. Hedhli, H. Zaafouri, M. Sabbah, E. Ben Brahim, A. Chadli Debbiche

*Department of Pathology, Charles Nicolle Hospital Tunis, Tunisia

Background & objectives: Deficient MMR proteins and PD-L1 expression have been shown to be prognostic factors[H1] and predictive biomarkers for anti-PD-1 immunotherapy in gastric adenocarcinoma(GA). We aimed to assess the expression of MMR proteins and PD-L1 in GA with clinicopathological features and survival.

Methods: This was a retrospective and descriptive study including 143 GA diagnosed at the Department of Pathology of Habib Thameur hospital (2001-2018). Evaluation of MMR proteins and PD-L1 status was carried out by immunohistochemistry. The combined positive score (CPS) was calculated for PD-L1 with a threshold of 1.

Results: The frequency of deficient MMR proteins (dMMR) GA was 30.1%. There was no significant association, in univariate and multivariate analysis, between MMR proteins and clinicopathological parameters. dMMR GA were PD-L1+ in 24%. The frequency of PD-L1+ GA was 21.7%. Medullary histological subtype according to World Health Organisation classification (p<0.001), intestinal subtype according to Lauren classification (p=0.014), lymphoid stroma reaction (p=0.004) were predictors of PD-L1+ status. Median survival was 16 and 18 months for patients with dMMR and PD-L1+ GA, respectively, with no significant association. PD-L1- status was associated with a poor prognosis.

Conclusion: One third of patients with GA have dMMR status. PD-L1- status is a factor of poor prognosis. Prognostic value of dMMR status should be investigated in a larger series.


Pathomorphological and molecular genetic features of serrated colorectal lesions

L. Mikhaleva*, R. Vandysheva, N. Shakhpazyan, A. Biryukov, M. Guschin

*A.P. Avtsyn Research Institute of Human Morphology, Russia

Background & objectives: Morphological diagnosis of serrated neoplasms today is based on the identification of a specific structure of formations in histological preparations. Therefore, the aim of our study was to identify molecular and biological features of serrated colorectal lesions (CSL).

Methods: We studied 481 cases of CSL (GP - 238, SSL - 201, and TSA – 42), stained with H&E and PAS-AB. For molecular-biological analysis, 69 observations of CSL were selected: SSL- 26, GP - 26, TSA - 17. The immunohistochemical panel included: CK20, Ki67, MUC2, MUC5AC, MUC6, MLH1, PMS2, MSH2 and MSH6. KRAS/BRAF/NRAS gene mutations were determined by real-time PCR.

Results: CSL show immunophenotypic signs of both colorectal differentiation (expressed expression of more than 50% of all cells of the markers MUC2 and CK20) and gastric differentiation (appearance of MUC5AC and MUC6 expression, focal positive PAS-AB staining). MUC6 expression is characteristic only for SSL. The malignisation pathway of SSL was associated with the presence of a BRAF gene mutation (53.8%) and high grade microsatellite instability (34.8%), while that of TSA was associated with a KRAS gene mutation (47.1%) and MSI-H (40%). The KRAS (15.4%), BRAF (38.5%) and MSI (60%) gene mutations detected in HP confirm their role in serrated carcinogenesis. The NRAS gene mutation was not detected in serrated colorectal lesions.

Conclusion: CSL show immunophenotypic signs of both colorectal differentiation and gastric differentiation. The malignisation pathway of CSL was associated with the presence of a BRAF gene mutation and MSI-H. Genetic mutations found in hyperplastic polyps, in combination with immunophenotype confirm their role in serrated carcinogenesis


Tumour infiltrating lymphocytes (TILs) relate to PD-L1 expression and provide prognostic value in stage II and III colon cancer patients

P. Azcue*, I. Encío, R. Vera, M. Mercado, M.L. Gómez-Dorronsoro

*Department of Health Science, Public University of Navarra (UPNA), France

Background & objectives: Tumour-infiltrating lymphocytes (TILs) and PD-L1 expression have been suggested as markers of immune-response in colon cancer also providing prognostic value. The aim is to assess TILs in the invasive primary tumour front, its relationship with PD-L1 expression and clinical outcomes.

Methods: In a cohort of 140 patients with stage II/III colon cancer, TILS were assessed according to the TILs Working Group standardized methodology and underwent automatic immunohistochemical staining for PD-L1. Clinical outcomes assess were disease-free survival and overall survival. The percentage of TILs score was categorized into low (≤ 5%), intermediate (≤ 10%), high (≤ 20%), and highest (> 20%).

Results: Assessment of TILs in the intermediate category and above in the tumour sample was statistically significantly related to PD-L1 expression. The higher percentage of TILs found, the stronger association with PD-L1 expression (p<0.001). There was no significant difference between TILs assessment and Stage II or III nor any other baseline characteristic. Category high TILs showed statistical significance for disease free survival HR=0.40 95%CI [0.23-0.95] independent of sex, age, TNM stage and localization.

Conclusion: Our results suggest that TILs and PD-L1 expression are closely related. In addition, the presence of TILs in the tumour microenvironment seem to provide a positive prognostic value in early stage colon carcinoma. Immunotherapies that aim to stimulate an immune response may benefit from TILs assessment as a predictor for PD-L1 expression, in addition to its prognostic value.


Novel patterns of V-set and immunoglobulin domain containing 1 expression in gastric cancer

C. Satala*, I. Jung, Z. Kovacs, R.I. Stefan-Van Staden, T. Bara, C. Molnar, A. Patrichi, S. Gurzu

*University of Medicine, Pharmacy, Science and Technology G. E. Palade, Targu Mures, Romania

Background & objectives: V-set and immunoglobulin domain containing 1 (VSIG1) is an intercellular-adhesion molecule expressed in the membrane of the normal gastric mucosa. The aim of this study is to report the possible significance of VSIG1 cytoplasmic translocation in gastric cancer.

Methods: Based on VSIG1 immunohistochemical (IHC) expression in tumour core and front in 94 cases, three patterns were established: homologous type I (membrane/membrane), homologous type II (null/null) and heterologous cases (membrane/cytoplasm or cytoplasm/null). VSIG1 status was correlated with clinico-pathological features (TNM stage and Dukes-MAC-like stage, molecular phenotype) and survival rate.

Results: From the 94 cases, 21.27% showed homologous type I expression, with the same percentage for cases with heterologous patterns. The rest of 57.46% demonstrated homologous type II pattern. Heterologous patterns were indicators of aggressive behaviour, such as advanced Dukes-MAC-like stage (p=0.02), lymph node metastases (p=0.03) and mesenchymal tumour phenotype, with loss of E-cadherin expression (p=0.004) and nuclear translocation of β-catenin (p=0.0007). Cytoplasmic expression was more frequently seen in poorly differentiated/undifferentiated carcinomas. In cases with heterologous pattern, overall survival was significantly poorer, compared to that of cases with homologous expression (p=0.0005).

Conclusion: In gastric carcinomas, VSIG1 cytoplasmic positivity might be an indicator of mesenchymal phenotype, and thereby, a marker for dismal prognosis, aggressive behaviour and shorter overall survival.

Funding: This work was supported by the Romanian National Authority for Scientific Research, Project no. 20-PCCF/2018


Serum and mucosal CD30 in paediatric inflammatory bowel diseases (IBD): useful biomarkers for diagnosis and disease activity monitoring?

O. Fabian*, A. Klocperk, T. Lerchova, P. Jencova, L. Stolova, M. Belhajova, D. Voriskova, D. Kazeka, A. Vicha, O. Hradsky, J. Bronsky

*Clinical and Transplant Pathology Centre, Institute for Clinical and Experimental Medicine, Prague, Czech Republic

Background & objectives: The underlying immune pathophysiology of IBD is incompletely understood, rendering quick diagnosis followed by tailored therapy difficult. The TNF superfamily receptor CD30 has been proposed as potential marker of ulcerative colitis (UC), and has been associated with elevated Th2 cells.

Methods: In this study we evaluate a cohort of 94 paediatric patients with UC and Crohn’s disease (CD) for serum soluble CD30 (sCD30) using ELISA, and expression of CD30 and subpopulations of Th1/Th2/Th17 lymphocytes in the gastrointestinal mucosa using flow cytometry (FCM). The dataset is supported by endoscopic and microscopic activity of disease and basic laboratory markers of inflammation.

Results: sCD30 was not associated with diagnosis of CD or UC. However, sCD30 levels correlated with levels of CRP, ESR, faecal calprotectin and albumin and also with clinical activity of the disease in both UC and CD patients. FCM was not helpful in evaluation of mucosal CD30, which was lowly expressed and not associated with diagnosis or disease activity. We show augmented Th2 and Th1/17 response in the terminal ileum and right-sided colon and decreased Th1/17 response in left-sided colon of UC patients. T lymphocyte subsets were also affected by anti-TNF treatment and patients’ age.

Conclusion: Neither sCD30 nor FCM evaluated mucosal CD30 were helpful in the diagnosis of paediatric UC. sCD30 seems to be marker of systemic inflammation and clinical activity of the disease.

Funding: This work was supported by the Grant Agency of Charles University in Prague project no. 728218, and the grant project NU 20-05-00282 issued by the Czech Health Research Council and Ministry of Health, Czech Republic.


Assessment of immune T cells expression in colorectal cancer

N. Boujelbene, I. Zemni, S. Dhouioui*, H. Ben Yahia, W. Babay, A. Ben Mansour, K. Mrad, H. Ouzari, I. Zidi

*Laboratory Microorganismes and Active Biomolecules, Sciences Faculty of Tunis, Tunisia

Background & objectives: Colorectal cancer(CRC) is the second most deadly cancer worldwide. CRC recurrence is at the origin of this high mortality.Resistance to chemotherapy remains one of the greatest challenges causing recurrence. Our study specifically addressed the expression of immune Tcells in CRC.

Methods: Immunohistochemical analysis was performed on a total of 23 patients with CRC and 18 adjacent normal tissues. Membranous expression of CD4, CD8 and nuclear expression of FOXP3 were analysed in T cells infiltrating the tissues in three different fields of the stained slides. Clinico-pathological characteristics were recorded.

Results: Patients mean age range was 63.9 years. CD4, CD8 and FOXP3 markers were significantly expressed in CRC tissues compared to normal tissues (Mann Whitney U test: CD4 p=0,0034 ; CD8 p<0,0001 ; FOXP3 p=0,0019). Interestingly, high percentage of CD8 positive expression was reported in CRC (near 30%) compared to CD4 and FOXP3 (not exceeding 4%) suggesting a high cytotoxic T cell infiltration. CD8high expression was found mostly in CRC without perineural invasion versus those with perineural invasion (p=0.042).

Conclusion: Altogether, our results showed the high expression of T cells infiltrating the tumour in CCR. Perineural invasion should be taken into consideration to design crucial strategies for CRC treatment.


HLA-E proteins expression and clinical relevance in colorectal cancer

N. Boujelbene, I. Zemni, S. Dhouioui*, H. Ben Yahia, W. Babay, M. Brahim, H. Ouzari, K. Mrad, I. Zidi

*Laboratory Microorganismes and Active Biomolecules, Sciences Faculty of Tunis, Tunisia

Background & objectives: Human leukocyte antigen(HLA)-E is a non-classical HLA class I molecule implicated in immune cells modulation. Many studies proposed HLA-E molecule as a predictive biomarker for cancers’ outcome. We aimed to characterize the HLA-E expression in colorectal cancer(CRC) according to clinicopathological characteristics.

Methods: HLA-E expression was studied in tumour tissues and adjacent normal tissues from 22 CRC patients by immunohistochemistry. HLA-E expression was found at the surface of cells. Labelled tumour cells percentage determined semi-quantitatively was correlated to clinico-pathological parameters.

Results: Patients mean age range was 64 years. HLA-E expression was significantly expressed in CRC tissues compared to normal tissues (100% and 86.6% respectively; Mann-Whitney U test: p < 0.0001). Moreover, high expression of HLA-E was revealed in patients exceeding 63 years, in those with early tumour stages (stage I and II), in those without lymph nodes metastasis, and in those with well differentiated tumours without significance.

Conclusion: Our results suggest that HLA-E is implicated in CRC promotion and could be proposed as a candidate biomarker for CRC. Its prognosis value remains to be confirmed through a wider study population.


Epstein-Barr virus infection in chemoradiotherapy-naïve gastric adenocarcinoma: relationship with PD-L1 Expression and survival

F. Sassi, R. Jouini, K. Ben Lazreg*, I. Helal, F. Khanchel, R. Hedhli, H. Zaafouri, M. Sabbah, O. Khayat, E. Ben Brahim, A. Chadli

*Habib Thameur Hospital Pathology department, Tunisia

Background & objectives: EBV has emerged as a prognostic biomarker in gastric adenocarcinomas (GA). EBV associated GA (EBVaGA) accounts for 10% of GA and comprises amplification of PD-L1.We aimed to assess EBV status and PD-L1 expression in GA with clinicopathological features and survival.

Methods: We performed tissue microarray slides from 143 GA patients treated with radical gastrectomy. PD-L1 expression was evaluated through immunohistochemistry (Combined Positive Score ≥1) and EBV status through chromogenic in situ hybridization. Differences in overall survival (OS) were assessed using the Kaplan-Meier method, log-rank test and Cox proportional hazards regression model.

Results: EBVaGAs accounted for 33.6%. They were associated with male gender (70.8%; p=0.020). The tumours were in the antrum (54.2%) with tumour size > 6 cm (52.2%). According to WHO classification, they were classified as poorly cohesive adenocarcinomas (43.8%). According to Lauren classification, they were classified intestinal subtype (45.8%), diffuse (43.8%) and indeterminate (10.4%). Perineural invasion was observed in 68.8% of cases and vascular emboli in 85.4%. EBVaGAs were associated to pT1/pT2 stage (p=0.031), low rate of lymph node metastasis (p=0.029) and PD-L1+ status (p=0.016). EBV+ status was predictor to PD-L1+ status (p=0.018). Median survival was 17 months in patients with EBVaGA compared to 16 months in EBV negative GA.

Conclusion: The prevalence of EBVaGA is high in Tunisian population (33.6%). It is more frequent in poorly cohesive than tubular adenocarcinomas. EBVaGAs were more common in the antrum which does not fit reports in the literature. EBV+ status was a predictive factor to PD-L1 expression which suggests that patients infected with EBV may respond to PD-1 checkpoint inhibitors. This incites to further investigation of EBV carcinogenesis for therapeutic targets to select high-risk patients.


Ckit mutations in patients with gastrointestinal stromal tumours

M. Ramadwar*, L. Rambadran, O.S. Shetty, M. Gurav, K. Deodhar, M. Bal, R. Kaushal, S. Yadav, V. Ostwal, M. Bhandare

*Tata Memorial Hospital, India

Background & objectives: CKIT mutations are oncogenic drivers for gastrointestinal stromal tumours (GISTs). They also function as predictive markers for response to imatinib. We studied various histologic types of GISTs, risk stratification, types of Ckit mutations and correlated with survival.

Methods: Histology and immunohistochemistry of 98 patients diagnosed with GIST were reviewed retrospectively. Histologic risk stratification was derived using CAP protocol. Sequencing results for Ckit exons 9,11, 13 and 17 were documented. Histologic and molecular parameters were correlated with survival. Clinical data was derived from electronic medical records.

Results: Immunohistochemistry for Ckit and DOG1 was positive in 98% of tumours; 2% being positive for DOG1 only. The histological risk groups were not associated with statistically significant differences in PFS (p=0.6) or OS (p=0.7). Ckit mutation rate was 73%, Exon 11 mutations were found in 75% of patients, exon 9 in 19%, exon 13 in 4.1 % and exon 17 in 5.5 % respectively. All patients with exon 9 mutations had consistent duplication c.1504_1509dupGCCTAT. Decision of dose escalation of imatinib or change to second line TKI was made in all patients with ckit exon 9 mutation. No statistical difference could be demonstrated among different mutational types when correlated with survival.

Conclusion: Overall Ckit mutation rate was lower (73%) than reported in literature, thus concluding that incidence of ckit mutations could be lower in Indian population. Ckit exon 9 mutations had consistent duplication while deletions and substitutions were found in in exon 11. Exon 9 mutations guided treatment decisions. However, no statistical difference could be demonstrated among histological risk stratification and different mutational types when correlated with survival which might be attributed to low numbers of low and intermediate category GISTs.


Molecular landscape of rectal cancer patients up to 5th decade – the preliminary results

P. Lewitowicz*, M. Kozlowska-Geller, M. Wawszczak-Kasza, W. Lewitowicz, S. Zieba

*Department of Clinical and Experimental Pathology, Institute of Medical Sciences, Jan Kochanowski University, Kielce, Poland

Background & objectives: An availability of modern molecular solutions provided us a new insight into cancer genetic variations. In the current study we intended to perform a molecular screening of rectal cancer in young patients and then numerical analysis of cancer stem cells.

Methods: The ruling in criteria were the patient's age before 50years old, no previous radio- and chemotherapy, and eligible DNA for the next generation sequencing. The DNA was isolated from FFPE tissue. We used a Hot-Spot Cancer Panel by Illumina harbouring 50 genes (700 amplicons). To assess stem cell population we intend to use CD133, SOX-2, and Lgr5 antibodies.

Results: The average patient's age was 43-year-old. The female/male ratio was 1.4:1. The mean follow up time is 38 months. We observed a following mutation frequency: TP53 61%, KRAS 58%, APC 47%, PIK3CA 19%, SMAD4 11%, FBXW7 11%, NRAS 9%, BRAF 7%, NRAS 5%, CTNNB1 4%, MLH-1 1%. We noted the co-occurrence KRAS/BRAF/NRAS mutation (p<0.05). Two cases did not present any mutation in the used panel. The immunohistochemistry and a statistical analysis is still in progress.

Conclusion: Our preliminary results indicate a rising number APC independent rectal cancer in a group of young patients. 40% of them presented multigene abnormality, where three to four mutations occurred the most common. Interesting is the fact of the evident contribution of the PIK3CA pathway and also 11% of SMAD4 which worsens the prognosis.


Evaluating PTEN expression using immunohistochemistry in neuroendocrine tumours of the digestive tract and correlations with tumour grade and location - a seven-year retrospective study

A. Plopeanu*, A. Dema, O. Popa, A. Pascu, R.A. Barna, B.R. Nataras, S. Taban

*Department of Microscopic Morphology-Pathology, ANAPATMOL Research Center, ''Victor Babes'' University of Medicine and Pharmacy of Timisoara, Romania

Background & objectives: Neuroendocrine tumours (NET) of the digestive tract are heterogeneous tumours, which despite similar morphology and grading have often distinct behaviour. We propose the use of PTEN immunohistochemical (IHC) expression for neuroendocrine tumours of the digestive tract as a prognostic factor.

Methods: 25 samples from patients diagnosed with neuroendocrine tumours from 2012 until 2018 were selected. All tumours were reclassified and graded based on 2019 WHO classification. Representative tissue sections were stained for Synaptophysin (clone 27G12; Leica), Chromogranin A (clone 5H7; Leica), Ki-67 (clone MM1; Leica), and PTEN (clone 6h2.1; Dako) using Leica BOND-MAX fully automated staining system.

Results: Out of 25 cases, 18 were graded based on Ki-67 expression as NET G1 (72%), 5 as NET G2 (20%), and only 2 as NET G3 (8%) tumours. 8 cases presented PTEN negative expression of which 2 were NET G3 (100%), 3 were NET G2 (60%) and 3 were NET G1 tumours (16%) (p=0.018). 9 cases were localized in the small intestine, 9 in the large intestine, 3 in the stomach, and 4 in the appendix. All appendix tumours had positive PTEN expression. 3 gastric tumours had negative PTEN (100%), 1 small intestine case was PTEN negative (12%) and 5 cases from the large intestine were PTEN negative (56%) (p=0.013).

Conclusion: Neuroendocrine tumours of the digestive tract have a distinct molecular profile that is still troublesome to understand regardless of grade, localization as well as other clinicopathological factors. We propose using PTEN expression as a significant negative prognostic factor for patients with NET of the digestive tract based on tumour grade and localization, but larger studies on more patients from each group are required in order to introduce PTEN expression in the treatment protocols.


Association between Immunoscore and budding in colorectal carcinoma

I. Helal*, K. Ben Lazreg, F. Khanchel, M. Ben Thayer, R. Hedhli, E. Ben Brahim, R. Jouini, A. Chadli

*Habib Thameur Hospital, Tunisia

Background & objectives: In colorectal carcinoma, the immunoscore (IS) and tumour cells budding (TCB) score are two emerging parameters that have an independent prognostic value. Our study aimed to research for significant association between IS and TCB score.

Methods: A total of 104 tumour specimens from patients after curative resection were reviewed. For the determination of the IS, we adopted a method described by Galon et al. It was scored into 2 groups through a semi-quantitative method. TCB score was rated according to the ITBCC criteria. Tumour buds were scored manually into 3 groups.

Results: The mean age of the patients was 61.6 years. The IS was low in 60.6%, and high in 39.4%. The TCB score at the invasive front was low, intermediate, and high in 53.8%, 22.1%, and 24% of cases, respectively. We used the Chi-squared test to assess the association between IS and TCB score. A significant association between these two scores was found with p=0.042 (<0.05).

Conclusion: Accumulating evidence suggests that adaptive immune response, represented by cytotoxic T cells, plays a crucial role in suppressing tumour invasion and metastasis. A few data have suggested that anti-tumour immune response may restrict tumour buds at invasive margins. Our results are in line with these findings. This significant association may explain the tendency towards a new combined budding-immune cell score.


Correlation between endoscopic appearance and histology in immune checkpoint inhibitors-induced gastritis

I. Ungureanu*, P. Bonnet, C. Julié, D. Parlier, P. Saiag, D. Lamarque, J. Emile, F. Elisa

*Department of Pathology, Ambroise-Paré Hospital, France

Background & objectives: Few publications have reported active lesions on gastric biopsies despite a normal endoscopic appearance in patients treated with immune checkpoint inhibitors (ICI). Our objective was to describe the correlation between histology and endoscopy of ICI-induced gastritis.

Methods: All the patients treated with ICI (Ipilimumab, Nivolumab, and/or Pembrolizumab) for metastatic melanoma in Ambroise-Paré Hospital who underwent gastric biopsies were retrieved from pathology laboratory files. Cases correspond to an eleven year-period (2010-2021). Endoscopic results were analysed and correlated with histology. Hematoxylin & eosin stained slides of gastric and duodenal biopsies were reviewed and immunohistochemistry was performed.

Results: 19/461 patients (4.1%) under treatment with ICIs were histologically confirmed with gastritis. 6/19 (31%) showed severe lesions of active gastritis on biopsies, while 13/19 (69%) showed mild/moderate gastritis. Histologically, all severe cases corresponded to diffuse chronic active gastritis involving the full thickness of the mucosa with different degrees of inflammation between the antrum and fundus. Important neutrophilic infiltrate was present with crypt abscesses, intra-epithelial lymphocytosis CD8+ and massive gland destruction with interstitial CD4+ predominant T lymphocytes. Immunohistochemistry for CMV and Helicobacter Pylori was negative. Among the six cases with severe gastritis, four patients had a normal endoscopic appearance of gastric mucosa/ minor lesions and two patients showed gastric erosions/ulcerations.

Conclusion: Severe damage in ICI-induced gastritis can be associated with a normal endoscopic appearance. It is characterized by a chronic active gastritis pattern with abundant infiltration of mucosa and epithelium by lymphocytes and neutrophils and massive gland destruction. Despite a normal endoscopic appearance, biopsies of both fundus and antrum should be performed, as histology can be contrasting in different gastric regions.


Diagnostic usefulness of p53 immunostaining in gastric cancer and dysplasia: a real-world clinical experience

M. Kim*

*Kyungpook University Hospital, Republic of Korea

Background & objectives: Stomach cancer are major threat to public health. A subset of gastric cancer harbour mutations of TP53. Gastric cancer with mutant TP53 gene usually accompanies morphologic changes. We have investigated the diagnostic utility of p53 immunostaining in real-world cases.

Methods: We retrospectively searched 50 stomach tumour and tumour-like lesion cases, wherein p53 immunostaining had a pivotal role in the diagnosis. P53 staining pattern was also analysed in association with clinicopathologic parameters.

Results: Mutant pattern p53 staining was significantly correlated with high-grade nuclear atypia (p<0.001), high-grade dysplasia and tubular adenocarcinoma (p<0.001), and MSS status (p=0.034). Furthermore, diagnostic application of p53 immunostaining was useful when 1) biopsy specimen contained only few tumour cells, 2) pathologic resection margin evaluation was difficult to due to the cauterization artifact 3) distinction of low-grade and high-grade gastric dysplasia.

Conclusion: p53 immunostaining can be helpful for the diagnosis of gastric tumour and tumour-like lesions, and accurate pathologic margin evaluation, particularly if the lesion shows intestinal-type differentiation and some degree of nuclear atypia.


Tumour area infiltration and absolute tumour cell counts in endoscopic biopsies of therapy-naive upper GI tract carcinomas - implications for predictive biomarker testing

A. Quaas*, H. Lamberty, A.H. Scheel, Y. Tolkach, F. Gebauer, B. Schoemig-Markiefka, T. Zander, R. Büttner, J. Rueschoff, C.J. Bruns, W. Schroeder

*Institute of Pathology, University Hospital Cologne, Germany

Background & objectives: Guidelines regulate how many biopsies should be taken to obtain reliable results of predictive biomarker tests. Little is known about how well these guidelines are applied, the number of biopsies correlates with the tumour area and the tumour cells counts.

Methods: The study included endoscopic biopsies of untreated carcinomas of the upper gastrointestinal (GI)-tract during the 2015-2020 review period. Archival (H&E)-stained histological sections were digitized, and the tumour areas were manually annotated. The tumour-bearing biopsy area and absolute carcinoma cell count per case were determined by image analysis.

Results: Biopsies from 253 patients were analysed. The following mean values were determined: a) tumour biopsy number: 6.5 (1–25, standard deviation (SD)=3,32, b) number of tumour-bearing biopsies: 4.7 (1–19, SD=2,80), c) tumour infiltrated area: 7.4mm2 (0.19 mm2–59.46 mm2), d) absolute tumour cell count: 13,492 (193–92,834) e) tumour cell count in a primary surgical specimen (tumour size: 6.7 cm): 105,200,176.

The guideline-recommended biopsy count of 10 was not achieved in 208 patients (82.2%), and the required tumour-bearing biopsy count of 5 was not achieved in 133 patients (52.6%).

Conclusion: Biopsies are often used to determine predictive biomarkers, like Her2/neu or PD-L1. Diagnostics standards to ensure representative material have been suggested in guidelines to reduce false-negative predictions. This is the first study describing the relationships between biopsy number, actual infiltrated tumour area, and carcinoma cell number. We advocate, that histopathological reports should indicate on which basis statements on therapy-relevant biomarkers were made. Digital pathology has the potential to objectively capture these parameters for documentation, quality assessment, and future clinical studies.


Immune microenvironment landscape shows treatment specific differences in rectal cancer patients

C. Graham Martínez*, Y. Barella, S. Kus Oztürk, M. Gorris, S. van Lent-van Vliet, C.A. Marijnen, I. Nagtegaal

*Radboud University Medical centre, The Netherlands

Background & objectives: Neoadjuvant therapy is the backbone of modern rectal cancer treatment. Insight into the biology of tumour response is needed to optimize organ-preserving approaches. The aim of this study is to explore treatment-specific responses of the tumour and tumour microenvironment.

Methods: Locally advanced rectal cancer patients were treated with chemotherapy (CT), radiochemotherapy (RCT), radiotherapy short wait (RTS) or long wait (LRT) or did not receive therapy (NT). 16 patients per group were included. Tumour patterns of response assessed on HE slides. IF-multiplex was performed for Tcyt (CD3+CD8+), Treg (CD3+FOXP3+), Thelper (CD3+CD8-FOXP3-), B cell (CD20+), DC (CD11c+) & Tumour (panCK+).

Results: A fragmented pattern was predominant in CT-and-RCT-treated patients, whereas shrinkage pattern was predominant in LRT-treated patients (p=0.02). Thelper cells were the predominant immune cell population across therapies and a higher immune cell density was observed in stroma compared to tumour region. The depletion of Tregs after therapy suggests a long and maybe permanent effect on the tumour microenvironment. Acute RT affects Tcyt infiltrate (p<0.01). Brachytherapy-treated patients show the lowest densities of stromal Thelper, Tcyt & Tregs (p <0.01). Synergistic effect of RCT induces stromal increase of Tcyt cells and depletion of Thelper cells compared to CT. All patients treated with some form of radiotherapy had a more homogeneous immune response.

Conclusion: We demonstrated treatment-specific differences in the immune microenvironment landscape of RC patients. Local treatment including RT lead to a more homogeneous immune response compared to NT and CT. Understanding the immune contexture in relation to specific treatments will inform future treatment decisions.

Funding: This work was supported by an Alpe d'HuZes/KWF program grant (KWF UL 2013-6311)].


H3K27me3 immunohistochemical loss predicts response to neo-adjuvant chemo-radiotherapy (CRT) in patients with locally advanced rectal adenocarcinoma

N. Caldonazzi*, P.C. Rizzo, S. Ammendola, G. Turri, C. Pedrazzani, A. Scarpa, V. Barresi

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

Background & objectives: Patients with locally advanced rectal cancer are treated with neo-adjuvant chemo-radiotherapy to improve resectability and decrease the probability of recurrence. This study aims to assess whether H3K27me3 immunostaining in pre-treatment biopsies of rectal adenocarcinoma may predict response to neo-adjuvant CRT.

Methods: We assessed H3K27me3 immunostaining in 43 pre-treatment endoscopic biopsies of locally advanced rectal carcinomas treated with neo-adjuvant CRT and correlated it with Tumour Regression Grade (TRG) measured using Dworak system in the following surgical specimen.

H3K27me3 immunostain was classified: i) retained (≥ 5% stained neoplastic cells); ii) lost (> 95% stained neoplastic cells); iii) inconclusive (unstained normal and neoplastic cells).

Results: H3K27me3 immunostaining was lost in 18 cases, retained in 17 and inconclusive in 8. All tumours with retained H3K27me3 expression had complete tumour regression (TRG 4/5). H3K27me3 loss was significantly associated with absent/partial tumour regression (TRG 0/1/2) in surgical specimen (P=0.0015)

Conclusion: Due to the lower probability to respond to neo-adjuvant CRT, a "watch and wait" approach to avoid side effect of surgery should be used with caution in patients with rectal carcinomas with H3K27me3 loss in the endoscopic pre-treatment biopsy.


NET G3 of the digestive system: clinico-pathological and molecular features of 7 cases

L. Casiccia*, R. Maragliano, M. Testa, F. Sessa, S. La Rosa, S. Uccella

*Institute of Pathology, Dept. of Medicine and Surgery, University of Insubria, Varese, Italy

Background & objectives: Grade 3 Neuroendocrine Tumour of the digestive system (DS-NET G3) is a recently recognized entity exhibiting well differentiated morphology and high proliferation rate. Our study aimed to analyse the morphophenotypical, molecular and clinical features of 8 DS-NETs G3.

Methods: We collected 8 DS-NETs G3 in our institution between 2015 and 2018. Clinical records and pathological samples were available for further analysis. The histopathological review was performed according to the upcoming WHO classification for Neuroendocrine Neoplasms. Immunohistochemistry for Chromogranin, Synaptophysin, INSM1, p53, Rb, p16, SSTR2A, DAXX/ATRX, Cyclin D1, Ki67 was performed. Next Generation Sequencing (NGS) was executed on 4 cases.

Results: Our cases included 3 pancreatic NETs (PanNETs) and 5 gastrointestinal NETs (1 gastric, 1 ileal, 1 appendicular, 1 caecal, 1 rectal). They were all characterized by a well differentiated morphology and a Ki67 index >20% (mean: 25%, range: 20-40%). General neuroendocrine markers were intensely and diffusely positive in all cells. SSTR2A showed membranous immunoreactivity in 6 of 8 cases. p53, Rb1 and p16 expression weren’t altered in any case, whereas Cyclin D1 was frequently overexpressed. Molecular analysis did not reveal any abnormality in key cancer genes, including TP53 and RB1 genes. The mean follow-up of patients was 24 months and no disease-related death was recorded.

Conclusion: Our case series recapitulates the clinicopathological and molecular characteristics of G3 NETs of the digestive system. This analysis highlights common features of these neoplasms, arisen in different sites, useful to distinguish them from neuroendocrine carcinomas occurring in the same locations.


PINK1 analysis in colorectal adenocarcinoma and their respective hepatic metastasis with clinical relevancy

J.C. Celis Pinto*, D. Corte-Torres, A. Vallina, A. Alonso Fernández-Velasco, I. Fernandez-Vega

*Hospital Universitario Central de Asturias, Spain

Background & objectives: PTEN-induced-kinase-1 (PINK1) is essential for maintaining mitochondria metabolism and survival in colon cancers. Higher PINK1 expressions were correlated with worsened survival. Our aim was to study the immunoexpression of PINK1 in samples from colorectal adenocarcinoma and their respective hepatic metastases.

Methods: Ninety consecutive patients with colon adenocarcinoma and subsequent hepatic metastasis surgically removed between 2005 and 2022 were studied. Tissue arrays were produced using a 2 mm diameter needle. Immunohistochemical studies were conducted and analysed by the H-Score method. Statistical analysis of these findings was carried out using the SPSSv25; p<0.05 program.

Results: Positive immunoexpression was detected in more than 95 percent of both primary tumours and their hepatic metastases with significant median differences (82,27 ± 48,75 vs 91,29 ± 49,59; p= 0,034). A positive correlation was identified for PINK1 immunoexpression between primary and metastatic tumours (r= 0,352; p=0.001). A cut-off of 100 points of PINK1 in primary samples and 110 in metastatic samples segregated patients into groups with a significant different prognosis. For instance, more than 110 points of PINK1 immunoexpression in patients with metastatic samples combined with more than 25 mitotic figures per 10 high-power-fields had a worse global survival (105,25 ± 14,04 vs 54,95 ± 10,47 months; p=0.018).

Conclusion: Overexpression of PINK1 was observed in hepatic metastasis versus primary colon adenocarcinoma with a positive correlation. Defined cut-off with clinical relevance for PINK1 immunoexpression in hepatic metastasis and primary colon adenocarcinomas was identified.

Funding: Sakura Finetek Spain


The usefulness of Cycline D1 in diagnostics of naive, CD117/DOG1 positive gastrointestinal stromal tumours (GISTs) – correlation with other histological and immunohistochemical factors

M. Lenarcik*, M. Polkowski, P. Wieszczy, A. Mróz

*CPME, Poland

Background & objectives: The aim of the study is to correlate Cycline D1 expression with size and mitotic activity assessment using PHH3 and Ki67 stainings in determining high risk tumours.

Methods: 116 postoperative GISTs were assessed in terms of histology and immunohistochemistry (CD117, DOG1, Ki67 and PHH3). The size of the tumours were based on macroscopic and radiological data. Statistical analysis included non-parametric Wilcoxon test was used to compare continuous variables and chi-square test to compare proportions. Spearman correlation coefficient was used to calculate correlation. All test were two sided.

Results: Cycline D1 was expressed in 72/116 cases (25 LG tumours according to ESMO criteria and 47 HG tumours ) – p>0,099. No correlation was found between cycline D1 expression and PHH3 expression (rho=-0,07, p=0,444) mitotic index (rho=-0,12, p=0,216). |The correlation was found between cycline D1 expression and size of the tumour (mean size of the tumour in cycline D1 positive group = 7,2cm vs cycline D1 negative group = 9,4cm; rho=-0,20, p=0,03)

Conclusion: The immunohistochemical expression of cycline D1 does not correlate with mitotic index and PHH3 and Ki67 immunohistochemical expressions in GISTs. It does not discriminate between LG and HG tumours. Hence it does not influence the risk stratification of GISTs as measured in tissue material.

The correlation between cyclineD1 expression and smaller size of the tumour is observed. It is to be established upon observational studies whether it may represents an independent risk factor as proved in others tumour.


Granulomatous appendicitis: a diagnostic challenge. Clinicopathological retrospective study of 60 cases

A. Pasco Peña*, A. Larrea, G. Aísa Rivera, M.I. Cevallos, M. Mercado, Á. Panizo Santos

*Hospital Universitario de Navarra, Spain

Background & objectives: Granulomatous appendicitis (GA) is an uncommon finding of appendectomies. The causes can be classified as infections or noninfectious. There is also a group where a cause is never identified (idiopathic). In this study, we report the clinicopathological features of GA.

Methods: We performed a single-centre, retrospective study of appendectomy specimens received by our department over a 26-year period of time (1995-2021). Clinical data were collected retrospectively from the medical records and the histological slides were reviewed. Additional stainings (Ziehl-Neelsen, Gram, PAS and Groccot) and molecular studies were performed.

Results: We identified 60 GA cases: 32 men and 28 women. Ages ranged 1-83 years (median 33.5yrs). 44 cases had transmural acute-subacute inflammation. Granulomas were found scattered throughout all layers of the wall: subserosa only (11.7%), submucosa+mucosa (65%) and transmural (23.3%). 12 GA were necrotizing granulomas. In 27 cases, granulomas were found in the lymphoid follicles with concomitant extrafollicular granulomas in 15. The presence of xanthogranulomatous inflammation was seen in 10 cases. Two cases were Ziehl-Neelsen positive and one case was PCR-TBC positive. The likely clinicopathological cause of GA: idiopathic (55%; 3 cases probably due to Yersinia), Crohn’s disease (21.7%), interval appendicitis (8.3%), tuberculosis (6.7%), foreign material (6.7%), and Actinomyces (1.7%).

Conclusion: The incidence of GA was very low: only 60 cases in 26 years. Idiopathic (primary) granulomatous appendicitis is the leading cause of in our series, followed by Crohn's disease. The site of occurrence in the appendix, the morphology of the granulomas and the clinical setting is often paramount in establishing the aetiology. In our series, special stains for infectious organisms in GA are of low diagnostic yield.


Impact of IL-17-positive lymphocytes and -197A/G SNP in development of gastric cancer

E. Aleksandrova*, J. Ananiev, K. Ivanova, M. Hadzhi, M. Galabova

*Trakia University, Bulgaria

Background & objectives: An expanding body of evidence implicates the dualistic role of IL-17, a pro-inflammatory cytokine, in both pro- and anti-tumourigenic processes. Elevated IL-17 expression has been observed in a variety of tumour tissues, including gastric cancer (GC).

Methods: We investigated immunohistochemically 45 GC patients with antibodies against IL-17. Genotyping for the -197A/G SNP in the IL-17A gene was performed via PCR-RFLP method. The clinicopathological parameters and survival were analysed retrospectively.

Results: We observed lower infiltration of IL-17-positive lymphocytes (-PL) in the tumour border in patients with low differentiated gastric cancer (Chi-square test, p=0.052). Also, we found a tendency that patients without distant metastasis had lower infiltration in the tumour border with IL-17-PL (Chi-square test, p=0.106), but in the tumour we found that non-metastasis patients had significantly lower infiltration with IL-17-PC (Chi-square test, p=0.024). Our results showed that the carriers of the G-allele for the -197A/G SNP were in advanced clinical stage of the disease (Chi-square test, p=0.072). The G-allele was also associated with the histology type of the tumour- the G-allele carriers had intestinal type tumours (Chi-square test,p=0.105).

Conclusion: Our results suggest that -197A/G SNP and IL-17-positive lymphocytes could be helpful to outline the progression for patients with gastric cancer.

Funding: This work was financially supported by the National Science Fund, Bulgarian, Research grant number KP-06-H23/2 from 17.12.2018


Prognostic value of natural killer cells in colorectal carcinoma

I. Helal*, K. Ben Abdallah, F. Khanchel, S. Fkih, R. Hedhli, E. Ben Brahim, R. Jouini, A. Chadli

*Habib Thameur Hospital, Tunisia

Background & objectives: Natural killer cells are considered to have prognostic impact in several solid tumours. In colorectal carcinoma, their role remains obscure. We aim to determine whether the presence of NK cells in tumour tissue is associated with a better overall survival.

Methods: A total of 104 tumour specimens from patients after curative resection were reviewed. Areas with the highest lymphocyte density in the centre and margins of each specimen were marked. Tissue microarrays regrouping cores extracted from marked areas were formed. Immunohistochemistry was carried out on slides resulting from the section of TMA blocks. Anti-CD56 antibody was used for NK cells identification.

Results: Mean age was 61.6 years. Gender ratio Male/Female was 1.36. 84.6% of tumours were Adenocarcinomas NOS, with a low differentiated grade in 80.7% of cases. 44.2% of patients were pTNM stage III or IV. Average follow-up period was 60 months. 41 deaths occurred. Mean number of NK cells in tumour centre and margins were 2.2 and 1.52 respectively. 31.7% of patients had a total of 4 NK cells or more. There was no significant difference in densities of NK cells between the centre and the margins of the tumour. Mean survival was 55.59 months. There was no correlation between density of NK cells in tumour tissue and overall survival (p=0.272).

Conclusion: In colorectal carcinoma, NK cells are present in very low density in tumour tissue. Their number in situ does not significantly impact overall survival.


Prognosison the pre-treatment immunological characteristics of neoadjuvant therapy in oesophageal squamous cell carcinoma

Y. Liu*, J. Li

*The Fourth Hospital, China

Background & objectives: The expression of PD-L1 and tumour-infiltrating lymphocytes (TILs) in tumour microenvironment can predict the prognosis in oesophageal squamous cell carcinoma (ESCC). This study aims to explore the prognosis of pre-treatment immunological characteristics in ESCC before the neoadjuvant therapy (NAT).

Methods: A total of 205 patients who receive 2-3 cycles of NAT and was surgically resected between 2015 and 2020.The expression levels of PD-L1, CD4+, CD8+, FOXP3+ and CD20+ cells before the NAT were observed. The patients were randomly divided into the training set and the validation set, and then the nomograms were drawn to predict the patient prognosis.

Results: The expression of PD-L1 before the NAT was positively correlation (P <0.05)with different types of TILs, with a positively related trends to the level of histological grade and lymph node metastasis status. The expression levels in TILs are positively correlated with postoperative histological grading, negatively connected to postoperative tumour size( P <0.05). We build two prognostic models and further verify it, results show that the predictive lymph node metastasis status model and the predictive depth of tumour infiltration model based on the expression level of PD-L1 and TILs of pre-NAT show a good consistency and have good predictive capabilities compared with ideal models.

Conclusion: The higher of TILs expression in ESCC, the higher of the expression of PD-L1, CD4+, CD8+, FOXP3+ and CD20+ cells. There is a raising trend of histological grade with a high-TILs in the pre-NAT ESCC patients, and the higher the chance of lymph node metastasis. Two predicted models have a moderate to good discrimination and consistency, which can provide the predictive value for the ESCC patients before the NAT, and it also can provide references for clinical precision treatment.


Adding a zero Buds group (BD0) in the tumour budding scoring system in colorectal carcinomas: is it relevant?

I. Helal*, M. Ben Thayer, F. Khanchel, S. Fkih, R. Hedhli, E. Ben Brahim, R. Jouini, A. Chadli

*Habib Thameur Hospital, Tunisia

Background & objectives: The validated Budding scoring system is made up of 3 groups: BD1(0–4 buds), BD2(5–9 buds), BD3(≥10 buds). A recent study suggested adding a fourth group with zero Buds(BD0). We aim to study the accuracy of the onset of BD0 category

Methods: Our study included retrospectively patients who were operated on for CCR during a four-year-period from 2013 to 2016. Patients who received neoadjuvant treatment were excluded. We performed conventional BD scoring for all cases. We divided the BD1 group into two groups BD0 (0 buds/0.785 mm2) and BD1*( 1–4 buds/0.785 mm2 ). We studied the differences between these two groups.

Results: Our sample of 133 patients consisted in 75 men and 58 women. According to the conventional BD scoring system, 67carcinomas were BD1, 33 were BD2 and 33 were BD3. The BD1 group was divided into two groups: 22 BD0 and 45 BD1*. Comparing the two groups, we found no statistically significant difference between BD0 and BD1* groups regarding age(p=0.5), tumour size(p=0.5), tumour grade(p=0.6), T stage, N stage(p=0.5),M stage(p=0.1), AJCC stage (p=0.2), vascular invasion(p=1), perineural invasion(p=0.7) and overall survival( p=0.9). However, when we compared the four groups(BD0, BD1*,BD2,BD3), there was a statistically significant association between BD and perineural invasion(p=0.02) , M stage (p=0.05), AJCC stage (p=0.03) and overall survival(p=0.02).

Conclusion: BD is ever arousing pathologists’ interest in the latest years. The onset of a BD0 category has recently been proposed. Our study revealed that there were no statistically significant difference between the two groups BD0 and BD1* regarding the main histoprognostic factors and the overall survival. These findings suggest that adding a fourth group BD0 wouldn’t have an additional relevance. However, further and larger studies are needed to assess the accuracy of adding the BD0 group.


Microsatellite instability (MSI) immunohistochemistry (IHC), colorectal cancer and Lynch syndrome (LS) genetic testing – one year experience at UK District General Hospital

A. Bommana*, R. Swamy

*Lister Hospital, United Kingdom

Background & objectives: BRAFV600E mutation followed by MLH1 promoter hypermethylation to identify LS in colorectal cancer is practised at our centre. This algorithm is followed for cases with loss of MLH1 IHC expression. We audited our practice to identify LS in colorectal cancers.

Methods: 209 colorectal cancers with their MSI IHC results reported during 2021 at our department were retrieved. Cases with loss of MLH1 IHC expression were identified. BRAF status analysed by New Genomic Sequencing (NGS) DNA panel and MLH1 promoter hypermethylation test results were recorded.

Results: 43 cases with loss of MLH1 and PMS2 IHC expression were identified. NGS showed wild-type BRAF in 9 cases. MLH1 promoter hypermethylation was present in 2/9 but absent in 4/9 cases with wild type BRAF. Thus 4/43 cases with loss of MLH1 IHC expression, wild type BRAF and no MLH1 promoter hypermethylation were identified as LS colorectal cancers. The subsequent results following MLH1 IHC testing were not available in time for discussion of treatment options at colorectal Multidisciplinary meeting (MDM) in 7 of these cases. Pathologist did not request MLH1 hypermethylation test in time for 3 cases and negative MLH1 hypermethylation result became available in 4 cases, after MDT discussion.

Conclusion: NICE and Royal College of Pathologists recommend MSI IHC testing in colorectal cancer to detect LS but awareness of its value to guide treatment decisions is lacking. People with LS have colorectal cancers that respond more favourably to immunotherapy and unfavourably to conventional chemotherapy regimens that include 5-Flurouracil. Pathways to facilitate timely MSI testing for district general hospitals which rely on external genomic laboratories for cancer molecular work-up needs to be addressed.


Morphological spectrum and clinicopathological correlation in appendiceal mucinous neoplasms (AMN) and pseudomyxoma peritonei (PMP): retrospective histopathology review of cases seen at a tertiary care institution between Jan 2012-June 2019

K. Deodhar*, A. Jadhav, S. Mokal, R. Kaushal, M. Bal, M. Ramadwar

*Dept. of Pathology, Tata Memorial Hospital, Mumbai, India

Background & objectives: We aimed to grade and stage AMN, PMP cases diagnosed between January 2012- June 2019 using AJCC 8th edition and WHO 5thedition criteria; to see if the three-tier system has bearing on prognosis, survival and find individual adverse prognostic factors.

Methods: After obtaining Institutional Review Board approval, slides of 81 patients (74 AMN and PMP; and 7 Goblet cell adenocarcinoma (GCA)) were reviewed and their clinico-radiological details were obtained. For grading and staging, 3-tier system was used. Survival analysis was performed to determine outcome of patients in different AJCC grades and to find individual adverse prognostic factors.

Results: For AMN and PMP, the median age was 56 years and median serum CEA was 14.45 ng/ml. There was a statistically significant difference in the Disease-free survival (DFS) of different grades and different histological entities of AMN (LAMN, HAMN, invasive adenocarcinoma, poorly differentiated mucinous adenocarcinomas) and PMP. Cytological atypia grade in appendix and pathological “M” stage showed statistically significant correlation with the DFS. The DFS of GCA was shorter than that of mucinous adenocarcinomas of appendix (G2 and G3). Tumour heterogeneity, scanty material on biopsy, inability to grade on cell block, discordance between biopsy and resection were some of the challenges faced during review.

Conclusion: Grading AMN in three-tier system is feasible and has bearing on the prognosis. Grade of cytological atypia in appendix and pathological M stage were important variables affecting the prognosis. The prognosis of GCA is worse than mucinous adenocarcinomas.


Histopathology and surgery in early-onset inflammatory bowel disease: a Colombian-based cohort study

R.D.P. Lopez-Panqueva*, D. Rubio-Cruz, J.F. Vera Chamorro, B. Mendoza de Molano, E. Londoño-Schimmer, R. García-Duperly, M. Mejía-Arango

*Department of Pathology and Laboratory Medicine Hospital Universitario Fundación Santa Fe de Bogotá/School of Medicine, Universidad de los Andes, Bogotá, Colombia

Background & objectives: Early-Onset Inflammatory Bowel Disease (IBD), both Crohn’s Disease (CD), and Ulcerative Colitis (UC) usually presents with greater severity and often need early surgical intervention. We aim to describe histopathological and surgical characteristics of IBD paediatric patients in a Colombian cohort.

Methods: Longitudinal retrospective study including all patients ≤18 years who had an established diagnosis of IBD from 1977 to 2021, at Fundación Santa Fe de Bogotá. Patients were divided in two groups: patients with and without surgical intervention. A Kaplan-Meier curve was performed to estimate the cumulative risk of needing surgery overtime. Variables were analysed as measures of central tendency.

Results: A total of 34 paediatric patients with IBD where found, 17 with CD and 17 with UC. 18 (58.8%) patients presented with severe disease activity confirmed by histopathology. Five (15%) patients developed low grade dysplasia, on average, after 6.7 years (σ: 2.5) and 20 (60%) required surgical intervention within the first 5 years, 75% with CD and 25% UC. Intestinal resection for fistula treatment, was performed in 50% of the cases, and 41.6% required surgical reintervention. A Kaplan Meier Curve estimated a cumulative risk, for the need of surgery, from the time of diagnosis of IBD with severe activity, of 15% at 1 year and 20% at 5 years.

Conclusion: This study shows severe clinical and histopathological presentation of Early-Onset Inflammatory Bowel Disease in our cohort. Surgical intervention requirement was high among this population and tend to increase overtime. Although, as in the literature, CD patients required more surgical procedures, need for surgery for UC was significant. A high percentage of reinterventions within the next years was also noted. Due to the severity of the disease, early suspicion, and diagnosis of paediatric or early-onset IBD could improve its natural course.


Expression of the ghrelin receptor in GIST

I.A. Spiridon*, S.E. Giusca, D. Ciobanu-Apostol, I.D. Caruntu

*U.M.F. "Grigore T. Popa" Iasi, Romania

Background & objectives: Ghrelin is a hormone exerting complex metabolic functions through coupling with its receptor (GHSR). This axis has documented effects in obesity-related neoplasia, with scarce knowledge on gastrointestinal stromal tumour (GIST) tumorigenesis. We describe the expression pattern of GHSR in GISTs.

Methods: The immunohistochemical (IHC) expression of GHSR was evaluated in 124 cases of GIST (78 gastric and 46 small bowel tumours) from adult patients, diagnosed based on histology and a standard panel of markers (CD117, CD34, DOG1). Statistical analysis was performed in order to correlate the tissue expression of the receptor with the classical clinicopathological factors.

Results: The semi-qualitative expression of GHSR revealed diffusely granular, homogenous, cytoplasmic staining and nuclear immunoreactivity that showed differentiated staining, with heterogenous intratumoral distribution in tumour core versus tumour periphery. An IHC score was developed to integrate these findings, differentiating between tumours with absent (score 0), low expression (score 1) and high expression (score 2) of GHSR. The GHSR score correlated with the presence of myxoid degeneration of the stroma (p<0.001) and showed a statistically relevant association with patient prognosis, assessed through AFIP and m-NIH criteria (p<0.03). Significant correlations with other clinicopathological factors were not confirmed.

Conclusion: Our results demonstrate that GISTs harbour spatial heterogeneity, with tumour cells in the periphery of the proliferation displaying a distinct phenotype when compared to the ones in tumour core. This feature could justify the differences in recurrence rates across tumours with various prognostic staging. The expression of GHSR in GIST is a potential indicator of the involvement of ghrelin axis in tumorigenesis, a promising result which can be further capitalized by molecular analysis of the isoforms GHSR1a and GHSR1b.


Evaluation of tumour-stroma ratio in gastric adenocarcinomas with semi-automatic digital imaging method and its relation with clinicopathological parameters and prognosis

B.E. Gözükara*, A. Gönültaş, A. Sürmelioğlu, F. Aker, M. Doğan

*Haydarpasa Numune Education and Research Hospital, Turkey

Background & objectives: The importance of tumour-stroma ratio (TSR) as a prognostic marker in some cancer types has been demonstrated. The aim of this study is to examine the relationship between TSR and histopathological parameters and survival in gastric adenocarcinomas.

Methods: The study group was formed with 155 cases of gastric resection specimens diagnosed with adenocarcinoma between 2011-2018. Stroma areas were calculated using semi-automatic digital imaging and software (EasyPath, Argenit, Turkey). The cases were grouped as low stroma (<36%) and high stroma (≥36%) with a threshold value of 36% determined. The clinicopathological findings, recurrence, and survival data were compared with TSR.

Results: According to this threshold value, 63.2% (n=98) of the cases had low stroma and 36.8% (n=57) had high stroma. A statistically significant negative correlation was found between TSR and overall survival, disease-free survival and recurrence rate (p:0.001; p<0.01). Of the clinicopathological parameters, only gender and perineural invasion (PNI) were found to be significantly associated with TSR (p=0.031; p<0.05). In Cox multivariable regression analysis, a significant statistical correlation was shown between overall survival and age, stroma ratio, histopathological subtype, LVI, and T stage (p<0.01). It was determined that tumour stroma ratio ≥36 increased the mortality risk of the cases by 2.253 (95% CI: 1.411-3.597) times.

Conclusion: TSR has a strong, independent prognostic value in gastric adenocarcinomas and would therefore be considered for integration into routine pathology practice after evaluation in validation studies with larger series.


Mismatch repair status and PDL-1 expression in gastric carcinoma

Z. Bayramoglu, S. Yilmaz Erozbek*, E. Kuzucular, F. Özden

*Istanbul Medipol University, Pathology Department, Turkey

Background & objectives: Gastric cancer (GC) is the 4th most common cancer worldwide and the 2nd leading cause of cancer-related death. In our study, the relationship between microsatellite instability and programmed death-ligand 1 (PDL-1) expression in GCs and clinicopathological parameters was investigated.

Methods: Immunohistochemical staining for PD-L1 (22C3 clone) expression was performed on 37 cases. Expression was scored in both the tumour and tumour-infiltrating immune cells. Furthermore, tumoral mismatch repair status (MLH1, MSH2, MSH6, PMS1) was evaluated.

Results: Twenty of our patients were male and 17 were female. The mean age was 62.5 (range 34-87). PD-L1 expression, either tumoral or tumour-infiltrating immune cells, was present in 8,10% (3/37) of GCs. Overall mismatch repair deficiency was seen in 27,02% (10/37) of the cases. PDL1 expression was observed in all mismatch repair-deficient cases (3/3).

Conclusion: We found PDL-1 positive in 30% of our patients with mismatch repair deficiency. Thus, gastric cancer patients with mismatch repair deficiency tend to show PD-L1 expression; this specifically indicated that mismatch repair deficiency could be prime candidates for PD-L1-directed therapy. Further studies in larger series are needed to confirm our findings.


Assessment of mucosal lymphatic vessels and regional lymph nodes of in-situ colorectal carcinoma

M.T. Rodrigo, K. Saez De Gordoa*, I. Archilla, S. Lopez-Prades, N. Vidal-Robau, G. Caballero, R. López del Campo, L. Rojo, A. Diaz, M. Cuatrecasas

*Pathology Department, Hospital Clínic, Barcelona, Spain

Background & objectives: Lymph node (LN) metastasis is an important prognostic factor in colorectal carcinoma (CRC). We aimed to demonstrate the presence of lymphatic vessels (LV) in the mucosa of in-situ CRC (pTis), and the analysis of regional LNs using a molecular method.

Methods: This is an observational and retrospective study of surgically resected in-situ CRCs. of LNs were assessed with both the One Step Nucleic Acid Amplification (OSNA) assay and H&E. The OSNA result, or total tumour load (TTL), is the amount of CK19 mRNA copies present in all LNs from a patient. D2-40 immunostaining was performed in both pTis and normal mucosa.

Results: We analysed 39 surgically resected in-situ CRCs. The mean age was 68.6 years-old, 23 (59%) were men, and 22 (56%) were located on the right colon. A median of 16 LNs were freshly dissected per patient. All cases were low-grade, pN0 with H&E and did not receive adjuvant therapy. At follow-up, all patients were alive without disease between 1 and 5 years. All tumours presented LVs in the lamina propria, being negative in normal colon mucosa. We detected 11/39 (28%) patients with positive LNs by OSNA. Positive tumours were more frequent in older men and located in the right colon. The TTL were low, from 400 to 4270 copies/μL.

Conclusion: Despite of pTis is considered to have little or no risk of LN metastasis, this study demonstrates the presence of LVs in the lamina propria of in-situ CRC, and of low amounts of tumour burden in regional LNs, only detected by molecular methods. Nevertheless, this positivity has been demonstrated to confer no clinical significance or risk of recurrence.


The impact of cancer stem cell markers in distal cholagniocarinoma

G. Fontinha*, J. Gama, F. Silva, P. Teixeira, R. Oliveira, M.A. Cipriano

*Centro Hospitalar e Universitario de Coimbra, Department of Pathology, Portugal

Background & objectives: Distal cholangiocarcinoma (dCCA) has a low incidence but exhibits a poor prognosis even in patients submitted to curative resection. This study sought to evaluate the prognostic value of cancer stem cell (CSC) markers in patients with dCCA, after surgical resection.

Methods: Retrospective cohort study with evaluation of all patients submitted to surgical resection due to dCCA, between 2008 and 2019. The primary endpoint is to assess the value of CSC markers in overall survival (OS) and disease-free survival (DFS). Immunostaining for CD44, ALDH1 and CD56 was performed. The study was approved by the local Ethics Committee (CHUC-123-20).

Results: 37 patients were identified, with 62.2% male and 37.8% female, with an average age of 69.19 (±9.92) years. After a median follow-up of 14±23.7 months, the OS was 16±2.8 months and the DFS was 14±5.2 months. CD44 and ALDH1 expression was observed in 34.8% and 26.1% of the evaluated tumours, respectively. No expression of CD56 was registered. In univariate analysis, CD44 (p=0.032) and ALDH1 (p=0.016) expression had influence in OS. Regarding DFS, no influence was verified. Multivariate analysis confirmed these findings: CD44 (HR=0.089, p=0.033) and ALDH1 (HR=9.24, p=0.037). ALDH1 expression was established as an independent worse prognostic factor concerning OS, with CD44 expression being associated with a better prognosis.

Conclusion: This study supports the role of CSC markers as predictors of OS in dCCA. ALDH1 expression was associated with worse OS, which is related to the more aggressive biological behaviour of these cells. CD44 was unexpectedly associated with a better OS, which may be explained by the fact that in our study the majority of CD44 positive tumours were small and at an initial stage (T1/T2). More studies are needed to clarify this role.


Agreement in tumour budding and poorly differentiated cluster detection in consecutive digitalized images with focus on mimickers, serial images and expertise

M. Jimeno*, R. López-Martos, J.P. Fernández, M.Á. Serrano, S. Roca, A. Sánchez-Gil

*Hospital Universitari Germans Trias i Pujol, Spain

Background & objectives: Tumour budding (TB) is a recognized adverse prognostic factor in colorectal cancer (CRC) but agreement on its quantification varies significantly. We aim to study interobserver agreement using digitalized slides with an emphasis in mimickers, serial images and observers’ experience.

Methods: Three consecutive H&E - CK AE1.3 - H&E digitalized images were obtained from 30 CRC cases, using a “hotspot” study area of 0.785 mm2 for each. Three observers with differing experience reviewed the 90 images, reporting the number of TB and poorly differentiated clusters (PDF) in addition to false positive images. For agreement analysis, the intraclass correlation coefficient was applied.

Results: Analysis showed moderate agreement values for TB and PDC detection in H&E images. Despite the presence of abundant mimickers in CK sections, TB and PDC detection was facilitated, and a higher correlation was observed, especially for cases with moderate or severe inflammation. No significant disparity in slide assessment was evident between the 3 observers.

Consecutive sections have shown a significant evolution of images with variable size and number of small groups of cells in only 10 microns thick. A notable number of cases have shown a significant progression towards a lower or higher TB/PDC number, that is enough to modify the final grade.

Conclusion: Despite reports of moderate to substantial agreement with respect to TB or PDC grade, agreement with respect to individual cell groups is moderate at best. Most studies published to date have selected representative slides but no specific area to be evaluated, leading to intrinsic selection bias. Cell morphology evolution observed in serial images evidence the need of algorithm-based analyses of larger areas to obtain an average value that could be translated into a powerful and reproducible prognostic tool.


Automated tumour budding quantification in T1 colorectal carcinoma H&E slides: association to lymph node metastasis

I. Nearchou*, Y. Kajiwara, M. Ueno, K. Kouzu, T. Nakamura, K. Lillard, H. Ueno

*Indica Labs, Inc., USA

Background & objectives: Tumour budding (BD) is a significant predictor of lymph node metastasis (LNM) in T1 colorectal cancer (CRC). However, the reproducibility of BD-scoring remains sub-optimal. This study aimed to automatically quantify BD on H&E slides and assess its association with LNM.

Methods: A deep-learning algorithm was applied on 197 T1 CRC H&E slides to automatically detect BD using HALO® and HALO AI™ image analysis platform. Univariate and multivariate logistic regressions were performed to assess the predictive value of BD and other features currently assessed in the clinic. The Akaike information criterion was used to identify the model with the highest predictive value.

Results: Various automated BD quantification methods were employed including recording the number and density of buds across the entire invasive front (IF) and at a hotspot area. BD number and density assessed on all slides containing the IF (p=0.001 and p=0.001) as well as on a single slide with highest BD (p=0.004 and p=0.013) were shown to be significantly associated with LNM when assessed using univariate logistic regression. The model with highest predictive value for LNM included BD density assessed on all slides, which was also the most significant feature (p<0.001), tumour grade (p<0.001) , lymphovascular invasion (p=0.285) and submucosal involvement depth (p=0.044).

Conclusion: Here, we demonstrate that the use of deep-learning algorithms could prove to be promising for the objective, standardised and reproducible BD quantification in H&E slides as well as for assisting pathologists during treatment decision making.

Funding: This study was funded by the Japan Society for the Promotion of Science. Indica Labs, Inc. provided in-kind resources.


Inter-observer concordance in the measurement of histological risk factors in pT1 colorectal adenocarcinomas

K. Saez De Gordoa*, I. Archilla, M. Rodrigo-Calvo, J.J. Aguirre, M. Camara Jurado, A. Canosa, F. Giner, M. Jimeno, I. Jurado, S. Landolfi, A. Lloret, I. Machado, C. Martínez-Ciarpaglini, E. Musulen, D. Naranjo, N. Papaleo, Ò. Rosiñol, R. Sanchez Yuste, G.T. Vázquez Benítez, M. Cuatrecasas

*Pathology Department, Hospital Clínic, Barcelona, Spain

Background & objectives: Identification of risk factors associated with lymph node metastasis (LNM) in pT1 colorectal carcinoma (CRC) is performed by histological evaluation. The aim of this study is to assess the inter-observer agreement in the evaluation of histological risk factors for LNM.

Methods: Scanned slides from 10 pT1 CRCs were assessed by 22 pathologists for submucosal infiltration depth (SID), lymphovascular invasion (LVI), histological grade (HG), and tumour budding (TB). The LVI, HG and TB concordance were calculated with percentage of agreement and Krippendorff’s alpha; intraclass correlation coefficient (ICC) for SID. An ulterior consensus meeting was held to analyse differences in histologic criteria assessment.

Results: Seven tumours were resected by endoscopy, and three by surgery. Four pT1 arose on pedunculated polyps, and six on sessile polyps. The evaluation of SID had moderate reliability, with an average ICC (two-way random-effects model) of 0.59 (95% confidence interval 0.12-0.88). LVI had the highest agreement rate (83.64%). TB had 71.36% of agreement and Krippendorff's alpha (α) of 0.13. HG had 64.44% of agreement and α = 0.26. In the consensus meeting the use of the measuring tool was corrected, as it generated discordances. An agreement on the criteria for assessing SID for pedunculated and sessile polyps was also reached, and the criteria for LVI, TB and HG were reviewed.

Conclusion: This is a pilot ongoing study. Lack of consistency in the evaluation of some prognostic factors by pathologists was mostly due to differences in measurement criteria, as well as the small number of cases evaluated by many pathologists.


Diagnostic utility of CK20, SATB2, CDH17, and Villin for the identification of gastrointestinal tumour origin: a tissue microarray study on 7,711 tumours of 117 tumour entities

D. Dum, P. Lebok, M. Lennartz, S. Weidemann, E. Burandt, C. Fraune, R. Simon, T. Krech, F. Büscheck, T.S. Clauditz, F. Jacobsen*

*University Medical Center Hamburg, Germany

Background & objectives: Adenocarcinomas of the gastrointestinal tract (GIT) represent a common source of liver and lung metastasis. Markers used to distinguish metastases from GIT tumours include CK20, SATB2, CDH17, and villin.

Methods: To comparatively assess the staining patterns of these markers across a broad range of different tumour entities, tissue microarrays containing 7711 neoplasms from 117 different tumour types and subtypes was analysed by immunohistochemistry.

Results: Positivity for CK20, SATB2, CDH17, and villin was seen in 94.4%, 88.1%, 98.3%, and 96.7% of colorectal, 36.4%, 22.4%, 52.8%, and 72.4% of gastric, 38.3%, 15%, 51.7%, and 65% of oesophageal adenocarcinomas, 49.1%, 20.8%, 69.8%, and 66% of adenocarcinomas of the ampulla of Vater, 19.4%, 1.4%, 40.3%, and 54.9% of pancreatic ductal adenocarcinomas, 8.7%, 2.2%, 2.2%, and 23.9% of hepatocellular carcinomas, and in 5.5%, 10.1%, 12.8%, and 12.8% of pulmonary adenocarcinomas. A positivity of ≥3 (or all 4) markers occurred in 96.2% (83.2%) of colorectal, 33.2% (13.3%) of gastric, 23.2% (11.7%) of oesophageal, 8.3% (0%) of ductal pancreatic, 0.9% (0%) of pulmonary adenocarcinomas, and in 0% (0%) of hepatocellular carcinomas.

Conclusion: All individual markers support the distinction of GIT adenocarcinomas from liver and lung cancer while a combined analysis of multiple markers may increase diagnostic accuracy. Characteristic staining patterns also occur in other entities. For example, Villin expression is linked to neuroendocrine neoplasms and yolk sac tumours. CDH17 is often positive in in neuroendocrine neoplasms. CK20 is linked to urothelial neoplasms and Merkel cell carcinomas. SATB2 occurs in Merkel cell carcinomas, renal cell carcinomas, and several mesenchymal tumour entities.


Nonconventional dysplasia in inflammatory bowel disease associated colorectal adenocarcinoma: a clinicopathologic study of twenty-four cases

T. Dot Gómara, S. Rázquin Lizarraga, T. Labiano Miravalles, G. Aísa Rivera, M.C. Llanos Chavarri, M. Bronte Anaut, I. Sánchez Sánchez, E.A. Sierra Arellano, R. Unciti Ruiz, Á. Panizo Santos*

*Navarra University Hospital, Spain

Background & objectives: Several types of nonconventional dysplasia have been recently described in inflammatory bowel disease (IBD). However, strict morphologic criteria are lacking, and their clinicopathologic features including potential association with conventional dysplasia and/or colorectal cancer are poorly understood.

Methods: A total of 24 IBD-associated CRC (IBD-CRC) colectomy specimens of 22 patients were reviewed. Seven morphologic patterns were recognized: hypermucinous dysplasia, traditional serrated adenoma-like, sessile serrated lesion-like and serrated lesion, not otherwise specified, Paneth cell differentiation and goblet cell deficient dysplasia. Lesions were classified according to the WHO 2019 criteria and literature review.

Results: We identified 149 dysplastic lesions and occurred with similar frequency in men and women (n=17 and n=5, respectively), with a mean age of 57 years (range: 34-82) with long history (mean: 9,6 years, range: 2-27) of ulcerative colitis (n=11, 42%) and Crohn's disease (n=13, 58%). All nonconventional dysplasia types were common (55% of the cases), present as focal or extended, pure or mixed, in peritumoral and remote mucosa. Tumours were more likely to be well-differentiated (43%), left-sited (58%), with mucinous features (33%) and signet-ring cell (12.5%). Many cases were deeply invasive (62% were pT3 or pT4) and 54% had lymph node metastasis.

Conclusion: Clinicopathological characteristics of IBD-associated CRC were significantly different from sporadic colorectal adenocarcinoma. Histopathological findings of nonconventional patterns of dysplasia were common in IBD-CRCs. Most cases had mixed and focal or extended features of all nonconventional dysplasia types. Nonconventional dysplasia occurred with similar frequency in ulcerative colitis and Crohn's disease. We did not find association between nonconventional dysplasia type and characteristics of IBD-CRC.


Clinicopathological study of retroperitoneal margin invasion in right radical hemicolectomy with colon cancer

M.F. Molina-Centelles*, P. Guerrero Antolino, M. Frasson, I. Rienda Martínez, N. Rausell Fontestad, C. Zac Romero, D. Ramos Soler, E. García-Granero Ximénez, F. Giner Segura

*Hospital Universitari i Politècnic La Fe de València, Spain

Background & objectives: There is high variability in relapse rate among series (3-14%) after right radical hemicolectomy. Retroperitoneal margin is a well-recognized parameter, frequently forgotten. The objective is to assess the invasion of retroperitoneal margin in pathologic specimens after radical right hemicolectomy.

Methods: Prospective histopathologic and clinical analysis of 79 patients who underwent right hemicolectomy (2017-2019). Retroperitoneal margin was inked and measured macro- and microscopically and was defined as affected when tumour distance was less or equal to 1mm. Association between retroperitoneal margin invasion and other histological and oncological results was analysed.

Results: Involvement of retroperitoneal margin was found in 15 cases (18.98%) and was significantly associated with more advanced degrees of dedifferentiation (G2: p=0,017; G3: p=0,037) tumour budding (intermediate grade: p=0.028; high grade: p=0.005), presence of poorly differentiated groups (p=0,039) and perineural invasion signs (p=0,044). The involvement of the retroperitoneal margin was associated with a worse overall survival (p=<0.01) and worse overall recurrence (p=<0.01).

Conclusion: Retroperitoneal surgical margin resection involvement could be considered an anatomopathological marker of poor prognosis and greater incidence of relapse after oncological right colectomy. Tumours involving this circumferential margin show features of aggressive behaviour such as increased degrees of dedifferentiation, tumour budding, presence of poorly differentiated groups and perineural invasion. A greater number of cases and longer follow-up term are needed to confirm the clinical significance of this parameter.


Haemorrhoidectomy specimens: incidental malignant lesions. An eight-year retrospective study with eleven case reports

G.G. Yange Zambrano*, G. Moreno-De-Juan, J.C. Yange-Zambrano, I. Gallego-Gutierrez, N. González-Ortega, M.d.M. Del-Barrio-Molina, M. Novell-Grau

*Hospital Mateu Orfila, Spain

Background & objectives: The prevalence of haemorrhoids is estimated at 4.4% (up-to 13-20%). Findings of malignant lesions in haemorrhoidectomy specimens are infrequent. The objective of this study is to be aware of the incidence of malignant lesions found in haemorrhoidectomy-specimens, not clinically suspected.

Methods: We carried out an eight-years retrospective study of haemorrhoidectomy specimens collected of the Mateu Orfila Hospital (Menorca), Spain. All samples coded as “haemorrhoidectomy” and/or “haemorrhoid” between 2013 and 2021, were selected from the Pathology Department database. Clinicopathological and demographic variables were collected from the electronic clinical records.

Results: 444 specimens from 246 men and 198 women, were analysed. The mean age was 45 years (range 20-84 years). Incidental findings were identified in 11 specimens (6 men, 5 women): 1.-Squamous Cell Carcinoma (SCC) and Anal Intraepithelial Neoplasia (AIN) III;2.-SCC-in-situ;3.-AIN II-III;4.-Neuroendocrine Tumour (NET) poorly-differentiated;5.-Adenocarcinoma well-differentiated and AIN-II;6.-AIN-II;7.-AIN I-II;8.-Malignant Melanoma (MM);9.-Squamous Cell Carcinoma (SCC) and AIN-III;10.-AIN-II;11.-AIN-I. The resection-margins were positive in three cases, negative in three and could not be determined in five cases. Cases 1 and 2 had HIV. Cases with only AIN and cases 1 and 3 had clinical follow-up. The SCC-in-situ, margin expansion was performed. Case 9 received QT-RDT. NET and MM cases were treated at another centre.

Conclusion: In our study the incidence of malignant lesions found incidentally in haemorrhoidectomy specimens was 2.47%. We consider that the surgical margins should be stained whenever possible and include a good representation of the material sent from the haemorrhoidectomy specimens, because although it is not frequent, we can find malignant lesions in these samples; and if so, we should try to provide as much histological information as possible.


Prognostic value of Decorin expression at the invasive front of colorectal adenocarcinomas

D. Bouklas, A. Kostopoulou, A.C. Lazaris, N. Kavantzas, D. Tiniakos*, A. Gklavas, E. Kapeni, D. Karandrea, G.E. Thomopoulou

*Newcastle University, Dept. of Pathology, UK; Nat. and Kapodistrian University of Athens, Greece

Background & objectives: Tumour budding (TB) and tumour-stroma ratio (TSR) are independent prognostic factors in colorectal carcinomas. Our aim was to assess the expression of small leucine-rich protein (SLRP) Decorin qualitatively and semi-quantitatively and to correlate this with TB, TSR and stromal desmoplasia.

Methods: Eighty-six cases of colorectal adenocarcinoma were used to generate tissue microarrays that were stained with Masson trichrome, reticulin, orcein, and immunostained with pankeratin, decorin, CD8, and PDL1. Information about mismatch repair proteins (MMR) and Eosinophil leucocyte density were known. Decorin mRNA was assessed with rtPCR.

Results: Cases were subdivided into 4 groups: Group Α 18% TB-HIGH/TSR-HIGH, Group B 14% TB-HIGH/TSR-LOW, Group C 21% TB-LOW/TSR-HIGH, Group D 40% TB-LOW/TSR-LOW. Decorin mRNA and protein expression were decreased in TB regions (p<0.003) and TSR regions (p<0.001). Decorin expression was also decreased in desmoplastic characterized areas. Decorin was a more sensitive indicator of stromal changes at the invasive front when compared with the morphologic assessment of collagen deposition and desmoplasia (p<0.001). There was no correlation between decorin expression and clinicopathologic parameters, MMR and Eosinophil density. Survival analysis showed no prognostic significance of Decorin protein and mRNA expression.

Conclusion: Decorin expression is decreased at the tumour front and in both the TB and TSR annotated regions. Decorin is a more sensitive indicator of the initial stromal changes that take place at the tumour-stroma borders compared to morphological assessment of desmoplasia. Decorin expression appears of no prognostic value in colorectal adenocarcinomas in our cohort.


Simulated digital gastric cancer endoscopic biopsies and surgical resections stained with PD-L1 IHC 22C3 pharmDx present similar PD-L1 expression when scored using combined positive score

D. Monaco, J. Quiroz, E. Olander*, K. Kulangara, S. Hund, L. Peltz, E. Manna, S. Tabuena-Frolli

*Agilent Technologies, USA

Background & objectives: The dynamic range of PD-L1 expression in endoscopic biopsies of gastric cancer (GC) is not as well-documented as that of surgical resections. This abstract aims to demonstrate the range of PD-L1 expression in simulated digital endoscopic biopsies (SBs) of GC.

Methods: PD-L1 expression in GC SBs (simulated via ImageScope software from surgical resections stained with PD-L1 IHC 22C3 pharmDx) was evaluated in whole-slide images using combined positive score. Scores are determined by dividing the number of viable PD-L1-stained tumour cells and tumour-infiltrating immune cells by the total number of viable tumour cells. Totals are multiplied by 100 and displayed as integers.

Results: Of the 231 GC (including both gastric and gastroesophageal junction adenocarcinoma) SB samples, the average denominator, determined using QuPath, was found to be 11,742 tumour cells. In contrast, the average denominator of the 20 resection samples was 231,623 cells. Despite this greater than 19-fold difference in number of tumour cells, the distribution of combined positive score (CPS) scores remained similar in both sample types. SBs and surgical resections demonstrated similar distributions of CPS scores in terms of minimum, maximum, 25th, 50th, and 75th percentiles. In addition, the average CPS scores for SBs and surgical resections were approximately CPS 24 and CPS 25, respectively.

Conclusion: These results suggest that, although the tumour cell counts of SBs are substantially lower than for resection samples, both present a dynamic range of PD-L1 expression. This suggests that GC SBs demonstrate a PD-L1 expression range similar to that of surgical resections, improving confidence in the evaluation of both small and large samples using CPS.


Impact of the Covid-19 crisis on digestive cancers operated on in a Tunisian hospital centre

M. Ben Thayer, F. Khanchel, I. Helal, S. Elfekih*, R. Hedhli, E. Ben Brahim, R. Jouini, A. Chadli

*Habib Thameur Hospital, Tunisia

Background & objectives: Several studies have assessed the impact of the Covid-19 crisis on cancers around the world. However, no similar study has been conducted in Tunisia. We aim to Study the impact of Covid-19 crisis on the anatomopathological characteristics of different digestive cancers.

Methods: We present a retrospective study conducted on cases of digestive carcinomas: colorectal (CRC), gastric (GC), pancreatic (PC) and gastrointestinal stromal tumours (GIST) operated on at Habib Thameur Hospital in between the first December 2016 and June 30, 2021.We subdivided these cases into two groups according to the date of their operation (before/during and after the lockdown) and we compared their histoprognostic characteristics.

Results: These were 270 cases, including 83 diagnosed during and after confinement (30.7%). These were 219 cases of CRC, 22 cases of GC, 13 cases of PC and 16 GIST. Among the 83 cases operated starting from the lockdown, 12 were grade 3, 72 stage T3 or T4, 37 had lymph node metastases, one had visceral metastases, 27 presented vascular emboli and 31 had perineural invasion. Carcinomas operated on starting from the lockdown were statistically significantly correlated with visceral metastasis (p=0.01) and a more pejorative histological grade (p<10-3). However we did not find any statistically significant difference concerning the stage T, Nstatus, vascular emboli, perineural invasion and stage.

Conclusion: From the onset of the Covid-19 crisis, the political and the health institutional policies were to avoid delaying management of cancerous pathologies. Our study showed that Covid-19 pandemic clearly affected the prognosis of cancer with significantly increased risk of high grade cases and metastases. This could be explained by the fact that patients avoided seeking for medical assistance for fear to be exposed toCovid-19 in health structures. Further studies are needed to assess the impact of this crisis on the survival of cancer patients.


Prognostic relevance of tumour budding and poorly differentiated clusters in gastric adenocarcinoma

É. Kocsmár*, L. Szalai, Á. Jakab, I. Kocsmár, I. Szirtes, A. Szijártó, Z. Schaff, A. Kiss, G. Lotz

*Department of Pathology, Forensic and Insurance Medicine, Semmelweis University, Hungary

Background & objectives: Tumour budding (TBing) is a prognostic factor in colorectal cancer and recent studies indicated similar role of poorly differentiated clusters (PDC). However, their prognostic value is still controversial in gastric adenocarcinoma (GAC), thus our aim was to determine it.

Methods: HE slides of 290 GAC patients was investigated. TB was defined as an isolated tumour cell or a tumour cell cluster of up to four cells, while PDC as a tumour cell cluster of five or more cells. TBs and PDCs were evaluated according to the International Tumour Budding Consensus Conference and divided into low- (Grade1+2) and high (Grade3) groups.

Results: Univariable (Kaplan-Meier) analysis revealed significant negative correlation of both TBing (p=0,0001) and PDCs (p=0,02) with overall survival (OS) in the total cohort and TBing in the intestinal GACs. No significant correlation was found regarding the diffuse and mixed Lauren type GACs. Significant negative correlation with OS was also found by multivariable (Cox) analysis of PDCs in the total cohort, moreover in case of both TBing and PDCs in the intestinal type GACs. Logistic regression analysis with backward selection showed a significant positive correlation of TBing with the presence of regional lymph node metastasis (p=0.009).

Conclusion: Both tumour budding and PDCs are promising prognostic markers of gastric adenocarcinomas. High tumour budding is associated with increased risk of nodal metastasis.

Funding: This study was partly supported by the New National Excellence Program (ÚNKP-21-4-I-SE-32) of the Ministry for Innovation and Technology, Hungary.


Microsatellite instability status and tumour regression grading on gastric and gastroesophageal junction adenocarcinoma – the first portrait of a Portuguese reality

R. Moiteiro da Cruz*, V. Lobo, C. Melo-Alvim, R. Luís

*Department of Pathology - Hospital de Santa Maria, CHULN; Faculdade de Medicina da Universidade de Lisboa, Portugal

Background & objectives: Locally advanced gastroesophageal junction and gastric adenocarcinoma (GEJGA) usually carry a poor prognosis; a molecular subgroup is associated with microsatellite instability (MSI), whose chemotherapy response has not been well characterized yet. Our aim was to evaluate tumour regression (TR), stratified by MSI status.

Methods: A retrospective and unicentric cohort study was assembled, encompassing patients submitted to surgery and perioperative chemotherapy (pQT) in 2015-2020. Clinical data regarding pre and post-treatment features, surgical approach, pQT schemes and follow-up was collected. All histological samples were reviewed by two observers; TR grading was reassessed, and mismatch repair proteins (MMRp) expression was evaluated through immunohistochemistry on tissue micro-arrays.

Results: Out of a total of 64 patients with available tissue samples, only 42 displayed residual tumour. Patients were mostly male (64%), ranging from 60 to 72-years-old (median:67). The series comprised 38,1%(n=16) were moderately differentiated adenocarcinomas and 42.9%(n=18) were poorly differentiated adenocarcinomas (G3) with 14,3%(n=6) were poorly cohesive, the majority of were located in the antrum (n=19); most tumours (57,1%) revealed less than 50% of TR (grade 3). From our sample, 40.5%(n=17) have anomalous MMRp expression and compared with tumours with preserved MMRp expression, the former group showed a statistically significant (p=0.024) worse tumour regression after pQT. However, there was no statistical difference between overall survival (p=0.603) and progression-free survival (p=0.823).

Conclusion: In our sample, defective MMRp neoplasms showed worse response to pQT, whereas anomalous and preserved MMRp groups did not seem to differ on prognosis. These new findings in the portuguese context, even if stemming from a limited series, are in line with emerging evidence suggesting the negative role of pQT on MSI tumours and might aid in prompting routine evaluation of MSI on GEJGA for therapy selection in the future.


Duodenal adenoma in familial adenomatous polyposis (FAP), a case series

M. Miralles*, C. Fumagalli, T. Vázquez del Olmo, A. Gallardo Alcañiz, M.C. Campos Mármol, A. Prat Cantoral, D. Piñol Ballús, J. Szafranska

*Hospital de la Santa Creu i Sant Pau, Spain

Background & objectives: Duodenal adenomas are rare tumours which mostly occur in individuals with familial adenomatous polyposis (FAP) and they can reveal abundant neuroendocrine or Paneth cells’ differentiation. Our goal is to describe clinical and pathological differences between sporadic and FAP-associated duodenal adenomas.

Methods: Twenty-eight cases of duodenal adenomas diagnosed at our institution (from 2010 to 2022) have been collected. Nine patients (pts) were in FAP (group 1), whilst in the other 19 cases FAP was not clinically suspected (group 2). We revised the histology and performed additional immunohistochemical stains for synaptophysin, chromogranin, CDX2, MUC2, MUC5 and MUC6.

Results: The mean age was 44.3 years in group1 and 71.5 years in group2(p=0.001). In group1, 67% of patients were women while in group2 73.7% were men. In both groups, duodenal adenomas were found more frequently in locations other than ampullary (67% and 73.7%, respectively), being multifocal in 56% of group1 and mostly unifocal in group2 (89,5%)(p=0,020). A moderate/high number of Paneth cells was found in 67% of group1, while the vast majority (84.2%) of group2-adenomas had an absence/few Paneth cells(p 0,013). 68.4% of group1-adenomas had moderate/high chromogranin expression compared to 31.6% of group2. Another finding was the presence of neuroendocrine hyperplasia in four cases, one of which was in FAP-group.

Conclusion: Duodenal adenomas associated with FAP usually occur in younger patients and are frequently multifocal. Histologically, they show a significantly higher number of Paneth cells and an increased chromogranin expression was found in our series. Occasionally, neuroendocrine hyperplasia within the adenomas can be observed. Nevertheless, data does not suggest a higher probability of existence of neuroendocrine tumours in adenomas associated with FAP.


Spasmolytic polypeptide-expressing metaplasia as a marker for gastric cancer risk prediction

S. Mozgovoi*, M. Stepanchenko, A. Gubanova, M. Parygina, J. Fedotova, M. Livzan

*Omsk State Medical University, Russia

Background & objectives: Assessment of chronic atrophic gastritis is the key factor in gastric cancer preventing strategy. Spasmolytic polypeptide-expressing metaplasia (SPEM) can be indicated as possible marker of extensive gastric atrophy with following association of gastric cancer development.

Methods: Resection specimens (n=30) from patients who had gastrectomy due to invasive gastric adenocarcinoma were obtained, processed routinely, stained with H&E. Pseudopyloric metaplasia (PPM) was recognised as antral-type glands in oxyntic mucosa. Microarray method was used to prepare specimens for IHC. Tissue specimens were stained with MUC6, TFF2 and pepsinogen-1 (PG-1) antibodies to reveal either PPM or SPEM accordingly.

Results: Widespread (involving 4-5 glands) PPM was detected in 14 cases. In 9 cases single (3-4 glands) areas were determined, in 7 cases pseudopyloric metaplasia wasn't detected. Foci of metaplastic changes were distributed as follows: PMM sites were found at distance of 4-7 cm from the border of invasive tumour. In 3 cases, a combination of epithelial dysplasia, intestinal metaplasia, and PPM was determined. Expression of PG-1 was found in 12 cases, in 1 case expression was artificial. TFF2+ glands were determined in 21 cases, including sites nearest to dysplasia foci. SPEM foci were designated as a part of true pyloric metaplasia (PPG-1 - ) and pseudopyloric metaplasia (PPG-1 +) .

Conclusion: Distribution of PPM wasn’t perifocal as expected, but found mostly at the distant zone of adenocarcinoma, although in some cases was nearby dysplasia foci. The expression of TFF2 and PG-1 didn’t always overlap and not all PPM glands could be considered a SPEM. The significance of these findings is yet to be understood, but recognising pseudopyloric/SPEM metaplasia in biopsy specimens could potentially help clinicians to reveal patients with considerably higher risk for gastric cancer.


Concurrent loss of MLH1, PMS2 and MSH6 immunoexpression in gastrointestinal cancer indicating a widespread dysregulation in DNA reparation processes

N. Reitsam*, S. Dintner, A. Probst, D. Vlasenko, J. Enke, B. Märkl, B. Grosser

*General Pathology and Molecular Diagnostics, University Hospital of Augsburg, Germany

Background & objectives: DNA mismatch repair proteins function as heterodimers (MLH1/PMS2; MSH2/MSH6), usually resulting in deficiency in one subsystem in microsatellite instable cancers. As the involvement of both subsystems is unusual, we aim to shed light on the molecular basis of this phenomenon.

Methods: We retrospectively analysed gastrointestinal cancers that underwent immunohistochemical testing for deficient DNA mismatch repair proteins during the last four years in our hospital to identify cases with a simultaneous deficiency in MLH1/PMS2 and MSH2/MSH6. To understand this unusual phenomenon, we performed further molecular testing (MSI-PCR, MLH1 promotor and/or BRAF status) and next-generation sequencing focusing on genes related to DNA reparation.

Results: In the cohort, we could identify 103 cases with deficient DNA mismatch repair proteins. In nearly all cases only one mismatch repair heterodimer - either MLH1/PMS2 or MSH2/MSH6 - was affected. However, five cases showed a concurrent loss of MLH1/PMS2 and MSH6. Whereas some cases seem to be sporadic with a MLH1 promotor hypermethylation and/or BRAF V600E mutation, we also could detect potential germ line mutations. Importantly, next-generation sequencing indicates that the concurrent loss of MLH1/PMS2 and MSH6 expression is associated with a dysregulation in DNA reparation as all of these cases showed additional mutations in genes coding for proteins like ATM, BRCA2, BARD1, CHEK1, FANCA, PALB2, RAD54L and RAD51D.

Conclusion: Our study suggests that the simultaneous loss of MLH1, PMS2 and MSH6 immunoexpression among different gastrointestinal cancers is associated with and potentially even based on more widespread alterations in DNA repair processes. Next-generation sequencing could reveal further mutations in additional DNA repair-related genes in the present cases. With the advent of drugs targeting DNA repair in clinical practice, especially PARP-inhibitors, and their potential indication extension for mutations other than BRCA1/2 such as PALB2 our results are of immediate clinical significance.


Intestinal spirochetosis: clinicopathological features of 22 year experience in a single institution

A. Pasco Peña*, M. Mercado, A. Larrea, C. Cerezo, C. Fuertes, I. Amat, A. Cordoba

*Hospital Universitario de Navarra, Spain

Background & objectives: Intestinal spirochetosis (IS) is an infrequent infection and its clinical significance in individual cases has remained unclear as their association with other diseases. We report the clinicopathological features of the patients diagnosed in our hospital in a 22 years experience.

Methods: Retrospective and descriptive study from the patients with a diagnosis of intestinal spirochetosis from 2000-2021 in We histologically reviewed paraffin-embedded section slides made from 2000 to 2021at Navarras University Hospital.

Clinical records were reviewed for each one of the patients for epidemiological features, treatment and follow up.

Results: We identified 29 cases of IS. Median age was 48 years old (25 - 76 years old). 79% were males. In 69% of the patients an endoscopic ultrasound was performed due to gastrointestinal symptoms.

The most common symptom was diarrhoea (44, 8%). 27, 6% of the patients were asymptomatic. 17,2% were in the colorectal cancer screening program.

48,3% of the patients were treated with antibiotic (metronidazole). 24,1% of the patients were HIV positive.

Transverse colon was the most common localisation (37,9%), 24,7% of the patients had more than one affected colonic segment.

Six patients (20,7%) had an associated polyp (3 tubular adenomas, 2 hyperplastic polyps and one juvenile polyp).

Conclusion: Intestinal spirochetosis is more frequent in young population and in male homosexuals. It presents as a symptomatic disease in immunosupresed patients and is very rare symptomatic in other the population except in children. The most common symptom is diarrhoea. Patients treated with antibiotics had a favourable outcome. The most common localisation was the transverse colon, but it can affect other colonic segments even in a discontinuous manner. Immunosuppressed patients have a highest risk of multi colonic segment affection.


Crypt epithelial apoptosis: sounds like IBD is on the horizon...

H. Berber*, A.T. Bol, E. Dicle Serbes, S. Kurt, Z. Kuloglu, A. Kansu, M. Toruner, B. Savas, A. Ensari

*Ankara University Medical School, Pathology Department, Turkey

Background & objectives: In inflammatory bowel disease(IBD), inflammatory crypt damage leads to shedding of epithelium, manifesting as increased crypt epithelial apoptosis which is usually overlooked. Here, we focused on crypt epithelial apoptosis and other histopathologic features of colitis in paediatric and adult IBD.

Methods: Initial diagnostic biopsies containing at least five different sites taken from adult(n=38) and paediatric(n=29) IBD cases were evaluated retrospectively. Histopathologic features of colitis, cryptitis in terms of extent and location (basal/midzonal/full-thickness), crypt abscesses (neutrophilic, mixed, apoptotic) and crypt epithelial apoptosis graded as (0:none,1:scattered, 2:countable, 3:uncountable) were evaluated in each biopsy. Statistical analysis was performed using Chi-square test and Spearman’s rank correlation.

Results: There were 29 paediatric cases(PC) comprising 20(69%) ulcerative colitis (UC), 9(31%) Crohn's disease (CD) while there were 26 (68.4%) UC, 12 (31.6%) CD in 38 adults. Apoptosis (97%) was the most frequent feature present throughout the colon in all cases in both UC and CD followed by cryptitis (93%) and crypt abscesses (60%) the majority of which were apoptotic or mixed. Apoptosis was significantly more common and more severe in UC compared to CD in left colon (p=0.017). There was positive correlation between grade of apoptosis and full-thickness cryptitis for left colon in PC (r=0.538,p=0.014), for right colon in adults (r=0.447,p=0.042) and for right colon in all cases (r=0.410,p=0,004).

Conclusion: Crypt epithelial apoptosis seems to be more common in UC compared to CD and is correlated with the extent of cryptitis in all age groups. These findings suggest that inflammatory damage to the crypt epithelium leads to apoptotic cell death in the active phase of the disease, since no correlation was found between the features of chronicity and apoptosis in our cohort. We believe that apoptosis may be an early sign of IBD, particularly when drugs are ruled out.

PS-08 | Poster Session Gynaecological Pathology


Heat artifact simulating serous tubal intraepithelial carcinoma: systemic histological analysis of prophylactic fallopian tube resection specimens

M. Fukunaga*, N. Fukunaga

*Shin-Yurigaoka General Hospital, Japan

Background & objectives: Prophylactic fallopian tube resection has been prevailing. Among pathological changes in tubular specimens, serous tubal intraepithelial carcinoma (STIC) is most important for patient management, and heat artifacts simulating STIC should be acknowledged in order to avoid misdiagnosis.

Methods: One thousand consecutive cases of prophylactic fallopian tube resection by laparoscopic excision using an electronic knife our hospital between 2015-2020 were examined, and the characteristic morphology and incidence of heat artifacts were analysed. Two blocks of the distal fallopian tube were prepared for examination in each case. The cases were benign uterine and ovarian disorders.

Results: Heat artifacts were observed in 530 of 1000 cases (53%). Marked changes were found in 118 cases (11.8%), moderate changes in 102(10.2%) and minor changes in 250 cases (25%). Eight cases were initially diagnosed as STIC. No patient had STIC. Histological findings of heat artifacts included cellular pseudo-stratification, a pronounced papillary arrangement and detachment of the epithelium from the connective tissue, mainly in the fimbria. Cytological changes included marked nuclear elongation and smudging, eosinophilic cytoplasm and obliteration of cell boundaries and lack of mitotic figures in the epithelial lining. These findings mimicked STIC. Immunostaining of p53, WT1 and Ki67 performed on 33 representative cases did not indicate STIC.

Conclusion: Heat artifacts from electronic knife usage are not uncommon. The marked papillary pattern of the epithelium was the principal histological characteristic leading to confusion with STIC, likely resulting from the structural characteristics of the tubal fimbria. Heat applied to tissue can produce nuclear elongation, hyperchromatism, smudging of nuclei, eosinophilic cytoplasm, and obliteration of cell boundaries. Awareness of this potential source of diagnostic error leads to its complete avoidance.


Intravenous leiomyomatosis of the uterus: a clinicopathological analysis of nine cases

F. Sassi*, F. Khanchel, R. Jouini, I. Helal, R. Hedhli, E. Ben Brahim, S. Sassi, A. Chadli Debbiche

*Department of Pathology, Charles Nicolle Hospital Tunis, Tunisia

Background & objectives: Intravenous leiomyomatosis (IVL) is a rare and benign smooth muscle neoplasm. It grows intravenously in the uterus and may invades cardiovascular and pulmonary system. The purpose of this study was to analyse clinicopathological data of 9 cases of IVL.

Methods: We retrospectively reviewed 9 patients treated for IVL and diagnosed at Pathology Department from 2008 to 2020. All patients underwent surgical treatment.

Results: Mean age of patients was 41.8 years (22-49 years). The 9 patients presented with no specific symptoms including menorrhagia (n=4), uterine mass (n=2), hypogastralgia (n=2) and dysuria (n=1). The diagnosis was made on myomectomy (n=5) and hysterectomy specimens (n=4). The diagnosis of IVL was suspected macroscopically in 4 cases by the presence of peripheral digitiform tabs or peripheral buds appearing to be endovascular. No intravenous leiomyomas were detected. Microscopically, all the cases showed a proliferation of benign smooth muscle within the vessels. Rare mitoses were noticed. Three patients were lost to follow-up. Six patients who were followed up are still alive and experienced no recurrence after a follow-up of 60months.

Conclusion: IVL can be easily misdiagnosed as symptoms are not suggestive clinically and it can mimic uterine leiomyoma. Radiologists should make an early detection of IVL when there is venous blood flow signals in fissure-like echoe. Hysterectomy is the treatment and myomectomy could be considered when there is fertility needs. Pathologists should perform careful sampling insisting in the surrounding uterine smooth muscle. IVL is yet a benign disease but is considered malignant due to its recurrency. Long-term follow-ups are crucial.


Immunohistochemical expression of neuroendocrine markers in a large cohort of primary ovarian tumours

M. Bártů*, K. Němejcová, P. Dundr, J. Laco, M. Flídrová, R. Jakša, J. Galko, I. Stružinská

*Institute of Pathology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic

Background & objectives: Expression of neuroendocrine markers in primary ovarian tumours without neuroendocrine morphology has only rarely been evaluated and data on its extent is limited. We have analysed neuroendocrine markers expression in tumours lacking neuroendocrine features and assessed its prognostic meaning.

Methods: The cohort consisted of 556 primary ovarian tumours, including serous borderline tumours (mSBT; 42), low grade serous carcinomas (LGSC; 100), high grade serous carcinomas (HGSC; 114), clear cell carcinomas (OCCC; 124), endometroid carcinomas (EOC; 52), mucinous borderline tumours (MBT; 80) and mucinous carcinomas (MC; 44). Immunohistochemical analysis was performed using TMA approach with antibodies against synaptophysin, chromogranin, CD56, and INSM1.

Results: The highest number of positive cases was observed in mucinous tumours and INSM1 marker (60/124; 48%), where the MBT and MC subgroups had similar proportions of positivity (MBT - 42/80; 53% vs. MC – 18/44; 41%). Mucinous tumours also showed the highest expression of synaptophysin (32/124; 26%) and chromogranin (51/124; 41%). In other tumour types, only weak or no expression of neuroendocrine markers was detected, except for HGSC, which showed the highest expression of CD56 (30/114; 26%). The data was statistically processed concerning the extent of neuroendocrine marker expression, and its association with clinicopathologic data was also investigated. Survival analyses showed that neuroendocrine markers have no prognostic significance.

Conclusion: This study examines the expression of neuroendocrine markers in primary ovarian tumours of non-neuroendocrine morphology, with a special focus on providing a comprehensive overview of the staining characteristics of individual tumour types, and the possible significance the expression of these markers could have for differential diagnosis. In terms of patient survival outcomes, the immunohistochemical expression of neuroendocrine markers seems to be of no clinical significance.

This work was supported by Ministry of Health of the Czech Republic (projects NV19-03-00007, RVO64165).


Immunohistochemical expression of NLRP3 inflammasome in endometrial carcinoma

M. Lambropoulou*, V. Papadatou, E. Pappa, S. Tologkos, T. Deftereou, C. Alexiadi, V. Lampropoulou, A. Karatza, T. ChatzIneophytou, G. Tripsianis, O. Pagonopoulou

*Histology-Embryology Lab., Medical Department, Democritus University of Thrace, Alexadroupolis, Greece

Background & objectives: NLRP3 belongs to the inflammasome family, an innate immune system complex. Recent studies have shown its role in the development of different malignancies. We aimed to investigate the NLRP3 expression in endometrial carcinoma and to correlate it with clinicohistopathological parameters.

Methods: Formalin-fixed, paraffin-embedded endometrial tissues from 46 samples were studied. 36 of them diagnosed with endometrial cancer (study group) and the other 10 (control group) came from total hysterectomies due to uterine prolapse. NLRP3’s expression was investigated immunohistochemically using a monoclonal anti-NLRP3 antibody and was also correlated with clinicohistopathological parameters.

Results: NLRP3 has been found to be expressed in endometrial tumour samples more frequently compared to control group. There was a statistically significant correlation between NLRP3 expression and patient’s age (p=0,009), degree of differentiation (p<0,001), stage of cancer (p<0,001), histological type and tumour depth (p<0,001). At the same time a decreased probability of survival (p=0,0003) and an increased mortality rate were clearly observed in samples with high NLRP3 expression (p<0,001).

Conclusion: Our research demonstrated that an overexpression of NLRP3 in endometrial malignancy can accelerate to advanced stage and tumour depth, resulting in poor prognosis and short survival. Although, more studies need to be performed to establish these results, as NLRP3 may be a useful diagnostic -prognostic factor or a therapeutic target in endometrial carcinoma.


Malignant struma ovarii with peritoneal implants: a report of 4 cases with molecular analysis on each site

S. Neyrand*, M. Decaussin-Petrucci, F. Descotes, J. Lopez, M. Devouassoux-Shisheboran

*Pathology Department, Centre Hospitalier Lyon Sud, Hospices Civils de Lyon, Pierre Bénite, France

Background & objectives: This study investigates genomic alterations in a series of 4 malignant struma ovarii (SO), including 2 Highly Differenciated Follicular Carcinoma of the Ovary(HDFCO) with their corresponding peritoneal implants, using nucleic acid sequencing to assess possible molecular profiles predicting clinical behaviour.

Methods: For each case, one representative block from ovarian tumour and one from peritoneal spread were selected. Genomic DNA was sequenced using high throughput sequencing on Illumina sequencer. RNA library preparation was performed following the Archer Fusion-Plex Protocol for Illumina. An in-house panel was used for NGS and RNA-Seq, targeting respectively 87 and 171 major oncogenes and deregulated tumour suppressor genes.

Results: In two cases, ovarian tumour was follicular variant of papillary carcinoma (FV-PTC) with BRAF G469A for one case and NRAS Q61K mutation for the second case. Peritoneal implants were histologically benign-looking and showed respectively BRAF G469A and no mutation.

The third case was composed of a histologically benign struma ovarii in the ovary. Recurrences included a benign struma ovarii on the contralateral ovary, with a TERT promoter deletion, and a benign-looking peritoneal implant without any mutation.

Last case was a histologically benign struma ovarii in the ovary with a peritoneal implant classified as follicular carcinoma and carrying NRAS Q61R mutation. Molecular analysis failed on the ovary.

Conclusion: This study shows that despite being benign-looking, peritoneal implants of struma ovarii can carry classical mutations of thyroid-type cancer: similar mutation to the initial histologically malignant struma ovarii or a novel one. Still, 2 out of 4 peritoneal implants of our series are not mutated.


Expression of programmed death ligand-1 and mismatch repair status in epithelial ovarian carcinomas

M. Rao*, M. Rajendran, P. Elhence, J. Naresh Bharti, P. Singh, G. Yadav, A. Nalwa

*All India Institute of Medical Sciences, Jodhpur, India

Background & objectives: Programmed death ligand -1 (PD-L1) is a co-regulatory molecule which suppresses the local immunity. Mismatch repair (MMR) deficiency has been implicated in the pathogenesis of many malignancies and has been reported to influence response to anti PD-L1 targeted therapy.

Methods: Expression of PD-L1 and MLH1, MSH2, MSH6 and PMS2 was assessed by immunohistochemistry (IHC) on 50 resected cases of epithelial ovarian carcinomas (EOCs).

Results: Mismatch repair deficiency was noted in 15 cases (30% of the cases). PD-L1 expression was noted in 20% of the cases (10 cases) in tumour cells and in 14% of the cases (7 cases) in the tumour infiltrating lymphocytes (TILs). A statistically significant inverse correlation was noted between PD-L1 expression in TILs and PD-L1 expression in tumour cells with extra-ovarian spread of tumour, and between TILs and lymphovascular invasion. There was no statistically significant association between MMR deficiency and PD-L1 expression in tumour cells or PD-L1 expression in TILs.

Conclusion: Approximately one third cases of EOCs showed MMR deficiency and PD-L1 expression was noted in only one sixth to one fifth of the cases. There was no statistically significant association between MMR deficiency and PD-L1 expression in the current study. However, more studies on a larger sample size are required to study relation between MMR status and PD-L1 expression in EOCs.


Oct-4 expression as a pluripotency factor and changes of apoptosis in the uterus during spontaneous and immune-dependent abortions in mice

P. Vishnyakova*, K. Artem’eva, I. Stepanova, I. Bogdanova, M. Boltovskaya, N. Yaglova, S. Obernikhi, E. Ponomarenko

*National Medical Research Center for Obstetrics, Gynaecology and Perinatology named after Academician V.I. Kulakov, Russia

Background & objectives: The balance of apoptosis and proliferation plays an important role in maintaining pregnancy. The aim: to study Oct-4, Bax and Bcl-2 expression in the uterus during spontaneous and immune-dependent abortions in mice.

Methods: 18 mice formed three identical groups. Сontrol-♀CBA/Lacx♂BALB/c, spontaneous abortions(SA)-♀CBA/Lacx♂DBA/2J; immune-dependent abortions(IDA)-♀CBA/Lacx♂BALB/c with intraperitoneal administration of β-heptyl glycoside muramyl dipeptide in dose of ≈1 mg/kg on days 5 and 7 of gestation (DG). Mice were taken out of the experiment on the 8 DG. The uterus were excised and immunohistochemically stained with antibodies to Bax(Δ21), Bcl-2, Oct-4.

Results: The level of embryos resorption in control was 12.5%, in SA- 34.8%, in IDA- 46.7%. Bax(Δ21) expression was detected in the decidua. Staining intensity varied from weak to moderate in control and from moderate to strong in SA and IDA. Bcl-2 was localized in the stromal part of the endometrium in control (intensity staining) and in IDA group (very weak staining), but no staining in SA. Oct4+ cells with nuclear or cytoplasmic staining are localized in the myometrium and perimetrium. The percentage of Oct-4+ nuclei in SA and IDA groups was less than in the control, with negative correlation between Oct-4+ nuclear staining and the level of embryo resorption (r=-0.99,95CI(-0.99;-0.88);p=0.0003)

Conclusion: Thus, the negative correlation between the level of embryo resorption and the percentage of Oct-4+ cells in the myometrium and perimetrium in SA and IDA is probably due to an insufficient increase in the volume of the fetoplacental unit under conditions of enhanced apoptosis.

Funding: The study was carried out within the framework of State Assignment No. 122030200534-4


The effect of a mesenchymal stem cell conditioned medium fraction on uterine healing after full-thickness surgical incision

P. Vishnyakova*, N. Tikhonova, A. Milovanov, V. Alexankina, T. Fokina, A. Alexankin, M. Boltovskaya, N. Nizyaeva, A. Sklifas, A. Temnov

*National Medical Research Center for Obstetrics, Gynaecology and Perinatology named after Academician V.I. Kulakov, Russia

Background & objectives: The mechanism of cesarean scar defect development is unclarified. Treatment of that complication is an important obstetric task. We assessed the effect of the conditioned medium obtained by culturing mesenchymal stem cells with low oxygen content (10%) on uterine healing.

Methods: Component from MSCs was used to treat uterine wound of Sprague Dawley rats (treated group=15, without treatment=11). Histologic examination was performed 5 and 15 days after surgical operation with Mallory staining and monoclonal antibodies to aSMA and CD34. The healing area between damage myometrium and the area of the blood vessels in the healing zone were calculated.

Results: By the 5th day, there was a complete closure of the uterine wall. The area between the separated muscle layers in the treated group was (p<0.05) smaller compared to the control in 5 (p<0.05) and 15 (p<0.05) days. In the control group, the healing area from the 5th to the 15th day increased unreliable (p>0.05). The area of the vessels in the healing zone of treated group was significantly lower compared to control in 5 (p<0.05) and 15 days (p<0.05). Blood vessel area of treated group decreased to 15th day in damage myometrium zone (p<0.05) but not damage perimetrium.

Conclusion: Protein composition obtained by culturing the cells under a reduced content of O2 (10%), significantly influences on size and vascularization of healing area between damage myometrium and perimetrium in uterine wall after full-thickness surgical incision. Area of connective tissue and fibrosis in healing uterine wall after treating the composition decreased that seems to improve uterine wall healing.


Morphological features of the placenta in women with confirmed SARS-CoV-2 infection

P. Vishnyakova*, N. Nizyaeva, E. Dolgopolova, N. Lomova, R. Shmakov, V. Frankevich

*National Medical Research Center for Obstetrics, Gynaecology and Perinatology named after Academician V.I. Kulakov, Russia

Background & objectives: It`s known that angiogenesis with vascular branching failure is characteristic of chronic intrauterine hypoxia. The aim was to assess the morphological features of the placenta in women with SARS-CoV-2+ infection, children whom have suffered from acute hypoxia in labour.

Methods: The main group of the study was women with positive test of SARS-CoV-2 gave birth to a live born children (n=42), and control group (n=40) with negative test to SARS-CoV-2. We performed histological(H&E staining) and immunohistochemical study on the placenta with antibodies to CD34.

Results: It was revealed that in 8.3% there was acute foetal hypoxia in second stage of the labour, which required operative delivery. We observed a higher percentage of infarctions, the presence of thrombosis of the chorionic vessels, intervillous and subamniotic hematomas in the main study group as compared to the control group (p<0,05). Histological examination revealed angiogenesis with a predominance of vascular branching (more than 10 blood vessels in one terminal villus, comparison 3-7 as normally) (p<0,05). Intrauterine hypoxia and depletion of compensation mechanisms, even with even week exposure (uterine contractile activity, umbilical cord compression, and others), there is a breakdown of adaptation mechanisms and the development of acute foetal hypoxia.

Conclusion: Thus, in physiological conditions, the compensatory capabilities of the placenta provide a high degree of resistance of the mother-placenta-foetus system to acute oxygen deficiency. The development of clinically foetal hypoxia during labour in most cases is due to morpho-functional disorders of the placenta that formed in the antenatal period.


PD-L1 IHC 22C3 pharmDx: Analytical validation on cervical cancer specimens

J. Musser*, T. Evans, A. Posch, J. Vasquez, S. Tabuena-Frolli, A. Apostolaki, K. Kulangara

*Agilent Technologies, USA

Background & objectives: PD-L1 IHC 22C3 pharmDx is a qualitative immunohistochemical assay being developed to detect PD-L1 expression in formalin-fixed, paraffin embedded (FFPE) cervical cancer tissue. We report analytical studies using PD-L1 IHC 22C3 pharmDx in cervical cancer specimens.

Methods: Blinded and randomized studies were performed on FFPE cervical specimens, including adenocarcinoma and squamous cell carcinoma. Studies included Sensitivity of PD-L1 IHC 22C3 pharmDx, Inter and Intra-Observer Scoring Precision and Combined Testing Precision (Inter-instrument/Inter-operator/Inter-day) using PD-L1 IHC 22C3 pharmDx. Positive/negative status was applied to scores using the Combined Positive Score (CPS) and binary diagnostic cutoff of CPS ≥1.

Results: A statistical analysis was performed to calculate Negative Percent Agreement (NPA), Positive Percent Agreement (PPA) and Overall Agreement (OA) with corresponding confidence intervals (CI). The acceptance criteria (AC) for precision studies were set such that the lower bound of the two-sided 95% CI computed on percent agreement must meet or exceed 85%.

Sensitivity of the assay was 68.5% in 130 specimens scored as CPS ≥1. Inter and Intra-Observer Precision on 50 specimens met AC for NPA (98.6%/97.7%), PPA (96.2%/97.8%) and OA (97.6%/98.2%), respectively. Combined Precision results for 36 specimens met AC for NPA (93.2%), PPA (90.2%) and OA (95.2%)

Conclusion: These studies demonstrated consistent and highly reproducible results using PD-L1 IHC 22C3 pharmDx in analytical studies for sensitivity and precision in cervical cancer specimens.

Funding: Merck & Co., Inc.


Number of FoxP3+ regulatory T-cells are associated with recurrence in vulvar squamous cell carcinoma

D. Arık*, T. Benli, E. Telli

*Eskişehir Osmangazi University, Turkey

Background & objectives: FoxP3+ Tregs suppress anti-tumour immune responses and contribute to the escape of tumour cells from the immune system. We evaluated FoxP3+ lymphocyte infiltration in tumour stroma and tumour cell islands separately in vulvar squamous cell carcinoma.

Methods: Cases diagnosed with vulvar SCC in our department between 2005 and 2021 were retrospectively reviewed. The paraffin blocks were selected, and immunohistochemical studies were performed in accordance with the manufacturer's instructions. Cell counts were made in areas with the highest concentration of FoxP3 positive lymphocytes. Positive cells detected in tumour stroma and within tumoral cell groups were counted separately

Results: We found a positive correlation between high FoxP3+ lymphocyte count and good prognostic parameters. There was less recurrence in the group with high FoxP3+ lymphocyte counts in tumoral cell islands. Overall survival was not statistically different between these groups. Less lymphovascular invasion was observed in the group with high lymphocyte count in the tumour stroma.

Conclusion: Tumour-infiltrating FoxP3+ Tregs not only influence tumour progression and clinical course of disease, but may also determine immunotherapy response. In vulvar SCC, FoxP3+ Treg infiltration into the tumour stroma and into tumoral cell islands is associated with good prognostic features. In these tumours, stage appeared as the only independent prognostic parameter. Studies to be conducted in larger series may reveal whether Tregs can be targeted in cancer treatment.


Diagnostic value of immunohistochemical staining in fumarate hydratase-deficient leiomyoma

A. Magnaeva*, A. Asaturova, A. Tregubova

*National Medical Research Center for Obstetrics, Gynaecology and Perinatology named after Academician V.I.Kulakov, Russia

Background & objectives: Fumarate hydratase-deficient (dFH) leiomyoma is a subtype of leiomyoma, that can be misclassified as atypical one due to a similar morphology. Consequently, we aimed to identify dFH leiomyoma, previously diagnosed as atypical leiomyoma with the help of immunohistochemical (IHC) staining.

Methods: Samples of 24 patients with atypical leiomyoma, who had been provided operation at the Research Center for Obstetrics, Gynaecology and Perinatology (Moscow, Russia) during the years 2016-2021, were recruited. Slides were analysed for special features suggesting dFH leiomyomas, as well as fumarase IHC staining was performed with controls. Statistical significance was analysed using Fisher’s test and Kruskal Wallis test.

Results: From the obtained data, it is apparent that of these 24 tumours, 42% were found to be deficient for FH. There were significant differences between patients' age (Me=31 years in dFH leiomyoma and Me=40 years in atypical leiomyoma, p < 0,05). There was also a significant positive correlation between hyaline globules and FH deficiency (p < 0,05), as well as severe nuclear atypia and FH deficiency (p < 0,05). At the same time, statistical tests did not show any significant differences of prominent nucleoli, perinuclear halo, staghorn vessels, and alveolar-part oedema between dFH leiomyomas and atypical ones.

Conclusion: This study has shown that diagnosis of dFH leiomyoma has potentially low reproducibility among pathologists due to the low correlation between histologic features and IHC results. Our findings suggest that the most representative morphologic appearance is only hyaline globules. Consequently, similar histologic features of two leiomyoma subtypes suggest to provide a differential diagnosis with the help of IHC staining. Besides, fumarate hydratase-deficient staining is of clinical importance in revealing a group of young patients for genetic counselling to exclude HLRCC.

Funding: State Assignment number 122020900122-7


Endometriosis cases of a tertiary centre. Retrospective evaluation of patients detected in a year

I. Bağci*, D. Arık

*Eskişehir Osmangazi University, Turkey

Background & objectives: Although endometriosis is a benign condition, its high prevalence, recurrence risk and association with malignancies make it an important health problem. In this study, we compiled the clinicopathologic features of cases diagnosed with endometriosis within a year in our department.

Methods: The biopsy materials evaluated in our clinic in 2021 were retrospectively analysed. Clinicopathologic data, in terms of age, localization, recurrence, operation type, comorbidity, parity were gathered. Gynaecological and non-gynaecological materials were classified. Type of the comorbidity were listed.

Results: There were 78 materials of 70 cases. The mean age of the patients was 39 years. Recurrence was seen in 4 patients. The most common pelvic location was ovary. Vaginal cuff involvement was observed in only 1 case. Eight cases were located in the extrapelvic region. 13 patients had multifocal disease and 4 patients had atypical endometriosis. There were ovarian neoplastic lesions accompanying ovarian endometriosis in eight cases. Endometrioid carcinoma of the uterine cavity was present in 3 cases. Benign uterine lesions such as adenomyosis, adenomyotic nodule and leiomyoma uteri were present in 34 cases.

Conclusion: As a result, 5,7% of the cases were recurrent. Endometriosis associated carcinomas accompanied 2,9% of the cases, and 5,7% of the cases were diagnosed with atypical endometriosis, which is accepted as precursor of the endometriosis associated carcinomas. 92,8% of the cases presented in pelvic location, and multifocal disease at diagnosis occurred in 18,5% of the patients. Careful microscopic examination, detection of multifocal disease, and identification of accompanying neoplastic lesions will enable endometriosis cases to be correctly diagnosed and treated appropriately.


Trend analysis as a quality measurement for biomarkers - mismatch repair application

A. Plotkin*

*Sunnybrook Health Sciences Centre; University of Toronto, Canada

Background & objectives: Universal screening of endometrial cancer (EC) for Lynch syndrome using mismatch repair (MMR) immunohistochemistry is becoming standard of care in many countries. We defined MMR-deficiency (MMRd) rates in EC and examined steadiness over time to identify trends and benchmark measurements.

Methods: Pathology reports of EC biopsy and curettage (2018-2021) and MMR immunohistochemical biomarker reports from Life Labs, Canada were audited. Two-sided logistic regression was used for sample size analysis and generalized linear models were used for trend analysis. 60 patients quarterly were identified as minimum case volume by sample size calculations required to establish benchmarks for testing proficiency (P≤0.05, 80% power).

Results: Our cohort reached case volume ≥60 cases starting from the fourth quarter of 2019, thus meeting this requirement. The 1181 ECs included in the study were classified into MMR-d (n=313, 26.5%) or proficient (n=868, 73.5%). MMRd was highest in high-grade and mixed EC that included endometrioid EC (EEC, n=34/63, 54%) compared to low-grade EEC (n=256/919, 28%) and high-grade non-EEC (n=23/209, 11%), P<0.001. Proportion of MLH1/PMS2 loss was 22.1% (95% CI: 19.8-24.6%), MSH2/MSH6 1.8% (95% CI: 1.1-2.7%), MSH6 2.2% (95% CI: 1.4-3.2%) and PMS2 0.8% (95% CI: 0.3-1.4%). For each protein, the proportion of MMRd cases was quarterly steady over the entire time period (P>0.05).

Conclusion: Large laboratories may have the minimum volume to establish benchmarks for internal MMR immunohistochemistry quality control, and follow up trends over time, but this volume is not attainable for smaller laboratories. This study underscores the importance of trend analysis and the need to ensure that pathologists have sufficient expertise in biomarkers reporting.


Expression patterns of the gonadotropin and sex hormone receptors in various cell types of human endometrium during the menstrual cycle

A. Magnaeva*, M. Shamarakova, A. Tsitrina, A. Asaturova, G. Tabeeva, A. Tregubova, L. Ezhova, V. Bozenko

*National Medical Research Center for Obstetrics, Gynaecology and Perinatology named after Academician V.I.Kulakov, Russia

Background & objectives: Expression alterations of oestrogen and progesterone receptors as well as follicle-stimulating and luteinizing hormone receptors lead to a number of reproductive issues. Therefore, understanding the physiological features of their expression and topical distribution is important for a endometrium comprehensive assessment.

Methods: Endometrial tissue samples of 7 healthy females, who participated in IVF programs at the Research Center for Obstetrics, Gynaecology and Perinatology (Moscow, Russia) due to male infertility were recruited. Fluorescent immunohistochemistry was used for Q-score evaluation of ER, PR, FSHR, and LHR expression levels during menstrual cycle in endometrial glands, stoma, endotheliocytes, and macrophages.

Results: The increased immunoreactivity of ER, PR, FSHR, and LGHR in the endometrial glands and stroma was initiated at the stage of early proliferation and reached its maximum level at the stage of late proliferation. Then, the expression of ER in the glands and stroma decreased during the early and middle stages of secretion, the immunoreactivity of PR in the stroma persists throughout the all stage of secretion, increased immunoreactivity of FSHR and LGHR in each component of the endometrium was observed in all secretion stages.

Conclusion: It was shown that there is a connection between studied receptors immunoreactivity and endometrial structural features during menstrual cycle. Increased ER, PR, FSHR and LGHR expression in proliferative phase occurs according to endometrial growth, while increased PR, FSHR and LGHR expression in secretory phase is associated with endometrial decidual transformation and optimal condition for embrio implantation in case of pregnancy.

Funding: State Assignment number 122020900122-7 The research was funded by RFBR and Moscow city Government, project number 16-29-07434


An EMT-based score model identifies poor prognosis endometrial cancer (EC) patients and highlights COL11A1 as an independent risk factor of adverse events

A. Mascaros Martinez*, A. Arroyo-Alvarez, E. Gonzalez, S. Tomas-Perez, B. McCormack, A. Herreros-Pomares, I. Marin-Ajado, J. Mari-Alexandre, J. Gilabert-Estelles, N. Santonja-Lopez

*Department of Pathology Hospital General Valencia, Spain

Background & objectives: Increasing evidence points to COL11A1 as a poor prognosis biomarker in several malignancies but not yet in EC. We aim to investigate the role of COL11A1 as a biomarker in EC and to study their relationship with epithelial-to-mesenchymal transition-related genes.

Methods: We included 82 tissue biopsies from patients with a histological diagnosis of EC who underwent surgery at our institution (2014-2017). For normalization purposes, a control group consisted of endometrial tissues from 16 non-cancer hysterectomized patients. Tissue specimens were obtained at surgery. Expression of genes of interest was assessed by RT-qPCR. Statistical analyses were performed with R (v.4.0.3) software.

Results: Results from the TCGA cohort (n=232) demonstrated that COL11A1 expression significantly associates (p<0.05) with that of cancer-related extracellular proteoglycans (VCAN), EMT triggers (POSTN), EMT-inducing transcription factors, (SNAI1, SNAI2, ZEB1) and mesenchymal markers (FN1). One step further, we developed an EMT-based prognostic algorithm:

(SNAI1x0.0984) + (COL11A1x0.5535) + (CDH2x0.0297) + (FN1x0.0008) + (VIMx-0.0014)

COLL11A1 predominantly contributes to the EMT-score model.

Further analyses by RT-qPCR on a validation cohort (n=82) determined that COL11A1 expression is significantly elevated in Type 2 vs Type 1 EC (p=0.006), G2-G3 vs G1 EC (p=0.011 and p=0.025), metastasis (p=0.043), progression (p=0.017), relapse (p=0.034) and EC-related exitus (p=0.007). Additionally, we obtained the same results with the EMT-score.

Conclusion: In light of these results and in agreement with the literature, it seems that diverse EMT biomarkers have prognostic connotations in different types of tumours. We have found that, in EC, COLL11A1 is related to EMT markers and tumours expressing COLL11A1 are usually more aggressive. Currently, these results are being validated by immunohistochemical staining.

Funding: This research was funded by the “Instituto de Salud Carlos III-Fondo Europeo de Desarrollo Regional” (ISCIII-FEDER), Spain (Grant number: PI17/01945), the “Generalitat Valenciana”, Spain (Grant number: GV/2020/200), the “Fundación para la Investigación del Hospital General Universitario de Valencia” (FIHGUV), Spain (Grant numbers: Prize FIGHUV 2019, 2020 and 2021) and the “Sociedad Española de Trombosis y Hemostasia” (SETH), Spain (Grant Number: Prize SETH) and E.G.-C. and J.M.-A. are supported by grants from Generalitat Valenciana ACIF/2020/216 and APOSTD/2019/087, respectively.


Mesonephric-like adenocarcinomas of the ovary: pathological and molecular characterisation of a case series

A. De Leo*, A.G. Corradini, F. Rosini, L. Di Sciascio, A. Orsatti, R. Ciudino, F. Chiarucci, M. Grillini, S. Coluccelli, T. Maloberti, G. Tallini, D. Santini, D. de Biase

*Pathology Unit, University of Bologna Medical Center, Italy

Background & objectives: Mesonephric-like adenocarcinoma (MLA) of the ovary is a rare malignant tumour of the female genital tract. In this study, we investigated the clinicopathological, immunohistochemical, and molecular features of 9 ovarian MLAs.

Methods: Clinical data and history were extracted from the patient's medical records (from January 2019 to December 2021). Immunohistochemistry (IHC) and targeted Next-Generation sequencing analysis were performed.

Results: MLA accounted for 1.9% of cases (9/269). In 6 cases, MLA was associated with coexisting ovarian lesions: seromucinous borderline tumour (3), polypoid endometriosis (1), endometrioid carcinoma (2). Synchronous endometrial MLA was found in 2 cases. Diffuse endometriosis was present in all cases. Microscopically, tumours showed varying proportions of architectural patterns: glandular 9/9; papillary 7/9; glomeruloid 8/9; sex cord-like 7/9; solid 5/9; and spindle cell 6/9. All cases were positive for PAX8 and GATA3 with heterogeneous expression of CD10 and TTF1. MMR proteins were preserved in all cases. NGS analysis revealed pathogenic variants of KRAS in 8 cases and BRAF in 1 case. Mutations in TP53, POLE, BRCA1/2 were not identified.

Conclusion: In our study, 8 patients presented with advanced disease, and in 2 cases early disease recurrence was observed after chemotherapy treatment. MLA represents a rare and novel histotype of ovarian carcinoma with distinct pathologic, immunohistochemical and molecular features and it can be added to the list of endometriosis-associated ovarian neoplasms. In consideration of the propensity for recurrence/metastasis MLA may be considered a potentially aggressive histotype despite its apparent low-grade morphology.


Neuroendocrine neoplasms of the female genital tract: correlation of molecular, immunohistochemical and clinical features in 24 cases

M. Testa*, C. Amaglio, R. Maragliano, E. Leoni, F. Sessa, D. Furlan, S. Uccella

*Pathology Unit, Dept. of Medicine and Surgery, University of Insubria, Varese, Italy

Background & objectives: Neuroendocrine neoplasms (NENs) are uncommon in the female genital tract. Due to their rarity, little is known about them. Aim of this study is to correlate their molecular and immunohistochemical features with clinical data and to investigate their prognostic value.

Methods: The electronic medical record of our Pathology Institute was utilized to identify women diagnosed with gynaecological NENs from 1983 to 2019. All cases underwent histological and immunohistochemical review. Patients’ clinical, demographic and treatment characteristics were searched. Molecular analyses were performed (including Next-generation Sequencing, real-time PCR, microsatellite instability evaluation). The association between tumour histology and survival was examined using Kaplan-Meier analyses.

Results: A total of 24 cases were identified. 42% of the NENs were cervical, 25% endometrial and 33% ovarian. Median age was 62 years (range 39–78 years), with site-specific differences. Only high-grade NENs were retrieved on cervix and endometrium: 1 Neuroendocrine-tumour (NET) G3, 8 Neuroendocrine-carcinomas (NECs) and 7 Mixed-Neuroendocrine-non-neuroendocrine-neoplasms (MiNENs), whereas the most common histological subtype in ovary was NET. 20/22 cases (91%) expressed Chromogranin-A and/or Synaptophysin and 17/23 cases (74%) expressed INSM1. Most of the cases didn’t show immunoreactivity for site-specific antibodies. HPV-infection and concomitant p16 overexpression was found in 8 cervical and 2 endometrial cases. 4 cases had altered immunohistochemical expression of DNA-mismatch-repair-proteins and associated microsatellite instability (MSI).

Conclusion: Gynaecological NENs represent a rare tumour entity and constitute a diagnostic challenge. They usually show immunoreactivity for traditional neuroendocrine markers (Chromogranin-A and Synaptophysin), but we recommend caution in INSM1 reliability, since it showed a minor sensitivity in our study; NENs resulted mainly negative for site-specific markers, therefore they cannot be used to rule out metastases. Limited data regarding gynaecological NENs pathogenesis are available; our study highlighted NENs might follow site-specific molecular pathways, like HPV-related infection, or NEN-specific pathways, like MSI.


Contribution of β-catenin in endometrial cancer progression

Z. Greally*, E. O’Regan, P. Lewitowicz, O. Adamczyk-Gruszka, A. Horecka-Lewitowicz, J. Gruszka, A. Strzelecka

*Student Science Club at Collegium Medium Jan Kochanowski University, Poland

Background & objectives: This study aims to investigate the influence of β-catenin on endometrial cancer progression. Currently the molecular subgrouping of EC proposed by the TCGA is a milestone. Furthermore, the driving mechanisms are vital to identify correlations between genes and their regulators.

Methods: A total of 103 White female patients with confirmed EC were enrolled. For the analysis, we used next-generation sequencing with Hot Spot Cancer Panel provided by Illumina Inc, San Diego, California, USA, and immunohistochemical analysis FOXA-1, FOXP1, oestrogen receptor, and β-catenin.

Results: Beta-catenin showed a correlation with AKT1 mutation (R = 0,2508 p = 0,04058 and surprisingly not with CTNNB1 mutation R = 0.5176 p = 0,000007). Moreover, beta-catenin expression was observed in TNM R = 0.2209, p = 0.0263, with prognostic impact RFS (R = 0,2049 p = 0,0388). Indirectly, the worse clinical outcome was observed in obese patients with beta-catenin expression (BMI R = 0.1931 p = 0.0510).

Conclusion: The study suggests a prognostic value of Β-catenin in EC. Our study confirmed that beta-catenin is a reliable biomarker in the prognosis of EC outcomes. It indicates that β-catenin can be used as a biomarker for the prognosis of patients with malignant cancers. Furthermore, there is no implementation of classification by molecular basis. Based on this medical research it is necessary to use biomarkers such as beta catenin in the prognosis of endometrial cancer.


Histopathological features of placentas of mothers who have had COVID19 infection during pregnancy

N. Yeldir*, G. Sönmez Ünal, H. Özer

*Sivas Cumhuriyet University, Turkey

Background & objectives: The COVID19 pandemic has been affecting the worldwide since 2020.The SarsCov2 can cause changes in many tissues. The aim is to investigate changes that occur in the placentas of women who have had COVID19 infection during any period of their pregnancy.

Methods: The study was performed prospectively with 24 women who had COVID19 infection at any period of their pregnancy and gave birth by cesarean section or vaginal. The data of the patients were evaluated for clinical, the pregnancy period of COVID19 infection and placental histopathological features.

Results: In 24 cases, the mean age was 28 years and the mean birth week of cases was 38th week.10 cases were vaccinated with two doses, others were unvaccinated. 3 cases were in the period of active covid infection and the mean week of COVID19 infection of other cases was 22th week. In histopathological examinations, calcifications, congestive chorionic villis, intervillous haemorrhages and increase in focal syncytial knots were common findings. Focal distal villous hypoplasia, hofbauer cells and focal hydropic villis were observed less. There are fibromuscular stenosis in 3 cases and marked distal villous hypoplasia in 2 cases.

Conclusion: It is believed that the SarsCov2 virus can cause pregnancy complications such as foetal malformations, foetal growth retardation and stillbirth. Placental abnormalities including foetal and maternal vascular malperfusion have been reported despite negative SarsCov2 tests of infants of mothers who had COVID19 infection during pregnancy. According to this study, COVID19 infection caused significant maternal vascular malperfusion findings in a few cases, although it did not cause significant histopathological changes of the placenta in many cases.


Expression of Caspase-8 and Bcl-2 in eutopic endometrium of women with chronic endometritis and endometriosis

A. Tregubova*, G. Tabeeva, M. Dumanovskaya, A. Magnaeva, A. Asaturova, S. Pavlovich

*FSBI «National Medical Research Center for Obstetrics, Gynaecology and Perinatology named after Academician V.I.Kulakov», Russia

Background & objectives: Apoptosis is characterized by a series of perturbations to the cellular architecture that contribute to cell death. This process involved in the pathogenesis of gynaecological diseases associated with chronic inflammation such as chronic endometritis and endometriosis.

Methods: The immunohistochemical expression of apoptotic markers Caspase-8 and Bcl-2 was evaluated in 50 patients divided into 3 groups: group 1 (normal endometrium, n=7), group 2 (chronic endometritis, n=20), group 3 (endometriosis, n=23). QuPath software was used to assess the level immunohistochemical staining intensity (optical density (OD) of staining). We used Kruskal-Walli’s test for statistical analysis.

Results: Expression of Caspase-8 in stromal and glandular cells of eutopic endometrium samples was significantly different across the three groups (p=0,016 and p=0,004 respectively). OD of gland cells in the first group: Me=0.113 (Q1-Q3: 0.086-0.119), second: Me=0.314 (Q1-Q3: 0.106-0.445), third: Me=0.101 (Q1-Q3: 0.09-0.142). OD of stromal cells in the first group: Me=0.057 (Q1-Q3: 0.05-0.06), second: 0.133 (Q1-Q3: 0.07-0.218), third: Me=0.056 (Q1-Q3: 0.05-0.06). Bcl-2expression in glandular cells of eutopic endometrium samples was significantly different across the three groups (p=0,004). OD of gland cells in the first group: Me=0.113 (Q1-Q3: 0.086-0.119), second: Me=0.314 (Q1-Q3: 0.106-0.445), third: Me=0.101 (Q1-Q3: 0.09-0.142).

Conclusion: Our findings suggest that there is an increase the activity of caspases-8 in stromal and glandular endometrial cells as well as an activity bcl-2 in glandular cells in chronic endometritis to compare with endometriosis and normal samples. These results might help to shed more light on the role of apoptosis in gynaecological disease associated with chronic inflammation.

Funding: State Assignment №121040600436-7


Mesonephric-like adenocarcinoma of the endometrium frequently associated with KRAS mutation and adenomyosis

Y. Chun*, Y.M. Kim, Y. Kim

*Guro Hospital Korea University, Republic of Korea

Background & objectives: Mesonephric-like adenocarcinoma (MLA), a rare subtype of endometrial carcinomas with aggressive behaviours, is histologically similar to the cervical mesonephric adenocarcinoma, but not associated with mesonephric remnants. It remains unclear whether the MLAs represent mesonephric origin or Müllerian neoplasm.

Methods: We studied the clinicopathologic, immunohistochemical and molecular features of 6 endometrial MLAs and 1 endometrial carcinoma with mixed mesonephric-like and endometrioid patterns.

Results: Four cases had initially been diagnosed as low-grade endometrioid endometrial adenocarcinomas. Histologically, MLAs showed a variety of morphologies including tubular, ductal, papillary, retiform, and solid. Five cases (71%) were associated with adenomyosis. All tumours showed negativity for ER and PR, p53 wild type and retained staining for the mismatch repair proteins. Most cases revealed positivity for GATA-3 (5/6), CD10 (6/7) and TTF-1 (5/6). Next generation sequencing documented mutations in KRAS (4/4), PIK3CA (1/4), ARID1A (1/4) and ERBB3 (1/4). One case showed copy number variations in MDM4 and AKT3. Two patients were in FIGO stage lll, 1 in stage IV and 4 in stage I-II. Three patients developed lung metastasis.

Conclusion: As MLAs demonstrate varied architectural patterns, it often could lead to misdiagnosis, especially on a small biopsy specimen. Endometrial MLA represents a distinct subtype of endometrial carcinomas associated with KRAS-mutations and characteristic immunohistochemical findings. Clinically, MLAs show an aggressive behaviour with a substantial risk for lung metastasis. Frequent association with adenomyosis, no mesonephric remnant, and mutations in PIK3CA and ARID1A suggest a Müllerian origin of the endometrial MLAs.


HPV-positive status and p53 alterations are associated with improved disease-free survival in patients with vulvar squamous cell carcinoma

A. Saco*, N. Carreras-Dieguez, C. Manzotti, M. del Pino, N. Rakislova, I. Trias, I. Ribera-Cortada, S. Alos, R. López del Campo, S. Diaz-Mercedes, L. Marimon, M.T. Rodrigo, J. Ordi

*Hospital Clínic Barcelona, Spain

Background & objectives: Three clinically distinct subtypes of vulvar squamous cell carcinomas (VSCC) have been described on the basis of human papillomavirus (HPV) status and p53 alterations. HPV-positive and p53 wild-type status have been reported to confer better disease-free survival and overall survival.

Methods: 193 surgically treated from 1975 to 2021 in a single institution in Spain were included. Median follow-up was 69 months (range 1–285). p53 was determined by immunohistochemistry (IHC) and HPV by p16 IHC. We assessed the survival of the patients stratified in three groups (HPV-positive, HPV-negative/p53 mutant and HPV-negative/p53 wild-type). Correlations with outcome were analysed using Kaplan-Meier survival curves.

Results: 35 patients (18.1%) had HPV-positive VSCC, 137 (70.9%) HPV-negative/p53 IHC abnormal and 21 (10.8%) HPV-negative/p53 wild-type carcinomas. Age at diagnosis of women with HPV-positive tumours was lower than those with HPV-negative/p53 abnormal and HPV-negative/p53 wild-type VSCC (median 62 years vs. 76 and 77 years, respectively, p=0.06). Patients with HPV-positive tumours showed lower rates of relapse than those with HPV-negative carcinomas (p<0.01). Remarkably, among women with HPV-negative VSCCs, patients with p53 abnormal IHC staining showed lower rates of relapse than those with p53 wild-type IHC (p=0.01). However, no differences in disease-specific survival or in FIGO stage (initial vs advanced) were identified among the three groups (p=0.40 and 0.37, respectively).

Conclusion: Our findings indicate that patients with HPV-positive VSCC, as well as patients with HPV-negative carcinomas with p53 abnormal IHC staining show improved disease-free survival, but these factors seem not to influence disease-specific survival. Patients with HPV-negative carcinomas, particularly those with p53 wild-type neoplasms may warrant wider margins and more strict surveillance after surgery due to their high risk of relapse.


Intraoperative pathologic evaluation of the ovary: a 21-year institutional practice review

T. André Sousa Oliveira*, M. Pinho, D. López-Presa

*Department of Pathology - Hospital de Santa Maria, CHULN, Portugal

Background & objectives: Intraoperative pathologic evaluation may assist in therapy management of ovary pathology by confirming diagnosis and cancer type and providing information about the extent of the disease. Herein, we reviewed our institution’s practice regarding this examination.

Methods: We analysed all reports of ovarian intraoperative pathology evaluations from a tertiary healthcare institution spanning a 21-year period [2001-2021], totalling 329 cases. Intra- and postoperative diagnosis, gross pathology features, lesion size, bilaterality and number of frozen blocs analysed were recorded. Of these, only 254 had intra- and postoperative responses available and were selected as our study cohort.

Results: Most patients were ≥45-years old (74.0%) and suspected of having an adnexal/ovarian neoplasm (n=205; 80.7%), an indeterminate cystic lesion (n=24; 9.4%) or another infrequent reason (n=25; 10.4%); 14 patients (5.5%) underwent examination for bilateral lesions. Frozen sections were done in 75.2%, averaging 1.9 sections/exam, with the remainder being only evaluated macroscopically. A concordant intra- and postoperative result occurred in 239 cases (96.5%), with discordant results arising from: overvaluing malignant potential (n=2; 0.8%), tumour misclassification (n=2; 0.8%), misinterpretation of artefactual tissue as carcinoma (n=1; 0.4%) and inadequate sampling (n=1; 0.4%). Three cases were deferred (1.2%). As higher-grade components cannot be consistently excluded, intraoperative results undervaluing malignant potential were considered concordant.

Conclusion: Ovarian intraoperative examination remains a reliable and useful tool for deciding therapeutic management. A systematic approach to grossing, sampling and clear communication between clinician and pathologist are encouraged to avoid misdiagnosis and wrongful treatment. When a precise diagnosis is not feasible, either the most probable diagnosis can be reported with an indication of other differential diagnosis which cannot be excluded, or the examination can be deferred when doubt remains after careful examination and consideration.


A new homologous recombination deficiency (HRD) test in ovarian cancer provides a high diagnostic yield

J. Kim*, G.Y. Kwon, S. Baik, S. Lee, E. Oh, J. Hong, S. Kim, K. Lee

*Department of Pathology, Kangnam Sacred Heart Hospital, Hallym University College of Medicine, Republic of Korea

Background & objectives: Homologous recombination repair (HRR) pathway deficiency (HRD) is involved in the carcinogenesis of ovarian cancer (OC) and poly(ADP-ribose) polymerase (PARP) inhibitors can be effective for patients with HRD. We investigated the HRD status and its clinical significance in OC.

Methods: We used a new HRD solution combining information from germline and somatic HRR mutations including BRCA1 and BRCA2 with a measure of genomic scarring. On next-generation sequencing in 39 OCs, BRCA mutations and amplification of associated genes were detected. Genomic integrity (GI) index was calculated and a GI status was concluded. By combining the results, HRD status was subsequently determined.

Results: HRD status in 34 (87.2%) OCs was successfully analysed. Five cases (12.8%) were undetermined due to inconclusive or rejected GI index. Twenty three (67.6%) out of 34 case were HRD positive and 11 (32.4%) cases were HRD negative. Tumours with BRCA1/2 mutation were 9 (26.5%) and tumours with positive GI index were 21 (61.8%). HRD was associated with high-grade serous carcinoma and high FIGO stage (≥IIIB) (p=0.032 and 0.028, respectively). Patients with high GI index (>9.7) displayed better disease-free survival compared to those with low GI index (p=0.012).

Conclusion: A new HRD solution beyond BRCA1/2 mutation detection provides expanded clinical information and improves the diagnostic yield of HRD in OC. This could be useful for personalized OC treatment.


Influence of histological and immunohistochemical features of endometrioid adenocarcinoma on the microvascular tumour density

M. Lyndin*, O. Kravtsova, V. Sikora, N. Hyriavenko, Y. Lyndina, Y. Soroka, R. Moskalenko, A. Romaniuk

*Sumy State University, Ukraine

Background & objectives: Endometrioid adenocarcinomas of the uterine body account for about 70-80% of all endometrial carcinomas. The most proven prognostic markers are age, stage of the disease, histological grade, microvascular density (MVD), and expression of prognostically important receptors.

Methods: The investigation was conducted on 30 samples of endometrioid adenocarcinomas. The presence of E-cadherin, VEGF, ER, PR, Ki-67, and р53 was detected by immunohistochemistry.

Results: The pronounced predominance of MVD in moderately and low-differentiated tumours over highly differentiated endometrioid adenocarcinomas was found (F=6.34, p=0.0055). A positive correlation was found between MVD and the expression of VEGF (r=0.47, p=0.0086) and Ki-67 (r=0.54, p=0.0023) in tumour cells; a negative – between MVD and PR (r=-0.45, p=0.012). Expression of ER, E-cadherin, and p53 had no effect on the MVD of neoplastic tissues (p>0.05).

Conclusion: The neovascularization degree and microvascular density of endometrioid endometrial adenocarcinoma tissues are enhanced by tumour dedifferentiation and VEGF overexpression. There is a positive correlation between the proliferative activity level of tumour cells and MVD; a negative – between the expression of PR and MVD.


Germline and somatic BRCA1 and BRCA2 mutational analysis in non-mucinous ovarian carcinomas. A retrospective and descriptive study of 80 cases

A.B. Moreno García*, I. Costa Trachsel, C.M. Blázquez-Mañá, A. Reques Llanos, R. Carrera-Salas, M.J. Martinez Reyes, A. Falgueras i Sanchez, M.d.C. Ramos-Guijo, Y. Garcia Garcia, L. Ribot Luna, A. Ruiz Nel·lo, J.C. Ferreres Piñas

*Department of Pathology. Parc Taulí Hospital Universitari. Sabadell; Institut de Recerca i Innovació Parc Taulí (I3PT), Universitat Autónoma de Barcelona, Spain

Background & objectives: BRCA1 and BRCA2 somatic or germline mutations in ovarian cancer (OC) have familial, prognostic and predictive significance. We analysed the mutational status of these genes in our cohort of patients and its correlation with several clinical and pathological parameters.

Methods: Next-generation sequencing of BRCA1 and BRCA2 was performed in 80 patients with non-mucinous OC, in tumour and peripheral blood, from January 2019: 62 high-grade serous (HGSC), 8 low-grade endometrioid, 3 high-grade endometrioid (HGECC), 4 clear cell (CCC), 1 low-grade serous carcinomas, 1 carcinosarcoma and 1 borderline serous tumour. Age, FIGO stage, histological type, clinical behaviour and molecular features were collected.

Results: Twenty-five pathogenic variants(PV) in 23/79 tumour-samples (30%) were detected: 16 BRCA1/ 9 BRCA2; 15 germinal(g) (60%) and 5 (20%) somatic(s) (5 blood pending); 16 "frameshift", 5 "nonsense", 3 "missense" and 1 "splicing” (identical results in germinal confirmed cases). Somatic medium allelic frequencies(AF) were 28% versus 68% in germinal cases. One additional germinal (no tumour analysis available) was detected. 22 were HGSC (15 BRCA1/7 BRCA2), one CCC (gBRCA1/sBRCA2), and one HGECC (BRCA1/ BRCA2, blood pending, tumourAF 54%/70%). BRCA mutated-patients were 55.7yo-medium and 20% stage I-II, at diagnosis; non-mutated were 65.5yo and 25% I-II. All BRCA mutated-patients are alive, 40% with no disease(AWND); 12% of non-mutated-patients are exitus and 20% are AWND.

Conclusion: The mutational study of BRCA1/BRCA2 in tumour-tissue is cost-effective, sensitive and specific for germinal and somatic line PV detection. Germinal, “frameshift” type and HGSC are the prevalent mutation-line, PV and histologic type, respectively. PV in tumour and blood in germinal confirmed cases were identical. Mutated cases are associated with younger onset age patients and better clinical behaviour, compared to non-mutated cases. This analysis is essential for an optimal management and for individual and family counselling.


Uterine smooth muscle tumours of uncertain malignant potential (STUMPs) with fumarate hydratase deficiency: report of 2 cases

C. Alves-Vale*, A. Beltran, A. Alves

*CUF Descobertas Hospital, Portugal

Background & objectives: Fumarate hydratase (FH) deficiency accounts for characteristic architectural and cytological features, which may result in erroneous classification of uterine neoplasms if not recognised. We report 2 cases of FH-deficient smooth muscle tumours of uncertain malignant potential (STUMPs), diagnosed after myomectomy.

Methods: Case 1 refers to a 42-year-old woman presenting with a solitary uterine transmural nodule with 95 mm, without relevant personal or family history. Case 2 refers to a 27-year-old woman, with multiple uterine nodules since the age of 21 and family history of “leiomyomas”, the largest with 111 mm, refractory to ulipristal acetate therapy.

Results: Microscopically, both tumours were composed of a spindle cell proliferation with focal areas of alveolar-type oedema and frequent vessels, sometimes with hemangiopericytoma-like pattern. Moderate to severe atypia was observed, including bizarre nuclei or prominent nucleoli with perinucleolar halo. Cytoplasm was eosinophilic, with rhabdoid inclusions focally. Immunoreactivity for FH was lost. Mitotic index was lower than 4/mm2. Necrosis of indeterminate nature was present in the first case, and both tumour cell necrosis and ischemic-type necrosis were detected in the second. The diagnosis of FH-deficient STUMP was made. Patients were referred to the Genetics consultation to exclude FH germline mutations, associated to hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome.

Conclusion: It is essential to recognize the prototypical morphology of FH-deficient neoplasms, in order to avoid overdiagnosis of malignancy in uterine smooth muscle tumours, with special impact on young women who desire to preserve fertility. Although close follow-up is recommended, the majority of STUMPs have benign behaviour. FH-deficient neoplasms may occur sporadically or associated to HLRCC syndrome, hence the importance of its prompt recognition to enable the early detection of aggressive forms of renal cell carcinoma.


High-risk HPV screening - A retrospective study of a Portuguese cervical cancer screening program

R. Moiteiro da Cruz*, T. André Sousa Oliveira, J. Boavida, M. Pinho, C. Ferreira, D. López-Presa

*Department of Pathology - Hospital de Santa Maria, CHULN; Faculdade de Medicina da Universidade de Lisboa, Portugal

Background & objectives: Cervical cancer is the fourth most common female malignancy. High-risk HPV molecular testing has been considered a useful screening option to identify women with cervical cancer. Herein, we aimed to correlate our institutional molecular results with the histological follow-up.

Methods: A retrospective and unicentric study was conducted, regarding the cervical cancer screening program of the Lisbon and Tejo Valley region, between October 2017 and December 2019. Women with HPV16 and/or HPV18 infection were considered; patients infected with the former strains and by other high-risk strains were also included; their clinical data and biopsy diagnosis were collected.

Results: From a total of 33567 high-risk HPV detection tests (cobas®), 3%(n=1138) were HPV16, and/or HPV18 positive, with/without co-infection with other strains: HPV16 positive–43,32%(n=493), HPV18 positive–13,26%(n=151), HPV16&18 positive–0,96%(n=11), HPV16&18 and other high-risk strains–1,76%(n=20). About 78,6%(n=895) were evaluated by colposcopy with posterior biopsy. From these, HPV16 group(n=399) was associated in 34,01%(n=136) with high-grade lesions [high-grade squamous intraepithelial lesion(HSIL)–95,59%(n=130); squamous cell carcinoma(SCC)–0,73%(n=1); adenocarcinoma(ADC) in situ-2,94%(n=4); ADC-0,73%(n=1)]; in HPV18 group(n=115), 6,56%(n=8) cases were associated with high-grade lesion (all HSIL). In HPV16/18 group(n=9), 77,78%(n=7) patients were associated with high-grade lesion (all HSIL). In the HPV16/18 with other high-risk strains group(n=13), 46,15%(n=6) cases were associated with high-grade lesions [HSIL-80%(n=5); ADC-20%(n=1)].

Conclusion: High-risk HPV molecular testing of our population has given us the opportunity to screen cervical cancer and to get the real incidence of HPV infection genotype 16 and 18. Despite the necessity of further studies to appreciate the specificity/sensibility of this test, namely considering patients infected with other high-risk strains besides 16 and 18, we conclude that patients with co-infection with various high-risk strains and HPV16 infection have the greatest association of high-grade lesions; HPV18 group is the least associated.


Disagreement in anatomopathological review reports in gynaecological pathology and its impact on treatment: the importance of the subspecialist pathologist in a cancer centre

B. Maran, L. de Brot, M. Corassa, R. de Paula*

*Ac Camargo Cancer Center, Brazil

Background & objectives: The anatomopathological report is crucial for clinical decisions. In this context, reference cancer centres often review external reports to reduce errors and inappropriate therapies. This study aimed to assess the degree of disagreement in reviews of external reports in gynaecology.

Methods: This is a single-centre, retrospective study, in which 219 cases of gynaecological pathology were reviewed between January 1, 2021 to December 31, 2021. The degree of disagreement was separated into “major” (when the discrepancy between the reports generated a change in the patient's therapy), "minor" (when there was no impact on patient management) and "no change" (when there was agreement).

Results: 219 cases were analysed. The median age of the population was 48 years. 201 cases were oncological and 18 cases were not related to cancer. Most of the cases represented primary gynaecological neoplasms (only 6 cases were from metastases or tissues outside the female genital tract). There was agreement (“no change”) in 70 cases (31.9%), "minor" disagreement in 107 cases (47.4%) and "major" disagreement in 42 cases (19.1%). When together, the major and minor disagreements correspond to 66.5% of the sample. The results were tabulated in tables and graphs.

Conclusion: The data presented, although showing a reality of only one year, are consistent with the perspective that in pathology, and especially in gynaecological pathology, the analysis of cases by a subspecialist is essential for the proper management of the patient. That is fundamental not only to ensure the provision of adequate treatment for patients, but also to reduce the risk of morbidities associated with aggressive cancer therapies.


Ovary intraoperative consultation – are we doing the best we can? – A ten-year retrospective analysis of our experience

C. Dahlstedt-Ferreira*, S. Carralas Antunes, R. Oliveira, D. Gomes Pinto

*Hospital Garcia de Orta, E.P.E, Portugal

Background & objectives: Ovarian tumours are frequently subjected to intra-operative consultation (IOC). These can be challenging cases and misdiagnoses are frequent[DGP1]. Our aim is to evaluate how our intraoperative diagnoses affected patient management in our institution to improve future performance.

Methods: We identified all the ovarian tumours subjected to IOC over a period of 10 years (2012-2022), which required intraoperative examination, in our institution. We analysed the concordance between intraoperative and definitive diagnosis by means to evaluate how our intraoperative diagnosis affected patient management. The dataset was based on the patients’ pathology reports and clinical files.

Results: In this period, we performed 81 IOCs for ovarian tumours. The average age of patients was 56 years. Sixty-one IOCs (76,3%) were concordant with the final diagnosis and twenty were discordant (23,7%). Four discordant cases changed from benign to borderline; five from benign/borderline to malignant and five from borderline to benign tumour. Histological type of six of the malignant tumours was changed. Discordant cases were more frequent in serous tumours (n=7; p=1, not significative) and six were of the mucinous type.

Conclusion: Intraoperative examination of ovarian masses is known to have a high accuracy and therefore provides some guidance to the surgeon’s conduct. It is also known to have many pitfalls posing a challenge to the pathologist. At our institution, the concordance between intraoperative diagnosis and definitive diagnosis is in line with the literature. Mucinous neoplasms pose a higher challenge compared to other histological types, although in our series we didn't find significant differences between IOC diagnoses of serous and mucinous tumours.


Intra-operative consultation of endometrial cancer – are frozen sections better than gross inspection alone? – A single-centre retrospective series over seven years

S. Antunes*, C. Dahlstedt Ferreira, R. Oliveira, D. Gomes Pinto

*Hospital Garcia de Orta, Portugal

Background & objectives: Endometrial cancer (EC) is the most common gynaecological malignancy in developed countries. By analysing intraoperative consultation (IOC) of EC cases, we want to assess our performance so far, aiming at determining the best approach for these cases.

Methods: We identified all cases of EC that underwent IOC, over the last 7 years at out institution. Data were gathered from pathology reports and patients’ clinical files. We focused on analysing the purpose of the IOC (diagnosis vs. staging), if frozen sections were performed, concordance of intra-operative with definitive diagnosis and how this result influenced the surgical procedure.

Results: Seventy-eight IOC in endometrial lesions were performed, 75 for staging and 3 for diagnosis. The average age of the patients was 67 years. Sixty-four (82,05%) cases were concordant, eleven (14,1%) were discordant and three (1,85%) were deferred. Twenty-seven IOC had frozen sections performed. Four discordant cases were upstaged in the surgical specimen due to myometrium invasion, two due to cervical stromal invasion and four due to serosa and/or adnexal involvement. No frozen sections were performed in the latter. There were no statistically significant differences between cases with and without frozen section (p=0,7362) in terms of concordance.

Conclusion: At our institution, concordance between IOC and definitive diagnosis is high, with only seven discordant cases (8,97%) with impact on the patient. Out results show that gross evaluation is mostly adequate for staging EC, but frozen sections might be of particular use on grossly borderline cases for myometrial and cervical stromal invasion. Serosal or adnexal nodules should be detected grossly, and frozen sections performed. IOC is useful in cases of EC, eliminating the need for reintervention in many patients.


PD-L1 and mismatch repair (MMR) protein expression in small cell carcinomas of the uterine cervix

R. Moiteiro da Cruz*, J. Almeida-Tavares, S. Lérias

*Department of Pathology - Hospital de Santa Maria, CHULN; Faculdade de Medicina da Universidade de Lisboa, Portugal

Background & objectives: Cervical neuroendocrine carcinoma(NEC) is rare with limited therapeutic options. Recent studies described abnormal p53, MMR deficient and PD-L1 expression in cervical NECs. Our aim was to evaluate p53, PD-L1 and MMR protein expression in NECs and its correlation to prognosis.

Methods: We selected 5 cases of NECs with known clinicopathological and prognosis data diagnosed over 22 years(2000 to 2022). Immunohistochemistry for p53, p16, PAX8, chromogranin, synaptophysin, TTF-1, PD-L1(22C3 clone) and MMR proteins was performed. PD-L1 was assessed using the combined positive score(CPS) with a threshold of ≥1 required for positivity and aberrant p53 (overexpression or null) was considered positive.

Results: Mean patient age was 58 years (range 33-71), all cases were diagnosed in FIGO stage IV and 2 died of disease. Three cases were pure small-cell NEC and 2 were mixed carcinomas, associated with adenocarcinoma (n=1) and squamous cell carcinoma (n=1), each. All cases demonstrated p16 positive staining, synaptophysin focally positive staining, chromogranin negative staining and PD-L1 negative. PAX8 was positive in 4 cases, TTF-1 in 3 cases, p53 mutant pattern was observed in 3 cases and no MMR deficiency was observed.

Conclusion: Our study showed strong nuclear staining for p16 in all cases, demonstrating the influence of high-risk HPV infection on its carcinogenesis. The variability of p53 expression demonstrate the diversity of genomic landscape of this tumour, being in accordance with the most common genetic alterations usually found. No expression of PD-L1, as well as no MMR deficient was found, suggesting a lower expression of PD-L1 in tumours with microsatellite stability.


Polymerase-ɛ exonuclease domain mutations predict excellent outcome among SWI/SNF-deficient undifferentiated and dedifferentiated endometrial carcinomas

B. Tessier-Cloutier*, M. Köbel, A. Momeni-Boroujeni, J. Benhamida, C. Stewart, M. Ladanyi, R. Soslow, C. Lee

*Memorial Sloan Kettering Cancer Center, USA

Background & objectives: SWI/SNF-deficient undifferentiated (UDEC) and dedifferentiated (DDEC) endometrial carcinomas are aggressive malignancies with poor treatment response. The prognostic role of the molecular classification is unknown in those malignancies. Here we review a molecularly and clinically annotated series of SWI/SNF-deficient UDEC/DDEC.

Methods: We collected a series of UDEC/DDEC with loss of expression of a core SWI/SNF protein (SMARCA4, SMARCB1, or ARID1B). The molecular subtype was assigned according to the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE), including hotspot POLE mutation testing and immunohistochemistry for mismatch repair proteins and p53. Kaplan Meier survival analysis was performed across all different molecular subgroups.

Results: We included 60 UDEC/DDEC, the median age of the cohort was 59 years (range: 37-82 years) and most cases were high stage at presentation (19/36). Of the 60 cases, 36 were MMR-deficient (MMR-d), 6 were POLE mutated (POLEmut), 4 p53 abnormal (p53abn) and 14 with no specific molecular profile (NSMP). The median overall survival of POLEmut cases was 26.1 months without any death, compared to 11.8 months in the rest of the cases (p=0.034). The cases classified as MMR-D, p53abn or NSMP had similar outcome (p=0.319).

Conclusion: All POLEmut cases had an excellent prognosis while the other three molecular subgroups (MMR-D, p53abn and NSMP) had poor outcomes. Molecular classification among the non-POLEmut cases was not informative of clinical behaviour in our study. These results highlight the importance of hotspot POLE mutation testing in SWI/SNF-deficient UDEC/DDEC to help guide management.


Contribution of Forkhead box A1 to endometrial cancer progression

E. O'regan*, Z. Greally, P. Lewitowicz, O. Adamczyk-Gruszka, A. Horecka-Lewitowicz, J. Gruszka, A. Strzelecka

*Jan Kochanowski University in Kielce, Poland

Background & objectives: Investigate the influence of Forkhead-box on endometrial cancer progression. The molecular subgrouping of EC proposed by the Cancer Genome Atlas is vital in precise molecular-based patient triage. The driving mechanisms are necessary to identify correlations between genes and their regulators.

Methods: A total of 103 White female patients with confirmed EC were enrolled. For analysis, we used next-generation sequencing with Hot Spot Cancer Panel provided by Illumina Inc, San Diego, California, USA, and immunohistochemical analysis FOXA-1, FOXP1, Oestrogen receptor. Participants underwent surgical treatment without previous radio-chemotherapy to conduct a credible comparative analysis of tumour characteristics, the treatment, and unchanged molecular profiling.

Results: we observed a negative correlation with FOXA-1 revealing that FOXA-1 silencing led to worse outcome based on the negative correlation with FOXA-1(test log-rank for FOXA1 2,031559, p = 0.04220 and HR 2.66, p = 0.06), The estimation of targeted protein frequency was conducted revealing that FOXA-1 occurred in 24 cases, especially in the FIGO IA stage . Furthermore, a correlation was found for FOXP-1 (R = 0,2872 p = 0.0041). This depicts Kaplan-Meier curves for FOXA-1 (p = 0.042). FOX proteins were closely correlated with TP53 and KRAS mutation. Oestrogen receptor expression was detected in all FOXP-1 positive cases and more than 90% of FOXA-1 positive ones.

Conclusion: Our study confirmed that FOXA-1 is a reliable biomarker in the prognosis of Endometrial cancer outcomes. The changes of FoxA downstream profiles in different cancers would provide more valuable clues for FoxA's biological function and could be considered as efficient biomarkers for cancer diagnosis or prognosis. The FOX transcription factor family is closely correlated to hormone-dependent carcinogenesis by interacting with steroid receptors. Thus, FOX binds the promoters of more than 100 genes, in turn, regulating many cellular functions.

PS-09| Poster Session Haematopathology


Flow cytometric characterisation of adult T-cell leukaemia/lymphoma (ATLL) and the associated cytogenetics and next generation sequencing (NGS) Findings

U. Edema*, J. Liu, Y. Wang, Y. Shi

*Montefiore Medical Center, Albert Einstein College of Medicine, USA

Background & objectives: ATLL, a rare aggressive mature T cell neoplasm is frequently positive for CD4/CD25 and shows loss of CD7 expression. Rarely, they present with unusual immunophenotypes therefore diagnostically challenging. We summarized the immunophenotypes of ATLL and analysed their associated cytogenetics/NGS findings.

Methods: In this study, we summarized the immunophenotypes of ATLL by flow cytometry and analysed the associated cytogenetics/next generation sequencing (NGS) findings. We retrospectively identified 117 patients with ATLL in a single institution in USA during a 19-year period (2003-2021). Of these 117 patients, 100 patients had flow cytometry tests, 70 patients had cytogenetics tests, and 43 patients had NGS tests.

Results: Out of the 100 patients with flow cytometry tests, 87 patients (87%) showed CD4+/CD7- immunophenotype, 7 (7%) patients showed CD4+/CD8+ immunophenotype, 2 (2%) patients showed CD4-/CD8- immunophenotype, 3 (3%) patients showed CD5- immunophenotype, 1 (1%) patient showed CD4-/CD7+ immunophenotype. The cases with unusual immunophenotypes frequently show complex cytogenetics/NGS findings with TP53, TBL1XR1 and NOTCH 1 being the most frequently mutated genes.

Conclusion: ATLL is associated with human T lymphotropic virus (HTLV-1) infection, usually in endemic areas such as Japan and Caribbean countries. CD4+/CD7- is the most common immunophenotypic findings of ATLL. ATLL can rarely show unusual immunophenotypes and frequently accompanied by complex cytogenetics/NGS findings. These cases can be incredibly challenging diagnostically. However, by combining the demographic features of the patients and typical clinical presentations, the possibility of ATLL should be raised and confirmed by HTLV-1 testing.


Primary thyroid lymphoma: a retrospective-observational study of 11 cases

R.A. Barna*, O. Vita, A. Dema, C. Lazureanu, R. Cornea, D. Brebu, O. Popa, A. Plopeanu, R. Covaci, M. Cornianu

*Department II Microscopic Morphology, Discipline of Morphopathology, ANAPATMOL Research Center, Victor Babes University of Medicine and Pharmacy, Timisoara, Romania

Background & objectives: Primary thyroid lymphoma (PTL) is an uncommon heterogeneous neoplasm diagnosed more frequent in women, commonly associated with autoimmune thyroiditis and account for less than 5% of primary thyroid malignancies. This study aimed to assess the clinico-pathological profile of PTL.

Methods: A retrospective observational study was conducted by analysing the medical records and examining the histopathological features of thyroidectomy and lobectomy specimens, from 11 patients diagnosed with PTL at the Emergency County Hospital from Timisoara. Clinical, pathological and immunohistochemical data (antibodies anti Ki67, CD20, CD10, CD30, CD5, Bcl-2, Bcl-6, cyclin-D1, ALK-1, kappa, lambda) were assessed.

Results: The mean age at the time of diagnosis was 69 years old. Eight women and three men were diagnosed with PTL resulting a female to male ratio of 2.7:1. Nine (81.81%) patients accused a painless and progressive growing tumour mass in the anterior cervical region, accompanied by local compression symptoms: dyspnea, dysphagia and dysphonia. All cases were diagnosed as non-Hodgkin, B-cell lymphomas (CD20 positive). The histological type consisted of six diffuse large B cell lymphoma, three MALT lymphoma, one follicular lymphoma and one case with follicular and diffuse lymphoma features. In 10 cases, PTL associated autoimmune lymphocytic thyroiditis. Seven cases (63,63%) were staged IE, two cases-IIE and two cases-IIIE.

Conclusion: This study reaffirms the clinico-pathological features of primary thyroid lymphoma, with a slight variation in female to male ratio. PTL are rare tumours and should be considered in the differential diagnosis of patients complaining of painless, progressive growing goiter or neck masses, and have a history of Hashimoto autoimmune thyroiditis. The prognosis of these patients is excellent and the overall survival is improved due to the advances in both diagnosis and treatment in recent years.


Molecular profiling of MYD88 and PIM1 genes in tissue samples from diffuse large B-cell non-Hodgkin’s lymphomas - defining element of their evolution and prognosis

M. Cristian*, M. Aschie, M. Deacu, C. Branzan, A. Cretu, A. Mitroi, G. Baltatescu, A.A. Serb, I. Poinareanu

*1) Clinical Service of Pathology, “Saint Apostle Andrew”” Emergency County Hospital, Constanţa, Romania. 3) Center for Research and Development of the Morphological and Genetic Studies of Malignant Pathology, “Ovidius” University of Constanţa, Romania

Background & objectives: Entire exome sequencing studies of DLBCL tissues have identified MYD88 and PIM-1 genes as involved in the development and signalling of this pathology. We aim to genotype profiles of MYD88 and PIM-1 genes and their implications on the prognosis.

Methods: We have conducted a retrospective study that included 50 paraffin-embedded tissues of DLBCL diagnosed at the Pathology Department of the Emergency County Clinical Hospital, Constanta, Romania, and Sacele Municipal Hospital, Brasov, Romania, between 2012 and 2021. The genotyping was analysed by RT-PCR using TaqMan® genotyping master mix and readymade TaqMan® genotyping assays for MYD88 (p.L252P) and PIM1 (p.G28A, p.L184V, p.V197F)

Results: Twenty-two patients were female and twenty-eight were male with ages ranging from 26 to 91 (mean:60,32 years).Twenty-four tumour samples were located in the lymph nodes and spleen, sixteen were located in the gastrointestinal tract (GI), four were located in the central nervous system and six tumours were located in the skin, testis, head and neck. Four cases (8%) were found to have mutation p.Leu252Pro in the MYD88 gene which occurs due to the transition of T>C at c.755 and only one case (2%) was found to have a mutation p.Gly28Asp in the PIM1 gene, as a transition of G>A at c.83.

Conclusion: In conclusion, our preliminary data suggests that the oncogenic mutations of PIM1 and MYD88 in our diffuse large B-cell lymphoma (DLBCL) cohort may improve diagnosis and prognosis of the patients with this pathology.

Funding: This study was funded by ”Grants competition in the bio-medical field 2021”, Contract no. 5/21.10.2021, registered at ”Ovidius” University of Constanta with no. 14453/21.10.2021. This research was performed in the Center for Research and Development of the Morphological and Genetic Studies of Malignant Pathology from ”Ovidius” University of Constanta.


T-cell lymphoma in bone marrow: morphologic and immunophenotypic study

H. Douik*, K. Tlili, G. Sahraoui, L. Charfi, I. Abbes, N. Boujelbene, B. Hedhli, I. Nasri, O. Chekir, K. Mrad, R. Doghri

*Salah Azaiz Institute of Cancer, Tunisia

Background & objectives: T-cell lymphomas (TCL) account for 10-15% of all lymphoproliferative disorders. Although commonly present, bone marrow involvement by TCL can be diagnostically challenging. Our aim was to establish a diagnostic approach to reliably identify bone marrow infiltration of TCL.

Methods: Retrospective study of 58 cases of bone marrow involvement by TCL collected in the pathology department over a period of 20 years (January 2001-December 2021). We analysed the morphological particularities of bone marrow infiltration by TCL.

Results: Our series included 41 males and 17 females with a median age of 45 years. The major subtype was TCL NOS in 58% followed by the angioimmunoblastic TCL in 18%. The anaplastic, the hepatosplenic and the NK subtype were diagnosed in 8% of cases for each one.

The infiltration pattern was interstitial in 72%, nodular in 15%, focal para trabecular in 10% and intrasinusoidal in 3% of cases highlighted with immunochemistry markers.

The infiltration percentage was less than 20% of cell population in 10 cases and more than 50% in 15 cases.

Conclusion: Bone marrow involvement in TCL is prognostically important for appropriate management. Morphology with immunochemistry can be reliably used to diagnose the bone marrow infiltration and to rule out the differential diagnosis.


Prevalence of Epstein Barr virus in de novo diffuse large B-cell lymphoma NOS in Algeria

N. Moulai*, M. Guermi, R. Bennoui, W. Ouahioune

*Department of pathology and Cancer Research Laboratory, Blida University Hospital, Saad Dahleb University, Algeria

Background & objectives: The prevalence of EBV in DLBCL accounts for <5-15% of DLBCL among Asian and Latin American patients and <5% among western patients. Our aim was to define the prevalence and clinicopathological features of EBV (+) DLBCL in Algeria.

Methods: A total of 162 cases of de novo DLBCL treated with R-CHOP were evaluated from January 2015 to August 2019. LMP1 and EBNA2 were performed for latency. The presence of EBV determined by EBER-ISH assay with 20% of cut-off. Clinical and pathological data were analysed.

Results: Of these 162 cases with DLBCL,13 (8%) showed EBV positivity. The median age of EBV(+)-DLBCL patients was 59, range from 22-87. 77% EBV(+)-DLBCL present in the nodal site . The ABC phenotype was found in 100% of our cases. 53.8% showed type III EBV latency. Clinically, EBV(+)-DLBCL presented an advanced clinical stage (84.6%), a high IPI (46.2%), and a low rate of response to treatment and survival (69.2%). Our results showed a significant difference between the two groups EBV(+)-DLBCL and EBV(-)-DLBCL on the response to R-CHOP treatment (p=0.01) and survival rate between the two groups. Log Rank (Mantel-Cox) p=0.0001

Conclusion: The prevalence of EBV in DLBCL is estimated at 8% in our series, joining the Asian series. This is still considerable compared to Western countries. Survival was significantly associated with EBV in our study.


The molecular spectrum of anaplastic large cell lymphoma (ALCL) - the wide next-generation sequencing profiling

A. Szumera-Ciećkiewicz*, A. Dansonka-Mieszkowska, J. Poleszczuk, O. Kuczkiewicz-Siemion, K. Kurek, E. Paszkiewicz-Kozik, M. Prochorec-Sobieszek, A. Tysarowski, J. Walewski, G. Rymkiewicz

*Narodowy Instytut Onkologii im. M. Sklodowskiej-Curie, Poland

Background & objectives: ALK-negative anaplastic large cell lymphoma (ALCL) ) is a malignant CD30-positive T cell neoplasm and clinically/molecularly three different subgroups have been outlined: DUSP22-rearranged, TP63-rearranged, and triple-negative cases (lacking DUSP22,TP63,ALK) with 5-year overall survival 90%, 17%, and 42%, respectively.

Methods: We investigated 26 ALCL cases: 17 ALK-negative and 9 ALK-positive (as a control group). The histopathological confirmation of the diagnosis (morphology and immunoprofile) with a consecutive 125-gene panel assay dedicated to lymphomas was performed. Samples and genes were clustered according to the Kendall rank correlation coefficient calculated on the percentage of reads value.

Results: The subgrouping of ALCL revealed: no(0) DUSP22-rearranged, three(3) TP63-rearranged, and sixteen(16) triple-negative cases. The prevalence of genes fusions (PGF) in the ALCL ALK-positive and ALK-negative groups included CCND1 and CCND3 (100%); the most frequently presented gene isoforms (PGI) in these two ALCL groups were BATF3 (70%) and ETV6 (60%). The profile of PGI in ALK-positive and triple-negative cases differed in LMO2, MUM1/IRF, MUC1 and were respectively 15%, <1%, 57% vs. 44%, 13%, 19%. The PGF including MYC was 14% in ALK-positive and 19% in triple-negative cases. Additionally, the TP63-rearranged cases showed a strong gene fusion connection between FOXP1 and EIF4E3.

Conclusion: The ALK-negative ALCL is a rare lymphoma, which is now classified as a separate entity that should be differentiated from primary cutaneous ALCL, and other T-cell/B-cell lymphomas with CD30 expression and anaplastic morphology. Comprehensive molecular studies under a larger cohort of ALK- ALCL are limited due to the low number of cases. Further investigation is needed not only to give more insight into the clinical significance of genetic alterations but also in search of new molecular targets for personalized therapy.

PS-10 | Poster Session Soft Tissue and Bone Pathology


Diagnostic utility of h3.3k36m immunostaining in chondroblastomas: a study of 16 cases from a tertiary cancer referral centre in India

V. Dave*, B. Rekhi

*Tata Memorial Hospital, India

Background & objectives: Chondroblastoma is a relatively uncommon benign bone tumour. At times, there is a challenge in differentiating it from its diagnostic mimics, especially on limited biopsies, with treatment-related implications.

To evaluate H3.3K36M immunostaining in chondroblastomas.

Methods: Ten cases were in the form of biopsy specimens and six were referred cases in form of paraffin blocks. Immunohistochemical staining for H3.3K36M (monoclonal, RM193, 1:100 dilution) was graded in terms of staining intensity(1+to 3+) and the percentage of tumour cells showing unequivocal nuclear staining.

Proximal tibia (5/16, 31.25%) was the commonest site, followed by the proximal humerus (4/16, 25%).

Results: We observed positive immunohistochemical staining for H3.3K36M in 15/16(93.75%) chondroblastomas, including all the tumour components, such as tumour cells and pink cartilage, but sparing the osteoclast-like giant cells. The percentage of tumour cells showing positivity ranged from 30% to 95%. We observed 3+ staining in 13/15((86.6%) cases and 2+ staining pattern in 2/15(13.3%) cases. A single case, which showed nuclear atypia displayed positive immunostaining for H3.3K36M in 80% of tumour cells with 3+ staining. None of the other 12 giant cell-rich lesions, including giant cell tumour of bone, displayed positive immunostaining for H3.3K36M.

Conclusion: Overall diagnostic sensitivity of H3.3K36M for chondroblastoma was 93.7% and specificity was 100%. This study, which is one of the first from our country, supports the diagnostic value of H3.3K36M for diagnosing chondroblastoma, including its distinction from its various diagnostic mimics on limited biopsy specimens.


Metalic-alloy wear debris after total hip arthroplasty is inducing periprosthetic tissue inflammation on subcellular level

S. Fokter*, R. Podlipec, E. Punzón-Quijorna

*University Medical Centre Maribor, Slovenia

Background & objectives: The metallic-associated adverse local tissue reactions (ALTR) and events accompanying worn implant materials are poorly understood on the subcellular level. Current immunohistochemical techniques lack chemical sensitivity to investigate causal relations between material and biological response on submicron scale.

Methods: A combination of photon, electron and ion beam microscopy-spectroscopy techniques including hybrid optical fluorescence and reflectance micro-spectroscopy, scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), helium ion microscopy (HIM) and micro-particle-induced X-ray emission (micro-PIXE) were applied on periprosthetic tissue obtained at revision surgery of a patient with osteoarthritis, who was treated earlier with a titanium-alloy total hip arthroplasty.

Results: Micron sized wear debris was found as the main cause of the tissue oxidative stress exhibited through lipopigments accumulation in the nearby lysosomes. This may explain the signs of chronic inflammation from prior histologic investigation. Furthermore, insights on extensive fretting and corrosion of the debris on nm scale and a quantitative measure of significant Al and V release into the tissue together with hydroxyapatite-like layer formation particularly bound to the regions with the highest Al content were revealed. Finally, by micro-PIXE we observed a wide spread of Ti- alloy debris throughout the whole tissue sample and confirmed selective metal leaching that corresponds to elevated concentrations in the patient’s serum.

Conclusion: The functional and structural information obtained at the subcellular level contributes to a better understanding of the macroscopic inflammatory processes observed on the tissue level. The established label-free correlative microscopy approach can efficiently be adopted to study any other clinical cases related to ALTR, as it can reveal more insights into implant rejection processes compared to the conventional histological examination further down on a submicron to single molecular scale.

Funding: This research was funded by Helmholtz European Partnering (CROSSING project, Grant-No: PIE-0007) and the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 799182 (TissueMaps project).


Extraadrenal soft-tissues Myelolipomas: clinicopathological study of 10 cases

M. Garcia Martos*, Y. Gómez Navarro, C.M. Vieru, A. Panizo Santos

*Hospital General Universitario Gregorio Marañón, Spain

Background & objectives: Myelolipoma is an uncommon benign tumour. It is most often found in the adrenal glands, although lesions in other unusual sites have also been described. The aim of our study is to describe the clinicopathological features of extraadrenal myelolipomas.

Methods: The pathology departmental archives of two University Hospitals were searched from 2005-2021 for patients originally diagnosed as extraadrenal myelolipoma. The cases were reviewed by the authors. Clinical parameters such as age, gender, tumour sites, and follow up were obtained from the existing medical records.

Results: Ten patients were identified: 7 males and 3 females, age ranged from 55 to 48 years. None of the patients had a previous history of malignancy or haematological diseases. All patients were diagnosed incidentally. The tumours involved posterior mediastinum (1), retroperitoneum (2), pelvic soft tissues (1), and paravertebral presacral (6). CT scan showed in all patients a well-demarcated tumour with fat density showing heterogeneous enhancement. So liposarcoma was suspected and surgical resection was performed. The tumours were well-circumscribed, encapsulated, with yellow to grey cut surfaces. Histologically, the tumours showed mature adipose tissue and hematopoietic elements, with concordant inmunohistochemistry. All patients went follow-up with no recurrence at the time of evaluation.

Conclusion: Myelolipoma is an uncommon benign tumour composed of adipose tissue and normal hematopoietic elements, which incidence is 0,08-0,2%. We present 10 cases of extraadrenal myelolipomas. Extraadrenal myelolipoma is a rare benign and asymptomatic tumour that may be misdiagnosed as a malignant lipomatous tumour on radiological studies. Most of them are incidentally detected during radiological investigation of unrelated symptoms. Surgery is the recommended treatment. The final diagnosis relies on pathologic findings.


Anastomosing haemangioma with unusual location and mimicking malignancy: a case series and literature review

E. Dicle Serbes*, E. Gedik, S. Yuksel, G. Kaygusuz

*Ankara University Medical School, Pathology Department, Turkey

Background & objectives: Anastomosing haemangioma (AH) is a relatively newly defined entity seen mostly in the kidney. Extrarenal localizations have also been reported. Herein, we report two non-renal cases, one of which is the second case in English literature involving anterior mediastinum.

Methods: Excisional biopsies were obtained from a 68-year-old woman with an anterior mediastinal mass (8 mm), and a 41-year-old man with a mass of 12.5 mm located between left adrenal gland and tail of pancreas. The latter had a pre-diagnosis of a malignancy. Both of them was incidentally found. Radiological examinations including CT, MRI and PET scans were also evaluated.

Results: Both lesions were un-encapsulated, well-circumscribed tumours consisting of predominantly small, anastomosing, splenic-like sinusoidal vascular structures which were lined by single layer of flat endothelial cells with occasional hobnail cells. Small fibrin thrombi was present in some vascular channels. No multilayering of endothelial cells, cytological atypia, necrosis, mitotic activity, extramedullary haematopoiesis or invasion into surrounding tissues was found. Immunohistochemically, tumour cells were diffusely positive with CD31, ERG and CD34 but negative for GLUT1, D2-40, Desmin or Cytokeratin. Ki67 proliferation index was lower than 5% in tumour cells. Cases were diagnosed as AH. Anterior mediastinal AH had positive surgical margins, the patient has been following for 2 years without any local recurrence.

Conclusion: Anastomosing haemangioma is a relatively newly described benign vascular tumour. It is a rare neoplasm which can have some overlapping histologic features with well-differentiated angiosarcoma. It was originally described in kidney and perinephric adipose tissue in 2009, but extrarenal sites including testis, spermatic cord, ovary, adrenal glands, gastrointestinal tract, mesentery, soft tissues and bone had also been reported. Recognition of this entity and knowing that it may occur in unexpected localisations may help it’s differential diagnosis from more aggressive lesions.


Three cases of Kaposi sarcoma as initial manifestation of HIV infection and AIDS

J. Jevtic*, L. Simic, R. Janković, S. Rajkovic, G. Djuricic, J. Sopta

*Institute of Pathology, Serbia

Background & objectives: Epidemic Kaposi sarcoma (KS), the most common subtype, is seen in the setting of HIV infection and related acquired immunodeficiency. We report three cases of KS as initial manifestation of AIDS.

Methods: We analysed medical records from Register of bone and soft tissue lesions biopsies in the Institute of pathology, Medical Faculty, University of Belgrade from 2006 to 2022.

Results: Only three patients had KS associated with HIV infection as initial manifestation of AIDS. All of them were males. One patient was 40-year-old with KS localized in tonsil, and two of them were 43-year-old with KS localized in cervical lymph nodes. All patients were presented with bulky masses and all of them underwent excisional biopsy. Histological analysis showed spindle cell tumour, without pleomorphism, mixed with inflammatory cells. Aforementioned spindle cells were immunohistochemically positive for vascular markers. Positivity for HHV-8 indicated diagnosis of KS and further clinical examinations in order to confirm/exclude AIDS were performed. All patients were found to be HIV positive, with developed AIDS.

Conclusion: Even though rare, KS could be initial manifestation of AIDS. In order to confirm/exclude the diagnosis of KS, when there are any histological suspicion, HHV-8 should be applied. Early diagnosis of KS and AIDS prevents complications and ensures better prognosis.


Pediatric chordomas: report of five cases with emphasis on poorly differentiated subtype and SMARCB1/INI1 deficiency

Z.B. Erdem*, A.M. Onenerk Men, S.N. Tuter, S. Ocak, N. Comunoglu

*Basaksehir Cam and Sakura City Hospital, Turkey

Background & objectives: Chordoma is a rare primary malignant bone tumour showing notochordal differentiation. They usually affect adults, and extremely rarely seen in paediatric age group. For children, such tumours present a different biology with aggressive histological features, and worse prognosis.

Methods: All chordomas diagnosed between January 2016-January 2022 in one institution were reviewed for the study (total number of 41). Five patients belonging to paediatric age group were found and included in the study.

Results: Patients ages ranged between 2 to 18 years(median:9 years); 3 females, 2 males. Tumour localizations were as follows: two cervical vertebrea, two clivus and one intracranial(cerebellopontin angle). On histomorphologic examination, three cases showed conventional chordoma features, one of them was compatible with chondroid chordoma and two of the cases had poorly differentiated morphology. On immunohistochemical analysis all tumours showed cytokeratin and EMA positivity. Four tumours showed S100 positivity with one of the poorly differentiated tumours being negative. Four of the tumours were brachyury positive (wasn’t applied to one of the conventional chordoma). SMARCB1(INI1) immunohistochemistry was applied to all and both of the poorly differentiated tumours showed loss of expression.

Conclusion: Currently, WHO classifies chordomas into three subtypes: conventional, poorly differentiated, dedifferentiated. Features in paediatric population are different from the corresponding adult tumours. Majority of “poorly differentiated” tumours are found in children and diagnostic feature of this subtype is loss of SMARCB1(INI1) expression. Demonstration of bracyury positivity with loss of SMARCB1(INI1) expession are easily applicable diagnostic tests. Although the limited data on poorly differentiated chordomas show that they have poor prognosis, proving SMARCB1(INI1) loss provides an opportunity for possible targeted therapies.


Does MYC-amplification help distinguishing primary from radiation-induced angiosarcomas?

B. Raposo Pulido*, J.M. Rodríguez Barbero, F.I. Camacho Castañeda, A.E. Ballén Barragán, M. Granados Almillo, J.P. Morillo Morillo, R. Granados Carreño

*Hospital Universitario de Getafe, Spain

Background & objectives: Angiosarcomas (AS) can be primary (PAS) or secondary (SAS) to radiation therapy or chronic lymphedema. Both share histological findings, but genetic and molecular differences have been reported. We compared these features in 8 cases of AS.

Methods: We retrospectively reviewed 8 cases of AS (4 males and 4 females) diagnosed at our hospital between 1996 and 2022. Clinicopathological, immunohistochemical (IHC) and molecular studies were performed in 5 cases of PAS and 3 of SAS.

Results: Eight patients with an age range of 18-79.

Three had SAS 3-30 years after irradiation for breast carcinoma (2 cases) or brain astroblastoma. Five PAS were from the skin, breast, pleura, popliteal artery and penis. Histopathological study revealed a high-grade malignant mesenchymal tumour with proliferating endothelial cells and varying degrees of atypia and growth patterns (epithelioid, spindled, or vasoformative) in all cases, not distinguishing PAS from SAS.

p53 overexpression was seen in all 5 PAS, but it was absent in the 3 SAS cases.

While c-myc overexpression was demonstrated by IHC in all but the penile AS, only 3 PAS and 1 SAS showed low-level MYC amplification (3-5 copies).

Conclusion: In our study, c-myc immunohistochemical overexpression did not correlate with MYC gene amplification in all the cases. Low-level MYC amplification was found in 50% of our cases. Interestingly we couldn’t demonstrate high-level MYC amplification (>10 copies) in any case. In our series, MYC and/or p53 alterations may play an important role to induce oncogenesis in AS, independently of prior radiation therapy, by increasing genomic instability.


Usefulness of novel SS18-SSX and SSX c-terminus antibodies for identification of specific fusion oncoprotein in sarcomas

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

*Medical University of Graz, Austria

Background & objectives: Chromosomal rearrangement can be identified by direct methods or immunohistochemical staining for a component of the fusion oncoprotein as a surrogate marker. We aim to gain insights into the staining profile of sarcomas using novel SS18-SSX and SSX c-terminus antibodies.

Methods: Retrospective analysis of 303 soft tissue sarcomas diagnosed at our Institution between 1999 and 2019 was performed, and tissue microarrays were constructed. Immunohistochemistry was conducted on the Benchmark Ultra platform with iVIEW DAB Detection Kit. Two different antibodies for SSX locus were used: SS18-SSX and SSX c-terminus. Ten whole-tissue sections of genetically confirmed synovial sarcomas were used as a control.

Results: In total, 19/303 (6.3%) and 23/303 (7.6%) sarcomas showed in most of the cases strong nuclear staining with SS18-SSX and SSX, respectively. In detail, from 21 synovial sarcomas, 19 (90.5%) stained positive for both antibodies. In 5 cases, nuclear staining for SSX antibody was weak. 9/10 (90%) control cases showed strong nuclear staining for SS18-SSX, and 3/10 (70%) were negative for SSX. Furthermore, SSX nuclear expression was also found in 4/56 (7.1%) myxofibrosarcomas. All other sarcomas, including various liposarcomas, angiosarcomas, undifferentiated pleomorphic sarcomas, leiomyosarcomas, and epithelioid sarcomas, were negative for both antibodies.

Conclusion: Novel SS18-SSX and SSX c-terminus antibodies are reliable diagnostic markers and can be used as surrogate markers to identify a specific fusion. The former antibody is more specific and shows strong nuclear staining in synovial sarcomas, whereas SSX can present with weak staining and is less specific. RNA-based NGS analysis should be performed in equivocal cases to confirm the specific rearrangement.


Chordoma: significance and correlation of the localisation, vascular density and Ki67 for the occurrence of chordoma recurrence

L. Simic*, J. Jevtic, M. Radojevic, D. Ribic, S. Rajkovic, G. Djuricic, D. Ristic, J. Sopta

*University of Belgrade, Faculty of Medicine, Institute of Pathology, Belgrade, Serbia

Background & objectives: Chordomas are malignant slow-growing, locally invasive tumours, usually localized in sacrum, vertebra and scull base, characterized by frequent relapses. The aim of the study is to examine the correlation between localization, blood vessels density and Ki67 value and disease recurrence.

Methods: All chordoma biopsies, taken in Institute for Orthopaedic Surgery Banjica in the period of five years (2017-2021), were analysed at the Institute of Pathology, in Belgrade. A total number of 21 cases were divided into two groups: patients with and without relapses. Ki67 value was counted on 100 cells in hot spot. Vascular density was estimated using CD34 immunostaining in 1mm2.

Results: Out of total 21 patients with chordoma only 9 had a relapse. The most affected bone was sacral bone, but without a statistically significant difference in the frequency of recurrence at different chordoma localizations. The average number of blood vessels in 1mm2 and value of Ki67 was statistically significantly higher in group of tumours with recurrence (p=0,015, p=0.033 respectively). Ki67 proliferative index values greater than 10% show a statistically significant difference in the frequency of relapses. The value of the proliferative index higher than 15% has a moderately high correlation with the occurrence of relapse.

Conclusion: Chordoma is a rare, but locally aggressive tumour, with high recurrence rate (35-40%). Based on our results, determination of the immunohistochemical expression of CD34 antigen in evaluation of vascular density, together with proliferative index Ki67 can be helpful to predict tumour recurrence.

PS-11| Poster Session Cytopathology


Pancreatobiliary cytopathology: the use of the Papanicolaou Society system in the transition towards a new era of standardised reporting

M. Hanks*, G.P. Aithal, A.M. Zaitoun

*Nottingham University Hospitals NHS Trust, United Kingdom

Background & objectives: Modern diagnostic techniques have allowed for a less invasive approach to pancreatic cytopathology sampling in combination with routine staining and immunohistochemistry. Our study aimed to compare the emerging Papanicolaou System with the C1-C5 grading system in a single UK institution.

Methods: We retrospectively assessed 723 cases of pancreatobiliary cytology with 142 cases demonstrating corroborative histology. The reported C1-C5 grade was reviewed by 2 independent pathologists and assigned a grade within the Papanicolaou Society of Cytopathology Guidelines for pancreaticobiliary cytology. We were then able to compare the 2 grading systems for diagnostic accuracy, sensitivity, specificity, false positive and false negative rates.

Results: All cases which were originally assigned to C1, C2, C4 and C5 categories maintained the diagnostic category assignment when comparing to the Papanicolaou Society system. The number of cases reported as atypical was reduced from 93 to 73 when using the Papanicolaou Society system; 5 cases were subsequently reported as IVA and 15 cases IVB.

Sensitivity was 98.3%, specificity 75%, false positive rate 2.5% and false negative rate 11.8% with a diagnostic accuracy of 91.5% with the C1-C5 system. The Papanicolaou Society guidelines showed a sensitivity of 99.1%, specificity of 79.1%, false positive rate of 2.5% and a false negative rate of 5% with a diagnostic accuracy of 94.3%.

Conclusion: The increasingly international approach to pathology highlights the need for a standardised reporting system to facilitate safe but effective communication whilst enabling national and international data comparisons. Our data supports the adoption of the Papanicolaou system recommended by the Royal College of Pathologists (UK); comparable results are achieved compared to C1-C5 whilst reducing the number of cases reported as atypical allowing for more informed diagnostic decisions to maximise patient benefit as we embark on the next generation of pathology.


Pancreatic neuroendocrine tumours: the shifting scales of lesion classification

M. Hanks*, M. Khan, D. Lobo, I. Beckingham, S. Ryder, A.M. Zaitoun

*Nottingham University Hospitals NHS Trust, United Kingdom

Background & objectives: Pancreatic neoplasms are classified using C1-C5 or Papanicolaou Society of Cytopathology; the latter groups lesions of variable malignant potential into a single category. The World Health Organisation proposes a new classification with pancreatic neuroendocrine tumours as malignant entities alongside adenocarcinomas.

Methods: We analysed 68 cases of pancreatic neuroendocrine tumours with corroborative cytology available in 47 cases over a 12 year period. We classified each case using C1-C5 Grading, Papanicolaou Society of Cytopathology and the proposed World Health Organisation classification for pancreatic lesions. We assessed the Ki-67 grading and presence of poor prognostic factors including vascular or perineural invasion and resection status.

Results: Cytological assessment reported 80.4% of lesions as IVB on Papanicolaou grading and 76% as C5 lesions. 73.1% of resected tumours were grade 1, 19.4% grade 2 and 7.5% grade 3. Overall staging using TNM8 showed 44.8% of lesions were T1, 31.3% T2, 19.4% T3 and 4.5% T4. 10 cases (14.9%) showed lymph node involvement. Complete resection was achieved in 83.6% of cases. Vascular invasion was seen in 29.9% of cases and perineural invasion in 9%. 32 biopsies had both cytology grade and histological grade reported and this was concordant in 87.5% of cases. 1 multi-focal tumour was reported which was non-concordant highlighting the importance of clinicopathological correlation following biopsy sampling.

Conclusion: Standardised reporting systems are an important tool facilitating effective communication on a national and international level. The Papanicolaou system uses a pragmatic approach to distinguish these lesions from more aggressive entities to offer flexibility in management however the updated WHO system removes this distinction. It may be beneficial to use the new WHO low risk and high risk pancreatic neoplasm categories to promote discussion on the risk to benefit ratio of surgery for these lesions whilst effectively risk stratifying patients.


What is the risk of malignancy associated with diagnostic categories of proposed World Health Organization international system for reporting pancreaticobiliary cytopathology?

P.U. Göçün, B. Simsek*, Ö. Ekinci, N. Ekmen, M. Arhan, T. Karakan, M. İbiş, M. Cindoruk

*Gazi Univercity Medical School Department of Pathology, Turkey

Background & objectives: The Papanicolaou Society of Cytopathology(PSC) reporting system for pancreaticobiliary cytology has 6 categories(I-VI) providing risk assessment and guidance for patient management. World Health Organization(WHO) proposed an updated reporting system. Risk of malignancy(ROM) of new categories of WHO system needs defining.

Methods: 420 pancreatic ESU-FNA materials from 410 patient from our archive from the last 12 years have been reviewed and categorized both according to the PSC and proposed WHO reporting systems. Histological diagnosis and/or clinical follow-up of patients were searched from hospital's database and risk of malignancy for both systems respectively were evaluated through statistical analysis.

Results: The absolute risk of malignancy for each diagnostic category of the proposed WHO system were as follows: 35% for insufficient/inadequate nondiagnostic category, 1.0% for benign/negative