Oral Free Paper Sessions

Sunday, 1 September 2013, 08.30–12.00, Room 5C

OFP-01 Oral Free Paper Session Breast Pathology


Fully automated FISH staining and digital analysis of HER2 in breast cancer: A validation study

E. van der Logt *, D. A. J. Kuperus, J. W. van Setten, M. C. van den Heuvel, J. E. Boers, E. Schuuring, R. E. Kibbelaar

*Pathologie Friesland, Dept. of Pathology, Noordeloos, The Netherlands

Objective: To validate the detection of human epidermal growth factor receptor-2 (HER2) status using the Leica HER2 fluorescent in situ hybridization (FISH) system and subsequent semi-quantitative analysis with the Menarini Benelux D-Sight digital imaging platform, in order to predict HER2-directed therapy in patients with invasive breast cancer.

Method: HER2 assessment was performed on 328 formalin-fixed/paraffine-embedded invasive breast cancer tumours on tissue microarrays (TMA) and 100 (50 selected IHC++ and 50 random IHC scores) full-sized slides of breast cancer resections biopsies obtained for diagnostic purposes. For digital analysis slides were pre-screened at 20× and 100× magnification for all fluorescent signals and semi-automated scoring was performed on at least two pictures with the D-Sight HER2 FISH analysis module. Results were compared to data obtained previously with the manual Abbott FISH test.

Results: The overall agreement with Abbott FISH data among TMA samples and in 50 selected IHC++ cases was 98.8 % (kappa = 0.94) and 93.8 % (kappa = 0.88), respectively. The results of 50 unselected IHC cases were concordant with previously obtained IHC and/or FISH data.

Conclusion: The combination of the Leica FISH system with the Menarini Benelux D-Sight digital imaging platform is feasible for the assessment of HER2 status in routine clinical practice in patients with invasive breast cancer.


A new quantitative in-situ immunohistochemistry method

H. Derand *, J. Lohse, K. Petersen, K. Jensen, R. Jørgensen

*Dako A/S, Research and Development, Glostrup, Denmark

Objective: There is a general need to make pathologic examinations less subjective and to support the accuracy required for companion diagnostics. The aim of this study was to develop a new IHC method based on bright field technology combining morphological information with quantitative assessment.

Method: By chemical manipulation of a visualization system, single antibodies are visualized as dots instead of a conventional stain. The dots can easily be counted by image analysis, and the number of dot reflects protein expression levels. Initial assay performance was evaluated using Her2 as a test system; breast cancer cell lines as well as breast cancer tissue specimen were included.

Results: The assay generates highly reproducible results and gives a linear assay with a larger dynamic range than the conventional assays. No overlap was seen between the different breast cancer cell lines. Moreover, evaluation of HER2 status in a number of breast cancers by the use of this new method showed a strong correlation with established methods.

Conclusion: The data suggest enumeration of dots can be direct related to protein expression levels. This represents a new and standardized way of objectively determining protein amounts in cells and tissue in situ.


Automated image analysis enables accurate enumeration of the Ki-67 labelling index of breast cancer

A. Laurinavicius *, A. Laurinaviciene, R. Meskauskas, I. Baltrusaityte, J. Besusparis, P. Herlin, B. Plancolaine, N. Elie, P. Belhomme, C. Bor-Angelier

*National Center of Pathology, Vilnius, Lithuania

Objective: Immunohistochemical Ki67 evaluation reflects proliferative activity and is regarded as important prognostic/predictive marker of breast cancer. However, its potential is hindered by lack of standardized and efficient methodologies to measure the Ki67 expression. Besides many other aspects, key element of the methodology remains accurate enumeration of Ki67-labelling index (LI). We investigated the accuracy aspect of automated image analysis (IA) approach.

Method: TMA (1 mm diameter spot per patient, n = 140) from invasive ductal breast carcinoma, stained for Ki67 and digitized by Aperio scanner, were used for the study. Reference values (RV) were obtained by counting the LI using stereological frame. IA was performed with Aperio Genie/Nuclear algorithms enabling automated selection of tumour tissue. The images were semi-quantitatively evaluated by 3 pathologists (P1,2,3).

Results: RV correlated strongly with IA (r = 0.95) and P1,2,3 (r = 0.86, r = 0.89, r = 0.93, respectively), p < 0.0001. Analysis of variance revealed no significant pairwise differences of the LI means of RV(40 %) versus IA(36 %), P2(43 %), or P3(44 %); however, the IA versus P2,3 differed, and P1(24 %) was significantly lower (p < 0.05). Regression analysis to predict RV revealed best performance for the IA results.

Conclusion: IA provided most accurate enumeration of the Ki67 LI. Validation against proper RV is a crucial step in setting up an IA tool.


Prognostic impact of Ki-67 in breast cancer assessed on tissue microarrays, core needle biopsies and surgical specimens

G. Knutsvik *, I. M. Stefansson, K. Collett, L. A. Akslen

*University of Bergen, Dept. of Clinical Medicine, Norway

Objective: Tumor cell proliferation in breast carcinomas is strongly prognostic and may also predict response to chemotherapy. However, no consensus exists concerning counting areas or cut-off values. Here, our aim was to assess the prognostic value of Ki67 counts from surgical excision specimens as compared with core needle biopsies (CNB) and tissue microarrays (TMA).

Method: We examined a retrospective, population-based series of breast cancer (n = 547). The percentage of Ki67 positive nuclei was evaluated by manual counting on TMAs, CNBs, and whole sections (WSs).

Results: The three evaluation methods were significantly correlated. The median percentage of Ki67 expression was 18.2 % (WS, hot-spot areas), 16.6 % (estimated average across WS), 12.8 % (CNB) and 7.0 % (TMA). Increased Ki67 expression by all evaluation methods was associated with aggressive tumor features (large tumor diameter, high histologic grade, ER negativity) and reduced survival.

Conclusion: Ki67 is prognostic over a wide range of cut-off points. However, absolute Ki67 values are influenced by choice of specimen, and the cut-off levels derived from studies of TMA sections are different from those obtained using specimens from a clinical setting. Our findings indicate that specimen specific cut-off values should be established for practical use.


Phenotypic and genotypic analysis of triple negative breast cancer with an emphasis on the identification of stem cells

M. Comanescu *, R. Ivan, G. Bussolati

*INCD Victor Babes, Pathology, Bucuresti, Romania

Objective: While targeted therapies have been developed for other types of breast cancers, triple negative breast cancers (TNBC) still represent a challenge for oncologists.

Method: We evaluated tumor specimens together with peritumoral tissue of 30 breast cancer patients. Besides the routine immunohistochemical analysis, other markers targeting stem cells (CSC) were used. In a first phase a gene array analysis was performes using Agilent Whole Human Genome 4 × 44 K Microarrays, which were further were scanned and signals were extracted using Feature Extraction 10.5.1. The data were analysed using the software GeneSpring GX. In a second phase, we investigated gene expression by real time PCR, using 3 types of 96-well plates: Breast Cancer Array, Human Stem Cell PCR Array and Human Stem Cell Signaling PCR Array from Qiagen. Interpretation of data was performed using Qiagen RT2 Profiler PCR Array data Analysis version 3.5.

Results: We identified significant differences in the immunophenotype of CSC between cases and together with the initial microarray based assay directed the study towards the investigation of stem cells.

Conclusion: Keeping in mind the need for improvement in the therapy of TNBC, we believe that the identification of genes involved in survival and self-renewal of cancer stem cells represents an area of interesrt Acknowledgments Project PN-II-RU-PD-2011-3-0248


Sentinel lymph node macrometastasis in breast cancer- is axillary lymph node dissection mandatory?

A. Figueiredo *, A. Ribeiro, M. Martins

*Dept. of Pathology, Hospital Curry Cabral, Lisboa, Portugal

Objective: Axillary lymph node dissection (ALND) is the current procedure in breast cancer patients with sentinel lymph node (SN) macrometastasis. The aim of this study is to determine which factors could be associated with axillary non-SN metastasis.

Method: Between 2011 and 2012, 134 patients underwent SN analysis with One Step Nucleic Acid Amplification (OSNA), of which 28 (20 %) had macrometastasis. We assessed tumors in 27 of these 28 patients (10 with non-SN involvement and 17 without). For the statistical analyses the Mann–Whitney test and logistic regression models were performed. A significance level of 0,05 was considered.

Results: We verified a significant crude association between non-SN involvement and primary tumor size (OR = 5,6; CI95 %: 1,1;30,9), tumor multifocality (OR = 7,5; CI95 %: 1,1;51,5) and maximum number of CK19 copies (MNCK19) (OR = 7,3; CI95 %: 1,2;46,2). This group had also higher median values of the ratio between MNCK19 and the SN weight (p = 0,001) and the number of positive SN (p < 0,001). We found no statistically significant association with age, the histological grade and subtypes and lymphovascular invasion.

Conclusion: The results suggest that new criteria to perform ALND of breast cancer patients with SN macrometastasis can be developed.


Immunohistochemical characterization of HER-2 tumors

A. Cuesta *, E. Honrado Franco, M. Baltasar Moreira, P. Castro Val, F. Izquierdo Garcia

*Hospital de Leon, Dept. de Anatomia Patologica, Spain

Objective: HER-2 positivity, defines a clinically challenging subgroup of patients with breast cancer with variable prognosis and response to therapy. The main aim of this study is to identify different immunohistochemical groups in HER-2 tumors and compare HER-2 amplified tumors with HER-2 polysomic or no amplified tumors.

Method: Tumors from 54 patients with IDC, 42 HER-2 amplified, 7 non-amplified and 5 polysomic were included in a tissue microarrays and stained for ER, PR, Ki67, p53, p63, ck5/6, Racemase, cyclin D1, p16 and for HER2 (immunohistochemistry and SISH silver in situ hybridization).

Results: HER-2 amplified tumors, are more frequently ER, PR, BCL2, P16 negative and do not express basal markers (ck5/6 and p63. P53, Ki67 and Racemase are positive. HER-2 polysomic tumors show overexpression of P53 and Racemase and PR negativity. On the other hand, HER-2 polisomic tumors, are similar to non-amplified tumors in the overexpression of basal markers (20 % versus 9 % in amplified tumors) and ER and the underexpression of Ki 67. We observed that all cases with polysomy of chromosome17 had an IHC score of 3+ except one 2+.

Conclusion: HER-2 amplified tumors, are a very homogeneous group with low expression of hormone receptors, and basal markers, and high expression of proliferation markers and Racemase. HER-2 polysomic tumors, might be a subgroup that share immunohistochemical characteristis of HER-2 amplified and non-amplified tumors, show HER-2 protein overexpression which probably has clinical differences.


Altered expression of cell cycle regulatory proteins in different types of phyllodes tumors of the breast

S. Bulimbasic *

*Dubrava University Hospital, Dept. of Pathology, Zagreb, Croatia

Objective: Phyllodes tumors (PTs) are rare fibroepithelial tumors of the breast with wide biological potential, classified as benign, borderline or malignant.

Method: In this study we assessed the prognostic value of histopathologic characteristics and immunohistochemical features across a panel of antibodies against cell cycle regulatory proteins (cyclin D1, p21, p27 and p53) in a study set of 90 PTs. Additionally, immunohistologic features of benign, borderline, and malignant PTs are compared in order to ascertain whether malignant progression is associated with differential marker expression and whether such differences may be diagnostically useful.

Results: Stromal overexpression of Cyclin D1, p27, p53 correlated significantly with tumor grade. No immunostaining differences were detected for p21. The number of altered proteins in stroma increased with higher grade and was accompanied by increased proliferation and increased mean vascular density (MVD). Correlations existed between stromal overexpression of Cyclin D1, proliferation index and MVD as well as p53, proliferation index and MVD. Inverse correlation was noted between stromal expression of p27, proliferation index and MVD.

Conclusion: According to logistic regression model, p53, proliferation index and MVD were the best predictors of malignant phenotype and can be used as additional methods for grading.


Breast tumours resembling Papillary Thyroid Carcinomas (PTC): Report of 10 cases with long term follow up

S. Foreid *, S. Popovska, V. Eusebi

*Hospital de Sao Jose, Dept. de Anatomia Patologica, Lisbon, Portugal

Objective: We report 10 cases of papillary carcinomas of the breast that histologically resemble PTC.

Method: These include 2 cases previously reported (Am. J. Surg. Pathol. 27, 1114–18, 2003) and 8 new cases.

Results: Patients were female of 54.7 years (mean age). All cases displayed neoplastic cells arranged in solid to papillary architectures and follicular structures resembling thyroid tissue. Cells evidenced eosinophilic granular cytoplasm and nuclei with clear chromatin with numerous grooves and occasional eosinophilic pseudoinclusions. All cases stained for mitochondria in >50 % of neoplastic elements. ER, PR, AR, Her-2, TTF1, Thyroglobulin were negative. Myoepithelial cells were absent in 8 cases. Cases were discovered mammographically, except for case 3 that had a lump present for 10 years. Sentinel nodes in 6 cases were negative. Case 3 had an intramammary metastatic lymph node. All patients are alive and well. No fewer than 3 had 10 year-FU.

Conclusion: Breast tumours resembling PTC have to be recognized to avoid misdiagnosis as metastatic PTC; they are triple negative tumours with indolent behaviour; the term tumour for these neoplasms seems appropriate as it is difficult, especially without immunohistochemistry, to establish their invasion.


Improved malignancy prediction by B3 breast lesions subclassification

F. de Beca *, C. Rasteiro, A. Correia, I. Amendoeira

*Porto, Portugal

Objective: Core-needle biopsy (CNB) of breast lesions can be classified in 5 categories according to lesion type and risk of malignancy. B3 category constitutes a challenging problem in clinical decision with most ending in excisional biopsy. This study aims at assess if subclassification (in B3a and B3b categories) according to the presence of atypia in otherwise B3 lesions better predicts malignancy on excision.

Method: 48 cases with diagnosis of B3 lesion on CNB and matched surgical excision specimen were included and subclassified in B3a and B3b categories to evaluate positive predictive value (PPV) and odds of malignancy in CNB.

Results: B3 category overall PPV for malignancy was 12.5 % with a significant low odds of malignancy of 0.14 (95 % CI: 0.06–0.34). When subclassified, B3b (lesions with atypia) demonstrated a higher PPV for malignancy (36.36 %) with a non-significant odds. Inversely, B3a (lesions without atypia) demonstrated a PPV for malignancy of only 5.41 % and a significant low odds of malignancy of only 0.06 (95 % CI: 0.01–0.24).

Conclusion: The low rate of malignancy in some of B3 lesions additionally reinforces the practice of avoiding surgical excision in selected patients and provides data that further supports B3 lesions subclassification according to the presence of atypia.


Adenoid cystic carcinoma of salivary glands and the breast: May their different clinical course be attributed to posttranscriptional divergencies?

O. Kiss *, J. Kulka, A.-M. Tokes, A. M. Szasz, S. Spisak

*Semmelweis University, 2nd Dept. of Pathology, Budapest, Hungary

Objective: Adenoid cystic carcinoma of the salivary glands and the breast (sACC and bACC) are histologically identical malignancies but their clinical course differ: sACC has a poor prognosis but bACC's clinical outcome is favourable. Our aim was to investigate sACCs and bACCs characteristics at the posttrascriptional level.

Method: Thirteen cases of sACCs and 10 cases of bACCs were investigated. Normal salivary gland (NS = 11) and normal breast tissues (NB = 9) were used as controls. Total RNA was isolated from FFPE tissues. Reverse transcription was carried out on each sample for 18 miRNAs. We performed quantitative PCR on 96-well plates.

Results: Relative expression of let-7b decreased in sACCs compared to NSs. In bACCs miR-20a decreased compared to NBs, while the expression of miR-17* and miR-27b was higher in bACCs than in NBs. The expression of miR-193b was higher in sACCs than in NSs but in bACC its expression was lower in bACCs than in NBs. The genes potentially regulated by the above mentioned miRNAs include CCND1, BCL2, VEGFA, and NOTCH1 which have central roles in the development, expansion and spreading of human malignant tumors.

Conclusion: The detected posttranscriptional differences might perhaps partially explain the diverse clinical course of bACC and sACC cases.

Sunday, 1 September 2013, 14.15–16.15, Room 5C

OFP-02 Oral Free Paper Session Surgical Pathology and Other Topics


Effect of unbalanced immune reactions on myocardial remodeling of dilatative cardiomyopathy patients with left ventricular assist device implantation

K. Wassilew *, E. Potapov, T. Krabatsch, M. Dandel, B. Song, A. Mickley, C. Schmuttermaier, A. Gratchev, R. Hetzer, J. Kzhyshkowska

*Deutsches Herzzentrum Berlin, Inst. für Herzpathologie, Germany

Objective: We investigated effects of chronic inflammatory disorders (CID) such as asthma and autoimmune reactions on immunopathological markers supporting alternatively activated macrophages in dilatative cardiomyopathy (DCM) patients with LVAD implantation and compared levels of interstitial fibrosis (IF) in LV apex from the site of LVAD implantation with anterior LV wall of the explanted heart.

Method: Paraffin sections of LV myocardium from 13 patients (8–62 years, 2 female, 9 with CID) were stained for a macrophage marker (CD68) and a M2 marker (stabilin-1). Subpopulations of macrophages were examined using double immunofluorescence/confocal microscopy. Findings were correlated with IF on Sirius-red-stained slides.

Results: Whereas circulating macrophages were solely CD68+, tissue macrophages showed combined CD68+/stabilin-1+ and myofibroblasts were actin+/stabilin-1+. A higher CD68+/stabilin-1+ cell count in the LV apex was associated with elevated IF in all but 3 patients. In patients without CID, mean IF (8.5 %) was within normal limits (<9 %); in CID patients, total IF was elevated (mean 14.6 %). Generally, there was less fibrosis in the anterior LV wall than in apical myocardium.

Conclusion: CID appear to initiate inflammatory responses and influence the repolarization of resting resident macrophages into M2, which are associated with larger areas of IF and therefore participate in myocardial remodeling.


Angiogenesis in symptomatic vascular malformations of skin and soft tissue relates to hemodynamic high flow features of the lesion

L. Meijer-Jorna *, C. M. van der Loos, O. J. de Boer, A. J. Horrevoets, J. R. Mekkes, C. M. van der Horst, A. C. van der Wal

*Symbiant/MCA, Dept. of Pathology, Alkmaar, The Netherlands

Objective: Vascular malformations are clinically categorized in high flow (HF) or low flow (LF) lesions. Episodic angiogenesis has been observed in arteriovenous malformations (AVMs) of skin and soft tissue. We investigated whether angiogenesis relates to different flow features.

Method: Resection specimens of 80 AVMs, of which 37 HF and 43 LF lesions were histopathologically screened for presence of angiogenesis, inflammation and/or thrombosis. Additional immunostaining was performed with anti-endoglin (CD105), a marker for endothelial activation, and anti-von Willebrand factor (vWF) for vascular leakage.

Results: 81 % of HF lesions versus 14 % of LF lesions showed angiogenesis (p < 0.005). In HF lesions, that were embolized prior to surgery (30 % of all), 88 % showed angiogenesis, 88 % inflammation and 33 % thrombosis. However, similar angiogenesis was also observed in AVMs without prior embolization (69 % HF versus 14 % LF, p < 0.005). Endoglin was more frequently expressed in HF lesions and extracellular vWF staining was found in nearly all HF and LF lesions.

Conclusion: Angiogenesis in AVMs appears to be strongly associated with HF features. This can be explained only to some extent by tissue damage initiated by previous embolization and/or (related) inflammation. Occurrence of angiogenesis in AVMs without prior embolization suggests that the high flow itself has angiogenic properties in AVMs.


A new ligand for the endothelial receptor, TIE-2, that enhances barrier integrity and prevents vascular leakage

C. Jackson *, N. Minhas, P. Naik, M. Xue

*Kolling Institute, Sutton Laboratory, Sydney, Australia

Objective: Activated protein C (APC), a natural anticoagulant with strong cyto-protective properties, is FDA-approved for treatment of severe sepsis and acts by promoting endothelial barrier protection. The aim of this study was to investigate the immediate effects of APC through the Tie2 receptor on endothelial barrier integrity.

Method: Methods include biacore, bioinformatics, (competitive) binding assays, and a mouse model of sepsis

Results: APC rapidly activated Tie2 with the ratio of phosphorylated(P)-Tie2:total(T)-Tie2 increasing by ~4.5-fold after 60 min of APC treatment. Docking studies revealed intermolecular H-bonds between APC and Tie2 indicating binding between the two proteins. Protein binding assay using Biacore confirmed strong binding affinity (Kd = 0.003 nM) between APC and Tie2, which was only slightly less than Angiopoietin (Ang)1-Tie2 (Kd = 0.002) and surprisingly, ~11 times higher than Ang2-Tie2 (Kd = 0.032 nM). In a mouse model of sepsis, APC significantly reduced lipopolysaccharide-induced vascular permeability in lungs and kidneys and this was reversed by inhibiting the Tie2 receptor.

Conclusion: In summary, APC is a new ligand for Tie2. By binding to and activating Tie2, APC rapidly enhances endothelial barrier function and prevents vascular leakage, which could explain its therapeutic action in sepsis.


Tumors and tumour-like lesions of the calcaneum: A study of 7 cases

F. Khanchel-Lakhoua *, K. Mrad, R. Dhouib, R. Doghri, M. Driss, L. Charfi, S. Sassi, I. Abbes, N. Ben Hamida, K. Ben Romdhane

*Salah Azaiez Institute, Pathology, Tunis, Tunisia

Objective: The calcaneum is an uncommon site for most bone tumors. The knowledge of the distribution of tumors in the calcaneus is important for an exact differential diagnosis and thereby for biopsy planning, diagnosis and therapy. The aims of our study is to evaluate tumors and tumor-like lesions of the calcaneum.

Method: We analyzed tumoral and tumour-like lesions of the calcaneum registered in the Department of Pathology Salah Azaiez Institut from 1992 to 2012.

Results: 7 cases were registered. There were 5 males and 2 females and their ages varied from 7 to 70 years with a median of 16. We registred 4 benign lesions, with one chondromyxoid fibroma, one angioma, one osteochondroma, one chondroblastomas. There were 3 malignant tumours (1 osteosarcoma, 1 Ewing sarcoma, 1 chondrosarcoma).

Conclusion: In our series, cartilaginous lesions were the main lesion of the calcaneum. In literature, cystic lesions such as simple bone cysts and aneurysmal bone cysts are the most common lesion. Other tumors in the calcaneus are mainly cartilaginous.


Histone deacetylase inhibitors as potential therapeutic approaches for chordoma: An immunohistochemical and functional analysis in MUG-chor1

S. Scheipl *, B. Lohberger, E. V. Froehlich, B. Rinner, A. Beham, F. Quehenberger, P. P. Varga, A. Lazary, J. Haybaeck, A. Leithner, B. Liegl

*Medical University of Graz, Orthopaedic Surgery, Austria

Objective: Chordomas are rare malignancies of the axial skeleton. Therapy is mainly restricted to surgery. This study investigates histone deacetylase (HDAC) inhibitors as potential therapeutics for chordomas.

Method: Immunohistochemistry (IHC) was performed using the HDAC 1–6 antibodies on 50 chordoma samples (34 primary tumours, 16 recurrences) from 44 patients (27 male, 17 female). Pan-HDAC inhibitors Vorinostat (SAHA), Panobinostat (LBH-589), and Belinostat (PXD101) were tested for their efficacy in the chordoma cell line MUG-Chor1 via Western Blot, cell cycle analysis, caspase 3/7 activity, caspase-3, and PARP cleavage. P-values below 0.05 were considered significant.

Results: IHC was negative for HDAC1, positive for HDAC2 in most (n = 36; 72 %), and for HDACs 3 to 6 in all specimens available (n = 43; 86 %). HDAC 6 expression was strongest. SAHA and LBH-589, but not PXD101 caused a significant increase of G2/M phase cells and of cleaved caspase-3 (p = 0.0003, and p = 0.0014 after 72 h, respectively), and a peak of caspase 3/7 activity. PARP cleavage confirmed apoptosis.

Conclusion: The presented chordoma series expressed HDACs 2–6 with strongest expression of HDAC6. SAHA and LBH-589 significantly increased apoptosis and changed cell cycle distribution in vitro. HDAC-inhibitors should be further evaluated as therapeutic options for chordoma.


Primitive mucosal melanoma: A review of 11 cases

M. Njima*, S. Ben Abdelkrim, N. laabaied, T. Tlili, S. Korbi, S. Hmissa

*Farhat Hached Hospital, Dept. of Pathology, Sousse, Tunisia

Objective: Melanomas are essentially cutaneous tumors; their mucosal location is rare accounting for 1.3 % of melanomas. The pathologist is often confronted with two problems, positive diagnosis and confirmation of the primitive character of the tumor.

Method: This is a retrospective study of PMM seen over a period of 15 years (1993–2007). Epidemiological and clinicopathological features were recorded.

Results: 11 cases were diagnosed during this period. There were 5 males and 6 females patients. The mean age was 58 years (range 22–75). The most common location was the eye in 4 cases (36 %), then genital sites (vagina, clitoris) in 2 cases and digestive sites (rectum, anus) were involved in two other cases. The tumor was stage II or IV in 50 % of cases it was T3 or T4 in 55 % of cases. Lymph node metastasis and distant metastases were observed in 27 % of cases each. Treatment was surgical in 7 cases, radiotherapy or chemotherapy in 1 case.

Conclusion: PMM is a rare and aggressive tumor. Its symptomatology is non specific. Definitive diagnosis is achieved after pathological analysis. Diagnosis is often delayed.


Quality control of deaths by pathologists: A systematic approach for improving death certificate completions, notification procedures and death statistics

G. C. Alfsen *, L. G. Lyckander, H. M. Eng, A. W. Lindboe

*Akershus University Hospital, Dept. of Pathology, Lørenskog, Norway

Objective: In order to improve the quality of death certificates and the notification procedures in deaths of presumed unnatural causes, all deaths at our institution have since 2008 been reviewed by two pathologists.

Method: Death certificates and medical records were examined continuously. Clinicians were contacted immediately if death certificates had illogical sequences of underlying death, or if cause of death was not in accordance with the clinical history. Changes were suggested and assistance in resubmittance offered. The Quality departement was notified if deaths by unnatural causes (of any kind) were suspected, and the clinicians contacted if notification procedures had not been followed.

Results: From 2008 to 2012, illogical death certificates decreased from 36 % to 20 %, certificates not in accordance with medical records from 24 % to 14 %. Unspecified “garbage” diagnoses were reduced by 79 %. Notification procedures in presumed unnatural deaths improved from 51 % to 86 %.

Conclusion: A centralized and systematic control of deaths improved the quality of death certificates and the handling of deaths by presumed unnatural causes. Being experienced in evaluating causes of death, having regular working hours and proximity to the morgue, the pathologists are best suited to perform this kind of quality control in hospitals.


Choice of method for manual Ki-67 determination

R. Røge *, S. Nielsen, M. Vyberg

*Aalborg University Hospital, Institute of Pathology, Denmark

Objective: Ki67 is a proliferation marker important for classification of malignant tumours such as breast carcinomas. However, guidelines for determination of Ki67 proliferation indices are heterogeneous. The aim of this study was to examine the current practices and interlaboratory variability of Ki67 scoring.

Method: As part of the NordiQC international immunohistochemical external quality assessment scheme, 370 laboratories were asked to score proliferation indices on 20 Ki67 stained breast carcinomas on a virtual slide. Additionally, participants were asked to elaborate on the method used for obtaining the indices: Method (‘Eyeballing’ or ‘manual counting of X cells’), Area examined (‘Hot spot’ or ‘Overall average’) and whether moderate and weakly stained tumour cells were counted as positive.

Results: 139 laboratories participated. Estimated proliferation indices varied greatly. For tumours with low Ki67 proliferation index, estimated indices varied between 0 and 10 %, while results for high expressing tumours varied between 40 and 100 %. Eyeballing was the preferred method (65 %). Less than 40 % examined ‘hot spots’ as opposed to overall average.

Conclusion: The need for standardization of both staining and interpretation of Ki67 immunohistochemical stains is underlined. Digital image analysis may be an important tool to accomplish this.


Initiation of a tumor tissue-bank in Malawi

S. Berezowska *, T. Tomoka, E. Borgstein, D. A. Milner, S. Kamiza, R. Langer

*Universität Bern, Inst. für Pathologie, Switzerland

Objective: Cancer in Africa has become a serious health problem with high mortality rates. Knowledge about epidemiology, pathogenesis and genetics is scarce. Pathology plays an important role in diagnostics and in cancer research, but the service is barely available. We initiated the project of building a tumor-biobank in Blantyre/Malawi, as a basis of structured, significant tissue-based research targeting cancer in sub-Saharan Africa.

Method: Routine diagnostics in Blantyre is based on formalin fixed paraffin embedded (FFPE) probes and allows diagnostic standard staining (HE, PAS, ZN). Application of further molecular analysis for routine or research is hampered by the lack of standardization of pre-analytic tissue handling (e.g. variable fixation times, unclear fixation agents).

Results: A basic approach for a biobank in the low resource setting encompasses: A) Structuring and standardization of tissue processing, allowing FFPE tissue to serve as a firm base for scientific projects. B) Evaluation of alternative fixation agents, which may offer alternatives for robust tissue preservation. C) Establishing a clinical data bank. D) Ethical issues (e.g. informed consent, cultural aspects).

Conclusion: Establishing a tissue-biobank in sub-saharan Africa is a valuable step towards tissue-based research for gaining insight into cancer in the African population, and a prerequisite for subsequent focused analyses (e.g. HPV-analyses).


The preliminary results of a residency satisfaction survey: “To love and not to love Pathology”

B. Pehlivanoglu *, H. Hassoy, I. Nalbantoglu, C. Calle, A. Dendooven, A. F. Okuducu, B. Doganavsargil

*Ege University, Dept. of Pathology, Izmir, Turkey

Objective: Pathology tends to be a less known discipline among medical school graduates. Multiple factors contribute to career choices such as individual’s personality, skills, academic and socioeconomic expectations. Unfortunately, the number of the residents who are not satisfied with their job is considerable. We conducted a survey to evaluate pathology residents’ perspectives on Pathology.

Method: The 42 item-survey was delivered via a web-based link and questioned participants’ personal and institutional background, workplace, training conditions and job satisfaction.

Results: Survey was answered by 101 participants from Turkey (n = 76), Europe (n = 16) and continental America (n = 9), 78,2 % of whom were female. Mean age was 29,7 years-old (range 25–40). Mean daily work and weekly grossing-hours of the participants were 10.1 ± 1.7 and 12.9 ± 5.1 h, respectively. Eighty-two percent of the participants were happy that they chose Pathology and 72,2 % foresaw a bright future ahead. Overall working conditions of their department were dissatisfying for 8.9 % of the participants. Most of the participants wanted to have better structured residency training programs and to interact more with experienced pathologists.

Conclusion: The study aims to provide an insight to residency period and is presented to increase the philosophical interest on the subject as well as announcing for more participants.

Sunday, 1 September 2013, 17.00–19.00, Auditorium II

OFP-03 Oral Free Paper Session Digestive Diseases Pathology I


Mutational analysis by next generation sequencing of preneoplastic intestinal metaplasia in patients with Barrett esophagus from endoscopic samples

S. Lagana *, Y. Yuan, T. Uehara, N. Jhala, T. Ganguly, Y. Liu, R. Brand, J. Sepulveda, G. Falk, A. Sepulveda

*New York Presbyterian-Columbia, Dept. of Pathology and Cell Biology, New York, NY, USA

Objective: To identify mutational biomarkers using next-generation sequencing (NGS) and endoscopic samples for stratification of Barrett Esophagus (BE) patients at increased risk of dysplasia.

Method: Ion Torrent AmpliSeq Cancer Panel was used to screen for mutations in 46 cancer genes. Sixteen samples were tested: Intestinal metaplasia from 6 patients (IM-P) with concomitant high-grade dysplasia (HGD)/adenocarcinoma (EAC) and HGD/EAC in 3 patients; IM of 7 patients followed for at least 2 years without any dysplasia (IM-N). Ion torrent suite software and ANNOVAR were used for analysis.

Results: The most frequent mutations in IM and HGD/EAC were detected in TP53 spanning codons 150 to 280. Four of 6 IM-P patients had TP53 mutations in IM samples and in HGD/EAC tested. None of the 7 IM-N patients had TP53 mutations. Sensitivity of TP53 mutation for presence of concomitant dysplasia was 67 % and specificity was 100 %.

Conclusion: DNA from routine endoscopic samples can be efficiently used to simultaneously detect multiple mutations by NGS. TP53 mutations were frequently detected in IM of patients with HGD/EAC but not in patients who did not progress to HGD/EAC, suggesting that TP53 mutational testing may be useful to identify IM-P patients who may benefit from closer surveillance.


Amplification but not translocation of ALK is a frequent event in esophageal cancer

P. Birner *

*Medizin. Universität Wien, Inst. für Pathologie, Austria

Objective: Translocations of ALK have been demonstrated in a variety of human malignancies, and the corresponding fusion-proteins are potential therapeutical targets. Aim of this study was to investigate ALK gene status in a large cohort of esophageal squamous cell carcinoma (SCC) and adenocarcinoma (AC).

Method: ALK status was investigated in 117 SCCs and 136 ACs by fluorescence in situ hybridization (FISH), and ALK protein expression by immunohistochemistry. Data on expression of ALK downstream effector tyrosine −705 phosphorylated STAT3 (pSTAT3) was available from a previous study.

Results: FISH was successfull in 251 cases. No ALK translocations were found, while 14/135 (12.1 %) of SCCs and 14/116 (10.4 %) of ACs showed ALK amplifications. Concomitant EML4 amplifications were present in 27/28 cases with ALK amplifications. Three cases showed EML4 translocations not involving ALK. None of the tumors with ALK amplification showed ALK protein expression, and no correlation with clinical parameters, survival or pSTAT3 expression was observed.

Conclusion: While ALK translocations are not present in esophageal cancer, ALK amplifications are common events. Since ALK amplified breast cancer cells were shown to respond to ALK inhibitors, ALK amplified esophageal cancers might be considered as possible candidates for therapies targeting ALK.


MALDI imaging mass spectrometry for proteomic segmentation of tumor heterogeneity in gastric cancer tissues

B. Balluff *

*Leiden University Medical Cent, The Netherlands

Objective: A high clonal diversity within a patient’s tumor is an important factor for the evolution of the disease and the clinical management of the patient with regard to cancer relapse and response to therapy (Marusyk, Nat Rev Cancer. 2012 Apr 19;12(5):323–34). The identification and molecular characterization of the tumor’s clonal diversity is therefore of high clinical relevance.

Method: In this study, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry was used to identify clinically relevant tumor subpopulations in gastric cancer. MALDI imaging allows the unlabeled in situ measurement of hundreds of molecules (like proteins) within their histomorphological context of tissue sections (Balluff, Gastroenterology. 2012 Sep;143(3):544–9.e1-).

Results: Spatially-resolved, tumor-specific proteomic data was acquired from 63 intestinal-type gastric cancer patients by MALDI imaging. The resulting data underwent a novel statistical procedure (Jones, PLoS One. 2011;6(9):e24913) which results in a spatial segmentation of areas within a tumor section based on their molecular similarity. Correlation of the molecular signatures of the segmented tumor areas with clinical data resulted in the identification of clinically relevant tumor subpopulation in terms of prognosis and metastasis.

Conclusion: Our results highlight the usefulness of MALDI imaging in combination with advanced statistical approaches for detecting novel and clinical relevant information from tumor tissues.


Increased tumor-budding/CD8+ lymphocytes ratio is associated with metastasis and venous invasion in Pancreatic Ductal Adenocarcinoma (PDAC)

E. Karamitopoulou *, I. Zlobec, A. Perren, B. Gloor, A. Lugli

*University of Bern, Institute of Pathology, Switzerland

Objective: T-Lymphocytes can be a major part of tumor microenvironment, especially at the area of tumor-host interaction where tumor progression, reflected by epithelial-mesenchymal transition (EMT) and its hallmark tumor-budding is taking place. Our aim was to determine the role of CD8+lymphocytes in correlation with tumor-budding in PDAC.

Method: Double immunostaining for AE1-AE3/CD8 was performed on a multipunch tissue microarray of 120 well-characterized PDACs. Tumor-buds, CD8+ and tumor-budding/CD8+ lymphocytes indices were evaluated and associated with clinico-pathological features, follow-up and adjuvant therapy information.

Results: There was a strong negative correlation between the number of buds and CD8+ counts (p = 0.01). Increased numbers of tumor-buds were associated with venous invasion (p = 0.0272) and reduced overall survival (p = 0.0147). Low CD8+ peritumoral lymphocytes showed only a marginal association with metastasis (p = 0.0683). Tumor-budding/CD8+lymphocytes ratio was strongly associated with venous invasion (p = 0.0078) and metastasis (p = 0.0452). No intratumoral CD8+lymphocytes were detected.

Conclusion: Low counts of CD8+ peritumoral lymphocytes in the micro-environment of PDAC promote EMT as reflected by tumor-budding and facilitate tumor progression, since tumor-buds seem to display metastatic potential only when coupled by decreased counts of CD8+lymphocytes. However, unlike to other tumor types, no prognostic effect was found, probably reflecting the fact of the absence of intratumoral CD8+lymphocytes.


Various carcinogenetic pathways may be involved in colorectal serrated polyposis

E. Courcet*, A. Aline-Fardin, P. Cervera, J.-F. Fléjou

*Hôpital Saint-Antoine, Service d’Anatomie Pathologiqu, Paris, France

Objective: Serrated polyposis (SP) is considered as a model leading to colorectal cancer (CRC) through the serrated pathway. However, the precise morphological and molecular steps are still incompletely understood. We aimed to characterize the morphological and molecular abnormalities in a series of SP, in CRC and in precancerous lesions.

Method: Fifteen consecutive patients with SP were selected in our files. Polyps (n = 258) were classified according to the World Health Organization classification (conventional adenomas (CA), sessile serrated adenomas/polyp (SSA/P), traditional serrated adenomas (TSA), hyperplastic polyps (HP)). CRCs (n = 13) and polyps (n = 31) were evaluated for mismatch repair proteins (MMR), BRAF mutated V600E protein, p53 and beta-catenin expression using immunohistochemistry.

Results: Residual adenomatous lesions (4 tubulo-villous CAs, 4 SSA/Ps) were identified beside CRCs. Polyp number per patient ranged from 6 to 51, with 89 % of serrated polyps (44 HPs, 130 SSA/Ps, 47 dysplastic SSA/Ps). Eight CRCs and 1 SSA/P with high and low-grade dysplasia were MLH1-/PMS2-. One CRC was MSH2-/MSH6-. Five of 24 SSA/Ps were BRAF V600E+.

Conclusion: SP seems to be a possible although rare presentation of Lynch syndrome with MSH2 mutation. Serrated adenomas and conventional adenomas seem to be both involved in carcinogenesis in SP.


Expression pattern of TrkB in colorectal cancer supports anoikis-resistance as a survival mechanism for tumor budding cells

H. Dawson*, I. Zlobec, V. H. Koelzer, E. Karamitopoulou, A. Lugli

*Universität Bern, Inst. für Pathologie, Switzerland

Objective: Tumor buds in colorectal cancer represent an aggressive subgroup of non-proliferating, non-apoptotic cells. We hypothesize that survival of tumor buds is dependent on resistance to anoikis. Here we investigate the role of TrkB, a promoter of epithelial-mesenchymal transition (EMT) and anoikis-resistance in facilitating a pro-tumor budding phenotype.

Method: TrkB immunohistochemistry was performed on a multiple-punch tissue microarray of 211 colorectal cancer patients. Cytoplasmic (cTrkB) and nuclear (nTrkB) staining were evaluated in tumor and buds. KRAS/BRAF mutations were investigated. Correlation with a panel of EMT-related proteins was performed.

Results: cTrkB and nTrkB were strongly inversely correlated in tumor (r = −0.38; p < 0.0001) and tumor buds (r = −0.41; p < 0.0001). cTrkB was associated with high-grade tumor budding (p < 0.0001), KRAS mutation (p = 0.0008) and expressed frequently in tumor buds (100/154 cases; p < 0.0001). nTrkB was expressed in low-grade budding cases (p = 0.0073), BRAF wild-type tumors (p = 0.0519) and expressed infrequently in tumor buds (34/154; p < 0.0001). cTrkB and nTrkB protein profiles corresponded to pro- and anti-budding phenotypes, respectively.

Conclusion: These results underline functional differences of TrkB dependent on cellular localization with cTrkB promoting a pro-budding phenotype. Moreover, our findings support the notion of anoikis-resistance as a survival mechanism for budding cells in colorectal cancer.


Primary Biliary Cirrhosis (PBC): A new histological scoring system allows a standardized and reliable evaluation of lesions with known prognostic significance

D. Wendum *, P.-Y. Boëlle, P. Bedossa, E.-S. Zafrani, F. Charlotte, M.-C. Saint Paul, S. Michalak, O. Chazouillères, C. Corpechot

*APHP-Hôpital St. Antoine, Dept. de Anatomie Pathologique, Paris, France

Objective: In PBC, an accurate evaluation of liver lesions predictive for progression or survival (fibrosis, lymphocytic interface hepatitis (LIH), ductopenia) is crucial. Presently there is no satisfactory system analyzing them reliably. We elaborated a semiquantitative scoring system and evaluated its intra/interobserver reproducibility.

Method: Fibrosis was classified into 4 stages (portal/periportal fibrosis, few septa, numerous septa, cirrhosis) and LIH into 4 grades. The bile duct ratio (BDR = portal tracts with ducts on total number of portal tracts) and Ludwig’s stage were also evaluated. 33 liver biopsies (HE, picrosirius red) were independently analyzed by 5 liver pathologists. Intra and interobserver agreement were assessed (multireader Light’s kappa, Washington intraclass correlation).

Results: The biopsies measured 23 mm [12–40 mm]. Five had numerous fibrous septa or cirrhosis and five had severe LIH. The mean BDR was 0.75. Intraobserver reproducibility was substantial for fibrosis (k = 0.78), LIH (k = 0.69) and BDR (ICC = 0.69). Interobserver reproducibility was moderate for fibrosis (k = 0.56), LIH (k = 0.59), BDR (ICC = 0.5). Ludwig’s staging had a fair intra and interobserver reproducibility (k = 0.26, k = 0.32 respectively).

Conclusion: This scoring system assesses the prognostic lesions with a substantial intraobserver and a moderate interobserver reproducibility. It is more reliable than Ludwig’s staging. It will likely improve the quality and robustness of the histopathological results in PBC.


E2F-1 immunophenotype is an independent marker of poor prognosis in human hepatocellular carcinoma

M. Palaiologou *, J. Delladetsima, M. Karanikolas, E. Fatourou, D. Karandrea, E. Antoniou, E. Felekouras, J. Koskinas, D. Tiniakos

*NKUA Medical School, Lab. of Histology and Embryology, Athens, Greece

Objective: E2F-1 transcription factor induces expression of genes controlling G1/S phase transition, DNA synthesis/repair, and apoptosis. E2F-1 activation depends on pRb phosphorylation. We have shown that E2F-1 is overexpressed and pro-apoptotic in hepatocellular carcinoma (HCC), while others have suggested that increased apoptosis in HCC could be indirectly oncogenic*. The prognostic significance of E2F-1 immunoexpression in human HCC has not as yet been clarified.

Method: Immunohistochemistry for E2F-1 and phospho(Ser795)pRB was employed on 57 surgically resected HCCs (grade I:8,II:24,III:16,IV:9). Patients were followed for 39.7 ± 27.2 months.

Results: Nuclear E2F-1 (nE2F-1) immunoexpression was observed in 32/57(54.3 %) HCCs and correlated with phospho(Ser795)pRB immunoexpression (p = 0.045), indirectly suggesting E2F-1 transcriptional activity. E2F-1 cytoplasmic immunoexpression (cE2F-1) was observed in 46/57(80.7 %) HCCs. There was no significant correlation of nE2F-1 or cE2F-1 with clinico-pathological parameters. Portal vein thrombosis and nE2F-1 immunophenotype were correlated with poor overall survival (p = 0.05 and p = 0.0001, respectively), but not with recurrence-free survival. Stepwise Cox regression analysis highlighted nE2F-1 as an independent marker of poor prognosis in HCC (95 % CI = 2.55–52.43, p = 0.002).

Conclusion: E2F-1 is expressed and transcriptionally active in the majority of human HCCs. E2F-1 immunophenotype is an independent marker of poor overall patient survival in a cohort of Greek HCC. * Virchows Archiv 2012;460:439–46

Sunday, 1 September 2013, 17.00–19.00, Room 5B

OFP-04 Oral Free Paper Session IT in Pathology


The Case Database of the European Congresses of Pathology: Current status and planned features

M. Lundin *, J. Szymas, J. Lundin

*Helsinki, Finland

Objective: The ECP Case database consists of Case presentations from the ECP/ESP congresses, years 2006–2012. The objective is to continuously expand and improve the contents and functionality of the database, and make it available for educational purposes.

Method: Slides of cases presented in the ECP slide seminars have been digitized and published online since 2006. The virtual slides together with case data (background, snapshots, diagnosis, handout) have been collected into a single repository. The database has been made available through a web site (http://www.webmicroscope.net/casedatabase) with interactive functions such as browsing by topic/working group, searching and a quiz/self test mode. The database is designed to be hosted on a network of campus servers

Results: After the ECP 2012 there were 634 cases with 3201 images included in the database. These cases have been contributed by 427 authors. The database will be expanded in 2013 with high-quality case presentations from the ECP in Lisbon.

Conclusion: The ECP Case Database is a significant educational resource, and continues to grow.


The first results of Latvian national tissue biobank and the novel elaborated biobank information database system: Status report after the first 3 years of experience

S. Isajevs *, I. Liepniece-Karele, A. Kirshners, N. Jeshkevics, A. Ruskule, J. Eglitis, M. Leja

*University of Latvia, Dept. of Pathology, Riga, Latvia

Objective: The objectives and goals of the first national Latvian biobank project are to develop, build and utilize cutting edge tissue biobank and databasae in Latvia containing samples of common malignant tumours and elaborate a multifunctional easy to use biobank database system.

Method: 1,655 patients with oncological and premalignant diseases were enrolled in the study. Biobank information systems run on the operating system MS Windows 7 Professional. Server runs on MS Server 2008 R2 Standard environment, MS Windows SQL Server 2008 R2 Standard served as a database management system.

Results: In total 1,655 patients with oncological and premalignant diseases were enrolled in the study during the 2010–2012. Our database comprised a total of 1,655 cases covering all types of frequent human malignancies: breast cancer (30.64 %), gastric (22.78 %), colorectal (15.52 %), prostate (17.38 %) and thyroid (13.78 %). The novel biobank information database was developed which fulfils all major biobanking criteria and extends these. The advantages of novel biobank information system are easy entry, retrieval and storage of information.

Conclusion: The first Latvian biobank including malignant disease, premalignant conditions and autoimmune disease was elaborated. The novel easy to use and reproducible biobank information database system was developed and adapted.


A Swedish government-funded project for distance diagnostics, consultative expert networks and multi-disciplinary team support

J. Lindholm *

*Djursholm, Sweden

Objective: A project aiming to create a system and workflow for distance diagnostics, consultative expert networks and multi-disciplinary team support. Three main scenarios: 1. Department that has long term or short term demand for analysis capacity. 2. Expert node with specific competence, serving a larger region. 3. Collaboration across sub-specialist areas in virtual multi-disciplinary teams to enhance the diagnostic quality and provide more effective treatment. The aim is complex as it transverse the organizational and sub-specialist boundaries that exists today enabling exchange of competence and capacity to provide the patient with the best care possible. The project also manages technic, regulations, reimbursement and patient security.

Method: 300 patient cases will be sent for pathology distance reading between two different counties in Sweden. Stockholm County Council—a large urban region and Blekinge County Council—a small region in the countryside. Much effort is being placed in risk-analysis and quality controls.

Results: The project is on-going. Distance reading is today being tested with real patient data between the two county councils. Simultaneously a prototype for multi-disciplinary team support is being put together.

Conclusion: Conclusions and results so far will be presented at ECP 2013 if abstract being accepted.


Next-generation tissue microarray (ngTMA) strengthens pathology biomarker research: An exemplary study of the tumor microenvironment using CD3, CD8, and CD45RO in six different solid tumor types

I. Zlobec *, V. H. Koelzer, H. Dawson, A. Perren, A. Lugli

*Universität Bern, Inst. für Pathologie, Switzerland

Objective: We defined next-generation tissue microarrays (ngTMA) as the combination of strategic TMA planning, histological expertise, digital pathology and automated tissue microarraying. The aim is to test ngTMA focusing on the immune-score (CD3, CD8 and CD45RO) within the tumor microenvironment of six tumor types.

Method: Ten cases each of malignant melanoma, lung, breast, gastric, prostate and colorectal cancers were reviewed. One representative H&E slide was scanned and uploaded onto a digital slide platform. Using different colors, 1 mm TMA annotations were placed directly onto the digital slide. Selected regions of normal tissue (n = 1), tumor center (n = 2), tumor front (n = 2), and tumor microenvironment (n = 2) were annotated. Donor blocks were loaded into an automated tissue microarrayer. Using donor block images and annotated digital slides, transfer of 420 tissue cores created two ngTMAs. CD3, CD8 and CD45RO immunohistochemistry were performed.

Results: Scanning time was <10 h; annotation time was 1 h. Punching of tissue cores and transfer took 12 s/core. ngTMA construction took 1.4 h. Desired histological regions including tumor-stroma interaction captured the T-cell response.

Conclusion: ngTMA is a well-defined process based on planning and design, digital pathology, histological annotations and automated tissue microarraying. ngTMA is a promising approach that clearly supports the role of pathologists in translational research.


Quantitative measurements of cell density and proliferative markers by image analysis

A. Kudaybergenova *

*RNCRCT, Pathology, St. Petersburg, Russia

Objective: Cell distribution and cell density are the main characteristic for tumor pathology. We do not yet understand how many cells inhabit a tumor from a 1 sq mm sample of histology slide. We analyzed a total number of tumor cells in breast cancer; renal cell carcinoma; lung carcinoma (NSC), and endometrial carcinoma and proliferative activity (% Ki67-positive cells) to establish the precise quantity of tumor cells per sq.mm of histological slide and the relations of this measure with proliferation.

Method: The study included 46 breast carcinomas; 44 RCC; 21 NSCLC, and 35 endometrial carcinoma.

Results: Mean absolute tumor cells in 1 mm2 of histology slide was for breast cancer: 4,160+/−251, Ki67–31 %; NSCL: 4,102+\\− 364 cells, Ki67–15 %; endometrial carcinoma: 7,073+\\− 614, Ki67–31.9 %; RCC 4,389+\\− 229, Ki67 11.62 %. There was moderate correlation between cell density and Ki67 for breast cancer r = 0.42 (p = 0.00018); for NSCL, r = 0,41 (p = 0,0,032), for endometrial carcinoma r = 0,48 (p = 0,0,059) and no correlation for RCC.

Conclusion: By analysis of these four types of cancer was established the quantity of tumor cells per mm2 and main the proliferative characteristics


Automatic detection of tumor areas by applying homology method

K. Nakane *, Y. Tsuchihashi

*Osaka University, Dept. of Medicine, Suita, Japan

Objective: Computer assisted pathological diagnosis is now an issue of importance in the current situations of shortage of diagnostic pathologists in Japan. In the present study we propose a simple mathematical model to identify tumor tissues from their normal counterparts utilizing changes in the Betti numbers in tumorigenesis.

Method: The Betti numbers, which are consisting of two numbers (b1 and b0), can express the degree of the connection of the figure. When the number of contact points between individual components increases, irrespective of their shape, b1 and a ratio b1/b0 significantly change. We hypothesize these numbers can be used as indices to represent the cellular “accumulation” which is one of the characteristics of tumor tissue.

Results: Hematoxylin and eosin stained mucosal biopsy sections of colonic tumors are used as test samples. We compare the values, b1 and b1/b0, of colonic tumor tissues with those of normal tissues. As the values of b1 and b1/b0, we put color stones on unit area. The area of abnormal, it can be seen that the stone is placed

Conclusion: The results obtained in the present study clearly indicate that the difference in the Betti numbers can differentiate tumor tissues from their normal counterparts.

Numerical Results:

figure a


Development of an automated 2D image reconstruction algorithm for multiple tissue blocks: Toward high-resolution whole organ 3D images

N. Hashimoto *, Y. Yagi

*Harvard Medical School and MGH, Pathology, Boston, USA

Objective: 3D image reconstruction from 100 s to 1,000 s of serial section Whole Slide Images (WSIs) from a single tissue block has been reported. Although the results were promising, high-resolution 3D images of whole organ offer more potential for new discoveries. There are a number of challenges in performing 3D image reconstruction of a whole tissue organ from WSIs. The objective of this study is to develop an algorithm to automatically reconstruct whole organ 2D images from WSIs of different tissue blocks, which were from the same slice of the tissue organ, since these WSIs may have different orientations and exact locations of tissue blocks are not known.

Method: We considered single slice of tissue organ. This slice is represented by WSIs of 5–15 blocks. Automatic 2D image reconstruction we have developed was implemented by finding the appropriate position and rotation of the images through template matching of low-resolution WSIs.

Results: Our 2D image reconstruction algorithm showed excellent results and the possibility to compare with gross or other modality images.

Conclusion: We developed the 2D image reconstruction method and confirmed its validity. It would be the important step to our goal “high-resolution whole organ 3D images”.


Display systems for digital pathology

T. Kimpe*, A. Xthona

*Barco, Healthcare Division, Kortrijk, Belgium

Objective: In digital pathology, pathologists make use of a display to look at scanned slides in order to come to a diagnosis. This paper presents clear guidelines of which specifications are needed for digital pathology displays.

Method: Image characteristics obtained when using current optical microscopes are examined. Special focus areas include luminance and color, stability and possible variation and instabilities among systems or over time. Characteristics of digital pathology scanners and display systems are also investigated and compared with current optical microscopes.

Results: When comparing characteristics of several displays, it has become clear that there is a very large variation in quality. Some (consumer) displays offer clearly lower image quality compared to optical microscope based systems, while other (typically medical) display systems specify similar or even clearly higher image quality compared to optical microscope based systems.

Conclusion: Pathologists make use of a display to look at scanned slides in order to come to a diagnosis. The display system therefore is a critical component, and it is important that certain minimum specification displays are used so that the same level of clinical performance can be obtained on digital systems as on optical microscopes.


Larynx virtual microscopy validation study

B. Sturm *, S. Fleskens, F. Bot, L. van Velthuysen, E.-J. Speel, P. Slootweg, J. van der Laak

*Radboud University Nijmegen, Dept. of Pathology, The Netherlands

Objective: Whole Slide Imaging (WSI) is a technology in which histopathological slides are optically scanned to produce digital images. Aim of the present study is validation of WSI for tissue samples of pathologic lesions in which subtle differences could have major clinical implications.

Method: Larynx biopsies were used containing preneoplastic lesions. Glass slides were scanned using an Olympus dotSlide system with 40× objective. WSI were displayed at a calibrated monitor and were reviewed according to the 2005 WHO classification system by three pathologists with head and neck pathology as a field of interest. Diagnoses were converted into a three grade system relevant for clinical management. WSI diagnosis concordance rates were calculated. Kappa statistics were calculated to assess the degree of inter- and intra-observer agreement.

Results: The overall interobserver agreement was comparable for both WSI and glass slide diagnoses (unweighted kappa 0.38 and 0.39, respectively). The intraobserver (glass vs WSI) agreement ranges from 0.41 to 0.48 and 0.52–0.57 for unweighted and weighted κ-values respectively. WSI showed overall slightly less (although not statistically significant) concordance with the consensus diagnosis compared to glass slides.

Conclusion: This study shows that difficult diagnostic cases could be diagnosed using WSI, without compromising diagnostic quality.


Students’ performance during practical examination on whole slide imaging using viewpath tracking

J. Szymas *, S. Walkowski, M. Lundin, J. Lundin

*University of Medical Sciences, Dept. of Clinical Pathology, Poznan, Poland

Objective: Introduction of whole slide imaging (WSI) to facilitate learning of oral cavity pathology by dental students has dramatically altered the way of teaching but little is known about students’ viewing behavior while interpreting WSI.

Method: We have decided to use tracking methods embedded in the WSI viewing system itself when viewing WSI by students during answering exam questions. Each time a student stopped panning and zooming for a for a time-period long enough for 100 % of the viewfield image data to be loaded, a record with coordinates in the virtual slide, timestamp and other metadata was sent to the database.

Results: Every student (N = 85) viewed 50 WSIs when answering 50 exam questions, generating in all more than 80,000 recorded viewfields. Mean number of viewfields/student was 1,600 and viewfields/WSI was 18.9. Correct students’ answers were 91.3 % (SD = 7.4 %). We calculated measures of total size of areas, dispersion of fragments and distribution of zoom levels viewed. Relationships were found between these measures and correctness of answers given by students.

Conclusion: The current tracking method during examination has provided us with a number of insights into the quality and effectiveness of WSI as a tool for learning and assessment.

Sunday, 1 September 2013, 17.00–19.00, Room 5C

OFP-05 Oral Free Paper Session Soft Tissue Pathology


Expression of P16 in osteosarcoma as predictive neo-adjuvant therapy factor

D. Borys *, R. Canter, A. Horvai

*University of California, Dept. of Pathology, Davis, USA

Objective: Osteosarcoma (OS) is a common malignant primary tumor of bone affecting adolescent and young adults. Osteosarcomas are high grade sarcomas with aggressive behavior. There are few if any molecular markers to predict behavior and prognosis of osteosarcoma. The objective of this study is to investigate expression of p16 in correlation with neo-adjuvant chemotherapy response in osteosarcoma.

Method: A tissue micro array was created using paraffin embedded samples from 40 pretreatment osteosarcoma cases from two institutuions UC Davis and UCSF. Immunohistochemistry was performed with commercially available p16 monoclonal mouse antibody (mtm laboratories AG, Germany). Expression for p16 was defined as nuclear staining in at least 30 % of cells. Percent tumor necrosis was measured in post-chemotherapy resection specimens with good response set at >90 % necrosis.

Results: Patients age ranged from 9 to 75 years (mean 20). Most common locations were tibia and femur. 21 patients were female and 19 male. The clinical and p16 results are summarized in table 1. P16 expression correlated positively with median percent necrosis and fraction of cases with good chemotherapy response (p = 0.004 and 0.003, respectively).

Conclusion: Immunohistochemical expression of p16 significantly correlates with good chemotherapy response in osteosarcomas. p16 immunohistochemistry may be useful adjunctive marker of prognosis in osteosarcoma.


RB1 and CDKNA2 gene deletions do not predict resistance to neoadjuvant chemotherapy in patients with osteosarcoma

D. Borys *, R. Canter, R. A. Schultz, A. Horvai

*University of California, Dept. of Pathology, Davis, USA

Objective: Pathologic response to neoadjuvant chemotherapy (NCH) predicts survival in osteosarcoma (OS) patients. The objective of the current study was to determine if copy number variations (CNV) of the CDKNA2 and Rb1 genes correlate with IHC expression of P16 and to determine whether CNV predict response or resistance to NCH.

Method: Pre-treatment genomic DNA was available from 31 pre-treatment cases of OS. CNV was determined by array comparative genomic hybridization (CGH). Clinical, pathologic, and copy number changes of the CDKNA2 and Rb1 genes were correlated for their association with good (>90 % necrosis)or poor (<90 % necrosis)response to chemotherapy.

Results: Array CGH was informative in 23 of 31 OS tumors. Among P16 negative tumors by IHC, CDKNA2 homozygous gene deletion was present in only 1 case (14 %), while the remainder had intact CDKNA2 or, in 2 cases, a gain, and LOH (loss of heterozygosity). Although homozygous Rb1 deletion was identified in 15 cases (65 %), there was no difference in the prevalence of Rb1 gene deletions between good (8/12) and poor responders (7/11) to chemotherapy (67 % vs. 64 %, P = 1.0).

Conclusion: Loss of P16 expression in OS is associated with resistance to chemotherapy, only a minority of cases result from deletion of the CDKNA2 gene. Rb1 gene deletions was not associated with response or resistance to NCH.


Dissecting spindle and pure pleomorphic variants in undifferentiated soft tissue sarcomas by gene expression profile

V. Canzonieri *, E. Pivetta, B. Wassermann, T. Perin, P. Spessotto, A. Colombatti

*CRO-IRCCS Aviano NCI, Dept. of Pathology, Italy

Objective: Soft tissue sarcoma (STS) clusters a heterogeneous group of tumors: more than 50 subtypes are currently diagnosed by genetic and morphological criteria. Among STS, Undifferentiated Soft Tissue Sarcoma (USTS) is a matter of discussion both for etiologic and diagnostic criteria. The aim of this study was to identify a new biomarker profile that could distinguish prevalent spindle vs prevalent pleomorphic USTS variants, as defined on the basis of morphologic evaluation.

Method: We carried out a gene expression analysis on 40 different USTS samples using Affymetrix microarray slides. A supervised hierarchical analysis on obtained microarray data, qRT-PCR and immunohistochemical analyses were performed.

Results: The two USTS variants (spindle, sUSTS and pleomorphic, pUSTS) clearly emerged from expression gene analysis. The number of the genes differentially expressed belonged to different categories (from translation regulators to ATP-binding proteins). qRT-PCR confirmed the analysis and immunohistochemistry for four proteins validated the obtained results.

Conclusion: Despite the outcomes require to be confirmed with a greater case study, we identified four genes (IGF2BP1, FAM133A, ABCA13, and EYA4) helpful in discriminating sUSTS vs pUSTS. This short panel of biomarkers could identify patient subsets with an especially unfavorable prognosis, thereby allowing stratification to a more aggressive and effective therapy.


TGFBR3 and MGEA5 rearrangements in pleomorphic hyalinizing angiectatic tumors of soft parts

J. Carter *, E. Montgomery, A. Folpe

*Mayo Clinic, Dept. of Pathology, Rochester, USA

Objective: Pleomorphic hyalinizing angiectatic tumor of soft parts (PHAT) is a rare, locally aggressive soft tissue neoplasm, typically occurring in the lower extremities. The putative precursor lesion of PHAT, termed “early PHAT” (EPHAT), shares many clinicopathological features with hemosiderotic fibrolipomatous tumor (HFLT). At the genetic level, HFLT, myxoinflammatory fibroblastic sarcoma (MIFS) and tumors showing hybrid features of HFLT and MIFS often show TGFBR3-MGEA5 rearrangements. To date, only a very small number of PHAT have been tested for this rearrangement; all have been negative.

Method: Four cases of PHAT, all containing EPHAT and lacking features of MIFS, were retrieved and evaluated for rearrangements in TGFBR3 and MGEA5 by FISH.

Results: The tumors occurred in adults (2M:2F; 37–55 years) and involved the foot, ankle and lower leg (2). Three of 4 cases were positive for rearrangements of TGFBR3 and MGEA5.

Conclusion: We report for the first time the presence of TGFBR3 and MGEA5 rearrangements in tumors showing mixed features of EPHAT and PHAT. The presence of such rearrangements, and the essentially identical morphological features of EPHAT and HFLT, strongly suggests that EPHAT/HFLT is related to both PHAT and MIFS, and that the latter two tumors may represent different morphological stages of a single entity in which only MIFS has acquired the capacity to metastasize.


SMARCB1 mRNA expression is regulated by microRNAs in epithelioid sarcoma

G. Papp *, Z. Sápi

*Semmelweis University, 1st Dept. of Pathology, Budapest, Hungary

Objective: About 10 % of epithelioid sarcomas have biallelic mutation of the SMARCB1 gene resulting in lack of this nuclear protein. Our previous studies have shown that neither the gene promoter nor the histone methylation was responsible for the functional loss of INI1 in epithelioid sarcomas. Moreover there was no SMARCB1 mRNA detected in the laser microdissected tumor cells. It was speculated that miRNAs (microRNAs) may contribute to SMARCB1 gene silencing in this tumor.

Method: We identified SMARCB1-specific and upregulated miRNAs and examined their functional effects using in vitro experiments.

Results: 8 potential SMARCB1-specific miRNAs were collected from online databases. Upregulations of miR-206/381/671-5p/765 were determined by quantitative real-time polymerase chain reaction in the 25 SMARCB1 negative cases. For in vitro tests 3 cell cultures showing normal SMARCB1 expression (HT1080, CaCo-2 cell lines and primary culture of human fibroblasts) were transfected with Pre-miR miRNA Precursors of abovementioned 4 miRNAs using electroporation. Three miRNAs (miR-206/381/671-5p) decreased the level of SMARCB1 gene expression in the cultured cells.

Conclusion: Our results suggest that miR-206/381/671-5p overexpression seems to be one important factor in the oncogenesis and progression in epithelioid sarcomas through SMARCB1 downregulation.


Preoperative intensity-modulated radiation therapy (IMRT) for high-risk soft tissue sarcomas of extremities and retroperitoneum: Histopathological response and molecular aspects

M. Straub *, K. Specht, H. Rechl, R. Eisenhart-Rothe von, B. Röper

*TU Munich, Pathology, Germany

Objective: Neoadjuvant radiotherapy is a promising therapeutic option for high-grade soft tissue sarcomas (hSTS). Histopathologic response following IMRT for hSTS was evaluated.

Method: Pretherapeutic biopsies and resection specimen of 41 hSTS (14 pleomorphic sarcomas, 9 myxofibrosarcomas, 5 synovial sarcomas, 7 dedifferentiated liposarcomas (DLS), 3 myxoid/roundcell liposarcomas, 1 epitheloid sarcoma, 1 rhabdomyosarcoma, 1 leiomyosarcoma) after preoperative IMRT were included. Percentage of vital tumor and resection margins were evaluated histomorphologically. P53 and p16 status was analyzed by methylation-specific PCR, sequencing and immunohistochemistry.

Results: Median age of patients was 57 year (24–89; M:F ratio 20/21). Anatomical locations were extremities (n = 36) and retroperitoneum (n = 5). Metastasis occurred in 11 patients, local relapse in n = 6. A clear resection margin (R0) was achieved in 31 patients, R1-resection in 6 patients (4 with retroperitoneal hSTS) and Rx-resection in n = 4. Percentage of vital tumor ranged from 5 to 80 % (median 45 %). In genomically highly complex sarcomas, median of vital tumor tissue was 30 % as opposed to sarcomas with intermediate genomic complexity (median 70 %) and translocation-associated sarcomas (median 60 %). Molecular and immunohistochemical analysis of p16 and p53 status is under way.

Conclusion: A high percentage of R0-resections can be achieved in STS of extremities after radiotherapy. Response rates vary widely, with certain subgroups (genomically complex sarcomas) displaying a better response.


Absence of CHOP translocation and MDM2 amplification in a case of lipoblastoma-like tumor of the vulva

C. A. Vásquez Dongo *, C. Delbene Azanza, B. Ferrer Fabrega, P. Jiménez León, Y. Rodriguez Diez, D. Bodet Castillo, C. Valverde Morales, S. Ramón y Cajal Agüeras, C. Romagosa Perez-Portabella

*Vall d’Hebron U. Hospital, Anatomical Pathology, Barcelona, Spain

Objective: Lipoblastoma-like tumor of the vulva (LBLTV) is a rare mesenchymal neoplasm that can mimic myxoid liposarcoma. Molecular changes which are important in the differential diagnosis of these tumors, such as CHOP translocation and MDM2 amplification haven’t been reported before.

Method: We described the case of a painful and well-circumscribed mass in the vulva of a 28 year-old woman and the main clues for differential-diagnosis including morphology, immunophenotype and FISH.

Results: The lesion was mainly myxoid, with clusters of mature adipose tissue. The tumor cells were spindle and bland. Some lipoblasts and a chicken-wire vascular network were also observed. The differential-diagnosis was mainly done between myxoid liposarcoma, dedifferentiated liposarcoma, lipoblastoma and the myxoid vulvar lesions. Immunohistochemicaly, the tumor cells were negative for CD34, smooth-muscle actin, desmin, neurofilaments and S100. FISH didn’t show CHOP translocated cells or MDM2 amplification. Once previous diagnoses had been ruled out, the final diagnosis was LBLTV which was surgical removed with security margins being free of disease 2,5 years after.

Conclusion: Although LBLTV is a rare diagnosis, it should be considered in the differential-diagnosis of myxoid and adipose vulvar tumors. The absence of CHOP translocation and MDM2 amplification can help to differentiate these benign tumors from their main malignant differential-diagnoses.

Monday, 2 September 2013, 14.15–16.15, Room 5B

OFP-06 Oral Free Paper Session Endocrine Pathology


Cellular retinoic acid binding protein I expression and cell proliferation in pancreatic neuroendocrine tumors

V. Delektorskaya *, G. Chemeris, N. Kozlov

*N.N. Blokhin Russian Cancer Res., Dept. of Pathology of Human Tumors, Moscow, Russia

Objective: The aim of the study was to investigate Cellular Retinoic Acid Binding protein type I (CRABP I) expression and its clinical significance in pancreatic neuroendocrine tumors (pNETs).

Method: Protein expression of CRABP I was investigated by immunohistochemical analysis in 71 pNETs of various histological subtypes, including well- and poorly differentiated neuroendocrine neoplasms. Relationship of CRABP I expression to clinical and pathological features, grading and cell proliferation (index Ki-67) was analyzed.

Results: Increased expression of CRABP I protein was detected in 43.7 % (31of 71) of cases, including 16.7 % (1 of 6), 42.1 % (24 of 57) and 75.0 % (6 of 8) of pNET G1, pNET G2 and pNEC G3, respectively. We found the significant relationship of increased CRABP I expression to histological differentiation (p = 0.003), primary tumor size (p = 0.024), lymph node and liver metastases (p = 0.017 and p = 0.024, respectively) and cell proliferation, evaluated on the basis of the Ki-67(MIB-1) antigen expression (p = 0.000). The protein expression was not associated with outcome and survival of pNET patients.

Conclusion: The increased protein expression and association with high-risk factors suggest the great potential prognostic and therapeutic interest of evaluation of CRABP I in pNETs. Further study is necessary to clarify the role of CRABP I protein in pNET pathogenesis.

CRABP I expression in the cell cytoplasm of pNET G2 (Ki-67–9 %).:

figure b


Succinate dehydrogenase subunit B- mutation-related pheochromocytomas and sympathetic paragangliomas: Gene analyses and clinicopathological study

N. Kimura *, K. Takekoshi

*National Hakodate Hospital, Dept. of Pathology, Japan

Objective: The aim of this study was to examine the clinicopathological features of succinate dehydrogenase subunit B (SDHB) mutation-related pheochromocytoma/paraganglioma (PCC/PGL).

Method: A total of 15 patients with SDHB-mutation were analyzed genomic DNA, clinical data on family history, catecholamine types, tumor metastasis, patient prognosis, and histology of the tumors. Histologic analyses were performed using the new classification named Grading of adrenal pheochromocytoma and paraganglioma (GAPP) classification that was made based on a nationwide Japanese survey. All tumors were scored from 0 to 10 points, and were also graded according to three differentiation types depending on the total score: well (0–2 points), moderate (3–6) and poor (7–10).

Results: Mean age was 32.3 ± 14.2, and sex ratio was 10M: 5F. Mutation sites were exon 2 (R46Q) in 6, exon 5 (L157X) in 4, and exon 3, 6, 7, intron 2 in one patient, respectively. Five patients had a family history. Catecholamine phenotypes were 12 in norepinephrine-, and three in nonfunctioning type. Nine patients had metastases. Histological grading of the tumors was 4.87 ± 0.99, corresponding to moderate type differentiation.

Conclusion: Tumors with SDHB mutations were characterized by moderate type differentiation with 53 % of a pseudorosette pattern.


Ki-67 antigen in pituitary adenomas: Role in the tumor behaviour prognosis

L. Mitrofanova *, U. Tsoy, P. Ryazanov, O. Beshchuk, V. Cherebillo, E. Grineva

*Almazov’s Centre, Dept. of Pathomorphology, St. Petersburg, Russia

Objective: To establish the relationship of the Ki-67 index with tumor size, hormonal activity and tumor behavior.

Method: We evaluated the expression of Ki-67 in 46 anterior pituitary adenomas. Tumors size and growth were verified by MRI. We used different criteria for invasive tumor definition: extrasellar growth, cavernous sinus infiltration, maximal diameter of the tumor >3 cm, tumor volume >5 ml.

Results: 14 adenomas were non-functioning and 32 functioning (25 growth hormone, 5 ACTH, 2 prolactin). In spite of applying different criteria for invasive tumor definition, there was no difference between the Ki-67 index in invasive and non-invasive adenomas. The Ki-67 index was higher in ACTH-secreting adenomas, though all of them were microadenomas, comparing with the rest ones (4,48 ± 1,54 % vs 1,05 ± 0,18 %, p < 0,002). In non-functioning adenomas positive correlation between the Ki-67 index and tumor volume was revealed (r = 0,64 p = 0,04). The Ki-67 index was significantly higher in adenomas which relapsed less than 2 years after the first operation (3,82 ± 1,8 vs 1,13 ± 0,19, p = 0,0013).

Conclusion: We did not get the data confirming evidence that the Ki-67 index can predict invasive growth of pituitary adenomas. Assessment of the Ki-67 index may be helpful in predicting tumor recurrence.


A study of GATA3 immunohistochemistry in parathyroid and thyroid lesions

G. Betts *, E. Beckett, D. Nonaka

*Christie Hospital, Dept. of Histopathology, Manchester, United Kingdom

Objective: GATA3 (GATA binding protein 3) is a transcription factor involved in tissue differentiation in the breast, kidney, thymus and parathyroid. Whilst thyroid tumours have been reported to be negative for GATA3, no previous reports have documented expression of GATA3 in parathyroid lesions.

Method: Fifty five parathyroid lesions were assessed using immunohistochemistry (IHC) for GATA3 including parathyroid adenomas(35), parathyroid hyperplasia(9), atypical parathyroid adenoma(3), parathyroid cyst(2) and parathyroid carcinoma(5) with one case of recurrent hyperplasia after autotransplantation in the arm. A tissue microarray was also immunostained for GATA3 containing 58 evaluable thyroid lesions including; follicular adenoma(8), hurthle cell adenoma(8), follicular carcinoma(10), hurthle cell carcinoma(2), papillary carcinoma(14), medullary carcinoma(8), anaplastic carcinoma(1) and insular carcinoma(7).

Results: All of the parathyroid lesions were positive for GATA3, 96 % (53/55) of cases displayed strong, diffuse nuclear positivity. An atypical parathyroid adenoma showed weak diffuse nuclear positivity and a parathyroid carcinoma, focal strong nuclear positivity. None of the thyroid lesions displayed any nuclear positivity, two insular carcinomas showed weak cytoplasmic positivity.

Conclusion: Nuclear positivity with GATA3 IHC is highly specific for parathyroid tissue when compared with thyroid tissue. GATA3 IHC may be a useful adjunct for histological and cytological discrimination of parathyroid and thyroid lesions.


Grading of Neuro Endocrine Tumors (NET): Mitosis and Ki-67 both essential?

M.-L. van Velthuysen *, T. Korse, A. van der Pol, M. Tesselaar

*AvL, Pathology, Amsterdam, The Netherlands

Objective: We investigated whether ki-67 and mitotic index assesment would lead to discordant grading and which of both is most informative to predict survival.

Method: Tumors of 252 patients with NET were analyzed for mitoses and Ki-67 index. Up till now survival data are available from 85 patients

Results: Of 252 cases, 88 showed discordance in grading between Ki-67 and mitotic index. In 37 cases this was due to a Ki-67 index ≥20, while mitoses were <20. In 25 of these cases mitotic index was <10, in 8 of these cases even <2. The ratio for biopsies and resection specimens was 1:1 for the cases with concordant mitosis and ki-67 index, while for the cases with discordant indexes this ratio was 7:3. Preliminary survival analysis of the 85 patients, showed that ki-67 index and mitotic index were both predictive for survival. However, for the patients with discordance between mitotic index and Ki-67 index (n = 31) the Ki-67 seemed to be the best parameter for survival.

Conclusion: Grading NET using Ki-67 and mitotic index results in conflicting data in 30 % of cases. Survival analysis suggests that Ki-67 is a better prognostic marker as compared to mitotic index. Correlation with survival analysis of all included patients is ongoing


Prognostic and predictive role of MGMT immunoreactivity and promoter methylation in sporadic pancreatic endocrine neoplasms

A. M. Schmitt *, M. Pavel, T. Rudolph, H. Dawson, E. Vassella, A. Perren

*University of Bern, Institute of Pathology, Switzerland

Objective: To determine the value of MGMT protein expression and promoter methylation in pNEN regarding 1. prognosis and 2. response prediction to temozolomide therapy.

Method: Two independent cohorts of pNEN were analyzed: 1. Prognostic cohort: Patients did not receive TMZ. Methylation specific PCR of the MGMT promoter (n = 52) and MGMT immunohistochemistry (n = 130) were performed. Results were correlated with survival data. 2. Predictive cohort: Primer extension based quantitative PCR of the MGMT promoter and MGMT immunohistochemistry were performed in 15 progressive metastasized NEN receiving TMZ chemotherapy and results correlated with radiological tumour response according to RECIST 1.1.

Results: 1. Prognostic cohort: Loss of MGMT protein expression, but not MGMT promoter methylation correlated significantly with a shortened disease free survival (p = 0.0054) and decreased disease specific survival (p = 0.0322). 2. Predictive cohort: MGMT promoter methylation, but not loss of MGMT protein expression correlated significantly with response (p = 0.036). All tumours showing hypermethylation of the MGMT promoter were of pancreatic origin and showed either stable disease or partial remission.

Conclusion: 1. Loss of MGMT expression is an adverse prognostic marker in sporadic surgically resected pNEN. 2. MGMT promoter methylation in sporadic metastasized pNEN might serve as a predictor of response to TMZ.


Application of molecular markers in cytological samples of thyroid nodules as pre-operative predictors of aggressiveness

M. Volante *, I. Rapa, J. Giorcelli, S. Aversa, P. Caraci, A. Fulcheri, A. Votta, F. Orlandi, M. Papotti

*University of Turin, Oncology at San Luigi Hospital, Orbassano, Turin, Italy

Objective: To test in thyroid cytology the role of molecular markers in predicting pathological features of potential impact in the pre-operative clinical management of thyroid neoplasms.

Method: A retrospective series of 201 consecutive FNA samples (from years 2004–2009) with a diagnosis of “thyroid neoplasm” (British Thyroid Association diagnostic categories: THY 3, 125 cases; THY 4, 27 cases; THY 5, 49 cases) with available histological diagnoses were selected. BRAF, N and H RAS mutations were screened using the pyrosequencing method onto DNA extracted from cell block preparations. Mutational profile was correlated with histological diagnosis and clinical-pathological characteristics of tumors at histology.

Results: BRAF mutations were detected in 37/201 (18 %) cases with overall sensitivity and specificity for malignancy of 31 % and 97 %, respectively. BRAF mutations were significantly associated with papillary carcinoma histotype, presence of multifocality and extra-thyroidal invasion but not with lymph node metastases. RAS mutations had low sensitivity and specificity and poor correlation with pathological features of aggressiveness, except for an inverse correlation with vascular invasion

Conclusion: the role of molecular markers in the pre-operative characterization of thyroid neoplasms at cytology is limited to BRAF, although the practical impact and cost-to-benefit ratio of such analysis still need to be addressed.


Expression of DNA repair and synthesis enzymes and tyrosine kinase inhibitor(s) targets in poorly differentiated and anaplastic thyroid cancer

M. Volante *, V. Monica, I. Rapa, S. Busso, J. Giorcelli, G. Scagliotti, M. Papotti

*University of Turin, Oncology at San Luigi Hospital, Orbassano, Turin, Italy

Objective: To analyze the expression of enzymes involved in DNA repair and synthesis and of VEGF and PDGF receptors in aggressive thyroid cancers as potential biomarkers of response to chemotherapy and tyrosine kinase inhibitors

Method: 35 poorly differentiated (PD) and 19 anaplastic thyroid carcinomas were analyzed for gene expression of ERCC1, RRM1, Topoisomerase IIa (TOPOIIa), thymidylate synthase (TS), VEGF-R types 1-2-3 and PDGF-R using real time PCR, as compared to normal thyroid tissue. Gene expression levels were compared to tumor type, type of well-differentiated associated component, presence of oncocytic features and genotype (RAS vs BRAF mutations)

Results: All genes investigated were significantly over-expressed in tumors vs normal thyroid. TS and ERCC1 were significantly over-expressed in PD as compared to anaplastic carcinomas. TS and RRM1 were significantly down- and up-regulated in BRAF-positive tumors, respectively. All VEGF-R subtypes were significantly over-expressed in PD as compared to anaplastic carcinomas, whereas the reverse occurred for PDGF-R. VEGF-R1 and PDGF-R were also significantly up-regulated and down-regulated, respectively, in oncocytic vs non-oncocytic tumors

Conclusion: All markers investigated showed heterogeneous expression profiles partially associated to pathological or genotypic tumor characteristics that might potentially reflect differences in profiles of responsiveness to chemotherapy or to selective targeted therapies


MiR-885-5p plays a causal role in the development of oncocytic phenotype in thyroid tumors

M. Dettmer *, M. Gandhi, M. Nikiforova, Y. Nikiforov

*Universität Bern, Switzerland

Objective: The molecular profile of oncocytic follicular thyroid carcinomas (oFTC) is largely unknown. We reported that miR-885-5p is upregulated in oFTC. The aim of this study was to explore the relationships between miR-885-5p overexpression and oncocytic phenotype of thyroid tumors.

Method: MiR-885-5p was stable overexpressed in human thyroid HTori-3 and TPC-1 cells. Cox4 gene is a part of the mitochondrial respiratory chain and was used to monitor the accumulation of mitochondria. Cells were stained with Cox4 antibody prior to FACS analysis (n = 5) and immunofluorescence and the amount of mitochondria was evaluated.

Results: Overexpression of miR-885-5p in both cell lines resulted in a significant increase of the Cox4 mitochondrial signal detected by the FACS analysis compared to controls (controls:58.2 ± 2.7 % vs. miR-885-5p:71.9 ± 1.2 %). Immunofluorescent analysis confirmed a significant increase in the Cox4 staining of the cytoplasm of the transduced cells, confirming the increase in the amount of mitochondria.

Conclusion: This study provides for the first time the experimental evidence that a single miRNA, miR-885-5p, is involved in the accumulation of mitochondria in thyroid cells, leading to the acquisition of the oncocytic features in human thyroid cells. Further studies are required to elucidate the target genes of miR-885-5p and the mechanisms behind this phenomen.


Galectin-3 and HBME-1 expression in follicular thyroid tumors, especially in so called “follicular thyroid tumors of uncertain malignant potential” and determination of their histological features

E. Tamponi *, G. Faa, G. Senes, M. L. Lai

*AOU Cagliari, Dipt. di Anatomia Patologica, Italy

Objective: Investigate expression and diagnostic rule in follicular thyroid tumors, especially in so called “follicular thyroid tumors of uncertain malignant potential” of galectin-3 and HBME-1. Investigate histological behaviour of follicular thyroid tumors of uncertain malignant potential especially in term of capsule invasion mode and venous or lymph invasion.

Method: A total of 924 follicular thyroid nodules were collected from our institution including 315 follicular adenomas (FA), 117 follicular thyroid carcinomas (FTC), 394 follicular variant of papillary thyroid carcinomas (FVPTC), 82 differentiated carcinomas, not otherwise specified (DCNOS), 17 well differentiated tumors of uncertain malignant potential.

Results: About 4 % of FA expressed HBME-1 and galectin-3, weakly. About 50 % of FTC expressed HBME-1 and galectin-3 with weakly or moderate stain. About 80 % of FVPTC expressed HBME-1 and galectin-3 with strong stain. About 65 % of DCNOS expressed HBME-1 and galectin-3 with almost strong stain. About 26 % of DCNOS shows venous invasion and all of them show the same way of capsule invasion of FTC.

Conclusion: DCNOS show histological features (capsule invasion mode and venous invasion) very similar to FTC and expression of galectin-3 and HBME-1 very similar to FV-PTC. We strong believe that DCNOS in a new entity and its histological and clinical behaviour has to be assessed.

Monday, 2 September 2013, 14.15–16.15, Room 5C

OFP-07 Oral Free Paper Session Head and Neck Pathology


Adult sinonasal sarcoma: A clinicopathologic analysis of 48 cases from the French Sarcoma Group ( http://www.conticabase.org )

V. Costes *, V. Szablewski, A. Neuville, A. Gomez Brouchet, M. Lae, D. Ranchere Vince, P. Terrier, G. d. Pinieux, J. M. Coindre

*Hopital Gui de Chauliac, Dept. of Biopathology, Montpellier, France

Objective: The aim of this study was to determine the frequency of primary sinonasal adult sarcomas, to identify the different histological subtypes, and to analyze prognostic factors.

Method: Forty-eight cases of adult sinonasal sarcoma from the French Sarcoma Group database (conticabase) were reviewed with immunohistochemistry and molecular characterization.

Results: The most frequent tumor types were alveolar rhabdomyosarcoma (ARMS) (33.3 %), embryonal rhabdomyosarcoma (ERMS) (14,6 %), unclassified sarcoma (14.6 %) and leiomyosarcoma (12,5 %). All “round cells” sarcomas were rhabdomyosarcoma. A total of 41.7 % of cases were grade 3.10.4 % of patients had previous medical history of radiotherapy (PHR). The 5-year OS, MFS and LRFS were 62.3 %, 73 % and 88.8 %. Statistical analysis retained histological type for predicting OS, MFS and LRFS with the worst prognosis associated with rhabdomyosarcoma, whatever the subtype alveolar or embryonal. Grade influenced OS and MFS. The predictive factor for complete response was surgery, whatever quality of resection margins.

Conclusion: Rhabdomyosarcoma represented the most frequent sarcoma in this topography and was associated with the worst prognosis, without significant differences between alveolar and embryonal subtypes. These results suggest that paranasal sinuses and nasal cavity should be considered as an unfavorable site for ERMS. Moreover, surgery should be always considered in treatment even if wide resection cannot be obtained.


T Lymphoma of the nasal cavities or paranasal sinuses (12 cases)

F. Bougrine *

*Military Hospital Tunis, Dept. of Pathology, Tunisia

Objective: T lymphomas of the nasal cavities or paranasal sinuses are unusual tumors, rarely seen in Occidental countries, where B cell lymphomas are the most common, They often lead to differential diagnosis problems with other destructive and necrotizing processes of the sinonasal tract.

Method: 12 cases of T lymphomas of the nasal cavities or paranasal sinuses have been retrieved at the department of pathology of Military hospital of Tunisia. during a period of 16 years, from 1995 to 2010. Follow-up was available for 10 patients

Results: The median age was of 36 year. Clinical presentation was dominated by nasal obstruction (11 patients), associated or not to nasal discharge (10 cases), epistaxis (3 cases), and nasal swelling (5 cases). The most frequent site of occurrence was the nasal cavity. The histopathological examination found: 4 cases of T/NK lymphomas and 8 cases of peripheral T lymphomas NOS. Detection of Epstein Barr virus was positive in10 cases. Outcome was marked by: Death occurrence (7 patients), a complete remission (3 cases).

Conclusion: T lymphomas of the nasal cavities are rare in Tunisia. They are agressive lymphomas, characterized by an heterogenous and little specific clinical presentation. Etiology is unknown, but there is an association between EBV and T lymphomas of the nasal cavities.


Differential occurrence of sinonasal Intestinal Type Adenocarcinoma (ITAC) and sinonasal non-ITAC in Finland and France and their association with wood dust exposure

H. Wolff *, I. Leivo, R. Holmila, D. Luce, K. Husgafvel-Pursiainen

*Finnish Inst. of Occup. Health, Laboratory of Pathology, Helsinki, Finland

Objective: The WHO classification (2005) distinguishes two types of sinonasal adenocarcinomas (SNAC) the intestinal-type adenocarcinoma (ITAC) and the non-ITAC. In the pathology literature ITACs but not non-ITACs have been strongly associated with hardwood dust exposure. We have studied the distribution of adenocarcinoma types and wood dust exposure in tumors from France and Finland. Hardwoods are typically used in France while in Finland softwoods dominate.

Method: Paraffin-embedded tissue (PET) samples were obtained from SNAC:s in Finland for the period 1989–2002. In France, cases were from the areas of Isere, Somme and Doubs, 1990–2002, Patients or next-of-kins were interviewed using a structured questionnaire

Results: Immunohistochemical (IHC) positivity for either CDX2 or CDX2 was used as a criteria for ITAC:s. Of 34 SNAC:s from France 29 were ITACs (85 %). while only 43 % of the SNACSs(n = 21) from Finland were ITACs. 87 % of ITACs and 46 % of non-ITACs were assosiated with wood dust exposure

Conclusion: Qualitative differences in wood dust exposure is a probable explanation for the differential frequencies of ITACs and non-ITACs in these countries. The association of non-ITACs with wood dust exposure is lesser than for ITACs. Use of IHC is recommended for differentiating ITACs from non-ITACs.


Diagnostic challenges of antrochoanal polyps

N. Choudhury*, A. Hariri, H. Saleh, A. Sandison

*Charing Cross Hospital, London, United Kingdom

Objective: Antrochoanal polyps (ACP) are benign, solitary, unilateral lesions. It is accepted that they are accurately diagnosed clinically, but ACPs may infact mimic a variety of other benign and malignant conditions. Patients require a full diagnostic work up including nasal endoscopy, radiology and histological analysis of the resection specimen. Clinically atypical lesions merit referral to a tertiary centre.

Method: A retrospective review of all patients with a clinical and radiological diagnosis of ACPs made over a 7 years study period was conducted. Their histology was reviewed and patients with a diagnosis other than ACP were further evaluated.

Results: 60 patients with a provisional diagnosis of ACP, who underwent endoscopic sinus surgery, were identified. Following surgery, 12patients (20 %) with discrepant histology were identified (7males and 5 females; mean age of 51.0+/−16.5 years). Of these, 10 had benign pathology including inverted papilloma, oncocytic papilloma, capillary haemangioma, angiofibroma and fungal ball. There were also 2 malignant cases including malignant melanoma and cycindical cell carcinoma.

Conclusion: ACP may mimic a diverse range of nasal pathologies. Our series has identified 20 % of patients with presumed ACP to have a different underlying diagnosis, following biopsy. Although individually these pathologies may be rare, careful histopathological analysis, with appropriate referral for specialist treatment, is necessary.


Insignificant differences in VEGF-C/D immunohistoexpression and lymphatic microvessel density between primary and recurrent salivary pleomorphic adenoma do not support the role of lymphangiogenesis in local spread of this tumor

I. Stárek *, R. Salzman, L. Kucerová, A. Skálová

*Medical Faculty Olomouc, ORL Clinic, Czech Republic

Objective: Recurrent pleomorphic adenoma (RPA) is typical by multiple nodules present far away from the primary site. One of the plausible explanation of this feature might be local lymphatic spread of tumor cells, promoted by lymphangiogenesis. In our study, immunohistochemical expression of lymphangiogenic factors VEGF-C and VEGF-D and lymphatic microvessel density (LVD) was tested in PA and RPA.

Method: 6 primary-non-RPA, 4 primary-to-recur PA and 10 RPA were tested. For the evaluation of VEGF-C and VEGF-D reactivity semiquantitative histoscore was used, LVD was determined as the number of D2-40 positive peritumoral lymphatic capillaries.

Results: No case showed VEGF-C positivity. VEGF-D reaction was found exclusively in epithelial tumor cells. VEGF-D histoscore and LVD in primary-non-RPA, primary-to-recur PA and RPA ranged from 4 to 6 (mean 5.17), 3–5 (4.25) and 4–5 (4.6), respectively, and 2–5 (3.17), 1–7 (3.75) and 1–7 (3.4), respectively. The differences were not stastically significant (p > 0.05). No correlation between VEGF-D histoscore and LVD was found (p > 0.56).

Conclusion: Our results suggest that lymphangiogenesis plays no role in local spread of RPA. Supported by the Instutitional Support of Ministry of Health, Czech Republic, Nr. 1RVO-FNOl.2013, and IGA Czech Republic, Nr. NT13701-4/2012.


In-situ and minimally invasive neoplasia in a series of 49 cases of salivary carcinoma ex-pleomorphic adenoma

M. Rito *, I. Fonseca

*IPO de Lisboa Francisco Gentil, Serviço de Anatomia Patológica, Portugal

Objective: Carcinoma ex-pleomorphic adenoma (Ca-ex PA) is defined by the presence of malignancy in a pre-existing pleomorphic adenoma. This study aims to review its morphological characteristics in a large series of salivary Ca-ex-PA and to evaluate the impact on disease progression.

Method: Forty-nine cases, diagnosed from 1988 to 2013, were classified for the malignant component(s) subtypes and assessed for the surgical margins status and extension of invasion, mitotic counts, hemorrhage, necrosis, neural and vascular invasion.

Results: All patients were treated surgically, 53 % with incomplete excision. 45 % of tumors had criteria of in situ and minimal invasion. 14 cases had low-grade and 35 cases high-grade histology. The predominant malignant components were: salivary duct carcinoma (47 %), myoepithelial carcinoma (22 %) and epithelial-myoepithelial carcinoma (16 %). 40 cases had follow-up information: 13 patients had disease progression and 6 died, all presenting widely invasive tumours. 5-year survival was 85 %. There was significant association of overall survival with wide invasion, mitotic counts, necrosis and perineural invasion.

Conclusion: In situ and minimally invasive Ca-ex-PA incidence in this series was higher (45 %) than previously reported and associates better prognosis irrespective of the histological subtype. Wide invasion, necrosis, perineural invasion and mitotic counts significantly impact on prognosis.


Clinicopathological characteristics and survival outcomes of patients with metastatic salivary gland tumors

C. Nagano *, C. Camillo-Coutinho, R. Iagñez, C. Lopes Pinto, I. Fonseca, S. Lourenço

*University of São Paulo, Stomatology (Dental School), Brazil

Objective: Malignant salivary glands neoplasms are rare and present a remarkable clinical, histological and biological diversity. Distant metastases are infrequent, mainly associated with high-grade neoplasms. We investigated metastatic salivary gland tumors in a Brazilian Institution, in order to identify metastatic risk factors.

Method: Medical records of all patients diagnosed with distant metastatic salivary gland tumors seen at Cancer Hospital A.C. Camargo, Sao Paulo–Brazil, from 1970 to 2012 were reviewed. Demographic, clinical, pathologic and treatment data were recorded.

Results: 37 patients were identified. 14 (37,8 %) were male and 23 (62 %) were female; median age was 48 years old. Lungs were the most common metastatic site (31 cases, 83,7 %). Adenoid Cystic Carcinoma was the most frequent histological type (24 cases, 64,8 %). Most primary tumors involved the parotid (18 cases, 48,6 %). 91,8 % (34 cases) of the patients underwent combined therapy (surgical resection, radiotherapy and/or chemotherapy) and 8,1 % (3 cases) were treated with non-surgical therapy (chemotherapy/radiotherapy). Factors significantly associated with poor survival were advanced stage, distant metastases, vascular invasion, and unresectable disease.

Conclusion: The histological type, tumor site and isolated treatment modalities are important factors associated with metastases and survival rate in patients with malignant salivary gland tumours.


Stem-cell markers in head neck squamous cell carcinoma and association with clinicopathological characteristics

C. Coutinho-Camillo *, F. d. Moraes, S. Lourenço, R. Ianez, E. d. Sousa, M. Silva, L. Kowalski, F. Soares

*Hospital AC Camargo, Dept. of Pathology, São Paulo, Brazil

Objective: To investigate the presence of stem cell markers in head and neck squamous cell carcinoma (HNSCC).

Method: The immuno-expression of integrin-β1, CD24, CD44, ALDH1 and CD133 proteins was analyzed in 52 HNSCC patients. The results were semi-quantitatively analyzed considering the patterns of distribution and intensity of staining.

Results: Seven out of 52 cases of HNSCC (13.5 %) showed positive cytoplasmic staining of ALDH1; integrin-β1 was expressed in 45 out of 50 cases (90.0 %); 30 out of 52 cases (57.7 %) showed positive membranous staining of CD44; CD24 was expressed in 44 out of 50 cases (88.0 %) and 3 out of 52 cases (5.8 %) showed positive membranous staining of CD133. Expression of CD24 and CD44 was associated with well-differentiated tumours (p = 0.04 and p = 0.04, respectively). Expression of integrin-β1 was associated with advanced stage tumours (p = 0.04) and expression of ALDH1 was associated with vascular invasion (p = 0.04). Five-year cancer-specific survival rates differed significantly between patients with negative and positive expression of CD44 (p = 0.052).

Conclusion: Our study provides the evidence of the expression of putative stem-cell markers in head and neck squamous cell carcinoma and association of these markers with patient’s outcome.


Activin A immunoexpression is useful to predict occult lymph node metastasis and overall survival in oral tongue squamous cell carcinoma

R. Coletta *, P. Rodrigues, N. Kelner, A. Bufalino, F. Fonseca, A. Santos-Silva, L. P. Kowalski, T. Salo, E. Graner

*UNICAMP, School of Dentistry, Dept. of Oral Diagnosis, Piracicaba, Brazil

Objective: Regional lymph node metastasis is the most impacting prognostic factor in patients with oral tongue squamous cell carcinoma (OTSCC). Approximately 30–50 % of OTSCC patients have regional metastasis at diagnosis, but the limited sensibility of the current diagnostic methods used for neck staging does not allow detection of all cases, leaving a significant number of undiagnosed metastasis (occult lymph node metastasis). Here we evaluated whether clinicopathological features and immunohistochemical detection of carcinoma-associated fibroblasts and activin A could be predictive markers for occult lymph node metastasis in OTSCC.

Method: One hundred and ten patients with primary TSCC, who were classified with early stage tumor and received surgical treatment with elective neck dissection, were enrolled in the study.

Results: Among all examined features, only activin A high expression was significantly associated with presence of occult lymph node metastasis (p = 0.006). Multivariate survival analysis showed that the activin A expression was an independent marker of reduced overall survival with a 5-year survival of 89.7 % for patients with low expression compared with 76.5 % for those with high expression (HR:2.44, 95 %CI:1.55–3.85, p = 0.012).

Conclusion: Our results demonstrate that activin A immunodetection can be useful for prognostication of OTSCC, revealing patients with occult lymph node metastasis and lower overall survival rates.


Odontogenic ghost cell tumours: A single center review

F. Menezes *, A. Polonia, D. Felizardo, M. Jacome

*Portuguese Institute of Oncology, Dept. of Pathology, Porto, Portugal

Objective: Odontogenic ghost cell tumours (OGCT) are rare lesions. Derived from epithelial and ectomesenchymal elements of the tooth-forming organ, OGCT are neoplastic proliferations of ameloblastomatous epithelium, variable amounts of dysplastic dentin and the key diagnostic ghost cells, thought to be a product of keratinization or coagulation necrosis of epithelial cells. Classically grouped as Calcifying odontogenic cysts, posterior revisions led to the 2005 WHO classification, splitting Calcifying cystic odontogenic tumours (CCOT), solid Dentinogenic ghost cell tumours (DGCT), and extremely rare Ghost cell odontogenic carcinomas. OGCT classification remains controversial though: further group subdivisions have been proposed, but no significant behavior differences within each group’s histopathologic spectrum have been found. Association with other odontogenic lesions is common; if ghost cells are overlooked, confusion with ameloblastoma is most likely. Our aim is to review OGCT aspects, and potential diagnostic pitfalls.

Method: Our institute database was browsed for all patients diagnosed with OGCT. Histological and immunohistochemistry aspects, patient follow-up and recent literature were reviewed.

Results: 4 cases (3 CCOT, 1DGCT) have been diagnosed by our department so far. The DGCT case was first misdiagnosed as recurrent ameloblastoma.

Conclusion: The aspects reviewed are consistent with recent literature. OGCT should be a differential diagnosis not to overlook when facing odontogenic tumours.

Monday, 2 September 2013, 17.00–19.00, Auditorium VIII

OFP-08 Oral Free Paper Session Joint Session–Neuropathology/Ophthalmic Pathology


Histological features and neo-angiogenesis in embolized meningiomas: Is there the risk of overgrading?

V. Barresi *, G. Branca, F. Granata, M. Caffo, C. Alafaci, G. Tuccari

*Policlinico G. Martino Pad D, Dip. Patologia Umana, Messina, Italy

Objective: Pre-operative embolization (POE) induces histological changes simulating malignancy in meningiomas. Our aims were to test for overgrading the scheme currently in use and to analyze whether the POE procedure may stimulate neo-angiogenesis in embolized meningiomas.

Method: The histological features of 41 embolized meningiomas were reviewed, compared with those observed in atypical non-embolized tumors, and considered for grading assessment. In the same cases, neo-angiogenesis was also quantified by the evaluation of microvessel density (MVD).

Results: In embolized meningiomas, necrosis and macronucleoli represented frequent findings. Nonetheless, differently from that observed in non-embolized meningiomas, necrosis mostly showed an abrupt line of demarcation from the viable tumour tissue, and macronucleoli were mainly restricted to peri-necrotic areas. Exclusion of these features resulted in increased specificity and positive predictive value of histological grading in the identification of recurring meningiomas. MVD showed a significant increase with the interval between POE and surgery and it was not correlated to malignancy in our cases.

Conclusion: The frequent evidence of necrosis and prominent nucleoli in embolized meningiomas may lead to overgrading, which may be avoided by excluding necrosis showing an abrupt line of demarcation and focal peri-necrotic macronucleoli in grading assessment. Also, as neo-angiogenesis may result from POE procedure, caution should be used in the interpretation of MVD as a prognostic factor in embolized meningioma.


Solitary fibrous tumour of the meninges: A case series

M. Cruz *, M. Ferreira, M. Teixeira, F. Pardal, A. Silva

*Hospital de Braga, Serviço de Anatomia Patológica, Portugal

Objective: Meningeal solitary fibrous tumours (MSFTs) are uncommon and malignant cases are even rarer. We report four cases of MSFTs diagnosed in our department during a 15-year period, including a locally invasive malignant case.

Method: The patients (3 F, 1 M) ranged from 48 to 77 years (mean 60). Tumours were tentorial in one case and supratentorial in three cases, one of these showing invasion into the brain, parietal bone and subgaleal soft tissues. All patients underwent surgery as initial treatment. Follow-up was obtained.

Results: Tumours consisted of spindle cells disposed in fascicles with a dense collagenous stroma. Two supratentorial cases had rare mitoses and no significant nuclear pleomorphism or necrosis. The tentorial case showed high cellularity, nuclear pleomorphism and frequent mitoses, but no necrosis. The invasive supratentorial case showed identical atypical histological features with additional necrosis. This patient received postoperative radiotherapy with no recurrence after 4 years of follow-up. All cases were diffusely positive for vimentin, CD34 and bcl-2 but negative for EMA and S100 protein.

Conclusion: Data on outcome for patients with MSFT are limited. This case series suggests that MSFTs can have a locally aggressive behaviour. MSFTs should, therefore, be excised as completely as possible and followed carefully in the long-term.


Sensitivity and specificity of IHC markers for diagnostics of variants of Non-small Cell Lung Carcinoma (NSCLC) in Brain Metastases (BM)

D. Rotin *, O. Paklina, O. Potapova, G. Kobyakov

*NN Burdenko Neurosurgery Institute, Dept. of Pathology, Moscow, Russia

Objective: Current modalities of target therapy in advanced Non-small cell lung carcinoma require diagnostic of exact variant of the tumor. Brain metastases are not rarely the first and only material in NSLC for histological evaluation. Aim: To determine on BM of NSCLC specificity and sensitivity of number of antibodies. To work out the optimal IHC panel for BM NSCLC

Method: Material from 96 patients (70 -male, 26- female), median age 58 (ranges 37–76) with BM NSCLC operated in NN Burdenko neurosurgery institute in 2004–201, was reviewed by two skilled pathologists and then stained with TTF-1, 1, CK7, CK5/6, CK18, p63, Napsin A etc. Specificity and sensitivity for each antibody was calculated.

Results: The highest rate of sensitivity and specificity for Adenocarcinoma was seen for Napsin A (94 %/91 %), following TTF-1 (79 %/86 %). For squamous cell carcinoma the best specificity and sensitivity demonstrated p63 and CK5/6–correspondently (91 %/98 %) and (87 %/94 %).

Conclusion: The optimal panel for diagnostic of the variant of NSCLC in BM includes 4 antibodies: TTF-1, Napsin A, p63, CK5/6.

Table of sencitivity and specificity:

Antibody TTF-1 Napsin A p63 CK5/6
Sensitivity 79 % 94 % 91 % 87 %
Specificity 86 % 91 % 98 % 94 %


Sebaceous gland carcinoma of the eyelid: Prognostic significance of nuclear survivin (BIRC5)

K. Mulay *, V. White, S. Shah, S. Honavar

*Center for Sight, Ophthalmic Pathology, Hyderabad, India

Objective: To evaluate the frequency of expression of nuclear survivin in sebaceous gland carcinoma ofthe eyelid and correlate its expression with histopathological features, clinical outcomes and expression of Ki-67 & p63.

Method: 55 cases of periocular sebaceous gland carcinoma with a minimum follow up of 1-year were retrieved from the archives. Patient files were assessed for clinical outcomes. Immunostaining for survivin, Ki-67 and p63 was performed on all cases. All tumours were assigned a nuclear survivin score (NS-Score) based on the intensity and percentage expression. The NS-score was correlated with the clinical outcomes, histopathological features and expression of Ki67 and p63.

Results: All tumours expressed survivin in their nucleus. 23 tumours (51 %) had a high NS score. A high NS-score was associated with a short disease free survival (p < 0.0001) and high Ki-67 activity (p < 0.001). No statistically significant association was seen with expression of p63 and histopathological features.

Conclusion: Survivin is expressed in the nucleus in sebaceous gland carcinoma of the eyelid. A high expression of survivin has a prognostic influence in sebaceous gland carcinoma of the eyelid.


Amyioid-ß and age-related macular degeneration

V. Ermilov *, A. Nesterova, O. Makhonina

*Volgograd State Medical University, Dept. of Pathology and Forensic Medicine, Russia

Objective: Age-related macular degeneration (AMD) is the leading cause of severe visual impairment in the elderly. The appearence of amyloid in eye tissues makes it possible to see in a new light the problem of eye amyloidosis.

Method: Histological, immunohistochemical, and electron microscopic studies of the 111 eyes with AMD revealed amyloid-β in the drusen, Bruch’s membrane, and between the basal membrane of the retinal pigment epithelium (RPE) and the internal collagen layer of Bruch’s membrane.

Results: Comparative analysis of morphological changes in tissues of the macular and paramacular areas and the incidence of amyloid-β incorporations in them permit us to propose that accumulation of local senile amyloid is conductive to development and aggravation of AMD. A relationship between the degree of RPE degeneration and accumulation of amyloid-β was revealed. Ultrastructural studies of Bruch’s membrane have shown that amyloid fibrills are localised in it with fragments of degrading RPE cells closely attached to them. Amyloid-β deposition was specific to drusen from eyes with AMD.

Conclusion: The authors put forward a hypothesis of the pathogenesis of AMD, in which the principal role in the formation and deposition of abnormal protein–amyloid-β, is played by degenerative cells of RPE.


The prognostic value of CXCR4-CXCL12 and CCR7 in uveal melanoma in patients with and without liver metastasis

R. Verdijk *, T. v. den Bosch, A. Koopmans, J. Vaarwater, A. d. Klein

*Erasmus MC University, Dept. of Pathology, Rotterdam, The Netherlands

Objective: To examine expression of the chemokine receptors CXCR4, CXCL12 and CCR7 in uveal melanoma with correlation to patient survival. Irrespective of primary treatment, nearly half of the patients develop liver metastases. Chemokine receptors are involved with different cell processes, including angiogenesis and trafficking of tumor cells in metastasis.

Method: Formalin-fixed paraffin-embedded primary uveal melanoma specimens were collected from non-metastatic (n = 30) and metastatic (n = 19) patients between years 1988–2008. Specimens have been examined for classical histopathological parameters and CXCR4, CXCL12 and CCR7 expression using immunohistochemistry. Single nucleotide polymorphism array were performed and chromosome fluorescence in situ hybridization of chromosomes 1,3,6, 8.

Results: CCR7 expression correlates with the epitheloid cell type (p = 0.006), tumor thickness (p = 0.000) and necrosis (p = 0.048). No copy number variations were found in the SNP array in the regions of the CXCR4 and CCR7 genes. In multivariate analysis CCR7 staining is inversely correlated to overall survival, and disease free survival whereas CXCR4 staining is not. SNP analysis showed no gains or losses in the regions of CCR7, CXCR4 or CXCL12.

Conclusion: CCR7 expression is correlated to adverse prognostic histopathological factors and is inversely correlated to overall survival, and disease free survival. CCR7 may be a potential target for therapeutic intervention.

Overall survival for CCR7 expression:

figure c


Immune infiltration in choroidal melanomas

D. Narasimhaiah *, R. Remark, E. Becht, D. Damotte, C. Legrand, P. d. Potter, P. Coulie, M. Vikkula, C. Godfraind

*Dept. of Pathology, Université Catholique de Louvain, Brussels, Belgium

Objective: In choroidal melanomas, the presence of CD8+ T lymphocytes is associated with bad prognosis. This is in contrast to most other solid tumors, where CD8+ T cell infiltrate is related to good prognosis. The objective of this study was to better understand the immune microenvironment in choroidal melanomas.

Method: Primary, untreated tumors were analyzed for gene expression using Affymetrix U133 plus 2.0 array (n = 15) and immunohistochemistry (n = 89).

Results: Gene expression profile analysis led to the identification of a gene signature consisting of 39 upregulated genes in a subset of choroidal melanomas. These genes were associated with: antigen processing and presentation, cell adhesion molecules, interferon-gamma and chemokine signaling pathways. On immunohistochemistry, signature positive tumors displayed a dense intra-tumoral infiltrate of HLA-DRA+CD163+ macrophages and CD3+CD8+ T-cells. It was mild to moderate in tumors lacking the signature. On this basis, additional tumors were analyzed by immunohistochemistry. In total, 19 melanomas had high immune infiltrate and 70 low. Kaplan-Meier plots demonstrated tumors with high immune infiltrate had shorter disease-free survival (log-rank P < 0.001).

Conclusion: We identified in choroidal melanomas an interferon-gamma induced gene signature associated with infiltration of macrophages and CD8+ T lymphocytes. The presence of tumor-infiltrating immune cells correlated with higher risk of occurrence of metastases.

Monday, 2 September 2013, 17.00–19.00, Room 5A

OFP-09 Oral Free Paper Session Paediatric and Perinatal Pathology


Increase of placental sensitivity to melatonin and the alteration to its local synthesis in hypertensive syndromes in pregnancy

R. Corrêa *, D. d. Resende Yamamoto, L. d. Resende Yamamoto, L. Penna Rocha, M. Antônia dos Reis

*Triângulo Mineiro Federal Universidade, ICBN, Uberaba, Brazil

Objective: To evaluate the relation between hypertensive syndromes and melatonin and its possible protective role against lesions due to hypertension.

Method: Placentas were classified into Gestational hypertension (GH), Chronic hypertension (CH), Pre-eclampsia (PE) and Pre-eclampsia superimposed on chronic hypertension (PSCH), and morphologically examined by Hematoxylin–Eosin and Periodic Acid Schiff methods. Immunohistochemistry was performed in order to detect tryptophan hydroxylase (TH) and melatonin receptor 1A (MR-1A).

Results: MR-1A expression was higher in all types of HSP, mainly in cases with GH, in Caesarean section delivery, preterm placentas and in the cases with alterations in the placental morphology, particularly those presenting inflammation. The expression of TH was higher in cases with CH when compared with the control. This expression was lower in primigestas, in cases of inflammation and with PE.

Conclusion: HSP therapies should be considered and studied, especially in cases of HSP associated with PE, in which the placenta is more sensitive as it has more receptors, but its synthesis ability is reduced. As for GH and CH, the possible benefits should be evaluated, since the local placental ability to produce melatonin still exists. Financial Support: CNPq, CAPES, FAPEMIG, FUNEPU.


Neuronal apoptosis in the neonates born to preeclamptic mothers

E. Ozer *, H. Cosar, H. Topel, E. Özer

*Dokuz Eylul University, Dept. of Pathology, Izmir, Turkey

Objective: The aim of this study is to investigate the biological significance of neuronal apoptosis in an experimental model of preeclampsia.

Method: Two of four pregnant Sprague–Dawley rats (preeclamptic group) were given water containing 1.8 % NaCl on gestation day 15 and 22 in order to establish the model of preeclampsia whereas other two (non-preeclamptic group) received normal diet. A model of perinatal asphyxia was established on the postnatal 7th day to one preeclamptic and one non-preeclamptic dam. Overall 23 pups born to overall four dams were decapitated to assess neuronal apoptosis by the TUNEL assay.

Results: Mean body and brain weights were significantly lower in the pups born to the preeclamptic dams (p < 0,001). The number of apoptotic neuronal cells was significantly higher in the preeclampsia groups in comparison with the control group (p = 0.006 and p = 0.006, respectively). It was also significantly higher in the asphyctic/non-preeclamptic group than the count in the control group (p = 0.01). There was also significant difference between both asphyctic groups (p = 0.003).

Conclusion: We conclude that preeclampsia causes small babies for the gestational age and cerebral hypoplasia. Both preeclampsia and perinatal asphyxia can cause increased neuronal apoptosis in the neonatal brains. However the prognosis for neurological outcome is much worse when the perinatal asphyxia occurs in newborns born to preeclamptic mothers.

Neuronal apoptotic cells labeled by the TUNEL assay.:

figure d


The role of placental NOTCH signaling pathway disturbances in preeclampsia

K. Pavlov *, E. Dubova, A. Shchegolev, G. Sukhikh

*Research Center for Obstetrics, Dept. of Pathology, Moscow, Russia

Objective: Notch signaling pathway is necessary for placental development and plays a critical role in placentation, trophoblast invasion and placental angiogenesis. Our aim was to evaluate patterns of Notch receptor placental expression in preeclampsia (PE) complicated pregnancies.

Method: We performed complex morphological and immunohistochemical study of 9 term placentas from mild preeclampsia (mPE) cases (1st group), 6 term placentas from severe preeclampsia (sPE) cases (2nd group) and 10 term placentas from uncomplicated pregnancies (control group). Immunohistochemical study was performed with rabbit polyclonal antihuman Notch-1 antibodies (Spring Bioscience).

Results: We revealed decreasing of Notch-1 expression in all placental compartments in mPE and sPE in compare to control. Marked decreasing was noted in syncytiotrophoblast (mPE–38.6 %, sPE–39 % lower) and extravillous trophoblast (mPE–33.3 %, sPE–34 % lower) (p < 0.05). Notch-1 terminal villi endothelial expression was also decreased (both mPE and sPE–26 % lower) (p < 0.05). We also noted insignificantly decreased villous mesenchimal cell Notch-1 expression (p < 0.05).

Conclusion: Revealed patterns of Notch-1 expression reflect the role of this receptor in PE development.


ACE mutation in the recurrent oligohydramniosis and neonatal death

J. Laine *

*TYKS-SAPA, Dept. of Pathology, Turku, Finland

Objective: Case: The girl was born at 32 weeks of gestation, with oligohydramniosis and severe lung hypoplasia. The ultrasound showed difference in the size of the kidneys. Despite intensive care the baby died at 7 h, with respiratory insufficiency and anuria. Previously the mother had had three deliveries at 32–33 weeks of gestation and all babies had died within 3 days, with similar symptoms as in the present case.

Method: Autopsy, immunohistochemistry and genetical testing were performed.

Results: The autopsy showed small lungs and urinary bladder. The fontanelles were large. In histology the kidney showed renal tubular dysplasia typical of that found in ACE mutation. The glomeruli were crowded and the cortical tubules were sparse, with narrow lumen and without brush border. In the immunohistochemistry the cortical tubuli were negative for CD10 and CD15 and positive for epithelial membrane antigen (EMA). Clusters of hemangiomatoid structures and thick-walled arteries were found in the cortex. The lungs showed findings in keeping with respiratory distress syndrome.

Conclusion: The findings are well in keeping with renal tubular dysgenesis that is a rare disease and due to ACE gene mutation (Lacoste ym. J Am Soc Nephrol 17: 2,253–2,263, 2006). The disease is autosomally recessively inherited and in this case has been unexpectedly manifested four consecutive pregnancies. The condition was confirmed by gene analysis.


Placental pathology supports stratification of third trimester births into gestational age subgroups

J. Stanek *

*Cincinnati Children’s Hospital, Dept. of Pathology, USA

Objective: To find if placental pathology supports the new clinical concept of dividing preterm and term births into gestational age subgroups associated with varying perinatal mortality and morbidity.

Method: 27 clinical and 43 placental phenotypes were retrospectively analyzed in 4,617 third trimester pregnancies: 1,332 preterm pregnancies (28–33 weeks), 1,066 late preterm pregnancies (34–36 weeks), 940 near term pregnancies (37–38 weeks), and 1,279 term pregnancies (39+ weeks).

Results: The Figure depicts statistically significant clinical and placental differences between the preterm and late preterm, and near term and term births. The placental acute inflammatory pattern was characteristic for both gestational age sides of the 3rd trimester, while clinical conditions linked to in-utero hypoxia (preeclampsia, diabetes mellitus, fetal growth restriction) and their placental counterparts (atherosis, membrane chorionic microcysts, chorangiosis, intervillous thrombi) were associated with mid-3rd trimester. Frequency of acute fetal distress (abnormal fetal heart tracing, and clinical and histological meconium) increased with gestational age until term.

Conclusion: Differences in placental pathology among the four subgroups of third trimester pregnancy not only challenge the use of an arbitrary cutoff point of 37 weeks separating the preterm and term birth, but also further support separation of late preterm births from preterm births, and near term births from term births.

Statistically significant differences in clinical and placental variables:

figure e


Up-regulated TNFa placental terminal villi expression as a predictor of early-onset neonatal sepsis

A. Shchegolev *, E. Dubova, K. Pavlov, E. Balashova, O. Ionov, D. Degtiarev, G. Sukhikh

*Research Center for Obstetrics, Dept. of Pathology, Moscow, Russia

Objective: Early-onset neonatal sepsis (EONS) is one of the most dangerous neonatal pathology with extremely high mortality. Our aim was to study correlation between abnormal placental TNFα expression and EONS development.

Method: 28 newborns with EONS and 15 healthy newborns were included in a study. White blood cells (WBC) and platelets (PL) count, serum C-reactive protein (CRP) and procalcitonin (PCT) level and complex morphological study of the placenta with immunohistochemical detection of TNFα were performed.

Results: WBC count, CRP and PCT serum levels were significantly elevated in neonates with EONS, whereas PL count was significantly decreased. In 10 EONS cases we revealed villous tree maldevelopment (excessive syncytial knotting) and persistent villous immaturity. Signs of placental inflammation were revealed in 11 cases of EONS: acute chorioamnionitis (6 cases), acute funisitis (3 cases), acute basal plate deciduitis (3 cases). We also revealed significant elevation of TNFα expression in syncytiotrophoblast and terminal villi endothelial cells (41.1 % and 45.3 % in compare to control, accordingly).

Conclusion: Up-regulated TNFα placental terminal villi expression could be used as a prognostic marker of early-onset neonatal sepsis development.


Fetal cardiovascular malformations: Clinical versus anatomopathological diagnosis

P. Serra *, A. Rodrigues, R. Pina

*CHUC- HG, Surgical Pathology, São Martinho Do Bispo, Portugal

Objective: Diagnostic correlation between fetal prenatal diagnosis (PD) results and autopsy findings on Cardiovascular malformations (CVM).

Method: We have analysed 772 fetal autopsies performed between January 2005 and December 2012. Only cases above 19 weeks gestation and whose CVM was not associated to cromossomopathies where selected, because these had PD with morphologic ultrasound and echocardiogram. We analysed the clinical records and autopsy diagnosis.

Results: There were 190 cases with CVM (24,6 %): 134 cases where above 19 weeks gestation but only 73 cases had a CVM not associated to a cromossomopathy. Our study was performed on the last group. The greatest results disparity was found on Ventricular septal defects (VSD) (7/14 cases), aortic arch coarctation (2/6), 2 cases of complete situs inversus, 1 Ebstein anomaly and 1 atrial septal defect. On complex cardiopaties, 8 cases showed minor disparity results. There was complete agreement on the hypoplastic left heart syndrome diagnosis (12 cases). On 1 case the CVM diagnosed on the echocardiogram was not found at the autopsy.

Conclusion: The CVM are a common indication for medical pregnancy termination and therefore they are common fetal autopsy findings. Only 17,8 % (13 cases) of CVM showed diagnostic disparities, mostly on VSD, because it were very small defects.


Innate immune response and expression of melatonin receptors in the lung of children

R. Corrêa *, J. G. Pacheco Olegário, M. Vinícius da Silva, J. Reis Machado, L. Penna Rocha, M. Antônia dos Reis

*Triângulo Mineiro Federal Universidade, ICBN, Uberaba, Brazil

Objective: To analyze the cytokines of the innate immune pulmonary response and the capacity for local response to melatonin according to the perinatal stress.

Method: 49 cases of pediatric autopsies were evaluated, divided according to cause of death, perinatal stress, gestational age (GA), and birth weight. The percentages of IL-6, C-reactive protein (CRP), IL-1β, TNF-α, and melatonin receptor were evaluated by immunohistochemistry.

Results: The IL-6 expression was higher in the children showing chronic stress (CS), anoxia, and infection. The IL-6 expression showed a progressive increase according to the relation between weight and GA. There was no significant difference in the expression of IL-1β and TNF-α. The CRP expression was higher in the cases showing CS and premature cases. The expression of melatonin receptors was significantly higher in the cases showing CS, being more evident in the cases showing infection.

Conclusion: The cause of death and the type of stress influence the expression in situ of melatonin and cytokines of the innate immune pulmonary response. The greater sensitivity of the lung to melatonin in these cases may indicate an attempt at controlling the immunological response, in an attempt to diminish the harmful effects of stress. Financial Support: CNPq,CAPES, FAPEMIG, FUNEPU.


Pediatric hepatocellular carcinoma of common type: Morphologic and immunophenotypic characterization of a monocentric series of 13 cases

C. Mussini*, E. Gonzales, M.-J. Redon, S. Branchereau, H. Martelli, E. Jacquemin, C. Guettier

*Hopital Bicetre, Pathology, Le Kremlin Bicetre, France

Objective: Hepatocellular carcinoma (HCC) represents 0,4 % of pediatric malignant tumors. Anti-HBV vaccination modified epidemiology of common type HCC with an increasing incidence of constitutional liver diseases. To study epidemiologic, morphologic and immunophenotypic profiles of pediatric HCC of common type.

Method: Between 2006 and 2013, 13 children had surgery for common type HCC. Clinical and follow-up data have been collected. Immunochemical study has been performed on 18 tumor nodules with EpCAM, CK19, AFP, b-catenin and glutamin synthase antibodies.

Results: Median age at diagnosis was 8,5 years (range: 1–15,25). Average AFP level was 105,313 (range: 15–909,000) HCC occured in 2 normal livers, 1 HBV cirrhosis and 10 genetic liver diseases (5 Progressive Familial Intrahepatic Cholestasis-type 2, 3 tyrosinemia type 1, 2 mitochondrial cytopathies). WHO grade was more often 2 or 3. Eighty-nine per cent (16/18) of HCC had a progenitor-like profile EpCAM+, AFP+including CK19+ (5/18) and CK19- (11/18). Nuclear expression of b-catenin was observed in 66,7 % (12/18) HCC, mainly at the periphery of the tumor. Diffuse expression of glutamin synthase was present in only 3/18 HCC.

Conclusion: Most pediatric HCC occurs on genetic liver diseases and their phenotype suggests an origine from hepatic progenitor cells.


Salivary gland tumours in children: A decade of experience and a review of current literature

A. Joannou-Coetzee *, A. Cohen, S. Whitaker, S. Di Palma

*Sheffield Children’s Hospital, Dept. of Histopathology, United Kingdom

Objective: Salivary gland tumours are rare in children. We describe our experience with these tumours from our institutions and review current literature.

Method: A search of the files from the Histopathology department at a paediatric referral hospital and from cases referred to one of the co-authors for histological review was performed between 2003 and 2013. The findings were compared to hitherto published evidence regarding diagnosis, prevalence and biology.

Results: We identified 5 pleomorphic adenomas; 3 mucoepidermoid carcinomas; 1 acinic cell carcinoma, 1 desmoid fibromatosis and 1 Burkitt’s lymphoma. In addition, cystic lesions that corresponded to a cystic hygroma (1 case) and a lymphatic malformation (2 cases) were also seen. Only 5 of these cases corresponded to a malignant neoplasia. In our experience, tumours arising in salivary glands of young patients have a high chance of malignancy (55.5 %).

Conclusion: Our results confirm the reviewed literature, that indicates that the incidence of malignant tumours in the salivary glands is much higher in children (55.5 %) than in adults (15 %–25 %). This may be the consequence of various genetic alterations in paediatric tumours. The rarity of salivary gland tumours at this age highlights the importance of the appropriate clinical and surgical management of these cases at the initial presentation.

Tuesday, 3 September 2013, 14.15–16.15, Auditorium VII

OFP-10 Oral Free Paper Session Gynaecopathology


The role of All Trans Retinoic Acid (ATRA) in ovarian cancer therapy

R. Januchowski *, K. Wojtowicz, M. Zabel

*University of Medical Sciences, Dept. of Histology and Embriology, Poznan, Poland

Objective: Ovarian cancer represents the most common cause of death among gynecological malignancies. Chemotherapy in this cancers is still not satisfactory because of drug resistance. The main mechanism of drug resistance results from the ability of cancer cells to actively expel therapeutic agents via transport proteins of the ABC family. Recent investigations indicate that aldehyde dehydrogenase (ALDH1A1) can also be involved in drug resistance of ovarian cancer.

Method: A chemosensitivity assay MTT test was performed to assess drug cross-resistance. Quantitative real-time polymerase chain reaction and Western blot were performed to determine mRNA and protein expression of genes involved in chemoresistance.

Results: We observed overexpression of ALDH1A1 in primary ovarian cancer cell lines resistant do topotecan and paclitaxel at mRNA and protein level. Pre-treatment with all-trans retinoid acid (ATRA) sensitised these cell lines to topotecan and paclitaxel. Treatment with ATRA leads to downregulation of ALDH1A1, ABCB1 and ABCG2 at protein but not mRNA level.

Conclusion: Our results indicate that ATRA regulate expression of ALDH1A1 ABCB1 and ABCG2 at post-translational level. Downregulation of these proteins is very important from therapeutic point of view. ATRA could be considered as a therapeutic agent in combination with cytostatics in ovarian cancer therapy.


Malignant ovarian epithelial tumors and coexistence with synchronous uterine carcinoma: A study of clinicopathological parameters and Her 2/neu protein expression

M. El-Shaer *, Z. S. Elalfy, Z. A. Shehab Eldin, F. Faten A. Ghazal, E. A. Omara

*National Research Centre Cairo, Dept. of Pathology, Egypt

Objective: To evaluate the oncogene Her2/neu in these ovarian tumors and its relation with synchronous uterine corpus carcinoma (SUC) and their association with different clinicopathologic parameters.

Method: Randomly selected retrospective 136 cases examined for the presence or absence of SUC,and all data were extracted

Results: Coexisting ovarian and SUC was found 11 % of malignant cases, 73.3 % of them were endometrioid ovarian adenocarcinoma with evident high percentage, 20 % of serous ovarian adenocarcinoma while only 6.7 % mucinous. The grades were found to be 13.3 % grade I,53.3 % grade II and 33.3 % grade III i.e. 86.6 % were moderately to poorly differentiated carcinomas. Staging was 41.7 % stage I, 0 % stage II, 50 % stage III & 8.3 % stage IV. About 27 % of ovarian tumors presented with SUC were stained positive for Her 2/neu, in comparison to only 6.3 % of isolated ovarian tumors without coexisting uterine primary tumor P value = 0.007.

Conclusion: The incidence of synchronous endometrial and ovarian cancer is not negligible, is commonly seen with malignant endometrioid ovarian tumors. Percentage of recurrence increased in the cases of ovarian tumor coexisting with SUC than in isolated ovarian tumor, ovarian tumors coexisting with (SUC) show a significant increase in Her2 over expression predicting an underlying molecular genetic defect causing this association.


Ovarian metastasis of endocervical adenocarcinoma

P. Caseiro Silverio *, H. Kannuna, D. Huber, J.-C. Tille

*Hôpital Universitaire Genève, Switzerland

Objective: Ovarian metastasis of endocervical adenocarcinoma is uncommon and can mimic primary ovarian neoplasm.

Results: A 51-year-old woman was hospitalized for thromboembolic disease progression despite anticoagulation. Computer tomography showed a suspicious pelvic tumor and an elevated CA 125. Exploratory laparoscopy confirmed a 12 cm left adnexal mass with moderate ascite and no other intra-abdominal lesions. Peritoneal biopsies were negative and peritoneal cytology revealed an adenocarcinoma. A primary cytoreduction surgery was performed (hysterectomy with bilateral oophorectomy, appendectomy, omentectomy). The ovarian tumor showed an endometrioid-like pattern, with elongated nucleus associated with numerous mitosis, apoptoses, and focally mucinous differentiation. No macroscopic lesion was visible on the cervix, but a CIN3 and endocervical adenocarcinoma was present, with the same morphology of the ovarian tumor. No lympho-vascular invasion was observed. At immunohistochemistry, both tumors were positive for p16, CEA and in 50 % for estrogen receptors. The PCR for HPV shows the presence of type 16 HPV.

Conclusion: The reported frequency of ovarian metastases in cervical adenocarcinoma is ~5 %. In absence of macroscopic cervical lesion, the ovarian metastasis could be misdiagnosed as primary ovarian tumor. The hybrid pattern and detection of HPV is useful for identification an endocervical origin. A possible way of dissemination is perhaps through transtubal spread.


Undifferentiated Connective Tissue Dysplasia Syndrome (uCTDS) and Genetically Determined Trombophyliae (GDT) influence on endometrium receptivity and reproduction function

T. Demura *, E. Kogan, D. Kolossovskiy, N. Fayzullina, A. Zanozin

*RCOGP, Dept. of Pathology, Moscow, Russia

Objective: Aim of investigation is a study of uCTDS and GDT influence on endometrium receptivity and pregnancy outcomes.

Method: Clinico-morphological analysis of 92 patients with diagnosis GDT was carried out. 54 (58.7 %) of these patients had GDT and uCTDS combination. Clinical study included analysis the following data: anamnesis, physical examination. Endometrium pipel-biopsies taken at 6-7th day after ovulation underwent a morphological investigation. Stepped paraffin sections were prepared. These sections stained by a hematoxiline & eosin and used for immunohistochemical determination the following markers: ER (DAKO, Denmark), PgR (DAKO, Denmark), PAI (Santa Cruz, USA), LIF (R&D Systems, USA), VEGF (DAKO, Denmark).

Results: In 83.1 % of all cases there was endometrium of either proliferation or early secretion phase in “implantation window”. In 84.3 % of all cases no mature pinopodies were found. Sclerosis and connective tissue disorganization occurred in 68.1 % cases with uCTDS and in 52.7 % cases without uCTDS; microcirculatory disturbances–in 48.9 % and in 38.9 % cases; mature pinopodies absence–in 87.2 % and in 80.6 % cases, abnormal ER/PgR ratio–in 80.9 % and in 69.4 % cases, consequently.

Conclusion: GDT and uCTDS combination can be considered as reproduction function disturbances negative prognosis factor.


Identifying prognostically relevant subsets of endometrial cancer using unsupervised clustering of immunohistochemical staining data

J. Huvila *, D. Laajala, P.-H. Edqvist, L. Talve, S. Grénman, F. Pontén, T. Aittokallio, O. Carpén, A. Auranen

*University of Turku, Dept. of Pathology, Finland

Objective: Multiple studies have suggested that biomarkers, such as ER and PR have a prognostic significance in EC. However, these biomarkers are currently not utilized as prognostic markers in clinical practice. Our aim was to assess the immunohistochemical risk profile of EC patients by performing unsupervised clustering of eight prognostic factors.

Method: We collected clinical, histopathological and follow-up data on 230 EC patients operated at Turku University Hospital during 2004–2007. A TMA was constructed for immunohistochemical analysis and the results were entered in an unsupervised hierarchical clustering analysis.

Results: The unsupervised clustering produced a cluster with three major subgroups of which one subgroup consisting of 47 (20.4 %) cases had a higher risk for relapse [OR 14.3 (CI 5.91–34.7, p < 0.001)]. Apart from one clear-cell cancer, all non-endometrioid cancers clustered in the high-risk subgroup; furthermore, 36 (16.5 %) of the adenocarcinomas clustered in the high-risk subgroup and had a higher relapse risk compared to the endometrioid cancers in the non high-risk subgroups [OR 12.2 (CI 4.72–31.6, p < 0.001)]. The association sustained when adjusted for FIGO stage and grade [OR 8.3 (CI 2.88–23.95, p < 0.001)].

Conclusion: Our results indicate that using a panel of immunohistochemical stainings could be a useful tool for risk assessment in EC.


eIF4E, a critical downstream target of the mTOR pathway, is overexpressed in high-grade dysplasia and carcinomas of the uterine cervix

A. Asimomytis *, M. Karanikou, A. Rodolakis, A. Vaiopoulou, P. Tsetsa, A. Antsaklis, E. Patsouris, G. Rassidakis

*University of Athens, First Dept. of Pathology, Greece

Objective: eIF4E, a critical regulator of the protein synthesis initiation, seems to recapitulate the oncogenic effects of the AKT/mTOR pathway in mouse models. In this study, we investigated the expression levels of eIF4E in precancerous lesions and cancer of the uterine cervix and their association with HPV infection.

Method: Uterine cervical biopsies from 73 patients were obtained including 40 fresh-frozen samples and 42 archival paraffin-embedded tissue specimens. eIF4E expression was analyzed by Western blot in whole protein extracts and by immunohistochemistry in archival tissues. HPV typing was performed using standard PCR methods.

Results: High expression levels of eIF4E was observed in all carcinomas and significantly correlated with the degree of dysplasia (p < 0.0005) in immunoblots. By immunohistochemistry, overexpression of eIF4E was seen in 17 of 21 (81 %) patients with high-grade dysplasia and carcinomas as compared to 2 of 20 (10 %) patients with low-grade lesions or normal histology (p < 0.0001). eIF4E expression did not statistically correlate with the presence of high-risk HPV or oncogenic HPV 16/18 types.

Conclusion: Our findings suggest that, eIF4E as a critical downstream effector of the mTOR pathway, may be involved in tumorigenesis of the uterine cervix, thus providing a novel target for investigational therapeutic approaches in these patients.


Endometrial carcinoma: Developments during the last 5 decades in Portugal

C. Bartosch *, B. Gomes, J. M. Lopes

*IPO-Porto, Pathology, Portugal

Objective: To characterize consecutive endometrial carcinomas (EC) treated at a Portuguese tertiary hospital, and evaluate changes comparing our series with previous Portuguese studies.

Method: Clinical-pathological review of all EC diagnosed (2000–2010) at CHSJ; PubMed, Scielo, IndexRMP search (“Portugal” and “endometrial carcinoma”/“carcinoma”,“endometrio”); statistical analysis.

Results: 215 EC identified (>60 years of age: 70.2 %), with clinical-pathological features consistent with recent reports. Two previous studies (1977 Study 1: 260 EC, 55.0 % with >60 years of age; 1995 Study 2: 57 EC, 57.0 % with >60 years of age) disclosed less elderly patients, and less comorbidities, like obesity (Study 1: 44.2 %; Study 2: 36.4 %), than in our series (54.3 %). More accurate EC distribution by stage, grade, and histological types is observed over the years. There was a trend (Study1 vs. our study) to increased use of surgical staging (surgery 78.2 % vs. 90.4 %; lymphadenectomy 16.9 % vs. 27.7 %) and decreased use of radiotherapy (89.6 % vs. 52.7 %); our series overall 5-year survival (78.4 %) is better than in Study 1 (57.3 %; absent in Study 2).

Conclusion: Substantial developments in diagnosis, treatment, and survival of patients with EC ensued over last 5-decade, at least in part due to better healthcare, but with more morbidities possibly caused by women longevity/risk factors in Portugal.


Tumor-free distance to serosa (TFD), depth of myometrial invasion (DOI) and 50 % cut-off of myometrial invasion (50 %MI) as predictors of metastasis at diagnosis and recurrence in endometrial carcinomas

C. Bartosch *, B. Gomes, J. M. Lopes

*IPO-Porto, Pathology, Portugal

Objective: To compare TFD, DOI, and 50 %MI as predictors of endometrial carcinoma (EC) metastasis at diagnosis/recurrence.

Method: Clinical-pathological review of all surgically treated EC at CHSJ during 2000–2010; univariate and multivariate analysis using logistic regression; and ROC curve analysis of single/model integrated prognostic parameters.

Results: 173 patients identified. Median age: 66 year. Tumour type: endometrioid: 80G1, 48G2, 22G3; non-endometrioid: 23. FIGO stage: 137I, 15II, 17III, 4IV. Median follow-up: 50.5 months; 25 recurrent; 25 deaths due to EC; 5 year survival: 83.3 %. TFD, DOI, and 50 %MI significantly associated to metastasis at diagnosis/recurrence/survival (univariate analysis). Prediction of metastasis was slightly better (not significant) for TFD (68.0 %AUC) compared to DOI (66.7 %AUC). Optimal cut-off values: 5 mmDOI and 8 mmTFD. In multivariate analysis of 8 mmTFD, 5 mmDOI, and 50 %MI separately, plus covariates (tumour type/grade/lymphovascular invasion), both 8 mmTFD (p = 0.01) and 50 %MI (p = 0.04), but not 5 mmDOI (p = 0.265) are independent prognosticators of metastasis. Prediction of metastasis at diagnosis/recurrence by the three multivariate models, showed slightly better (not significant) performance of 8 mmTFD (87.2 %AUC) vs. 5 mmDOI (86.2 %AUC) and 50 %MI (86.1 %AUC).

Conclusion: TFD and 50 %MI seem to be similar predictors of metastasis at diagnosis/recurrence of EC. However, tumor-free distance to serosa seems a simpler reproducible parameter in the diagnostic evaluation of endometrial carcinoma.

Tuesday, 3 September 2013, 14.15–16.15, Room 5A

OFP-11 Oral Free Paper Session Uropathology


High RBM3 expression is an independent prognostic marker in operated prostate cancer and tightly linked to ERG activation and PTEN deletions

K. Grupp *, J. Wilking, K. Prien, C. Hube-Magg, H. Sirma, R. Simon, J. R. Izbicki, G. Sauter, S. Minner, T. Schlomm, M. Tsourlakis

*UKE Hamburg-Eppendorf, Allg. Chirurgie und Pathologie, Germany

Objective: The RNA-binding motif protein 3 (RBM3) has been suspected as a prognostic biomarker in several cancers.

Method: To explore the prevalence and clinical significance of RBM3 expression in prostate cancers.

Results: RBM3 expression was analyzed by immunohistochemistry on a tissue microarray containing 11,152 prostate cancers. RBM3 expression was more often detectable at various intensities in prostate cancers compared to benign prostate predominantly localized in the nucleus of the cells. RBM3 immunostaining was found in 64 % of the interpretable prostate cancers and was considered strong in 25.6 %. High RBM3 expression was linked to advanced tumor stage, high Gleason score, positive nodal involvement and positive surgical margin status (p < 0.0001 each). There was a remarkable accumulation of strong RBM3 expression in ERG positive prostate cancers and tumors harboring PTEN deletions (p < 0.0001 each). Moreover, RBM3 staining was tightly related to early biochemical recurrence if all tumors or subgroups of ERG negative and ERG positive cancers were analyzed (p < 0.0001 each). In multivariate analysis, including RBM3 staining, Gleason grade, pT stage, PSA, surgical margin status, and nodal status, the prognostic impact of RBM3 staining retained statistically significance (p = 0.0084).

Conclusion: Our observations indicate that high RBM3 expression is an independent prognostic marker in prostate cancer.


Nephrogenic adenoma/metaplasia: A multicenter clinicopathological study of 106 cases

A. Corominas-Cishek *, M. Schiavo-Lena, L. Macías, A. Yagüe, R. Guarch, R. Tardanico, J. I. López

*Cruces University Hospital, Dept. of Pathology, Barakaldo, Spain

Objective: To analyze the clinicopathological characteristics of a large series of nephrogenic adenomas and metaplasias, lesions supposedly resulting from the displacement and implantation of renal tubular cells along the urinary tract

Method: Retrospective review of 106 cases collected in 4 different medical institutions in Spain and Italy. Sex, age, location, specimen type, histology, coexisting lesions, and main clinical settings were recorded. Immunostaining with PAX8, p63, PSMA, S100A1, CEA, CB1, CD117 and e-cadherin was performed in selected cases

Results: Males predominated in the series (84 M/22 F) with an average age of 65 (range 14–88). Bladder was the commonest location (66 %) but lesions were found from renal pelvis to urethra. Histologically, pure tubular (37.7 %), papillary (12.2 %) and flat (3.7 %) patterns, or combinations of them (46.8 %), were found. The most frequent concurrent lesions were urothelial carcinoma (16 %), cystitis (10.3 %), lithiasis (4.7 %) and stenosis (2.8 %). Urothelial carcinoma was the clinical antecedent in 88 % of the recorded cases. Average time between cancer resection and the diagnosis of nephrogenic adenoma/metaplasia was 16 months (range 1–168)

Conclusion: Nephrogenic adenoma/metaplasia is a underdiagnosed benign lesion with diverse morphology that may appear at any site along the urinary tract. The condition must be distinguished from urothelial carcinoma with which, moreover, is very frequently associated


Significance of cribriform pattern of invasive prostate cancer

G. Kir *, B. Cosan, C. S. Topal, F. Zerenler, S. Cetin

*Umraniye Hospital, Dept. of Pathology, Istanbul, Turkey

Objective: There is now an increased understanding that invasive cribriform carcinoma is a relatively aggressive disease. With the revision of the Gleason system at an International Socitey of Urological Pathology (ISUP) consensus conference (2005), there was consensus that most cribriform glands should be classified as pattern 4. In some recent publications, recommendation is that all cribriform patterns be diagnosed as Gleason pattern 4 rather than Gleason pattern 3.

Method: We assessed the cribriform pattern associated with more definitive pattern 3, 4 and 5 elsewhere and evaluated the association of cribriform pattern with extraprostatic extension (EPE) and pathologic stage on the histopathologic evaluation of 185 radical prostatectomy specimens,

Results: Cribriform pattern was more frequently observed in cases with definitive patterns 4 and 5 than pattern 3. The cases with Gleason score 3 + 3 with cribriform foci, were more frequently associated with EPE and stage pT3 than the cases without cribriform foci.

Conclusion: Our results demonstrate that, to diagnose all cribriform patterns as Gleason pattern 4, would significantly impact on further therapeutic options and prognosis. However as many of these modifications are emprical and supported only by few sudies, long term follow-up studies with clinical end points are necessary to validate these recommendations.


EGFR mutational status in patients with penile carcinoma

I. Cunha *, A. Silva, B. Lisboa, R. Rocha, S. Zequi, G. Guimarães, J. Vassalo, F. Soares

*Hospital A.C. Camargo, Dept. of Pathology, São Paulo, Brazil

Objective: Penile carcinoma (PC) is a worrisome issue in developing countries such as Brazil. Therapeutic options are limited, with poor prognosis, especially for patients with metastatic disease. Therefore, new therapeutic targets are warranted. The purpose of the present study was to search for mutations in EGFR tyrosine-kinase (TK) domain 18–21, frequently mutated in non-small cell lung carcinoma (NSCLC). This could have therapeutic implications with TK inhibitors

Method: Frozen tumor specimens from 29 patients with PC were submitted to direct sequencing of the EGFR-TK domain, using the polymerase chain reaction method. Sequences were analyzed with the CLC Bio software and compared with the EGFR sequence.

Results: None of the sequenced tumor samples showed relevant alterations in the four exons studied. However, 19 out of the 29 cases exhibited an alteration in exon 20 (p.Q787Q), which has been described as a single nucleotide polymorphism in other tumor types, without clinical significance.

Conclusion: In PC, unlike NSCLC, different alterations might play a role in EGFR expression and activation. As the knowledge on the molecular mechanisms of PC is still limited, because of the relative rarity of this tumor in wealthier regions of the globe, further studies are necessary to support new therapeutic alternatives.


A modified point-count method as a practical approach to assessing tumor volume and percentage of prostate gland involvement by carcinoma

D. Athanazio *, P. R. Athanazio, A. Carvalho dos Santos, L. A. Rodrigues de Freitas

*Federal University of Bahia, Dept. of Pathology, Salvador, Brazil

Objective: To evaluate a modified point-count method for quantifying carcinoma in prostatectomy specimens (n = 142), adapted from Billis et al., Int Braz J Urol. 2013.29:113–120.

Method: The basal/apical margins were sampled using the cone method. The remainder of the gland was divided into 12 quadrant-shaped regions which had two slices sampled. Eight equidistant points were marked on the coverslip over each fragment. The points inside the tumoral areas were counted and expressed as the percentage of prostate gland involvement by carcinoma (PGI) and as the tumor volume (TV).

Results: A significant correlation between the preoperative PSA level and each of the three quantitative estimations was found, but it was higher for both values (PGI and TV) obtained using the point-count method, viz.: number of slices involved (NSI) (r = 0.32), PGI (r = 0.39) and TV (r = 0.44). When the data sets were stratified into three categories, all three methods correlated with Gleason scores ≥7, primary Gleason scores ≥4, perineural/angiolymphatic invasion, extraprostatic extension, seminal vesicle invasion and positive margins.

Conclusion: All three quantitative methods were associated with morphologic features of tumor progression. The results obtained using the modified point-count method correlated better with the preoperative PSA levels.

Eight equidistant points were marked directly on the coverslip over each fragment.:

figure f


Differential expression of genes related to tumor immunity in penile carcinomas with or without association to the human papillomavirus infection

I. Cunha *, M. Pavanello, A. Busso, S. Rogatto, G. Guimaraes, S. Zequi, R. Rocha, F. Soares, J. Vassallo

*Hospital A.C. Camargo, Dept. of Pathology, São Paulo, Brazil

Objective: Penile carcinoma (PC) presents a high incidence in Brazil. Around half of the cases are associated with human papillomavirus infection (HPV). Immunity to tumor seems to play a role in the pathogenesis of neoplasia. In spite of many studies on tumor immunity in other tumor types,no experience has been published on PC immunity. The present study aims to identify genes related to immunity in PC samples according to HPV status, based on a high throughput gene analysis approach.

Method: Four non-tumoral glans, 11 HPV positive, and 25 HPV negative PC were obtained from frozen tissue biorepository of our institution. Gene expression profiles (44 K, Agilent Tecchnologies) of the three groups were compared and those related to immunity were analyzed using the Ingenuity Pathway Analysis (IPA–http://www.ingenuity.com/).

Results: In silico functional characterization revealed higher expression of gamma-interferon (IFNG) gene (log2ratio = +2.40) in PC samples. In HPV positive cases, 3 out of 17 immune canonical pathways were significantly altered, namely, target cell apoptosis by T cytotoxic lymphocytes, chemokine signaling and Nur77 signaling in T lymphocytes.

Conclusion: Similar with other tumor types, including those related to HPV, INFG revealed to be higher expressed in PC. This result may support further studies, which might unravel its use as a therapeutic target.


Analysis of alterations in the VHL gene and clinical significance in renal clear cell carcinoma cell type

M. Alves *, W. Costa, F. Silva, D. Carraro, I. Cunha, S. Zequi, G. Guimarães, F. Soares, R. Rocha

*AC Camargo Hospital, Anatomic Pathology, São Paulo, Brazil

Objective: Provide useful predictive or prognostic information in patients with renal clear cell carcinoma cell type (ccRCC) through VHL mutational status.

Method: VHL protein expression was analyzed by immunohistochemistry in a TMA containing 148 samples and validated in 62 cases of RNA by qRT-PCR. The mutation profile was assessed in 91 cases by Sanger sequencing.

Results: VHL was found mutated in 57.1 % of cases, being missense mutations present in 28.8 %, nonsense in 5.7 %, intronic mutations in 15.3 %, deletions in 44.2 %, indels in 9.6 %, duplication in 9.6 % and insertion in 1.9 % of cases. The prevalence of mutations by exon was: exon 1, 53.8 %; exon 2, 30.7 %; and exon 3, 15.3 %. Concerning VHL protein expression, our data showed high frequency of positivity (78.9 %) but no significance between protein expression, clinical data and survival were achieved. Importantly, 86.8 % of strong positive cases represented 45 muted cases of the 91 samples evaluated by sequencing.

Conclusion: According to our study, it is clear that a positive VHL immunostaining would not necessarily indicate a wild-type VHL status and that the examination of the VHL protein itself, downstream proteins/pathways and additional genes may provide more insights into the functional consequences of particular genetic changes and their clinical relevance.


Handling of prostatectomy specimens: Simulation of partial embedding methods

A. Alves *, G. Borrecho, P. Luís, L. Correia, A. Costa-Silva

*Hospital de Santa Maria, Dept. de Anatomia Patologica, Lisbon, Portugal

Objective: Documenting staging parameters and margin status in radical prostatectomy specimens (RPS) is a key component in patient management. A variety of partial embedding methods are available with the aim of reducing costs and time constraints. Our goal is to determine the potential loss of prognostic information associated with different sampling protocols, by evaluating a commonly used tumor diagram associated with the pathology report.

Method: The tumor diagrams of 106 totally embedded (TE) RPS were retrospectively studied using an image editing software. Different protocols were simulated: alternate slice embedding, starting with the 1st (A1) or 2nd (A2) slice; total posterior embedding (PE); alternate slice+peripheral rim embedding (APR). Prostatic apex and base were always embedded. Tumor volume (TV), margin status and number of tissue blocks were determined.

Results: TV for each protocol was: 12,69 %-TE; 12,61 %-A1; 13,06 %-A2; 13,01 %-PE. In 49 cases with positive margins, the false-negative rate was: 22,5 %-A1; 26,5 %-A2; 12,24 %-PE (3 not evaluated). Block number reduction was: 42 %-A1; 48 %-A2; 36–58 %-PE; 36 %-APR.

Conclusion: Considering the importance of margin status, as compared to tumor volume, we think that the alternate slice+peripheral rim embedding is far superior to the other methods and is the most reliable partial embedding method, allowing prognostic information maintenance and saving of resources.


Xp11.2 translocation renal cell carcinoma: Mutational analysis of KRAS and BRAF status and correlation between TFE3 immunostaining and fluorescent in situ hybridation

M. Classe *, F. Escande, V. Gregoire, J. Le Nobin, A. Villers, S. Aubert, X. Leroy

*CHRU Lille, Dept. of Pathology, Lille Cedex, France

Objective: Our aims were to study the KRAS and BRAF status of translocation renal carcinoma and to compare TFE3 expression by immunohistochemistry and gene rearrangement analysis by fluorescence in situ hybridation (FISH).

Method: 26 cases were studied. Immunohistochemistry was performed with TFE3 and TFEB antibodies and break apart dual color FISH assay was performed with TFE3 probe. KRAS and BRAF mutation status was analysed by PCR followed by pyrosequencing on DNA extracted from paraffin embedded samples.

Results: The 26 patients ranged from 7 to 76 years old (median: 31). 13 were staged pT1, 6 pT2, 4 pT3 and for 3 patients the information wasn’t available. In the follow-up 2 patients died of the disease. No KRAS or BRAF mutations were found. 22 tumors showed at least a weak (8), moderate (10) or intense (8) expression of TFE3 or TFEB (1/23) and all tumours for which FISH was interpretable, were proved to have a specific gene rearrangement in 10 % to 80 % of tumor cells.

Conclusion: It is the first work studying BRAF and KRAS genes in translocation renal carcinoma and reporting no mutations. We also observed a good correlation between TFE3 immunostaining and TFE3 rearrangement by FISH.

Tuesday, 3 September 2013, 17.00–19.00, Auditorium VI

OFP-12 Oral Free Paper Session Joint Session–Digestive Diseases/Molecular Pathology


Is Src homology 2 domain of Cten essential for cell motility?

M. Akhlaq *, M. Ilyas

*University of Nottingham, Dept. of Molecular Medical Sciences, United Kingdom

Objective: Cten is a known oncogene in colorectal cancer. It is the smallest member of tensin gene family and has a role in enhancing cell motility. Cten is a fascinating protein because of its interaction with integrins and the presence of a Src Homology 2 (SH2) domain. The objective of this study is to investigate role of SH2 domain in Cten’s ability to enhance motility in cells.

Method: Site directed mutagenesis used to introduce a point mutation in an important Arginine residue (at site 474) in the SH2 domain of Cten. The effect of forced expression was evaluated by transwell migration and wound healing assay. Western Blot performed to evaluate levels of related proteins.

Results: Decrease in motility observed when transfection with SH2 mutant and Cten-GFP was compared to control Cten-GFP (p < 0.05). On western blotting, ILK and FAK levels were also decreased and N-cadherin and E-cadherin levels were affected showing an epithelial mesenchymal transition (EMT switch) with Cten-GFP but not with SH2 mutant Cten-GFP.

Conclusion: It is concluded that effect of Cten on cell motility is in part modulated through its SH2 domain. This leads to interaction with other proteins involved in signal transduction such as integrin linked kinase (ILK) and focal adhesions kinase (FAK).


Failure of induction of the oncomiRic cluster miR-182/miR-96 in HCV-induced non-metastatic hepatocellular carcinoma tissues renders them as markers for metastasis

R. Assal *, H. Mohamed El Tayebi, K. Adel Hosny, G. Esmat, A. Ihab Abdelaziz

*German University in Cairo, Molecular Pathology Unit, New Cairo, Egypt

Objective: miR-182 and miR-96 are expressed as a single cluster with miR-183. They are intergenic and are located on chromosome 7q32.2. Both microRNAs were shown to be upregulated and induce metastasis in hepatocellular carcinoma (HCC). However they have never been investigated in non-metastatic HCC. We aimed at investigating the expression profile of miRNAs miR-182 and miR-96 in non-metastatic HCC.

Method: Total RNA was isolated from 23 Hepatitis C virus (HCV) induced non-metastatic HCC tissues and 12 healthy liver tissues. Expression of both miRNAs 182 and 96 was analyzed using qPCR after reverse transcription and normalization to RNU6B. The obtained Ct values were statistically analyzed using Mann–Whitney method.

Results: miR-182 and miR-96 share the same seed sequence (ACGGUU) which suggests that they can potentially target the same mRNAs thus regulating cellular functions. miR-182 and miR-96 screening in HCV-induced non-metastatic HCC liver tissues did not reveal any significant change when compared to liver tissues from healthy controls.

Conclusion: miR-182 & 96 are promising prognostic tissue markers for metastasis in HCC.


Expression of histone modifying enzymes and histone post-translational marks in colorectal cancer

A. Polónia *, M. Coimbra, S. Reis, A. Tavares, A. Raimundo, L. Santos, C. Jerónimo, R. Henrique

*IPO Porto, Dept. of Pathology, Portugal

Objective: Characterization of the expression of histone modifying enzymes (HMEs) and their respective post-translational histone marks (HMs) in a series of colon cancer (CC) patients and determination of their value as biomarkers of prognosis and predictive of therapeutic response.

Method: A series of 98 CC cases were randomly selected from the archives of Portuguese Oncology Institute-Porto between March 2003 and November 2006. The expression of HMEs (EZH2, SMYD3, SETDB1 and LSD1) and HMs (H3K9me3 and H3K27me3) was determined by immunohistochemistry and assessed using a modified Histo-score.

Results: SETDB1 was overexpressed in left-sided CCs. EZH2, H3K9me3 and H3K27me3 were overexpressed in more invasive CCs as well as in CCs with regional lymph node metastases. LSD1 was overexpressed in less invasive CCs. Concerning response to treatment, we observed: higher survival rates in Stage I/II patients disclosing lower SETDB1 expression; higher survival rates in patients treated with Folfiri disclosing high LSD1 and H3K9me3 expression; and higher survival rates of patients treated with 5-FU/Leucovorine disclosing high H3K9me3 and H3K27me3 expression.

Conclusion: This work accomplished the identification of new biomarkers that might be useful in clinical practice to predict response to different treatment modalities and points to new directions in the study of colon carcinogenesis.


New proteomic platform for utilization of formalin-fixed, paraffin-embedded samples in biomarkers search

P. Ostasiewicz *, J. R. Wisniewski, D. Zielinska, K. Dus, P. Ziólkowski, M. Mann

*Wroclaw University of Medicine, Pathology, WrocBaw, Poland

Objective: The aim of the study was to develop technology that would allow for effective exploration of tissue archives (paraffin blocks) with the modern proteomic tools in order to search for biomarkers.

Method: First, using newly developed protocol, we isolated proteins from fresh and from the formalin-fixed paraffin-embedded (FFPE) mouse liver and compared the results of the subsequent proteomic analysis. In second step we applied the protocol to the analysis of microdissected FFPE samples of human tissues. We compared the proteomes of colorectal carcinoma with patient-matched normal mucosa. All isolates were analyzed with tandem mass-spectrometry (MS/MS, Orbitrap-Velos) and raw data were evaluated with MaxQuant software.

Results: Protein identifications obtained for fresh and FFPE samples were the same with respect to both quality and quantity. It is also possible to analyze post-translational modifications in FFPE. The long-stored samples can be also analyzed. We quantified more than 6,000 proteins from carcinoma and paired healthy mucosa. Analysis of the data revealed that several proteins were expressed differently in normal versus neoplastic mucosa; many of them are not considered as potential biomarkers yet.

Conclusion: For the purpose of proteomic investigations FFPE material is equivalent to the fresh one in all aspects. Proteomics is a method of a great potential in biomarker search.


Sensitivity and standardisation of multiplex molecular assays

J. Frampton *, A. Mulligan, C. Thorne, J. Goodall, K. Schmitt

*Horizon Discovery, Diagnostics, Cambridge, United Kingdom

Objective: As multiplex molecular assays are rapidly being integrated into genotype testing, it is critical to understand their sensitivity to ensure an accurate interpretation of results. Although multiplex assays offer significant advantages for mutation detection, there remain challenges as each platform presents an inherent chemistry bias and specific preparation steps. A method for standardisation and verification is therefore required.

Method: Quantitative Multiplex Genomic DNA and formalin fixed paraffin embedded (FFPE) cell line samples were used in this study. The multiplex standard covered the following mutations: B-Raf (V600E), K-Ras (G12D, G13D), cKIT (D816V), EGFR (G719S, ΔE746-A750, T790M, L858R and L861Q), N-Ras (Q61K), PI3KCA (E545K), PI3KCA (H1047R), and the allelic frequencies were verified using digital PCR.

Results: Using the multiplex reference standards, the sensitivities of the Sequenom, Ion Torrent and Mi-Seq platforms were analysed. The platforms were able to detect the majority of the mutations at the expected allelic frequencies. However, there were clear discrepancies when using the multiplex assays and some mutations were inconsistently detected.

Conclusion: These data illustrate both the benefits and challenges of deploying multiplex assays for complex applications such as tumor profiling. The workflow for multiplex analysis is broad and there are a number of areas which need optimization.


STAT signaling is deregulated in malignant pleural mesothelioma: Are microRNAs the reason?

L. Arzt *, F. Quehenberger, I. Halbwedl, H. Popper

*Medizin. Universität Graz, Inst. für Pathologie, Austria

Objective: The STAT signaling pathway is totally deregulated in malignant pleural mesothelioma (MPM), an aggressive cancer due to former asbestos exposure. Protein expression is controlled by noncoding RNAs, e.g. microRNAs (miRNA) and we investigated the possible impact of miRNAs on the STAT signaling pathway in MPM.

Method: RNA was obtained from 52 formalin-fixed and paraffin-embedded tumor tissue samples. MiRNAs possibly regulating the STAT signaling pathway were identified via in silico target prediction tools. Quantitative real-time PCR was used to assess miRNA expression levels. Protein expression levels were detected by immunohistochemistry.

Results: STAT1 is upregulated whereas STAT3 is downregulated. The negative regulators SOCS1 and SOCS3 are totally missing. MiR-30d* regulates pSTAT1(Ser727) expression. MiR-19b, miR-30b, miR-30c and miR-222 are upregulated (target: SOCS1/SOCS3). MiR-21 (target: STAT3), a well-known oncomir, is extremely upregulated in MPM.

Conclusion: Absence or downregulation of proteins can be explained by upregulation of miRNAs. We currently investigate if SOCS1 and SOCS3 are expressed after downregulating the corresponding miRNAs. With these experiments we aim to answer if the STAT1/3 signaling cascade can be restored to a physiological level. Understanding the role of STATs in MPM could be the first step into the development of a targeted therapy for these tumors.


MicroRNA portraits in human vulvar carcinoma

B. Maia *, A. Lavorato Rocha, I. Rodrigues, C. Coutinho, G. Baiocchi, M. Stiepcich, R. Puga, L. Lima, F. Soares, R. Rocha

*AC Camargo Cancer Hospital, Dept. of Molecular Morphology, São Paulo, Brazil

Objective: Our study aimed to characterize microRNA in vulvar tumors through an expression profile of 754 miRNAs, relating this with clinical and anatomopathologycal data, and presence of HPV infection.

Method: Twenty (20) HPV negative and 20 HPV positive samples, genotyped for high-risk HPVs (HPV16,18,31,33) and a pool of seven nomal vulvar skin samples were used for the identification of differentially expressed miRNAs by TLDA qRT-PCR.

Results: Twenty-five (25) differentially expressed microRNAs between HPV positive and HPV negative groups were obtained. A network between microRNA expression profiles and target mRNAs previously demonstrated as relevant in vulvar carcinomas, such as TP53, RB, PTEN, and EGFR was constructed. Downregulation of both miR-223-5p and miR-19-b1-5p were correlated with the presence of lymph node metastasis; downregulation of miR-100-3p and miR-19-b1-5p were correlated with presence of vascular invasion; overexpression of miR-519b and miR-133a were associated with advanced FIGO staging.

Conclusion: Our study demonstrates that microRNAs may be clinically important in vulvar carcinomas and our findings may help for further studies on functional implications of miRNA dysregulation in this type of cancer.

Interactions network between differentially expressed microRNAs and their target mRNA, with relevance in vulvar carcinogenesis:

figure g

Tuesday, 3 September 2013, 17.00–19.00, Room 5A

OFP-13 Dermatopathology


Cutaneous Melanoma: A case series of a Portuguese hospital

R. Sampaio *, J. Palla Garcia, C. Peixoto, J. Ramon Vizcaíno, T. Almeida

*Oporto’s Hospital Centre, Dept. of Surgical Pathology, Porto, Portugal

Objective: The latest data provided by Portuguese Regional Cancer Registry in 2005 placed the incidence of melanoma in 6–8 cases per 100,000 inhabitants and an increasing incidence was observed comparing with 2001. The aim of this study was to assess, clinical factors and histological predictors of survival, in skin melanoma cases of Pathology Department of Oporto’s Hospital Center.

Method: A retrospective review between years 2004 to 2011 was conducted. All cases of skin melanoma (n = 106), were evaluated in their clinical factors (age, sex, local, metastasis, lactate dehydrogenase, survival rate) and their histological factors (histological type, mitotic rate, ulceration, Clark’s level, necrosis, TNM staging, tumour thickness, regression and infiltrating lymphocytes). The association between survival and clinical/pathologic factors were analyzed using Cox regression. Predictive factors of distant metastasis were evaluated by logistic regression.

Results: The presence of distant metastasis was the only independent factor associated with survival (p = 0,022). Histological type, tumour thickness, Clark’s level, necrosis, ulceration were independent predictive factors of distant metastasis at presentation.

Conclusion: The purposes of pathologic examination are to provide an accurate diagnosis of melanoma and an useful prognostic information in the clinical management of the patient. In the near future, pathologic reports can also be used to predict responses to therapy.


Correlation between diagnostic shave biopsies and therapeutic excisions done for Lentigo Maligna (LM) cases: A 4-year audit study

R. Ramakrishnan *, J. Lee, S. Fernandez, J. Weir

*Charing Cross Hospital, Dept. of Cellular Pathology, London, United Kingdom

Objective: Diagnostic shave biopsies are routinely performed at our centre for lentigo maligna bearing cosmesis in mind. The aim of the study was to evaluate the different categories of diagnostic biopsies for LM, look for sampling/diagnostic inadequacies related to procedure & correlate with post-excision histology.

Method: LM cases diagnosed at our hospital(s) over 4 years were retrieved. Information pertaining to nature of biopsy, primary diagnosis, diagnosis at excision and margin clearance (in mm) were noted.

Results: 70 clinical suspicious cases of LM who underwent biopsies were retrieved from database. 30 (43 %) were incisional ellipses, 8 (11.5 %) punch & 32 (45.5 %) shave biopsies. Histologically, 46 (65.5 %) were LM, 9 (13 %) LM with insitu melanoma (LMis), 6 (8.5 %) lentigo maligna melanoma (LMM) & 9 (13 %) solar/actinic lentigo/non-diagnostic of LM. Re-excision resulted in 3 cases being reclassified as radial growth phase melanoma in situ (4 %) and another 3 (4 %) as LMM. None of the 9 non-diagnostic cases had suspicious lesions on follow-up

Conclusion: Shave biopsies have an excellent diagnostic pick-up rate (almost 100 %) for LM or higher grade lesions with no false negativity recorded in our series. Subsequent re-excision biopsy showed a small proportion of high-grade lesions(6/70, 8 %) not seen in the original biopsy.


Inflammatory infiltrate in melanoma with regression as prognostic parameter

S. Zurac *, G. Negroiu, R. Andrei, S. Petrescu, T. Tebeica, M. Petre, M. Neagu, C. Constantin, V. Chitu, C. Salavastru, F. Staniceanu

*Colentina University Hospital, Pathology, Bucharest, Romania

Objective: Regression in melanoma has unknown biologic significance. Our previous studies demonstrated different immunophenotype in areas of regression (RM) and nonregressed areas (NRM) in melanoma, the latter being similar to melanoma without regression (absence of regression–AR), thus identifying regression as expression of intratumor heterogeneity. This study characterized inflammatory infiltrate in different areas of melanoma.

Method: We analyzed inflammatory infiltrate in 102 cases of melanoma (in AR, NRM and RM) by immunohistochemical stains for CD3, CD5, CD7, CD4, CD8, CD25, FOXP3, CD20, CD23, CD138, CD1a, Langerin.

Results: The majority of tumor infiltrating lymphocytes (TIL) were T-cells, the morphologic estimation of TIL overlapping to IHC identification of T-cells (P < 0.001); slight predominance of CD4+ T-cells (CD4:CD8 > 1:1) was present, without correlation with pT level, ulceration or regression. B-cells (CD20+) and plasma cells (CD138+) were more frequent in advanced tumors, without statistic correlation. Langerhans cells (LCs) were more frequent in superficial spreading melanomas than nodular ones (P < 0.001); LCs were constantly associated with thinner tumors (P = 0.007); nodular distribution of LCs in RM associated frequent LCs in NRM (P = 0.038).

Conclusion: LCs infiltration is a promising parameter to be evaluated in melanomas for both predicting progression and therapeutic possibilities. Acknowledgments: project partially supported by Postdoctoral Program POSDRU/89/1.5/S/60746


Fluorescence in Situ Hybridization (FISH) assay in melanoma prognostication

G. Dyduch *, A. Sinczak-Kuta, J. Szpor, M. Bialas, S. Demczuk, K. Okon

*Jagiellonian University, Dept. of Pathomorphology, Kraków, Poland

Objective: A 4-color fluorescence in situ hybridization assay (FISH) with probes targeting 6p25, centromere 6, 6q23 and 11q13 are being used to differentiate melanoma from melanocytic nevi. The significance of the assay in the discriminating melanomas with metastatic potential from those with an indolent course is a matter of controversy

Method: 50 cases of malignant melanoma with 5-year follow-up, including 13 with lymph node and 9 with distant metastases were analyzed using Melanoma Vysis FISH Probe Kit.

Results: Of 50 melanomas 40 were FISH positive according to the previously defined criteria (82 % sensitivity). Metastatic melanomas showed lower positivity in comparison with non-metastasic ones (77 % vs.82 %). In 10 FISH negative cases 4 were associated with distant and 1 with lymph node metastasis. There were no significant differences in patient survival between FISH-positive and FISH-negative cases. Among local prognostic factors ulceration was positively correlated with percentage of tumor cells with increased number of 6p25 copies.

Conclusion: Melanoma FISH assay seems to be of limited value in predicting the course of malignant melanoma, nevertheless further analysis of larger number of cases is needed to elucidate the issue.


Melanoma MAPK pathway proteins and associated tumour suppressors: p16 is an independent prognostic biomarker by tissue microarrays

J. Lade Keller *, R. Riber-Hansen, P. Guldberg, H. Schmidt, T. Steiniche

*Aarhus University Hospital, Institute of Pathology, Aarhus C, Denmark

Objective: To produce a combined evaluation of changes in the immunohistochemical (IHC) expression of key melanoma drivers in order to assess their impact on progression and prognosis of melanoma.

Method: Tissue microarrays were constructed from a cohort of primary melanomas (n = 355) and evaluated for IHC expression of MAPK pathway activators (c-KIT; BRAF V600E oncoprotein; pERK; MITF) and related tumour suppressors (p16; p53). The results were correlated with clinicopathological parameters and clinical outcome. Median follow-up time was 9 years.

Results: In univariate analysis, absent p16 expression and reduced MITF expression were both associated with ulceration (p = 0.009 and p < 0.0001, respectively), tumour stage III (p < 0.0001 and p = 0.001, respectively), and increasing Breslow thickness (both p < 0.0001), as well as with a poor melanoma-specific survival (p = 0.002 and p = 0.05, respectively), overall-relapse-free survival (p < 0.0001 and p = 0.003, respectively), and distant-metastasis-free survival (p < 0.0001 and p = 0.02, respectively). In addition, absence of p16 expression predicted overall-relapse-free (p = 0.02) and distant-metastasis-free (p = 0.04) survival, independently of Breslow thickness, ulceration and tumour stage. Expression of c-KIT, BRAF V600E oncoprotein, pERK, and p53 did not consistently predict adverse prognosis.

Conclusion: IHC determined p16 expression is an independent prognostic marker of potential value in daily melanoma practice.


An aggressive hypoxia related subpopulation of melanoma cells is TRP-2 negative

D. Lenggenhager *, A. Curioni-Fontecedro, M. Storz, O. Shakhova, D. Widmer, B. Seifert, H. Moch, R. Dummer, D. Mihic-Probst

*Kantonal Hospital St. Gallen, Inst. für Pathologie, Switzerland

Objective: TRP-2 is an enzyme involved in melanin biosynthesis and known as a melanoma differentiation antigen. In mice Trp-2 was shown to be expressed in melanocyte stem cells of the hair follicle; therefore it was also considered to be an indicator of stemness. The potential role in tumor progression suggested TRP-2 as a target for vaccination. Therefore, there is a need to further investigate on the role of TRP-2 in melanoma as a stem cell or differentiation marker.

Method: We analysed the expression of TRP-2 in over 200 melanoma biopsies and cell lines. In order to define if TRP-2 can be considered as an indicator of stemness, we analysed the mouse hair follicle for Trp-2 expression.

Results: We found an association between TRP-2 expression and differentiation. We identified a hypoxia related TRP-2 negative proliferative subpopulation which is associated with tumor thickness and disease progression, indicating a less favourable tumor specific survival. Also in mice we could show that Trp-2 is expressed in differentiated melanocytes.

Conclusion: Our findings underline that TRP-2 is a differentiation antigen whose expression decreases during tumor progression, therefore TRP-2 vaccination strategies would not target the hypoxia related proliferative, undifferentiated subpopulation of melanoma, indicating the need for combined approaches.


CD8+ T-cells in invasive and in situ squamous cell carcinoma of the skin and actinic keratosis

H. Kourea *, A. Stravodimou, V. Tzelepi, H. Papadaki, C. Scopa

*Uniersity of Patras, Dept. of Pathology, Greece

Objective: CD8+ Τ-cells participate in tumor surveillance and impede tumor growth, a function extensively studied in cutaneous melanoma. We examined the presence of C8+ T-cells in actinic keratosis (AK), in situ (IS) and invasive (IN) squamous cell carcinoma (SCC) of the skin.

Method: CD8+ Τ-cells were identified using immunohistochemistry and recorded using image analysis in 25 cases of INSCC and 18 cases of IS (and their adjacent IS, AK or benign tissue (BN), when present). Statistical analysis was performed using the paired T-test. p-values < 0.05 were considered statistically significant.

Results: In INSCC, peritumoral (PT) CD8+ Τ-cells were more numerous than the intratumoral (IT) CD8+ Τ-cells (n = 25) (mean 277 vs. 129, p < 0,001) and the adjacent ΒN (n = 21) (mean 277 vs. 118, p < 0,001). Significant differences in INSCC, were not observed between PT CD8+ T-cells and those in the adjacent IS and AK. In ISSCC, PT CD8+ Τ-cells were more numerous around IS compared to the adjacent ΒN (n = 15) (mean 238 vs. 54, p < 0,001) and in AK compared to BN (n = 11) (177 vs. 54, p = 0,031), respectively.

Conclusion: CD8+ Τ-cell infiltration of the skin increases early from the precancerous AK, indicating early involvement of the immunologic response in the prevention of development of SCC.


Early B cell differentiation in Merkel cell carcinomas indicates cellular ancestry

A. Zur Hausen *, D. Rennspiess, V. Winnepenninckx, E.-J. Speel, A. K. Kurz

*Maastricht University Medical Centre, Dept. of Pathology, The Netherlands

Objective: To assess the cellular origin of Merkel cell carcinomas.

Method: We tested 21 MCCs for the expression of MCPyV, TdT, PAX5, IgG, IgM, IgA, kappa and lambda by immunohistochemistry and assessed IgH and Igk rearrangement in all 21 MCCs.

Results: All of the MCCs revealed specific expression of PAX5 and 72.8 % of the MCCs expressed TdT. In addition, most of the MCC revealed specific expression of one or more Ig subclasses and kappa or lambda. One MCC did reveal monoclonal IgH and Igk rearrangement next to 2 other MCCs showing Igk rearrangement.

Conclusion: Since co-expression of TdT and PAX5 under physiological circumstances is restricted to pro/pre- and pre-B cells we propose–based on our results–that the cell of origin of MCC is a pro/pre- or pre-B cell rather than the postmitotic Merkel cells. MCPyV infection and transformation of pro-/pre- B cells is likely to induce the expression of simple cytokeratins as has been shown for SV40 in other non epithelial cells. This model of cellular ancestry of MCC might impact therapy and possibly helps to understand why approximately 20 % of MCC are MCPyV negative. zur Hausen A, et al., Cancer Res. 2013, in press.

Tuesday, 3 September 2013, 17.00–19.00, Room 5B

OFP-14 Oral Free Paper Session Nephropathology


Preimplantation analysis of kidney biopsies from Expanded Criteria Donors (ECD)

A. Sagasta Lacalle *, A. Sánchez Escuredo, F. Oppenheimer, M. Solé

*Hospital Clinic Barcelona, Dept. de Anatomía Patológica, Spain

Objective: Preimplantation analysis of kidney biopsies in ECD is commonly used, with little consensus on management standards. Aim: To analyze the concordance between different observers and techniques for the parameters used in routine according to Remmuzzi score (Rs).

Method: Retrospective analysis of 92 biopsies (2000–2010). Frozen sections (FS) were blindly revised by a trained observer and compared with original reports (interobserver correlation). Frozen and paraffin sections (PS) analysis by the same observer were also compared (correlation between techniques). For correlation analysis Kendall’s Tau b (KTb) was calculated.

Results: Agreement between observers using FS was weaker than correlation between techniques in all examined parameters (KTb Rs 0.104 vs 0.306). Glomerulosclerosis was the parameter with better correlation (KTb FS vs PS 0.358), and tubular atrophy was the one with the worst (KTb FS vs PS 0.1579). Combined tubulo-interstitial score did not improve correlation (KTb IF/TA 0.346/0.15 vs combined 0.157) According to Rs, PS and FS analysis would have result in higher rate of organ rejection (12 and 14, respectively) than the original report (6).

Conclusion: Correlation between FS and PS is weak, but better than interobserver agreement. Given the relevance of the observed differences in organ acceptance, specific training is advisable irrespective of the technique used.


Kidney allografts with biopsy features of chronic mixed rejection reflect poorer survival than those with pure chronic antibody-mediated rejection

D. Dobi *, Z. Bodo, K. Boda, E. Kemeny, E. Szederkenyi, B. Ivanyi

*University of Szeged, Dept. of Pathology, Hungary

Objective: The lesions of chronic antibody-mediated rejection (CABMR) and T-cell-mediated rejection (TCMR) can coincide. Since the clinicopathologic relevance of this pattern, termed chronic mixed rejection (CMR), has not been entirely elucidated, it was subjected to analysis.

Method: The lightmicroscopic, immunohistochemical (C4d, HLA-DR) and ultrastructural features of 61 consecutive biopsies displaying transplant glomerulopathy and/or peritubular basement membrane multilayering were re-evaluated, applying the Banff 09 categories. Arterial fibrointimal thickening with lymphocytes/macrophages received a distinct score (cv ly/ma). Hierarchical cluster analysis, Cox proportional hazards and Kaplan-Meier curves served to determine the significance of lesions relating to graft survival.

Results: 42.6 % of the cases exhibited CMR. The cluster analysis identified cv ly/ma and peritubular capillaritis as TCMR lesions; and glomerulitis as an antibody-mediated lesion. The median survival in CMR was significantly lower than that in pure CABMR cases (9 vs 26 months, respectively). On Cox regression, acute TCMR grades Ia/Ib, cv ly/ma, calcineurin inhibitor toxicity and tubular atrophy had significant negative effects on survival.

Conclusion: CMR was a common phenotype in our series; cv ly/ma represented chronic active TCMR, as indicated by Banff 09; CMR carried an inferior prognosis than pure CABMR, independently of the pattern of the T-cell-related component (supported by TAMOP 4.2.2.A-11/1/KONV-2012-0035 grant to B.I.).


Chronic transplant glomerulopathy: Clinical and morphological characteristics

A. Perkowska-Ptasinska *

*Warsaw Medical University, Transplantology and Nephrology, Poland

Objective: Chronic transplant glomerulopathy (TG) is for a long time recognized type of kidney graft injury, but apart from glomerular capillary lesions the morphology of kidney grafts with TG has not been yet described. The aim of the study was to characterize the morphology of kidney grafts with TG and to analyze the impact of studied lesions on the graft survival.

Method: 159 TG cases were retrospectively compared with non-TG group comprising 85 recipients with IFTA and/or chronic arteriolo- or arteriopathy.

Results: TG group was characterized by a higher incidence of some of glomerular lesions (e.g. mesangiolysis, glomerulitis), tubulitis, C4d positivity, PTC-itis and interstitial inflammation. Among vascular lesions endarteritis, proliferative arteriopathy (arteriosclerosis without elastica multiplication), arteriolosclerosis, and hypertrophy/hyperplasia of arteriolar SMCs were significantly more common in TG group. Although the mean time interval between transplantation and TG recognition was 6 years, an active AMR with C4d deposition in PTCs was found in 43 %, and an acute T-cell rejection in 20 % of TG cases. Among lesions studied proliferative arteriopathy and glomerular thrombi were found to have an independent negative impact on graft survival.

Conclusion: TG is associated with a spectrum of inflammatory and structural changes in the renal transplant microvasculature, interstitium and arterial tree.

Morphological characteristics of TG, and non-TG groups:

TG vs non-TG cases
light microscopic evaluation Banff criteria + additional parameters the incidence in TG group the incidence in non-TG group p
C4d in PTCs 42.86 % 1.3 % <0.0001
PTC-itis 40.88 % 11.76 % <0.0001
PTCs’ dilatation 35.85 % 10.59 % <0.0001
acute interstitial inflammation (“i”) 30.82 % 10.59 % <0.0001
total interstitial inflammation (“ti”) 81.76 % 68.24 % 0.02
the percantage of globally or segmentally sclerosed glomeruli 30.21 ± 21.71 19.59 ± 21.15 <0.0001
mesangiolysis 38.99 % 11.76 % <0.0001
increase in mesangial celullarity 38.99 % 8.24 % <0.0001
increase in mesangial matrix volume 48.43 % 27.06 % 0.002
glomerulitis 61.01 % 7.06 % <0.0001
tubulitis 31.45 % 10.59 % 0.0002
endarteritis 9.79 % 0 0.003
arteriosclerosis without the multiplication of elastic lamina 18.88 % 3.61 % 0.0009
arteriosclerosis with the multiplication of elastic lamina 28.67 % 22.89 % NS
arteriolar sclerotization 47.17 % 18.82 % <0.0001
arteriolar SMCs hyperplasia 39.62 % 11.76 % <0.0001
arteriolar wall hyalinization 86.79 % 77.65 % NS


Categorization of the diabetic nephropathy by Tervaert classification in clinical setting

F. Moreno *, A. Pinho, R. Dias, J. R. Vizcaino

*Centro Hospitalar do Porto, Dept. de Anatomia Patológica, Portugal

Objective: To assess the reliability and prognostic value of the Terveaert classification system in renal biopsies (RN), usually performed in type 2 diabetes mellitus patients with an atypical presentation of renal disease in clinical setting.

Method: Single-center study in a tertiary referral center for renal pathology. Three pathologists (1 senior reader: >10 year of experience; 1 intermediate reader: 3 year of experience; and 1 junior reader: first year of practice) evaluated retrospectively consecutive biopsies and categorized diabetic nephropathy (DN) blinded for inter-observer assessment and clinical outcome.

Results: Among 710 RN evaluated, there were 12 pureDN and 8 DN coexisting with other types of pathology (mixedDN). The mixedDN forms were associated to IgA nephropathy (3), transplant glomerulopathy (2), light chain disease (1), amyloidosis (1) or HIV-Associated Nephropathy (1). As it was previously reported, the inter-observer reproducibility for Terveaert classification was good (K = 0.82). The estimated 5-year renal survival rate was 98.4 % in ClassesII, 54.3 % in ClassIII, and 36.2 % in ClassIV (p = 0.04).

Conclusion: These findings corroborate the results from experimental centers: in fact, Tervaert classification seems to be user friendly, accurate and clinically useful in DN. Future studies are therefore recommended, in order to be consistently applied in clinical practice.


Clinicopathological characteristics of segmental and global active subclasses of class IV lupus nephritis

M. Wagrowska-Danilewicz *, M. Danilewicz

*Medical University of Lodz, Dept. of Nephropathology, Poland

Objective: The prognosis of lupus nephritis is predicted by the class, activity and chronicity of the glomerular and interstitial pathology. The purpose of the study is to compare the clinical and laboratory data and the severity of glomerular active features in segmental active subclass of class IV lupus nephritis (IV-S/A) and global active subclass (IV-G/A)of class IV lupus nephritis.

Method: A retrospective analysis of 34 patients with IV class of lupus nephritis was performed. Clinical and laboratory data were available in all patients selected.

Results: Of 34 patients with class IV lupus nephritis, 25 were classified as having class IV-G/A lesions, and 9 patients class IV-S/A lesions. Nephrotic syndrome and hypertension were significantly more frequent in patients with IV-G/A lesions, whereas hematuria and low grade proteinuria were significantly more frequent in patients with IV-S/A. Class IV-S/A showed predominant mesangial deposits, and less prominent immune deposits on immunofluorescence in comparison to class IV-G/A. Fibrinoid necrosis of glomerular tufts were significantly more evident in class IV-S/A. Endocapillary hypercellularity and hyaline deposits on light microscopy were more frequent in class IV-G/A.

Conclusion: Results of the study point to the clinical and morphologic differences between class IV-S/A and class IV-G/A lupus nephritis.


Role of renal biopsy in silent lupus nephritis

M. E. Guerra *, Y. Arce, M. M. Díaz, P. Moya, J. Ballarín, F. Algaba

*Hospital Central de Asturias, Dept. of Pathology, Oviedo, Asturias, Spain

Objective: To evaluate the frequency of silent lupus nephritis (SLN) in protocol renal biopsy (PRB) after 2 years of complete clinical remission

Method: Prospective and descriptive study of patients diagnosed of lupus nephritis with criteria of complete renal remission.

Results: 9 patients, 7 women and 2 men with a mean age 24.2 years. Results of first biopsies were: 1 patient was class II, 1 patient class III, 5 patients class IV and 2 patients class V with a mean actitivity and chronicity index of 5.5 and 0.87, respectively. In PRB 5 patients were class II, 1 patient class V and 3 patients a mixed pattern (II or III+V) with a mean activity and chronicity index of 0.62 and 1.12, respectively. Considering the result of PRB, mantainance treatment was decreased in 3 patients, in 5 patients remained and was increased in 1 patient.

Conclusion: After PRB no patients achieve complete histological remission: membranous pattern remains, endocapillary proliferation decreases and in all of them, mesangial proliferation holds. Activity index decreases, meanwhile chronicity index increases. SLN is highly prevalent in patients with systemic lupus erythematosus, being renal biopsy the gold standard for diagnosis. In our study, PRB implicates a change in therapeutic decision in 44.4 % of cases.

Table 1.:

1 V 0 0 V 0 3 no change
2 III 7 1 III+V 2 1 increase
3 IV 13 0 II+V 0 1 no change
4 II 1 2 II 0 2 decrease
5 II+V 0 0 II+V 1 1 no change
6 IV 9 4 II 2 0 no change
7 IV 5 0 II 0 0 no change
8 IV 9 0 II 0 1 decrease
9 IV II 0 0 decrease


Hypocomplementemic urticarial vasculitis syndrome in kidney biopsies

A. Vizjak *, J. Mraz, T. Avcin, A. Hocevar, J. Lindic, D. Ferluga

*Faculty of Medicine Ljubljana, Inst. of Pathology, Slovenia

Objective: Hypocomplementemic urticarial vasculitis syndrome (HUVS) is a rare, not yet fully explored immune-complex small vessel systemic vasculitis involving kidney in about 50 % of patients.

Method: Using light, immunofluorescence and electron microsopy, we studied histopathologic changes in 14 kidney and 8 skin biopsies of 8 HUVS patients.

Results: HUVS was diagnosed in 4 children, 2 were siblings (mean age 6.9 years) and 4 adults (mean age 50.3 years). In 3 patients, HUVS developed into SLE, manifesting with positive anti-DNA antibodies, after the first kidney biopsy. Light microscopy in the initial kidney biopsy showed mild mesangial glomerulonephritis (GN) (n = 3), focal proliferative GN (n = 3) combined with membranous GN in one case, and diffuse proliferative GN (n = 2) combined with membranous GN in one case. Granular full-house immune deposits in glomeruli, extraglomerular vessels and extensively in the tubulointerstitial compartment were seen in all cases by immunofluorescence, confirmed by electron microscopy. Fingerprint ultrastructure of electron dense deposits was noted in 5 cases. Skin biopsy revealed immune-complex leukocytoclastic vasculitis in all 8 cases.

Conclusion: Our study showed that light, immunofluorescence and electron microscopy histopathologic features of HUVS are very similar to SLE, in accordance with the not infrequent clinical and immunoserological overlapping of the two diseases.


Pharmacological nephroprotection in a novel mouse model of hereditary nephrotic syndrome

I. Simic *, M. Tabatabaeifar, H. Denc, T. Wlodkowski, G. Mollet, C. Antignac, F. Schaefer

*University Children’s Hospital, Pediatric Nephrology, Heidelberg, Germany

Objective: Inducible knock-in mice carrying the R140Q podocin mutation develop nephrotic-range proteinuria and focal-segmental glomerulosclerosis. The aim of our study was to test the nephroprotective efficacy of RAS antagonists in this mouse model.

Method: C57BL/6 mice with Nphs2Flox/R140Q, Cre+genotype were injected with Tamoxifen for 5 days to induce hemizygosity for R140Q-mutant podocin. The animals were treated prophylactically with ramipril (R), candesartan (C), the combination of ramipril and candesartan (R+C), amlodipine (A) or remained untreated with either tamoxifen induction (sick controls) or vehicle injections (healthy controls). Histopathological changes were examined after 4 weeks.

Results: Animals treated with RAS antagonists scored lower glomerular sclerosis indices (R, 1.00; C, 1.13; R+C, 0.91; A, 1.20; sick controls, 1.47; healthy controls, 0.25). The average number of podocytes per glomerulus was reduced by 50 % in sick animals, but was preserved in R+C animals (R, 70; C, 72; R+C, 79; A, 62; sick controls, 39; healthy controls, 75). Despite preserved podocin mRNA expression, Western blot analysis showed subtotal loss of podocin protein in all induced animals irrespective of pharmacological treatment.

Conclusion: The administration of RAS antagonists markedly attenuates podocyte loss and delays glomerulosclerosis in mice carrying the most common human podocin mutation. Our findings suggest that RAS blockade provides effective pharmacological nephroprotection in this hereditary podocytopathy.


Warfarin related nephropathy in human and experimental animals

S. Brodsky *, A. Satoskar, L. Hebert, T. Nadasdy

*Ohio State University, Dept. of Pathology, Columbus, USA

Objective: We had reported that excessive anticoagulation in patients on warfarin therapy (INR > 3.0) can result in acute kidney injury (AKI). Morphologic findings included glomerular hemorrhage and renal tubular obstruction by red blood cell (RBC) casts. The clinical outcome in these patients was unfavorable. We named this condition warfarin related nephropathy (WRN). The aim of this study is to reproduce WRN in an animal model.

Method: CKD was modeled by 5/6 nephrectomy (5/6NE) in rats. Control and 5/6NE were treated with different warfarin doses at different stages of CKD development (3, 9 and 19 weeks after the surgery). Serum creatinine and hematuria were measured; the kidney pathology was evaluated.

Results: Treatment with warfarin resulted in a dose-depended increase in prothrombin time and was associated with elevated Scr and hematuria in 5/6NE, but not control. SCr increase was correlated with CKD progression. Vitamin K prevented Scr and hematuria increase. Morphologically, RBC tubular casts were present, similar to those seen in humans (Figure 1).

Conclusion: WRN can be modeled in 5/6NE model of CKD. Excessive anticoagulation with warfarin results in increased SCr and hematuria, and RBC in tubules, resembling findings in humans. The pathogenesis of WRN can be studied in 5/6NE rats.

Figure 1:

figure h


A case of glomerular basement membrane lamellation associated with mutation in the MYO1E gene and not with Alport syndrome

S. Leh *, D. Brackman, I. Mujic, H. Boman, E. Svarstad, T. Fiskerstrand

*Haukeland University Hospital, Dept. of Pathology, Bergen, Norway

Objective: Glomerular basement membrane lamellation is the hallmark of Alport syndrome, but is occasionally also found in other hereditary renal diseases. This case was at first erroneously diagnosed as Alport syndrome due to pronounced basement membrane changes.

Method: A 10 year old boy developed hematuria and proteinuria. A kidney biopsy showed mesangial changes, segmental sclerosis in few glomeruli, interstitial foam cells and by electron microscopy thickening, lamellation and splitting of the glomerular basement membrane. A diagnosis of Alport syndrome was made. The boy had normal hearing, no eye abnormalities, and sequencing of the COL4A5 gene revealed no pathogenic sequence variants. There was no family history of kidney disease, but of parental consanguinity, which prompted us to investigate the possibility of an autosomal recessive disease. We performed a 250 K Affymetrix SNP-array on DNA, to look for areas of homozygosity.

Results: The largest region of homozygosity (20.8 Mb) on chromosome 15 contained one known podocyte gene MYO1E, encoding a membrane-associated non-muscle class I myosin. By RNA analysis, we detected homozygosity for a large deletion (c.1905_2049del145), encompassing exon 19 of MYO1E, and revised the diagnosis to MYO1E associated focal segmental glomerulosclerosis.

Conclusion: The differential diagnosis of glomerular basement membrane lamellation includes MYO1E associated focal segmental glomerulosclerosis.

Basement membrane thickening, lamellation and splitting:

figure i

Tuesday, 3 September 2013, 17.00–19.00, Room 5C

OFP-15 Oral Free Paper Session Cytopathology


Performance of ProEx C in detecting cervical squamous intraepithelial lesions among women with ASC cytology

R. Alaghehbandan *, S. Ratnam

*Memorial University of Newfoundland, Dept. of Anatomic Pathology, St. John’s, Canada

Objective: The clinical usefulness of the ProEx C test (BD) for the detection of cervical intraepithelial neoplasia (CIN) among women with atypical squamous cells (ASC) cytology was determined in a multicentre study carried out in Canada.

Method: The study population comprised of women representing five of the ten Canadian provinces referred to colposcopy for further assessment of cervical cancer risk and follow-up, and those routinely screened. Cervical specimens were collected in PreservCyt and cytology was performed using the ThinPrep method (Hologic). Histology confirmed CIN served as the disease endpoint to assess the test performance.

Results: The study analysis was based on a total of 354 women (mean age 30.6 years) with 75 (21.0 %) having CIN 2+. Overall, ProEx C was positive in 33.3 % (119/354). ProEx C detecting CIN was best found at CIN2+ with a concordance rate of 74.3 % (95 % CI: 73.1–75.5). The sensitivity of ProEx C in detecting CIN2+ lesions was 68.9 % (95 % CI: 67.7–70.2) while the specificity was 75.7 % (95 % CI: 74.6–76.9).

Conclusion: In conclusion, ProEx C was reasonably specific but less sensitive in detecting CIN2+ lesions. ProEx C may have the potential to serve as a useful adjunct test for the detection of CIN2+ lesions in cervical cancer screening.


Accuracy of diagnosing pathological changes of glandular epithelium in cervical smears of primary screening

Z. Pohar Marinsek *, V. Kloboves Prevodnik, S. Uhan Kastelic

*Institute of Oncology, Dept. of Cytopathology, Ljubljana, Slovenia

Objective: Determine accuracy of diagnosing pathological changes of glandular epithelium of the uterine cervix in primary screening.

Method: Study included 573 cervical smears diagnosed as atypical glandular cells (AGC), severly atypical glandular cells/adenocarcinoma in situ (SAGC/AIS) or invasive adenocarcinoma (IA) during primary screening between 2005 and 2009 at the Institute of Oncology, Ljubljana (IO). All smears were reviewed and final diagnoses obtained from histology or follow-up were compared to those from primary screening and to those from the review.

Results: Follow-up revealed 70 glandular neoplasias, 79 squamous neoplasias and 413 negative cases. 85 % of AGC and 33 % of SAGC/AIS from primary screening were negative on follow-up. 14 % of AGC, 36 % of SAGC/AIS and 8 % of IA from primary screening were squamous neoplasias. On review 61 % of true negative cases were corecty diagnosed and only 1.5 % of cases diagnosed as AGC or glandular neoplasia were in fact squamous neoplasia.

Conclusion: Between 2005 and 2009 there was a high procentage of cytological overdiagnosis concerning cervical glandular epithelium and poor distinction between glandular and squamous neoplasia in primary screening. Improved results on review are probably due to education of screeners and cytopathologists prior to organized cervical screening in Slovenia.


Digital holographic microscopy as screening tool for cervical cancer

H. Sevestre *, N. Benzerdjeb, P. Camparo, C. Garbar

*CHU, Pathology, Amiens, France

Objective: To identify optic criteria of normal and abnormal cervical cells upon examination with a digital holographic microscopy (DHM) and software (Holocyt Intelligence System (HIS).

Method: Analysis performed with the DHM, vial “c-Box”, software (OZONE®) provided by HIS. Residual material of LBC from three labs, poured into a “c-Box” subjected to DHM. At least 20 normal or abnormal looking cells reconstructed selected per vial. Criteria: maximum height nuclear (MHN), normalized nuclear to cytoplasm height (NHN), nuclear diameter (ND) and nucleo-cytoplasmic ratio (NCR). Values expressed as mean ± SD. Mann–Whitney and ROC curves (threshold p < 0.05).

Results: 32 specimens with normal Pap test, 1,333 cells analyzed with DHM. 21 specimens with abnormal Pap test, 494 cells analyzed. For the three observers or each observer whatever criterion a significant difference between normal and abnormal specimens observed (except for MHN in PCA p = 0.56. Areas under the ROC curves (95 % CI): 0.82[0.79–0.84] for NHN, 0.85[0.82–0.87] for MHN, 0.94[0.93–0.96] for ND, 0.97[0.96–0.98] for NCR.

Conclusion: The criteria analyzed with DHM had good area under ROC. Large scale validation study will provide clear cut automated delineation of normal and abnormal cells without adulteration of fixed cells.


Preliminary validation of a liquid based cytology technique (kabcyt) using conventional screening and imaging system

H. Sevestre *, T. Brun, B. Karkouche

*CHU, Pathology, Amiens, France

Objective: To describe the evaluation of a liquid based cytology technique (Kabcyt): confection of slides with Thinprep residual material, comparative screening by pathologists, conventional and assisted by the Thinprep Imaging System (TIS, Hologic).

Method: After Thinprep (TP) technique and screening, conventional and assisted by TIS, the residual material of 95 consecutive patients (excluding ASC-US and ASC-H) was used to perform the Kabcyt (KB) technique. KB slides were also double screened. The technique was performed in five rounds by a in-training medical student. The initial (TP) and second (KB) slides were classified separately by two pathologists according to Bethesda system.

Results: No difference of diagnostic categories observed in 92 pairs of cases (96.8 %). One pair: ASC-H and repair (TP) vs atrophic menopausal status (KB); on hysterectomy specimen: no cervical lesion. One pair: LSIL (TP) vs normal (KB). One pair: the reverse; after consensus evaluation, LSIL in these four slides. TIS evaluation was satisfactory in 100 % of TP and 74 % of KB slides.

Conclusion: The KB liquid based cytology method was succesful in this « real life» validation process. This manual technique was quickly learned by a medical student; it provided slides accepted by the TIS in most cases.


Morpholgy versus flow cytometry of cerebrospinal fluids

M. Lund-Iversen *

*Oslo University Hospital, Dept. of Pathology, Norway

Objective: To compare morphological findings with flowcytometric phenotyping (FCP) of cells in cerebrospinal fluid (CSF).

Method: In 2010 we investigated, as part of rutine diagnostics, 165 CSF samples from 131 patients aged 7–86 years with the following clinical queries: Lymphoma [93 (56,3 %)], metastatic carcinoma [2 (1,2 %)], unspecific neurological or radiological findings [64 (38,8 %)] and none [6 (36,4 %)]. Within 4 h after collection, 100 μL were prepared as DiffQuick stained cytospin, and the remaining volume were phenotyped using antibodypanels according to morphological findings.

Results: Eight samples were excluded. Microscopy revealed malignant cells in 8 samples, 2 carcinomas and 6 lymphomas. Ep4+ cells and pathological lymphoid phenotype confirmed morphological findings respectively. Five samples contained monoclonal B-cells consistent with low grade B-cell lymphomas, and 8 samples had light chain restriction of uncertain nature. Atypical cells were not found in these samples.

Conclusion: Microscopy of cells in CSF can confirm cerebromeningeal malignant affection, but FCP is superior to microscopic evaluation in detection of cerebromeningeal affection of low grade lymphomas. FCP is both a confirmatory supplement and an independent diagnostic tool, but one have to be aware of false positivity due to blood contamination, as well as the fact that clonal expansion not necessarily is consistent with hematological neoplasia.


Multiprobe FISH for the elucidation of equivocal pancreatobiliary cytology

T. Vlajnic *, G. Somaini, S. Savic, A. Barascud, L. Degen, E. Obermann, L. Bubendorf

*University Hospital Basel, Institute of Pathology, Switzerland

Objective: Endoscopic fine-needle aspiration is the standard method for the diagnosis of tumors of the pancreatobiliary tract. We explored the utility of FISH-improved diagnostic stratification between reactive and malignant cells in cases of equivocal cytological atypia.

Method: The multiprobe FISH assay UroVysion was used for copy number enumeration of chromosomes 3, 7, 17, and the 9p21 locus and applied to Papanicolaou-stained specimens with a diagnosis of equivocal atypia (n = 48), adenocarcinoma (n = 31), or no evidence of malignancy (n = 14). We captured images of the atypical cells and saved the coordinates on an automated stage prior to hybridization. A positive test was defined as increased copy number (>2) of at least 2 chromosomes or homo-/heterozygous loss of 9p21.

Results: FISH confirmed all 31 cytological diagnoses of pancreatobiliary adenocarcinomas and was negative in all 12 patients with no clinical evidence of malignancy. Among the 52 cases with equivocal atypia FISH detected 19/33 cases with a final diagnosis of adenocarcinoma and was negative in all 19 cases with no final evidence of malignancy (sensitivity 58 %, specificity 100 %, PPV 100 %, NPV 56 %).

Conclusion: Multiprobe FISH combined with automated relocation of atypical cells is a powerful technique to distinguish between reactive atypia and malignancy in pancreatobiliary cytology.


Robinson’s cytological grading and correlation with Bloom Richardson histological grading on touch imprint cytology from core needle breast biopsy

B. Babic *, A. Guzijan

*Clinical Center Banja Luka, Pathology, Bosnia and Herzegovina

Objective: Cytological grading of malignant breast tumors is a useful method for making decision about the therapy and prognosis.

Method: 37 patients with breast cancer are cytological graded based on 6 morphological parameters by Robinson method: uniformity and size of the cells, nuclei membrane and chromatin, cell discohesion and nucleolus. For all of them, correlation was made with histological grade by Bloom Richardson methods. Cytological samples are performed by touch imprint cytology from core needle biopsy.

Results: Most of malignant tumors are cytological grade 2, 19 cases and the least grade 1,7 cases. Grade 3 is found in 11 cases. Histological grade 2 is found in 28 cases and grade 3 in 3 cases. Histological grade 1 is found in 6 cases. In grade 1 correlation with histological grade is nearly perfect. Discrepancy was found in only 1 case. Disagreement between grade 2 and 3 is found in 13 cases. Concordance between this two methods was 62,7 %.

Conclusion: Nuclei grading on cytological samples correlates with histological grading. Cytological grade is an essential predictive factor for histological grade and in combination with other diagnostic methods can give us useful data about size, grade and type of the tumor.


Reliability of flow cytometric analysis on cytological samples in subclassification of small B cell lymphomas

U. Klopcic *, V. Kloboves Prevodnik, J. Lavrencak

*Institute of Pathology, Dept. of Cytopathology, Ljubljana, Slovenia

Objective: To establish if typical patterns of expression of antibodies CD10, CD5, CD23 and FMC7 enable correct classification of small B cell lymphomas (SBCL) by flow cytometry.

Method: We analysed flow cytometric measurments of 157 fine needle aspiration samples of primary SBCL: 19 marginal zone lymphomas (MZL), 55 follicular lymphomas (FL), 30 mantle cell lymphomas (MCL), and 53 chronic lymphocytic leukaemia/small lymphocytic lymphomas (CLL/SLL). Typical antigen patterns for differentiation of different types of SBCL were: FL (CD10+, CD5−, CD23−), MZL (CD10−, CD5−, CD23−), MCL (CD5+, CD10−, CD23−) and CLL/SLL (CD5+, CD23+, FMC7−).

Results: 88 % of SBCL in our series had typical immunophenotype (96 % of CLL/SLL, 63 % of MZL, 100 % of low grade FL, 76 % of high grade FL, and 93 % of MCL). The results are summarized in the Table 1.

Conclusion: Flow cytometry of FNAB samples enables precise classification in the great majority of SBCL. Correct classification is problematic in CD5+ MZLs, since the same immunophenotype can also be obtained in some CLL/SLLs and MCLs. Furthermore, the diagnosis of FL is difficult in CD10- cases, where immunophenotype overlaps with MZL. When morphology does not correlate with immunophenotype, and in doubtful cases, additional immunocytochemical markers (e.g. Cyclin D1, Bcl-2, Bcl-6) and/or molecular techniques for detecting cytogenetic abnormalities should be used to increase the accuracy rate.

Table 1:

  CD10+ CD10− CD5+ CD5− CD23+ CD23− FMC7+ FMC7−
MZL 0 19 6 13 2 17 8 11
FL1 12 0 0 12 2 10 6 6
FL2 22 0 0 22 6 16 16 6
FL3 17 4 0 21 4 17 15 6
MCL 0 30 28 2 0 30 25 5
CLL/SLL 0 53 53 0 52 1 1 52


Lymphoplasmacytic lymphoma: A difficult diagnosis in lymph node fine needle aspiration cytology

P. Luís *, A. Alves, D. Lopéz-Presa, C. Ferreira

*Hospital de Santa Maria, Dept. de Anatomia Patológica, Lisboa, Portugal

Objective: Fine needle aspiration cytology (FNAC) is a powerful tool in the diagnosis of many conditions in lymph nodes it can be quite challenging to make a definitive diagnosis.

Method: We present a case of a 55-year-old male, with history of tuberculosis in 2011, which had recent cervical lymph node enlargement, asthenia and sweating. A FNAC of a cervical lymph node was made, and material was collected for smears and culture. Because the smears raised suspicion, FNAC was repeated, and material was collected for cell block and flow cytometry.

Results: The smears revealed a polymorphous population, with predominant small to intermediate sized lymphoid cells, some of them with plasmacytoid morphology, plasma cells with Dutcher bodies and histiocytes; no morphological features of tuberculosis were found. Flow cytometry identified a 68 % lymphoid population with a phenotype compatible with mature B-cell neoplasm. In cell block the lymphoid population showed immunoreactivity for CD20 and bcl-2 and negativity for CD3, CD5, CD10, CD23, CD43 and cyclin D1; the plasmacytic cells were monoclonal with restriction to kappa light chain and expressed cytoplasmatic IgM. Final diagnosis was Lymphoplasmacytic lymphoma, posteriorly confirmed by lymph node biopsy.

Conclusion: Lymphoma characterization by FNAC is sometimes difficult, but keeping in mind all the technical possibilities with FNAC material, more precise diagnoses can be made.

Cell Block H.E. 400×:

figure j

Wednesday, 4 September 2013, 08.30–12.00, Room 5C

OFP-16 Oral Free Paper Session Joint Session—Pulmonary/Thymic and Mediastinal Pathology


Hyaluronidase, hyaluronan synthase, E-cadherin and TGF-ß profile in lung adenocarcinoma subtypes and squamous cell carcinoma of smokers/nonsmokers

V. de Sá *, L. Carvalho, A. Alarcão, P. Couceiro, V. Sousa, V. Capelozzi

*Faculdade de Madicina, Dept. of Pathology, São Paulo, Brazil

Objective: We examined Hyal1 and 3, HAS1,2 and 3, e-cadherin and TGF-β profile in lung adenocarcinoma (AD) subtypes and squamous cell carcinoma (SqCC) of smokers/nonsmokers.

Method: Fifty-six patients, mean age 64 years, who underwent lobectomy for AD (n = 31) and SqCC (n = 21) were included in the study. We used immunohistochemistry and morphometry to evaluate Hyal, HAS, e-cadherin and TGF-β expression.

Results: SqCC expressed Hyal3 predominant by stromal cells, whereas acinar predominant AD expressed strongly HAS3. In situ, papillary and solid patterns presented a weak expression of the immunomarkers. A positive association was found between smoking history and increased HAS1, HAS3 and Hyal1, while predominant acinar AD were negatively associated to HAS1, HAS3 and Hyal1. The following positive associations were found: TGF-β vs E-cadherin in stroma, TGF-β in tumor vs HAS2. Negative correlation occurred between TGF-β and HAS3 vs Hyal1, TGF-β vs HAS3 and Hyal1. E-cadherin was more frequent in the samples without lymphonode metastasis.

Conclusion: A profile of Hyal, HAS, E-cadherin and TGF-β has impact on AD subtypes and SqCC of smokers/nonsmokers, appearing as promise markers to guide treatment options.


Detection of IGF1R in non-small cell lung carcinoma

G. Vlacic *, I. Kern

*University Clinic of Golnik, Dept. of Pathology and Cytology, Slovenia

Objective: The purpose of this study was to determine the presence of insulin-like growth factor-1 receptor (IGF1R) in tumour cells of non-small cell lung cancers (NSCLC).

Method: We collected 225 cases of NSCLC and used the paraffin-embedded blocks to perform immunohistochemistry to determine the expression of IGF1R and dual silver in situ hybridization (SISH) to quantitatively detect IGF1R genomic gain. IHC was assessed as positive or negative, SISH was analyzed in 40 nuclei and was regarded as positive if the ratio between IGF1R gene copy numbers and CEP 15 was higher than 2.

Results: In 34 cases there was not enough material to perform the study. There were 102 adenocarcinomas, 67 squamous cell carcinomas (SCC) and 22 NSCLC NOS. SISH was performed on 38 cases of which 13 were adenocarcinomas, 18 squamous cell carcinomas and 7 NSCLC NOS. Immunohistochemically we detected positivity in 85,1 % of SCC, in 79,4 % of adenocarcinomas and in 63,6 % of NSCLC NOS which gave us the overall result of 79,1 %. Gene amplification was detected in only 10,5 % of cases which were all SCC.

Conclusion: The expression of IGF1R in tumour cells of NSCLC can be detected in the majority of cases while gene amplification is rare.


ALK rearrangement, EGFR, KRAS mutations and MET amplification are not exclusive in bronchial-pulmonary adenocarcinomas

L. Carvalho *, M. Silva, A. Alarcão, T. Ferreira, M. J. d’Aguiar, A. F. Ladeirinha, V. Sousa

*Faculty of Medicine Coimbra, Inst. of Anatomical Pathology, Portugal

Objective: ALK encodes a tyrosine kinase receptor and its rearrangement-ALK+was found in 2–7 % of lung carcinomas, resulting a constitutively active and oncogenic protein reported as exclusive of EGFR and KRAS mutations and associated with resistance to EGFR inhibitors but highly sensitive to treatment with ALK inhibitor crizotinib.

Method: Sections of 126 bronchial-pulmonary carcinomas surgical specimens of all histological types were screened for ALK positivity by FISH with break-apart dual color probe (Abbott) and immunohistochemistry(IHC)–monoclonal antibody clone 5A4 (Leica); EGFR and KRAS mutations were determined by DNA direct sequencing and EGFR and MET genes amplification, by FISH (Abbott).

Results: IHC was applied in FISH ALK+cases and of the 126 screened tumors, 9 adenocarcinomas (7 %) were FISH ALK +, 3+ and 2+ score in IHQ. Among these 9 FISH-ALK+cases, 3 had EGFR mutations and 2 had both EGFR and MET gene amplifications in FISH; KRAS was wild type.

Conclusion: IHQ correlates with FISH for ALK gene rearrangement, not excluding EGFR and MET alterations. ALK status has to be tested in advanced bronchial-pulmonary adenocarcinoma, as it could be responsible for TKI-resistence of EGFR mutated tumors that benefit from ALK-targeted agents. EGFR mutations search revealed again to be necessary in this context of targeted therapy.


p53 mutated protein screened by p53 Ab-1 (Clone PAb 240) may be reliable to mutations search and targeted therapy in bronchial-pulmonary carcinomas

L. Carvalho *, A. Alarcão, P. Mota, J. R. Costa, J. Fonseca, A. Ladeirinha, T. Ferreira, M. J. d’Aguiar

*Faculty of Medicine Coimbra, Inst. of Anatomical Pathology, Portugal

Objective: DO-7 expression in neoplasias relates with p53 overexpression/non-functional protein synthesis; also p53 Ab-1-clone PAb 240-identifies mutant p53 protein in Pathology routine.

Method: DO-7 and PAb 240 were applied to 48 Bronchchial-Pulmonary Carcinoma sections: Adenocarcinomas(ADC)-31biopsies/17surgical specimens(SS); 53 Epidermoid Carcinomas(EC) -44 biopsies/9SS; 21 Small Cell Lung Carcinomas(SCLC)-biopsies and 26 cases of Polymorphic Carcinomas(PC)-Large Cell, Basaloid and Pleomorphic Carcinomas, Adenosquamous Carcinomas and Carcinomas CK7+/CK5.6+ 21 biopsies/5SS.

Results: Strict linear behaviour for PAb 240-positivity/DO-7positivity and negativity/positive–negative DO-7, grosso modo in the common score of 0, 1(<10 %), 2(10–50 %) and 3(>50 %) positive nuclei–DO-7 3/mutant p53 from 1 to 3: ADC: 21/48 positive, EC 28/53, SCLC 8/21 and PC 14/26 cases. DO-7 negativity/positivity together with mutant p53 protein negative expression results were similar: 27/48, 25/53, 13/21 and 12/26 respectively.

Conclusion: It is the first time to the best of our knowledge: PAb 240 was applied to BPC with DO-7 positivity and clear negativity independent of DO-7positive/negative expression and BPC typing, ready to be used isolated in routine to support trials of P53 mutations and targeted therapy.


Membranous Filamin A protein expression is associated with poor overall survival in patients with non-small-cell lung cancer

M. Gachechiladze *, V. Kolek, J. Klein, L. Radova, Z. Kolek, J. Skarda

*Palacky University, Clin. and Molec. Pathology, Olomouc, Czech Republic

Objective: An actin-binding protein Filamin A (FLNA) connects the actin filament network to cell membrane receptors, and acts as a scaffold for various signaling pathways related to cancer growth and progression. Different cellular localization of FLNA protein, detected by immunohistochemistry, has been shown to have a different clinical significance. We aimed to investigate the correlation between differential expression of FLNA and clinicopathological factors in patients with non-small-cell lung cancer (NSCLC).

Method: We performed FLNA protein immunohistochemistry on FFPE tissue samples from 82 NSCLC patients, using EP2405Y antibody against C-terminus of FLNA (LSBio). Cytoplasmic, membranous and nuclear staining were evaluated semi-quantitatively and correlated with all available clinicopathological factors.

Results: Cox regression analysis of survival showed that overexpression of membranous FLNA negatively correlates with overall survival (HR = 0,9,972; 95 % CI (0,9945; 0,9998); P < 0,05), as well as disease free survival (HR = 0,9976; 95 % CI (0,9951; 1); P < 0,06) in all stages of NSCLC. No significant correlations were found between FLNA cytoplasmic and/or nuclear expression and other clinicopathological factors.

Conclusion: According to our study results FLNA membranous positivity might serve as useful prognostic marker in patients with NSCLC, although more extended study is necessary.


Simultaneous KRAS and EGFR mutations: A single laboratory’s experience in 1,384 lung adenocarcinoma samples

K. Éles *, E. Tóth, J. Szoke

*National Institute of Oncology, Surgical & Molecular Pathology, Budapest, Hungary

Objective: KRAS mutation occurs in approximately 20–35 % and EGFR mutation occurs in 5–10 % of NSCLC. Previous reports have shown that EGFR and KRAS mutation are mutually exclusive. Our aim was investigate the frequency of simultaneous mutation of these two genes in lung adenocarcinoma.

Method: KRAS exon 2 and EGFR exon 19,21 mutations were tested on 1,384 lung adenocarcinoma samples between 2008 and 2012. The mutation analysis was performed by real- time PCR followed by Sanger sequencing of the EGFR exon 19,21 and KRAS exon 2 genes.

Results: KRAS mutations were detected in 36,4 % EGFR mutations were detected in 9 % of adenocarcinomas. Double mutations were found in 10 cases of 1384 patient (0,7 %). 7 patients had EGFR 19 exon deletion and 3 had EGFR exon 21 point mutation.

Conclusion: Our results show that KRAS mutation may coexist with EGFR mutation. There are few reports in the literature about concomitant EGFR and KRAS mutation in lung adenocarcinoma. It is important to use sensitive detection methods, as data shows that minor KRAS mutant tumour cell population may influence negatively the therapeutic effect of EGFR inhibitors even in the presence of tyrosine-kinase inhibitor sensitive mutations. Concomitant EGFR and KRAS mutation are most propably related to the multiclonal character of the tumour.


Prognostic significance of p63, TTF-1 and maspin in non-small cell lung carcinomas

B. Yaman *, D. Nart, G. Çok, A. Veral

*Ege University Medical Faculty, Pathology, Izmir, Turkey

Objective: Antiapoptotic genes and protease inhibitors take an important role in lung cancers.

Method: 80 patients with non-small cell lung cancer were selected. Tumour characteristics, immunoreactivity with p63, TTF-1, maspin, and follow up datas were evaluated.

Results: Patients were predominantly male (n = 71, 88.8 %) and mean age was 59. Fourtyfive (56.3 %) tumours were adenocarcinoma (AC), 23 (28.8 %) squamous cell carcinoma (SCC), four (5 %) large cell carcinoma (LCC), six (7.5 %) large cell neuroendocrine carcinoma (LCNC), and two the others. The mean diameter of tumours was 4.06 cm (1.5–11). Patients with advanced TNM stage and ≥3 cm tumour had poor survival (p < 0.005). Immunohistochemically 87.5 % of SCCs, 4.3 % of ACs, 25 % of LCCs, and 16.7 % of LCNCs stained with p63 (p < 0.0001). The positivity with maspin in SCC was 66.7 %, and in AC 17.4 % (p < 0.0001). Immunoreactivity of TTF-1 in ACs was 84.8 %, whereas no staining in SCCs (p < 0.0001). The negativity of maspin in ACs has a positive prognostic effect (p = 0.048).

Conclusion: The results of this study indicate that p63 and TTF-1 are useful markers in differential diagnosis of SCC and AC, but they have no significance in survival. And maspin can be used as a prognostic marker in ACs.


Has subtyping of mucinous adenocarcinomas of the lung any significance?

H. Popper *, U. Gruber-Moesenbacher, C. Manzl, A. Geles

*Medizin. Universität Graz, Inst. für Pathologie, Mol. Lung und Pleura Pathologie, Austria

Objective: Adenocarcinomas (AC) of the lung were reclassified in 2011, but mucinous ACs were all lumped together into one category. Mucinous AC can also be subtyped for predominant morphological pattern. This might have implications for molecular markers and subsequently for targeted therapy.

Method: We retrieved 76 mucinous pulmonary ACs from our biobank. KRAS mutation analysis was done in all cases. A TMA was created for molecular analysis, especially KRAS signaling pathways RAS-MAPK-ERK, RAS-RAL, and RAS-PI3K-AKT-mTOR. The type of mucin produced by the tumors was also analyzed and categorized. Clinical data were available for all patients.

Results: The majority of mucinous ACs presented as either predominant acinar or papillary variants, micropapillary was often combined with papillary, cribriform was associated with acinar pattern. Other variants were solid, signet ring cell, and colloid components, whereas pure forms of colloid, signet ring cell and micropapillary ACs were rare. Half of the cases showed mutation of the KRAS gene in codons 12, 13, and 61, double mutations did occur. KRAS mutation did not correlate with any of the subtypes. However, progression free survival correlated with the subtypes.

Conclusion: Subtyping of mucinous AC is encouraged, and might open new insights into the molecular signaling pathways of this group of AC.


KRAS, EGFR, PDGFR-alpha, KIT and COX 2 status in Carcinoma Showing Thymus-like Elements (CASTLE)

L. Veits *, P. Hufnagel, R. Penzel, P. Ströbel, K. Kaserer, S. Schröder, N. Neuhold, K. W. Schmid, P. Schirmacher, A. Hartmann, R. J. Rieker

*Klinikum Bayreuth, Abt. Pathologie, Germany

Objective: CASTLE is a rare malignant neoplasm of the thyroid resembling lymphoepithelioma-like and squamous cell carcinoma of the thymus with different biological behaviour and a prognosis superior to anaplastic carcinoma of the thyroid. We retrospectively investigated 6 cases of this very rare neoplasm in order to investigate the mutational status of KRAS, EGFR, PDGFR-α and KIT, as well as the immunohistochemical expression pattern of CD117, EGFR and COX 2.

Method: The material was investigated using immunohistochemical staining as well as mutational analysis of EGFR, PDGFR, KIT and KRAS. Diagnosis was confirmed by a moderate to strong expression of CD5, CD117 and CK5/6, whereas Thyreoglobulin, Calcitonin and TTF-1 were negative in all cases.

Results: COX 2 expression was moderate to strong in almost all cases. In four cases single nucleotide polymorphisms (SNPs) could be detected in exon 12 of the PDGFR-α gene (rs1873778), in three cases SNPs were found in exon 20 of the EGFR gene (rs1050171). No mutations were found in the KIT and KRAS gene.

Conclusion: Nearly all tumors showed a moderate to strong COX 2 expression and a wild-type KRAS status. No activating mutations in the EGFR, KIT and PDGFR-α gene could be detected. Our data may indicate a potential for targeted therapies, but if these therapeutic strategies are of benefit in CASTLE remains elusive.


Thymoma with molecularly verified “conversion” into T lymphoblastic leukemia/lymphoma over 9 years

V. D. Ertel *, M. Früh, A. Guenther, T. Cerny, C. Fretz, S. Cogliatti

*Cantonal Hospital, Pathology, St. Gallen, Switzerland

Objective: Thymoma is an epithelial tumor accompanied by non-neoplastic lymphocytes.

Method: Histological and molecular analysis of thymoma in a 62-year old male patient during a 9 years course.

Results: Initially a combinded B1/B2 thymoma was diagnosed in Masaoka stage IVa based on morphologic and immunophenotypic features. Over the years, suffering from recurrent disease, the patient developed combined B2/B3 thymoma. With sudden onset after 9 years, the disease switched dramatically to aggressive dynamics, when rebiopsies revealed at that time a T lymphoblastic leukemia/lymphoma. After dismal terminal course, the patient died within a few months by physical exhaustion. For verification of our hypothesis of secondary development of a precursor T lymphoblastic neoplasm following thymoma, we performed PCR analyses that revealed a clonal rearrangement of the T-cell receptor-gamma_genes in the V-gamma_10–12 and a biclonal peak in the V-gamma_1–8 primer setting. When we retrospectively analysed thymoma specimens, we found an identical biclonal peak in V-gamma 1–8 in combined B2/B3 thymoma and a polyclonal pattern in all previous tissue samples.

Conclusion: Our results support the assumption of a transforming process of polyclonality to oligoclonality in non-neoplastic cortical thymocytes in combined B1/B2 thymoma and B2/B3 thymoma, respectively and finally to monoclonality in secondary T lymphoblastic leukemia/lymphoma.

Wednesday, 4 September 2013, 14.00–16.00, Auditorium VI

OFP-17 Oral Free Paper Session Haematopathology


The diagnostic value of miR17-92 microRNA cluster in diffuse large B-cell lymphoma

A. Fassina *, R. Cappellesso, F. Marino, M. Siri, F. Simonato, M. Benetti, M. Crescenzi, M. Trento, L. Ventura, M. Fassan

*University of Padua, Dept. of Medicine, Padova, Italy

Objective: Diffuse large B cell lymphoma (DLBCL) can present as de novo or can arise through the transformation of indolent lymphomas, including follicular lymphoma (FL). The morphological differentiation between germinal center-DLBCL (GC-DLBCL) and high grade (grade 3) FL could be challenging: new diagnostic tools are needed to assure the clinical-pathological follow-up of these patients. Recent expression profiling studies reported microRNAs (and miR17-92 cluster, in particular) as useful tools in differentiating DLBCL and FL.

Method: We investigated the expression profile of six members of the miR17-92 cluster (i.e., miR18b, miR19b, miR20a, miR92, miR93, and miR106a) by quantitative reverse transcription-polymerase chain reaction in 36 cases of GC-DLBCL and 18 cases of high-grade non-transforming FL, confirmed on clinical, histological, and immunohistochemical data.

Results: All the considered miR17-92 cluster miRNAs were significantly over-expressed in GC-DLBCL. The ROC estimated thresholds miR17-92 cluster miRNAs displayed a sensitivity level higher than 0.80 in achieving the GC-DLBCL diagnosis. The classification tree built on the six thresholds allowed the correct identification of 35/36 GC-DLBCL (97.2 %).

Conclusion: The miR17-92 cluster can represent a reliable, standardizable diagnostic tool for the sub-classification of large B cell lymphoid neoplasm for differentiating GC-DLBCL from high grade FL.


The alteration of lipid metabolism in Burkitt lymphoma identifies a novel marker: Adipophilin

G. De Falco *, M. R. Ambrosio, P. P. Piccaluga, C. Doglioni, B. J. Rocca, M. Onorati, V. Malagnino, K. Naresh, S. Pileri, L. Leoncini, S. Lazzi

*University of Siena, Dept. of Medical Biotechnology, Italy

Objective: Recent evidence suggests that lipid pathway is altered in many human tumours. In Burkitt lymphoma this is reflected by the presence of lipid droplets which are visible in the cytoplasm of neoplastic cells in cytological preparations. These vacuoles are not identifiable in biopsy section as lipids are lost during tissue processing.

Method: We investigated the expression of genes involved in lipid metabolism, at both RNA and protein level in BL and in other B-cell aggressive lymphoma cases.

Results: Gene expression profile indicated a significant over-expression of the adipophilin gene and marked up-regulation of other genes (FASN, SCD5, USF1) involved in lipid metabolism in BL. These findings were confirmed by immunohistochemistry on a series of additional histological samples: 45 out of 47 BL cases showed strong adipophilin expression, while only 3 out of 33 not-BL category showed weak adipophilin expression.

Conclusion: Our preliminary results suggest that lipid metabolism is altered in BL, and this leads to the accumulation of lipid vacuoles. These vacuoles may be specifically recognized by a monoclonal antibody against adipophilin, which may be a useful marker for BL because of its peculiar expression pattern. Moreover this peptide might represent an interesting candidate for interventional strategies.


Lymphoplasmacytic myeloma: A rare variant of plasma cell myeloma. A study of 14 cases

M. Fameli*, A. Tasidou, E. Parasi, S. Giannouli, N. Stavrogianni, E. Stefanoudaki, G. Kokkini, E. Terpos, M. A. Dimopoulos, T. Papadaki, L. Marinos

*Evaggelismos, Hematopathology, Athens, Greece

Objective: Recently, two subgroups of cyclin D1+ plasmacytic myeloma (MM) are recognized: conventional (C-MM) and lymphoplasmacytic (LPL-MM) MM. LPL-MM is characterized morphologically by small plasma cells mimicking lymphoplasmocytoid lymphocytes and co-expression of both plasma cell (CD138) and B-cell (CD20, PAX-5) markers.

Method: 14 Bone Marrow Trephines (BMT) from 14 patients with cyclin D1+ (first diagnosis), with morphological and immunohistochemical (IM) features of LPL-MM. BMT study: morphology (Ε), IM: broad antibody panel.

Results: Among 686 new MM cases we identified 182/686 cyclin D1+ (~26,5 %). LPL-MM subgroup comprised ~2,04 % (14/686) of total MM cases and ~7.6 %(14/182) of cyclin D1+ cases (Table 1).

Conclusion: LPL-MM represents a rare MM subtype (2,04 % of new MM cases) and is characterized by Almost exclusive male predominance (13/14), “bipolar” (<30 % & >80 %) percentage of infiltration Exlusively γ-heavy chain and predominantly λ-light chain restriction. Difficulties in the differential diagnosis from lymphoplasmacytic lymphoma (2/14 cases had been previously diagnosed as such).

Table 1. Results:

M/F age % infiltration CD56+ CD20+ PAX-5+ Light chain only Total κ/λ Heavy chain
13/1 71.25 <30 % 5/14 2/14 100 % 14/14 6/14 5/9 γ = 8/14
  57–83 >80 % 8/14   9/14   κ = 1, λ = 5   α = 0
   fibrosis 1/14   60–90 %     


Primary mediastinal large B-cell lymphoma: Immunohistochemical features and differential diagnostics

A. Artemyeva *, G. Frank

*Gerzen Institute, Dept. of Pathology, Moscow, Russia

Objective: Primary mediastinal large B-cell lymphoma (PMBL) may share common morphological and phenotypical features with mediastinal grey zone lymphoma (MGZL), Hodgkin lymphoma (HL) and diffuse large B-cell lymphoma (DLBL) with mediastinal involvement. Their differential diagnosis might represent a challenge requiring a specific immunohistochemical (IHC) analyses.

Method: Formalin-fixed paraffin-embedded tissue by 58 cases PMBL, 10 cases MGZL, 17–HL and 13–DLBL were examined for H&E staining and LCA, CD20, CD3, CD15, CD30, CD10, CD23, EMA, ALK, PAX-5, BOB.1, OCT-2, mum 1, p65, MAL, Ki67 IHC expression. The final diagnosis was established according to the WHO classification criteria.

Results: All cases were diagnosed as PMBL showing strong positive reaction with PAX-5, BOB.1 and OCT-2, 26 (45 %)–p65 nuclear expression, 40 (69 %)–exspressed CD23, 2 (3.4 %)–CD10, 39 (67 %) were MAL-positive. In cases of MGZL 8/10 showing CD23, PAX-5, BOB.1, OCT-2 and MAL expression, p65–only cytoplasmic staining. In 1 HL case was observed staining with MAL.

Conclusion: Only complex usage of such IHC markers like transcriptional factors (PAX-5, BOB.1, OCT-2), membrane associated protein MAL and p65 enables to give the diagnosis of PMBL. Estimation of p65 true nuclear expression complicated by background cytoplasmic staining, that restricted this marker usage.


Combined inhibition of BCR/ABL- and CD27-signaling eradicates chronic myelogenous leukemia stem cells

C. Schürch *, C. Riether, A. Ochsenbein

*Medizin. Universität Bern, Inst. für Pathologie, Switzerland

Objective: Chronic myelogenous leukemia (CML) originates from leukemia stem cells (LSCs) harboring the oncogenic BCR/ABL tyrosine kinase. Tyrosine kinase inhibitors (TKIs) have revolutionized CML therapy; however, definite cure remains unachievable. LSCs are resistant to TKIs and there is a significant risk of disease progression to acute leukemia. Consequently, future therapies must aim at the elimination of LSCs. We recently documented in a murine CML model that LSCs express the costimulatory molecule CD27. Blocking CD27 on LSCs inhibited LSC proliferation and attenuated disease progression. Here, we investigated a combination treatment using TKIs and CD27-signaling blocking monoclonal antibody.

Method: Co-treatment was applied in vitro to BCR/ABL-expressing human leukemia cells and in vivo to mice harboring a CML-like disease induced by transplantation of BCR/ABL-transduced bone marrow.

Results: In vitro, co-treatment acted synergistically and significantly reduced leukemia cell growth compared to single treatments. In vivo, co-treatment significantly improved survival of CML mice and induced long-term cure in >50 % of animals, whereas mock- or single-treated animals all succumbed to CML. Analysis of bone marrow from CML mice and secondary transplantation experiments revealed that co-treatment specifically eradicated LSCs and leukemia progenitor cells.

Conclusion: Combining TKI treatment and blocking CD27-signaling may represent an attractive strategy to directly target LSCs in CML.


Cytotoxic CD8+ T cells regulate myelopoiesis by cytokine signals to bone marrow stromal cells

C. Schürch *, C. Riether, A. Ochsenbein

*Medizin. Universität Bern, Inst. für Pathologie, Switzerland

Objective: Cytotoxic CD8+ T cells (CTLs) are crucial for protection against primary infection with intracellular pathogens. However, complete pathogen clearance and return to homeostasis requires a regulated interplay between innate and acquired immunity. CTL-secreted interferon-gamma (IFN-g) directly regulates innate immune cells. In addition, IFN-g has been shown to modulate hematopoiesis, but the exact mechanisms remain unclear.

Method: To assess the effect of CTL-secreted IFN-g on myelopoiesis, we either infected wild-type mice with lymphocytic choriomeningitis virus (LCMV) or adoptively transferred TCR-transgenic CTLs specific for LCMV-gp33 into mice ubiquitously expressing LCMV-gp33 on MHC class I. Hematopoietic stem (HSCs) and progenitor cells were analyzed at different time points.

Results: LCMV-infection or adoptive CTL transfer did not affect early HSCs but induced the proliferation of multipotent progenitors (MPPs), resulting in increased production of mature myeloid cells. IFN-g did not affect MPPs directly, but stimulated mesenchymal stromal cells of the bone marrow HSC niche to produce hematopoietic cytokines including interleukin-6.

Conclusion: Bone marrow stromal cells (BMSCs) have an important documented role in the regulation of HSC maintenance and quiescence. Our study now demonstrates that BMSCs are central in the regulation of myelopoiesis in response to infection and that CTLs exert an indirect positive feedback on myeloid progenitors during viral clearance.


Retrospective description of previous biopsies in patients diagnosed of angioimmunoblastic T-cell lymphoma

O. Balagué *, D. de Jong

*NKI/AL, Pathology, Amsterdam, The Netherlands

Objective: Investigate the histopathological features of previous biopsies in patients diagnosed of angioimmunoblastic T-cell lymphoma(AITL)

Method: We have retrieved the 45 cases of AITL diagnosed between 2007 and 2012 in our region and selected those patients with previous biopsies to the diagnose of AITL. We revised the previous diagnoses and performed complementary analysis to investigate whether the lymphoma had been missed and why.

Results: 10 patients (22 %) had previous biopsies. The time between the first biopsy and the diagnose ranges from 3 to 36 months. AITL was suggested in 3 cases, but not concluding because of the presence of germinal centers,absence of dendritic expansion, lack of typical clinical features or clonality could not be demonstrated. Follicular hyperplasia was the diagnose in 4 cases (1 case with features of progressively transformed germinal centers). 3 patients were diagnosed of lymphoma (1 ATCL and 2 DLBCL). 9 of 10 cases were revised at the time of diagnose, 6 of them were then interpreted as AITL.

Conclusion: The diagnose of AITL is relatively often delayed. The absence of the typical clinial features, the presence of germinal centers and blastic B-cell populations seem to be reasons that difficult us an early diagnose.


The therapeutic potential of targeted p53 activation by the MDM2-inhibitor nutlin-3a in anaplastic large cell lymphoma in relation to the ALK-expression status

E. Drakos *, V. Sinatkas, K. Psatha, A. Eliopoulos, H. Papadaki, G. Z. Rassidakis

*University of Crete, Medical School, Dept. of Pathology, Heraklion, Greece

Objective: p53 is rarely mutated in anaplastic large cell lymphoma (ALCL). We have shown that nutlin-3a, an MDM2-inhibitor, can induce cell cycle arrest and apoptosis in ALK+, ALCL cells harboring wt-p53. However, the therapeutic potential of nutlin-3a in ALK- ALCL, in comparison to ALK+ ALCL, has not been thoroughly studied.

Method: ALK+ ALCL (SUP-M2), and ALK-, ALCL (Mac2A and Mac1) cells harboring wt-p53, were treated with nutlin-3a. p53 activation and the biologic effects were investigated by western blot analysis, colony formation and MTT assays, trypan blue, annexin-V and PI staining. Also, combined treatments including mTOR, or BCL2-inhibitors were used.

Results: Nutlin-3a treatment induced activation of p53,and inhibition of growth and cell-cycle progession of both ALK+ and ALK−, ALCL cells. Nutlin-3a-induced apoptotic cell death of SUPM2 and Mac1 cells was accompanied by Puma upregulation. However, Mac2A cells, characterized by high levels of BCL2, were resistant to apoptosis. Combined treatment of Mac2A cells with nutlin-3a and the BCL2-inhibitor, YC-137, or rapamycin,or torin-2 resulted in dramatically decreased cell viability.

Conclusion: A subset of ALK- ALCL tumors, characterized by increased BCL2 expression levels, may be resistant to nutlin-induced apoptosis. Combined activation of p53 and BCL2, or mTOR inhibition may result in enhanced therapeutic potential in ALCL.


HIV-related plasmablastic lymphoma of the head and neck: Proposal for a diagnostic algorithm

S. Boy *, P. Willem

*University of Pretoria, Oral Pathology and Oral Biology, South Africa

Objective: The literature concerning plasmablastic lymphoma (PBL) is incomplete and current knowledge mainly based on single case reports and small case series. This rare neoplasm is extensively investigated yet no specific diagnostic criteria currently exist. Distinguishing PBL from other high-grade B-cell and terminal B-cell neoplasms with plasmablastic features, especially in HIV/AIDS patients in third world circumstances is challenging and urges the development of a diagnostic algorithm. In South Africa more than 10 % of the population is HIV positive and PBL more commonly diagnosed than anywhere else in the world.

Method: Fifty eight cases diagnosed as PBL were subjected to a series of morphological and molecular investigations (Fish analysis: MYC BA, IGH BA, BCL6 BA, t(8: 14), t(11;14) and t(14;18); Immunohistochemistry: CD20, CD10, CD79a, Bcl6, CD45, CD38, CD138, IRF4/MUM1, PRDM1/BLIMP1, CD56, CD117, CCND1, Ki67, ALK, HHV8; In situ hybridisation: EBER, Immunoglobulin light chains). Available clinical features were noted. Results were compared to those of cases of high-grade B-cell lymphoma with plasmablastic differentiation and confirmed cases of multiple myeloma with plasmablastic morphology.

Conclusion: From the results we propose a diagnostic algorithm for HIV-related PBL to distinguish it from other high-grade B-cell lymphomas and plasma cell neoplasms with plasmablastic differentiation within the HIV setting.


Pathological characteristics of 105 cases of HIV associated lymphoma included in the French ANRS CO16 LYMPHOVIR cohort study

S. Prévot*, C. Besson, D. Costagliola, C. Chassagne-Clement, C. Delattre, B. Fabiani, C. Laurent, T. Lazure, B. Petit Jean, M. Raphaël

*Antoine Béclère Hospital, Pathology, Clamart Cedex, France

Objective: Introduction of combined antiretroviral therapy has reduced the incidence of NHL but not the incidence of HL in the HIV-infected population. This study analyzes the pathological characteristics of HIV-related lymphoma (L) collected in 4 years (2008–2012) in 32 centers among the prospective French national cohort of HIV-related L.

Method: Pathological material of 105 cases was reviewed according to the 2008 WHO classification with complementary techniques performed when necessary.

Results: 43 % were classical HL and 57 % B cell NHL. A predominance of mixed cellularity subtype HL was observed (33/45) and all but 3 were EBV positive. The distribution of the 60 NHL was: diffuse large B cell L 42 % (39 % EBV associated, 61 % of non GC subtype and 39 % of GC subtype), Burkitt L 28 %, plasmablastic L 12 %, marginal zone L 5 %, PTLD-like L 5 %, primary effusion L 5 %, anaplastic large cell L and unclassifiable because of necrosis (1 case of each).

Conclusion: This present study points out: (1) the high proportion of HL among HIV infection with L in the cART era, all of the classical type and the vast majority being EBV associated; (2) the heterogeneity of HIV-related NHL, all of B cell phenotype.

Wednesday, 4 September 2013, 14.00–16.00, Room 5B

OFP-18 Oral Free Paper Session Digestive Diseases Pathology II


Limitations of the UICC/AJCC postoperative staging system for esophageal adenocarcinomas after neoadjuvant chemotherapy: Adding tumor regression grade enhances prognostic significance

R. Langer *, K. Becker, L. Sisic, M. Büchler, F. Lordick, J. Slotta-Huspenina, W. Weichert, H. Höfler, M. Feith, K. Ott

*Medizin. Universität Bern, Inst. für Pathologie, Switzerland

Objective: For tumors treated with neoadjuvant chemotherapy (CTX), postoperative staging classifications initially developed for non-pretreated malignancies may not accurately predict prognosis. We tested whether a TNM-based histopathologic prognostic score (PRSC), which additionally applies to tumor regression, may improve estimation of prognosis of esophageal adenocarcinomas treated by neoadjuvant chemotherapy, compared to the current UICC/AJCC TNM staging system (TNM7).

Method: 360 resection specimens were classified according to TNM7. For the PRSC, three factors were assigned a value from 1 to 3 (ypT0-2 = 1point; ypT3 = 2points; ypT4 = 3points; ypN0 = 1point; ypN1-2 = 2points; ypN3 = 3points; <10 % residual = 1point; 10–50 % residual tumor = 2points; >50 % residual tumor = 3points). TNM7 and the three-tiered PRSC basing on the sum value of these factors (group A: 3–4; group B: 5–7; group C: 8–9) were correlated with survival.

Results: Both TNM7 and PRSC showed highly significant prognostic impact (p < 0.001 each). However, TNM7 showed limitations especially in the lower staging groups while PRSC could accurately identify three prognostic different groups. Moreover, the PRSC showed better discrimination than TNM7 in multivariate analysis (for PRSC: p < 0.001; HR = 1.93; for UICC staging: p = 0.009; HR = 1.25).

Conclusion: The proposed PRSC clearly identifies subgroups with different outcomes and may be more helpful for guiding further therapeutic decisions than the current UICC/AJCC TNM staging system


SYT and EWS abnormalities are recurrently occurring in poorly differentiated gastroesophageal cancers

X. Sagaert *, E. Van Cutsem, S. Tejpar, H. Prenen, P. Nafteux, K. Haustermans, T. Tousseyn, G. De Hertogh, S. Palmans, R. Sciot, M. Debiec-Rychter

*University Hospitals Leuven, Dept. of Pathology, Belgium

Objective: Being confronted with a case of a poorly differentiated gastroesophageal carcinoma with sarcoma-like features arising within high-grade Barrett-dysplasia, we investigated the immunohistochemical and molecular-genetic features in a (retrospective) series of this disease entity.

Method: 42 poorly differentiated gastroesophageal cancers were investigated by (1) immunohistochemistry for presence/absence of selected carcinoma/sarcoma markers (prekeratin, NCAM, vimentin, CD99) and by (2) FISH for presence/absence of abnormalities of the SYT and EWS genes, that are frequently rearranged in sarcomas. Twenty five well/moderately differentiated GE cancers were used as a control.

Results: 13/42 cases displayed an aberrant immunophenotype, with moderate/strong expression of NCAM, vimentin, and/or CD99. FISH did reveal a substantial number (14/42 = 33 %) of SYT/EWS-related genomic anomalies: 3 EWS-rearrangements, 3 SYT-rearrangements, 1 SYT and 1 EWS focal amplification, and 8 cases with SYT/EWS polyploidy. No immunohistochemical nor FISH abnormalities were found in the control group.

Conclusion: Our data show that SYT and EWS abnormalities are recurrently occurring in poorly differentiated gastroesophageal cancers. In addition, these abnormalities seemed to target cancers that are at risk of not responding well to the conventional chemoradiotherapy. Of interest, the one patient that started this study went into complete remission after being administered a sarcoma therapy, hence emphasizing the need for a prospective study.


Resection margin status and lymph node yield in pancreaticoduodenectomy specimens at a single tertiary-care oncology centre

M. Bal *, M. Ramadwar, K. Deodhar, M. Goel, S. Arya, P. Patil, R. Engineer, S. Shrikhande

*Tata Memorial Hospital, Dept. of Pathology, Mumbai, India

Objective: To assess resection margin (RM) status and lymph node yield in pancreaticoduodenectomy (PD) specimens

Method: Data from all PD resections (2010–2011) were retrieved. A standardized pathology reporting protocol was used wherein transection [common bile duct (CBD), pancreatic neck (PN), proximal and distal enteric] and circumferential RMs (medial, retroperitoneal, posterior and anterior surfaces) were colour-coded and sampled. A 1 mm cut-off was defined as R1 (microscopic disease at margin).

Results: Study comprised 133 patients. Median age was 54 years. Tumours’ epicenter was in the periampullary (PA) region (88), pancreatic head (28), bile duct (5) and retroperitoneum (1); 11 had no tumour. Seven patients received neo-adjuvant chemo/radiotherapy (NACT/RT). R1 rate for pancreatic ductal adenocarcinoma (PDAC) and PA tumours was 71 % and 7 %, respectively; overall R1 rate was 17 %. R1 rate for posterior, retroperitoneal, PN, CBD, anterior, medial, proximal and distal enteric margins was 36 %, 30 %, 17 %, 9 %, 4 %, 4 %, 0 % and 0 %, respectively. Mean nodal yield was 13.6 (median, 11.5; range 0–34); following NACT/RT, mean nodal yield was 5.4.

Conclusion: Incidence of PA tumours was higher than PDAC in our PD specimens. R1 rate for PDAC resection was significantly greater than for PA tumours; posterior margin being the commonest involved. Neo-adjuvant therapy might influence nodal yield.


Loss of Raf-1 Kinase Inhibitor Protein (RKIP) promotes Epithelial Mesenchymal Transition (EMT) and correlates with aggressive phenotype in Pancreatic Ductal Adenocarcinoma (PDAC)

E. Karamitopoulou*, I. Zlobec, A. Lugli, B. Gloor, A. Perren

*University of Bern, Institute of Pathology, Switzerland

Objective: RKIP has emerged as a significant metastatic suppressor in a variety of human cancers and is known to inhibit the Ras/Raf/MEK/ERK signaling. By suppressing the activation of NFkB/SNAIL circuit, RKIP can regulate the induction of EMT. The aim of this study was to evaluate RKIP expression and to determine its association with clinic-pathological features, including EMT in form of tumor budding in PDAC.

Method: Staining for RKIP was performed on a multipunch Tissue Microarray (TMA) of 120 well-characterized PDACs with full clinico-pathological, follow-up and adjuvant therapy information. RKIP-expression was assessed separately in the main tumor body and in the tumor buds. Cut-off values were calculated by receiver operating characteristic curve analysis.

Results: RKIP expression was lost in 61.4 % of the PDACs and was significantly lower in the tumor buds compared to the main tumor body (p < 0.005). RKIP loss in the tumor was associated with higher tumor grade (p = 0.0389) and high-grade peritumoral budding (p = 0.0118). RKIP loss in the buds was associated with increased T stage (p = 0.035). No correlation with M- and N-stage or patient survival was found.

Conclusion: Loss of RKIP correlates with aggressive features in PDAC, especially characterized by the presence of EMT in form of tumor budding.


Correlation of mitotic grade and Ki-67 index grade between histological and scrape cytological specimens in pancreatic neuroendocrine tumor

K. Hirabayashi *, G. Zamboni, G. Bogina, E. Bongiorno, L. Bortesi, P. Castelli, A. Pesci, N. Nakamura

*Tokai University, Dept. of Pathology, Isehara, Japan

Objective: The purpose of this study was to correlate mitotic grade (MG) and Ki67-index grade (KG) between histological and scrape cytological specimens in 61 resected primary pancreatic neuroendocrine tumors (P-NETs).

Method: Ki67 index was calculated as a percentage of 2000 cells in ‘hot spots’ on histological specimens and as a percentage of 100, 500, 1,000, and 2,000 cells in ‘hot spots’ on scrape cytological specimens. Mitotic count was calculated in the high-density area of mitoses per 10 high power fields by scanning 50 fields on histological specimens and 10, 20, or 50 fields on scrape cytological specimens. MG and KG were classified according to ENETS/WHO grading system.

Results: Kappa values of scrape cytological KG per 500, 1000, and 2000 cells showed a substantial agreement with histological KG, whereas KG per 100 cells only showed a fair agreement. Furthermore, when the cases were subdivided into KG1-2 and KG3, kappa value showed an almost perfect agreement. In contrast, kappa values of scrape cytological MG showed a fair agreement with histological MG.

Conclusion: Our results indicate that scrape cytological KG, determined in a small number of cells (e.g., 500 cells), accurately predicts histological KG, whereas scrape cytological MG does not predict histological MG.


The CD8/CD45RO immunoscore in biopsy specimens of colorectal cancer predicts histopathologic features of the matched resection specimen and survival outcome

V. Koelzer *, I. Zlobec, H. Dawson, M. Borner, D. Inderbitzin, A. Lugli

*Universität Bern, Inst. für Pathologie, Switzerland

Objective: Reliable biomarkers based on pre-operative biopsies of colorectal cancer (CRC) are missing. We hypothesize that an immunoscore of CD8+ T-effector and CD45RO+ memory T-cell infiltrates in biopsies may predict histopathological features and survival outcome of CRC patients.

Method: Intraepithelial and stromal (s) CD8+ and CD45RO+ T-cells were quantified in 5 high-power-fields each of the highest density of infiltration in biopsy specimens of 130 well characterized CRC patients. Using Classification and Regression Tree (CART) analysis, CD8+ and CD45RO+ infiltrates in each zone were assessed for clinical relevance.

Results: High total (t) numbers of infiltrating CD8+ T-cells in biopsies strongly predicted the absence of nodal metastasis (p = 0.0182) and lymphatic invasion (p = 0.0201) in the matched resection specimen. High numbers of sCD45RO+ T-cells predicted earlier T-stage (p < 0.0001), lower tumor grade (p = 0.0223) and absence of distant metastasis (p = 0.0177). 100 % specificity and PPV for the prediction of pN was reached by combined analysis of tCD8 and tCD45RO. Strong immune infiltration in biopsies was highly prognostic [tCD8: HR (95 %CI) = 0.29 (0.1–0.7); p = 0.0061; sCD45RO: HR (95 %CI) = 0.43 (0.2–0.9); p = 0.0364].

Conclusion: An immunoscore of CD8 and sCD45RO in pre-operative biopsy specimens allows prediction of full TNM-stage of the matched resection specimen and survival outcome in the pre-operative setting.


Hypermethylation of ZEB2 represents a novel mechanism of colorectal cancer progression and is a highly unfavourable prognostic factor in KRAS wild-type tumors

I. Zlobec*, M. Helbling, H. Dawson, V. H. Koelzer, E. Diamantis Karamitopoulou, A. Lugli

*Universität Bern, Inst. für Pathologie, Switzerland

Objective: A majority of colorectal cancers exhibit de-regulation of WNT pathway signaling. This may include loss of E-cadherin expression through transcriptional regulation of ZEB1 and ZEB2. Here, we investigate the methylation status and prognostic effect of ZEB1 and ZEB2 in colorectal cancer.

Method: 77 primary colorectal cancers from patients with full clinicopathological and survival time data underwent methylation analysis for ZEB1 and ZEB2, and mutational analysis of KRAS and BRAF using pyrosequencing.

Results: ZEB1 was unmethylated in all cases. Frequent methylation of ZEB2 was observed (average 32.6 %; range 1–99 %). ZEB2 hypermethylation was significantly greater in patients with KRAS wild-type tumors (p = 0.0397) and BRAF mutated cancers (p = 0.055). Stratified by KRAS status, hypermethylation of ZEB2 in wild-type tumors was highly associated with more advanced pT stage (p = 0.0463), lymph node positivity (p = 0.0032), distant metastasis (p = 0.0192), venous invasion (p = 0.0193), lymphatic invasion (p = 0.0025) and unfavorable survival (p = 0.0155; HR (95 %CI): 3.2 (1.2–8.6)), but not with tumor budding. No associations were observed in KRAS mutated cases.

Conclusion: These findings underline a role for ZEB2 hypermethylation as a novel mechanism of colorectal cancer progression. Moreover, hypermethylation of ZEB2 identifies a highly aggressive subgroup of colorectal cancer patients in the context of a KRAS wild-type background only.


Investigation of IL23 (p19, p40) and IL23R highlights nuclear IL23p19 expression as a marker of indolent tumor features and favorable outcome in colorectal cancer patients

I. Zlobec *, M. Helbling, V. H. Koelzer, H. Dawson, R. Langer, U. Nitsche, E. Diamantis Karamitopoulou, A. Lugli

*Universität Bern, Inst. für Pathologie, Switzerland

Objective: IL23 is involved in chronic inflammation but its role in cancer progression is not fully elucidated. Here we characterize IL23 subunits p40, p19 and IL23 receptor (IL23R) in the normal-adenoma-carcinoma-metastasis cascade of colorectal cancers and their relationship to clinicopathological and outcome data.

Method: Immunohistochemistry for IL23R, IL12p40, IL23 and IL23p19 (monoclonal) was performed on a multi-punch tissue microarray (n = 213 patients). Expression differences between normal-adenomas-cancers-lymph nodes were evaluated. Correlation with clinicopathological and outcome data was undertaken. Results were validated on an independent cohort (n = 341 patients).

Results: An increased expression from normal-adenoma-cancer was observed (p < 0.0001; all) followed by a marked reduction in lymph nodes (p < 0.0001; all). Cytoplasmic and/or membranous staining of all markers was unrelated to outcome. Nuclear IL23p19 staining occurred in 23.1 % and was associated with smaller tumor diameter (p = 0.0333), early pT (p = 0.0213), early TNM (p = 0.0186), absence of vascular (p = 0.0124) and lymphatic invasion (p = 0.01493) and favorable survival (univariate (p = 0.014) and multivariable (p = 0.0321) analysis). All IL23p19 positive patients were free of distant metastasis (p = 0.0146). Survival and metastasis results could be validated in Cohort 2.

Conclusion: The presence of nuclear IL23p19 is related to indolent tumor features and favorable outcome supporting a more ‘protective’ role of this protein in colorectal cancer progression.


LGR5 positivity defines stem-like cells in colorectal cancer

T. Gaiser *, T. Ried, A. Marx, D. Hirsch

*Universität Mannheim, Inst. für Pathologie, Germany

Objective: Like normal colorectal epithelium, colorectal carcinomas (CRC) are organized hierarchically and include cells with stem-like properties. Leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5) is highly expressed in these stem cells. However the precise function of LGR5 in CRC remains largely unknown.

Method: Here, we analyzed the functional and molecular consequences of RNA-mediated silencing of LGR5 in CRC cell lines SW480 and HT-29. Additionally, we exposed Lgr5-EGFP-IRES-Cre-ERT2 mice to azoxymethane/dextrane sodium sulfate (AOM/DSS) which induces inflammation-driven colon tumors. Tumors were then flow-sorted into fractions of epithelial cells that express high or low levels of Lgr5 and were molecularly characterized using gene expression profiling and array comparative genomic hybridization.

Results: Silencing of LGR5 reduced proliferation, migration and colony formation in vitro, and tumorigenicity in vivo. In accordance with these results, Notch signaling was down-regulated upon LGR5 silencing. In the AOM/DSS-induced mouse colon tumors Lgr5 high cells showed higher levels of several stem cell-associated genes and higher Wnt signaling than Lgr5 low tumor cells and Lgr5 high normal colon epithelial cells.

Conclusion: Our data elucidate mechanisms that define the role of LGR5 as a marker for stem-like cells in CRC.

Poster Sessions

Sunday, 1 September 2013, 09.30–10.30, Pavilion 2

PS-01 Poster Session Digestive Diseases Pathology I: Upper Gastrointestinal Tract


Morphological features of eosinophilic esophagitis in patients with asthma

L. Mikhaleva *, T. Barkhina, V. Golovanova, N. Shchegoleva, N. Gracheva

*Moscow City Hospital, Dept. of Pathology, Russia

Objective: To study the morphological features of the mucosa of esophagus in patients with eosinophilic esophagitis during bronchial asthma

Method: We used biopsies of esophageal mucosa of patients with asthma. Morphological study was performed by using hematoxylin and eosin staining, according to Mallory and by holding Schick-reaction. We studied a cellular composition of the inflammatory infiltrate by immunohistochemistry using monoclonal antibodies to CD4, to CD8, to CD16, to CD20, to CD68, a CD117. The histologic sections were examined in a light microscope. We conducted the morphometric study and the statistical analysis of the results.

Results: We identified an intraepithelial eosinophilic infiltration of the mucosa in the proximal part of esophagus (15 to 55 in a field of view), eosinophilic “microabscesses” and a significant subepithelial sclerosis. According to morphometry the ratio CD4/CD8 was significantly less than “1”. We confirmed by morphometric and statistical studies that the intraepithelial infiltrate consisted mainly in macrophages and “natural killer” cells. We noted an early formation of strictures and stenosis of esophagus in cases of eosinophilic esophagitis during asthma.

Conclusion: The study revealed morphological features of the esophageal mucosa, typical for cases of eosinophilic esophagitis with asthma. Further study of the pathogenesis of this comorbidity will help to optimize treatment strategy for these seriously ill patients.

An intraepithelial eosinophilic infiltration of the mucosa in the proximal part of esophagus (15 to 55 in a field of view):

figure k


Interobserver reproducibility in interpretation of columnar lined esophagus

F. Grillo *, L. Mastracci, N. Piol, F. Pitto, L. Molinaro, R. Fiocca

*University of Genoa, DISC, Histopathology, Italy

Objective: Terminological confusion regarding the histological interpretation of columnar lined esophagus (CLE) has kept pathologists wading in murky waters for many years. Histology however is mandatory for the confirmation of endoscopically suspected esophageal metaplasia (ESEM). Aim of this study is to evaluate interobserver variability in the interpretation of CLE between pathologists.

Method: Thirty pathologists (10 gastrointestinal pathologists; 10 general pathologists; 10 pathology trainees) distributed nationwide and working in community and teaching hospitals were invited to review three 10-case sets of CLE. A first set was coupled with endoscopy with no precise evaluation of ESEM extent; the second and third sets were coupled with descriptive endoscopy (Prague CM criteria); national guidelines (Fiocca et al. DigLivDis 2011;43 Suppl 4:S319-30) were distributed to participants before evaluation of the third set. Agreement was statistically assessed using Randolph’s free-marginal multirater kappa.

Results: Concordance between pathologists was lower for the first set (K = 0.34) compared to the second (K = 0.44) and the third set (K = 0.63). Reproducibility was generally higher among GI pathologists compared to general pathologists and trainees. Interpretation of intestinal-type epithelium was less problematic than cardia and oxyntic-types.

Conclusion: Precise endoscopic description and use of guidelines increase consistency in interpretation and reporting of CLE in esophageal biopsies.


Selected molecular markers of disease progression in Barrett Esophagus

J. Ehrmann *, P. Luzna, V. Divoky

*Faculty of Medicine, Olomouc, Czech Republic

Objective: Barrett esophagus (BE) is an acquired preneoplastic condition that is characterized by replacement of a stratified squamous epithelium of the distal esophagus by a columnar epithelium. BE represents a permanent risk for developing esophageal adenocarcinoma (EAC). Thus, there is a strong need for specific molecular markers of BE progression that would enable early diagnosis of developing EAC.

Method: In our study we followed accumulation of gamm-H2AX, a marker of the DNA damage response (DDR) pathway activation, together with expression of active β-catenin and two inflammatory cytokines, IL-8 and IL-1β.

Results: Our results show that the activation of the DDR in BE increases with disease progression and that gamma-H2AX accumulates mainly in EAC. Active β-catenin is found in cytoplasm in all disease stages whereas its membrane localization is exclusively associated with EAC. While the level of IL-1β increased accordingly with tumor progression, IL-8 was expressed constitutively in all disease stages.

Conclusion: We conclude that genetic alterations associated with premalignant stages of BE do not have capacity to evoke DDR and that gamma-H2AX is a useful biomarker of disease progression of BE to EAC. Grant support LF_2012_016, LF_2012_019, NT 13585-3/2012


Unravelling novel mechanisms of Barrett’s esophagus progression: Centrosome amplification precedes the onset of invasion

M. Mesquita *, C. Lopes, A. Cunha, A. Pereira, P. Chaves

*Inst Português de Oncologia, Anatomia Patológica, Lisboa, Portugal

Objective: Barrett’s esophagus (BE) malignant progression involves major changes in cellular processes controlled by the centrosome, the primary microtubule-organizing centre in animal cells. Though centrosome dysfunction is associated with several neoplastic pathways, its role in BE tumorigenesis is still unknown. Our aim was to evaluate the centrosome profile in Barrett’s progression.

Method: Dysplasia-negative BE biopsies, esophagectomy specimens and established cell lines were analyzed. The percentage of cells with amplified centrioles (>4 centrioles/cell) was assessed by immunofluorescence.

Results: The percentage of cells with amplified centrioles increased from metaplasia to neoplasia: 0.0 % ± 0.0 %-dysplasia-negative BE biopsies, 0.46 % ± 0.69 %-BE associated with ADC, 2.46 % ± 1.73 %-ADC, 8.25 % ± 4.32 %-lymph node metastasis. Despite the high variability between patients, an increase of cells with amplified centrioles during progression was observed in all patients. These results were confirmed in cell lines: 1.7 %-metaplasia, 33.3 % ± 7.1 %-high-grade dysplasia, 10.8 % ± 10.5 %-ADC, 8.0 % ± 5.9 %-metastasis.

Conclusion: Our findings suggest that centrosome abnormalities are related to Barrett’s progression and precede the onset of invasion. Correlation between these changes and the well-known genetic profile of the cell lines provides new clues into the permissive genetic background for such abnormalities (like p53 loss) and may impact on BE diagnosis, prognosis and treatment.


Lymphoepithelioma-like carcinoma of the esophagogastric junction with microsatellite instability

S. Gurzu *, Z. Szentirmay, I. Jung

*University of Medicine and Pharmacy, Dept. of Pathology, Tirgu-Mures, Romania

Objective: To present an extremely rare tumor of the esophagogastric junction that can be associated with Epstein-virus infection

Method: A 60-year-old male presented with symptoms suggesting a gastric cancer and total gastrectomy was performed. The surgical specimen was submitted for histopathological examination.

Results: Microscopically, the ulcero-infiltrative tumor of the esophagogastric junction was a poorly differentiated adenocarcinoma intermingled with dense lymphoid infiltration predominantly composed of T-cell lymphocytes. The tumor cells infiltrated the submucosa, muscularis and subserosal layers of the stomach respectively the esophageal adventitia. No metastases were noticed in the 58 regional lymph nodes. Based on the histopathological features, the diagnosis was lymphoepithelioma-like carcinoma, pT3N0 stage. In situ hybridization for Epstein-Barr virus showed no nuclear signal in tumor cells. The p53 expression was observed in fewer than 10 % of the tumor cells. Real-time PCR analysis showed microsatellite instability without K-ras mutation in codon12. No recurrences or metastases were reported 2 years after surgical intervention. No adjuvant therapy was performed

Conclusion: Diagnosis of lymphoepithelioma-like carcinoma remains difficult, especially in those cases that lack glandular differentiation. Microsatellite instability seems to play an important role in its histogenesis and associates good prognosis even in late stages


Clear/glassy cell change of gastric mucosa: A diagnostic pitfall?

J. Pinheiro *, E. Rios, F. Carneiro

*Hospital de Sao Joao, Dept. de Anatomia Patologica, Vila de Cucujaes, Portugal

Objective: Glassy gastric cells (GGCs) were first described 27 years ago in gastric mucosa, characterised by a uniform glassy, eosinophilic or clear vacuole that pushes the nucleus towards the apical pole. Since then, the literature dedicated to this topic is quite scarce. GGCs constitute a diagnostic pitfall being frequently misinterpreted as malignant signet-ring cells.

Method: Two gastrectomy specimens and 12 endoscopic biopsies of stomach containing GGCs were retrieved from the files of our department. Slides were stained with haematoxylin-eosin and PAS-D.

Results: GGCs were identified in 14 patients (10M/4F; 50–85 years; mean age: 66.2 years), in the setting of chronic gastritis (n = 6), gastric carcinoma (n = 2), hyperplastic polyps (n = 1), fundic gland polyps (n = 2) and normal mucosa (n = 1). GGCs were observed in the antrum and body, in compact glands lined by a single row of cells. PAS-D staining was negative in the glassy/clear vacuoles, being restricted to a thin rim of apical cytoplasm.

Conclusion: GGCs can be found in the setting of a broad spectrum of gastric conditions and mimic malignant signet-ring cells (a diagnosis that is excluded by the negative PAS-D staining of the glassy/clear vacuoles). Awareness of the morphologic and histochemical features is necessary for proper interpretation.


An “unclassified” polyp of stomach

E. Basar *, B. Doganavsargil, R. Vardar, M. Sezak, M. Tuncyurek

*Ege University, Dept. of Pathology, Izmir, Turkey

Objective: Gastric polyps are a heterogeneous group of tumours including more common non-neoplastic fundic gland, hyperplastic or hamartomatous polyps and less frequent neoplastic polyps. However there also exist a considerable number of “reactive or unclassified” protrusions which are readily defined as “polyps” endoscopically but not as easily or reproducibly classifiable pathologically. We present a subgroup of such “polyps” which are not well documented in literature in detail.

Method: Twenty-one polyps of 18 patients which were reported as “polyp, not otherwise specified” in a 2 years period were included in the study.

Results: The mean age of the patients were 59 ± 12,6 years old, (Range: 32–80 y.o). Male/female ratio was 7/13, the localizations were antrum (85 %, 17/20), corpus (10 %, 2/20) and cardia (5 %, 1/20), The mean diameter was 1.14 ± 0.3 cm (Range: 0.5–1.7 cm). Identifiable common histological variations were marked foveoler hyperplasia (33,3 %), thick and irregular muscularis mucosa (23.8 %), prominent eosinophilic infiltration (38.0 %), intraepithelial lymphocytes (47.6 %), mild to severe stromal edema and congestion in all cases.

Conclusion: There exist a certain type of “unclassified/distal antral polyp” the most distinctive features of which are antral gland hyperplasia and hypertopy of muscularis mucosa. The cases were discussed in respect to nomenclature and differential diagnosis from other benign polyps.


Gastric Dieulafoy’s lesion or caliber-persistent artery: Report of two cases

I. Jung *, C. Copotoiu, S. Gurzu

*University of Medicine and Pharmacy, Dept. of Pathology, Tirgu-Mures, Romania

Objective: To present a vascular malformation of the gastrointestinal tract, incidentally diagnosed under microscope

Method: Two patients were hospitalized with hematemesis. Case 1 was a 62-year-old-male endoscopically diagnosed with chronic gastric ulcer for that laparoscopic suture was performed. Due to recurrent bleeding total gastrectomy was the treatment of choice but the patient died due to septic shock. Case 2 was a 75-year-old-male that underwent a Billroth-II gastric reconstruction for a peptic ulcer 38 years ago. At the present hospitalization, a stump gastric cancer was endoscopically diagnosed and total removal of the gastric remnant was performed, with favorable evolution

Results: In both cases 1 and 2, corresponding to the ulcerated lesion (case1), respectively near to the tumor, that was an early cancer (case 2), the histological examination revealed abnormally thick-walled enlarged vessels in the submucosa and muscularis propria, some of them being thrombotic. Focally, these oversized tortuous vessels protruded through the muscularis mucosae in the mucosal layer, some of them being eroded. Based on these characteristics and the hematemesis, the final diagnoses were fatal hemorrhage due to Dieulafoy’s lesion (case 1) respectively early gastric stump carcinoma associated with Dieulafoy’s lesion (case 2)

Conclusion: Dieulafoy’s lesion is a vascular malformation that still remains largely undiagnosed


Melatonin reduces intensity of morphogenetic signs of hyperacid gastritis, experimented on animals

G. Mylovydova *, G. Gubina-Vakulik, N. Kolousova

*Laboratory of Pathology, and Exp. Surgery, Kharkiv, Ukraine

Objective: Study microscopic features of a mucous coat of stomach of the rabbits staying in the conditions of continuous lighting within 5 months and estimate the effect of melatonin injections course on intensity of morphogenetic symptoms of hyperacid gastritis.

Method: For 5 months rabbits were under constant illumination. Then eight of them received 10 injections of melatonin 2.5 mg/kg. 5 animals received 10 injections of saline. 5 rabbits of the same age were intact. Their gastric mucosa microslides were stained by gallocyanine (by Einarsson) for total nucleic acids. Parietal cells and their nuclei sizes, as well as the optical density of the main cell cytoplasm (Axiostar-plus-Zeiss) were determined.

Results: Experiment led to formation of chronic atrophic-hypertrophic gastritis with erosions, parietal cells hyperplasia. Course of melatonin injections resulted in a significant decrease in the number of parietal cells. There were the signs of decreasing morphofunctional activity and enhanced apoptosis of parietal cells. A major cell cytoplasm contains an increased amount of RNA (0,406 ± 0,015 and 0,474 ± 0,018 conditional units of optical density, p ≤ 0,05).

Conclusion: Long permanent lighting leads to the development of chronic atrophic-hypertrophic gastritis in animals. The course of injections of night hormone melatonin causes improvement of morphofunctional pattern of mucous coat of stomach.


H. pylori and chronic gastritis: A histopathologic study

A. Kurt *, S. A. Özmen, I. Çalik, I. Gelincik

*Bölge Egitim ve Arastirma Hastesi, Dept. of Pathology, Erzurum, Turkey

Objective: The aim of this study was to determine the level in the application of the Sydney Classification and to find out whether this grading system is reproducible in routine histopathological practice.

Method: Hematoxylen and Eosin, Alcian blue pH 2.5, PAS and Giemsa stained sections of 210 gastric biopsy specimens were evaluated. These were mucosa of antral biopsy, diagnosed as chronic gastritis during routine histopathological examination within April- May 2012. Each observer graded chronic inflammation, neutrophyl leucocyte, atrophy, intestinal metaplasia and Helicobacter pylorii density in the antrum on a scale 0–3. The measurement of on the histopathological grades was examined by “measures of agreement” and statistics respectively.

Results: It was found out that the proportion of overall agreement was on 19 % for atrophy, 17 % for lymphoid follicles, 67 % for neutrophyl leukocyte, 15 % for intestinal metaplasia in the antrum.

Conclusion: In this study, it was found out that there was a suitable relationship between lymphocyte inflammation, neutrophyl leukocyte and Hp. There was no relationship between Hp and glandular atrophy, intestinal metaplasia and lymphoid follicular formation. It was concluded that the results are in accordance with the referance generally. In conclusion, we are in the opinion that Sydney System is useful in diagnosing gastritis.


Interobserver agreement of gastritis staging by OLGA and OLGIM system between general pathologists

S. Isajevs *, I. Liepniece-Karele, D. Janciauskas, G. Moisejevs, K. Funka, I. Kikuste, A. Vanags, I. Tolmanis, M. Leja

*University of Latvia, Dept. of Pathology, Riga, Latvia

Objective: The aim of our study was to compare interobserver agreement in the staging of gastritis by OLGA and OLGIM system between the general pathologists.

Method: 839 patients undergoing upper endoscopy were enrolled in the study. Three general pathologists graded biopsy specimens according to the Sydney classification, OLGA and OLGIM staging system. Interobserver agreement was analyzed by kappa statistics.

Results: Overall, 280 (33.4 %) and 167 (19.9 %) patients were classified as stage I–IV according to OLGA and OLGIM, respectively. Interobserver agreement for atrophic gastritis was moderate in antrum and incisura angularis (respectively, kappa = 0.53 and 0.57, p < 0.0001), but fair for atrophic gastritis assessement in corpus (kappa = 0.38). However, interobserver agreement was almost perfect for intestinal metaplasia assessement both in antrum, incisura angularis and corpus (respectively, kappa = 0.82, 0.80 and 0.81, p < 0.0001). The interobserver agreement for displasia was moderate (kappa = 0.58, p < 0.0001).

Conclusion: Gastritis staging systems (both OLGA and OLGIM) convey prognostically important information on the gastritis-associated cancer risk. However, OLGIM staging system characterized with a highest interobserver agreement. Supported by ERDF Nr.2010/0302/2DP/


What summons eosinophils to gastric mucosa?

B. Doganavsargil *, M. Sezak, B. Sarsik, B. Yaman, R. Vardar, M. Tuncyurek, S. Bor, DISPEN Study Group

*Ege University, Dept. of Pathology, Izmir, Turkey

Objective: The factors affecting eosinophil trafficking to Gastrointestinal tract, which shows a higher eosinophil density (ED) than many tissues, is not fully uncovered. We searched the relationship between ED and gastritis.

Method: One thousand cases were retrieved randomly from DISPEN-database and re-evaluated. Special forms of acute and chronic gastritis and biopsies which were unsuitable for atrophy assesment were excluded. ED was assessed as number of eosinophils per one high power field (HPF) in hot spots (by Olympus BX50 microscope, ×40 magnification:0,54 mm diameter, Ocular: ×10). Gastritis was evaluated according to modified Sydney classification and correlated by non-parametric tests.

Results: Distribution of cases with normal mucosa, atrophic and nonatrophic gastritis were 13.4 %, 7 %,79,6 % respectively. Chronic active gastritis, intestinal metaplasia and H. Pylori positivity observed in 35.4 %, 89.2 %, 68.2 % of cases. Mean antral and corporal ED’s were 3.88 ± 6.15 (Range: 0–70/HPF) and 2.32 ± 1.00 (Range:0–45/HPF). Sixty cases showed considerable (≥10/HPF) eosinophils. Increased ED was correlated with atrophy, H.pylori colonisation and active inflammation (p < 0.001, each). The difference between atrophic and nonatrophic gastritis was remarkable.

Conclusion: Eosinophils are responders of inflammation thus affected by active gastritis and H. Pylori colonisation. However, the relationship between eosinophil infitration and atrophy, deserves special attention and should be further investigated for possible pathogenetic aspects.


Solitary gastric hamartoma: A case report

N. Abid*, R. Kallel, N. Gouiaa, H. Mnif, S. Ellouze, M. Ksentini, S. Makni, T. Boudawara

*Habib Bourguiba University Hospital, Pathology, Sfax, Tunisia

Objective: Gastric hamartoma is considered to be a rare entity and is frequently associated with polyposis syndromes. Sporadic hamartoma of the stomach is uncommon. We described a new case; our aim is to study the histological features of this entity and insist on its differential diagnosis.

Method: A 53-year-old man with a story of urinary lithiasis was proposed to be treated with extracorporeal shock wave lithotripsy. Pretreatment imaging by noncontrast computed tomography showed focal eccentric mural thickening at the wall of the greater curvature of the stomach. Esophagogastroduodenoscopy revealed a pedunculated polypoid mass with ulcerated mucosa measuring 1 cm in diameter. The polyp was endoscopically removed then fixed in formalin and embedded into paraffin. Serial sections stained with hematoylin-eosin were examined and immunohistochemical staining was perfomed for vimentin, desmin and smooth muscle actin

Results: Histological findings were consistent with hamartoma, containing glands with cystic formation, smooth muscle, vasoformative tissue and fat cells.

Conclusion: Gastric hamartoma is a rare occurrence in patients without familial polyposis coli. Clinically, solitary gastric hamartoma is asymptomatic and tend to be found incidentally,or may cause mild upper digestive symptoms. The final diagnosis depends on the pathological findings; however, gastric hamartomashould be histologically distinguished from gastritis cysticaprofunda.


Juvenile polyposis with prominent gastric involvement: Case report

H. Baldaia *, M. Guimarães, J. Magalhães, F. Carneiro

*Centro Hospitalar S. João, Dept. of Pathology, Porto, Portugal

Objective: Juvenile polyposis is a familial cancer syndrome characterized by multiple juvenile polyps throughout the gastrointestinal tract. Three types are described: juvenile polyposis of infancy, juvenile polyposis coli and generalized juvenile polyposis.

Results: We present the case of a 50-year-old male with no relevant family history, submitted to a proctocolectomy 7 years before for “Familial Adenomatous Polyposis”, APC negative. He presented with weakness and vomiting; the upper endoscopy revealed multiple polyps throughout the stomach. A total gastrectomy was performed which showed carpeting of gastric mucosa with sessile lobulated polyps, the largest 4,0 cm in diameter. Histologically, these polyps were hamartomatous, with a smooth or multilobulated surface, a prominent inflammatory stroma and tortuous and dilated glands. There were no dysplastic features. The histological evaluation prompted a review of the previous proctocolectomy specimen which revealed similar hamartomatous polyps, some with low grade dysplasia. A diagnosis of juvenile polyposis was confirmed by SMAD4 mutation.

Conclusion: This case documents a prominent gastric involvement in generalized juvenile polyposis, a condition that must be included in the differential diagnosis of polyposis syndromes affecting the stomach. The combination of clinical data and histological and molecular features are essential for the diagnosis.


Carcinoma in hyperplastic polyps of the gastrointestinal tract: A report of two cases

J. Orlowska *, D. Jarosz, J. Pachlewski, M. Rupinski, J. Kupryjanczyk, J. Regula

*Oncology Centre Warsaw, Dept. of Pathology, Poland

Objective: Hyperplastic polyps of the gastrointestinal tract (GIT) were considered innocuous for many years. At present malignant transformation of gastric hyperplastic polyps to carcinoma is well documented (WHO, 2010), however, its incidence is very low (2.1 %) 1. On the other hand, it is estimated that colorectal serrated lesions may be precursors of 15 %–25 % of colorectal carcinomas, particularly localized in the proximal colon.

Method: Two unusual cases of focal carcinoma in hyperplastic polyps (stomach and ascending colon) are described.

Results: CASE 1: Gastroscopy performed in 85-year-old woman due to anaemia, revealed a 13 mm pedunculated polyp of the corpus which was endoscopically removed. Histologically it was partially typical for hyperplastic polyp, and partially for conventional adenoma with focal tubular adenocarcinoma (G2) intermingled with signet ring cell carcinoma. CASE 2: Screening colonoscopy performed in a 60-year-old man revealed 16 pedunculated and semipeduculated polyps of 3 to 15 mm distributed throughout the colon which were endoscopically removed. Two of them were hyperplastic, and all but one remaining were tubular adenomas with low grade dysplasia. One 8 mm ascending colon polyp showed a structure typical for hyperplastic polyp with sharp transition to conventional adenoma showing focal invasive adenocarcinoma (G3) with signs of angioinvasion. 1. Orlowska J., et al.: Malignant transformation of benign epithelial gastric polyps. Am J Gastroenterol 1995;90:2,152–2,159.


Gastric adenocarcinoma associated with hyperplastic polyposis

S. Rammeh*, N. Sabbegh Znaidi, A. Blel, R. Aloui, R. Zermani

*Charles Nicolle Hospital, Pathology, Tunis, Tunisia

Objective: We present a case report of a gastric adenocarcinoma associated with a diffuse hyperplastic gastric polyposis.

Method: A 56 years old tunisian male was investigated for dysphagia. The Esophago-gastro-duodenoscopy revealed a mass in the fundus and the antrum stomach associated with numerous gastric polyps filling the entire stomach. He had a total gastrectomy.

Results: Histological exmination showed a moderately differentiated adenocarcinoma associated with numerous hyperplastic type polyps.

Conclusion: The clinical and endoscopic features are important to consider in order to distinguish hyperplastic polyps from hamartomatous polyps, Coden disease and Cronkhite-Canada syndrom.


PTP4A3 (PRL-3) expression correlate with lymphatic metastases in gastric cancer

K. Guzinska-Ustymowicz *, A. Pryczynicz, K. Niewiarowska, W. Famulski, A. Kemona

*Medical University of Bailysto, Pathomorphology, Bialystok, Poland

Objective: Gastric cancers frequently metastasize to the nearest lymph nodes, involving retroperitoneal and left supraclavicular lymph nodes as well as distant organs. Early detection of metastases greatly improves the patients’ prognosis. Recent literature has proved that protein tyrosine phosphatase type IVA member 3 (PTP4A3, PRL-3) plays a major role in the metastasis of gastric cancer, especially to local lymph nodes. The objective of the current study was to assess the expression of PTP4A3 in gastric cancer in correlation with selected anatomoclinical parameters and patients’ survival.

Method: The study was conducted on a group of 71 patients treated surgically for gastric carcinoma and divided according to Lauren’s, Goseki’s, Bormann’s and Kubo’s classifications. The PTP4A3 expression was determined using the immunohistochemical method with mouse monoclonal anti-PTP4A3 antibody

Results: We demonstrated a statistically significant correlation between the expression of PTP4A3 and Kubo’s classifications (p = 0.0454) and on the verge of statistical significance with Lauren’s classification (p = 0.0503). The positive expression of the protein was associated with the poorly-differentiated mucoid carcinoma and diffused-type carcinoma (58 % of cases). Also a very significant correlation was found between local lymph node involvement and positive PTP4A3 expression in primary tumour (p < 0.001).

Conclusion: Our investigations seems to confirm that PTP4A3 may have a significant impact on the lymphatic spread of gastric carcinoma.


Gastric metastases of breast cancer may mimic primary gastric cancer

A. Djikic Rom *, J. Jotanovic, M. Andrejevic, O. Skrobic, M. Micev

*Clinical Centre of Serbia, Dept. of Histopathology, Belgrade, Serbia

Objective: The stomach is an infrequent site of breast cancer metastasis. Metastatic spread to the stomach may occur many years after the initial treatment for breast cancer and it’s very difficult to distinguish it from primary gastric cancer. It is important to make this distinction because of tretment differences between these two entities.

Method: Routine histological and ancillary immnohistochemical examination.

Results: We present a case of 66-years-old woman who had a diagnosis of breast cancer 12 years ago and had left radical mastectomy with subsequent radiochemotherapy. Last 2 years she had dysphagia and regurgitation. Endoscopic examination revealed stenosis of the upper thoracic esophagus and diffuse erosions of mediogastric region. Histologically gastric mucosal biopsy showed microfocal cancerous infiltration mostly in superficial propria which rarely could be seen in the deeper parts of the mucosa. Some of carcinomatous cells revealed obvious signet-ring cell type morphology. Imunohistochemically, most tumoral cells expressed steroid receptors but were negative for GCDFP-15 and mammaglobin. We concluded that tumoral infiltration was consistent with metastatic breast cancer infiltration.

Conclusion: Previous history of other malignancies should be always considered before histopathological examination.


Gastrointestinal lymphomas: 10 years experience of a single center

F. Unal Yildirim *, B. Doganavsargil, N. Ozsan, M. Sezak, M. Tunçyurek, M. Hekimgil

*Ege University Medical Faculty, Dept. of Pathology, Izmir, Turkey

Objective: One-third (30–40 %) of extranodal lymphomas occurs in gastrointestinal (GI) tractus. Although all histological types may develop, small and large B-cell lymphomas predominate, T- and extranodal NK/T- cell lymphomas are less common. Approximately 60–75 % of cases occur in the stomach. Frequency of small intestinal lymphomas shows geographic variation.

Method: We reviewed 177 cases diagnosed between 2002 and 2011, re-classified according to the recent World Health Organization classification.

Results: There was a slight male predominance (female/male ratio was 0,71; 73/104) The median age was 54 years old (Range: 3–90 y.o). Eighty-two percent (n = 145) of cases had only endoscopic biopsies, where 18 % (n = 32) also had resection materials. The most commonly involved site was stomach (n = 126; 71 %), followed by small intestine (n = 26; 15 %) colon (n = 16; 9 %), rectum (n = 6; 3 %), appendix (n = 2; 1 %) and esophagus (n = 1; 0.5 %). The most common subtypes were diffuse large B-cell lymphoma (n = 109; 62 %), MALT lymphoma (n = 31; 17 %) and Burkitts lymphoma (n = 18; 10 %). Rare subtypes were mantle cell lymphoma (n = 6; 3 %), T-cell lymphomas (n = 3; 3 %), follicular lymphoma (n = 1; 0.5 %), plasmablastic lymphoma (n = 1; 0.5 %) and anaplastic large cell lymphoma (n = 1, 0.5 %).

Conclusion: The distribution of histological types and localization were consistent with the literature. Despite geographic predisposition no immunproliferative small intestinal disease (IPSID) cases were identified.


Heterogeneities of HER2 expression and amplification in gastric carcinomas

M. Bamba *, H. Sugihara, S. Takemura, K. Fukuda, M. Masuyama, T. Shigematsu, G. Kato, R. Kushima

*Saiseikai Shiga Hospital, Dept. of Pathology, Ritto, Japan

Objective: Recently, HER2 becomes a molecular target for treatment of gastric carcinomas with a monoclonal antibody trastuzumab. However, evaluation of HER2 expression are not easy because of their intratumoral heterogeneities. In this study, we analyzed heterogeneities of HER2 expression and amplification in gastric carcinomas.

Method: This study was based on the analysis of 72 surgically resected gastric carcinoma cases. First, we performed fluorescence in situ hybridization to select HER2-amplified carcinomas. Then, H&E stain, immunohistochemical stain with HER2 antibody (4B5) and dual color in situ hybridization for HER2 gene were applied in selected carcinomas.

Results: Twelve out of 62 informative cancer lesions were HER2-amplified either homogenously (6 lesions) or heterogeneously (6 lesions). Of these 12 lesions, HER2 overexpression was homogeneous in 2 lesions, heterogeneous in 8 lesions and negative in 2 lesions. Although 3 out of 6 heterogenous HER2-amplified lesions included some non-expressed areas, its amplification was detected not only in virtually all the areas positive for its expression, but also in 18 out of 25 areas (from 10 cases) without its expression.

Conclusion: These findings suggest that even if HER2/neu overexpression cannot be detected anywhere in the lesion, it may possibly be valuable to examine HER2 amplification to extend the application for trastuzumab-therapy in gastric carcinomas.


HER2 amplification in gastric cancer is a rare event restricted to the intestinal phenotype

A. Gamboa-Dominguez *, C. Cruz-Reyes

*INCMNSZ, Dept. of Pathology, Mexico City, Mexico

Objective: Identify HER2 prevalence in gastric cancer and correlate it with location, phenotype and follow-up.

Method: Consecutive gastric cancer patients with tissue blocks, gross and follow-up data, were submitted to immunohistochemistry(IHC) with Herceptest. Chromogenic and fluorescent in-situ hybridization (CISH/FISH) was performed in IHC +ve tumors.

Results: 269 patients were included with a median age of 61 years. In 172 gastrectomized patients histotypes were diffuse (72/41.8 %), intestinal (63/36.6 %), and mixed (37/21.5 %). Her2 IHC expression was 0 in 167, 2+ in 2 and 3+ in 3 tumors. Only endoscopic biopsies were available in 97 patients and Her2 IHC expression was 0/88, 1+/3, 2+/4 and 3+/2 patients. 10/269 (3.7 %) had Her2 amplification. Amplified tumors were intestinal adenocarcinomas located throughout the different regions of the stomach. Heterogeneity was documented in four widely sampled tumors.

Conclusion: Her2 amplification was restricted to the intestinal phenotype; is a rare event and, its screening should be driven by gastric cancer histotype.


Automated HER2 expression and FISH detects all variables in gastric carcinoma for economical quickness targeted therapy

L. Carvalho *, M. R. Silva, A. Alarcão, A. Ladeirinha, T. Ferreira, M. J. d’Aguiar

*Faculty of Medicine Coimbra, Inst. of Anatomical Pathology, Portugal

Objective: Anti-HER2 transtuzumab benefit in Her2 overexpression and amplification in 10–20 % of gastric carcinoma(GC) implies testing. Two scoring systems for immunohistochemistry-IHC and FISH runs in the same fully automated staining system were compared to validate quickness and economical sparing.

Method: Sections of 49 biopsies of GC were either fully automatically stained with monoclonal antibody-based OracleHER2 Bond IHC kit and registered after the breast scoring for GC results and tested for amplification with dual-colour LSI HER2/CEP17 Dual Probe in the automated system BOND-MAX™.

Results: HER2 FISH amplification and overexpression–six cases (13 %) IHC 3+ also positive for FISH; of 2 cases (4 %) IHC 2+, one was FISH positive (HER2/Chr17 = 2.8) and the other was FISH negative (HER2/Chr17 = 1.0) due to polysomy of chromosome 17; 2 cases IHC 1+, were FISH negative (HER2/Chr17 = 1.6 and 1.5).

Conclusion: The automated BOND-MAX™ staining system for IHC and FISH demonstrated overall good quality in observed slides. Our study identified 6 cases with aberrant in situ hybridization polysomy of chromosome 17, negative for HER2 FISH amplification. Described heterogeneous tumor FISH patterns of chromosome 17 aneusomy appear as Her-2/CEP17 low ratio and were classified as negative. The applied methodology revealed to be economical with less manual discrepancy for routine in Pathology.


Minimum biopsy set for Her-2 evaluation in gastro-esophageal adenocarcinoma

F. Grillo *, L. Mastracci, I. Gullo, N. Piol, L. Molinaro, M. Fassan, M. Rugge, R. Fiocca

*University of Genoa, DISC, Histopathology, Italy

Objective: Patients with advanced or metastatic HER2-positive adenocarcinoma of the stomach or gastro-esophageal junction (GEC) can be treated with Trastuzumab. In these patients HER2 assessment is often based only on endoscopic biopsies; immunohistochemical HER2 expression in GEC has however been shown to be highly heterogeneous. The aim of the study was to identify the minimum endoscopic biopsy set required to confidently evaluate HER2-status in GEC.

Method: 103 cases of resected GECs were selected. For each case two different sections were immunostained for HER2 and slides were digitally scanned. By using digital image analysis 10 random virtual biopsies (VB) (2.6 mm diameter), were taken for each case from the luminal part of the sample. HER2-status as defined by biopsy sets composed of an increasing number of VBs was compared to the overall HER2-status.

Results: A minimum of 5 biopsies was shown to be the most accurate in predicting overall HER2-status (sensitivity: 91.89 %; specificity: 96.97 %). Fewer biopsies did not influence the level of specificity while sensitivity varied from 61.54 % for 1 biopsy to 89.19 % for 4 biopsies. Above 5 biopsies no increase in accuracy was seen.

Conclusion: A minimum set of 5 biopsies is required for maximum HER2 assessment accuracy in GEC.


Her2 expression in Turkish gastric carcinomas: Immunohistochemistry and dual-color single in situ hibridization

S. Erdamar *, C. Sonmez, E. Demirhan, N. Kepil

*Cerrahpasa Medical College, Dept. of Pathology, Istanbul, Turkey

Objective: A recent randomized controlled trial (Trastuzumab for Gastric Cancer [ToGA] study) established standard scoring criteria of human epidermal growth factor receptor 2 (HER2) for gastric cancer and demonstrated the efficacy of trastuzumab for treating metastatic gastric cancer. We aimed to evaluate the frequency of her2 positivity in advanced gastric carcinomas.

Method: A total of 218 patients were included in this retrospective study. All tumor samples were examined for HER2 expression by immunohistochemistry (IHC),and HER2 amplification by dual color in situ hybridization (DISH).

Results: HER2-positive tumors were identified in 47 patients (21.5 %). HER2-positive cases were more frequently found in the proximal localization and intestinal histological type (P = 0.04 and 0.030, respectively). There was 98 % concensus between IHC3+ cases and DISH positivity. Only 2 cases were positive in pure signet ring cell carcinomas. 78 % of IHC2+ cases were DISH+.

Conclusion: This study indicate that frequency of HER2 positivity in our series is similar with TOGA study. IHC3 positivity is strongly demonstrate DISH positivity, but the antibody chosen for HER2 IHC is important for analysis.


Does biopsy reliably identify grade in gastro-entero-pancreatic neuroendocrine tumors?

F. Grillo *, M. P. Brisigotti, M. Albertelli, C. Rossi, R. Fiocca, L. Mastracci

*University of Genoa, DISC, Histopathology, Italy

Objective: A new proliferation-based grading system (ENETs/WHO) has proved important in establishing prognosis and guiding therapy in gastro-entero-pancreatic neuroendocrine tumors (GEP-NETs). Biopsies are often performed for grading but little is known about their reliability in assigning grade. The aim of this study was to determine the accuracy of grade identification in virtual biopsies (VBs) of GEP-NETs.

Method: Twenty cases of resected GEP-NETs were selected. VBs of different lengths (3–20 mm) and widths (0.5–1.5 mm) were performed randomly. Number of neoplastic cells and Ki67 index were quantified in up to 10 different areas for each case and for different sizes of biopsies (classified as large, medium or small). Ki67 index in biopsies was compared with that assessed in the whole samples.

Results: Out of all (186) VBs performed, 50.7 % of them undergraded G2 lesions. In all 11 G1 cases all VBs of large size (>2,000 cells) correctly identified grade. In 3 G1 cases the small Vbs (containing <2,000 cells) overgraded the cases as G2.

Conclusion: Clinicians must be aware that grade identified on biopsy material can undergrade G2 cases (likely due to intratumor heterogeneity) or overgrade G1 cases (when little tissue is available for evaluation).


Grade increases in gastro-entero-pancreatic neuroendocrine tumor metastases compared to the primary tumor

F. Grillo *, M. P. Brisigotti, M. Albertelli, T. Borra, L. Mastracci, R. Fiocca, D. Ferone

*University of Genoa, DISC, Histopathology, Italy

Objective: The neuroendocrine tumor (NET) proliferation-based grading system (ENETs/WHO) has proved reliable for prognostic stratification. Whether grade changes between the primary site and metastases has never been investigated. Our aim was therefore to evaluate Ki67-index changes between the primary tumor and its metastases.

Method: A total of 256 GEP-NETs (1993–2011) were identified from the Histopathology archives; 45 of them had tissue from the primary tumor and from local/distant metastases (28 synchronous; 8 metachronous). Immunohistochemistry for Ki67 was performed on both primary and metastatic tumors.

Results: Out of 28 patients with primary NET and synchronous metastases, 22 (78,6 %) presented exactly the same grade. Six (21,4 %) cases showed discrepancy in Ki67-index and grade, 5 changed from G1 in the primary to G2 in the metastasis (nodal, hepatic and mesenteric) while 1 changed from G2 to G3 in the metastasis. Eight patients underwent surgical excision of metachronous metastases during follow up. Four (50 %–2 nodal; 2 hepatic) patients showed an increase in Ki67-index in the metastatic site and a change in grade, from G1 to G2.

Conclusion: Primary NETs and their synchronous/metachronous metastases not infrequently show differences in grade and evaluation of Ki67-index at all sites may be relevant for patient management.


Stromal tumors: GIST (about 13 comments)

L. Beddar *, K. Boudaoud, Z. TEBBI, M. Kout

*CHU Benbadis, Dept. of Pathology, Constantine, Algeria

Objective: GIST represent less than 1 % of all tumors of the gastrointestinal tract. Including the estimated impact is close to two new cases per 100,000 inhabitants per year. The purpose of our work is to study the pathological features and prognostic factors establish stromal tumors

Method: We report 13 cases whose average age is 56 years, with no particular medical history. The location is gastric in 80 % of cases. CT scan examination reveals the presence of large training with irregular contours, often compressing the vascular structures. The treatment is surgical.

Results: It is malignant mesenchymal tumors with cell fuso sometimes epithelioid differentiation, mitoses are always present from (<5 mitoses/50 + fields 10/50 fields at magnification 400). The immunohistochemical study in search of diagnostic certainty shows: -CD117positif in 90 % of cases, intense staining + + cells in 100 %. -Desmin-: negatif -Ki67: positive reaction.

Conclusion: GIST represent a specific nosological entity since the discovery of their relationship with “CAJAL cells,” they are often associated with a mutation in the gene Kit. Prognostic factors are essentially tumor size and mitotic index. Gastrointestinal stromal tumors usually occur in advanced stages because of their silence clinic before they can lead to local complications and sometimes metastasis.

GX10 GIST: immunostaining shows: CD117positif:

figure l


Differential diagnosis of gastrointestinal stromal tumors: Sampling cannot be overemphasized

O. Daum *, K. Peckova, M. Michal

*Faculty Hospital Plzen, Sikl’s Dept. of Pathology, Czech Republic

Objective: To highlight an important pitfall in the differential diagnostics of mesenchymal tumors of GIT.

Method: 79-years-old man underwent a right-sided hemicolectomy due to intestinal obstruction. The resection specimen was routinely processed for histological investigation. In the mesocolon, 17 lymph nodes were found.

Results: Grossly, the tumor sized 13 × 15 × 13 cm showed the typical “fish-meat” sarcomatous appearance. Histological investigation confirmed sarcomatous morphology of the tumor composed of spindle-shaped cells positive only for vimentin and focally for KIT. However, 2 lymph nodes were infiltrated by adenocarcinoma. Furthermore, both KIT and PDGFRA were wt. Therefore, the whole tumor mass was processed in 86 paraffin blocks and finally a focus of adenocarcinomatous differentiation (1 cm in diameter) was found.

Conclusion: Sarcomatoid carcinoma is a great mimicker of GIST. Extensive sampling, although primitive, is a more efficient tool in these cases, than modern sophisticated diagnostic methods.


Syncronous adenocarcinoma and gastrointestinal stromal tumor of the stomach: Report of a case

C. I. Bassorgun *, K. Balaban, M. A. Turkoglu, G. O. Elpek

*Akdeniz University, Pathology, Antalya, Turkey

Objective: “Collision” tumours consist of different neoplasms coexisting within a single lesion. We describe an exceptional case of a combined gastric adenocarcinoma and gastrointestinal stromal tumor(GIST).

Method: We report the case of a 66-year-old male patient who was diagnosed with the continius development of a GIST and adenocarcinoma of the stomach, in which the carcinoma cells focally invaded the GIST.

Results: On histopathologic examination, two completely different tumors were recognized in the stomach. One tumor was a moderatly differentiated adenocarcinoma characterized by glandular structures associated with lymphoid infiltration of the stroma. The other tumor was found to have proliferated in the wall of the stomach, with diffuse palisade spindle-shaped cells. It was diagnosed with the continius development of a GIST and adenocarcinoma of the stomach, in which the carcinoma cells focally invaded the GIST.

Conclusion: Gastric collision tumors are uncommon, and the combination of adenocarcinoma and GIST is extremely rare. According to the literature reviewed to our knowledge there are a few reported cases of synchronous occurrence of adenocarcinoma and gastrointestinal stromal tumors of stomach.


Gastrointestinal stromal tumor risk grade evaluation criteria relationship with disease progression

L. Poskiene *, D. Pangonyte

*LUHS, Pathology, Kaunas, Lithuania

Objective: To determine gastrointestinal stromal tumor (GIST) risk grade distribution based on tumor size and mitotic activity in different area (20, 22 and 50 high power fields (HPF)) and to set optimal area for mitosis counting.

Method: 131 of GIST were studied. Mitosis counted in the 20 HPF (5 mm2) according to WHO and ESMO recommendations; 22 HPF (5,3 mm2) according to data of Miettinen- Lasota/Armed Forces Institute of Pathology; 50 HPF (12,3 mm2) according to data of other studies and recommendations. Mitotic count were divided: I group ≤ 5, II group–6–10, III group > 10 mitosis. Risk grade was established. Outcome data consisted of recurrence or distant metastasis.

Results: The distribution of mitosis groups were identical in the 20 and 22 HPF. There were more cases in the III group of 50 HPF compared to 20 HPF (p = 0.03). While assessing the degree of risk, based on tumour size and mitoses, there were more patients that belonged to the high risk group, evaluating mitoses 50 HPF (p = 0.003), but the progression of the disease in both groups was the same.

Conclusion: The area of 20 HPF (5 mm2) is sufficient for mitotic assessment. One-third of the patients belong to high risk group and only in this group 31,7 % patients disease progressed (13 of 41).


Primary extra gastrointestinal stromal tumors

A. C. Iorgescu *, E. Stoica-Mustafa, C. Pechianu, V. Tomulescu, I. Popescu, V. Herlea

*Fundeni Clinical Institute, Pathology, Bucharest, Romania

Objective: Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms of the gastrointestinal tract which express most frequently KIT protein detected by immunohistochemistry stain for CD117 antigen. Outside the gastrointestinal tract they are called extra-gastrointestinal stromal tumors (EGIST) and they bear the same histopatological and immunohistochemical aspects as their counterparts in the gastrointestinal wall. EGIST are rare neoplasms most of them being detected on the omentum, mesentery and retroperitoneal.

Method: We report a series of ten cases of extra-gastrointestinal stromal tumors, all diagnosed and treated in Fundeni Clinical Institute between 2005 and 2012.

Results: Our patients were 5 females and 5 males with ages between 34 and 74. Six tumors were located retroperitoneal, two in the mesentery and one peritoneal, most of them being high risk forms. IHC tests have been performed and several correlations have been made.

Conclusion: Primary EGISTs are rare, some of them with an aggressive behavior and a very poor prognosis. More data is needed for a proper characterization of the pathogenesis, prognosis and treatment options.


The prognostic value of angiogenesis in Gastro-Intestinal Stromal Tumors (GIST)

R. Basilio-de-Oliveira*, V. L. Pannain, P. E. Portari, C. A. Basilio-de-Oliveira

*UNIRIO, Pathology, Rio de Janeiro, Brazil

Objective: To assessment the angiogenesis by immunohistochemistry with vascular endothelial growth factor (VEGF), intratumoral microvascular density (MVD) by CD105 and CD31 and the Ki67 proliferation index.

Method: The immunohistochemical study (CD34, CD105, KI67 antibodies) was performed in 54 GIST.

Results: GIST showed the following Miettinen’s risk: 3 no risk, 4 very low, 10 low, 14 intermediate and 23 high. Of the 23 tumors with Ki67 > 5 %, 19 showed a reduction in overall survival. VEGF strongly positive was associated with a reduction in overall survival and its absence was found mainly in the tumors with a good prognosis (18/33). Both MVD of CD31 (>2.5 %) and the CD105 (>1.2 %) were related to reduced overall survival. All factors studied showed statistical significance in univariate analysis. Undergoing index Jacard, MDV CD105 showed the best result.

Conclusion: The angiogenic factors showed prognostic ability, especially the CD105, which combined with others such as Ki67 can bring important contribution in understanding the prognosis of this tumor.


Plexiform fibromyxoma of the stomach

K. Stevanovic *

*LNS, Pathology, Luxemburg, Luxemburg

Results: Plexiform fibromyxoma, also known as plexiform angiomyxoid myofibroblastic tumour (PAMT), is a very rare and benign mesenchymal tumour of the antral part of the stomach. We report one case of plexiform fibromyxoma in Luxemburg. A 21-year-old woman presented with symptoms of nausea and stomach pain for a year and a half. An esophagogastroduodenoscopy was performed and revealed a submucosal antral mass. A computed tomography scan of the abdominal region described an invasive tumour suspicious for malignancy. The patient underwent a partial gastrectomy. On the gross examination, the tumour had a multinodular but well circumscribed aspect and measured 6,5 × 3 × 3 cm. Histologically it was composed of bland-looking spindle cells embedded in a myxoid matrix rich in small, thin-walled vessels. The tumour infiltrated the mucosa, the muscularis propria and the subserous tissue of the antrum. The tumour cells expressed alpha-smooth muscle actin and CD10. No expression of CD117 (c-KIT) or CD34 was observed. The proliferation index estimated by Ki67 was low (<2 %).

Conclusion: We present here an interesting case of plexiform fibromyxoma in a young female patient. The main differential diagnosis is the myxoid type of gastrointestinal stromal tumour.


Primary gastric synovial sarcoma: Case presentation and review of the literature

H. E. Torres Rivas *, M. S. Fernández García, M. F. Fresno Forcelledo

*HUCA, Pathology, Oviedo, Spain

Objective: Five to 10 % of the soft tissue sarcomas are Synovial Sarcomas (SS), and only 10 % of these have an unusual location without compromising the limbs/joints. To date, 14 gastric SS have been reported. We have added a case and reviewed the published data.

Method: A 45 year-old male with a large gastric tumor rising from lesser curvature. Hematoxylin-eosin stain revealed a solid neoplasia composed by spindle cells, with elongated nuclei and well-defined cytoplasm.

Results: The immunohistochemical phenotype was consistent with SS (Focal positivity: Cytokeratin AE1/AE3 and EMA. Negative: CD34, c-Kit, Desmin and S100) The molecular study for specific SS translocation X:18 was positive.

Conclusion: Uncommon gastric location represents a challenge to pathologists in order to exclude a gastro–intestinal stromal tumor (GIST), especially when dealing with the monophasic SS subtype, because of their histologic similarities. The number of reported cases of gastric SS still remains small.

Gross features of gastrectomy and neoplasia. (a) Polylobulated yellowish tumor mass, emerging from the lesser curvature, (b) Post-fixation court observing the solid-fiber pattern of the neoplasm.:

figure m


Gastrointestinal leiomyomas of muscularis mucosae–12 cases report with complete histological and immunohistochemical characterization

F. Staniceanu *, C. Popp, S. Zurac, G. Micu, A. Bastian, L. Nichita, E. Gramada, R. Andrei, C. Socoliuc, A. Stoica

*Colentina University Hospital, Pathology, Bucuresti, Romania

Objective: Gastrointestinal leiomyomas of muscularis mucosae (GILMM) are rare benign lesions usually representing incidental findings during endoscopic evaluation or surgical exploration of digestive tract. Although they are the most frequent type of digestive mesenchymal tumors, there are reported only small series of GILMM.

Method: We report 12 (male:female ratio 5:1) consecutive cases of GILMM (3 gastric and 9 colonic) with complete histological and immunohistochemical characterization. We performed routine hematoxylin-eosin and van Gieson’s stains and immunohistochemical assays for smooth muscle actin, S100, c-kit (CD117), CD34, Ki67, p53 and p21.

Results: Except one lesion, all were incidental findings: 2 necroptic findings (one woman with Cowden’s syndrome and multiple benign tumors and one man with cardiac disease and no other tumoral lesions); 2 surgical findings (one associated with an extranodal malignant large B cell lymphoma of the stomach) and 8 endoscopic findings (7 routine investigations and one for rectal bleeding associated with the leiomyoma).

Conclusion: Although rare, GILMM are an important entity, especially since the differential diagnosis with GISTs, leiomyosarcoma and neurofibromas of gastrointestinal tract has a great impact for the patient.


Mixed adenoneuroendocrine carcinoma of duodenum

B. Andrejic *, M. Živojinov, P. Miloševic, T. Boškovic

*Medical Faculty Novi Sad, Dept. of Histology and Embryology, Serbia

Objective: Mixed adenoneuroendocrine carcinoma (MANEC) is a rare, special type of tumour, characterised by an intimate mixture of two histologically different tumours, a neuroendocrine carcinoma (NEC) and adenocarcinoma.

Method: A 50-year-old man presented with a obstructive jaundice. An endoscopic retrograde cholangiopancretography (ERCP) with percutaneous transhepatic cholangiography (PTC) and implantation of endoprothesis was performed. Four days after ERCP/PTC a duodenum with tumor-like mass of the ampulla of Vater was resected along with liver metastases.

Results: Tumor showed two distinct, partly mixed components. The part of the tumor in the mucosal and submucosal layer was a well-differentiated adenocarcinoma. Tumot component, infiltrating mucosa, submucosa and muscular duodenal layer, consisted of a moderately to poorly differentiated round cells (about 60 % of entire tumor mass). Immunoprofile of this component was strikingly chromogranin-positive, moderately synaptophysin-positive with ki67 proliferation rate >50 %, confirming it as high grade NEC. Liver metastases had same immunoprofile. Staining for CK20 was strongly positive in adenocarcinoma and the negative in NEC. According to the current World Health Organization criteria the final pathological diagnosis was poorly differentiated MANEC of the duodenum.

Conclusion: Since follow-up data are scarce the prognosis of patients with MANECs is ill defined, and needs to be evaluated in further studies.


Metachronous metastized duodenal carcinoma in a Lynch syndrome patient: An unusual presentation

J. Ferreira *, R. Cabrera, C. Fidalgo, I. Rosa, P. Lage, M. J. Parreira, A. Bettencourt, I. Claro, A. Dias-Pereira, R. Fonseca

*IPO Lisboa, Serviço de Anatomia Patológica, Portugal

Objective: The prevalence of small intestine carcinoma in Lynch Syndrome, is higher than in the general population but duodenal carcinoma remains very rare. We report a case of duodenal adenocarcinoma in a patient with Lynch Syndrome.

Method: A 71-year-old-woman, with a family history of cancer, carrying a pathogenic mutation in the MSH2 gene, was followed at our institution since 2008. She had been previously diagnosed with an urothelial carcinoma (age 65) and an endometrial adenocarcinoma (age 69). In September 2012, as part of her annual follow-up, an upper gastrointestinal endoscopy revealed a fungating lesion in the duodenal ampulla. Abdominal CT showed a nodule in the nefrectomy space. The patient underwent a Billroth II gastrectomy and resection of the pararenal lesion. She is currently well.

Results: The surgical specimen contained a duodenal polypoid 3 cm tumor. Histology revealed a poorly differentiated adenocarcinoma arising in a tubulovillous adenoma. The tumor invaded the submucosa, had venous invasion and lymph node metastasis. The pararenal mass had identical histology. Immunohistochemical analysis revealed the loss of expression of MSH2 and MSH6 proteins in tumor cells.

Conclusion: According to medical literature, only 3 cases of duodenal adenocarcinoma in Lynch Syndrome have been reported to date.


Adenocarcinoma in solitary Peutz-Jeghers polyp in the duodenum

L. Spasevska *, V. Janevska, N. Jankulovski, B. Dukova, G. Ristovski, A. T. Zrmanovska

*Institute of Pathology, Skopje, Macedonia

Objective: Solitary Peutz-Jeghers-type hamartomatous polyps are rarely diagnosed in duodenum and have lower risk of malignancy. It is consider as a separate clinical entity from Peutz-Jeghers Syndrome (PJS) in relation with risk of cancer in other organ systems. There are few reported cases of focal adenocarcinoma within solitary Peutz-Jeghers-type hamartomatous polyps.

Method: The specimens from standard dissected operative material were formalin fixed and paraffin embedded, stained with hematoxylin-eosin, and immunohistochemically with smooth muscle actin.

Results: We present a case of 68 year old man with preformed pancreaticoduodenectomy (Whipple-resection) and finding of whitish polypoid lesion with nodular surface in duodenum measuring 5 cm in length. Routine dissection revealed malignant neoplasm infiltrating the wall of duodenum and the head of pancreas. Histopathology analysis showed polyp with branching smooth muscle bundles, which were immunohistochemically positive for smooth muscle actin, covered with hyperplastic mucosa and areas with adenomatous component. It also contained well differentiated adenocarcinoma with pushing border pattern involving pancreatic tissue. There was no lymphovascular invasion or metastasis in regional lymph nodes.

Conclusion: Duodenal solitary hamartomatous polyps although associated with low risk of malignancy, we have diagnosed a cancerous polyp. Patients who exhibit solitary Peutz-Jeghers-type hamartomatous polyps should be treated by surgical resection and need additional screening.


HepPar-1 and arginase-1 in small intestinal and ampullary adenocarcinoma

S. Lagana *, S. Hsiao, F. Bao, A. Sepulveda, R. Moreira, J. Lefkowitch, H. Remotti

*New York Presbyterian-Columbia, Dept. of Pathology and Cell Biology, New York, NY, USA

Objective: HepPar-1 and Arginase-1 are urea cycle enzymes used to distinguish hepatocellular carcinoma from other carcinomas. HepPar-1, but not Arginase-1, is known to stain normal small intestine. This study was performed to define the immunohistochemical staining patterns of HepPar-1 and Arginase-1 in adenocarcinomas of the small intestine and ampullary region.

Method: HepPar-1 and Arginase-1 immunostaining was performed on 20 non-ampullary small intestinal adenocarcinomas and 32 adenocarcinomas from the ampullary region. Ampullary adenocarcinomas were divided into intestinal morphology (15), pancreatobiliary (14), and unclassifiable (3). Non-neoplastic small intestinal mucosa and colorectal adenocarcinoma were also stained.

Results: HepPar-1 stained 12 of 20 non-ampullary adenocarcinomas with a median of 63 % of cells staining in positive cases. It also stained 11 of 15 ampullary carcinomas with intestinal morphology, median of 75 % of cells staining in positive cases. Two of 14 ampullary carcinomas with pancreatobiliary morphology were positive for HepPar-1. Arginase-1 was positive in two ampullary region carcinomas in 1 duodenal adenocarcinoma. Two of 22 colorectal carcinomas stained for HepPar-1 with none positive for Arginase-1.

Conclusion: HepPar-1, but not Arginase-1, is often positive in small intestinal adenocarcinoma and ampullary adenocarcinoma with intestinal morphology.


Histiocytic sarcoma of the small bowel

R. Colling *, J. Pawade, N. Wong

*University Hospitals Bristol, Dept. of Histopathology, United Kingdom

Objective: We present a case report of a 48 year old previously fit and well lady with right sided acute abdominal pain.

Method: Ultrasonography showed a 7 cm, highly vascular, soft tissue mass filling and compressing the lumen of the mid ileum. Computed tomography of this lesion was consistent with a gastrointestinal stromal tumour (GIST) and regional lymphadenopathy was also present. A small bowel resection was performed of a polypoid lesion with a fleshy and haemorrhagic cut surface.

Results: Histology revealed a solid tumour arising within the submucosa, invading through the muscularis propria and onto the serosal surface. The tumour was primarily composed of large ovoid cells with abundant eosinophilic cytoplasm and folded irregular nuclei. There were numerous giant cells present. The tumour cells showed positive immunostaining with CD163, CD68, lysozyme, CD45, HLA-DR, CD11c, vimentin and S100. The MIB-1 proliferation fraction was 40 %. There was negative staining for CD21, CD35, CD1a, langerin, HMB45, MelanA, EMA, ALK-1, CD56, CD30, CD3, CD2, CD20, CD79a, desmin, CD117, Dog1, and pan-cytokeratin. Seven lymph nodes were identified and all showed tumour involvement.

Conclusion: A diagnosis of histiocytic sarcoma was concluded and the patient is receiving radiotherapy, for which there has been a partial response so far. Histiocytic sarcoma is a rare, aggressive malignancy which may mimic a wide range of entities clinically and radiologically. The small bowel is an extremely rare site of origin, can be misdiagnosed as GIST radiologically and can present a challenging range of differential diagnoses to the cellular pathologist. The importance of accurate histological assessment and immunohistochemistry in these cases is therefore apparent.

Sunday, 1 September 2013, 09.30–10.30, Pavilion 2

PS-02 Poster Session Endocrine Pathology


Expression of Decoy receptor 3 in diffuse sclerosing variant of papillary thyroid carcinoma: Correlation with M2 macrophage differentiation and lymphatic invasion

W.-C. Chang *, J.-Y. Chen, C.-H. Lee, A.-H. Yang

*Mackay Memorial Hospital, Dept. of Pathology, New Taipei City, Taiwan

Objective: The diffuse sclerosing variant of papillary thyroid carcinoma (DSV-PTC) is a unique variant of papillary carcinoma characterized by extensive lymphovascular invasion of tumor cells. The lymphatic emboli contain tumor cells as well as macrophages. The aim of this study was to determine the relationship between the expression of Decoy receptor 3 (DcR3), recruitment of tumor-associated macrophages (TAMs) and lymphatic invasion in DSV-PTC.

Method: We retrospectively examined 14 cases of DSV-PTC using immunohistochemistry studies. The density of TAMs, lymphatic vessel density, lymphatic invasion, tumor emboli area and DcR3 expression were assessed. Statistical analyses were performed.

Results: The lymphatic tumor emboli contained relatively higher density of TAMs than stroma and classical PTC (CPTC) areas. In addition, the number of lymphatic invasion and tumor emboli area were positively correlated with the number of M2 TAMs. Moreover, DcR3 was expressed only in lymphatic tumor cells and squamous metaplastic tumor cells but not in macrophages and CPTC. In addition, the preferential expression of DcR3 in tumors was associated with higher levels of M2 TAMs and lymphatic invasion.

Conclusion: Our findings suggest that DcR3 expression in DSV-PTC tumor cells may promote the polarized macrophage differentiation towards the M2 phenotype. This phenomenon may further promote lymphatic invasion of DSV-PTC tumor cells.


Ectopic thyroid tissue in the adrenal gland: Report of two cases

J. M. Cameselle Teijeiro *, A. Romero Rojas, M. R. Bella Cueto, I. A. Meza Cabrera, A. Cabezuelo Hernández, D. García Rojo, H. Vargas Uricoechea

*Clinical University Hospital, Dept. of Anatomic Pathology, Santiago de Compostela, Spain

Objective: To describe two very unusual cases of ectopic thyroid tissue in the adrenal gland (ETTAG).

Method: We present the clinicopathologic, immunohistochemical, in situ hybridization, and molecular findings of 2 cases of ETTAG.

Results: The 2 cases presented as incidental cystic adrenal masses in adult females, one having a congenital hernia of Morgagni. The ETTAG was histologically indistinguishable from normal orthotopic thyroid tissue, and its follicular nature was confirmed by immunohistochemical positivity for thyroglobulin (Figure 1), thyroperoxidase, thyroid transcription factor-1, CK AE1/AE3, CK 7, pendrin, human sodium iodide symporter, paired box gene 8 and FOXE1, as well as positivity for the messenger RNA of the thyroglobulin gene by in situ hybridization analysis. No C cells (negativity for calcitonin, cromogranin and synaptophysin) were present. Neither BRAF nor KRAS mutations were detected with real-time polymerase chain reaction analysis. Further work-up did not show evidence of thyroid malignancy.

Conclusion: Although ETTAG pathogenesis remains unknown, the lack of C cells together with the coexistence of a congenital defect of the anterior diaphragm (hernia of Morgagni) in one of our patients could suggest an over-descent of medial thyroid anlagen derived cells in the origin of this heterotopia.


p40 immunoexpression in thyroid

C. Eloy *, R. Celestino, P. Soares, J. Cameselle-Teijeiro

*IPATIMUP, Dept. of Cancer Biology, Porto, Portugal

Objective: In thyroid, primary squamous cell carcinoma (SCC) and mucoepidermoid carcinoma (MEC) disclose squamous differentiation and p63 expression. SCC and MEC are thought to arise from solid cell nests (SCN) or from squamous metaplasia (SM) of the follicular epithelium. SCN and SM are often difficult to distinguish from each other and both express p63.p40 was reported to be more specific than p63 in the diagnosis of lung squamous cell carcinoma but its expression has not yet been reported in thyroid.

Method: We studied p40 immunoexpression in cases of incidentally found SCN (n = 10) and SM (n = 2), and cases of SCC (n = 2) and MEC (n = 2).

Results: p40 was expressed in all SCN, SM, SCC and MEC cases, and was not detected in the remaining follicular epithelium in any of the 16 samples.

Conclusion: In thyroid, p40 immunoexpression is, like p63 immunoexpression, a sensitive and specific tool for the detection of SCN and lesions with squamous differentiation; it does not help, however, in the distinction between SCN and SM, not even to understand their role in the histogenesis of SCC and ME. p40 expression is not exclusive from lung squamous cell carcinoma and appears to be a marker of differentiation rather than an organ specific marker.


Papillary microtumor of the thyroid gland: A validation of the Porto proposal

J. M. Cameselle Teijeiro *, M. J. Ladra González, R. M. Reyes Santías, I. Abdulkader Nallib, F. Barreiro Morandeira

*Clinical University Hospital, Dept. of Anatomic Pathology, Santiago de Compostela, Spain

Objective: Papillary microtumor (PMiT) was proposed in 2003 (Porto Cancer Meeting) to describe an apparently benign group of incidental thyroid carcinomas, ≤10 mm in adults. The aim of the Porto proposal was to avoid the word carcinoma as well as unnecessary treatments. We test this proposal.

Method: Clinicopathological study of 191 papillary microcarcinomas (PMiC) and 103 PMiT (2000–2008, Galicia, Spain), followed for 83.6(48–146) months.

Results: PMiC (191): 34 males, aged 49.37(18–84) years; tumor size 33(4–10) mm; thyroid capsular invasion 3.1 %, vascular invasion 3.7 %; total thyroidectomy 77 % and I131 39.3 %; lymph node metastasis in 14 patients (7.3 %), distant in 2(1.04 %), and 2 deaths. PMiT (103): 17 males, aged 51.72(20–80) years; size 33(3–10) mm; no capsular nor vascular invasion; total thyroidectomy 62.1 % and I131 4.9 %; no metastasis nor deaths. No sex differences comparing the proportion of PMiC/PMiT in relation to the number of thyroidectomies; no increase in prevalence regarding age. Rise in cancer thyroid incidence during period studied due to the PMiC.

Conclusion: PMiT, a clinically benign neoplasm, confirms the Porto proposal. Lack of sex differences in patients with PMiC/PMiT suggests biological differences with clinical thyroid cancer. PMiC is responsible for the increase in incidence of thyroid carcinomas.


A very unusual case of thyroid fat containing lesion: A nodule with papillary hyperplasia and focal atypia

C. Cacchi *, H. M. Arnholdt, A. Bartolazzi

*Klinikum Augsburg, Abt. Pathologie, Germany

Objective: Thyroid fat-containing lesions are not common. Sometimes however, cytological and architectural features suspicious for papillary thyroid carcinoma (PTC) can be detected in benign lesions.

Method: We report a case of 51 years old man referred to surgery for multinodular goiter, showing cystic nodules up to 3 cm. Formalin fixed paraffin embedded blocks were stained with routine Haematoxylin and Eosin and representative blocks were selected for immunohistochemistry.

Results: Microscopically one of them showed papillae with a delicate stroma with recognizable a prominent intralesional mature fat, having focal areas with enlarged, clear nuclei, with grooves and overlapping, without nuclear pseudoinclusions. The lesion didn’t show complete capsule and merges within the rest of thyroid tissue. The nodule expressed a positive reaction for Gal-3 and for HBME-1 immuostaining matching exactly the cells with cytomorphological atypicalitiy. On the basis of that we ruled out the diagnosis of PTC and concluded for an adenomatous nodule with fat-rich stroma and papillary atypical hyperplasia (focal cytological atypia) with molecular evidence of incipient transformation.

Conclusion: The presence of adipose tissue in the thyroid is rare but its presence doesn’t exclude malignancy per se. Therefore accurate integration of morphology and ancillary tests such immunohistochemistry are mandatory for a correct diagnosis.


VEGF, VEGFR1 and microvessel density in papillary thyroid cancer

K. Ivanova *, J. Ananiev, E. Onal, T. Vlaykova, M. Gulubova

*Trakia University, Faculty of Medicine, Dept. of Gen. and Clin. Pathology, Stara Zagora, Bulgaria

Objective: Vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis. Up-regulated VEGF and VEGF receptor1 (VEGFR-1) expression in papillary thyroid cancer (PTC) have been shown to correlate with worse prognosis.

Method: Thirty nine PTC, 8 follicular (FTC) and 10 anaplastic (ATC) thyroid cancer samples were investigated immunohistochemically with antibodies against VEGF, VEGFR-1, CD31, CD1a and CD83-positive dendritic cells (DCs).

Results: We demonstrated that VEGF expression was significantly (p = 0.025) more prevalent in PTCs (92.3 %) than in ATC (60.0 %). CD31+ microvascular density (MVD) was statistically significantly higher (42.3+/−30.3) in PTCs than in FTCs (75.6+/−15.5) (p = 0.044) and in ATCs (34.3+/−20.8) (p = 0.407). PTCs with less CD31+MVD count showed better survival. In PTCs where VEGF expression in tumor cells was higher the immature DCs (CD1a+) were lower in number (2.53+/−4.0) in tumor stroma. Similarly, mature (CD83+) DCs were higher in number in tumor stroma in cases with negative VEGF expression in tumor cells (4.12+/−6.1) (p = 0.104).

Conclusion: In summary, the expression of VEGF and VEGFR-1 in tumor cells of PTCs was related to increased CD31+MVD, poor survival and with decrease of DCs. Therefore, except stimulating tumor angiogenesis VEGF might suppress DCs recruitment or proliferation in tumor stroma, and so to maintain the immune tolerance in thyroid cancer.


Tryptase- and chymase-positive mast cells in thyroid cancer

E. Onal *, K. Ivanova, J. Ananiev, A. Zdraveski, H. Stoyanov, T. Vlaykova, M. Gulubova

*Trakia University, Faculty of Medicine, Dept. of Gen. and Clin. Pathology, Stara Zagora, Bulgaria

Objective: The prognostic value of mast cells in the malignant tumors has not been yet demonstrated even though their hyperplasia was reported mainly at the periphery of neoplastic areas.

Method: We investigated immunohistochemically tryptase- and chymase-positive mast cells (MCT and MCC) in 31 papillary (PTC), 7 follicular (FTC), 7 anaplastic (ATC) and 8 oncocytic thyroid cancer (OTC). CD31+ microvessels (MVD) were also identified.

Results: We demonstrated that mast cell (MCT) density was higher in the stroma of ATC as compared to PTC and FTC. Converslly, in tumor stroma of FTC–MCC number was elevated, followed by ATC, PTC and OTC (FTC vs. PTC, p = 0.023). The MCC number in the stroma of PTC showed tendency for inverse correlation with CD31+ MVD (R = −0.309, p = 0.091) Patients with lower numbers of MCT in tumor stroma (p = 0.066) and in tumor border (p = 0.055) tended to have worse survival. In PTC, MCT and MCC numbers were significantly more as compared to the surrounding normal thyroid structure (p < 0.001). Mast cell role in VEGF expression and tumor development was discussed.

Conclusion: In conclusion, we may state that mast cells in thyroid cancer prevailed in those originating from folicular epithelium, and that their lower numbers may serve as a marker of worse prognosis.


Clinicopathologic features of primary thyroid lymphomas: A single center experience

H. Aki *, D. Uzunaslan, C. Saygin, H. Demir, A. Salihoglu, B. Ferhanoglu, N. Tuzuner

*Cerrahpasa Faculty of Medicine, Dept. of Pathology, Istanbul, Turkey

Objective: Analysis of clinicopathologic features of all PTL patients diagnosed at Cerrahpasa Faculty of Medicine.

Method: Pathology reports, hematoxylen-eosin and Bcl-2, Bcl-6, CD10, MUM1 and Ki-67 stained slides of seven patients diagnosed between years of 2000 and 2012 were retrieved and re-evaluated. Age, gender, year of diagnosis and follow-up data were recorded.

Results: Clinicopathologic features of 7 cases are summarized at Table 1. All of the patients had DLBCL as a histologic type. Three of them (3/7) were GCB subtype and four of them (4/7) were non-GCB. Male to female ratio was 2:5, and the median age at diagnosis was 52.8 years. Median follow-up was 40.8 months. Immunohistochemical features of all patients are summarized at Table 2. All specimens stained positively with CD-20, but none of them was Bcl-2 positive. Two patients were positive for CD 10, four had bcl-6, and three had MUM-1 positivity. All patients had a high proliferation index with Ki-67 immunostaining (mean, 92.14 ± 11.13).

Conclusion: Due to the rarity of PTL, current knowledge on this variant is limited to the data extracted from small case series. Large patient series and prospective clinical studies are necessary to elucidate their true clinical behavior and optimal treatment regimens.


Table 1

Case Gender Age Age at diagnosis Histologic type Overall Survival
1 Female 47 2000 Peripheral Large B Cell
2 Female 82 2004 Diffuse Large Cell 22 month, exitus
3 Male 58 2005 Peripheral Large B Cell, aktive 7 year, alive
4 Male 34 2006 Diffuse Large B Cell 6 year, alive
5 Male 43 2007 B Cell Non-Hodgkin Lymhoma 1 month, exitus
6 Female 43 2007 Diffuse Peripheral Large B cell 5 year, alive
7 Female 63 2012 6 month, alive

Table 2

Case CD20 Bcl-2 Bcl-6 CD10 Mum-1 Ki67 (%) Type
1 (+) (−) (−) (+) (−) 100 GMB
2 (+) (−) (−) (−) (−) 70 Non-GMB
3 (+) (−) (−) (−) (+) 100 Non-GMB
4 (+) (−) (+) (+) (−) 95 Non-GMB
5 (+) (−) (+) (−) (+) 100 Non-GMB
6 (+) (−) (+) (−) (+) 95 GMB
7 (+) (−) (+) (−) (−) 80–90 GMB


Metastatic tumors of the thyroid gland profile: A 15 years study

D. G. Ciobanu Apostol *, C. Preda, A. Grigorovici, R. Danila, L. Ionescu, C. Vulopoi

*UMF”Gr. T. Popa”, Dept. of Pathology, Iasi, Romania

Objective: To evaluate the clinical and pathologic impact of metastases to the thyroid gland (MT).

Method: We studied the pathologic features of 21 patients with MT over a 15-year period (1999–2013) diagnosed in the Department of Pathology of the St. Spiridon Hospital, investigated by fine-needle aspiration biopsy (FNAB), frozen section, routine histopathological exam and immunohistochemical staining.

Results: Eighteen (85.7 %) of the 21 patients presented with an enlarged mass in the thyroid. The average age was of 58.1 years, and sex ratio was F/M = 10/11. FNAB represented a method of preliminary diagnosis in a ratio of 71.42 %, and detected an 80 % malignancy rate. The MT could be identified macroscopically in 18 patients. Lesions were often solitary (9) rather than diffuse (5) or multiple (7). Microscopic examination revealed moderate or poorly differentiated tumors: squamocellular carcinoma (8), digestive and pulmonary adenocarcinoma (7), renal cell carcinoma (2), melanoma (1), and malignant lymphoma Hodgkin and nonHodgkin (3).

Conclusion: MT is uncommon and may be a diagnostic problem. The prognosis of a patient with a secondary malignant tumor to the thyroid is determined by the underlying primary tumor, and if metastatic disease is limited to the thyroid gland, surgery can prolong survival.


Cavernous hemangioma of the thyroid gland: A case report

F. Yilmaz*, A. Kilitci, O. Büyükasik

*Abant Izzet Baysal University, Medicine Faculty, Bolu, Turkey

Objective: Hemangioma is a benign neoplasm of capillary proliferation and stroma that can be found in all organ systems. We report a case of a primary thyroid hemangioma, with nonspesific ultrasonographic(US) appearance. Cytologic dianosis was benign, final diagnosis was made at pathologic examination of he hemithyroidectomy specimen.

Method: Case report: A 24-year-old euthyroid female presented for evaluation of a symptomatic, slowly growing neck mass. She had no history of trauma, fine-needle aspiration(FNA) or other neck procedures. Ultrasound scan revealed a solitary hipoechoic nodule of the right thyroid lobe. Surgery in the form of right hemithyroidectomy was done

Results: At macroscopic examination, most of the right lobe was occupied by a reddish, solid, 3,8 cm lesion. Histologically, the tumor was encapsulated with fibrous tissue and contained many cavernous thin walled vessels, some which were thrombosed. The vascular nature of the structures was confirmed by immunohistochemistry: the cells were positive for the endothelial markers CD34, SMA, Factor8 related antigene and negative for TTF-1. The definitive diagnosis of a benign and isolated hemangioma of the right thyroid lobe was made.

Conclusion: Secondary hemangiomas of the thyroid after cytologic examination are possible, primary thyroid hemangiomas are rare. It is difficult to differentiate cavernous hemangiomas of the thyroid gland from other typical thyroid diseases due to the similar pattern they exhibit on ultrasound. An effort should be made to dissect the thyroid without rupture of these lesions in order to ensure a bloodless procedure.


Anaplastic Hürthle cell thyroid carcinoma with rhabdomyoblastic component: Case report

S. Tatic *

*Institute of Pathology, Belgrade, Serbia

Objective: Anaplastic carcinoma of the thyroid gland is highly malignant tumour. It may have sarcomatoid features and it can simulate many different sarcomas like fibrosarcoma, leiomyosarcoma or rhabdomyosarcoma

Method: We present the case of 49 years old male with anaplastic Hurthle cell carcinoma with rhabdomyoblastic component diagnosed after total thyroidectomy.

Results: Macroscopically, goitre, weighing 300 g, with a solid, partly cystic and haemorrhagic whitish node, 70 × 50 × 50 mm dimensions, occupying both, predominantly left lobe was described. Microscopically, tumour was composed of two components: epithelioid and mesenchymal. Oncocytic cells, in epithelioid component, were CK AE1/AE3+, CK7+, CK19+, TgA+, TTF−1 +, CK20−, CK18+, EMA +/− positive. Mesenchymal cells, with scant eosinofilic cytoplasm, showed intensive vimentine, desmin, INI-1 and myogenin positivity. Proliferative factor Ki67 was 10 time higher in mesenchymal than epitheloid component (50 % versus 5 %). Tumour infiltrates pseudocapsule and surrounding muscle and adipose tissue.

Conclusion: Hurthle cell thyroid carcinoma with anaplastic rhabdomyoblastic component is very rare and has a poor prognosis, which was confirmed in our case, where after total thyroidectomy a relapse occurred after 1 month.


Hyperfunctioning poorly-differentiated thyroid carcinoma: An extremely rare neoplasm

D. Sakonlaya *

*Thammasat University, Faculty of Medicine, Dept. of Pathology, Pathumthani, Thailand

Objective: To report a case of hyperfunctioning poorly-differentiated thyroid carcinoma with metastasis to bones.

Method: Case report of hyperfunctioning poorly-differentiated thyroid carcinoma with radiological and histological studies.

Results: A 43 year-old Thai female had suffered from hyperthyroidism with neck mass and significant weight loss for 1 year. Physical examination revealed multiple irregular, hard nodules involving right thyroid with right cervical lymphadenopathy. Thyroid function test showed thyrotoxicosis. Thyroid uptake was high normal. Functioning bone metastases at right scapula (size 9 × 8.7 × 8 cm) and right pelvis (size 12 × 7.7 × 7.5 cm) were demonstrated by thyroid scan with suspicious liver metastasis. Total thyroidectomy specimen revealed multiple, irregular, tan-white masses with extensive necrosis involving right lobe and isthmus. Histological examination showed multifocal infiltration of moderately to mildly dysplastic cells with endocrine features, predominantly arranging in trabecular with focal insular pattern and extensive necrosis. Vascular invasion and extrathyroidal extension was present. Scattered microfollicles with colloid were occasionally observed. Radical neck dissection specimen showed nodal metastasis and jugular vein invasion. Tumor cells were immunoreactive to: thyroglobulin(100 % of cells); TTF-1(100 %); AE1/AE3(80 %); Ki-67(30 %); cyclin D1(60 %); and Bcl2. After radioactive iodine treatment, patient still suffered from hyperthyroidism due to functioning bone metastases.

Conclusion: A case of hyperfunctioning poorly-differentiated thyroid carcinoma was reported.


Anaplastic thyroid carcinoma and primary thyroid lymphoma: The role of miRNA

A. Fassina *, F. Simonato, M. Siri, R. Cappellesso, F. Marino, L. Ventura, M. Crescenzi, M. Pivato, M. R. Pellizzo, M. Fassan

*University of Padua, Dept. of Medicine, Padova, Italy

Objective: Thyroid lymphoma (TL) and Anaplastic Thyroid Carcinoma (ATC) are rare, accounting for 5 % and 2 % respectively of thyroid malignancy. The majority of TL are of B-cell origin and present in elderly women; ATC are of the follicular origin, occur most frequently in men over 65, and are aggressive. The differential diagnosis of ATC and FL could be challenging and a precise diagnosis is therefore crucial. Recent studies evidenced a specific miR signature able to discriminate ATC from benign lesions.

Method: We retrieved cytological thyroid smears of 22 ATC and 14 TL and we extracted total RNA. We performed RT-PCR on the following miRNA panel: miR222, miR221, miR146b, miR26a.

Results: miR 221, miR 222 and miR26a were significantly over-expressed in ATC respect to TL (both P < 0.01). The ROC analysis estimated the optimal cut-off threshold for discriminating ATC vs TL, for all considered miRs, except for miR 146a, with a sensitivity level higher than 0.80. All the miR ROC thresholds were included in a classification tree analysis, and correctly identified 14/15 ATC (93 %).

Conclusion: Expression profiling of miR 146b, miR 26a, miR 221 and miR 222 is a promising tool for differential diagnosis of ATC and FL, also in scant cytological material.


BRAF V600E mutation is associated with aggressive behaviour in the papillary variant of papillary thyroid carcinoma

A. Corominas-Cishek *, M. Gonzalez, A. Pérez, V. Caamaño, G. Muñiz, N. Cerda, A. Gaafar, J. I. Lopez

*Cruces University Hospital, Dept. of Pathology, Barakaldo, Spain

Objective: To evaluate the impact of BRAF V600 mutations in the prognosis of papillary thyroid carcinoma.

Method: BRAF mutational status was analyzed in paraffin material of a series of 100 consecutive papillary carcinomas diagnosed by the same pathologist in our institution. The study was carried out using cobas® 4,800 BRAF V600 mutation test (Roche). Clinical and pathological data were obtained from the patient’s clinical histories.

Results: Thirty-one cases were mutated. Average age of patients with mutated tumors was 56 years (range, 45–75). Twenty-five mutated tumors displayed the conventional papillary phenotype (83 %), showed a tendency for extrathyroideal extension and presented a higher rate of loco-regional lymphoid involvement. Sixty-nine cases were non mutated, 45 (65 %) of them showing follicular histology. Non mutated group of tumors displayed a less aggressive clinical behavior, 89 % (62/69) of them being organ-confined lesions at the time of diagnosis (43 % were pT1 and 24 % were pT2).

Conclusion: BRAF V600 mutations are associated with conventional papillary variant of papillary thyroid carcinomas. In addition, this mutation is related with an aggressive clinical behavior.


Detection of Epstein-Barr virus-infected B lymphocytes with surface thyrotropin receptor antibodies in the peripheral blood from Graves’ disease patients

K. Nagata *, K. Higaki, Y. Nakayama, H. Miyauchi, Y. Kiritani, K. Kanai, M. Matsushita, I. Murakami, H. Kimura, K. Hayashi

*Tottori University, Dept. of Molecular Pathology, Yonago, Japan

Objective: Graves’ disease is an autoimmune hyperthyroidism caused by thyrotropin receptor antibodies (TRAbs). Because Epstein-Barr virus (EBV) persists in B cells and is occasionally reactivated, we hypothesized that EBV contributes to TRAbs production in Graves’ disease patients by stimulating the TRAbs producing B cells. Several reports have suggested a relationship between plasma cell differentiation of B cells and EBV reactivation. If EBV stimulates the antibody producing cell, the EBV must be present in that cell. We examined whether there were EBV-infected (EBV(+)) B cells with TRAbs on their surface as the membrane immunoglobulin (TRAbs(+)) in the peripheral blood of Graves’ disease patients.

Method: We developed a labeling system for detecting cell surface TRAbs. We stained the peripheral blood mononuclear cells from Graves’ disease patients by this labelling system and fluorescent EBER1 in situ hybridization. We sorted TRAbs(+) EBV(+) double positive (DP) cells by flow-cytometry and confirmed by confocal laser microscopy

Results: We observed granular signals of EBER1 in the nucleus, and red spot of TRAbs on the cell surface. TRAbs(+) EBV(+) DP cells really exist in peripheral blood from Graves’ disease patients.

Conclusion: These results suggested EBV had the possibility to stimulate the host cell’s TRAbs production


Clonal analysis of multifocal papillary thyroid carcinomas using human androgen receptor gene-based assay (HUMARA)

T. Nakazawa *, T. Kondo, N. Oishi, K. Mochizuki, T. Kawasaki, R. Katoh

*Yamanashi University, Dept. of Pathology, Chuo, Japan

Objective: Papillary thyroid carcinomas (PTCs) occasionally form non-continuous tumor foci in the same thyroid gland. It still remains controversial whether individual PTC nodules arise independently (multi-centric occurrence) or result from spread via lymph vessels (intra-glandular metastasis). To clarify which possibility gives an appropriate explanation, we investigated the clonal origin of multifocal PTC.

Method: We examined X-chromosome inactivation patterns of 32 PTC foci using a human androgen receptor gene-based assay (HUMARA) from 14 Japanese women. All PTC foci measured more than 3 mm and were macroscopically evident.

Results: HUMARA revealed to be informative in 13 cases and not in one. Of the 13 informative cases, 11 cases (84.6 %) showed inactivation of the same allele of the androgen receptor (AR) gene between two and three different PTC foci, suggesting a concordant X-chromosome inactivation pattern. Other two cases (15.4 %) revealed that inactivation of AR gene were not demonstrated in same allele, suggesting a discordant X-chromosome inactivation pattern. Statistically, these results indicated that the probability that each tumour shared the same progenitor cells was at least 69.2 % in this cohort.

Conclusion: The higher proportion of concordant X-chromosome inactivation pattern suggests that multifocal PTC can be made more frequently by intra-glandular metastasis than by multi-centric occurrence.


Implied risk of malignancy in a large series of thyroid fine needle aspiration cytology specimens based on the Bethesda system

M. R. Bella Cueto *, R. Orellana Fernandez, N. Combalia Soriano, M. Prenafeta Moreno, S. Barcons Vilaplana, S. Perez Aguilera, G. Marques Villacampa, M. R. Escoda Giralt, I. Capel Flores, M. Rigla Cros, M. Rey Ruhi

*Parc Taulí Sabadell H.U/UDIAT, Dept. of Pathology, Spain

Objective: To determine the risk of malignancy in a series of thyroid fine needle aspiration cytology (FNAC) specimens categorized by Bethesda system, with special interest in subdividing categories III and IV.

Method: In our Department we have been using for many years an equivalent system to Bethesda’s. We present the risk of malignancy obtained by cyto-histological correlation in a series of 896 satisfactory FNAC specimens obtained between September 1989 and March 2013, subdividing category III in Atypia of Undetermined Significance (AUS), Follicular Lesion of Undetermined Significance (FLUS) and Follicular Oncocytic Lesion of Undetermined Significance (FOLUS), and category IV in Follicular Neoplasm or Suspicious (FN), and Follicular Oncocytic Neoplasm or Suspicious (FON).

Results: Risk of malignancy has been: 1,26 % for category II; 20,17 % for category III; 48 % for category IV; 81,8 % for category V; and 100 % for category VI. Subdividing category III showed a risk of malignancy of 50 % for AUS; 16,90 % for FLUS; and 18,03 % for FOLUS; and subdividing category IV, the risk has been 55,76 % for FN, and 30,43 % for FON.

Conclusion: Implied risk of malignancy in our series has been higher than referred in reference literature except for category II, and there are significant differences in categories III and IV when they are subcategorized.


Can subclinical hypothyroidism influence lipid serum levels and thus increasing the cardiovascular disease risk?

F. A. Oliveira *, A. C. Silvestre Morais, H. S. Goetz, R. S. Goetz

*IPTSP/UFG, Setor de Patologia, Goiânia, Brazil

Objective: The present study described the prevalence of subclinical hypothyroidism (SCH) in a Brazilian population sample and the correlation between serum levels of Thyroid Stimulating Hormone (TSH) and lipid fractions.

Method: Medical records from a private practice in Goiania, Goias, Brazil, between February 2008 and October 2012, were retrieved and analyzed. Patients over 18 years old and who were not taking thyroid hormones, medications for thyroid diseases or to control lipid levels were included in the study. Data about age, sex, serum total cholesterol (CT), low density lipoprotein (LDL), high density lipoprotein (HDL), very low density lipoprotein (VLDL), triglycerides (TG) and TSH were collected.

Results: The prevalence of SCH found in the study sample was 8.4 %. No difference of prevalence between the sex groups or increase of TSH levels in the age ranges was observed. In the main group a significant and positive correlation was found between the TSH levels and the CT, LDL, VLDL and TG (p < 0.05). Comparing the euthyroid group with the patients with SCH a significant increase in CT, LDL and HDL was found in the latter group (p < 0.05).

Conclusion: This study showed that a higher level of TSH increases lipid levels resulting in risk of cardiovascular disease. Financial Support: FAPEG, IPTSP/UFG.


Cribriform-morular variant of papillary thyroid carcinoma in a patient with familial adenomatous polyposis. A case report

A. Borda *, E. Szasz, N. Berger, A. Loghin, R. Catana, M. Decaussin-Petrucci

*University of Medicine, Histology, Tirgu Mures, Romania

Objective: Cribriform-morular variant (CMV) is a rare subtype of papillary thyroid carcinoma (PTC) that usually occurs in the setting of familial adenomatous polyposis (FAP).

Method: Our patient, a 41-year-old woman, underwent a total colectomy for FAP 9 years ago. She presented at consultations for a bilateral thyroid mass, and mild swallowing disorders. Neck ultrasound identified multiple nodules in both thyroid lobes. Fine-needle-aspiration was refused by the patient. Consequently a total thyroidectomy was suggested.

Results: On macroscopy multiple whitish well-circumscribed nodules of various sizes were present both in left and right lobes. On microscopy the nodules showed a mixture of architectural features of which the cribriform and dense cellular morules were the most salient features. Most cells were elongated and their nuclei showed cytological and architectural features of PTC. The neoplasm did not show angioinvasiveness, extracapsular extension or lymph-node metastases. CK7+/CK20− immunohistochemical pattern excluded a colorectal origin. Although the thyroglobuline was negative, patient history, architectural/cytological and some immunohistochemical features (TTF1+, HBME1+ in the morules, galectine 3+, CK19+, oestrogen and progesterone positivity) guided the diagnosis to CMV of PTC.

Conclusion: This indolent variant of PTC should be considered in any situation in which a cervical mass appears in a patient with FAP.


Multinucleated giant cells significance: A study of 64 cases of papillary thyroid carcinoma

N. Sabbegh Znaidi *, S. Rammeh, A. Blel, Y. Zidi, F. Ferah, N. Kourda, R. Aloui, R. Zermani

*Hoptal Charles Nicolle, Pathology, Tunis, Tunisia

Objective: Multinucleated giant cells (MGC) are often detected in cases of papillary thyroid carcinoma (PTC). Their origin and significance, however, has not been established.

Method: A series of 64 cases of papillary thyroid carcinoma (PTC) including 33 cases of conventional CPT and 31 cases of follicular variant).

Results: Overall, we identified MGC in 21/33 (63 %) of the conventional CPT versus 8/31 (25 %) of the follicular variant (p = 0.0023). The mean age of patients having PTC with MGC was 39,4 ± 16,14 years versus 43,52 ± 15,86 (p = 0,32). The mean sizes of PTC in cases with MGCs was 29,85 ± 15,05 mm vs 43.05 ± 86,3 (p = 0.03). The cases of PTC with MGCs had higher multifocality rate 11/29 (37 %) vs 4/35 (11 %) (p = 0.012), nodal metastasis (13/23 (56 %) vs 8/23 (34 %) (p = 0.13) and extrathyroidal extension (12/29 (41 %) vs 5/35 (14 %) (p = 0,0001).

Conclusion: Cases of PTC with MGC are significantly more frequent in conventional CPT than in the follicular variant and have more extrathyroidal extension and multifocality.


Gastroenteropancreatic neuroendocrine tumors: A casuistic study

F. Costa *, A. Coelho, J. R. Vizcaíno

*Centro Hospitalar do Porto, Braga, Portugal

Objective: Gastroenteropancreatic neuroendocrine tumors (GEPNETs) represent a heterogeneous group of neoplasms with variable clinical findings and biological behavior, arising from several different neuroendocrine cell types. Many classification systems have been proposed for stratifying the malignant potential of GEPNETs. WHO updated its classification in 2010 and all GEPNETs, either G1, G2 and G3 (Neuroendocrine Carcinoma (NEC)) were categorized as malignant tumors.

Method: We collected and reviewed all the cases diagnosed as GEPNETs from the files of Centro Hospitalar do Porto from 2004 to 2012 and follow-up information was obtained. Immunohistochemical expression of Ki-67 labeling index was assessed and the tumors were recategorized according to 2010 WHO Classification.

Results: Fifty seven cases of GEPNETs were retrieved (age range 18–81 years, mean 55 years; male:female ratio of 1:1,28): esophagus 0 %, pancreas 21,1 %, stomach 26,3 %, small intestine 26,3 %, colorectal 12,3 % and appendix 14,0 %. The distribution of tumors according 2010 WHO Grading Classification was NET G1-70,2 %, NET G2-22,8 % and NEC-7,0 %. Malignant behavior was found in 15,0 % of NET G1, 69,2 % of NET G2 and 100 % of NEC.

Conclusion: Assessment of pathological parameters is crucial in understanding biological behavior of the tumor as well as predicting prognosis of patients with GEPNETs.


Shorter distance of Papillary microcarcinoma (Pmc) to the thyroid pseudocapsule and its relation with recurrence and metastases

A. Gamboa-Dominguez *, V. Rosas-Camargo, F. Candanedo-Gonzalez

*INCMNSZ, Dept. of Pathology, Mexico City, Mexico

Objective: Identify the effect of tumor distance to the thyroid pseudocapsule in microcarcinoma papillary (mcP).

Method: Tumor size, location and its distance to thyroid pseudocapsule was measured. Demographic, clinical, surgical, and 131I administration were obtained from charts.

Results: 1,343 thyroid specimens were evaluated and 511 PTC were identified. 59 (4.39 %) were mcP. Male/female ratio was 1:14 affecting mainly young patients, with multiple tumors (51 %) and involving bilateral lobes (30 %). Median tumor size was 7 mm and cervical metastases observed in 42 % without distant dissemination. Subtotal thyroidectomies were achieved in 50, 18(36 %) with lymph node dissection. All but four patients received 131I. Tumor contact with the pseudo capsule was documented in 13 patients. Ultrasonographic localization of a suspicious nodule was possible in 12. After 10 y follow-up, 55 patients had uneventful course; three presented local recurrences and one patient mediastinal/lung metastases. The four patients who had tumors in contact with the pseudo capsule and presented with local recurrences/distant metastases received 131I and underwent thyroidectomies with lymph node dissection (3 patients)/lobectomy(1 patient). mcP contact with pseudo capsule (p = 0.0016) and extra thyroidal disease at initial presentation (p = 0.02) were adverse factors.

Conclusion: mcP located close to the thyroid pseudo capsule is an adverse histologic finding, and should be explored in larger series using multivariate models.


The value of intra-operative frozen section in thyroid surgery. An analysis of 1110 cases

R. Jouini *, N. Abdessayed, W. Koubâa, A. M’rabet, M. Bel Haj Salah, I. Msakni, I. Harigua, O. Khayat, N. Labbene, E. Ben Brahim, A. Cahdli Debbiche

*Habib Thameur Hospital, Pathology, Tunis, Tunisia

Objective: To review our own experience with frozen section (FS) in thyroid surgery and to assess its value in the management of patients with thyroid disease.

Method: This retrospective study examined the results of 1,110 frozen sections of thyroid specimens analyzed over the 10 year period from 2003 through 2012and their correlations with the final histological examination. Deferred responses were not taken into account for statistical calculations.

Results: In our series, frozen section and final histopathological diagnosis agreed in 85,4 % and disagreed in 5,5 %. 9,1 % of the cases were deferred. The global specificity and sensitivity of frozen section analysis for all histological subtypes were 99,3 % and 64,7 % respectively. Its sensitivity for papillary carcinoma was 61,7 %, 83,3 % for follicular carcinoma and 100 % for anaplastic carcinoma. Discordances were due to 6 false-positive diagnoses and 55 false-negative (FN) diagnoses. 50 % of FN were represented by papillary microcarcinoma. The positive predictive value (PPV) of the frozen section examination was 94,4 % and its negative predictive value (NPV) was 93,9 %.

Conclusion: Our data supports the utility of intraoperative frozen section in the confirmation of malignancy of thyroid nodules. It’s correlated with a high degree of specificity and an acceptable rate of sensitivity. Most of the discordance between frozen section and final histopathological diagnosis was explained by papillary micocarcinoma.


Poorly differentiated carcinomas of the thyroid gland: A review of a series of 65 cases

F. Z. Benserai *, S. AIT Younes, F. Benserai, F. Asselah

*Mustapha Bacha Hospital, Algiers, Algeria

Objective: Controversial entity Spectrum heterogeneous of variants

Method: Review of 65 consecutive cases diagnosed at the departmentof Histopathology from 1997 to 2012 IHC selected cases (LSAB/Tg, CT,ChromogranineA, KI67,TP53, cycline D1, TTF1, Bcl2, Ecadherine)

Results: Invasive thyroid carcinomas 569 cases/Poorly differentiated carcinomas 65 cases (11,4 %) Clinical features: Rapidly growth masses or history of recent growth in a long multinodular thyroid. 44 womenF and 21 men, median age at the diagnosis (49 to 80 old years) The tumours measured 3 to 14 cm (m: 8 cm). Three different histologic patterns insular (37 cases) trabecular (15 cases) and solid (17 cases). Immunophenotypic profile All cases are negative for calcitonin, chromogranin and variable positivity for thyroglobuline.

Conclusion: A histologically poorly differentiated carcinoma subset Showing insular, solid or trabecular patterns predominant has distinct morphological and clinical features. This features often present as high stage tumours with a clinically aggressive course. No morphological or immunophenotypic parameters were of prognotic significance. This carcinomas can occur de novo or preexisting well differentiated carcinomas.


Solid variant of papillary carcinoma: Clinicopathologic features in the Algerian population retrospective study of 30 cases

F. Z. Benserai *, S. Ait Younes, R. Kassa, D. Chilla, F. Asselah

*Mustapha Bacha Hospital, Algiers, Algeria

Objective: Study of Characteristics: epidemiological, morphological, scalable and prognostic contribution of immunohistochemistry

Method: Revaluation of 30 cases of solid variant of papillary carcinoma collected from January 2005 to January 2013.

Results: 4 % of papillary carcinoma. This rare variant was found in 30- of our patients. These are 22 women and 08 men. The average age of patients was 29.5 years _G extreme ages of 14–75 years_G. Half of patient had presented pulmonary and bone metastasis. The tumor developed on a hyperthyroidism, with a toxic multinodular or Graves’ disease. The average tumor size was 4 cm in diameter _G 2.5 cm–8 cm _G with extra thyroidal extension for all patients The histological appearance is specific; characterized by solid sheets and typical nuclear of papillary cancer with wide areas of necrosis.

Conclusion: This variant of papillary carcinoma is dominated by solid sheets of tumor cells with typical nuclear features of papillary carcinoma, is a rare but aggressive.


Are RET and HRAS mutation status in medullary carcinoma and its metastasis always homogeneous? Preliminary results

C. Cacchi *, H. M. Arnholdt, C. J. Haas

*Klinikum Augsburg, Abt. Pathologie, Germany

Objective: The aim of this study was to investigate the prevalence of the mutational status (RET and HRAS) in medullary carcinomas (MTC) and their metastases.

Method: We retrieved 11 cases of MTCs (5 sporadic, 4 hereditary and 2 still unclear) with lymph node metastasis from 2000 to present. Microdissection of representative tissue was performed then DNA was extracted and, after PCR-amplification, the mutational Status of RET (p.M918T) and HRAS (codon 61) was determined by Sanger-sequencing.

Results: We found HRAS mutation neither in primary tumor nor in the lymph node metastases. One case of sporadic MTC and an unclear MTC showed a RET (p.M918T) mutation in both primary tumor and metastasis. In one case o RET wild type MTC the lymph node metastases showed a mutation. Morever in a case of multiple metastases by a still unclear MTC, two metastases were wild type and one of them was mutated.

Conclusion: HRAS mutation is reported to be common in sporadic MTCs lacking RET mutation, however our preliminary results showed no mutation in these cases. Interestingly, the detection of a discordant RET mutation status between primary tumor and metastases indicate that the possibility of heterogeneity may be observed.

Medullary Carcinoma (H&E):

figure n


Predictive value of the Bethesda System Reporting for Thyroid Cytopathology (BSRTC): Should we keep categories III and IV?

R. Silva *, H. Barroca, I. Amendoeira, C. Eloy

*Centro Hospitalar de São João, Dept. de Anatomia Patologica, Porto, Portugal

Objective: BSRTC establishes 6 diagnostic categories associated with a determined risk of malignancy. Our aim was to determine the predictive value of BSRTC in Hospital de Sao Joao.

Method: We collected all the cases of thyroid cytology performed during 2011. The cases were classified according the BSRTC and the cytological diagnoses were compared with those made by the histological examination of the respective surgical specimen.

Results: Of the 1,384 identified patients, 307 underwent surgery. The thyroid cytology of the 307 patients was classified as: 37(12.0 %) I-Unsatisfactory; 125(40.7 %) II-Benign; 36(11.7 %) III-Atypia of Undetermined Significance/Follicular Lesion (AUS/FLUS); 30(9.8 %) IV-Follicular Neoplasm; 12(3.9 %) V-Suspicious for Malignancy; and 67(21.8 %) VI-Malignant. The percentages of cases in which a histological diagnosis of malignancy was made were the following for each diagnostic category: 18.9 % of the I-Unsatisfactory, 3.2 % of the II-Benign, 44.4 % of the III-AUS/FLUS, 40.0 % of the IV-Follicular Neoplasm, 83.3 % of the V-Suspicious for Malignancy and 100 % of the VI-Malignant.

Conclusion: In our series, the BSRTC had a positive predictive value of 1.00 and a negative predictive value of 0.97. The sensitivity was 0.94 and the specificity was 1.00. The similar malignancy risk for group III-AUS/FLUS and IV-Follicular Neoplasm reinforces the impression that it may prove useless keeping both categories.


Diffuse lipomatosis of thyroid: A case report

J. Costa *, J. Pardal, V. Máximo, F. Gonçalves, C. Eloy

*Centro Hospitalar S. João, Serviço de Anatomia Patológica, Porto, Portugal

Objective: Diffuse lipomatosis of thyroid (DLT) is a rare and benign condition characterized by infiltration of the gland by fat tissue.

Method: We report the case of a 46-year old male with hypothyroidism that underwent partial thyroidectomy at age 3. Now, he presented a slow growing mass in the neck. Ultrasound examination disclosed a solid, hypoechoic mass in the right thyroid region that measured 15.0 × 6.5 × 4.9 cm. Fine-needle aspiration biopsies were inconclusive. The patient underwent excision of the mass that weighted 250 g, was yellowish and displayed a lobulated appearance.

Results: Histologically, it was composed by thyroid parenchyma extensively infiltrated by mature adipocytes without atypia. Congo red staining was negative. SDHA immunostaining highlighted the mitochondrial content in follicular cells while SDHB was not expressed. Mitochondrial DNA was sequenced and no mutations were found in any of these genes. The diagnosis of DLT was based on the morphological similarity observed in the specimen of the first surgery (43-years ago) and in the last one.

Conclusion: DLT must be distinguished from others conditions with fat infiltration such as amyloid goiter, adenolipoma and papillary carcinoma. DLT may be related to non-structural molecular mitochondrial alterations at variance with multiple symmetric lipomatosis that can be associated with mitochondrial DNA mutations.


Sarcoid reaction in thyroid and regional lymph node diseases. A peculiar form of tumour and non-tumour response in four cases

A. Hens Perez *, M. Moreno, A. Calvo, M. Añón, G. Muñoz, M. García, Mª d. C. Vázquez, D. Martinez

*Hospital Puerto Real, UG A Patológica Bahía de Cádiz, Puerto Real (cádiz), Spain

Objective: Sarcoidosis is a multisystemic idiopathic disease with clinicopathologic criteria well defined that rarely expressed in the thyroid environment. Sarcoid reaction, as a granulomatous non-systemic lesion similar to those seen in sarcoidosis, exceptionally involves thyroid gland. We report four cases of thyroid and regional lymph node sarcoid reaction in the context of tumour and non-tumour thyroid lesions

Method: We reviewed 2,535 thyroid and regional lymph node surgical specimens registered in our service in the last 15 years, finding four cases of thyroid or regional lymph node with sarcoid reactions, along with a case of systemic sarcoidosis thyroid involvement, which information is presented,

Results: We report four cases of women aged 38–57 years, two of them with a history of papillary thyroid carcinoma, whose main clinicopathologic findings are shown in the table below. All of them enjoy good health with no evidence of tumour or granulomatous disease

Conclusion: Although the association between sarcoid reaction and malignancy is known, a very few cases with papillary thyroid carcinoma are mentioned in the literature, being also exceptional the coexistence with other thyroid lesions or perithyroid lymph node involvement, like the four reported cases

Patient Dates:

1 57 + + +
2 43 + + + +
3 38 + + +
4 55 + + +


Single parathyroid adenoma in a patient with secondary hyperparathyroidism: A case report

D. G. Ciobanu Apostol *, C. Cristea, L. Ionescu, G. Savin, A. Covic, R. Danila

*UMF”Gr. T. Popa”, Dept. of Pathology, Iasi, Romania

Objective: We report the case of a 57 year old women, referred to surgery due to non-dialysis dependant renal hyperparathyroidism.

Method: The pathology diagnosis consisted of frozen sections and routine histopathologic exam. Tissue sections were fixed in 10 % buffered formalin, embedded in paraffin, sectioned at 4-μm, stained with Hematoxylin and Eosin (HE), and trichromic Van Gieson (VG).

Results: An thorough open surgery neck exploration revealed an 3/2/1.5 cm nodular enlargement of the right inferior PT while the other PT glands had a normal aspect. After frozen section confirmation the enlarged right inferior PT was removed and a biopsy was taken from the right superior PT. On multiple sections the first sample presented a thin incomplete capsule separating the adenoma from a rim of uninvolved atrophic gland. Inside the nodule the adipocytes were absent, the pattern of growth was diffuse, mostly with dark chief cells. The second one contained normal parathyroid tissue with chief, oyphil cells and scattered adipocytes. The value of PTH normalised postoperatively.

Conclusion: Although a diffuse hyperplasia involving all PT glands is a feature of renal hyperparathyroidism, in this rare case the macroscopic and microscopic aspects revealed a single PT adenoma, resembling primary hyperparathyroidism.


Clear cell parathyroid adenoma

W. Rekik*, A. Heifa, H. Nfoussi, R. Ben Hammouda, A. Zehani, I. Chelly, A. Kdous, S. Haouet, N. Kchir

*Tunis, Tunisia

Objective: Water-clear cell hyperplasia is a rare but well-documented cause of primary hyperparathyroidism. Parathyroid adenomas of water-clear cell type are exceptionally rare, and only six case reports are available at present in the medical literature.

Method: We report an additional case and the differential diagnoses are discussed.

Results: We report histologic findings of a clear cell parathyroid tumor in a 60-year-old patient. Ultrasound examination of the neck showed a mass region in the posterior aspect of the left lobe of the thyroid. The excised parathyroid was a large mass measuring 3.8 × 2.8 × 1 cm in diameter. It was soft, covered with a thin capsule, did not infiltrate the thyroid parenchyma, and showed no evidence of malignant process. Histopathological examination showed large polyhedral cells with well-defined cell membranes and clear cytoplasm with a small amount of eosinophilic granular material. These clear cells were positive for pancytokeratin and PTH immunohistochemical stains. These results favored a diagnosis of parathyroid Clear Cell Adenoma.

Conclusion: Parathyroid adenomas composed exclusively of water-clear cells are exceptionally rare. Thus, in spite of its low occurrence, this diagnosis must be considered after rejection of the most frequent parathyroid clear cell hyperplasia and parathyroid carcinoma, or depending of the location, clear cell thyroid tumor and clear cell renal carcinoma metastasis.

figure o


A case of ectopic Cushing’s syndrome: Neuroendocrine tumor of the cecum

I. Voronkova *, L. Gurevich, E. Marova, L. Rojinskaya

*Research Center of Endocrinology, Dept. of Pathomorphology, Moscow, Russia

Objective: Neuroendocrine tumors (NETs) of cecum are considered rare tumors. The prevalence of cecum’s NETs is 3,47 % of all gastrointestinal NETs. More rare these NETs have hormone’s hyper secretion. In the available literature, we did not find a case report about a NET of the cecum with ectopic ACTH secretion (EAS).

Method: A 52-year-old woman with EAS after computer tomography of the organs of the abdominal cavity the formation has been revealed in ilcocecal corner. Right hemicolectomy with the tumor and lymphadenectomy were made.

Results: Microscopic examination of the tumor showed NET of the cecum, with the infiltration of all its layers, valvula Bauhini and the mesenteric adipose tissue. The metastasis of a similar structure was found in 4 mesentery lymph nodes. The immunohistochemistry was positive for synaptophysin and chromogranin, ACTH, serotonin. There were 2 types of cells: one with the expression of serotonin, and the other ACTH. Tumor cells were negative for calcitonin, prolactin, corticotrophin-releasing, luteinizing and follicle-stimulating, parathyroid and growth hormones. Index of Ki-67 was equal to 0, despite the aggressive behavior of the tumor. That is why we have not shown the grade of the NET.

Conclusion: In the future we observed for the patient for 1 year with disease remission

NET of the cecum with ectopic Cushing’s syndrome. Expression ACTH (Fig. A) and serotonin (Fig. B) in tumor cells ×20:

figure p


Galectin-3, cyclin D3 immunohistochemistry and tumour dimensions in oncocytic follicular lesions of the thyroid: Definitve results of retrospective study

C. Cacchi *, H. M. Arnholdt, C. J. Haas, M. Bruno

*Klinikum Augsburg, Abt. Pathologie, Germany

Objective: The differential diagnosis between oncocytic follicular adenomas (OAs) and oncocytic follicular carcinoma (OCs) may be challenging on the routine practice. The aim of this study was to evaluate the hypothesis that a combination of the factors tumour-diameter, immunohistochemical expression of Cyclin D3 and Gal-3 is helpful to distinguish between OCs and OAs.

Method: We retrieved, re-examined and reordered the diameter of 40 cases (14 OCs and 26 OAs) from1995 to 2007. Immuhistochemical analysis with Galectin-3 and Cyclin-D3 antibody was performed for each lesion.

Results: The mean value (in cm) of OCs was greater than in OAs (4.1 ± 2.3 vs. 2 ± 0. 8) (P < 0,001). OCs and OAs differ in the percent of positive cells for Galectin-3 25 ± 25 vs. 3 ± 8 (P < 0,001) and for Cyclin D3 46 ± 37 vs 8 ± 13 (P < 0,001). A combination of the two markers (Gal-3 + OR Cyclin D3+) demonstrated an excellent sensitivity (100 %) and a good specificity (85 %).

Conclusion: OCs and OAs differ from tumor dimension. Moreover, our study indicates that the association of Gal-3 and cyclin D3 has an excellent sensitivity and a good specificity, (100 % and 85 % respectively) and suggest these as helpful indicators to distinguish OCs from OAs.

Cyclin D3 in Follicular Oncocytic Carcinomas:

figure q


Sitagliptin treatment prevented pancreatic lesions evolution in a rat model of type 2 diabetes–proposal of antioxidant, antiapoptotic, anti-inflammatory and proproliferative mechanism

C. Mega *, H. Vala, J. Oliveira, F. Teixeira, R. Fernandes, E. Teixeira-Lemos, F. Reis

*Faculty of Medicine Coimbra, Lab. Pharmacology & Exp. Therap., Portugal

Objective: This study aimed to elucidate mechanisms underlying the protective effects of sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, against pancreatic lesions, in an animal model of T2DM.

Method: Male obese diabetic ZDF (fa/fa) rats, 20-weeks-old, were treated with vehicle or sitagliptin (10 mg/kg BW/day) for 6 weeks, and compared with lean control littermates (n = 8/each). Biochemical parameters and lipid peroxidation were evaluated in serum/blood/tissues. Pancreatic lesions were assessed semiquantitatively by routine histopathological and PAS staining methods. Expression in mRNA of apoptotic (Bax, Bcl2, caspase 9), inflammatory (TNFα, IL-1β, IL6), proliferative (PCNA) and angiogenic (VEGF) mediators was assessed by RT-qPCR. Immunohistochemical methods were used to confirm Bax/Bcl2 protein expression. Results are means ± s.e.m. ANOVA and Post-hoc tests were used (P < 0.05 was considered statistically significant).

Results: Sitagliptin treatment of diabetic ZDF (fa/fa) rats, ameliorated biochemical serum/blood parameters, pancreatic lipid peroxidation and diabetic lesions. Immunohistochemistry confirmed antiapoptotic effect observed by reduced expression of Bax/Bcl2 ratio by RT-qPCR. Caspase 9, IL-1β mRNA expression was decreased and proliferative and angiogenic factors overexpressed.

Conclusion: Sitagliptin, in this animal model of T2DM, may derive its protective pancreatic effect by antioxidant, antiapoptotic, anti-inflammatory and proproliferative/proangiogenic mechanisms.


Erythrocytes as target cells of diabetes types 1 and 2

T. Pavlova *, K. Prashchayeu, N. Pozdnyakova, V. Bashuk, A. Selivanova

*BelSU, Dept. of Pathology, Belgorod, Russia

Objective: Is to provide morphofunctional characteristics of erythrocytes s in case of diabetes mellitus (DM) (types 1 and 2).

Method: 37 patients from 28 to 71 years old. Light microscopy, scanning electron microscopy, scanning probe microscopy.

Results: In case of DM 1, only 10–15 % of discocytes were unchanged. 20 % discocytes had multiple excrescences, mulberry-shaped cells were also found among such discocytes. There were discocytes with crest ridges, spherical discocytes, including spherical erythrocytes with excrescences. About 5 % of red blood cells had the shape of deflated balls and were burned-out. In addition, both microcytes (5.49 μm) and macrocytes (8,36 μm) were observed, which was indicative of poikilocytosis. In average, cells (6.5 ± 0.5 μm) were smaller than those of the standard size (7.5). Erythrocytes of the patients with DM 2 were mostly in the form of dicocytes. Poikilocytosis appeared in a lesser degree than in the previous group and was indicated mostly by macrocytes (8.72 μm). Average size of cells was 7.0 ± 0.5 μm. Pic. 1

Conclusion: Erythrocytes are the target cells of DM. The data obtained about morphofunctional changes should be taken into account for therapy prescription, and their dynamics can serve as an option for therapy efficiency control.

Erythrocytes Diabetes mellitus type 1. A. х 6,000 B.х 20,000 Scanning Electron Microscopy. C.D. Atomic force microscopy:

figure r


Ki-67 index assessment in Neuro Endocrine Tumors (NET): Image analysis and pathology routine compared

M.-L. van Velthuysen *, T. Korse, J. Sanders, F. Prins

*AvL, Pathology, Amsterdam, The Netherlands

Objective: We investigate the accuracy of the Ki-67 index assesment for grading NET.

Method: Ki-67 index of 82 tumor specimens of NET was assessed by two pathologists as in routine practice with the eyeballing method and twice by image analysis using ImageJ. For image analysis, photomicrographs of Ki-67 stained slides taken at a magnification of 20 and 40 were used.

Results: The correlation coefficient between pathologists was 0.897. Correlation for the measurements using image analysis was 0.956. Correlation of the pathologists with ImageJ, were 0.955 (20×) and 0.935 (40×) for one pathologist and 0.905 (20×) and 0.922(40×) for the other pathologist. If the Ki-67 index was translated to a grade (<3 % = grade 1, 3–20 % = grade 2 and ≥20 % = grade 3), pathologist 1 assigned a higher grade as compared with image analysis in 10 cases and pathologist 2 assigned a higher grade in 15 cases. Image analysis (20 vs 40) showed discordant grade in 5 cases. These discordances were due to differences in scoring around the cut-off values.

Conclusion: Assessment of Ki-67 index in routine practice by a pathologist correlates very well with Ki-67 index calculated by image analysis. However, grade assignment would be lower if image analysis is used.


Collagen-based dressings loaded with neurotensin repair ulcers in diabetic mice

L. Carvalho *, L. Moura, T. Ferreira, M. J. d’Aguiar, A. Dinis, E. Suesca, S. Casadiegas, M. Fontanilla Duque, H. C de Sousa

*Faculty of Medicine Coimbra, Inst. of Anatomical Pathology, Portugal

Objective: Diabetes foot ulcers (DFU) lead to amputations. Neuropeptides, substance P and neurotensin (NT) act as inflammatory modulators improving healing. Natural polymers, collagen and hyaluronic acid, have been used in wound healing applications. This work aimed to use collagen as a platform for the NT delivery into the wound site.

Method: Diabetes was induced by intraperitoneal injections of 50 mg/kg of streptozotocin during 5 days. Collagen, NT(50 ug/wound/per day), collagen loaded with NT(50 ug/wound/per day) or PBS were placed daily on two dorsal 6 mm excision wounds. Histological analysis(H&E and Masson’s Trichrome) of skin, at days 0, 3 and day of total wound clousure (df), was done.

Results: Day 0: hialin acellular collagen/higher number of cells in skin layers of diabetic mice compared to controls. Day 3: collagen+NT treatment did not contract skin in diabetics and collagen alone stimulated loose collagen synthesis. Day df: NT treatment stimulated PMN/lymphocytes to wound site while collagen dressing stimulated migration of fibroblast/higher deposition of loose collagen. NT+collagen treatment induced higher collagen deposition and recruitment of PMN in diabetics.

Conclusion: NT+ collagen dressing increased cellularization of skin tissue maintaining inflammatory status. Also, higher deposition of loose collagen was observed. In conclusion, NT+collagen dressing could be a possible wound dressing for human DFU treatment.


Expression of vascular endothelial marker CD 31 and carbonic anhydrase IX in carcinoid tumours

K. Kuracinova *, M. Štofíková

*Faculty of Medicine, Bratislava, Slovakia

Objective: Local hypoxia in tumour tissue and level of vascular supply are two interconnected dynamic factors playing a role in behaviour of the tumour. Neuroendocrine tumours in different locations were evaluated for density of vascular endothelial marker CD 31 and expression of carbonic anhydrase IX (CA IX), marker of tumor hypoxia.

Method: Immunohistochemical staining of differentiation antigen CD31 and CA IX membrane protein was performed on 25 neuroendocrine tumours with consequent morphometric analysis and comparison according to their location.

Results: Density of CD 31 positivity corresponded to level of vascularization of the tissue. The average vascularization in the gastrointestinal tract neoplasm was 3.52 %. Neuroendocrine tumours of lung tissue exhibit low degree of vascularity of the average value 1.15 %. From 11 lung carcinoids 81.8 % were positive for CA IX. Gastroenteropancreatic carcinoids expressed CA IX in a much lesser extent, from 14 examined specimens CA IX expression was detected in only 1 case.

Conclusion: Lung carcinoids have low density of vascularization and strong positivity of hypoxic marker. Gastrointestinal carcinoids have higher capacity to initiate neovascularization process than lung carcinoid. It is the task for future research to elucidate the causes of different CA IX expression and vascularization in carcinoids. Supported by grant UK/346/2013. Supported by ITMS 26240220052.


Neuroendocrine neoplasms of the Ampulla of Vater: Clinicopathological analysis of 9 cases

L. Pérez Casanova *, C. Meléndez, T. Serrano, C. Villabona, C. Valls, A. Teulé, J. Busquets, E. Condom

*Hospital de Bellvitge, Pathology, L’hospitalet de Llobregat, Spain

Objective: The aim of this study is to evaluate the prognostic significance of the ENETS grading, TNM staging system and the 2010 WHO classification in neuroendocrine neoplasms (NEN) of the ampulla of Vater (AV).

Method: Nine patients with NEN of AV treated with surgery were retrospectively analysed. Based on grading scheme with mitotic count and Ki67, the ENETS identifies three tumor categories: grade 1 (Ki67 <2 %), grade 2 (Ki67 2–20 %) and grade 3 (Ki67 >20 %). The 2010 WHO classification distinguish between well-differentiated neuroendocrine tumors (NET) grade 1 or 2, and poorly-differentiated neuroendocrine carcinomas (NEC) grade 3.

Results: Two cases showed areas with glandular differentiation and focus of adenocarcinoma were found in 3 cases. Seven patients had lymph nodes metastasis and only one case had liver metastasis. Six patients were classified as NEC and three patients as NET. Half of the patients with ampullary NEC (3/6) (two T2N1, one T3N1) died of disease (mean, 15 months). All three patients with ampullary NET had no evidence of disease after resection (mean follow up 4,6 years).

Conclusion: Both the ENETS grading system and the 2010 WHO classification are valid instruments for ampullary neuroendocrine neoplasms prognostic assessment but not TNM-based staging. The association with adenocarcinoma could suggest a common pathway in the carcinogenesis of these 2 neoplasms.


Tumor-to-tumor metastasis: Uterine leiomyoma with metastasis of neuroendocrine carcinoma

A. Dema *, M. Decaussin-Petrucci, S. Taban, D. Onet, N. Hrubaru, A. Vaduva, R. Cornea, D. Crstici, M. Derban, E. Lazar

*University of Medicine and Pha, Pathology, Timisoara, Romania

Objective: Metastasis of one tumor to another is a rare phenomenon with fewer than 200 reported cases. The aim of this paper is to describe the case of a 59-year-old woman with metastases of large cell neuroendocrine carcinoma (LCNEC) to a uterine leiomyoma.

Method: Nine months before the diagnosis, the patient underwent left lobectomy for thyroid nodules. During surgery preparation for thyroid nodules, a 3 cm pulmonary lesion was discovered, hepatic metastases and increased neuron-specific enolase, calcitonin and serotonin serum levels. While trying to identify the primary site of the tumor, a suspicious lesion was found in the uterus and the patient underwent hysterectomy.

Results: Gross exam revealed a uterine leiomyoma with poorly demarcated, hemorrhagic nodular areas. Microscopically, these areas were composed of undifferentiated large tumor cells, immunohistochemically positive for NE markers.

Conclusion: To our knowledge, this is the first reported case of metastases from LCNEC to a leiomyoma. The diagnosis of LCNEC can be very difficult and it requires additional clinical, laboratory, radiological and immunohistochemical information. This case emphasizes the difficulties in classifying a tumor without NE morphology but with serum and immunohistochemical markers of NE differentiation. Such a phenomenon (tumor-to-tumor metastasis) must be kept in mind to avoid diagnostic errors in unusual looking uterine tumors.


Renal cell carcinoma metastatic to pancreatic endocrine neoplasm: A second case described in world literature

K. Bednarek-Rajewska *, D. Breborowicz, A. Wozniak

*University of Medical Sciences, Chair of Clinical Pathology, Poznan, Poland

Metastatic tumors in the pancreas are rare but well documented events. The tumor- to- tumor metastasis within the pancreas is even more unusual possibility. We report a case of 64- year old male who presented with a tumor of the pancreatic head visualized in CT scan of the abdomen. He had nephrectomy 12 years prior the examination. The patient underwent partial pancreatectomy. Frozen section diagnosis of the pancreatic tumor was endocrine neoplasm. Histopathological examination of paraffin blocks revealed a tumor composed mainly of pancreatic endocrine neoplasm present mostly a the periphery. The central part showed lobules and tubules of clear cells surrounded by delicate branching vessels. The results of immunochistochemical stains were felt to be consistent with a metastatic renal cell carcinoma surrounded by pancreatic endocrine neoplasm and therefore representing an unusual case of renal cell carcinoma metastatic to pancreatic endocrine neoplasm.


Neuroblastoma in adults: A case series

H. Sartelet *, M. Decaussin, M. Latour, V. Combaret, R. Fetni, M. Peuchmaur

*CHU Sainte Justine, Dept. of Pathology, Montreal, Canada

Objective: Neuroblastoma is one of the most common malignancies in children. It most commonly originates in the adrenal medulla, but may arise anywhere within the sympathetic nervous system. Adult neuroblastoma is a exceptionaly rare and poorly recognized entity among tumors originating in the retroperitoneum and abdomen. We present a case series of four neuroblastomas in adults.

Method: The adult neuroblastoma group included two men and two women of 22–53 years of age (mean 34 years). Of the 4 cases, one tumor was situated in the retroperitoneum and three in the adrenal gland. One had stage 1 disease, one had stage 2 disease, and two had stage 4 disease at diagnosis.

Results: Two tumors were classified as poorly differentiated neuroblastoma, one as differentiated neuroblastoma and one as nodular ganglioneuroblastoma. All tumors presented a low mitosis-karyorrhexis index. These cases often showed immunoreactivity for multiple neural markers such as CD56, synaptophysin, neurofilament, and neuron-specific enolase. Fluorescence in situ hybridization analysis was also performed and showed no MYCN oncogene amplification.

Conclusion: Neuroblastoma should be considered as a diagnosis for adult patients with a suprarenal mass and, if confirmed, it should be classified and treated according to pediatric guidelines.


Algerian pediatric adrenal cortical neoplasms: An institutional retrospective study

F. Z. Benserai *, R. Kassa, D. Chilla, F. Benserai, F. Asselah

*Mustapha Bacha Hospital, Algiers, Algeria

Objective: Compare the histological criteria of malignancy in pediatric Adrenal Cortical Neoplasms used the Armed Forces Institute of Pathology (AFIP) score and Weiss’s score.

Method: An institutional retrospective review (2002 to 2013) identified 11 patients ˂ 19 years old with adrenocortical carcinoma (ACC) and adenomas (ACA).

Results: All the patients (6 girls, 5 boys) presented hormone-related symptoms. The tumors ranged in size from 3 to 15 cm (mean: 7.6 cm), the tumors ranged in weight from 25 to 990 g (mean:310 g). According to the Weiss’s score: 03 ACA, 08 ACC; According to the AFIP system: 02 benign, 02 uncertain malignant potential and 07 malignant tumors. There was reactivity with Vimentin, Melan A and Ki-67. All patients underwent adrenalectomy. The follow-up ranged from 10 years to 02 months.

Conclusion: Pediatric Adrenal cortical neoplasms are rare, Their biologic behavior are distinct. Pediatric ACA and ACC can exhibit overlapping morphologic features, which makes distinguishing benign and malignant adrenocortical tumors difficult. The AFIP proposed system may represents a useful method for assessing the behavior of adrenocortical neoplasms in pediatric patients.


A rare case of juxtadrenal schwannoma

E. Souka*, E. Kyrodimou, Z. Kontogianni-Miller, A. Kostopoulou, E. Papaliodi

*G.H.A. G. Gennimatas, Dept. of Surgical Pathology, Athens, Greece

Objective: Schwannoma can rarely develop in the retroperitoneum (0.5–1.2 % of benign retroperitoneal tumors). We present an unusual case of juxtadrenal schwannoma and a mini review of the literature.

Method: A 68 year old asymptomatic female presented with a left adrenal incidentaloma, at abdominal ultrasonography. Physical and endocrinological examinations were unremarkable. She was diagnosed with a non-functioning left adrenal tumor on computed tomography and underwent left adrenalectomy. The macroscopic examination revealed an encapsulated, solid, fibroelastic, yellow-white tumor, in the periadrenal fat, measuring 4 × 3.6 × 3.3 cm. The adrenal gland was tumor free and normal in appearance. On microscopy the tumor was noninvasive and comprised of Antoni type A and B areas, a fibrous capsule and a lymphocytic cuff. The tumor cells were immunoreactive for S-100 and Vimentin. Ki-67 labeling (MIB-1antibody) of tumor cells was 1–2 %.

Results: The diagnosis was Juxtadrenal Schwannoma. A subgroup of the retroperitoneal schwannomas is located in the periadrenal fat (juxtadrenal schwannoma) and is often diagnosed preoperatively as non-functioning adrenal tumor. Macroscopically they are separate from the adrenal tissue, which appears normal. Microscopically and immunohistochemically, they are similar to the usual schwannomas. Differential diagnosis includes extra- gastrointestinal stromal tumor, solitary fibrous tumor, leiomyoma, neurofibroma, nodular fasciitis and schwannoma of the adrenal medulla.


Angiosarcoma of the adrenal gland: A case report

A. Dimitriadi *, E. Kyrodimou, C. Karabogias, G. Koutsonikas, T. Choreftaki

*General Hospital of Athens, Surgical Pathology, Greece

Objective: We report a rare case of primary adrenal epithelioid angiosarcoma.

Method: A 62 year old male patient presented with abdominal pain. Computed tomography and Magnetic Resonance Image revealed a large mass of right adrenal gland. The patient underwent adrenalectomy.

Results: Macroscopically, the mass had a brownish color with median diameter 10 cm, was cystic with extensive hemorrhage and central necrosis. Microscopically, neoplastic cells were recognized in limited areas with a diffuse pattern and sparsely formed tubules. The mitotic rate was up to 5/10 High Power Field. Immunohistochemically,these cells were reactive for vimentin,CKAE1/AE3,CK7,CD31. Differential diagnosis includes a variety of vascular neoplasms (primary and metastatic), carcinoma, melanoma, adenoma with extensive hemorrhage, as well as reactive processes with fibroblasts.

Conclusion: Angiosarcoma of the adrenal gland is a rare vascular that poses differential diagnostic dilemma with primary or metastatic tumor, especially when there is extensive hemorrhage in an adrenal mass.


Oncocytic adrenocortical tumors: Study of four cases

E. Minaidou *, A. Dimitriadi, E. Souka, E. Kyrodimou, T. Choreftaki, E. Papaliodi

*General Hospital of Athens, Surgical Pathology, Greece

Objective: We report four cases of oncocytic adrenocortical tumors (OACTs): two cases were malignant, one indeterminate and one was an adenoma, according to Weiss criteria (modified by Aubert).

Method: All patients, of age 50 to 62 years old, presented with non-functional “incidentalomas”. After resection, the tumors weighted 39–68 g with median diameter 5–9,5 cm.

Results: Microscopically, the neoplasms consisted of variably atypical cells with abundant granular eosinophilic cytoplasm and prominent nucleoli. Necrosis, vascular invasion, high mitotic index were not observed. Immunohistochemistry was performed for anti-mitochondrion, inhibin-A, Mart-1, SYP, CgA, Calretinin, Cam5.2, CKAE1/E2, EMA, Ki-67 (MIB-1), p53 and TopoII and histochemistry for reticulin.

Conclusion: OACTs are a very uncommon and heterogeneous group some of which may be overdiagnosed as carcinomas, as Weiss score 3 is attained easily given their innate morphology. In the last years, there is a big interest in these types of tumors as in their pure form are low grade tumors with a more indolent clinical course as compared with conventional adrenocortical carcinomas, while reticulin seems to help in non underestimating malignancy.


Adenomatoid tumor of the adrenal gland: Case report

N. Chaleplidis*, C. Karabogias, E. Kyrodimou, G. Zografos, T. Choreftaki, E. Papaliodi, Z. Kontogianni-Miller

*General Hospital of Athens, Surgical Pathology, Greece

Objective: We present a case of an adenomatoid tumor (AT) of the adrenal gland.

Method: An incidental mass was found in the left adrenal gland of a 30-year-old male patient who underwent a Computed Tomography for abdominal pain. We received an adrenalectomy specimen, 6,5 cm in diameter, weighing 15 g. After sectioning, a circumscribed, whitish, soft, solid, focally micro-cystic tumor was recognized, 3,7 cm in diameter in the cortex. The rest of the adrenal gland retained its medullo-cortex architecture.

Results: Microscopically, the tumor consisted of anastomosing tubular, adenoid, solid, cystic and papillary formations, which extended to the peri-adrenal adipose tissue. They were lined by flattened or, mostly, epithelioid cells with abundant eosinophilic, focally micro-vesicular, cytoplasm. Cytological atypia, nuclear pleomorphism, or evident mitotic activity, were not observed. The tumor showed strong immunoreactivity for Calretinin, CKAE1/AE3, CK7, and Vimentin, weak and focal reactivity for CK5/6, and was negative for CD31, CD34, FVIII, CK20, EMA, CEA, Desmin, Inhibin, CD10, RCC, TTF-1, WT-1, p53, HBME-1. Proliferative index Ki-67 (MIB-1) was estimated at 2–3 %.

Conclusion: AT’s are benign tumors of mesothelial origin, rarely encountered extra-urogenitally, that have a broad differential diagnosis, including vascular neoplasms, mesothelioma and metastatic adenocarcinoma.


Ganglioneuroma of the adrenal gland: Report of two cases

V. Theodorou *, B. Christoforidou, P. Xirou, E. Goupou, N. Vladika, F. Patakiouta

*Katerini, Greece

Objective: Adrenal ganglioneuromas are rare, benign neoplasms of the primordial neural crest tissue.

Method: A 36-year old female presented with low-back pain and a 68-year old male with abdominal pain/constipation and weight-loss. Abdominal CT and MRI at both patients revealed a mass at left adrenal gland and adrenalectomy was performed. Grossly, both tumors (maximal diameter 12 cm and 6 cm) were well-circumscribed, encapsulated with homogenous, rubbery to gelatinous consistency and gray-white colour.

Results: Microscopically, both tumors consisted of spindle cells with wavy nuclei in a fascicular arrangement within variable amount of collagen admixed with single or small nests of mature ganglion cells. Myxoid stroma and mature fat tissue cells were also present. Immunohistochemically, in both tumors spindle cells were S-100 protein positive and ganglion cells were NSE, Synaptophysin and Neurofilament proteins positive.

Conclusion: Adrenal ganglioneuromas, due to their presentation as “incidentalomas” with non-specific clinico-radiological features, represent a pre-operative challenge. Surgical excision and histopathological confirmation are the gold standard for treatment and diagnosis.


Epitheliod angiosarcoma of the adrenal gland: A case report

I.-P. Efstratiou *, E. Pazarli, S. Pervana, D. Alataki

*Papageorgiou General Hospital, Dept. of Pathology, Thessaloniki, Greece

Objective: We report a rare case of a primary adrenal angiosarcoma.

Method: A 70-years-old woman presented with abdominal pain and sciatica leading to the discovery of an 11 cm diameter right adrenal mass. The tumor was compressing the renal pelvis resulting in hydronephrosis. She underwent right nephrectomy and adrenalectomy. The adrenalectomy specimen consisted of a well circumscribed ovoid mass with extensive hemorrhage surrounded by a rim of flattened adrenal tissue.

Results: Histological examination revealed epithelioid angiosarcoma arising in the adrenal gland. Immunohistochemically the large neoplastic cells showed positive reactivity for the vascular marker CD31,vimentin and pankeratin AE1/AE3. The ki67-proliferation index was 25 %. The postoperative course was uncomplicated. Three years after the initial surgery the patient is free of metastatic disease or recurrence. No chemotherapy was administered.

Conclusion: Primary angiosarcomas of the adrenal gland are very rare vascular tumours. Since the first description in 1988 about 30 cases have been published. The age range of the patients was 39–85 years with a mean of about 60 years. Because of cytokeratin positivity epithelioid angiosarcoma can mimic a metastatic carcinoma. Although generally angiosarcomas are considered as highly aggressive malignancies, angiosarcomas of the adrenal gland have a variable prognosis.


A case of giant myxoid adrenal cortical carcinoma with lipomatous metaplasia

S. Gurzu *, T. Bara, T. j. Bara, I. Jung

*University of Medicine and Pharmacy, Dept. of Pathology, Tirgu-Mures, Romania

Objective: To present a rare variant of adrenal cortical carcinoma diagnosed in a young women and the criteria used for its diagnosis

Method: A 36-year-old female presented with symptoms suggesting Cushing’s syndrome. The abdominal computed tomography revealed a tumor located in the left adrenal gland. Left adrenalectomy was performed

Results: Macroscopic examination of the surgical specimen revealed a 17 × 11 × 4 cm-sized encapsulated tumor weighing 1.2 kg, with a nodular structure, necrosis and hemorrhages on cut section. Histopathologic examination showed a heterogeneous aspect, the tumor cells being either arranged in clusters and cords or presenting diffuse architecture. Most of the cells were large, polygonal-shaped, with eosinophil cytoplasm. About 30 % of the tumor stroma had a myxoid aspect. Among the tumor cells, focal metaplastic lipomatous zones were evidenced. Immunohistochemically, the tumor cells were positive for Vimentin, Inhibin, CD56, and Melan A and negative for Chromogranin, Keratin AE1/AE3 and Epithelial membrane antigen. The molecular examinations did not detect K-ras or EGFR mutations. The final diagnosis was myxoid adrenocortical carcinoma with lipomatous metaplasia

Conclusion: In case of myxoid adrenocortical carcinoma, the diagnosis is mainly based on correlation of clinical aspects with hematoxylin eosin being only confirmed using immunohistochemical staining


Incidental adrenal neuroma: A case report

N. Can *, E. Tastekin, S. Isler, M. Azatcam, Z. Peglivanoglu

*Trakya University, Pathology, Edirne, Turkey

Objective: Neuroma represents proliferation of peripheral nerve fibers in which ratio of axons to Schwann cell fascicles approaches 1:1 and it is usually seen in soft tissues. Adrenal gland is a rare location of neuroma. Herein, we report an incidental adrenal gland located neuroma.

Method: A 65 years old male patient presented with abnormal kidney functions due to left sided grade 3 hydronephrosis and he was performed left simple nephrectomy. Grossly, nephrectomy specimen was surrounded by an adrenal tissue sized 5 × 3 cm along the upper pole. Microscopical examination of the renal tissue revealed chronic pyelonephritis but multiple unencapsulated spindle cell proliferations without Anthoni-A and Antoni-B areas were remarkable in the slides of adrenal medullary tissue. These spindle cell proliferations revealed S-100 and NSE positivity and there was no reaction with SMA, Melan A, Chromogranin A in immunohistochemical studies. So that, these unencapsulated spindle cell nodules were diagnosed as neuromas.

Conclusion: Neuromas are benign soft tissue tumors originated from peripheral nerve fibers. They are usually located in soft tissues and also mucosal neuromas can be seen, rarely. According to our knowledge soft tissue tumors such as Schwannoma can be found in adrenal tissue but neuroma is very rare. So that, we represent a rare localisation of neuroma and a rare soft tissue tumor originated in adreanal tissue in this report.


Crosstalk between pituitary adenomas blood vessels and their hormonal profile

B. Balinisteanu *, A. M. Cimpean, R. A. Ceausu, M. Raica

*Deta, Romania

Objective: To correlate pituitary blood vessels phenotype, microvessel density and the immunohistochemical hormonal profile as well as the hormone values in serum and cerebrospinal fluid.

Method: Thirty nine specimens obtained from patients with pituitary adenomas were included in our study. Immunohistochemical stains with specific antibodies against each of the pituitary hormones were performed on the selected specimens. The endothelial cells were labelled with anti CD34 antibodies and the perivascular cells with smooth muscle actin antibodies (SMA).

Results: Microvessel density was highest in GH secreting adenomas and lowest in prolactinomas. Based on a double immunostainig CD34/SMA, a predominance of immature and intermediate blood vessels was observed. We found a significant correlation between the immature blood vessels and the expression of tissue prolactin assessed by immunohistochemistry (p = 0,044) and partial correlations between serum (p = 0,036), cerebrospinal prolactin values (p = 0,006) and immature and intermediate blood vessels and between mature blood vessels and cerebrospinal fluid values of prolactin (p = 0,008).

Conclusion: Our study sustains the effect of prolactin on endothelial and perivascular cells, thus prolactin being directly involved in pituitary adenomas angiogenesis. The assessment of pituitary adenomas blood vessels phenotype correlated with their hormonal profile might partially explain the clinical and biological discrepancies found in these tumors.

Sunday, 1 September 2013, 09.30–10.30, Pavilion 2

PS-03 Poster Session Head and Neck Pathology


Carcinosarcoma arising in a pleomorphic adenoma of lacrimal gland: A rare tumor

H. Rodrigues *, C. Oliveira, M. J. Julião, M. F. Xavier Cunha

*CHUC, Anatomia Patológica, Pardilhó, Portugal

Objective: Tumours of the lacrimal gland are rare (<1/million individual year), half of them have epithelial origin. Pleomorphic adenoma is the most frequent (50 %) and the malignant transformation rarely reported.

Results: A 78 years male with retro-orbitary lesion in the left eye known since 2002 clinicaly suspicious of an “inflammatory pseudotumour”. In 2011 he presented with pain and sudden proptosis. Biopsy reveals a malignant mesenchymal neoplasia. Total exenteration of the left orbit was perfomed and a 4,3 cm tumour whitish and firm was found. Microscopic examination showed a neoplasm with areas of pleomorphic spindle and polygonal cells, areas of chondrosarcoma and carcinoma. Sections of the periphery of the tumour showed pleomorphic adenoma with transition to malignant areas and a small amount of lacrimal gland tissue. Epithelial component showed immunoreactivity to CK7, AE1/AE3 and Cam 5.2; Vimentina and focally SMA were positive in the mesenchymal component. Diagnosis was carcinosarcoma arising in a pleomorphic adenoma.

Conclusion: Carcinosarcoma of the lacrimal glands is an extremely rare tumour. We performed a literature review and to our knowledge only three cases are reported. Aggressive surgical excision is recommended with adjuvant radio and chemotherapy. Our patient died shortly after surgery.


Intranasal encephalocele: Presentation of two cases

N. Can *, E. Tastekin, E. Isler, A. Aslan, M. Azatcam

*Trakya University, Pathology, Edirne, Turkey

Objective: Nasal Encephalocele (NE) represents herniation of brain tissue and leptomeninges through bony defect of skull. NE is an uncommon diagnosis of nasal polyps and usually presents in older children and adults with equal gender distribution. Herein, we report two cases diagnosed as NE by histological, radiological and clinical evidence.

Method: Case 1: A 38 years old female patient presented with headache and rhinorrhea during the last 2 years. MR imaging revealed herniation of meningeal tissue through the wall of sphenoid sinus and polypoid lession in ethmoid sinus. The patient was performed anterior-posterior ethmoidectomy. Histological examination exhibited sheets of neuroglial tissue which revealed immunohistochemical reactivity with S-100 and GFAP. Case 2: A 56 years old female patient presented with rhinorrhea during the last month. MR imaging was reported as a right paramedian mass accompanied by fronto-nasal encephalocele. The patient was performed anterior-posterior ethmoidectomy and endoscopical excision of the mass. Neuroglial tissue which revealed immunohistochemical reactivity with S-100 and GFAP was seen in microscopical examination

Conclusion: Although NE is very rare the pathologists should be aware in the evaluations of nasal polyps.


Respiratory epithelial adenomatoid hamartoma in maxillary sinus: A case report

K. Koulia *, A. Tsavari, E. Moustou, E. Arkoumani, A. Zizi-Sermpetzoglou

*General Hospital Tzaneio, Dept. of Pathology, Piraeus, Greece

Objective: Respiratory epithelial adenomatoid hamartoma (REAH) is a rare lesion. Until today fewer than 50 cases have been reported in the literature. These lesions commonly site in the nasal cavity, but may occur in the nasopharynx and paranasal sinus. The exclusive involvement of the maxillary sinus is quite rare. This benign lesion originate from the Schneiderian epithelium, but not from the seromucous glands. Over 80 % of the patients with REAH are males, aging from third to ninth decate of life.

Method: A year-old male presented with a history of 2 years of cacosmia and recurrent rhinosinusitis. The patient underwent endoscopic sinus surgery to remove polyps. The speciment was sent for biopsy.

Results: Histologically, the lesion was composed of small to medium sized glands, separated by thickened, hyalinized stromal tissue. The glands were round to oval and were composed of multilayered, ciliated respiratory epithelium. A diagnosis of REAH was set.

Conclusion: Maxillary sinus localization is very rare, thus very important to distinguish a REAH from schneiderian pappilomas of the inverted type and adenocarcinomas. The treatment for REAH is complete local excision. Our patient is free of recurrence symptoms in 18-month follow-up.


Cell-cycle protein expression in respiratory epithelial adenomatoid hamartoma

M. Rito *, F. B. Santos, R. Fonseca, I. Fonseca

*IPO de Lisboa Francisco Gentil, Serviço de Anatomia Patológica, Portugal

Objective: Respiratory epithelial adenomatoid hamartoma (REAH) is a rare lesion, its putative neoplastic nature being recently suggested. Our objective is to review their clinicopathological profile and investigate immunophenotypic expression of proteins known to be involved in the tumorigenesis of other sinonasal tract neoplasms (p16, p53, MLH1 and MSH2).

Method: Eight cases of REAH were retrieved (2002 to 2010). Clinical data was obtained from the patients charts. Histological features were reviewed and p16, p53, MLH1 and MSH2 immunoexpression was evaluated.

Results: Patients mean age was 66 years with female predominance (n = 6). Lesions were unilateral in 5 cases and were associated with inflammatory polyps in 4 and with Schneiderian papilloma in 1. All patients underwent conservative local resections and no recurrences were recorded (mean follow-up time: 15 months). The 8 cases exhibited positive immunostaining for p16 (focal = 3, moderate = 4, diffuse = 1) and p53 (focal = 4, moderate = 4). MLH1 and MSH2 were normally expressed in all cases.

Conclusion: Our findings confirm REAH clinical features and benign course. Cell cycle-related proteins expression supports the previous evidence of the non-hamartomatous nature of this entity.


E-cadherin, b-catenin, p53, p16 and COX-2 expression in 98 intestinal-type sinonasal adenocarcinomas

B. Vivanco *, J. Perez-Escuredo, J. L. Llorente, M. Hermsen, F. Fresno

*HUCA, Dept. of Pathology, Oviedo, Spain

Objective: Intestinal-type sinonasal adenocarcinoma (ITAC) is an infrequent epithelial cancer. The aim of this work is to study the expression of several proteins in ITAC and in healthy tissue controls, and to figure out its prognosis implications.

Method: Immunohistochemical analysis of 98 ITAC was conducted on a tissue microarray and in healthy tissue samples.

Results: According histological subtype, p53 expression is less frequent in mucinous ITAC (p = 0,001) and weak E-cadherin expression is more frequent in this subtype (p = 0, 05). Loss of b-catenin expression and high p16 expression are associated with shorter overall survival (Fisher-exact test: p = 0,015 and 0,003/Multivariate Cox Regression: p = 0,011 [4,548HR (CI: 1,321–8,983)] and p < 0,001 [5,073HR (CI:2,044–12,594)]) and higher metastatic frequency (p = 0,008 and 0,044). P53 and COX-2 expression are related (p = 0,005) as well as b-catenin and E-cadherin expression (p = 0,012). Healthy tissue shows normal E-cadherin and b-catenin expression, and infrequent p53, p16 and COX2 expression.

Conclusion: Mucinous ITAC shows different immunohistochemical profile. Loss of E-caherin and b-catenin axis and high p16 expression are independent prognostic factors. P53 and COX2 are thought to be related to inflammatory-mediated carcinogenesis.


High-risk human papillomavirus is transcriptionally active in a subset of sinonasal squamous cell carcinomas

L. Alos *, B. Larque, S. Hakim, A. Nadal, A. Diaz, M. del Pino, L. Marimon, I. Alobid, A. Cardesa, J. Ordi

*Hospital Clinic Barcelona, Dept. of Pathology, Spain

Objective: The aim of the study was to assess the HPV active transcription in a series of sinonasal carcinomas, in correlation with the HPV DNA identification and the p16 immunohistochemistry.

Method: Seventy patients with squamous cell carcinomas of the sinonasal tract were included in the survey. All tumors were investigated for HPV through the HPV DNA detection by polymerase chain reaction (PCR), using the SPF10 primers and by in situ hybridization (ISH), using the high-risk GenPoint probe (Dako). HPV16 E7 mRNA transcripts detection was performed by PCR in 27 cases. The immunostaining for p16 was performed in all cases.

Results: Fourteen carcinomas (20 %) were positive for high-risk HPV by PCR: 13 HPV16 and one HPV35. ISH showed a dotted nuclear positivity in all these cases. HPV16 E7 mRNA was detected in 7 tumors harbouring HPV16; in the remaining HPV-positive cases RNA did not reach the quality for analysis. Strong, diffuse positivity for p16 was observed only in the HPV- positive cases.

Conclusion: We have shown that HPV is the etiological agent of a subset of sinonasal carcinomas demonstrating the transcriptionally-active HPV in these tumors. Immunostaining for p16 can be used as a surrogate marker to identify these tumors.


Expression of Pax5 and TTF-1 in Olfactory Neuroblastoma (ON)

P. Czapiewski *, P. Adam, J. Dzierzanowski, K. Okon, W. Biernat

*Medical University of Gdansk, Dept. of Pathology, Poland

Objective: Pax5 and TTF-1 play role in development of B-lymphocytes, and thyroid and lungs, respectively. They are expressed in human fetal brain, several brain tumors and some high grade neuroendocrine carcinomas. ON is an uncommon tumor of the nasal cavity that derives from olfactory neuroepithelium and shows features of neuroendocrine differentiation. The aim of our study was to identify expression pattern of Pax5 and TTF-1 in ON.

Method: We included 11 cases of ON (7M, 4 F, aged 46–86) into the study and analysed immunohistochemical expression of TTF-1 and Pax 5.

Results: TTF-1 was shown in 3/11 (27,3 %) cases as a nuclear pattern with mild to strong intensity, present in less than 1 % (n = 1) up to 30 % (n = 2) of cells. Pax5 was not identified in any of 9 analysed cases.

Conclusion: TTF-1 can be expressed in ON and, therefore, may present a diagnostic problem in metastasizing cases. Its clinical value requires further evaluation.


Dermoid cyst: A rare entity of the parotid gland

S. Toprak *, T. Ozgur, H. Gokce, C. Cevik, E. Akbay, E. Atik

*Mustafa Kemal University, School of Medicine, Dept. of Pathology, Hatay, Turkey

Objective: Dermoid cyts of the head and neck are benign lesions and they are extremely rare in the parotid gland. The clinical and radiologic presentation of dermoid cysts are ambiguous and preoperative diagnosis is difficult.

Method: A 53-year-old male presented with a 1-year-old swelling of the area of the right parotid gland to the Otorhinolaryngology Department outpatient clinic. Physical examination showed a soft, non-fluctuant mass in the inferior portion of the parotid gland. Computed tomography revealed a well-defined nodular mass with 2 × 2 cm diameter that was hypodense in the central region.

Results: The patient underwent right superficial parotidectomy and the material has been sent to pathology laboratory. In macroscobic examination there has been a soft 2.5 × 2 × 2 cm tan cystic mass holding on to the inner surface of the parotid gland with yellow oily material inside. Microscobic features included a cyst lined by stratified squamous epithelium and there has been skin appendages in the subepithelial stroma confirming the dermoid cyst diagnosis.

Conclusion: The differential diagnosis of dermoid cysts is large and includes mucous retention cyst, benign mesenchymal tumors, benign salivary gland tumors like pleomorphic adenoma, Hodgkin disease. To avoid recurrence dermoid cysts need careful surgical intervention.


Dermoid cyst of parotid gland: A case presentation

S. Mocan *, A. Iacob, D. Milutin, T. Mezei, D. Nekula, C. Petrovan

*Emergency Clinical Hospital, Dept. of Pathology, Targu Mures, Romania

Objective: Among different type of cystic lesion of the major salivary gland, dermoid cysts are described generally occurring midline in the floor of the mouth, with rare reported lesions in the parotid gland.

Method: We present a case of 26 years old women with a slow growing mass in the left parotid region. After a clinical and computed tomography evaluation a fine needle aspiration was performed. She underwent a superficial lobectomy of the left parotid gland and the lesion was surgically removed.

Results: Cytological specimen obtained from fine needle aspiration showed abundant eosinophilic debris some of which appeared to be keratin, with numerous squamous and foreign body giant cells. Also, fragments of hair were easily recognized. The resected specimen contained a collapsed, cystic lesion, with partially disrupted wall, of 3,5 cm diameter. Microscopic examination revealed a dermoid cyst with squamous epithelium on the surface and abundant skin adnexa. In the disrupted wall, the epithelium was replaced by many foreign body giant cells.

Conclusion: Although dermoid cysts can be easily recognized by microscopic examination, fine needle aspiration cytology can be helpful in recognizing this entity before surgical intervention. This case report shows the challenges in diagnosis and gives a short review of the literature.


Perineurioma of the parotid gland

A. Yesilirmak *, K. Kosemehmetoglu

*Hacettepe University, Dept. of Pathology, Ankara, Turkey

Objective: Mesenchymal soft tissue tumors are uncommon in the salivary gland. A 22-year old woman presented with a slowly growing unilateral parotid gland mass that was excised completely by partial parotidectomy.

Method: Routine pathological examination and immunohistochemistry were performed.

Results: Partial parotidectomy revealed 1,5 × 1 × 0,7 cm sized, grey-white, firm, well circumscribed, but unencapsulated, solitary nodule. Microscopically, tumor, arising in the parotid gland and extending to the peripheral fat tissue, was composed of spindle cells forming storiform pattern around peripheric nerve bundles and vascular structures. The tumor cells had eosinophilic bipolar cytoplasm and ovoid, bland nuclei without atypia and were associated with thick collagen fibers. Immunohistochemically, neoplastic cells were diffusely positive for EMA and focally positive for GLUT-1, collagen IV and SMA. S100, CD34, desmin and PR were completely negative. Differential diagnosis included perineuroma and meningioma. The absence of intranuclear inclusions, negativity for PR and GLUT-1 positivity supported the diagnosis of perineuroma, instead of meningioma. Electron microscopy was requested.

Conclusion: To our knowledge, this case represents the second reported case of soft tissue perineurioma in the salivary gland. Previous reports of parotid meningiomas should be reviewed for a possible misdiagnosis of perineurioma, although discrimination may be arbitrary.


Cytomorphology of mammary analogue secretory carcinoma of salivary glands: Report of two cases

S. Tommola *, I. Kholová, R. Karikoski

*Fimlab Laboratoriot Oy, Dept. of Pathology, Tampere, Finland

Objective: Mammary analogue secretory carcinoma is a recently described salivary gland neoplasm. Only small series and case reports on cytological features have been reported to date.

Method: Case 1: 56-year-old female presented with 1,5 cm parotid gland tumorous mass. Case 2: 34-year-old male revealed mass in submandibular gland of 1.2 cm diameter. Fine needle aspiration was performed twice under ultrasound control in both cases. Centrifuged material was stained with Papanicolaou stain. Additionally, immunocytochemistry was performed on cell-block material. The final histopathological diagnosis was revealed from resected gland.

Results: Aspirates were mildly to hypercellular. Cells formed tubular structures, smaller groups and nests. Cells varied slightly in size and shape. Cytoplasm was mildly vacuolated. Focally, oncocytic cytoplasm was detected in case 1. No myxoid material on background was noticed. Immunocytochemically, cytokeratin-7, EMA, vimentin, mammaglobin and S-100 were positive. MIB-1 proliferation index was 3 % (case 1) and less than 1 % (case 2). Low-grade salivary gland neoplasm was suggested diagnosis. The surgical specimen final diagnosis was mammary analogue secretory carcinoma.

Conclusion: Mammary analogue secretory carcinoma is a newly described salivary gland entity. Cytological differential diagnosis includes acinic cell carcinoma, mucoepidermoid carcinoma and pleomorphic adenoma.


Invasive carcinoma arising in sclerosing polycystic adenosis of the salivary gland

R. Canas Marques *, A. Félix

*Rio de Mouro, Portugal

Objective: Sclerosing polycystic adenosis (SPA) is a rare salivary gland lesion, recently regarded as a neoplastic lesion of low-grade malignant potential but to date no invasive carcinoma, metastasis or associated mortality has been reported. We report the first case of an invasive carcinoma arising in a recurrent SPA lesion.

Results: A 55-year-old man, with a past history of radiotherapy in childhood for a right lower lip lymphangioma, and three surgical excisions on the right parotid gland for recurrent lesions of SPA was submitted to a total right parotidectomy with ipsilateral neck dissection. Morphologically, the gland was occupied by multinodular lesions of SPA, with similar features as previous excised lesions. Arising in one of the nodules an invasive adenocarcinoma, with an in situ component was identified. Immunostains confirmed invasion by the absence of myoepithelial cells. The surgical margins were free of tumor and no nodal metastases were found. The patient was treated with additional local radiotherapy and, currently, is well and without recurrence, after 4 years of follow-up.

Conclusion: This case represents the first documented invasive carcinoma arising within a relapsing SPA highlighting the awareness of the potential malignant behaviour of this recently described neoplastic lesion.


Xanthogranulomatous sialadenitis: A potential mimicker of salivary gland malignancy

A. Agaimy *, B. Märkl, J. Zenk, M. Michal, A. Skalova

*Universität Erlangen-Nürnberg, Inst. für Pathologie, Germany

Objective: Xanthogranulomatous inflammation is an uncommon tumor-like subtype of chronic inflammation that has been mainly reported in kidney, gallbladder, testis and other less common sites. To date, only a few case reports exist on xanthogranulomatous sialadenitis (XGSA).

Method: We retrospectively reviewed our files for cases of XGSA. Inflamed cystadenolymphomas, cases post-FNA and re-resections with secondary inflammation were excluded.

Results: Nine patients were identified (6 males and 3 females aged 43–84 year; mean: 58 year). The parotid gland was affected in 8 cases and the submandibular in one. None was bilateral. Clinical diagnoses were mostly suspicious parotid gland tumor or swelling. Most lesions measured ≥2 cm and had poorly circumscribed margins. One lesion was confined to an intraparotid lymph node. Two cases showed no evidence of a preexisting salivary gland lesion/cyst (idiopathic XGSA). The remainder showed variably sized cystic ductal lesions suggesting cyst rupture as a trigger of secondary XGSA.

Conclusion: XGSA is probably significantly underreported. A thorough search for a pre-existing lesion is mandatory for appropriate classification. Idiopathic XGSA should be distinguished from other systemic granulomatous diseases and from salivary gland malignancy, particularly hematological neoplasms.


Epithelial predominant carcinosarcoma of salivary gland misdiagnosed as achromic melanoma

F. Baderca *, H. Urechescu, M. Pricop, E. Lazar, C. Solovan

*Timisoara, Romania

Objective: Carcinosarcoma of the salivary glands is a rare and aggressive tumor first described by Kirklin in 1951. We describe a case of a 52-y-o man who presented himself in January 2012 for an enlargement of the left parotid gland.

Method: The tumor appeared 1 month before and grew progressively. The clinical inspection revealed a 3 cm diameter mobile nodule, relatively well delimited.

Results: The microscopic evaluation showed islands of spindle and epithelioid malignant cells with pleomorphic vesiculous nuclei, eosinophilic macronucleolus and intranuclear inclusions in a mixoid stroma. Mitotic index was 45 mitoses/10HPF. Lack of a sarcomatous component and the development of tumor in proximity of skin, raised the suspicion of achromic melanoma. Immunohistochemical reactions sustained the diagnosis of carcinosarcoma versus melanoma. After 8 months, patient returned with a 1.5 cm diameter nodule at the site of surgical incision, with clinical suspicion of granuloma. The histopathology showed a tumor resembling the initial lesion but with areas of sarcomatous differentiation (condroid and osseous formation).

Conclusion: Even if the diagnosis of carcinoma of salivary gland is usually straightforward, the importance of immunohistochemical reactions in cases of epithelial predominant carcinosarcoma of salivary gland remains.


Isolated late metastasis from testicular seminoma presenting as a parotid gland mass: Case report and review of the literature

J. Künzel *, A. Agaimy, S. Krause, M. Vieth, C. Alexiou

*Universtätsklinik Erlangen, Germany

Objective: Parotid metastases from non head and neck cancers are rare and may represent a diagnostic and therapeutic challenge. A late metastasis from a seminoma to the parotid gland is presenting an unusual manifestation of disease.

Method: Case report and review of the literature

Results: A 45-year-old male patient with a history of testicular seminoma 5 years previously presented with a rapidly progressive parotid mass. Ultrasonography and computed tomography showed a space-occupying lesion at the angle of the right jaw. The mass was infiltrating into the parotid gland and into the parapharyngeal space. A primary parotid neoplasm was suspected, and panendoscopy with combined open biopsy was performed. Histological examination confirmed a seminoma metastatic to the parotid gland, and comparison with the primary tumour showed identical histology. The patient received chemotherapy for recurrent seminoma in accordance with the PEI protocol (cisplatin, etoposide, ifosfamide). After a total of four courses of chemotherapy, salvage radical parotidectomy with removal of all suspicious residual tumour tissue was then performed.

Conclusion: This case illustrates the difficulties that may be encountered in the differential diagnosis of parotid gland masses and underlines the necessity for a detailed clinical history and the need for strong interdisciplinary collaboration between oncologists and pathologists in order to correctly diagnose cases with such an unusual presentation.


Metastasis of Renal Cell Carcinoma (RCC) to large salivary glands: Description of 4 cases

H. Majewska *, K. Radecka, A. Skalova, D. Stodulski, C. Stankiewicz, W. Biernat

*Medical University of Gdansk, Dept. of Pathology, Poland

Objective: Metastatic tumors involving salivary glands arising from non-head and neck area are very rare. Renal cell carcinoma (RCC) is known for its high propensity for early metastasis. RCC metastasis to the maxillofacial area is a scarce event (16 %), however metastasis to the salivary gland are extremely rare. We report 4 cases of such pattern of dissemination.

Method: Four cases of metastatic RCC were selected from the historic files of salivary gland neoplasms operated between 1992 and 2012 in the Departments of Otolaryngology and Maxillofacial Surgery, Medical University of Gdańsk. The origin of the tumors was confirmed immunohistochemically, as vimentin, CD10 and RCC antigen were positive.

Results: The group included 3 females and 1 male. The age ranged from 66 to 97 years (mean 76,7 years). The tumours involved: parotid gland (3 cases) and submandibular gland (1 case). The size of tumours ranged from 2,6 to 5 cm. Total parotidectomy with selective neck dissection (2 cases) or simple resection of the tumor was performed (1 case). Histologically, the tumors were clear cell carcinomas. Differential diagnosis mainly included clear cell myoepithelial tumors, oncocytic lesions and clear cell adenocarcinoma.

Conclusion: The parotid was the initial manifestation of renal malignancy in 3 cases (1 status unknown). RCC, albeit rare, should be taken into differential diagnosis in clear cell tumors of the salivary glands.


Study of INI1 expression in four cases of myoepithelial carcinoma of the salivary glands

M. Caldas *, D. Felizardo, M. Jácome

*Ist. Português de Oncologia, Dept. de Anatomia Patológica, Porto, Portugal

Objective: Myoepithelial carcinoma of the salivary glands is defined as a neoplasm composed almost exclusively of cells with myoepithelial differentiation. A lesion with similar differentiation also occurs in the soft tissue. In myoepithelial carcinomas of soft tissue, INI1 expression loss is described in up to 40 % of cases in children and in up to 10 % of cases in adults and has not been detected in benign myoepithelial neoplasms. The aim of our study is to determine the pattern of INI1 expression in myoepithelial carcinomas of the major salivary glands.

Method: Cases of myoepithelial carcinoma of the major salivary glands diagnosed between 1998 and 2012 were retrieved from the files of the Department of Pathology, Portuguese Oncology Institute, Porto. Available HE and immunohistochemical slides were reviewed. Immunohistochemical staining for INI1 was performed on representative sections.

Results: 4 cases of myoepithelial carcinoma were retrieved, all located in the parotid gland. The patients were 3 male and 1 female, age range 76–85 years. All cases showed nuclear expression of INI1.

Conclusion: In our series, INI1 expression is preserved in myoepithelial carcinomas of the salivary gland.


Carcinoma ex pleomorphic adenoma: A clinicopathologic review of ten cases

A. Yagüe Hernando*, J. M.ª Elizalde Eguinoa, P. Fernández Seara, E. Almudévar Bercero, A. Echegoyen Silanes, Y. Ruiz de Azúa Ciria, S. Rázquin Lizarraga

*Complejo Hospitalario Navarra, Surgical Pathology, Pamplona, Spain

Objective: Carcinoma ex Pleomorphic Adenoma (CEPA) develops from primary and recurrent Pleomorphic Adenoma (PA) and constitutes about 12 % of all malignant tumors of salivary glands. The benign mixed tumor component is often hialinized, whereas the malignant component can be of different histologic type, usually undifferentiated or adenocarcinoma NOS. We retrospectively review ten cases of CEPA diagnosed at our institution in the past 6 years.

Method: Of the ten patients aged between 42 and 80 years old, 7 were women and 3 were men. Primary PA was located in the parotid gland (6 cases), submaxilary gland (3 cases) and palate minor salivary gland (1 case).

Results: Adenocarcinoma NOS (4 cases) and Mioepithelial carcinoma (3 cases) were the most common histologic subtypes followed by salivary duct carcinoma (2 cases) and sarcomatoid (1 case). High histologic grade was found in 50 %. Half of the cases showed capsular invasion, whereas two of them where minimally invasive (<1.5 mm of capsular invasion) and three of them showed no capsular invasion.

Conclusion: The term Carcinoma ex Pleomorphic Adenoma is not sufficient as a stand-alone diagnosis. Recommendations for reporting these tumors should include: histologic type/grade, % of carcinoma and extend of invasion of the carcinomatous component (intracapsular, minimally invasive and invasive).


Carcinosarcoma ex-pleomorphic adenoma of the submandibular gland: Case report and review of the literature

E. Borg*, V. Attard, B. Alexandra, P. M. Speight

*Mater Dei Hospital, Dept. of Histopathology, Msida, Malta

Objective: Carcinosarcomas of the submandibular gland are extremely rare. They can arise either de novo or from malignant transformation of a pleomorphic adenoma (carcinosarcoma ex pleomorphic adenoma).

Method: We report a case of an 89 year old lady who presented with a longstanding, slowly growing swelling in the right submandibular area. A fine-needle aspirate was suggestive of a pleomorphic adenoma. The submandibular gland was subsequently excised. The literature was searched for cases of carcinosarcoma of salivary glands and the data was tabulated.

Results: The resected mass was solid with a pale, firm cut surface. Microscopy and immunohistochemistry showed a carcinoma with foci or residual pleomorphic adenoma and areas of atypical spindle cell proliferation consistent with sarcoma. A diagnosis of carcinosarcoma ex pleomorphic adenoma was rendered. The literature search revealed 101 reported cases of carcinosarcoma with no sex predilection. Of these only 22 cases had arisen in the submandibular gland. Most cases arose de novo with only a small minority representing malignant transformation of a pre-existing pleomorphic adenoma.

Conclusion: Carcinosarcoma ex pleomorphic adenoma of the submandibular gland is an extremely rare tumour associated with a poor prognosis. Most patients will experience metastasis, recurrence and a poor prognosis despite optimal therapy.

Macroscopic appearance of Carcinosarcoma ex pleomorphic adenoma Submandibular gland:

figure s


Genomic profile study of pleomorphic adenoma and carcinoma ex-pleomorphic adenoma by array-comparative genomic hybridization

F. V. Mariano *, L. P. Kowalski, O. P. de Almeida, R. D. Coletta, A. C. Victorino Krepischi, A. Altemani

*Faculty of Medicine—UNICAMP, Pathology, Campinas, Brazil

Objective: In this study we analyzed pleomorphic adenomas (PA) without malignant transformation and carcinoma ex-pleomorphic adenomas (CXPA) composed of tumors in different stages of carcinogenesis (intracapsular, minimally and frankly invasive), and classified according to histopathological subtypes by array-comparative genomic hybridization (array-CGH).

Method: Five PAs and eight CXPAs were evaluated. Tumor and reference DNA were hybridized to a 180k oligonucleotide array (Human CGH Microarray Kit 4 × 180K, Agilent).

Results: The PAs showed prevalence of gain in chromosome 8, loss in 17q and 19q. All alterations in PAs were maintained in CXPAs, with additional gain and losses. A different genomic profile were found in all CXPAs and within of same histological subtype. It was not observed progression of alterations with increase of tumor invasiveness. The most repeated alterations in CXPAs were gain and losses in chromosomes 2, 5, and 8.

Conclusion: In comparison to PAs, CXPAs showed an increase of number genomic alterations and the events in chromosomes 2 and 5 can be important for malignant transformation process. It was not possible to identify a particular pattern genotypic for each histological subtype and for stage of carcinogenesis. However more cases need to be investigated.


In vitro influence of extracellular matrix in myoepithelial cells from pleomorphic adenoma stimulated by epidermal growth factor

V. Montalli *, A. Altemani, V. C. Araújo, N. S. Araújo, E. F. Martinez

*State University of Campinas, Pathology, Brazil

Objective: The extracellular matrix-cell interactions as well as growth factors are known to be essential not only for the normal development but also its role in the tumorigenesis process.

Method: The present study aimed to in vitro evaluate the effect of EGF, in different concentrations (5, 10 and 20ŋg/mL) on cell morphology as well as the viability of benign myoepithelial cells, under the influence of different extracellular matrix proteins (matrigel, type I collagen, fibronectin). It was also evaluated the immunophenotype of these cells by α-smooth muscle actin and vimentin immunoexpression.

Results: The myoepithelial cells exhibited polyhedral morphology in all extracellular matrix conditions independently of the growth factor supplementation, except in the presence of EGF on polystyrene surface, where the cells exhibited a spindle-shaped morphology. In addition, no significant statistical alteration on cell proliferation was observed in the myoepithelial cells under influence of the studied doses of EGF. SMA was immunostained in the cells independently of EGF supplementation.

Conclusion: The extracellular matrix did not exert an important role on morphology, proliferation nor immunophenotype of the myoepithelial cells, even under the influence of EGF. These results may suggest that EGF might act in the neoplastic process (Grants from FAPESP 2011/10366-7, 2011/51112-8)


Polymorphous low-grade adenocarcinoma remains a challenging diagnosis: Our experience at a cancer institute

D. Felizardo *, A. Luis, F. Menezes, N. Coimbra, M. Caldas, A. Galaghar, M. Jacome

*Portuguese Oncology Institute, Dept. of Pathology, Matosinhos, Portugal

Objective: Polymorphous low-grade adenocarcinoma (PLGA) is a unique tumor which arises mainly in the minor salivary glands. Adenoid cystic carcinoma (ACC) is one of the most relevant differential diagnoses. Regrettably, discriminating value of immunohistochemistry remains controversial. Herein we report our experience, highlighting distinctive morphological and immunohistochemical characteristics.

Method: Cases of intra-oral PLGA and ACC diagnosed between 2006 and 2012 were reevaluated. All PLGAs were compared against an equal number of ACC cases. Key distinctive morphological aspects; ki-67 (using computer-assisted analysis) and calponin staining were assessed.

Results: In a total of 24 minor salivary gland malignant neoplasms, we retrieved 3 PLGA cases (13 %). One case was locally aggressive and had nodal metastasis. Ki-67 staining was lower in PLGAs (mean value 1.8 %; 9.1 % in ACCs). Calponin was focally positive in 1 and negative in 2 PLGAs and it was positive in all ACCs. Noteworthy histological distinctive features of PLGA were architectural diversity and bland cytology.

Conclusion: Most PLGAs are diagnosed in General Hospitals and only a small fraction arrives to specialized institutions. This would justify our lower percentage of PLGA (13 %) versus literature described proportion (26 %). Some PLGAs present unusual aggressiveness. Finally, immunohistochemistry might be helpful, but conventional histological evaluation remains crucial to formulate a correct diagnosis.


Evaluation of AKT pathway in polymorphous low-grade adenocarcinoma

N. A. Silva Lascane *, B. T. Sedassari, F. M. Martins, J. L. Saturno, S. C. O. Machado Sousa

*University of São Paulo, Oral Pathology, Brazil

Objective: The aim of this study was to investigate the role of AKT pathway in polymorphous low-grade adenocarcinoma (PLGA) of the salivary glands.

Method: PLGA were classified according to morphological characteristics as lobular, trabecular, tubular, cribriform and papillary. Immunohistochemistry was performed against antibodies pAKT, pmTOR and pS6 in 14 cases diagnosed as PLGA and scored as 0: negative cells, 1: 1–5 %, 2: 5–50 % and 3: >50 %.

Results: The most predominant morphological pattern were tubular and trabecular. pAkt and pmTOR were positive in 7 cases each one and 4 were scored as 2 and the other, 3 cases were scored as 3. Only 2 cases expressed pS6 positively, both scored as 1. pAKT and pS6 presented a nuclear expression and pmTOR showed expression in cell membrane, cytoplasm or both. Lobular was the subtype mostly positive followed by tubular subtype, 8 cases and 7 cases, respectively.

Conclusion: The results suggest a possible correlation of the proteins expression and PLGA subtypes lobular and tubular and showed that the AKT pathway plays a role in PLGA tumorigenesis, and this knowledge might benefit patients with target chemotherapy.


E-cadherin expression in mucoepidermoid carcinoma of oral & maxillofacial complex

F. A. Oliveira *, G. M. Carmo, B. C. Jham, A. C. Batista, E. F. Mendonça, E. C. Barroso Duarte

*IPTSP/UFG, Setor de Patologia, Goiânia, Brazil

Objective: The present investigation sought to evaluate E-cadherin immunohistochemical expression in mucoepidermoid carcinoma of oral and maxillofacial region.

Method: Thirty oral and maxillofacial mucoepidermoid carcinoma cases, diagnosed between 1996 and 2011, were retrieved from the medical files of the Head & Neck HAJ/ACCG, Goiania, Goias, Brazil. Immunohistochemistry study was performed on histological sections of formalin-fixed paraffin-embedded tissue samples (5 m-thick) using primary antibody anti-E-cadherin. Qualitative analysis of E-cadherin expression in the mucoepidermoid carcinoma was determined using a ×40 objective lens.

Results: In the present study around 73.3 % of Mucoepidermoid carcinoma cases presented a weak intensity of E-caderin expression, while a higher intensity expression was observed in 26.7 % of cases when compared with normal salivary glands. Patients presenting increased intensity of E-cadherin expression had better overall survival (93,4 mounths) when compared to those with low expression (55,5 mounths).

Conclusion: Our study demonstrated a correlation between E-cadherin intensity expression and overall survival. In most of cases the intensity of E-cadherin expression was reduced when compared with normal salivary glands that could be related to poor overall survival of patients. Financial Support: IPTSP/UFG, FAPEG.


Correlation among histologic grading, tumor size and lymph node metastasis in mucoepidermoid carcinoma of the salivary glands

B. T. Sedassari *, N. A. Silva Lascane, M. I. Fares Franco, S. C. O. Machado Sousa

*University of São Paulo, Oral Pathology, Brazil

Objective: The aim of this study was to correlate histologic grading, tumor size and nodal metastasis in mucoepidermoid carcinoma (MEC) of salivary glands.

Method: Seventeen cases diagnosed as MEC of salivary glands were selected from our files and data on topography and tumor size were analyzed. Slides stained with H&E were examined to confirm the diagnosis and histologic grading was performed according to WHO. Then, histologic grading was correlated with tumor size and nodal metastasis.

Results: Of the 17 cases, 11 were classified as low-, 3 intermediate- and 3 were high-grade. Tumor size ranged from 0,3 to 8,0 cm, with the smaller tumors classified as a low-grade and the largest as a high-grade carcinoma. Perineural invasion was detected in 7 cases, 2 low-, 2 intermediate- and 3 high-grade tumors. Angiolymphatic invasion was found in only one case, classified as a high-grade. Nodal metastasis was found in two cases, both low-grade.

Conclusion: Our study suggests that histologic grading may be correlated with tumor size, but not with perineural invasion and nodal metastasis, probably reflecting the genetic potential of the malignant neoplastic cells in invading and metastasizing, as well as events related to the tumor microenvironment may not correlate with morphological appearance.


Pulmonary metastasis of mucoepidermoid carcinoma synchronous with typical carcinoid tumor of the duodenum: An autopsy case report

B. T. Sedassari *, N. A. Silva Lascane, S. C. O. Machado Sousa, M. I. Fares Franco

*University of São Paulo, Oral Pathology, Brazil

Objective: Our aim is to report an autopsy case of metastatic mucoepidermoid carcinoma (MEC) synchronous with a typical carcinoid tumor (TCT) of the duodenum.

Method: A 76-year-old male patient experienced acute dyspnea, lung congestion and died 10 months after a parotidectomy for MEC. An autopsy was performed.

Results: On external examination, edema and neoplastic infiltration on the left side of the cervical region were observed associated with submandibular fistula. Generalized visceral congestion was observed upon the opening of the thoracoabdominal cavity. A mass in the lower lobe of the right lung was noted associated with chronic focal pneumonia, vesicular emphysema and edema. The aorta showed severe atherosclerosis. The GIT exam revealed duodenal wall thickening, diverticulosis with diverticulitis in the large intestine and polyp in colonic mucosa. The histological exam confirmed the diagnosis of metastatic MEC in the lung and TCT in duodenum. Acute pulmonary edema was considered the immediate cause of dead.

Conclusion: CME is the most common salivary gland carcinoma and its synchronic occurrence with a duodenal TCT has never been reported. Patients with MEC, as well as in other tumors, need to be monitored for a long period. Surgery and, whenever possible, adjuvant therapies should be employed in order to cure.


Morphological alterations in labial salivary gland in patients who underwent radiotherapy

S. Bologna *, T. H. N. Teshima, T. B. Nunes, M. D. Durazzo, M. M. Simonsen Nico, S. V. Lourenço

*HC-FMUSP, Dermatologia, São Paulo, Brazil

Objective: Radiotherapy is a modality of treatment largely used for head and neck malignancies. However, high doses of radiation in this area frequently results in permanent and severe salivary gland dysfunction which causes significant hyposalivation and xerostomia. Thus, the main purpose of this study is to evaluate the histopathological aspects of minor salivary glands affected by head and neck radiotherapy.

Method: Nineteen patients previously diagnosed with head and neck cancer submitted to radiotherapy were included in the study. Incisional biopsies of their labial minor salivary gland were performed pre and post-radiotherapy for histopathological analysis.

Results: Almost 80 % of all cases exhibited morphological alterations. Twelve cases showed acinar atrophy and three showed the presence of periductal hyalinization most probably due to radiotherapy. Other features as acinar fibrosis and replacement of glandular parenchyma by adipose tissue. All patients with these histopathological findings also demonstrated clinical signs and symptoms of xerostomia.

Conclusion: Association between radiotherapy and actinic damage in salivary glands result is xerostomia; further studies are necessary to develop radiation prevention and to prevent loss of function of salivary glands.


Sjogren’s syndrome: Histopathological evaluation of thrombosis in specimens of minor salivary glands

S. Bologna *, S. V. Lourenço, T. H. Teshima, S. Pasotto, M. M. S. Nico

*HC-FMUSP, Dermatologia, São Paulo, Brazil

Objective: Sjogren’s syndrome (SS) is an autoimmune exocrinopathy characterized by lymphocytic infiltration of exocrine glands in multiple sites. Progressive focal lymphocytic infiltration of salivary gland parenchyma with initial focal sialadenitis and late fibrosis are well described. Antiphospholipid antibodies (aPL) are observed in 2 % to 37 % of SS patients and may be responsible for thromboembolic events. Nevertheless, thrombosis may occur in the absence of aPL. Thomboembolic events have been described in organs such kidneys and central nervous system. Reports on the presence of thrombosis in salivary glands are still lacking. We investigated the presence of thrombosis in specimens of salivary glands from patients diagnosed with SS.

Method: Samples derived from 27 female patients with xerostomia, diagnosed with SS were histologically evaluated.

Results: Salivary gland alterations were detected in all cases. They included diverse levels of atrophy, acinar metaplasia and fibrosis. Presence of lymphocytic foci and linfo-myoepithelial ductal aggression were also observed; in 10 cases, it was associated with periductal hyalinization. In 17 cases, thrombosis of intra-glandular blood vessels was detected; from these case, 9 presented severe stromal fibrosis.

Conclusion: Salivary gland thrombosis is a histological feature that could be included as an additional criterion in the microscopic evaluation and diagnosis of SS.


The role of intrauterine growth retardation in parotid gland remodeling

S. Morozov *, O. Reshetnikova

*State Medical University, Lugansk, Ukraine

Objective: Salivary gland pathology results in the oral health status disturbances. Complicated pregnancy of mother may stimulate impaired salivation in their children later. The objective of this study was to determine the effects if experimental systemic intrauterine growth retardation (IUGR) in rats on their parotid gland remodeling.

Method: Sixty five samples of the newborn rats parotid glands, including 35 with experimental systemic growth retardation and 30 controls after uncomplicated pregnancies, were studied morphologically. Histological slides, stained with hematoxylin and eosin, were studied microscopically, and then their stereometric parameters were revealed.

Results: Results indicated morphological features of glandular acini and ducts immaturity. The area of pathologic changes in the glandular tissue increased. The volume fraction (VF) of parenchyma was significantly lower compared with controls (respectively-25,00 ± 6,96 and 51,72 ± 7,04 %,P < 0,05). The stroma VF in IUGR group increased to 35,67 ± 4,29 % (in control group-23,70 ± 3,25 %,P < 0,05).

Conclusion: Studies have confirmed the direct relationship between systemic IUGR in rats and delayed maturation of parenchymal components of the parotid gland. Theses may cause the insufficiency of the glands functional activity. The resultant oral homeostasis imbalance predispose to various dental pathologies, including caries, parodontitis, etc.


Clinicopathologic features associated to CD44/CD24 expression in benign and malignant salivary gland neoplasms

A. Ribeiro-Silva *, D. Soave, J. P. Oliveira-Costa, G. Silveira, R. Ianez, L. Oliveira, S. Lourenço

*Ribeirão Preto Medical School, Dept. of Pathology, Brazil

Objective: The present study aims to evaluate the CD44/CD24 expression in association with clinicopathologic features in benign and malignant salivary gland neoplasms.

Method: Immunohistochemical stains for CD44 and CD24 were performed on tissue microarrays containing SGN samples from 219 patients (128 pleomorphic adenomas, 22 Warthin tumors, 11 mucoepidermoid carcinoma, 34 adenoid cystic carcinoma, 4 acinic cell adenocarcinoma, 5 NOS adenocarcinoma, 3 polymorphous low-grade adenocarcinoma, 5 basal cell adenocarcinoma, 4 carcinoma ex-pleomorphic adenoma and 3 salivary duct carcinoma). The CD44, CD24 and CD44/CD24 expression phenotypes were correlated to patient clinicopathologic features and outcome.

Results: In benign neoplasms CD44 expression was associated with Warthin tumors when compared to pleomorphic adenomas (p = 0.0001). CD24 was associated with Warthin tumors when compared to pleomorphic adenomas (p = 0.0001). In malignat neoplasms CD44 expression was associated with the primary site of neoplasm (p = 0.046). CD24 was associated with clinical stage III/IV, T stage and lymph node (p = 0.008, p = 0,27, p = 0,001 respectively). The CD44/CD24 profiles were associated with the primary site of injury, lymph node and T stage (p = 0.005, p = 0.011, p = 0.023 respectively).

Conclusion: Our investigation demonstrates that CD44/CD24 expression is related to benign histologic types. CD44 and CD24 immunoexpression may provide prognostic information associated to clinicopathologic features in salivary gland malignant neoplasms.


Caspases involvement with ductal lumen formation during human salivary gland morphogenesis

T. Teshima *, S. Bologna, R. Iañez, C. Coutinho-Camillo, S. Lourenço

*University of São Paulo, Dept. of Oral Pathology, Sao Paulo, Brazil

Objective: Caspases represent a large family of proteases which possess an essential role in controlling apoptotic process. Although not completely understood, apoptosis can be related to the development of human salivary glands, mainly with the opening of the ductal lumen, and caspases would be possibly involved with that mechanism. Thus, the main purpose of this study is to elucidate a potential intervention of caspases 3, 6, 7 and 9 in human salivary gland morphogenesis, relating their expression to each stage of development.

Method: Samples derived from about 20 fetuses of natural miscarriages obtained from the Medical School of the University of Sao Paulo were submitted to immunohistochemical reactions for caspases 3, 6, 7 and 9.

Results: Only caspases 6 and 9 illustrated a positive expression during all stages of the gland development, being stronger in terminal phase. Nuclear staining of epithelial cells was observed in more than 65 % of the analyzed specimens. At the latest stage, cytoplasmic expression was also observed in epithelial and ductal portions.

Conclusion: Apoptotic proteins expressed in different stages of human salivary gland morphogenesis evidence an interaction among them during this process, especially guiding the formation of the ductal network, and the understanding of that mechanism will contribute to improve knowledge of salivary gland diseases.


Expression of PI3K/Akt pathway in adenoid cystic carcinoma and pleomorphic adenoma of salivary glands

R. Ianez *, C. Camillo-Coutinho, C. Pinto, A. Silva, F. Soares, S. Lourenço

*A.C. Camargo Hospital, Sao Paulo, Brazil

Objective: Pleomorphic adenoma (PA) and adenoid cystic carcinoma (ACC) are the commonest benign and malignant salivary gland neoplasms, respectively. They are originated from the intercalated duct region and are composed by luminal structures and myoepithelial cells. In a previous study we detected that protein c-kit is involved in the process of salivary gland morphogenesis and PA; this protein appeared to be related to pluripotent cells. Additionally, recent reports have shown that alterations in KIT gene are present in ACC. Based on this evidence, we further investigated the expression of PI3K/Akt pathway involved in the Kit signaling cascade in ACC and PA.

Method: The PI3K/Akt pathway was investigated in 50 cases of PA and 50 cases of ACC using immunohistochemistry

Results: In PA c-kit was positive in isolated luminal cells, and myopithelial cells were positive for alpha and beta PI3k, phospo-Akt and phosphor-mTor. In ACC, neoplastic luminal structures were positive for c-Kit and the other proteins showed positivity in myoepithelial cells especially in cribriform areas.

Conclusion: Expression of the downstream proteins of the c-kit cascade in myoepithelial cells in both PA and ACC may be indicative that these neoplastic cells share common proliferative pathways, despite being benign or malignant.


Advanced adenoid cystic carcinoma treated by imatinib mesylate: Report of five cases

M. Vozmitel *, J. V. Guljaeva, S. Rjabceva, T. I. Nabebina, A. Dubrovskij

*Minsk, Belarus

Objective: Adenoid cystic carcinoma (ACC) is a KIT-positive salivary glands tumor with frequent local recurrences and distant metastases. We report here our experience of using imatinib mesylate 400 mg/daily for treatment of 5 patients with unrespectable and/or extensively metastatic ACC.

Method: Clinical data, histologic features of tumors. Formalin-fixed paraffin blocks of 5 cases were available for immunohistochemistry (KIT; Dako) and RT-PCR (MYB-NFIB fusion and KIT gene mutations).

Results: Patient’s information: 3 man/2 women; median age 44 years. Disease status: multiple lung metastases (n = 3), massive liver metastasis (n = 1), wide local progression (n = 1). All tumors were KIT-positive. Exons 9, 11, 13 mutations of KIT were not revealed by PCR in all cases, whereas MYB-NFIB fusion transcript was detected in 2 of 5 tumors. Two patients had progressive disease, one of them dead of disease in 3 month after treatment start. Three patients had stabilization of disease for 1, 3 and 5 months, as the best response. In one of them after-treatment lesion established extensive degenerative changes of tumor tissue.

Conclusion: ACC shows doubtful treatment response to anti-c-kit targeted therapy. KIT gene mutations are not specific for ACC, while MYB-NFIB fusion is more frequent and may be a future candidate for targeted therapy.


Study of microRNA expression in malignant salivary gland tumours

F. Passador-Santos *, D. Soave, A. Silva, A. Soares, N. Araujo, V. Araujo

*São Leopoldo Mandic Institute, Pathology, Campinas, Brazil

Objective: Malignant salivary gland tumours comprise approximately 5 % of all head and neck malignancies. Adenoid cystic carcinoma (ADCC) is considered one of the most common malignancies of the salivary glands. Recently, a new class of small RNAs named microRNAs (miRNAs) has been described and applied for diagnostic and prognostic purposes. The aim of this study was to investigate the expression of miRNA 15a/16 and150 in ADCC

Method: Thirty-five cases ADCC of the salivary glands were retrieved from the files of the departments of Pathology of Sao Leoplodo Mandic Institute and Research Centre and Faculty of Medicine of the University of Sao Paulo (USP-Ribeirao Preto). Twenty-five cases from minor salivary glands, six cases from parotid and four cases from submandibular gland were collected. Total RNA was extracted using a standard Trizol method and expression of miRNAs 15a/16 and 150 was evaluated using real time PCR profiling

Results: Nineteen cases presented solid histopathologic pattern, 12 showed the cribriform histopathologic pattern while four cases had a tubular histopathologic pattern. Our preliminary results showed different expression of miRNAs 15a/16 and 150 in ADCC when compared to normal salivary glands

Conclusion: Our preliminary results indicate that deregulation of miRNA expression may be involved in the pathogenesis of ADCC


Cytokeratin immunoprofile of parotid region Squamous Cell Carcinoma (SCC): Is it possible to distinguish a salivary gland from cutaneous origin?

N. A. Silva Lascane *, B. T. Sedassari, M. I. Fares Franco, D. S. Pinto-Jr, S. C. O. Machado Sousa

*University of São Paulo, Oral Pathology, Brazil

Objective: The aim of this study was to compare the expression of cytokeratins (CKs) 7, 8, 13 and 14 in squamous cell carcinoma (SCC) from major salivary glands and from the parotid region (probably an infiltration of a cutaneous SCC).

Method: Twelve cases diagnosed as SCC in parotid gland were microscopically separated in primary SCC of the salivary gland (PSCC) and SCC of the parotid region (SCCPR). Immunohistochemistry was performed against CKs 7, 8, 13 and 14 in 8 cases of PSCC and in 4 cases of the SCCPR.

Results: All but one case of PSCC were negative for CK7. CKs 8 and 13 were positive in only two cases of PSCC, whilst CK14 was positive in all cases. One of these cases presented only a few cells positive to CK14 and this case was also positive to CKs 8 and 13. On the other hand, all cases of SCCPR showed positivity to CKs7 and 14, and 3 cases to CK8.

Conclusion: There is no correlation of the cytokeratins studied and the tumor site. The proximity of both lesions to excretory duct might explain the potential of these cells to differentiate in glandular-like or squamous-like or intermediate cells.


Profile of the lipid droplets in salivary gland carcinomas

F. V. Mariano *, H. T. Santos, V. A. Montalli, A. Altemani

*Faculty of Medicine—UNICAMP, Pathology, Campinas, Brazil

Objective: To verify the quantity of cytoplasmic lipid droplets (CLDs) in salivary carcinomas due to its association with tumor aggressiveness.

Method: In 68 salivary carcinomas (17 adenoid-cystic, 12 epithelial-myoepithelial, 11 mucoepidermoid, 10 acinic cell, 7 myoepithelial, 6 polymorphic low-grade and 5 salivary duct) the CLDs were stained for adipophilin. The quantity of CLDs in tumor cells was classified as absent (0 %–5 %), focal (>5 %–50 %) and diffuse (>50 %).

Results: 11.7 % of the carcinomas presented CLDs in more than 50 % of tumor cells as follows: myoepithelial (57 %), acinic cell (30 %) and epithelial-myoepithelial (8.3 %). In 53 % of the tumors, CLDs were not detected. In acinic cell carcinomas, the increased number of CLDs was associated with vacuolated appearance of the tumor cells.

Conclusion: In salivary carcinomas, CLDs are not a common event and its occurrence is not necessarily associated with high grade carcinomas. In acinic cell carcinomas, the upregulated lipogenesis reflects in the tumor morphology.


A 74–year-old caucasian woman with Kimura’s disease

Y. Gorbacheva*, I. Kazantseva, A. Nikitin, E. Stepanova

*M.F. Vladimirsky Moscow Region, Dept. of Pathology, Russia

Objective: Kimura disease (KD) is a tumor-like chronic inflammatory disorder with angiolymphatic proliferation, usually affecting young men of Asian race, but it is rare in other races. The etiology of KD is still unknown.

Method: We report a rare case of KD involving the left salivary gland with surrounding lymph nodes and subcutaneous tissue. A 74-year-old woman presented with solid edema left parotid and orbital area, hearing and vision imparing.

Results: Computed tomography showed bilateral neck lymphadenopathy, subcutaneous tissue multiple infiltrations. Laboratory tests revealed that peripheral eosinophils (83 %) and serum IgE levels (400 ME/mL) were markedly increased. An excision biopsy of lymph node and salivary gland showed KD specific morphological features: lymphoid and eosinophil infiltration, the proliferation of high endothelial venules with slit-like lumina, eosinophillic microabscesses, fibrosis. The differential diagnosis between KD and leukemia with eosinophilia was performed: PDGFRA, PDGFRB and FGFR1 genes mutation was not revealed, bone marrow examination showed only eosinophilia.

Conclusion: Thus, such a clinical, instrumental, laboratory and morphological features combination allowed to provide KD diagnosis.

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Parapharingeal solitary fibrous tumor in a 57-year-old female

F. Moreno *, R. Dias, A. Duarte

*Centro Hospitalar do Porto, Dept. de Anatomia Patológica, Portugal

Objective: Solitary fibrous tumor is an uncommon mesenchymal tumor that usually arises from the pleura. There are occasional reports of extrapleural sites, including the parapharingeal space. Most cases are benign and cured with complete resection but malignant potential was reported. Microscopically, they are well circumscribed but non-encapsulated tumors with a characteristic patternless arrangement of narrow spindle-cell cords interspersed by thick collagen bundles. Most have hypercellular and hypocellular areas and might show prominent haemangiopericytoma-like appearance. Immunohistochemically the cells are uniformly positive for CD-34 and Bcl-2 and negative for S-100 protein.

Method: We report the case of a 57-year-old woman proposed for study of a right parotid swelling, xerostomy, persistent right tinnitus and temporomandibular pain with 2 month evolution.

Results: The magnetic resonance study showed an expansive, well defined mass located in the right parapharingeal space. The patient underwent local surgical recession and the microscopic examination revealed a benign parapharingeal solitary fibrous tumor with the characteristic morphologic and immunohistochemical features described above. There is no evidence of local recurrence 1 year after surgery.

Conclusion: This case illustrates the typical histological and Immunohistochemical presentation of this type of tumors. The follow-up of these patients is mandatory, since there are reported cases of recurrent disease with malignant characteristics.


A case of imported paracoccidioidomycosis

H. Coelho *, J. Vaz de Castro, D. Brito, J. Oliveira e Neta, M. J. Aleixo, C. André, L. Antunes, M. J. Brito

*Hospital Garcia de Orta, Dept. de Anatomia Patológica, Almada, Portugal

Objective: The purpose of this study is to report a case of infection by Paracoccidioides brasiliensis, a fungus endemic in South America.

Method: A Brazilian 63-year-old man, with marked alcoholic and smoking habits, working in Portugal as gardener for the last 8 years, presented in the emergency department with odynophagia in the previous month associated with dysphagia in the last week. Observation of the oral and oropharyngeal cavities revealed an irregular mucosa with granular surface. On the left buccal mucosa, an ulcerated lesion was identified extending towards the inferior alveolar border, friable and painful on palpation. Endoscopy showed that the tongue base also harboured a lesion with the same characteristics, obliterating the left vallecula.

Results: Histologic examination identified microorganisms compatible with P. brasiliensis in the samples obtained from oropharyngeal mucosa.

Conclusion: Globalization and migratory patterns have increased the number of imported diseases. It is thus important that pathologists and clinicians, in Europe, become aware and familiarized with the clinical presentation of this rare disease, endemic to South America.


A malignant Perivascular Epithelioid Cell neoplasm (PEComa) of the carotid bifurcation in a 19-year-old man

D. Koumoundourou*, T. Stathas, D. Mpatsoulis, V. Zolota

*University Hospital of Patras, Pathology, Greece

Objective: PEComas, are unique mesenchymal tumors characterized by a mixed myogenic and melanocytic phenotype. Examples of this neoplasm originating in the head and neck region are limited.

Method: A 19-year-old man was submitted to our hospital with a painless hard swelling of the right upper neck. The patient underwent a surgery with ipsilateral cervical lymph node dissection. A soft grayish tumor mass measuring 35 mm in greatest diameter was resected

Results: Microscopically the tumor consisted of solid sheets of medium sized spindle cells arranged in fascicles and nests with high cellularity, foci of coagulative necrosis and exceptional mitoses. The cytoplasm was either clear or acidophilic and tumor cells had a prominent eosinophilic nucleolus. An adjacent 30 mm lymph node was also affected. Immunohistochemically tumor cells were HMB-45+++, S-100+, MyoD1+, myogenin+. According to the criteria proposed for the classification of PEComas (Folpe) the diagnosis was a malignant PEComa. The patient received radiation therapy.

Conclusion: PEComas have been reported mainly in the abdominopelvic cavity and rarely in parenchymatous organs, skin, and soft tissues. None of the reported cases was situated in the carotid bifurcation. The present case serves to emphasize the potential of PEComa for aggressive behavior and the importance of distinguishing this tumor from other epithelioid neoplasms.


Metastatic epithelioid hemangioendothelioma of the palate region

S. Mocan *, S. Comisel, D. Milutin, T. Mezei, A. Iacob, C. Petrovan

*Emergency Clinical Hospital, Dept. of Pathology, Targu Mures, Romania

Objective: Metastatic tumors of the oral region are challenging because are very rare and frequently can mimic a benign lesion.

Method: We present a case of a 58 years old male, with a tumor in the palate region and multiple metastases. Three years previously, the patient was diagnosed with epithelioid hemangioendothelioma localized in the deep soft tissue of the lower extremity and he was treated by surgery and chemotherapy. After clinical evaluation, the tumor form the posterior region of the soft palate was removed.

Results: The tumor was localized deep in the mucosa, with an infiltrative growth pattern and extensive necrosis. The tumor was composed of short strands of small round epithelioid and slightly spindle cells set in a myxohyaline stroma, with abundant eosinophilic cytoplasm with prominent vacuolization. The number of mitoses was 4–6 mitoses/10 HPF. Immunohistochemically, the tumor cells stained positively for CD 31, CD 34 and Factor VIII-related antigen, and focally for Cytokeratin AE1/AE3.

Conclusion: Epithelioid hemangioendothelioma is considered as a borderline endothelial tumor. There aren’t strict criteria for malignancy that can be used to predict recurrence and metastases. The case illustrates the aggressive behavior of the tumor and the difficulties in diagnosis, especially because the tumor appears in an unusual location.


Peripheral Ossifying Fibroma (FOP): Clinico-pathological and immunohistochemistry findings in a series of 13 patients

J. M. Suárez Peñaranda *, H. Lázare, P. Gándara Vila, J. J. Carrera Álvarez, J. Caneiro, M. Pérez Sayáns, A. García García, J. M. Gándara Rey

*Hospital Clinico Santiago de, Compostela, Dept. de Patalogia, Santiago de Compostela, Spain

Objective: We report the findings of 18 biopsies from 13 patients with the diagnosis of FOP, including including the immunohistochemical study.

Method: Clinical data have been collected retrospectively from the files of the patients and the slides and paraffin blocks from the files of the Department of Pathology of the Clinical and University Hospital of Santiago de Compostela. Immunohistochemistry was performed for smooth muscle actin (SMA), CD68 and CD34 using the Dako Autostainer Link 48 and EnVision.

Results: 9 patients were females and 4 males. Ages ranged from 10 to 70 years. Most tumors were below 2 cm and only two were larger: 2,5 and 5 cm, respectively. Four patients presented relapses (30.7 %), from 1 to 8 years after surgery, one of them three times. Histopathological examination revealed the presence of a benign mesenchymal proliferation with variable degrees of bone formation: lamellar bone in 12 cases, woven bone in 11 cases and cementum in 11. Immunohistochemistry was performed in 14 biopsies: 10 showed stain for SMA and 3 for CD68, while CD34 was negative.

Conclusion: POF has showed a distinct predilection for women between the 2nd and 4th decades of life. The relapse rate has been 30 %, higher than expected according to the literature. The immunhistochemistry study confirms the myofibroblastic nature of the condition.


Primary central chondrosarcoma of craniofacial bones: Report of 5 cases

W. Ouahioune *, N. Moulai, N. Bettaz, N. Medjber, A. Mansour

*University of Blida, Dept. of Medicine, Algeria

Objective: Primary central chondrosarcoma (PCCS) accounts for about 20 % of malignant bone tumors. It’s characterized by the formation of cartilaginous matrix. The most common skeletal sites are the bones of the pelvis. PCCS is rarely found in craniofacial bones. The aim of this study is to report 5 new cases of PCCS of head and neck with analysis of the clinicopathological characteristics of this tumor.

Method: It’s a retrospective study of 5 PCCS collected over 10 years at our department. The follow up ranges from 02 to 10 years.

Results: On the 21 PCCS diagnosed in our service, 05 of them were localized at craniofacial bones. There were 03 males and 02 females; age ranged from 25 to 49 years. The sites of involvement were jaw bones (03 cases), temporal bone (01 case) and occipital bone (01 case). Radiologically, the diagnosis of PCCS was not raised in any case. The diagnosis of PCCS was established after histological examination. 4 of 5 patients were at first treated by a wide en-bloc surgical resection then they underwent radiotherapy. 03 of those who were treated had recurrences.

Conclusion: Although this tumor is rare in the craniofacial bones, PCCS must always be kept in mind.


Familial tumoral calcinosis: Clinico-pathological findings and confocal laser scanning microscopy of hard and soft tissues lesions

E. Maiorano *, L. Limongelli, A. Tempesta, A. P. Cazzolla, V. De Falco, D. Di Venere, G. Favia, A. Napoli

*Università degli Studi di Bari, Dipt. di Anatomia Patologica, Italy

Objective: We report on the clinico-pathological features of Familial Tumoral Calcinosis (FTC), a rare disease of childhood and adulthood, caused by mutations in FGF and GalNAc transferase 3, consisting in a bone metabolism disorder with abnormal phosphate and calcium (calcinosis) deposits around the joints, in viscera and soft tissues.

Method: A 17 year-old girl, complaining for long-standing leg pain, resistant to FANS therapy, was diagnosed with osteogenesis imperfecta and therefore undergoing bisphosphonates therapy. She was referred to our Dental Clinic for diffuse dental abnormalities, maxillary hypoplasia and tooth roots inclusions and underwent combined surgical and orthodontic treatment. The surgical samples were used for conventional and Confocal Laser Scanning Microscopic (CLSM) examination.

Results: Microscopically, several metaplastic micro and macro-calcification in soft and periodontal tissue location were detected, along with typical islands of homogenous, non tubular, dentino-osteoid calcified structures in dentinal tissues. Also, dentinal dysplasia with osteoid-like material, without incremental lines but with strong basophilia, intermingled with remnants of mature mucous connective tissue, were demonstrated. The diagnosis of FTC was confirmed by genetic analysis.

Conclusion: CLSM helps to demonstrate distinct odontoblast and osteoblast anomalies in FTC that lead to the accumulation of atypical calcified tissues, responsible for the several clinical signs detected in the patient and formerly attributed to osteogenesis imperfecta.

CLSM features of Tumoral Calcinosis:

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Podoplanin expression is enhanced in pseudoepitheliomatous hyperplasia associated with bisphosphonate osteonecrosis of the jaw

J. Zustin *, D. Reske, A. Koenemann, A. Assaf, H. Scheuer, R. Friedrich

*UKE Hamburg, Institute of Pathology, Germany

Objective: Pseudoepitheliomatous hyperplasia has been observed in bisphosphonate osteonecrosis of the jaw (BRONJ) lesions. Podoplanin expression has been linked with oral squamous cancer cell migration and invasion. Because podoplanin expression has further been reported in inflamed gingival epithelium, we examined podoplanin expression in pseudoepitheliomatous hyperplasia in BRONJ.

Method: Archival cases of BRONJ were re-reviewed in order to identify cases showing pseudoepitheliomatous hyperplasia. Six positive cases were simultaneously analyzed using immunohistochemical antibodies for cytokeratins (AE1/AE3) and podoplanin (D2-40).

Results: All six cases of pseudoepitheliomatous hyperplasia associated with BRONJ showed irregular epithelial proliferations both at the site of necrotic bone and within an inflamed granulation tissue. All lesions showed strong diffuse positive reaction with AE1/AE3 antibody. The pseudoepitheliomatous hyperplasia lesions revealed strong expression of D2-40 antibody in the basal epithelial cells and weak reaction in the parabasal cell layers.

Conclusion: We observed a D2-40 expression in benign pseudoepithelial hyperplasia associated with BRONJ. We suggest that podoplanin expression might be induced in benign pseudoepitheliomatous hyperplasia by chronic inflammation associated with BRONJ similar to chronic periodontitis. Even though malignancies have exceedingly rarely been reported in association with BRONJ, every precaution should be taken in the interpretation of the dignity of epithelial proliferations positive for podoplanin.

Pseudoepitheliomatous hyperplasia associated with BRONJ:

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Recurrent squamous odontogenic tumor: A case report

S. Manojlovic *, J. Budimir, L. Manojlovic, D. Macan

*University Hospital Dubrava, Dept. of Pathology, Zagreb, Croatia

Objective: Squamous Odontogenic Tumor (SOT) is exceedingly rare benign odontogenic epithelial neoplasm. Recurrences are exceptional. The present case is the third report of recurrent SOT so far.

Method: Case report: A 50-year old female presented with a swelling in the mandible. Radiographic images disclosed a radiolucency in premolar area. Local excision was performed. Histopathological analysis revealed a squamous odontogenic tumor. Two recurrences arose at the same localisation,1 and 4 years later, respectively. Histopathological pattern was in both recurrences identical to the primary tumor.

Results: SOT is extremely rare tumor, around 45 cases having been reported to date. Most of them arose intraosseally, near the periodontal ligament. Recurrence has been described in two cases. The etiology and pathogenesis of the SOT are unknown. Differential diagnosis includes a variety of odontogenic cysts and desmoplastic ameloblastoma. The most important differential diagnosis is intraosseous squamous cell carcinoma (SCC). Conservative surgical procedures are considered adequate treatment for SOT.

Conclusion: The present case is, to our knowledge, the third report of the recurrent SOT. At least one case of SOT transformed into a carcinoma. Therefore, more extensive surgical excision and a long-term follow-up is essential for such lesions.


Podoplanin in ameloblastoma: Its relevance and distribution in different clinicopathologic subtypes

C. H. Siar *, K. H. Ng

*University of Malaya, Dept. of Oral Pathology, Kuala Lumpur, Malaysia

Objective: Human podoplanin, a specific lymphatic endothelial cell marker, is biologically related to cell migration and invasion. The ameloblastoma is a benign but locally-invasive odontogenc epithelial neoplasm. The aim of this study was to determine the distribution pattern of podoplanin in ameloblastoma and to speculate on its relevance.

Method: Immunohistochemical staining for podoplanin was performed on 18 unicystic(UA), 20 cases solid/multicystic(SMA), 4 desmoplastic(DA) and 20 recurrent ameloblastoma(RA).

Results: Podoplanin was detected in most ameloblastoma subtypes(UA = 18/18; SMA = 13/20; DA = 2/4 and RA = 17/20). Distribution pattern was heterogeneous, and indistinctive between tumour centre and periphery. Protein localization was pre-ameloblast-like>stellate reticulum-like cells, membranous and cytoplasmic. Stromal fibroblasts, lymphatic endothelium, nerves, osteoblasts and salivary glands were variably podoplanin-positive.

Conclusion: Although ameloblastoma subtypes are distinctive in their clinicopathologic presentations and behaviour, their podoplanin distribution patterns are not different. This suggests that this protein molecule might not be a major factor influencing the progression of these different subsets.

Recurrent ameloblastoma and podoplanin expression:

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Ameloblastoma originated from maxillary sinus

E. Dogan *, Z. A. Abidin, O. Ipci, E. Akoglu

*Mustafa Kemal University, School of Medical Pathology, Hatay, Turkey

Objective: Ameloblastoma is a locally agressive tumor which arises from odontogenic epitelium. Origin of sinonasal tract is very rare and few cases have been reported.

Method: A 61-year-old male patient presented with epistaksis for 2 months. Hemorrhagic degenerative mass has seen in physical examination at the level of the left middle concha nasal cavity. CT scans has revealed a mass originated from left maxillary sinus. Multiple biopsies were taken from the mass.

Results: Macroscopically, the largest 1.5 × 0.6 × 0.5 cm in size 3 pieces covered with the mucous surfaces of irregularly shaped fragments were observed. Microscopically,in the form of islands, cords, which palizading at periphery, often monotonous, narrow oval-round nuclei and eosinophilic cytoplasm by the tumoral cells was observed.

Conclusion: Sinonasal ameloblastomas are rare tumors which arise from odontogenic epithelium. Small biopsies may be superficial and may not show typical histological findings. And this tumor could kept in mind in differantial diagnosis with aother tumor sharin similar morphology in small biopsies.


Activation of the AKT/mTOR pathway in dentigerous cysts, odontogenic keratocysts and ameloblastomas

R. Chaisuparat *, S. Yodsanga, S. Montaner, B. Jham

*Chulalongkorn, Dept. of Oral Pathology, Bangkok, Thailand

Objective: To investigate the activation of Akt/mTOR in dentigerous cysts (DC), odontogenic keratocysts (OKC) and ameloblastomas, and to correlate the findings with clinical and histopathological parameters.

Method: A total of 90 cases (30 DC, 30 OKC and 30 ameloblastomas) were studied. Patient records on age, sex, lesion location, symptoms, and radiographic and histopathologic features were collected. Immunohistochemical study of the Akt/mTOR pathway (p-Akt-S473, p-Akt-T308 and p-RPS6) was studied on tissue microarrays. Immunohistochemical reactivity was graded according to the percentage of positive tumor cells. Statistical significance was considered at p < 0.05.

Results: Over 90 % of OKC and ameloblastoma cases stained positive for p-Akt (Ser473), while 60 % of DC cases were found positive for p-Akt (Ser473). Ameloblastomas displayed the highest number of cases with positive p-Akt (Thr308) (73 %), followed by OKCs (40 %) and DC (20 %). Similarly, p-RPS6 was detected most frequently in ameloblastomas (83 %), followed by OKCs (76 %) and DCs (53 %). Statistical analysis showed significant differences in phosphorylation levels of all three proteins (p < 0.05). However, there was no significant correlation between those phosphorylation levels and the clinicopathologic features of the lesions.

Conclusion: We have shown that the Akt/mTOR pathway is upregulated in DCs, OKCs and ameloblastomas, suggesting a role for this pathway in the development and progression of the lesions.


Morphological evaluation of experimental temporomandibular joint disorder

K. Semenov*, O. Reshetnikova

*Dnepropetrovsk Medical Academy, Ukraine

Objective: Temporomandibular joint disorders (TMJD) clinically presented with chewing muscles and joints pathology. Although it is known that physical stress on the structures around the joint may result in TMJD, in many cases the cause of TMJD remains unknown. The aim of present study was to find out morphological changes in chewing muscles and joint structures in case of experimental TMJD.

Method: Ten mature outbred 9-month male rats were the material for study. Five rats had standard diet with no pathology of the TMJ (controls). Other five rats were used for the TMJD modeling. The musculoskeletal blocks from the TMJ area of the injured side as well as from the intact TMJ area were taken for histology and morphometry.

Results: The results have shown the hypertrophy of the masseter muscle, foci of collagen fibers distruction with inflammatory infiltrates in tissues around the TMJ with experimental TMJD. The articular disk revealed small lesions. Morphometry showed the increased area, width, length and perimeter of the masseter muscle fiber section at the TMJD side.

Conclusion: These findings present experimental evidence of an association of TMJD symptoms with the hypertrophy of the chewing muscles, pathomorphological changes both in muscles and joint structures.


Morphogenesis of teeth and pathology of thyroid gland

T. Pavlova *, E. Peshkova, I. Goncharov, D. Kolesnikov, K. Prashchayeu, V. Markovskaya

*BelSU, Dept. of Pathology, Belgorod, Russia

Objective: Study of endocrinopathies is important in health care. The impaired production of thyroid hormones (thyroid gland-TG) affects bones’ mineral content, particularly, reticular tooth plate.

Method: The following combinations were studied: teeth with caries from the patients with hypo- or hyperthyreosis; teeth without caries from healthy patients. Molars were extracted from 18 to 50 years old patients. The scanning microscope was used to determine the structure and the microelements.

Results: The patients with TG pathology have more carious lesion than healthy people. Caries is localized in cervical region; the progression is faster. Nanoscale pictures of conditionally healthy teeth were examined and found caries before the appearance of external changes of enamel. In enamel with carious lesion, calcium decreased to one-fourth (10,52 %) of the normal enamel–40,37 %; in dentine, calcium decreased to one-sevenths (5,7 %). In enamel with caries, phosphorus decreases by a half; in dentine, phosphorus decreases to one-fourth. Due to caries, the amount of oxygen is increased: two times in enamel, 1,2 time in dentine. Pic.1.

Conclusion: We identified changes in the structure of hard tissues teeth in patients with pathology of TG.

Pic.1. Tooth enamel in caries. A. Scanning Electron Microscopy x 1000. B. Atomic force microscopy:

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Nodal involvement in advanced laryngeal squamous carcinoma is associated with Ep-CAM but not E48 expression

A. Nadal *, C. Romeu, X. Farré, A. Cardesa

*Hospital Clínic Barcelona, Dept. of Pathology, Spain

Objective: The analysis of Ep-CAM and E48 expression and their relationship with differentiation and development of nodal metastases in laryngeal carcinoma.

Method: Conventional immunohistochemical analysis with monoclonal antibodies against Ep-CAM and E48 in a series of 66 laryngeal carcinomas in stage 3 or 4 and in adjacent non-neoplastic epithelia. Comparison with tumor histologic differentiation according to Broders grading and with presence of nodal metastases.

Results: Ep-CAM expression is higher in carcinoma than in normal epithelia. 96 % of carcinomas show Ep. CAM expression. High levels of expression (>50 % of cells) are associated with nodal metastases (p = 0.001) and poor differentiation (grades 1–2 vs 3–4, p = 0.003). E48 expression is higher in normal epithelia than in carcinoma. Poorly differentiated carcinomas had less positive cells than well to moderately differentiated carcinomas (p = 0.001).

Conclusion: Ep-CAM and E48 expression are associated with cell differentiation but in opposite ways. Ep-CAM could participate in the development of nodal metastases in laryngeal squamous carcinoma according to the association observed between expression and nodal metastases. Selection of satges 3 and 4 prevents a biass against nodal metastases caused by stages 1 and 2.


Is there any relationship between Helicobacter pylori and laryngeal carcinoma?

I. Yilmaz *, U. Berber, Z. Kucukodaci, G. Narli, A. Haholu, D. Demirel, E. Erkul

*Gata Heh, Dept. of Pathology, Istanbul, Turkey

Objective: Helicobacter Pylori (HP) infection is a major risk factor for the development of gastric cancer. Several studies have been done to investigate the role of HP in laryngeal disorders. The results of the studies on the presence of HP in benign and malignant laryngeal tissue are conflicting. The purpose of this study was to investigate the presence of HP in laryngeal squamous cell carcinoma by a sensitive method Real time polymerase chain reaction assay (RT-PCR).

Method: Formalin-fixed-paraffin-embedded tissue samples from 74 patients with laryngeal cancer who underwent total/partial laryngectomy in our hospital between 2005 and 2011 were used in this study. A sensitive RT-PCR assay was used to detect the presence of HP in the laryngeal carcinoma.

Results: HP was detected in only one case. The positive case was also investigated for the existence of HP with histopathologic evaluation and HP immunohistochemistry. However, we could not detect HP in this case with both methods. We suggested that HP in this case may contaminated from stomach to laryngeal tissue during surgery.

Conclusion: This study revealed that HP might not contribute to the pathogenesis of laryngeal carcinoma. Further studies with more patients are needed to clarify the role of HP in laryngeal carcinomas.


Detection of P 16 INK4a in laryngeal papillomatosis and its relationship with dysplasia

E. Ben Brahim *, A. Ben Salem, R. Jouini, W. Koubâa, I. Msakni, O. Khayat, M. Belhaj Salah, A. Souissi, A. Gharbi, C. Mbarek, A. Chadli

*Habib Thameur Hospital, Pathology, Tunis, Tunisia

Objective: Laryngeal papillomatosis (LP) is a rare and benign neoplasm, caused by human papillomaviruses (HPVs) especially HPV 6 and 11. The diagnosis of dysplasia in these tumors is still a source of pathologist’s disagreement. In cervical dysplasia, the use of anti p16INK4a (p16) antibody in immunohistochemistry allows having a better inter- pathologists reproducibility. We therefore sought to evaluate the diagnostic utility of anti P16 in detection of dysplasia in LP leading to better inter-pathologists reproducibility.

Method: A total of 17 samples of LP were analyzed for p16INK4a protein (p16) expression by immunohistochemistry and for human papillomavirus (HPV) infection using in situ hybridization (ISH) and polymerase chain reaction (PCR).

Results: Using ISH and PCR, 27 low risk HPV (LR-HPV) infections, 10 high risk HPV (HR-HPV) infection and one infection with an undetermined risk’s HPV were detected. The immunohistochemical study using anti P16 showed a non significant staining in all cases even the ones diagnosed histologically as dysplastic lesions or having a HR-HPV infection.

Conclusion: Our study find that anti P16 is a non specific marker of dysplasia in laryngeal lesions and that different molecular events, in comparison to the cervical ones, could be involved in progression of LP to the carcinoma.


Laryngeal laser excision mounted in cucumber. Our experience

M. Giles Lima *

*The James Cook University Hospital, Cellular Pathology, Middlesbrough, United Kingdom

Objective: Excisional biopsy is currently the preferred treatment option for early malignant laryngeal lesions. Assessment of margins is challenging in such small specimens. Using a mounting media can improve its accuracy. We present our experience using the described -cucumber technique-

Method: Cases of laser excision of vocal cord lesions were retrospectively retrieved from Head and Neck departmental database. Those whose specimens were placed and glued to a de-hydrated piece of cucumber were identified in the Pathology database and reports obtained. All specimens had been conventionally processed and stained.

Results: 44 cases were selected, 29 were reported as invasive squamous cell carcinoma, 13 as dysplasia or in situ carcinoma and 2 had no tumour conditions. 71 % of the cases of invasive squamous cell carcinoma, dysplasia or in situ carcinoma had the peripheral margin reported and often specified along with other prognostic factors as depth of invasion. In 7 % of the cases the margins were not assessable and 21 % of the cases were suboptimal but reportable.

Conclusion: The use of cucumber as mounting support allows a confident diagnosis of involvement of specific margins in small biopsies as laryngeal laser excisions.


Actinic cheilitis: Epithelial thickness changes and the presence of mutated p53 protein

C. Fabiana Joca de Arruda *, G. Sanchez Nagata, K. Lopez Ortega, M. Trierveiler Martins

*FOUSP, Oral Pathology, São Paulo, Brazil

Objective: Actinic cheilitis (AC) is a potentially malignant disorder caused by ultraviolet (UV) radiation which shows some specific epithelial characteristics, among them, variation in the epithelium thickness. It is known that the TP53 gene is a target of UV radiation. The objective of this research was to determine the variation of epithelium thickness in AC and to correlate it to the expression of mutated p53 protein.

Method: The presence of acanthosis, atrophy, extreme atrophy or normal epithelial thickness was evaluated in 456 AC cases. After this, 20 cases of each of these thicknesses were selected and submitted to the PAb240 antibody against mutated p53 protein by means of immunohistochemistry.

Results: Among the 456 cases studied, 38.8 % showed acanthosis, 34.8 % atrophy, 6.1 % extreme atrophy and 20.1 % normal epithelial thickness. The presence of the mutated p53 protein was seen in 40 % of the cases with acanthosis, 35 % with atrophy, 40 % with extreme atrophy and 24 % with normal thickness. The difference was not statistically significant.

Conclusion: Most of the cases showed epithelium thickness changes (acanthosis or atrophy) but this does not correlate to the genetic alterations in the TP53 gene caused by UV radiation.


Human papillomavirus detection in archived histology samples of oropharyngeal squamous cell carcinomas: Correlation of in situ hybridization and polymerase chain reaction

K. Vesely *, R. Tachezy, H. Binkova, Z. Horakova, E. Foltynova, M. Salakova, J. Klozar, R. Kostrica

*Brno, Czech Republic

Objective: Human papillomavirus (HVP) has been recognized as an etiologic and prognostic factor in head and neck squamous cell carcinomas (HNSCC). In our study we analyzed archival (2001–2010) formalin-fixed paraffin-embedded tissues from patients with primary oropharyngeal squamous cell carcinomas for comparison of different detection methods.

Method: HPV detection was done by means of in situ hybridization (ISH) and polymerase chain reaction (PCR), the detection of indirect marker of active viral infection p16 was evaluated by immunohistochemistry.

Results: In our set of samples ISH was more sensitive than PCR with general primers most likely due to the degradation of nucleic acids caused by less careful fixation procedures. The positivity for HPV as assessed by ISH was strongly correlated both with the results of p16 immunohistochemistry and PCR HPV DNA detection.

Conclusion: We concluded that ISH is a clinically relevant method for molecular testing of HPV infections in archived biopsy material of HNSCC.


Oropharygeal squamous cell carcinoma (OPSCC): A clinicopathological study of 102 cases

M. C. Etxezarraga *, G. Cancho, L. Ortega, J. C. Lopez Duque, N. Liaño, J. Bilbao, M. M. Ramirez, C. Ereño

*Hospital Universitario Basurto, Dept. de Anatomía Patológica, Bilbao, Spain

Objective: To study the prognosis significance of p16 in patients with OPSCC and to evaluate the relationship between p16 and human papillomavirus (HPV).

Method: A total of 102 of OPSCCs diagnosed during 10 years were randomly selected, their histological sections were revaluated and follow-up was retrieved from medical files. P16 and P53 immunostaining was performed. HPV status was determined by PCR and in situ hybridation. Kaplan-Meier (KP) log- rank test was performed using SPSS software.

Results: The median follow-up period was 4 years. The 53.9 % (55/102) results p16(+) and 46 % (47/102) were p16(−). The 92 % (51/55) of p16(+) cases were in males with an average of 59,4 years (range 38–84). 73 % of p16(+) tumors were well-moderately differentiated SCCs, mostly located in tonsillar fossae. Viral DNA was present in 40 % (22/55) of p16 (+) cases, being HPV 16 type the most frequent. Five years overall survival was 63 % for p16(+) versus 36 % for p16(−) patients: p < 0,05 by KP log- rank test.

Conclusion: In OPSCC p16 expression predict a better overall survival. In our series HPV positive cases rate was lower than other published series.


Human papillomavirus, EGFR mutation and loss of heterozygosity at 17p13 and 9q22 in tonsillar squamous cell carcinoma

N. Kim *, S. J. Cho, Y. S. Roh

*Gachon University, Gil Medical Center, Pathology, Incheon, Republic of Korea

Objective: Head and neck squamous cell carcinoma (SCC) is the sixth most common malignancy with an increasing incidence of tonsillar SCC in several countries, and epidemiologic evidences demonstrate that up to 50 % of head and neck SCCs harbor human papillomavirus (HPV). This study was performed to determine the clinical significance of microsatellite alterations at 17p13 and 9q22, mutations in the epidermal growth factor receptor (EGFR) along with their association with HPV infections in tonsillar SCCs.

Method: The HPV was detected by type-specific polymerase chain reaction. Loss of heterozygosity (LOH) was analyzed by markers flanking chromosome 17p13 and 9q22.

Results: Thirty-four male and six female patients of tonsillar SCCs were included. LOH was detected in 57.5 % (23/40) at 17p13 and 45 % (18/40) at 9q22 with EGFR mutation (17.5 %, 7/40) and HPV-positivity (45 %, 18/40) the most common genotype was HPV 16 and 18 (6/18 and 6/18, 33.3 % and 33.3 %, respectively). Ten percent of HPV-infected tonsillar SCCs were multiple infection by HPV 11, 18, 40, 45, 52, 58 and 70.

Conclusion: Albeit small series, our results showed EGFR mutation and LOH were not associated with HPV-status, which confers a better prognosis. Further investigation is needed to clarify its association.


Predictive clinicopathological factors and immunohistochemical markers of locoregional control in patients with early-staged tongue cancer treated by brachytherapy

J. Laco *, I. Sirák, L. Tucek, M. Hodek, P. Paluska, L. Kašaová, S. Paulíková, M. Vošmik, M. Cvanová, M. Halámka, J. Petera

*Faculty of Medicine, Dept. of Pathology, Hradec Kralove, Czech Republic

Objective: The aim of the study was to investigate predictive clinicopathological factors and immunohistochemical markers of locoregional tumor control in patients with early-staged squamous cell carcinoma of tongue treated by brachytherapy.

Method: Twenty-four patients treated during the years 2001–2010 were enrolled in the study. In every tumor, TNM status, grade, resection margin, and depth of invasion were recorded. Using immunohistochemistry, both expression (percentage of positive tumor cells) and expression intensity (mild, moderate or strong) of p16 protein, epidermal growth factor receptor, nuclear factor-kappaB, hypoxia-inducible factor-1alpha, human epidermal growth factor receptor-2, Ku-80, cyclooxygenase-2 and vascular endothelial growth factor (VEGF) were evaluated. Correlation between disease-free survival and abovementioned parameters was performed using the NCSS 2004 software.

Results: Median follows-up was 37 months. The estimated 5-year local control was 80 % and locoregional control was 62 %. Only depth of tumor invasion (median 4 mm; range 1–20 mm) and expression intensity of VEGR (mild in 6, moderate in 17, and strong in 1 case) were significantly predictive of worse disease-free survival in Cox multivariate analysis (p = 0.018; p = 0.016).

Conclusion: Strong expression of VEGF and deep tumor invasion may be significant negative predictive markers of disease-free survival in tongue cancer patients treated by brachytherapy alone.


Tumor budding and depth of invasion predict the prognosis of patients with early stage oral tongue squamous cell carcinoma

A. Almangush *, I. Bello, J. Hagström, Y. Soini, P. Koivunen, R. Grénman, I. Leivo, T. Salo

*Haartman Institute, Dept. of Pathology, University of Helsinki, Finland

Objective: Oral (mobile) tongue squamous cell carcinoma (OTSCC) is characterized by poor survival even in cases of low stage (T1-T2). This multicentre study examined the value of tumor budding, depth of cancer invasion, histologic risk score (HRS), and cancer-associated fibroblasts (CAFs) in predicting the disease-specific survival of T1N0M0 and T2N0M0 OTSCC.

Method: A whole population-based patient material from all five university hospitals in Finland was collected for this study. Hematoxylin and eosin-stained slides were used to assess tumor budding, depth of cancer invasion, and histologic risk score in 233 cases of OTSCC. For identification of CAFs, immunohistochemical staining for α-SMA was carried out.

Results: Tumor budding and depth of tumor invasion predict disease-specific survival (p = 0.009 and 0.02, respectively). On the other hand, HRS and CAFs did not classify patients with early OTSCC into low and high-risk groups. However, worst pattern of invasion (WPOI), a component of HRS proved to be an independent prognosticator (P = 0.005).

Conclusion: Tumor budding, depth of invasion and worst pattern of invasion demonstrated the best predictive value in early OTSCC. Our findings may provide an approach for individualized patient management of early OTSCC.

Histological appearance of tumor budding:

figure ab


Stromal myofibroblasts in tongue squamous cell carcinoma of young patients

A. R. Santos-Silva*, F. P. Fonseca, R. D. Coletta, M. A. Lopes, O. P. Almeida, R. Carlos, A. M. P. Soubhia, A. C. P. Ribeiro, K. D. Hunter, P. M. Speight

*Piracicaba Dental School, Dept. of Oral Diagnosis, Brazil

Objective: The increase of oral squamous cell carcinomas (OSCC) affecting young people has been demonstrated worldwide. However, little is known about the biological nature and course of the disease. Therefore, the aim of this study was to evaluate the presence of myofibroblasts in the stromal compartment of OSCC affecting young patients.

Method: In this international multi-centre study, 29 patients younger than 40 years old affected by tongue SCC were retrieved from 4 different oral pathology services and investigated for the presence of myofibroblasts by immunohistochemical reactions against αSMA. The results obtained were correlated to ploidy status of the tumors and compared to the expression observed in cases of OSCC affecting older patients previously matched for tumor site and AJCC grade.

Results: 58.6 % of the young patients presented stromal myofibroblasts, whereas 70 % of the older patients revealed positivity for αSMA. No significant difference could be seen when comparing both groups and no correlation could be obtained with higher tumor grades and DNA content.

Conclusion: No significant difference exist between the presence of stromal myofibroblasts of tongue SCC affecting young and old individuals, suggesting that this stromal component would not be responsible for the clinical behavior of OSCC in young people.


Usefulness of Human Papilloma Virus (HPV) status in Squamous Cell Carcinoma (SCC) of the Upper Aerodigestive Tract (UADT): A clinicopathological study of 406 cases

M. C. Etxezarraga *, M. Zufiaurre, J. A. Nieto, N. Liaño, J. C. Lopez Duque, J. Bilbao, M. M. Ramirez, C. Ereño

*Hospital Universitario Basurto, Dept. de Anatomía Patológica, Bilbao, Spain

Objective: To study the prognosis value of HPV status in patients with SCC of the UADT and try to establish the clinicopathological features and overall survival (OS) differences between positive versus negative patients.

Method: A total of 406 SCCs of UADT diagnosed during 10 years were randomly selected, were revaluated histologically and follow-up was retrieved from medical files. P16 and P53 immunostaining was performed. HPV status was determined by PCR and in situ hybridation. Kaplan-Meier (KP) log- rank test was performed using SPSS software.

Results: p16 expression was found in 50 % (204/406) of all UADT-tumors. HPV was detected in 75(36 %) cases, being types 16 and 33 the most frequent. Among HPV-positive cases, 35(46 %) present diffuse nuclear and cytoplasmic (DNC) staining. Koilocytosis was present in 8 (66 %). The more most common site was supraglottic larynx (12/35). Mean age was 60,1 years (20–91 range). Male predominate (360/406). The median follow-up period was 4,2 years. OS analysis between 75-HPV(+) vs. 129-HPV(−) patients was statistically significant: p < 0,01 by KP log-rank test.

Conclusion: Ours results prove the value of HPV-positive status to predict a better OS survival. The pattern of p 16 DNC immunostaining is associated with viral status.


Cytokine profile changes after oral squamous cell carcinoma cells and macrophages co-culture

D. Antunes *, D. M. Guimarães, M. B. Lima, F. D. Nunes

*São Paulo, Brazil

Objective: Inflammation as a key player in the development of different cancers, especially of epithelial origin, has been object of studies for several years. Macrophages are important components of this process and assume different roles in cancers as, for instance, increased cytokine production. Changes in the production of inflammatory cytokines may lead to altered cell survival and proliferation. Objective: to investigate differences in the expression pattern of inflammatory cytokines when macrophages are co-cultured with oral squamous cell carcinoma (OSCC) cells.

Method: One macrophage (U937) and two oral squamous cell carcinoma cells lineages (SCC4 and SCC9) were cultured alone and together. Analysis of the cytokine profile was performed by enzyme immunoassay using the Luminex® xMAP™ system.

Results: All studied cytokines were expressed in OSCC cells and macrophages at variable levels. IL-6, IL-8 and GM-CSF were expressed at very high levels in both lineages of OSCC. Macrophages also expressed very high level of IL-8. When both OSCC cell lineages were co-cultured with macrophages significant increase of IL-1β, IL-2, IL-4, IL-6, TNF-α and G-CSF was observed.

Conclusion: The results show a significantly modified expression profile of inflammatory cytokines when OSCC cells and macrophages are cultured together. The increased cytokines were previously associated with a modified neoplastic behavior.


Low prevalence of Human Papilloma Virus (HPV) infection in biopsies of oral leukoplasia

J. M. Suárez Peñaranda *, P. Gandara Vila, C. Aliste, M. Perez Sayans, J. M. Gandara Rey

*Hospital Clinico Santiago de, Compostela, Dept. de Patalogia, Santiago de Compostela, Spain

Objective: We have tested the presence of HPV DNA in biopsies from oral leukoplasias to evaluate its role as a contributor in the early steps of oral cancer development

Method: DNA was obtained from 43 biopsies and tested for the presence of HPV DNA with the method CLART HPV2 (Genomica). Histopathology evaluation of dysplasia was made independently by two pathologists, according to the WHO classification.

Results: HPV DNA was present in only 10 biopsies: HPV 16 in 5, HPV 6 in 2, HPV 51 in 2 and HPV 33, 43 and 56 in one each. Cases with positive for HPV showed moderate dysplasia (1), severe dysplasia/in situ carcinoma (2) or infiltrating carcinoma (2). Cases negative for HPV showed mild dysplasia (7), severe dysplasia/in situ carcinoma (3) or infiltrating carcinoma (2 cases). No statistical correlation could be found between the presence of HPV DNA and the degree of dysplasia or the presence of carcinoma.

Conclusion: The prevalence of HPV infection in oral leukoplasias is low in our patients and not related with the degree of dysplasia or the presence of carcinoma. HPV infection does not seem to be an important etiopathogenic factor in the early stages of development of oral squamous cell carcinoma.


Tumor deposits in head and neck squamous cell carcinomas

S. Sarioglu *, S. Iplikci, N. Akbulut, B. Aydin, E. Dogan, M. Unlu, H. Ellidokuz, E. Ada, F. Akman, A. O. Ikiz

*Dokuz Eylul University, Dept. of Pathology, Izmir, Turkey

Objective: Tumor deposits (TDS) were described for colorectal and gastric carcinomas with prognostic implications. The aim of this study is to evaluate TD in head and neck squamous cell carcinomas (SCC).

Method: Sections from neck dissection specimens from 84(60 %) laryngeal, 31(22,1 %) oro/hypopharyngeal, 25(17,9) oral SCC cases were reevaluated according to the criteria described for the TDs of colorectal carcinomas. The relation of TDs with clinicopathological findings were analyzed statistically.

Results: Among 140 cases (63,66+54,78 months follow up), 24(17,1 %) had tumor deposits [12(4,3 %) laryngeal, 5(20 %) oro/hypopharyngeal, 7(22,6 %) oral cases (p = 0,53)]. Disease recurrence was identified in 40(28,6 %) of the patients, this was more frequent in cases with TDs [13(13,4 %) versus 11(27,5 %) p = 0,048] and especially for cases with distant metastasis [15(12,9 %) versus 9(42,7 %) p = 0,003)]. Fifty one (36,4 %) died during follow up and of these 38 were related to disease. Death was more frequent in cases with TDs. At 12 and 24 months 94,7 % and 85,2 % of patients without TDs were alive but this was 64,1 % and 37,9 % for cases with TDs respectively (p = 0,005).

Conclusion: TDs may be very important prognostic factors in head and neck SCC.

A tumor deposit and overall survival:

figure ac


The role of HOX genes in Head and Neck Squamous Cell Carcinoma (HNSCC)

K. Hunter *, F. Hakami, L. Darda, D. Lambert, C. Murdoch, R. Morgan, P. Woll

*University of Sheffield, Oral Pathology, United Kingdom

Objective: Molecular alterations in HNSCC are variable and related to the site and stage of disease. Our previous HNSCC microarray data showed differential expression of a number of HOX genes. These are developmental patterning genes whose expression is altered in many cancers.

Method: Expression of all 39 HOX genes was assessed by qPCR in HNSCC cells. The 2 most differentially expressed were assessed by WB and IHC in oral cells and tissues. Expression was altered by transient over-expression and siRNA into low and high-expressing cells respectively. The phenotype was assessed by MTS, fibronectin adhesion and transwell migration assays. Agilent microarray analysis was performed to identify novel targets and differentially activated pathways.

Results: HOXD10 and HOXB9 were expressed at low levels in normal cells and precancerous cells and at high levels in most primary tumours. HOXD10 expression was very low in lymph node metastases. Over-expression or silencing affected proliferation, adhesion, and migration. Direct/indirect targets of HOXD10 identified by microarray analysis include miR146a and may explain some of these effects.

Conclusion: The elevated expression of HOXD10 and HOXB9 allows tumours to grow whilst HOXD10 loss in metastases may promote cell survival. These 5-prime HOX genes may represent novel therapeutic targets in HNSCC.


Adenoid dysplasia of the oral mucosa

B. Bunn *, K. Hunter, S. A. Khurram, W. van Heerden

*University of Pretoria, Oral Pathology & Oral Biology, Johannesburg, South Africa

Objective: To present the proposed in-situ component of the rare mucosal acantholytic variant of squamous cell carcinoma (ASCC). An immunohistochemical appraisal of intraoral lesions displaying features of “adenoid dysplasia” is reported. The immunoprofile is compared with that of invasive ASCC, typical dysplasia and conventional keratinising squamous cell carcinoma (SCC).

Method: Immunohistochemical staining for E-cadherin, β-catenin, EMA, AE1/3, p63, vimentin and SMA was performed on formalin-fixed, paraffin-embedded tissue sections of adenoid dysplasia, ASCC, conventional moderately differentiated, keratinising SCC and moderate epithelial dysplasia.

Results: The immunoreactivity in cases designated as adenoid dysplasia recapitulates the immunoprofile detected in invasive ASCC. E-cadherin and β-catenin were most useful in this regard with little to no E-cadherin staining present within the acantholytic cells and upregulation of β-catenin. This contrasted starkly with the surrounding epithelium and in cases of conventional SCC and dysplasia.

Conclusion: Cases of intra-oral adenoid dysplasia were morphologically and immunophenotypically similar to invasive ASCC. ASCC represented an aggressive variant of SCC with an increased propensity for distant metastasis. It is postulated that adenoid dysplasia be associated with a greater risk of malignant transformation.


EMA expression and DNA ploidy in oral epithelial dysplasia

B. Bunn *, W. van Heerden

*University of Pretoria, Oral Pathology & Oral Biology, Johannesburg, South Africa

Objective: Microscopic assessment of histological grade is the most reliable method of determining the malignant transformation potential of dysplastic lesions. This study aimed to determine whether a relationship exists between EMA expression, DNA ploidy and histological grade in oral epithelial dysplasia.

Method: EMA immunohistochemical staining and DNA ploidy analysis by means of high resolution image cytometry was performed on formalin-fixed, paraffin embedded tissue sections previously diagnosed as mild (n = 11), moderate (n = 11) and severe (n = 13) dysplasia. Lesions were classified as either diploid or aneuploid while EMA immunoreactivity was semi-quantitatively assessed. In addition, the staining pattern and distribution were also documented. Normal oral epithelium and invasive squamous cell carcinoma served as negative and positive controls for EMA expression.

Results: EMA immunoreactivity increased in dysplasia, the intensity and distribution of which correlated with histological grade and DNA ploidy. Basal cell and/or suprabasal positivity were more frequently noted in lesions of a high-grade nature and were associated with an increased incidence of aneuploidy. EMA positivity within the suprabasal cells correlated with a lower histological grade in lesions which were more frequently diploid.

Conclusion: The immunolocalisation, intensity and distribution of EMA staining correlated well with histological grade and DNA ploidy. Both EMA expression and DNA ploidy analysis represented useful adjuncts to histological grading for determining the malignant potential of dysplastic oral epithelial lesions.


Acetylsalicylic acid efficacy on in vitro proliferation inhibition of oral squamous cell carcinoma

R. Moura *, D. Santos Pinto Junior

*University of Sao Paulo, Oral Patology, Brazil

Objective: The aim of this study was to analyze the effect of the Acetylsalicylic acid on squamous cell carcinoma cell line (OSCC9) through analysis of the cell viability and death in OSCC9 cells treated with acetylsalicylic acid.

Method: The SCC9 cell culture was performed and the cells were plated when the cell monolayer reaches 70 % confluence. The Acetylsalicylic acid cytotoxicity was analyzed by [3-(4,5-dimethylthiazol-2-yl)-5-(4- sulfophenyl)-2H-tetrazolium (MTS) cell proliferation assay. Viable cells reduce tetrazolium compounds into colored formazan products that were detected as an absorbance change with a spectrophotometer. The amount of formazan color produced is directly proportion to the number of viable cells. The standard of apoptosis was evaluated using the TdT-mediated dUTP nick-end labeling (TUNEL) assay, detecting apoptotic cells that undergo DNA degradation during the late stages of apoptosis. Untreated cells were used as control.

Results: Dose–response curves were generated. Based on obtained IC50 (50 % inhibitory concentration) values, the Acetylsalicylic acid concentration were determined the in 40μM. TUNEL assay shows Acetylsalicylic acid causes apoptosis in dose of 40 μM in 24 h.

Conclusion: Acetylsalicylic acid can be an effective therapeutic agent since it has shown great efficacy in the cellular proliferation inhibition on the OSCC9 cell line.


Methylation profile and amplification of homeobox genes in oral squamous cell carcinoma

F. Nunes *, C. Esteves, M. F. Rodrigues, F. Xavier

*FOUSP, São Paulo, Brazil

Objective: Genetic and epigenetic alterations have an important role in carcinogenesis, especially in allowing the overexpression of oncogenes and silencing the tumor suppressor genes. It is well known that deregulation of genes controlling growth and embryonic development is implicated in carcinogenesis, including homeobox genes. The methylation profile and amplification of HOXD10, HOXD11, PROX1, ZHX1, IRX4, HOXC13, HOXA5, HOXA7, HOXA9, HOXB5 genes were studied in oral squamous cell carcinoma

Method: DNA methylation was evaluated in 40 fresh tumor and non-tumor samples by qPCR using a customized plate for quick and precise detection of CpG islands methylation. Gene amplification was analyzed on the same samples by qPCR.

Results: All homeobox genes showed a low percentage of methylation. However, when amplification was related to clinical parameters PROX1 and IRX4 genes were associated with an increased risk of death associated with disease, whereas HOXA5 was associated with lymph node involvement, and a low HOXB5 with the occurrence of metastasis. Patients who showed methylation of the latter gene presented short-term survival.

Conclusion: The findings presented in this study provide new information about homeobox genes as potential biomarkers for diagnosis and predictive of tumor behavior for oral squamous cell carcinomas.


Expression of adhesion molecules in an in situ model of tumorigenesis

E. Martinez *, I. de Souza, A. P. Demasi, N. S. Araújo, V. C. Araújo

*São Leopoldo Mandic Institute, Oral Pathology, Sao Paulo, Brazil

Objective: Cell-cell interactions are important regulators of both normal and abnormal biological processes. Catenins interact with an intercellular protein (Ecadherin) regulating a strong cell-cell cohesion. Both proteins, in concert with the extracellular matrix are involved in many cell-signaling pathways such as proliferation and migration during the neoplastic process. Thus, the aim of this study was to analyse cell-cell interactions on sites that mimic an in situ situation when malignant squamous cells are surrounded by benign myoepithelial cells from pleomorphic adenoma.

Method: Benign myoepithelial cells from pleomorphic adenoma were cultured with non-filtered malignant conditioned medium from squamous cell carcinoma (CAL27, ATCC) in fibronectin substratum. βcatenin and Ecadherin immunoexpression were examined in these in situ areas by indirect immunofluorescence.

Results: The carcinoma malignant cells strongly expressed βcatenin and Ecadherin in the cell surfaces, mainly in further periods. In contrast, βcatenin and Ecadherin were not immunoexpressed in the neoplastic benign myoepithelial cells from pleomorphic adenoma during the neoplastic process.

Conclusion: The present results suggested that βcatenin-Ecadherin complex, in the proposal in vitro model, has had an important role in the maintenance of the carcinoma malignant cell clusters, highlighting their importance in the tumoral process under the influence of the extracellular matrix. (Grants from FAPESP 2011/14053-3)


In vitro influence of substance P on squamous cell carcinoma

I. Souza *, V. Montalli, M. H. Napimoga, F. Passador-Santos, V. Araújo, E. Martinez

*São Leopoldo Mandic Institute, Oral Patology, Guarulhos, Brazil

Objective: In the neoplastic process, the tumor microenvironment plays a fundamental role in the cancer behavior. Among the components related to tumor progression, studies have been indicated substance P as an important factor contributing for tumoral behavior. Thus, the purpose of the present study was to evaluate the in vitro influence of substance P, at different doses (10–8M and 10–6M), in the cell proliferation, invasion and the tissue matrix inhibitors (TIMPs) expression on squamous cell carcinoma cells.

Method: Cells from squamous cell carcinoma of the tongue (ATCC, CAL 27) were used. For the invasion assay, the cells were cultured in transwell chambers and the lower surface was then counted. TIMP-1 and TIMP-2 levels were evaluated by ELISA.

Results: There was a reduction on cell proliferation in all studied concentrations. However, substance P influenced the cell invasion, as well as, the levels of TIMP-1 and TIMP-2. The number of invaded cells was higher and TIMP-1 and TIMP-2 levels decreased in the presence of substance P.

Conclusion: The present in vitro results demonstrated that substance P increased the invasive potential of squamous cell carcinoma cells indicating its influence on tumor behavior. (Grants from Fapesp 2011/14053-3)


Immunohistochemical expression of the PI3K-AKT-mTOR pathway in epithelial dysplasia, reactive epithelial lesions and squamous cell carcinomas

F. Martins*, S. de Sousa, E. Santos, D. dos Santos Pinto Jr, S. B. Woo, M. Gallottini

*University of Sao Paulo, Oral Pathology, Brazil

Objective: To evaluate the immunohistochemical expression of the AKT-mTOR pathway in oral lesions diagnosed as low-grade (LGD) and high-grade dysplasia (HGD), squamous cell carcinoma (SCC), normal mucosa (NM) and frictional keratosis (FK).

Method: 186 cases, divided into 5 groups. Clinical information of the lesions was compiled. The labeling pattern of pAKT, pmTOR, PS6, and p4EBP1 antibodies were investigated by two pathologists and quantified by images using a photomicroscope (AxioCam®, Carl Zeiss MicroImaging GmbH, Germany). The variables were tested using chi-square, ANOVA F and univariate logistic regression analysis.

Results: Among all the cases of NM, positivity was found only pS6 (50 %); in FK cases there was positivity for pS6 (54.8 %) and 4EBP1 (22.6 %). In LGD, immunoreactivity was observed for pS6 (67.4 %), pAKT (56.2 %), p4EBP1 (41.7 %) and pmTOR (29.2 %), in the HGD group, positivity was found for pS6 (74 %), pAKT (68 %), p4EBP1 (44 %) and pmTOR (28 %). In SCCs, immunoreactivity was found for pAKT (83.3 %), PS6 (77.4 %), p4EBP1 (50 %) and pmTOR (50 %). Statistically significant differences were observed between all study groups and the proteins, except for pS6.

Conclusion: All the studied proteins are potential biomarkers to differentiate normal tissues from dysplastic lesions and SCC, but only pAKT and pmTOR proteins seem to be related to oral carcinogenesis.


Analysis of PTEN/AKT in the development of human oral tissue and oral squamous cell carcinoma: Role of malignant transformation

M. Buim *, S. Lourenço, F. A. Soares

*Hospital A C Camargo, Pathology, São Paulo, Brazil

Objective: Oral squamous cell carcinoma (OSCC) is a prevalent public health problem and is characterized by high degree of local aggression and lymph node metastasis. Signaling through the PTEN/AKT pathway is responsible for balancing survival and apoptosis cell. The goal of this study was to analyze the association of the PTEN and AKT expression with clinicopathological feature of the OSCC patients

Method: Expression of PTEN and AKT gene/protein was investigated in 68 cases of OSCC, obtained from the files of Anatomic Pathology Department from A.C. Camargo Hospital (Brazil), by qRT-PCR and IHC.

Results: Low PTEN gene and protein expression occurred in 66 % and 63 %, respectively, OSCC cases. However, no association was observed between this molecule and clinicopathological parameters. High phosphorylate AKT (pAKT) protein expression was associated with poor/moderated differentiated histological grade (p = 0.029); no associated was observed between pAkt protein expression and others clinicopathological parameters. AKT gene expression, by qRT-PCR, was associated with clinical stage (0.034), perineural infiltration (p = 0.014) and tumor size (p = 0.037).

Conclusion: Our results suggest that downregulation of PTEN can be important to OSCC tumorigenesis and can contribute to oncogene activation, and high pAKT expression might be an unfavorable prognostic marker in OSCC.


Metallothionein is not associated with metastatic behaviour in oral squamous cell carcinoma

S. Cardoso *, P. R. Faria, A. C. Campos, L. B. Muniz, A. M. Loyola

*Federal University of Uberlând, Pathology, School of Dentistry, Uberlândia, Brazil

Objective: Overexpression of metallothionein (MT) is associated to worst prognosis for many human cancers, including oral squamous cell carcinoma (OSCC). Recent papers reported association of MT with metastatic behavior of OSCC, but there are conflicting results from other works. The aim of this study was to compare the immunohistochemical reactivity of MT in samples of metastasizing or nonmetastasizing primary OSCC (PM and PNM, respectively), as well as in respective metastatic lymph node carcinomas (N) in order to clarify the role of MT in the biological behavior of OSCC.

Method: Fifteen PNM, 15 PM OSCC, and 14 N were submitted to immunohistochemistry to detect MT. It was semiquantified according to cytoplasmic and nuclear reactivity in different areas of adjacent non-neoplastic epithelium, invasive carcinoma, and metastatic implants. Statistical tests were used to compare these findings with metastatic behavior.

Results: None criteria for MT reactivity was not associated with the metastatic behavior of primary OSCC. It was also not associated with the primary or metastatic nature of the samples.

Conclusion: The present results do not indicate a role for MT on the metastatic phenotype of OSCC, nor support the usefulness of MT immunohistochemical analysis as a predictive tool for metastasis in this disease. Financial support: CNPq, FAPEMIG, CAPES.


Association of intrauterine growth retardation and fetal teeth germs structural remodeling in rats

S. Morozov *, O. Reshetnikova

*State Medical University, Lugansk, Ukraine

Objective: It is widely accepted that fetal intrauterine growth retardation (IUGR) results in various diseases in later life. Preterm and low birth weight newborns are at risk of severe childhood caries. But the relationships between growth restrictions and structural parameters of newborn’s teeth germs are still unclear.

Method: Morphologic and morphometric peculiarities of the newborns teeth germs in 65 rats (including 35- with experimental systemic growth retardation and 30–controls) have been analyzed. Histological slides, stained with hematoxylin and eosin, were studied microscopically, then they were analyzed by point count method and using the computer morphometry of the enamel thickness.

Results: Results have shown the delayed growth and maturation of the teeth hard tissues. Volume fractions of have shown the delayed growth and maturation of the teeth hard tissues. Volume fractions of dental papilla, enamel organ and dentin with enamel were reduced compared with control parameters (respectively 8,33 ± 0,66; 4,77 ± 1,09; 3,46 ± 0,75 % compared with 10,00 ± 1,02; 8,98 ± 1,60; 6,76 ± 0,88 % in controls, P < 0, 05). The average enamel’s thickness also decreased to 2,98 ± 0,19 mkm (in controls-4,24 ± 0,77; P < 0,05).

Conclusion: These findings present the evidence of an association between IUGR and impaired growth and maturation of the newborns teeth germs. Immature hard tissues of teeth germs may predispose to early development of the tooth decay.


Extramedullary plasmacytoma in tongue: A rare entity

E. Tastekin *, N. Can, M. Azatçam, F. Oz Puyan, A. Arslan

*Trakya University, Pathology, Edirne, Turkey

Objective: Extramedullary plasmacytoma is a variant of multiple myeloma and relatively rare. Approximately 80 % of them occur in the upper aerodigestive tract.

Method: Case: A 56-year-old female patient has been follow-up for an ulcerated lesion in tongue since 2010. The successive four incisional biopsies were performed. All biopsies were diagnosed as ulceration, acute inflammation and hyperemia. Before the fifth biopsy physical examination was shown severe granular appearance nodular lesion revealing severe granular apperance on oral side of tongue. Incisional biopsy was performed. Microscopically epithelial ulceration and subepithelial diffuse plasma cell infiltration were detected. These plasma cells were positive stained with CD138, kappa and IgG antibodies immunohistochemically. There was no reaction with lambda, other Ig antibodies and LCA. We diagnosed this case as “Atypical plasma cell infiltration” and we suggested performing of serum protein electrophoresis. There was no spike in any type of plasma proteins. These histomorphological and laboratory findings suggested to the diagnosis of “extramedullary plasmositoma in tongue”.

Conclusion: Surgical pathologists should evalute ulcerated and diffuse inflammatory lesions very carefully and must avoid from the only descriptive diagnosis. Extramedullary plasmositoma is an important entity for differential diagnosis.

Sunday, 1 September 2013, 09.30–10.30, Pavilion 2

PS-04 Poster Session Ophthalmic Pathology


Androgen receptor in sebaceous gland carcinoma of the eyelid: Does it have a prognostic significance?

K. Mulay *, V. White, S. Shah, S. Honavar

*Center for Sight, Ophthalmic Pathology, Hyderabad, India

Objective: To evaluate the frequency of expression of Androgen receptor (AR) in sebaceous gland carcinoma of the eyelid and correlate its expression with histopathological features and clinical outcomes.

Method: 55 cases of eyelid sebaceous gland carcinoma with a minimum follow up of 1-year were retrieved from the archives. Patient files were assessed for clinical outcomes. Immunostaining for androgen receptor, Ki-67 was performed on all cases. All tumours were assigned an androgen receptor score (AR-Score) based on the intensity and percentage expression. The AR-score were correlated with the clinical outcomes, histopathological features and expression of Ki-67.

Results: All tumours (100 %) expressed AR. A high AR-score was significantly associated with lower disease free survival (p < 0.0001), high Ki-67 activity (p < 0.001) and early recurrence (p < 0.001). There was no statistically significant association between AR-score and histopathological features.

Conclusion: Our results confirmed the expression of androgen receptor (AR) in sebaceous gland carcinoma. It also suggested a prognostic influence of AR expression in sebaceous gland carcinoma of the eyelid.


Choroidal extramedullary hematopoiesis as a chance finding in abusive head trauma

R. Verdijk *

*Erasmus MC University, Dept. of Pathology, Rotterdam, The Netherlands

Objective: To investigate the frequency of extramedullary hematopoiesis in post mortem eyeball specimens sent for evaluation of abusive head trauma.

Method: Extensive unilateral extramedullary hematopoiesis of the choroid was observed in an otherwise clear cut case of abusive head trauma with bilateral retinal hemorrhage, optic nerve sheath hemorrhage and peripapillary intrascleral hemorrhage. Subsequently, 60 consecutive cases of suspected abusive head trauma examined since 2005 were evaluated for the presence of extramedullary hematopoiesis.

Results: Review of 60 archival cases of suspected abusive head trauma identified two more examples of extramedullary hematopoiesis in abusive head trauma that initially had been diagnosed as uveitis.

Conclusion: Extramedullary hematopoiesis of the choroid may be observed in 5 % of post mortem histopathologic examinations of eyes sent for evaluation of abusive head trauma. It may be a chance finding or can have a relation to premature birth, possible anemia or may be a reaction to intraocular bleeding due to earlier trauma. Choroidal extramedullary hematopoiesis may easily be confused for choroiditis and interpreted as a sign of infectious disease. Awarenes of the possibiliy of extramedullary hematopoiesis of the choroid should prevent misdiagnosis.


Raised numbers of IgG4-positive plasma cells are a common histopathological finding in orbital xanthogranulomatous disease

R. Verdijk *, P. Heidari, R. Verschooten, P. L. van Daele, H. J. Simonsz, D. Paridaens

*Erasmus MC University, Dept. of Pathology, Rotterdam, The Netherlands

Objective: To determine the relation of orbital xanthogranuloma to IgG4-related disease. Xanthogranuloma is a benign, tumor that rarely affects the orbit. The response to immunosuppressive therapy and relapse rate may be dependent on as yet unknown immunological reaction patterns that may involve IgG4 related disease.

Method: We searched our charts for orbital xanthogranuloma diagnosed over the last 25 years. Patient files were reviewed for clinical and follow up data. Histopathological classification was re-assessed. Sixteen cases of orbital xanthogranuloma were evaluated. Immunohistochemical stains for IgG and IgG4 were performed.

Results: Eight out of 16 cases (50 %) showed raised numbers of IgG4-positive plasma cells in the tissues, scored as >50 per high-power field, with IgG4/IgG ratio >0,40. This seemed to be associated with recurrent disease. The groups were too small to reach statistical significance, however. The morphological features of the cases with high IgG4-positive plasma cell counts conforming to the Boston Consensus Criteria lacked the criteria of storiform fibrosis and phlebitis. None of the patients developed systemic manifestations of IgG4 related disease.

Conclusion: Raised numbers of IgG4-positive plasma cells are a common finding in histopathological specimens of xanthogranulomatous disease of the orbit and are not indicative for IgG4 related systemic disease.

Clinical, histological (H&E) and immunohistochemical images of IgG and IgG4 staining of the different xanthogranulomatous diseases.:

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Atypical pleomorphic adenoma of the lacrimal gland: Report of a case

M. Sofopoulos *, N. Mylona, N. Koufopoulos, A. Vacarciuc, V. Petrou, N. Arnogiannaki

*Agios Savvas Hospital, Dept. of Pathology, Athens, Greece

Objective: We present a case of an Atypical Pleiomorphic Adenoma of the lacrimal gland.

Method: A 68 year old man with a small painless slow growing mass situated on his right lacrimal fossa. Clinical examination revealed a firm nodular, non pulsatile mass whose overlying conjuctiva was freely movable. Total excision was performed and a biopsy was sent to our laboratory.

Results: The specimen consisted of numerous irregular, ill-defined whitish pieces of tissue deprived of capsule. Histological examination revealed a neoplasm with intimate admixture of epithelial and mesenchymal elements. The latter is focally hyalinized and chondromyxoid. The epitelial component consists of cuboidal cells with enlarged nuclei, arranged in a trabecular fashion forming glands with an associated myoepitelial layer. These cells bear large, slightly atypical nuclei, with prominent nucleoli. Mitoses were rare and necrotic debris within the neoplasmatic lumen were found.

Conclusion: The diagnosis was atypical mixed tumor of the lacrimal gland. Atypical Pleiomorphic Adenoma is a rare premalignant condition which exhibits at least one of the following: capsule infiltration, hypercellularity, hyalinization, necrosis or cellular anaplasia. The treatment protocol recommends complete en-bloc excision of the tumor without a preliminary biopsy since incisional biopsy is related to an increased rate of recurrence due to incompletely excised mixed tumors.


Fine-needle aspiration biopsy as prognostic tool in uveal melanoma

M. Mera *, L. Blaga

*Univ. of Medicine and Pharmacy, Dept. of Pathology, Cluj-Napoca, Romania

Objective: Fine-needle aspiration biopsy (FNAB) becomes increasingly a valuable tool not only for diagnosis but for prognostication before treatment in an effort to avoid the much distressing enucleation. Our study is meant to ascertain whether sufficiently accurate prognosis information could be gleaned from fine needle aspiration biopsies as this is not used routinely yet in our area.

Method: We have included in our study 29 cases with FNAB prior to enucleation, including cytopathologic features as well as conventional histologic assessment.

Results: We have found that enough material for reliable cytodiagnosis by FNAB has been obtained in 27 (93 %) of cases. By means of appropriate statistical analysis we have found an excellent correlation between the presence of epithelioid melanoma cells observed on FNAB samples and the other histologic features obtained after enucleation. As further research we intend to add flow cytometry and molecular genetics workups to assess the prognostic reliability of a FNAB alone.

Conclusion: Fine-needle aspiration biopsy becomes increasingly compelling in order to assess a patient’s prognosis so that the most appropriate and the least distressing treatment can be applied. The ultimate goal is to improve the clinical practice in our area, where enucleation is still the procedure of choice.


Angiolymphoid hyperplasia with eosinophilia (epitheloid hemangioma) of the orbit

A. Demirovic *, I. Veliki-Dalic, R. Ivekovic, B. Kruslin, L. Pazanin

*University Hospital Centre, Dept. of Pathology, Zagreb, Croatia

Objective: Angiolymphoid hyperplasia with eosinophilia (ALHE) is the same condition as epitheloid hemangioma. It is an uncommon vascular tumor that occurs in the orbit extremely rarely. The main differential diagnosis is Kimura’s disease (KD). We present a case of an ALHE of the orbit and discuss main differences between ALHE and KD.

Method: Case presentation

Results: An 83-year-old patient presented with a subcutaneous mass of the left lower eyelid. Magnetic resonance imaging revealed a tumor in the left orbit and lower eyelid. An inferior orbitotomy was performed with tumor excision. Histopathological evaluation revealed a lobulated tumor measuring 2.5 × 2 × 0.8 cm, composed of proliferated blood vessels of varying caliber, lined by plump endothelial cells with large cytoplasmic vacuoles. The stroma contained numerous lymphatic follicles with prominent germinal centers and large proportion of eosinophils. The diagnosis of ALHE was made.

Conclusion: ALHE was first characterized by Wells and Whimster and the term epitheloid hemangioma was introduced by Enzinger and Weiss. ALHE shares some features with KD. However, they can be distinguished microscopically: ALHE has swollen, vacuolated endothelial cells; endothelial cells in KD are attenuated, without cytoplasmic vacuoles. Moreover, KD has systemic manifestations. ALHE is treated with surgical exision with approximately 33 % recurrence rate.

Sunday, 1 September 2013, 09.30–10.30, Pavilion 2

PS-05 Poster Session Paediatric and Perinatal Pathology


Morphological characteristics of thyroid gland of the fetuses from HIV-infected mothers

I. Sorokina *, S. Sherstyuk, T. Ospanova

*Nationa Medical University, Dept. of Pathologic Anatomy, Kharkiv, Ukraine

Objective: Thyroid diseases are now emerging in the first place among all endocrine diseases in children. Information on immunohistochemical features of thyroid gland in stillbirths from HIV-infected mothers, despite the steadily growing number of HIV-infected women of childbearing age, in the available literature has not been identified. The purpose of research is identification of immunohistochemical features of thyroid gland of the stillbirths from HIV-infected mothers

Method: In this research was used the thyroid glands of the fetuses from HIV-infected mothers. Immunohistochemical study was performed using monoclonal antibody to T3 and T4.

Results: Immunohistochemical study of the thyroid glands of the fetuses from HIV-infected mothers reveal the increased intensity of luminescence of the thyrocytes, compared to the control group, to T4 and T3 monoclonal antibody but light intensity of T4 was higher than T3. Apparently, the increase of the secretory activity of the thyroid gland of the stillbirths from HIV-infected mothers, compared to control group, is a manifestation of compensatory-adaptive mechanisms aimed to maintain homeostasis in the difficult conditions of maternal HIV infection.

Conclusion: In the thyroid gland of the stillbirths from HIV-positive mothers revealed a marked increase of secretory activity.


Autopsy findings in congenital somatic overgrowth consistant with Beckwith Wiedemann syndrome

M. Kos *, T. Lenicek

*KBC, Dept. of Pathology, Zagreb, Croatia

Objective: To make as accurate postmortem diagnosis as possible in a prematurely born neonate showing features of congenital somatic overgrowth.

Method: An autopsy was performed on a male neonate born at 32 weeks gestation after the lethal outcome in the 2nd day of life. The infant was mechanically ventilated, clinical informations were that he developed a right sided pneumothorax and respiratory distress syndrome. Except for acidosis, the laboratory findings also showed hypoglycemia, other results were unremarkable.

Results: The neonate weighted 3,030 g (normal 1,543+/−519 g), the lenght was 50 cm (normal 38.9+/−5.7 cm), at autopsy there was generalized visceromegaly. Microscopically, pancreas showed endocrine hyperplasia, both kidneys contained foci of nephroblastomatosis, both adrenals showed cytomegaly and cystic changes of the definitive cortex. An ectopic focus of adrenal tissue was found in the left lung. Other findings were consistant with prematurity; partial atelectasis, interstitial emphysema, hyaline membranes in the lungs and internal and external hematocephalus. The neonate also exhibited midfacial hypoplasia, macroglossia and ear lobe grooves.

Conclusion: There is a number of syndromes characterized with congenital somatic overgrowth. The findings in this case are most consistant with Beckwith-Wiedemann syndrome. The more sophisticated diagnostic methods (genetic analysis) were not available.


Case report: Congenital pulmonary lymphangiectasis in fetal autopsy

G. Tasova Yilmaz *, S. Toru, G. Ozbilim, C. Y. Sanhal

*Akdeniz University, School of Medicine, Dept. of Pathology, Antalya, Turkey

Objective: Congenital pulmonary lymphangiectasis (CPL) is a rare developmental disorder involving lung. CPL is a poorly documented disease, characterized by prominent lymhatic dilatation of septal-subpleural-peribronchial tissue of the lung.

Method: Here in we report a 17-week-gestation fetus with anhydramnios. In light microscopy there were marked dilatated channels in the subpleural -peribronchial-subseptal region of the lungs. The channels were lined with flattened cells which were expressing CD 31, negative for CD34. Although pulmonary interstitial emphysema (PIE) was considered an important differential diagnosis, a giant cell reaction surrounding the interstitial cystic lesions, a histologic hallmark of PIE. The morphological and immunohistochemical findings confirmed to a primary form of CPL, Noonan Group 3.

Results: CPL is characterized by dilatation of the pulmonary lymphatic vessels and occurs as a congenital anomaly. Noonan classified it into three goups. Primary developmental defect of pulmonary lymphatics is group 3. Group 3 is called also as congenital pulmonary lymphangiectasis, normal regression of the connective tissue elements fails to occur after the 16th week of fetal life, associated with an agressive clinical course, poor prognosis.

Conclusion: We report a rare developmental disorder diagnosed in fetal autopsy examination which was clinically and radiologically not detected.


Corticotropin releasing factor neuropeptides and binding sites in human fetal lung development and pathology

M. Lambropoulou *, E. Chouridou, M. Koureta, I. Balgouranidou, M. Simopoulou, N. Tsesmetzis, T.-E. Deftereou, V. Tsoulopoulos, N. Papadopoulos, E. Chatzaki

*Medical School, Democritus University of Thrace, Alexandroupolis, Greece

Objective: The CRF system (neuropeptides CRF, Ucn I, II, III and binding sites CRF1, CRF2, CRF-BP) is responsible for stress regulation and homeostasis of an organism. Here, we study CRF system expression in human normal and pathological fetal lungs.

Method: Lung tissues from 46 archival human fetuses were divided in Group A (normal), Group B (chromosomal abnormalities) and Group C (congenital disorders). Expression of the CRF system elements was evaluated using immunohistochemistry and was correlated to pathology, lung developmental stage and clinicopathological characteristics.

Results: Immunoreactivity for all antigens was found in both epithelial and mesenchymal lung cells of bronchi and alveoli. Ucn I and CRF1 were more frequently expressed in Group A. Ucns were more frequently expressed in the pseudoglandular stage. There was a positive correlation between the expression of the CRF neuropeptides and between CRF1 and CRF. Two fetuses with lung malformations showed no or low expression.

Conclusion: We demonstrated the expression of CRF system in human fetal lungs, being correlated to pathology and developmental stage. Our results comply with findings in experimental animal models, implicating the CRF system to fetal lung development, being more significant in the early stages.

CRF1 expression in fetal lung X200:

figure ae


Cri du Chat syndrome with gingival fibromatosis: Histopathological analysis with confocal laser scanning microscopy

G. Favia *, S. Franco, S. Miccoli, V. De Falco, M. G. Lacaita, D. Di Venere, M. Falagario, E. Maiorano

*University of Bari, Dept. Odontostomatology and Surgery, Italy

Objective: Cri du Chat Syndrome (CCS) is a rare chromosomal disorder resulting from deletion of the short arm of chromosome 5, characterized by distinctive catlike cry. We report on still unreported clinic-pathological features CCS: diffuse Gingival Fibromatosis (GF) with Confocal Laser Scanning Microscopic (CLSM) examination.

Method: Case Presentation: A 21-year-old female, affected by CCS showed diffuse GF on the palatal gingiva in the molar region, which was surgically removed by Diode Laser, formalin-fixed, stained with hematoxylin-eosin and picrosirius red and analyzed atCLSM Nikon E-600 with double Laser inducing fluorescence (green and red).

Results: Microscopically, the lesion consisted in large parallel collagenous fibers, abundant blood vessels with plump endothelial cells and chronic inflammatory reaction, and large polygonal cells in the vascular interstitial spaces, with large nuclei, resembling undifferentiated totipotent mesenchymal cells (stem-like cells). At CLSM large and variably oriented collagenous fibers displayed intense fluorescence due to cross-linking between such fibers, which generally characterize fibromatosis, and the vascular structures. The latter showed lower fluorescence intensity, and were surrounded by loose collagenous fibers and trapezoidal large mesenchymal cells.

Conclusion: GF seems a characteristic feature of CCS, with typical fluorescent pattern at CLSM, in which purported totipotent mesenchymal cells may play a pathogenetic role.

Confocal Laser Scanning Microscopy:

figure af


Tuberous sclerosis: Histological analysis with confocal laser scanning microscope of gingival angiofibromatosis

M. L. Caruso *, A. Tempesta, S. Miccoli, V. Lacarbonara, M. G. Lacaita, M. Corsalini, G. Favia, E. Maiorano

*IRCCS De Bellis Castellana, Pathology, Castellana Grotte Ba, Italy

Objective: We report on a case of Tuberous Sclerosis (TS) with gingival angiofibromatosis (GA), diagnosed by histopathological analysis with Confocal Laser Scanning Microscopy (CLSM) and treated with High-Power Diode Laser gingivectomy.

Method: Case presentation: The patient underwent gingivectomy and gingivoplasty with High-Power Diode Laser in pulsed modality and the surgical sample was formalin-fixed, paraffin-embedded and stained with hematoxylin-eosin and Pricrosirius red.

Results: Microscopically, thickened acanthotic epithelium with elongated rete ridges, densely packed, whorly collagen fibers, fibroblasts, variably sized vascular structures, and a few chronic inflammatory cells were detected. At CLSM examination, (Nikon Eclipse E-600 with green/red Laser inducing fluorescence) the collagen fibers, showing intense fluorescence, also manifested variable spatial orientation, due to cross-links among the bundles, ad typical of fibromatosis. Also, variably sized blood vessels and large and polygonal interstitial cells displayed fluorescence of lower intensity. The vascular component consisted of small groups of venous-like structures, frequently showing dilated lumina, thin walls and plump endothelial lining.

Conclusion: The histopathological analysis with CLSM of GA occurring in TS highlightes distinctive features, such as low fluorescence areas and a typical vascular component which may represent distinctive features of such lesion.

Confocal Laser Scanning Microscopy:

figure ag


Correlation of computed tomography post-mortem imaging findings and autopsy findings regarding a case of congenital toxoplasmosis

C. Tomikawa *, R. Schultz, P. H. N. Saldiva

*FMUSP, Dept. of Pathology, Sao Paulo, Brazil

Objective: Post-mortem imaging is progressively used for autopsies, mainly in the forensic area, regarding this, we propose to correlate computed tomography (CT) post-mortem imaging findings and autopsy findings regarding a case of congenital toxoplasmosis.

Method: A female newborn with 33 weeks of pregnancy, admitted to emergency service with signs of intrauterine hypoxia,dying shortly after delivery, was submitted to the autopsy service. Whole-body CT and standardized autopsy examination were performed. An experienced clinical radiologist evaluated the imaging data without knowledge of autopsy findings.

Results: The CT and autopsy reports had few discrepancies. These were mainly regarding minor diagnosis not directly related to the cause of death. The autopsy report was, above all, more detailed with histological and immunohistochemical studies determining involvement extension and intensity on the organs.

Conclusion: Well established for forensic autopsies, the CT post-mortem imaging may be a complement to traditional autopsy also on the context of a newborn/stillborn–however, it is not yet capable of replacing it.

Central nervous system calcifications on CT:

figure ah


Prenatal diagnostic approach to fetal skeletal dysplasia

H. S. Toru *, G. Tasova Yilmaz, I. H. Ozbudak, B. Nur, C. Y. Sanhal, K. Karaali, O. Alper, I. Mendilcioglu, E. Mihci, S. Karaveli

*Akdeniz University, Pathology, Antalya, Turkey

Objective: Skeletal dysplasias (SDs) constitute a group of heterogeneous disorders affecting growth-morphology of the chondro-osseous tissues. Prenatal diagnosis of SD is a considerable clinical challenge due to phenotypic variability.

Method: The archive of fetal autopsies was reviewed between January 2006 and December 2012. Fifty-four cases were detected which were diagnosed by ultrasonography. Among them 17 cases underwent amniocentesis, one case underwent chorionic villus sampling and three cases underwent molecular analyses.

Results: One of the cases had a diagnosis of Trisomy 18, terminated at gestational age of 14thweek. Three cases had specific gene mutations as following: p. Arg248Cys, and p. Gly370Cys missense mutations at FGFR3 and homozygous c.1878delA/c.1878delA mutation at Dymeclin gene. In FGFR3 mutated cases, ultrasonographic findings were detected at gestational ages of 20th and 25th weeks. Case with dymeclin gene mutation had a sibling with Dyggve-Melchior-Clausen syndrome which was terminated at gestational age of 16thweek. Thirty-one of cases were associated with at least one congenital anomaly.

Conclusion: This study showed that SD is usually detected clinically after 20th gestational week. This gestational age could be considered as late to plan termination of pregnancy because of the psychological and clinical complications. Performing genetic analyses for SD can provide early diagnosis and management.


Twin reverse arterial perfusion syndrome: A case report

S. Toprak *, E. Atik, A. G. Okyay, O. Ipci, T. Ozgur, H. Gokce

*Mustafa Kemal University, School of Medicine, Dept. of Pathology, Hatay, Turkey

Objective: Twin Reverse Arterial Perfusion (TRAP) Syndrome is rarely seen in %1 of monochorionic twins. We aimed to present a rarely seen case.

Method: 28 year old woman at 28th gestation week with a history of two aborts and healthy triplet babies referred to clinic. Doppler USG revealed monochorionic plasenta and reversal flow in umbilical artery. Amorphous twin and plasenta send for autopsy.

Results: Macroscopically there were gastroschisis and two arteries,one vein and colon in edematous umbilical cord. Plasenta was monochorionic. At recipient twin deformed feet with oligodactyl, partially developed lumbar and thoracic spine was observed while the cervikal spine, head, heart, upper limbs and external genitalia was not observed. Abdominal organs were not well developed. TRAP syndrome was diagnosed.

Conclusion: TRAP syndrome is one of the rarest complications of monochorionic twins. Structurally normal, the pumping twin, perfuses acardiac twin. Reverse flow in the umbilical artery selectively to iliac vessels of the recipient twin is the key pathology. As the oxygenation of lower segment of the body is well, lower limbs grow better than upper segments. This leads to anomalies extending to amorphogenesis of upper segments of body. This case highligts the importance of prenatal diagnosis and planning treatment.


Neural tube defects and associated anomalies in fetal and perinatal autopsy series

H. S. Toru *, C. Uzun, G. A. Gokhan Ocak, C. Y. Sanhal, I. Mendilcioglu, S. Karaveli

*Akdeniz University, Pathology, Antalya, Turkey

Objective: Neural tube defects (NTDs) are heterogeneous and complex group of congenital anomalies. NTDs are major congenital malformations affecting the brain and spinal cord. NTDs often result in fetal death, death early in life, or in developmental disabilities among surviving infants and children.

Method: The archive of fetal autopsies was reviewed between January 2006 and June 2012. Sixty-two of them had NTD. For all fetuses we carried out macroscopic assessment, photographic records, gestational age, maternal age, clinical findings.

Results: Twenty-two of 62 cases with NTDs are associated with at least one anomaly. Thirty-two cases were chranioschisis, 28 cases were spina bifida and 2 cases were craniospinal rachischisis subtype. The most common anomalies associated with NTD is renal-urinary system anomalies(16.1 %), followed by skeletal (9.7 %), pulmonary (4.8 %)and cardiac anomalies(4.8 %).

Conclusion: The overall prevalence of associated malformations, observed in 35.5 %, emphasizes the need for a thorough investigation of infants with NTDs. Routine screening for other malformations, especially renal-urinary system, skeletal and pulmonary anomalies and cardiac defects may need to be considered in infants and fetus with NTDs, and referral for genetics evaluation and counseling seems warranted in most of these complicated cases. Prenatal early diagnose of the NTD may reduce the psychological and clinical complications of termination.


Fetal mediastinal teratoma- a rare cause of nonimmune hydrops fetalis. Report of one case

A. Rodrigues *, L. Monteiro, S. Ramos

*Hospital Egas Moniz, Pathology, Lisbon, Portugal

Objective: Fetal mediastinal teratomas are rare congenital germ cell tumors that can compress mediastinal structures and cause nonimmune hydrops.

Method: The authors present a case of a 36 years old woman, gravida 1, para 0111. Ultrasound evaluation at 21 weeks of gestation revealed fetal hydrops and suggested a diaphragmatic hernia with severe bilateral lung hypoplasia. An amniocentesis was performed and showed normal chromosomes, 46, XY. At 22 weeks of gestation a medical interruption of pregnancy was performed.

Results: The post-mortem examination revealed a massive tumor of the anterosuperior mediastinum with cardiac and bilateral lung compression and fetal hydrops. The tumor was solid, encapsulated, 34,59 g of weight and 5 × 4,5 × 3 cm. Histological examination revealed a solid teratoma composed of mature tissues admixed with immature neuroglial elements.

Conclusion: This rare condition, that could have an adverse outcome, should be included in the diagnostic evaluation of any case of second trimester nonimmune hydrops, associated with severe lung hypoplasia.


Heterotaxy syndromes: A case report of Ivemark syndrome

C. Callé *, A. Rodrigues, A. Teixeira, L. Caceres Monteiro

*IPO Lisboa, Anatomia Patológica, Portugal

Objective: The heterotaxy syndromes are rare diseases with an incidence of 1–1.5/10,000 live births. These complex entities are associated with numerous malformations, in particular, complex cardiac and extra-cardiac malformations that have an important impact on the prenatal and postnatal course. Right isomerism has a high postnatal mortality due to the more complex type of cardiac defects.

Method: We report a case of medical interruption of pregnancy at 25 week of gestation, due to severe cardiac abnormalities incompatibles with life. The echocardiography revealed a fetus with total anomalous pulmonary venous drainage, atrioventricular septal defect, and right side truncus arteriosus of aortic type. No other malformations were detected in morphologic ecography.

Results: The autopsy confirmed the complex heart defects and revealed bilateral trilobed lungs and asplenia, settling the diagnosis of Ivemark Syndrome.

Conclusion: The Ivemark complex (OMIM 208530) is a malformation with bilateral right-sidedness, with a Mendelian autosomal recessive inheritance, included in the group of Heterotaxy syndromes. The exact diagnosis of this entity is mandatory for adequate genetic counselling of the parents and for planning the postnatal care in a new pregnancy.


Contribution of fetal autopsy for diagnosis of Beckwith-Widemann Syndrome (BWS)

M. E. Jo Velasco *, I. Guerra, G. Perez de Nanclares, P. Morales, I. Garin, J. J. Aguirre, Z. S. Quintero, C. Gomez

*Hospital Txagorritxu, Dept. of Pathology, Vitoria-Gasteiz, Spain

Objective: Omphalocele is recognized as congenital malformation with a high mortality. It is associated with other congenital malformations.29 % has abnormal karyotype. The aim of the present study is to show the importance of perform the foetal autopsy in the legal interruption of the pregnancy with fetal diagnosis for scan of omphalocele.

Method: Fetal autopsy,histopathologic examination and molecular analysis were performed in a male fetus:46 XY karyotype,therapeutically aborted at 21 weeks of gestation with scan of omphalocele.

Results: Gross examination showed a male fetus with omphalocele and his weight corresponded to 24 weeks of gestation. Histologic showed adrenocortical cytomegaly which suggested the presence of BWS.We revised again grossly the fetus and we found that BWS was confirmed by the existence of his 3 criteria;macroglossia, omphalocele,macrosomy. Accompanied by minor signs: hemihypertrophyfacial,ear-creases, hepatomegaly. Molecular analysis revealed loss of methylation at IC2 (KvDMR); the most common genetic alteration associated with BWS.

Conclusion: BWS is an overgrowth disorder with variability in clinical manifestations and molecular causes. Their manifestations are highly variable with some cases lacking one or more of the hallmark features. In our case the existence of the 3 criteria help us in our diagnosis. The pathognomonic histologic feature in BWS is the adrenocortical cytomegaly. We present one BWS case diagnosis by a meticulous autopsy and our molecular diagnosis is consistent with that described thus far. We want to highlight the importance of the fetal autopsy and its histological study.


Microscopic features of kidneys in newborns born to mothers with preeclampsia

I. Sorokina *, V. Markovsky, M. Myroshnychenko, T. Ospanova, I. Korneyko

*Nationa Medical University, Dept. of Pathologic Anatomy, Kharkiv, Ukraine

Objective: The purpose of the study is to identify the microscopic features of kidneys in newborns born to mothers with complicated pregnancy by mild preeclampsia.

Method: The material for this study was the tissue of kidney, stained with hematoxylin and eosin according to van Gieson. All material was divided into two groups: 1 group–controls (3 cases) comprising the newborns from mothers with physiological pregnancy, 2 group-study group (7 cases) comprising newborns born to mothers whose pregnancy was complicated by mild preeclampsia.

Results: In kidneys of newborns of the second group embryonic glomeruli predominated; small areas of focal dysplasia and some glomeruli with the cyst formation were present; sclerotic processes in the glomeruli and interstitial tissue increased; the features of blood circulation disturbance characterized by hyperemia of peritubular and glomerular capillaries were noted. Mainly in the newborns of group 2, small foci of marked sclerosis with solitary lymphoid cell infiltrates were determined in cortical area of kidneys. Renal tubular epithelium of children from group 2 was characterized by focal degenerative changes.

Conclusion: Structural changes, identified in the kidneys of newborns born to mothers with complicated pregnancy may create the conditions for development of nephropathology in these children in future ontogenesis.


Placental pathology in cases of pregnancy, complicated with uterine leiomyoma

O. Reshetnikova *, O. Burgelo

*State Medical University, Lugansk, Ukraine

Objective: It is now known that uterine leiomyoma (UL) has a negative effect on women’s reproductive health contributing to a variety of gestational complications. It increases risk of perinatal lethality of a child. Pregnancy with this tumor is frequently accompanied by clinical and morphological manifestations of placental insufficiency. The aim of this study was to examine peculiarities of placental and fetal growth in case of gestation, complicated with UL.

Method: Thirty five placentas from pregnancies, complicated with UL and ten placentas of women with physiological pregnancy (controls) were studied by organometry, histology and computer morphometry.

Results: The results have shown that the weight of the placenta, maternal surface square and fetal- placental ratio in the main group of observations exceeded the results in control (p < 0.05). Placental volume for women with UL did not reach the control value (p < 0.05). The fetal weight did not have statistically significant differences. Placental pathology correlated with the place of placentation and neighborship to the leiomyoma.

Conclusion: Further study needed to evaluate the structural remodeling of the placental membrane depending of the tumor sites in the uterus and the area of placentation.


Villous trophoblastic expression of transcription factor FOXO1 in preeclampsia

J. Stanek *, R. Sheridan, C. Belludi, J. Khoury, S. Handwerger

*Cincinnati Children’s Hospital, Dept. of Pathology, USA

Objective: There is little information about FOXO1 (forkhead box protein O1) expression in pathologic placentas.

Method: Paracentral sections from grossly unremarkable areas of 10 placentas each from patients with mild preeclampsia (PE), severe PE and gestational age-matched controls were double immunostained for FOXO1 and E-cadherin, the latter distinguishing villous cytotrophoblasts (CTB) from syncytiotrophoblasts (STB). Cells counts were performed blindly on same ten 40× objective fields by three observers. Log transformed counts were analyzed using a generalized linear model.

Results: In mild PE, counts of FOXO1 positive STB were statistically significantly decreased and negative STB were significantly increased compared to controls. CTB positive cells were not statistically significantly different, while CTB negative cells were significantly increased. In contrast, FOXO1 positive and negative STB and CTB counts in severe PE were not statistically significantly different from controls (Figure).

Conclusion: Abnormal FOXO1 expression was observed in STB and CTB in mild but not severe PE placentas. Since FOXO1 is critical for placental cellular morphogenesis, abnormal FOXO1 expression may in part contribute to the abnormal trophoblast differentiation in mild PE. The differences in FOXO1 expression in mild and severe PE are consistent with other studies suggesting that the two forms of PE are different disease processes.

FOXO1 expression in villous trophoblasts:

figure ai


Increased apoptotic activity on human preeclamptic placentas: An immunohistochemical study

M. Lambropoulou *, N. Tsesmetzis, E. Chatzaki, P. Ypsilantis, O. Pagonopoulou, E. Kontomanolis, V. Tsoulopoulos, E. Chouridou, M. Koureta, E. Papadopoulos, N. Papadopoulos

*Medical School, Democritus University of Thrace, Alexandroupolis, Greece

Objective: The aim of this study was to assess the apoptotic activity in placentas of preeclamptic pregnancies.

Method: Paraffin-embedded placental tissue specimens collected from preeclamptic (group A, n = 174) and normal pregnancies (group B, n = 30) were examined by conventional histology and by immunohistochemistry using monoclonal antibodies against caspase 8, caspase 9, M30, and the DeadEnd Colorimetric TUNEL system.

Results: In preeclamptic placentas, the expression of all apoptotic markers was significantly higher compared to those from normal pregnancies (p < 0.05). Specifically, expression levels of Caspase 8, 9 and M30 in group A were significantly higher than in group B. TUNEL staining also confirmed that placental tissues from group A had significantly increased levels of apoptotic cells.

Conclusion: Overexpression of apoptotic markers was observed in placentas complicated with preeclampsia in comparison with normal ones, indicating that preeclampsia leads to increase of programmed cell death in placental tissue.

Preeclamptic placenta, M30X200.:

figure aj


Umblical cord haemangioma: A case report

T. Ozgur*, A. G. Okyay, H. Gokce, S. Toprak, E. Atik

*Mustafa Kemal University, School of Medicine, Hatay, Turkey

Objective: Umblical cord tumors are rare occurences with various pathologic features and prenatal differentiation is often very difficult. Haemangiomas are the most common of these lesions defined in the literature.

Method: 22 year-old woman gravida 1, para 0 was admitted at a gestational age of 38 weeks with urgent pelvic pain to Gynecology and Obstetrics outpatient clinic. Ultrasonography revealed a mass associated with umblical cord and there has been no other anomalities in fetus. The patient underwent cesarean delivery with high fetal weight and undefined umblical cord mass and gave birth to a female neonate with Apgar 9/9. The placenta has been sent to pathology laboratory.

Results: In macroscobic evaluation the umblical cord was long and contained a solid mass with 6 × 6 × 5 cm diameter and gray-brown cut sections. Sections of the mass had branching blood vessels with variable degrees of endothelial proliferation surrounded by loose myxomatous connective tissue. CD31 stain confirmed the vascular origin and the diagnosis was haemangioma.

Conclusion: Umblical cord haemangiomas should be remembered when an echogenic mass is visualized within the umblical cord which is located near the placental insertion site. Color doppler ultrasonography and serum AFP levels are helpful to distinguish from other lesions.


Placental insufficiency in placentas with viral infection

A. Kolobov *, E. Musatova, D. Niauri, V. Karev, V. Zinserling

*St. Petersburg, Russia

Objective: Infectious pathology plays important role in etiology of placenta insufficiency. HIV and herpes viruses may either cause primary or latent infection during pregnancy.

Method: The 63 placentas were collected from three groups of patients: Group 1–from women with herpes-virus infections (31 placentas–13 with HSV-1, 15 with CMV, and 3 with combined HSV-1 + CMV); Group 2–from HIV-infected women (12 placentas); Group 3–20 placentas from women without any infection and placental insufficiency. Expression of receptors was determined by monoclonal antibodies against CD68 (КР1 clone, Dako), CD31 (Novocastra), bFGF (Santa Cruz Biotechnology), TGF-β1 (Novocastra) HSV (I and II) and CMV (Diagnostic BioSystems), HIV-infection was confirmed antibodies against р24 (Dako).

Results: In all cases with viral infection was diagnosed placenta insufficiency. Expression of CD68 was the highest in Group 1 (13.07 ± 0.83 %), followed by group 2 (0.91 ± 0,02 %) when compared with Group 3 (2.02 ± 0.60 %, р < 0.05 for both comparisons). In placentas with HIV was noted increased expression of bFGF (5.49 ± 1.48 %) and TGF-β1 (11.2 ± 3.6 %). In placentas from Group 1 expression of bFGF was decreased–1.21 ± 0.5 %.

Conclusion: Thus, the mechanism of insufficiency in placentas with viral infection includes damage of vasculosyncytial membrane.


Placental metabolic markers expression in assisted reproductive technologies-achieved pregnancies

A. Shchegolev *, E. Dubova, K. Pavlov, N. Aleksandrova, O. Baev, G. Sukhikh

*Research Center for Obstetrics, Dept. of Pathology, Moscow, Russia

Objective: The placenta evolved to support development of the fetus, and so potentially plays a key role in the etiology of developmental programming through its impact on nutrient transfer.

Method: Our ami was to evaluate placental metabolism (transmembrane–SNAP 23, annexin 3; energy–Light Ferritin Chain–LFC, ATP-5J; carbohydrate–GLUT 1, GLUT 3) in women conceived after assisted reproductive technologies (ART). Immunohistochemical study with rabbit polyclonal antibodies SNAP 23, annexin 3, ATP-5J, GLUT1 and GLUT3; goat polyclonal antibodies LFC.

Results: Higher rate of obstetrical complications (OR = 1.1–2.33, p < 0.05 such as preeclampsia, GD, pregnancy loss, preterm delivery etc.) after ART (in comparison with spontaneous conception) is associated with positive somatic (OR = 1.4–3.22, p < 0.05), obstetric and gynecological history (OR = 1.14–4.0, p < 0.05) and also multiple pregnancy. In placentas of women conceived by ART the significant reduction of annexin3, ATP5J (markers of transmembrane and energy exchange) is revealed. Expression of GLUT3 is significantly lower in placentas after ART.

Conclusion: Finding data could be determined more by higher rate of previous extragenital and gynecological pathology than ART procedure itself. Future study is needed to understand the roles of the differentially expressed proteins in ART placentas.


The significance of acetylcholinesterase stain in diagnosis of Hirschsprung disease

T. Koletsa *, P. Pantelidou, G. Karayannopoulou, G. Karkavelas, I. Kostopoulos

*Medical School AUTH, Dept. of Pathology, Thessaloniki, Greece

Objective: The histologic diagnosis of Hirschsprung disease (HD) is challenging. A relatively reliable diagnostic method is the histochemical stain for acetylcholinesterase (AChE). The accuracy of the method is investigated in infants and neonates.

Method: 104 cases with clinical suspicion of HD were retrieved from the archives of our Department. In all cases, stain for AChE was performed in frozen sections of colon biopsies. Most cases were represented by at least two biopsies taken from different colon sites. There were also repeated biopsies in 18 cases. The histological findings were analyzed.

Results: Only eight patients were over 1 year of age. The male:female ratio was 2:1. Out of 104 cases, 20 cases (19.23 %) were diagnosed as HD. In all 20 cases marked staining of AChE-positive nerves in the lamina propria was observed. Moreover, 68 (65.38 %) cases were negative, whereas in 12 cases (11.54 %) the histological findings raised the suspicion of neuronal intestinal dysplasia. Four cases (3,85 %)were diagnosed as ultrashort HD. There was no false positive HD diagnosis.

Conclusion: AChE stain is considered to be an accurate method for diagnosing HD, restricting or eliminating false positive cases. The increase of AChE-positive nerves in the colonic mucosa confirms the diagnosis of HD.


Disseminated congenital melanoma associated with defective function of CD8 T-cells and NK cells

E. Gradhand *, R. Lucy, L. E. Newell, K. Neumann, P. Ramani

*University Hospitals Bristol, Dept. of Histology, United Kingdom

Objective: There are only 26 reported cases of congenital malignant melanomas (CMM) in the English scientific literature since 1925. The aim of this study was to characterise a CMM associated with an immunodeficiency by its histology, immunohistochemistry and molecular biology.

Method: A newborn boy presented with a large periorbital mass. He also had a generalised papular rash. We received skin punch biopsies to exclude molluscum contagiosum and a bone marrow trephine. In addition to routine histology and immunohistochemistry, we performed molecular genetic testing on the blocks of the primary lesion for Braf, cKit, Nras, Kras and Egfr mutations.

Results: The routine stains of the bone marrow and skin biopsy showed similar infiltrates of atypical cells of a CMM which stained positive for melanoma markers. The molecular testing revealed no mutations in any of the investigated genes. Immunological investigations demonstrated defective function of CD8 T-cells and NK cells.

Conclusion: Due to the disseminated metastases, we performed a molecular analysis to look for any treatment options and a possible unique mutation in CMMs. Unfortunately, the results were negative, but surprisingly, the child is now 2 years old showing a normal development and relatively stable health but with disseminated metastases of a CMM.


Brain pathology in three children with Leigh syndrome and different mutations found

K. Jandova *, H. Skalova, T. Honzik, M. Tesarova, J. Duskova

*General University Hospital, Institute of Pathology, Prague, Czech Republic

Objective: Leigh syndrome is an early onset, often fatal progressive neurodegenerative disorder caused by mutations in the mitochondrial or nuclear DNA. Until now, mutations in more than 35 genes causing Leigh syndrome have been reported, indicating an extreme genetic heterogeneity for this disorder.

Method: CNS lesions in three children with various mitochondrial disorders who died at the age of 8 months, 18 months and 6 years are described.

Results: In all three children defect of cytochrome C oxidase was proven. Subacute necrotizing encephalomyelopathy at different stages corresponding with the age of the patients was observed. Mutations in SURF1 gene in 6 years old girl, in AIMF1 gene in 18 months old boy and in COX10 gene in 8 months old girl were detected.

Conclusion: Irrespective of the different mutations found the morphology damage of the brain remains relatively monotonous reflecting rather the survival interval.


Cell-death factors expression in neuroblastoma

L. Brcic *, M. Bogovic, M. Misic, S. Seiwerth

*Univ. of Zagreb School of Med., Institute of Pathology, Croatia

Objective: Neuroblastoma is among the most common neoplasms in children usually diagnosed in higher clinical stages with uncertain outcome. Search for reliable prognostic and predictive factors is still underway.

Method: Apoptosis plays important role in tumor suppression/prevention. Fas and FasL are membrane proteins involved in one of several different ways to activate the cascade of reactions resulting in apoptosis. CTL (cytotoxic T lymphocytes) via the Fas/FasL system eliminate tumor cells, and vice versa, tumor cells can use this system to induce apoptosis of CTL. Another cytolytic activity of cells is mediated by perforin, released from the killer cells, creating pores in the membrane of target cells causing necrosis or apoptosis and independently through Hsp-70.

Results: We tested expression of Fas, FasL, perforin and Hsp-70 by immunohistochemistry in 50 tumor samples of children diagnosed between 1994 and 2013 at the University Hospital Center Zagreb and their correlation with molecular, histological and biological characteristics (favorable and unfavorable histology, MKI, N-myc status, INSS and INRGSS stage, risk groups based on POG criteria, metastases localization).


Metanephric stromal tumor: A pediatric renal neoplasm

F. Khanchel-Lakhoua *, R. Dhouib, L. Charfi, R. Doghri, I. Abbes, S. Sassi, M. Karima, M. Driss, D. Kacem, K. Ben Romdhane

*Salah Azaiez Institute, Pathology, Tunis, Tunisia

Objective: Metanephric stromal tumor (MST) of kidney is a pediatric benign stromal specific renal neoplasm. A few cases have been reported. In this communication we describe the gross and microscopic features of MST with emphasis on diagnostic criteria.

Results: We report a case of right renal tumor in a 3-year-old boy which was radiologically suspected of being a Wilms tumor. The patient had a neoadjuvant chemotherapy and a right radical nephrectomy was performed. Microscopically, on low power examination, alternating areas of high and low tumor cellularity were noted. Entrapped tubules and glomeruli were commonly seen within the lesion. The tumor cells were spindle to stellate in shape and arranged in a nodular pattern. Characteristically, there were onion skin-like concentric cuffs of tumor cells around entrapped tubules. The small intratumoral vasculatures showed irregular thickening (“angiodysplasia”).

Conclusion: MST is usually centered in the renal medulla. It has characteristic microscopic appearance which differentiates this lesion from congenital mesoblastic nephroma and clear cell sarcoma of the kidney. In most cases complete excision alone is curative. Recognition of this entity can spare a child potentially toxic adjuvant chemotherapy that might be used for lesions in its differential diagnosis.


Four cases with congenital pulmonary airway malformation

H. S. Toru *, G. Tasova Yilmaz, I. H. Ozbudak, I. Mendilcioglu, A. Aslan, G. Ozbilim

*Akdeniz University, Pathology, Antalya, Turkey

Objective: Congenital pulmonary airway malformations (CPAM) are a rare hamartomatous lesion of the lung, typically diagnosed in the antenatal period. CPAM is separated in to five major types based on clinical and pathological features.

Method: Here in we report four cases with CPAM. Three of them were postnatal lung segmentectomy material and one of them was fetal autopsy specimen. All of them were classified into histological subtypes according to five layered classification system of Stocker.

Results: Among four cases three of them were CPAM type II, one of them was CPAM type I. All of the cases were detected antenatal in ultrasonographic examination after gestational age of 22nd week. Only one family wanted termination, in the autopsy evaluation of this case CPAM type II is detected on the upper left lobe, compressing distal parts of the lung, lateralizing the heart to the right. Other three cases underwent surgical resection. Two of them were CPAM type II, one of them was CPAM type I.

Conclusion: Ultrasonography has been demonstrated to be highly useful modality in the in utero diagnosis of CPAM. Recognizing the imaging features of these abnormalities is necessary for prenatal counseling and appropriate prenatal and postnatal management.


Congenital salivary gland anlage tumor of nasopharynx in a neonate

L. Mascarenhas Lemos *, M. Mafra

*Lisbon Central Hospital, Dept. of Pathology, Lisboa, Portugal

Objective: Nasal and upper respiratory tract obstruction in the neonatal period is a potenctially life-threatening condition that can result from inflammatory conditions, hamartomas, congenital malformations and tumors. Salivary gland anlage tumor, also referred to as congenital pleomorphic adenoma, is a rare, benign congenital tumor of the nasopharynx, which may produce nasal obstruction.

Method: Case report with literature review.

Results: A male neonate presented with complaints of nasal obstruction and feeding difficulties. During the common clinical diagnostic approach was found a friable, pediculated mass with origin on the nasopharynx and protusion to the oropharynx. Radiographic evaluation demonstrated a circumscribed pediculated mass with 8 mm that did not communicate with the intracranial compartment. Pathology revealed a hamartomatous lesion with a biphasic nature demonstrated by the admixture of epithelial groups of duct-like and squamous structures and bland stromal cells.

Conclusion: Congenital salivary gland anlage tumor of nasopharynx is usually not suggested as a possible cause in the setting of nasal obstruction from a mass in the neonatal period because it is uncommon. The different clinical significance of other developmental masses and neoplastic lesions requires having congenital salivary gland anlage tumor considered on the differential diagnosis.


Analysis of the prevalence of Wilms’ tumor in Bulgarian children diagnosed with malignant tumors for a period of 10 years (2001–2010)

D. Serteva *

*N.I. Pirogov University Hospital, General and Clinical Pathology, Sofia, Bulgaria

Objective: The aim of this study is to show the prevalence of Wilms’ tumor (WT) in Bulgarian children, operated at University General Hospital for Active Medical Treatment and Emergency Medicine “N.I. Pirogov”, Sofia, Bulgaria, for a period of 10 years, compared to the total number of malignant tumors in children, diagnosed at the same hospital and the distribution of WT according to gender and age.

Method: Conventional histological staining–hematoxylin and eosin, immunohistochemical staining (WT antigen), archive of the department of pathology.

Results: A total number of 396 cases of malignant tumors in children were diagnosed and operated at the hospital for the 10-year-period, 49 of them have a confirmed histological diagnosis of WT (12.37 %). Number of males with WT–29/49 (59.18 %),number of females with WT–20/49(40.82 %). Mean age at diagnose: males–3 y10 m; females–3 y9 m; general age distribution: up to 2 year of age–11/49 (24.49 %), from 2 year to 5 year of age–24/49 (46.94 %), from 5 year to 15 year of age–14/49 (28.57).

Conclusion: WT makes a significant part of the malignancies in children, having a slight prevalence in males (59.18 % vs 40.82 %). The mean age at diagnose shows no gender difference. From the general age distribution–highest prevalence at the 2–5 year-of-age interval.

Sunday, 1 September 2013, 09.30–10.30, Pavilion 2

PS-06 Poster Session Soft Tissue Pathology


Malignant melanoma: Cutaneous metastasis frequency and survival rate

C. Tamas *, L. Popa, A. Lazar, C. Stanescu, C. Morosanu, D. Radulescu, D. Turliuc

*University of Medicine Iasi, Dept. of Plastic Surgery, Romania

Objective: We studied the variation in locoregional metastasis frequency and the relation between clinical form and survival in malignant melanoma (MM).

Method: We used the Kaplan-Meier survival curves and Cox proportional hazards model to calculate the survival rate for 263 patients with MM, operated between 2000 and 2010.

Results: Half of the patients (51 %) developped limbs MM. Patients with nodular melanoma(98) had a higher risk for cutaneous metastasis(61,22 %) than the rest and a low survival rate(36,73 %). There is a relation beween the number of metastatic limph nodes and the survival rate. This rate decreases with 13,64 % if more than 4 limph nodes are involved:from 16 months,in a single affected limph node,to 10 months for more than 4.

Conclusion: The presence of cutaneous metastasis in MM is associated to a very low survival rate. The patients with stage I MM had the longest survival period. In the third stage,the survival rate depends on the number of the metastatic limph nodes(57,14 % IIIA;43,50 % IIIB;12,50 %IIIC). In a small number of cases(4 %) the metastatic tumor was the first sign of MM. Key words: MALIGNANT MELANOMA,CUTANEOUS METASTASIS,LIMPH NODES,SURVIVAL


A retroperitoneal dedifferentiated liposarcoma with low-grade and high-grade heterogeneous components

I. Jung *, D. Milutin, S. Gurzu

*University of Medicine and Pharmacy, Dept. of Pathology, Tirgu-Mures, Romania

Objective: To present a retroperitoneal dedifferentiated liposarcoma with heterogeneous differentiation.

Method: A 50-year-old female presented with a 20-cm-sized tumor of the retroperitoneum. The surgical specimen was submitted for histopathological examination

Results: Macroscopic examination of the surgical specimen revealed an inhomogeneous aspect on cut section, the gelatinous areas being intermingled with solid zones with whirling-pattern, hemorrhages and necroses being also observed. Histopathologic examination confirmed the heterogeneous aspect. The areas of well-differentiated liposarcoma were admixed with tumor areas that presented histological features characteristics for pleomorphic malignant histiocytoma, osteosarcoma, fibrosarcoma, myofibroblastic and rhabdoid patterns being also seen. Immunohistochemically, the heterogeneous components were confirmed using specific antibodies such as Vimentin, Smooth Muscle Antigen, Desmin, S100, and CD68. Focal positivity was also observed for ALK, CD34 and CD105 but C-KIT antibody did not mark the tumor cells. No recurrences and/or metastases were reported 6 months after surgery.

Conclusion: Dedifferentiated liposarcoma seems to have a very heterogenous origin and the correct diagnosis is based on examination of several tumor sections


Possible predictive value of correlation between synovial angiogenesis and serum level of adiponectin in patients with rheumatoid arthritis

I. Jung *, R. Melinte, B. Dorcioman, S. Gurzu

*University of Medicine and Pharmacy, Dept. of Pathology, Tirgu-Mures, Romania

Objective: To present the particularities of synovial angiogenesis and serum level of adiponectin in patients with rheumatoid arthritis (RA) compared with those with osteoarthritis (OA).

Method: The immunohistochemical expression of the antibodies VEGF (Vascular endothelial growth factor) and CD31 was analyzed in 20 cases with RA and 10 with OA. The synovium specimens were obtained at the time of total knee joint arthroplasty. For a proper quantification of angiogenesis, the necrotic areas and those with high inflammatory activity were eliminated. In all patients the serum level of adiponectin was also preoperative determined using ELISA kits.

Results: The average serum level of adiponectin was significantly high (19718.45 UI) in patients with RA compared with those with degenerative OA (4836.25 UI). VEGF expression was diffusely observed in the synoviocytes of patients with RA but focal positivity was revealed in OA. The number of vessels was also significantly high in RA (9.03 vessels/HPF) compared with OA (4.75 vessels/HPF).

Conclusion: High adiponectin levels seem to be correlated with increased synovial VEGF expression. Intraarticular administration of antiangiogenic drugs associated with anti-inflammatory substances could inhibit both local angiogenesis and inflammation. It could prevent the severe synovial damages, usually observed in rheumatoid arthritis.


Reduced INI-1 expression in gastrointestinal stromal tumors

G. Karagkounis *, I. Themeli, M. Chantziara, T. Argyrakos, C. Vourlakou, K. Lariou, D. Rontogianni

*Evaggelismos Hospital, Dept. of Pathology, Athens, Greece

Objective: The loss of the SMARCB1/INI1 tumor suppressor gene protein characterizes malignant rhabdoid tumors, epitheliod sarcomas, myoepithelial carcinomas and renal medullary carcinomas while it is reduced in synovial sarcomas. We study the immunohistochemical expression of INI1 protein in Gastrointestinal Stromal Tumors (GISTs).

Method: Twenty GISTs (CD117+, CD34+) were classified according to the WHO/2010 criteria. Immunohistochemical detection of INI1 protein (clone MRQ-27) was performed. Staining was interpreted as: complete loss, partial loss (in more than 10 % of the tumor cells) and reduced (weak expression in more than 10 % of the cells). SPSS 17.0 was used for the statistical correlation of the results.

Results: 1/20 (5 %) of cases showed complete loss, 2/20 (10 %) showed partial loss, 4/20 (20 %) showed reduced expression while 13/20 (65 %) retained the expression of INI1. There was no statistically significant correlation between the reduced/lost expression of INI1 and 1. the possible biological behavior of the neoplasm, 2. the prognostic group as specified by WHO/2010, 3. the size of the tumor and 4. the mitotic count (p > 0.05).

Conclusion: 35 % of GISTs showed reduced expression or loss of INI1 protein. No correlation of the INI1 protein status with the prognostic characteristics of the tumors was proven.


Immunohistochemical evaluation of VEGF, its receptors and HIF1a expressions in Dupuytren’s disease

A. Coer *, L. A. Holzer, G. Holzer

*University of Primorska, Faculty of Health Sciences, Izola, Slovenia

Objective: Dupuytren’s disease (DD) is a benign progressive fibromatosis of the hand and fingers that leads to a permanent and irreversible flexion contracture of the fingers. We hypothesized that hypoxia and/or angiogenic factors are involved in the pathogenesis of DD, so the aim of our study was to analyse the expression of VEGF and its receptors VEGFR1 and VEGF2 in tissue samples obtained from patients with DD.

Method: An immunohistochemical study of tissue samples from 32 patients with DD was performed. Frozen tissuesections were analysed and expressions of VEGF, VEGFR1, VEGFR2, and HIF1α were determined immunohistochemically.

Results: Nodules in involutionary phase of DD with αSMA positive myofibroblasts were found in 15 samples. In 17 cases almost acellular tendon-like cords were present (residual phase of DD). At least one of the analysed angiogenic proteins was expressed in 9 out of 15 αSMA positive cases. In the involutionary phase, myofibroblasts showed overexpression of VEGF2 and HIF1α in comparison with samples from residual phase.

Conclusion: Overexpression of VEGFR2 and HIF1α in the αSMA positive fibroblast-rich nodules indicates that angiogenesis might play a role in the pathogenesis of DD.


Ossifying fibromyxoid tumor of soft parts: Histological, immunohistochemical and ultrastructural characteristics of the case presenting novel cytogenetic findings

J. Rys *, M. Iliszko, M. Vogelgesang, K. Knapczyk, A. Kruczak, B. Lackowska, J. Lasota, J. Limon, M. Miettinen

*Center of Oncology, Tumor Pathology, Cracow, Poland

Ossifying fibromyxoid tumor (OFMT) is a rare but morphologically distinctive soft-tissue tumor. Since 1989 approximately 450 cases of OFM were described, however cytogenetic characteristics of OFMT have been reported only in eight tumors (of six patients). In all of them clonal chromosomal aberrations have been detected by cytogenetic analysis and the short arm of chromosome 6, in particular 6p21, was frequently involved in OFMT. Notably, a balanced or unbalanced t(6;12)(p21;q24) translocation seemed to be characteristic for OFMT. We present a peculiar case of OFMT localized in the subcutaneous tissue of the thigh presenting the novel cytogenetic findings. The karyotype of the tumor cells exhibited the following numeric and structural aberrations: 44–48,X,−X[3],add(12)(p1?1)[3],der(12)t(12;15)(p13;q15)[5],−13[4],−15[3],−22[4],+r[6],inc[cp12]/46,XX[18] To our knowledge, this is the first reported case in which such complex karyotype with clonal chromosomal abnormalities including the unbalance translocation der(12) t(12;15)(p13;q15), ring chromosome and loses X, 13, 15 chromosomes has been shown.


Retroperitoneal leiomyosarcomas

C. Pereira *, C. Melendez, X. Sanjuan, J. Fabregat, L. Secanella, V. Romagosa

*Hospital Bellvitge, Anatomia Patologica, Hospitalet Llobregat, Spain

Objective: Leiomyosarcoma (LMS) is one of the most frequent retroperitoneal tumours, that appears as large mass at diagnosis. Complete surgical resection is often difficult and they are related with high rate of recurrence, metastasis and death.

Method: Were reviewed clinicopathologic and immunohistochemical features of retroperitoneal LMS treated at our institution between 1995 and 2012 and graded following French system.

Results: A total of 15 cases were reviewed (9 female, mean age: 60 years). Two were low grade (G1) and 13 high grade. Immunophenotype of G1 maintained all muscle markers, while G3 express actin and caldesmon but lose desmin. Vascular invasion was identified in 3 cases, all graded as 3 of mitosis. Of these, 2 had metastases and died shortly. The recurrence rate was 16.6 % while metastasis was 41.6 %.

Conclusion: 1) Caldesmon is more sensitive than desmin to identify high-grade LMS. 2) Desmin negativity does not exclude LMS. 3) LMS scored 3 of mitoses are associated with vascular invasion. 4) Recurrences and metastases occur in any grade, but surgical resection achieve prolonged survival in low grade. 5) The recurrence rate in our series is low and could be related to correct radical excision of primary tumor. 6) Death of progressive LMS is related with high-grade tumors.


Expression of epidermal growth factor receptor in soft tissue tumours

K. Kuracinova *, A. Janegova, P. Janega, P. Babal

*Faculty of Medicine, Bratislava, Slovakia

Objective: Epidermal growth factor receptor (EGFR) is member of the Her-family of transmembrane receptor tyrosine kinases. The expression of the EGFR protein in different subtypes of soft tissue tumours has not been known as thoroughly as in epithelial tumours.

Method: Immunohistochemical positivity of the EGFR in 69 samples of soft tissue tumours was evaluated by light microscopy and compared according to their localization.

Results: No staining was in liposarcomas, nodose fascitis, inflammatory myofibroblastic tumour, fibromyxoid sarcomas, malignant fibrous histiocytoma, angiosarcoma, chondrosarcoma, synoviosarcoma. Positive membranous staining for EGFR was seen in 23 cases. Ewing sarcoma (3/1), undifferentiated sarcoma (3/1), neurofibrosarcoma (5/5), malignant peripheral nerve sheath tumor (4/1), fibrosarcomas (3/1), leiomyosarcoma (8/2), embryonal rhabdomyosarcoma (18/12), alveolar rhabdomyosarcoma (7/1). Our measurement shows high intensity and positivity in embyonal rhabdomyosarcomas and neurofibrosarcomas in one case of Ewing sarcoma and in two cases of leiomyosarcomas from uterus. From alveolar rhabdomyosarcomas was one sample positive, but this tissue had the mutation in PAX/FKHR gene. These tumours behave more likely as embryonal rhabdomyosarcoma.

Conclusion: Our results demonstrated that IHC staining for EGFR shows membranous positivity in multiple subtypes of soft tissue tumours. The results open the possibility to evaluate the effect of EGFR blocking therapies. Supported by ITMS 26240220052.


Tumours and tumour-like lesions of the hip in the paediatric age

F. Khanchel-Lakhoua *, K. Mrad, R. Dhouib, L. Charfi, I. Abbes, S. Sassi, M. Driss, R. Doghri, R. Ben Ghorbel, K. Ben Romdhane

*Salah Azaiez Institute, Pathology, Tunis, Tunisia

Objective: Bone tumours and tumour-like lesions of the hip in children are rare. The aim of our study is to assess the histological type of tumors in children hip.

Method: We reviewed all tumors or tumor-like lesions occurred in the pelvis or proximal femur, involving the hip in patients less than 17 years old in Department of Pathology Salah Azaiez Institut from the 1st of January 1994 to 31st of December 2012.

Results: We registred 23 tumours or tumor-like lesions in 12 girls and 11 boys with a mean age of 13 years. Lesions occurred specially in the pelvis (78 %). In respectively 13 % and 8 % of cases they touched the sacrum and proximal femur. The majority of tumors (73 %) were malignant tumours, dominated by Ewing sarcoma (23 %). Only 21 % were benign tumour (8,6 % chondroma. 8,6 % chondroblastoma, 4,3 % osteoma). One tumor-like-lesion was registred (anevrysmal cyst).

Conclusion: Our results showed the large predominance of sarcoma, especially Ewing sarcoma. They are different from the Rizzoli institute series which is dominated by tumour-like lesions and benign tumours.


Retroperitoneal liposarcoma: The experience of a Tunisian cancer institute

R. Hamrouni*, L. Charfi, R. Dhouib, K. Mrad, R. Doghri, I. Abbes, N. Boujelbene, M. Driss, S. Sassi, N. Ben Hmida, K. Ben Romdhane

*Salah Azaiez Hospital, Pathology, Tunis, Tunisia

Objective: The aim of this study was to analyse the clinicopathological features of different subtypes of retroperitoneal liposarcoma.

Method: A retrospective study about 20 cases of retroperitoneal liposarcoma (RLPS) diagnosed over a period of 18 years (1995–2012) to evaluate clinical results and prognostic factors for patient survival.

Results: We report 20 cases (12 men and 8 women, the mean age was 57.42 years) of RLPS. None of our patients had a percutaneous biopsy of his tumour. Surgery was indicated to all patients. Resection was performed in all cases (we performed a bloc resection in 17 cases and a simple tumour resection in 3 case). Maximum tumor diameter was 250 mm(range,65–350 mm). Histological classification included well-differentiated type in 11 cases, dedifferentiated type in 3 cases, myxoid/round cell type in 5 cases and pleomorphic type in 1 case. Resection of contiguous organs was required in 85 %. Three of patients had an adjuvant therapy. All patients had recurrent disease, and recurrence developed at a mean of 13.23 months after primary or repeated surgical treatment. The 3-year survival rate was 46.98 %

Conclusion: RPLS grow slowly and silently. Its prognosis is poor compared to the other histological subtypes of retroperitoneal sarcomas. Only complete excision provides a hope of a cure, this is often difficult.


Paraspinal juxta-articular myxoma: An unusual benign mesenchymal lesion readily mistaken for malignancy

C. Beggan *, J. Kinsella, M. Leader

*Carrickmacross, Ireland

Objective: We present a case of juxta-articular myxoma, a myxoma variant occurring around joints. The main histological differential is myxofibrosarcoma. For management and patient prognosis, separation of these is essential.

Method: A 55 year old female presented with a palpable neck mass. A poorly circumscribed cellular lesion, composed of ovoid and spindle cells within a myxoid stroma was observed. Given the absence of mitoses, necrosis, hyperchromasia or pleomorphism a diagnosis of juxta-articular myxoma was made. The cellularity, whilst unusual in a myxoma, is described in juxta-articular myxoma.

Results: Myxoma describes any bland hypocellular gelatinous neoplasm. Most are intramuscular and microscopy reveals a paucicellular lesion with round and stellate cells intermixed with abundant myxoid extracellular stroma containing sparse capillary sized blood vessels. No pleomorphism or mitoses are seen. Juxta-articular myxoma is a cellular lesion with increased vascularity. Distinction from a cellular myxoma is made based on location adjacent to joints and lack of GNAS1 mutation. Distinction from low-grade myxofibrosarcoma requires assessment of pleomorphism, hyperchromasia, mitoses and necrosis.

Conclusion: We describe the 1st reported case of paraspinal juxta-articular myxoma. Diagnosis requires excision rather than core biopsy, as atypical features may be focal. Complete excision is the mainstay of treatment. Local recurrences are described with incomplete resections.


Spinal hydatidosis: Report of four cases

U. Aykutlu *, B. Yaman, B. Doganavsargil, M. Argin, M. Sezak, M. Zileli, F. Öztop

*Ege University, Dept. of Pathology, Izmir, Turkey

Objective: Hydatidosis is a tapeworm infestation, caused by mainly Echinococcus granulosus (Cystic echinococcosis) or less commonly, Echinococcus multilocularis (Alveolaer echinococcosis). Liver (50–70 %) and lungs (20–30 %) are the primarily affected organs. Spinal involvement is seen in less than 1 % of cases.

Method: We present three cystic and one alveoler echinococcosis cases located in spine.

Results: Cystic echinococcosis cases were 2 men and 1 woman aged 61, 40 and 13 years-old respectively. The sites of involvement were T11-12, L4-5-sacrum with medullary cavity extention and T8 vertebrae. Alveoler echinococcosis was located in L10-L12 vertebrae of a 37 years-old woman from an endemic region of Turkey. Back pain and leg numbness were the presenting symptoms in all cases. Lytic, expansive, cystic masses and vertebra corpus destruction were observed radiologically. Histologically no protoscolices but prominent cuticular membranes were identified. Alveolaer echinococcosis case was initially misdiagnosed as granulomatous osteomyelitis due to extensive necrosis and palisating histiocytes and absence of cuticular membranes or laminated layers in the first biopsy. All cases recurred in the time.

Conclusion: The cases are presented because of their rarity and potential risk of miscibility with tumors radiologically or other types of spondylitis histologically especially when typical laminated layers are tenuous.


Parosteal osteosarcoma: Clinicopathological study of 10 cases

W. Ouahioune *, N. Bettaz, M. Guermi, S. Boukhalfa, L. Benrezki

*University of Blida, Dept. of Medicine, Algeria

Objective: Parosteal osteosarcoma (POS) is the most common type of osteosarcoma of the surface of bone. This rare tumor accounts for about 4 % of all osteosarcomas. POS involves the surface of long bones. We report 10 cases of POS diagnosed in our service during a period of 10 years.

Method: On the 81 osteosarcomas diagnosed in our department, 10 of them were POS.

Results: Age ranged from 14 to 68 years. The sex ratio was of 6 females/4 males. In 06 cases out of 10, the site of involvement was the femur. For the other cases, the sites were humerus (1 case), tibia (1 case) and the iliac fossa (1 case). Radiologically, diagnosis was raised in 03 cases. All the patients were treated by wide excision. Histologically, diagnosis was easily made in 07 cases. For the remaining cases, some difficulties were faced. 03 patients had recurrences.

Conclusion: Parosteal osteosarcoma is a malignant tumor, but still of good prognosis if diagnosed and excised on time.


Reticular soft tissue perineurioma

M. Bel Haj Salah*, N. Abdessayed, I. Msakni, W. Koubaa, O. Khayat, A. Chadly-Debbiche, R. Jouini

*Habib Thameur Hospital, Pathology, Tunis, Tunisia

Objective: To describe clinical and histological features of reticular soft tissue perineurioma

Method: We report the case of a 60-years-old men who presented with a long lasting tumor of the thigh. Macroscopically, tumor was nodular and measured 14 cm in its greatest dimension. It was well limited with a firm whitish cut section. Microscopic examination showed a proliferation of variable cellularity made of slender fibroblast-like cells with small nuclei forming a net-like pattern and setting in a myxoid or occasionally denser collagenous matrix. Mitotic activity was absent. Tumoral cells were reactive with EMA and CD34. Diagnosis of reticular benign soft tissue perineurioma was made.

Results: Perineuriomas are very rare nerve sheath tumors that display pure perineurial cell differentiation. Two main groups are described: intraneural and soft tissue perineurioma. Soft tissue perineuriomas, although more common than their intraneural counterparts, is still relatively uncommon and probably underrecognized. Histologically, they display variable pattern and they often respond to a sclerosing perineurioma. Reticular perineurioma is extremely rare,it is characterized by a lace-like arrangement of cells resulting in the formation of microscopic cysts.

Conclusion: Although their histological distinctive features, the diagnosis of soft tissue perineuriomas is usually based on immunohistochemical demonstration of EMA and GLUT1 positivity.

Sunday, 1 September 2013, 09.30–10.30, Pavilion 2

PS-07 Poster Session Thymic and Mediastinal Pathology


Thymoma: Evaluation of clinical and pathological characteristics of 27 cases

A. Lovrenski *, D. Tegeltija, M. Panjkovic, Z. Eri, I. Jelicic

*Institute for Lung Diseases, Dept. for Pathology, Sremska Kamenica, Serbia

Objective: Analysis of patients demographic features, simptoms, morpholigical charasteritic any histological types of tumor.

Method: Retrospectively we studied 27 medical records of patients reffered to the Institute of pulmonary diseases of Vojvodina, in Sremska Kamenica between the January of 2005 and December of 2011.

Results: All types of thymoma more often occured in males, accept type B. The average age in patients with type A was 57 and with type B2 30 years. Patients with type A thymoma and B1 were mainly asymptomatic, while patients with type B2 and AB presented with dyspnea, dysphagia, pain and cough. Myasthenia gravis often was associated with type A (40 %). The most common histologic type was type B1. The largest number of patients, 14 of them (51 %) had a tumor diameter between 5 and 10 cm. Five patients had malignant thymoma, and the most common types of thymoma that showed signs of capsula invasion or pleural and pericardial implants were type AB (60 %) and B2 (50 %).

Conclusion: Thymomas occurs mainly in people older than 40 years and are slightly more frequent in females. There is no correlation between the incidence of malignant thymoma and patients age.

Thymoma type A, HE x 20:

figure ak


Primary mediastinal (thymic) large B-cell lymphoma: A case report and review of treatment modalities

K. Diamantopoulou *, P. Korkolopoulou, G. Piagkos, A. Stamatelopoulos, P. Mihos, H. Mahera

*Athens, Greece

Objective: Primary mediastinal large B-cell lymphoma (PMBL) is a rare subtype of diffuse large B-cell lymphomas. We present a case of PMBL with partly overlapping immunophenotype of classic Hodgkin lymphoma (CHL) and gray zone mediastinal lymphoma in an attempt to highlight potential therapeutic approaches.

Method: A 48-year-old female presented with superior vena cava syndrome, due to a large mediastinal mass with invasion of the right lung, and an enlarged supraclavicular lymph node. Formalin-fixed paraffin embedded tissue obtained from fine needle biopsy of the mass was examined with histology and immunohistochemistry.

Results: Microscopically, a diffuse lymphoid neoplasm was observed. Tumor cells were medium to large-sized with pale cytoplasm, round nuclei and occasionally prominent nucleoli. Immunohistochemically, neoplastic cells were CD20(+), CD79a(+), PAX-5(+), CD30(+), Bcl-2(+), Bcl-6(+), MUM-1(+), CD23(+/−), CD15(+ rarely), CD10(−), Ki67(30–40 %). Thymic islets were demonstrated with EMA and CK19. The diagnosis was PMBL, although CHL and gray zone mediastinal lymphoma also entered the differential diagnosis.

Conclusion: PMBL is an aggressive B-cell lymphoma (WHO 2008) with unique clinical and molecular features. Treatment approaches, such as R-CHOP, as our case received, with or without radiotherapy, could be implicated with high cure rates.


Mediastinal thymoma coexisting with intrapulmonary mature teratoma

N. Mylona *, M. Sofopoulos, S. Tsitsiou, N. Koufopoulos, S. Ntoumou, N. Arnogiannaki

*Saint Savvas Hospital, Dept. of Pathology, Athens, Greece

Objective: We report a case of a mediastinal thymoma and a simultaneous primary teratoma of the lung.

Method: A 72-year-old man with a lobulated mass 7 × 4 × 2,5 cm in the anterior mediastinum and a second lesion 4 mm in the upper left lobe of his lung.

Results: Histopathologic examination revealed a B3 thymoma as far as the anterior mediastinal mass and an intrapulmonary mature teratoma concerning the lung’s lesion. The first consisted of multiple nodules separated by fibrous tissue. The epithelial tumor’s cells had medium, oval shaped nuclei without prominent nucleoli. Atypia and mitotic activity were also observed. The lymphoid component comprised of immature T-lymphocytes. The latter lesion consisted of mature bone, adipose tissue and bone marrow, whereas the surrounding pulmonary parenchyma showed histological findings of bronchiolitis.

Conclusion: Thymomas and teratomas are two of the four most common anterior mediastinal tumors. However, primary intrapulmonary teratomas are rare and it is thought that these tumors originate from the third pharyngeal pouch which is the anlage of the thymus. Their presence may be due to displacement or separation of the thymus during early embryogenesis and they usually occur in the left upper lobe, as in this case. The treatment for teratomas is resection and the prognosis is excellent.


Typical carcinoid tumor arising in mediastinal mature teratoma

M.-M. Koumpanaki *, N. Vladika, P. Xirou, B. Christoforidou, F. Patakiouta

*Thessaloniki, Greece

Objective: Mature cystic teratomas (MCT) are common neoplasms of the anterior mediastinum. They are composed of mature tissues derived from two or three germinal layers. Malignant transformation is rare (1 %) and usually refers to squamous cell carcinoma. Primary carcinoid tumor occurring in MCT is extremely rare.

Method: We report a case of a 57-year old male presenting with a mediastinal mass showing teratomatous features in CT scan. No metastases or lymph node involvement were found. The tumor was excised and we received a cystic thick-walled lesion, sized 6.2 × 4 × 3 cm, filled with yellow-tan material.

Results: On microscopic examination, the neoplasm comprised of a variety of tissues from all three germinal layers. In certain areas of the cyst wall aggregations of small uniform cells were seen having morphologic and immunohistochemical features consistent with a typical carcinoid tumor.

Conclusion: Only four neuroendocrine tumors arising in mediastinal MCT have been reported. Complete surgical excision was curative in all cases. It is essential to differentiate typical carcinoid within MCT from other types of malignant non-germ cell components and immature neuroepithelial elements with known metastatic potential. In our case, 3 years later the patient is alive and well with no signs of metastases.


Soft tissue aneurysmal bone cyst in thoracic cavity

T. Canpolat *, F. Bolat, N. Bal

*Baskent University, Pathology, Adana, Turkey

Objective: Primary aneurysmal bone cyst is a benign, locally aggresive and expansile tumor that typically occurs in the long bone or vertebral bodies of children and young adults. It was believed to occur exclusively within bone. ABC devoloping in soft tissue is extremely rare but in resent years a few cases presented of soft tissue.

Method: We describe a case ABC of the soft tissue in thoracic cavity of 15 years old man. Although the tumor initially was radiologically considered terotoma, clinically cyst hidatic the microscopic features were identical to those faund in clasic ABC We report the imaging and pathological findings of soft tissue (STABC) and discuss its differantial diagnosis.

Results: 15- year-old man presented with chest pain and dyspnea. Chest X-ray showed a homogenous mass lesion in the right thorosic cavitiy. Computed tomography (CT) scan of the thorax was done, showed a large predominantly cystic, well-defined mass It had multible enhancing septations and irregular areas of calcification. The ribs were normal The mass was mixed with cystic solid and calcific components and associated without rib. It was adherent to lung parenchyma.

Conclusion: Many tumors in the thoracic cavity during childhood should be considered in the differential diagnosis of ABC.


Pure primary mediastinal yolk-sac tumor in an adult female patient: A case report

M. Cardoso *, A. Alves, C. Carvalheiro, N. Guerra, J. Caldeira, M. Ferreira

*Centro Hospitalar Lisboa Norte, Anatomia Patológica, Portugal

Objective: Pure primary mediastinal yolk sac tumor is a rare entity and occurs in male patients and prepubertal women. This is the first case report with histological confirmation in the surgical specimen in an adult fertile woman.

Method: A 28-year-old woman presented with an acute and intense retrosternal pain radiating to the left shoulder, dyspnea and a history of mild fever. Chest CT scan revealed a lobulated tumor in the left mediastinum (107 × 87 mm) and elevated levels of αFP (84505kU/L) were found. She was submitted to a mini-sternotomy with tumorectomy and left thymic lobectomy.

Results: Grossly, the tumor had a smooth surface with multiple firm white nodules and areas of hemorrhage. Histologically there were solid, microcystic and micropapillary patterns, Schiller-Duval bodies, occasional hyaline globules, extracellular eosinophilic bands and areas of necrosis. Immunohistochemically there was diffuse positivity for alpha-fetoprotein.

Conclusion: Extragonodal germ cell tumors are thought to derive from primitive germ cells that mismigrated along the urogenital ridge during embryogenesis. Histological, serological and cytogenetic characteristics of mediastinal yolk sac tumors are similar to their gonadal counter-parts, with major differences in clinical behavior. Although extremely rare, they can also occur in adult female patients, associated with other germ cell tumors or in a pure form.


The anatomoclinical particularities of thymomas in the center of Tunisia

S. Hmissa *, S. Korbi, N. Missaoui, S. Mestiri, H. Landolsi, S. Ben Abdelkarim, A. Ben Abdelkader, M. Mokni

*Farhet Hached Hospital, Dept. of Pathology, Sousse, Tunisia

Objective: Thymomas are rare tumors. Here, we studied their clinicopathologic features.

Method: We performed a retrospective study of 25 thymomas diagnosed in the Pathology Department of Farhet Hached Hospital of Sousse from January 1993 to December 2012.

Results: The mean age was 41.7 years (23–60 years). Clinically, we observed an alteration of the general state in 20 % of cases, a myasthenia (24 %), a chest pain (36 %), respiratory symptoms including cough, dyspnea, and sputum (60 %). The average time before diagnosis was 3 months. In biology, an inflammatory syndrome was observed in 52 % of cases. Chest computed tomography showed a solid mass located in the thymic in 24 cases. The histological diagnosis was a single case of thymoma A, 2 cases of thymoma AB, 5 cases of thymoma B1, B2 thymoma 3 cases, 5 cases of thymoma B3, and 8 cases of thymoma C. Stages III and IV were the most frequent (72 %). The evolution is marked by five deaths, two recurrences, and two recurrent myasthenic syndrome.

Conclusion: Thymomas were diagnosed as aggressive forms (5 cases of B3 thymomas and 8 cases of thymoma C) and at advanced stages (72 %) in the center of Tunisia.


Late recurrence of a type AB thymoma after primary resection

J. S. Stadie *, M. Pasic, T. Drews, N. Solowjowa, R. Hetzer, K. Wassilew

*German Heart Institute Berlin, Section Heart Pathology, Germany

Objective: Thymomas are rare epithelial tumors, but the most common neoplasms of the mediastinum. Case reports of local recurrence or distant metastasis of type AB thymomas are exceptional, even if they presented as Masaoka stage II or III tumors at time of diagnosis.

Method: We investigated a case of an 84-year old man with a history of thymectomy and myasthenia gravis, with incidental preoperative finding of a mediastinal tumor in context of an elective heart valve replacement.

Results: Radiologically, a peculiarly shaped centrally calcified tumor (80 mm in largest dimension) was noted right antero-laterally of the aorta and the pericardial reflection in the anterior mediastinum. Intraoperatively, the encapsulated tumor showed adhesions to both pleura mediastinalis and visceralis. Microscopically, the mass lesion consisted mainly of bland spindle-shaped epithelial cells arranged in primarily whorled, partly storiform pattern and rosettes with focal invasion of the mediastinal fat. Other fractions of the tumor showed a more lymphocyte-rich component, leading to the diagnosis of a minimally invasive Masaoka stage II type AB thymoma, which was confirmed by the immunophenotype.

Conclusion: The presented case highlights the need for long-term follow-up even after resection of a thymoma type tumor, which is considered to recur rarely.

Monday, 2 September 2013, 09.30–10.30, Pavilion 2

PS-08 Poster Session Cardiovascular Pathology


Mitochondrial cardiomyopathy

S. M. Vesterkaer *, U. Baandrup

*Sygehus Vendsyssel, Dept. of Pathology, Hjørring, Denmark

Objective: The mammalian mitochondrial genome (mtDNA) is a double-stranded DNA molecule, located in multiple copies within the mitochondria of each cell. Despite a contribution of less than 1 % of the total cellular nucleic acid content, it is fundamentally important for the function of every human tissue. The primary function of mitochondria is to support aerobic respiration and produce, by oxidative phosphorylation, the bulk of cellular ATP. Mitochondria also control cytosolic calcium concentration, regulate apoptosis and host other important biochemical pathways. Mitochondria are under the dual genetic control of both the mitochondrial and nuclear genomes, mutations within either DNA molecule may result in respiratory chain deficiency.

Results: I will focus on mtDNA mutations in human disease, by presenting and illustrating two case reports regarding Mitrochondrial myopathy, Encephalopathy, Lactic Acidosis and Stroke-like episodes (MELAS) and Maternally Inherited Diabetes and Deafness syndrome (MIDD), respectively. Mitochondrial diseases have a broad range of clinical manifestations and a highly variable course. Identical mutations may result in different diseases, and different mutations can cause identical disorders. This makes the diagnosis and counseling, of mutation carriers, challenging. Unexplained cardiomyopathy, together with multiple organ diseases, should however lead to a suspected diagnosis. Unfortunately no specific treatment is available.


Intravital and postmortem morphological studies and magnetic resonance imaging of 80 cases of arrhythmogenic right ventricular dysplasia

L. Mitrofanova *, N. Mitrofanov, D. Lebedev, M. Gordeeva

*Almazov’s Centre, Dept. of Pathomorphology, St. Petersburg, Russia

Objective: To improve verification of Arrhythmogenic Right Ventricular Dysplasia (ARVD)

Method: Morphometric, histological and immunohistochemical analysis were carried out on 47 endomyocardial biopsies (EMB) from the right ventricular (RV) and on the hearts of 33 patients with ARVD died unexpectedly. Intravital and post-mortem cardiac MRI scans were performed.

Results: All the cases showed the atrophy of muscle fibres, lipomatosis and fibrosis in the RV. The average residual cardiomyocyte area was 39,7 ± 12,8 %, the average cardiomyocyte diameter was 10,4 ± 1,5μm. The average thickness of RV anterior wall was 0,14 ± 0,07 cm. 44 % of the patients had myocarditis, 33 % of the patients had myocardial fibre disarray in RV and 60 % had focal γ-catenin expression in muscle fibres. Intravital MRI showed RV dissinchrony in 82 % of the cases, signs of adipose infiltration of RV in 65 % of the cases and mitral valve prolapse in 42 %.

Conclusion: To make the diagnosis of ARVD on EMB not only the residual area of RV muscle fibres should be determined but the average cardiomyocyte diameter as well. As to MRI the most important criterion of ARVD is the RV dissinchrony. In some cases we observed the combination of ARVD with mitral valve prolapse and myocardial fibre disarray in RV.


Left-sided arrhythmogenic ventricular cardiomyopathy with sudden-death in a young man: A case report

L. De Carvalho *, G. Pozzan, G. Monteiro, F. Bolfarini, R. Penny, G. Ciconelli Del Guerra

*Centro Universitário Lusíada, Pathology, Santos, Brazil

Objective: Arrhythmogenic ventricular cardiomyopathy is a clinically and genetically heterogeneous heart muscle disorder associated with ventricular arrhythmias and risk of sudden death. The classic form of the disease has an predilection for the right ventricle and is characterized by ventricular myocardial fibrofatty replacement. Biventricular arrhythmogenic cardiomyopathy and left dominant arrhythmogenic cardiomyopathy forms had been included in the spectrum of disease.

Method: 21-year-old male presented collapse during the sleep. The heart presented on gross examination a subepicardial, circumferential hemorrhagic lesion at left ventricle.

Results: The microscopy showed a myocardial replacement by fibrous tissue with capillaries and scattered lymphocytes. There were no remarkable changes in the wall thickness in both ventricles and coronaries abnormalities.

Conclusion: Arrhythmogenic ventricular cardiomyopathy/dysplasia is one of the leading causes of sudden-death in young people. The left-dominant subtype of the disease is still under-recognized and it may precede the onset of significant right ventricular changes,as we reported in this case.


Myocarditis associated sudden and non-sudden death: The testimony of a series

R. Henriques de Gouveia *, C. Cordeiro, B. S. Silva, F. Corte Real, D. N. Vieira

*INMLCF, I.P., Dept. of Pathology, Coimbra, Portugal

Objective: “Myocarditis” are still responsible for important morbility and mortality (5 %) worldwide, whether death setting is natural (sudden and unexpected or not) or non-natural. Authors report the experience of a National Institution.

Method: Demographic/circumstancial/toxicological and morphological data of 1,141 autopsies with anatomo-pathological examination (performed in 2011, territorial area = 29.206Km2, inhabitants = 2.595.540) were reviewed.

Results: Twenty-three autopsies (2,02 %) presented Myocarditis. They refered to 18 males with mean age of 53,8 years [21–75 years] and to 5 females whose mean age was 77,8 years [40–96 years]. Myocarditis was lymphocytic(n = 6), mixed cellularity (n = 14) and neutrophylic (n = 3). Etiology was infeccious, immunologic, toxic, hypersensitivity. Death occured at home, hotel, restaurant, prison, hospital, ambulance, river. It occured either suddenly (n = 16, 69,6 %); unexpectedly, of natural cause, but not suddenly (n = 1); complicating car accident with hospitalization (n = 4); complicating after explosion burn with hospitalazation (n = 1); or due to a fall (n = 1).

Conclusion: 1) Myocarditis remain an importante cause of Sudden Death. 2) Death due to myocarditis may have legal relevance in insurance disputes, medical negligence suspicion. 3) Knowing the real ‘Health Status’of a country, in what concerns pathologies, causes and death circumstances, both intra and extra-hospitalar data is required. 4) Promoting “Disease National Registries” is of utmost importance and demands multidisciplinary cooperation.


The structure of pre-transplant heart disease in Belarus

S. Rjabceva *, O. Yudina, A. Smolensky, Y. Ostrowskiy, P. Yudin, M. Vozmitel

*City Pathological Bureau, Pathology, Minsk, Belarus

Objective: The main indications for heart transplantation were cardiomyopathy (CMP) arising from end-stage chronic ischemic heart disease, dilatation of the heart cavities and other heart diseases.

Method: We examined 58 hearts of recipients which were operated in RSPC “Cardiology” from 2009 to 2011 years by macro- and microscopically.

Results: By clinical examinations our patients were with dilated cardiomyopathy (79.3 %), ischemic cardiomyopathy (15.6 %) and chronic rheumatic heart disease (5.1 %). The structure of pre-transplant heart disease has changed after the histological study of heart tissue. We diagnosed a dilated cardiomyopathy (67.2 %), ischemic cardiomyopathy (13.8 %), restrictive CMP (8.6 %). Mitochondrial CMP found in 5.2 % cases, a giant cell myocarditis–in 1.7 %, acute myocardial infarction–in 1.7 %, congenital disorders of connective tissue–in 1.7 % patients.

Conclusion: Morphological study showed that myocardial disease of recipients with a heart dilation are a heterogeneous group in Belarus, in which an important place occupied by patients with mitochondrial disorders (5.2 %).


Sudden death in the young due to myocarditis: The experience of two Iberian forensic centers

R. Henriques de Gouveia *, J. Lucena, J. Carvalho, B. S. Silva, F. Corte Real, D. N. Vieira

*INMLCF, I.P., Dept. of Pathology, Coimbra, Portugal

Objective: “Myocarditis” is responsible for 3.5 % of cardiac arrest in the young (1–35 years) occurring outside Health Institutions and eventually leading to Sudden Death. Two ‘Iberian Forensic Centers’ compared their 1-year experience based on the fact that Mediterranean countries share backgrounds, diets and lifestyles.

Method: Demographic, circumstantial, toxicological, microbiological and morphological data of autopsies with anatomo-pathological examination (performed in 2011) were reviewed. The ‘Spanish Center’, responsible for a population of 1.917.097 inhabitants, performed 850 autopsies. The ‘Portuguese Center’, serving a population of 2.595.540 inhabitants, performed 1141 autopsies.

Results: Both Centers had one case of Sudden Death in young persons due to Myocarditis. Spain: a 6 year-old female deceased of acute fulminant myocarditis, presenting positive microbiological analysis for Influenza B, Parainfluenza 4 and Rhinovirus. Portugal: a 33 year-old male unexpectedly died after diner due to acute Myocarditis. He was on anti-influenza therapy.

Conclusion: 1) Spanish and Portuguese experience, in these Centers, is similar concerning Myocarditis as a cause of Sudden Death in the Young. 2) It occurs both in the very young and in young adults. 3) A thorough postmortem examination is crucial for the correct diagnosis.


A rare case of epicardial involvement in Erdheim-Chester disease

J. S. Stadie*, M. Pasic, K. Hauptmann, R. Hetzer, K. Wassilew

*German Heart Institute Berlin, Section Heart Pathology, Germany

Objective: Erdheim-Chester disease (ECD) is a rare non-Langerhans cell histiocytosis with heterogeneous systemic manifestations. Cardiovascular involvement is considered an important but rare, and often overlooked, finding. Cardiovascular involvement in ECD mainly occurs as periaortic ‘fibrotic change’, with pericardial involvement and pericardial effusion. Venous, myocardial or epicardial affections and involvement of the coronary arteries are noted less frequently.

Method: We report on a case of a 36-year old man with a tumorous cardiac lesion, diagnosed preoperatively as an incidental finding not related to the cause of his hospital admission.

Results: Radiologically, an ill-defined pericardially centered lesion, extending to the ascending aorta, the left pulmonary artery and the epicardial fat, encasing both left and right coronary arteries, was noted. Histological and immunhistological examination of the extensively sampled epicardial lesion and of samples from the gluteal tumor diagnosed later during the clinical course showed similar findings, namely diffuse infiltration of plentiful cytologically bland, S-100-negative, CD68-positive foamy histiocytes, admixed with multinucleate giant cells, set in a fibrous stroma.

Conclusion: The synopsis of clinical, pathological and radiological findings in this case allows the diagnosis of Erdheim-Chester disease with uncommon primary epicardial manifestation.


Autonomic innervation of pulmonary veins: Morphological substrate of atrial fibrillation

I. Kholova *, R. Leinonen, A. Mäkinen, O. Jääskeläinen, T. Paavonen

*Fimlab Laboratories, Dept. of Pathology, Tampere, Finland

Objective: Atrial fibrillation is the most common sustained tachyarrhythmia. Morphological substrates of atrial fibrillation were found also in myocardial sleeves around pulmonary veins. This study aims to examine immunohistochemically autonomic nerve distribution in pulmonary veins and venous-atrial junctions.

Method: Immunohistochemical staining was performed in 48 pulmonary veins and 9 atria samples using antibodies to tyrosine hydroxylase, choline acetyltransferase and GAP-43. The morphometrical analysis was performed with computer-assisted system (ImageJ).

Results: Adrenergic and cholinergic nerves and ganglia were often co-located. Their density varied at different compartments. The nerve structures were present both within myocardial sleeves and in surrounding fibro-fatty tissue.

Conclusion: Adrenergic and cholinergic nerves and ganglia may play the role in atrial fibrillation triggers from pulmonary veins. However, selective targeting of either vagal or sympathetic nerves is due to co-localization impossible.


Congenital vascular malformations: Cerebral lesions differ from extracranial lesions by their immune expression of the glucose transporter protein GLUT1

L. Meijer-Jorna *, E. Aronica, C. M. van der Loos, D. Troost, A. C. van der Wal

*Symbiant/MCA, Dept. of Pathology, Alkmaar, The Netherlands

Objective: Cerebral vascular malformations were investigated for the presence of the glucose transporter protein GLUT1, which is normally expressed in endothelial cells of pre-existing microvasculature of the brain and absent in vasculature of the choroid plexus and extracranial vasculature without a barrier function. Extracranial arteriovenous malformations (AVMs) are known to show an absence of GLUT1 expression which distinguishes them from infantile hemangiomas of skin and soft tissue. The expression of GLUT1 in cerebrovascular malformations is not systematically investigated.

Method: Paraffin sections of 12 cerebral AVMs, including one choroid plexus AVM, 10 cerebral cavernous malformations (CCMs) and 10 extracranial (facial) AVMs were immunostained with anti-CD31 and anti-GLUT1 in an immunodoublestaining procedure which was further analyzed with the use of spectral analysis software.

Results: All 22 cases of cerebral vascular malformations (AVMs and CCMs) showed co-localization of GLUT1/CD31 of endothelial lining of the vessels (malformed arteries and veins) within the malformation. In the 10 extracranial AVMs expression of GLUT1 was completely absent.

Conclusion: Cerebral AVMs differ from extracranial AVMs by their endothelial immunoexpression of GLUT1, indicating that the vessels of these malformations retain the endothelial phenotype of the local vascular beds from which they are derived during embryogenesis.


Rupture and dissection in pulmonary artery aneurysms: A case report

S. Rjabceva *, A. Gerasimovich, E. Adolf, M. Vozmitel

*City Pathological Bureau, Pathology, Minsk, Belarus

Objective: Pulmonary artery aneurysm (PAA) is an uncommon disorder. Dissection mostly develops in a pulmonary artery aneurysm associated with pulmonary hypertension and/or connective tissue disease. The aim of this study was to describe the histopathological features of PAA.

Method: We studied one case of a 44-year-old woman with PAA. We used Hart’s method for elastic fibers and van Gieson stain.

Results: A 44-year-old woman was admitted to hospital with chest pain and shortness of breath. She had some features similar to Marfan syndrome: arachnodactylia, pectus deformities and thoracolumbar scoliosis. She died from cardiac sudden death after 11 days. At necropsy, we found the enlarged main pulmonary artery (a diameter of 6.6 cm) with a dissection and a rupture of walls and hemorrhage in pericardial cavity with massive effusion. Coronary artery compression was a reason of myocardial infarct and death of patient. Histologically, there are myxoid degeneration of media, irregular arrangement elastin and collagen fiber in pulmonary artery aneurysm wall.

Conclusion: We diagnosed an idiopathic pulmonary artery aneurysm in this patient with Marfan-like phenotype. The diagnosis of PAA is often difficult, the natural history of the disease is not completely understood. Recognition of pulmonary artery aneurysms is important because of the high morbidity and mortality rates of rupture.


New reference tables for predicted heart weight

J. Vanhaebost *, M. Faouzi, P. Mangin, K. Michaud

*Curml, Lausanne, Switzerland

Objective: Knowledge of normal heart weight ranges is important information for pathologists. Comparing the heart weight to reference values is one of the key elements used to determine if the heart is pathological, as heart weight increases in certain cardiac pathologies. Current tables are outdated and were established on different populations. The purpose of this study is to establish new tables relevant to the local population and to determine the best predictive factor for normal heart weight. We also aim to provide technologic support to calculate the predictive normal heart weight.

Method: The reference values are based on a study including 288 adult patients without any obvious pathologies who were autopsied in western Switzerland from 2007 to 2011. The statistically analysed parameters were age, gender, height, body weight, BMI and body surface area.

Results: Heart weight is statistically correlated with all of the parameters studied. Body surface area is the best predictor of normal heart weight.

Conclusion: New references tables for predicted heart weight are presented as well a web application that enables the comparison of heart weights observed at autopsy with reference values. This application can also calculate the BMI and body surface area.


Postmortem CT-angiography of coronary arteries in cases of sudden cardiac deaths

K. Michaud *, S. Grabherr, P. Mangin

*University of Lausanne, CURML, Switzerland

Objective: Cardiac diseases are the most frequent causes of death in developed countries. The most common clinical finding associated with sudden cardiac death in general population is ischemic heart disease related to atherosclerotic coronary artery.

Method: At the University Center of Legal Medicine of Lausanne, postmortem CT-scanning and postmortem multiphase CT-angiography are routinely used since 2009 to investigate violent deaths but also natural deaths in which cardio-vascular lesions such as coronary artery diseases are suspected. This technique allows the visualization of the vascular system similarly to clinical investigations.

Results: In our experience, the exploration of coronary arteries by postmortem CT-angiography is very useful to analyze and document their anatomy and to detect stenoses, occlusions or calcifications. It also shows advantages in investigating deaths following cardiac surgery or vascular interventions.

Conclusion: Interpretation of post-mortem cardiovascular radiology is still a new field and we have to learn to interpret correctly the technique-related artefacts and those caused by post-mortem changes in order to distinguish them from pathological lesions (such as eroded plaques, coronary vasculitis). At the same time, post-mortem radiological examination followed by a complete autopsy by is very promising to further the understanding of interpretation of radiological examination in living patients.


Splenic lymphangioma with endothelial papillary proliferation in a patient with gastric carcinoma

N. Dias *, D. Oliveira, M. R. Silva, M. J. Julião

*Coimbra, Portugal

Objective: Lymphangiomas of the spleen are rare benign vascular tumours. They are classified as simple (capillary), cavernous and cystic (hygroma). Papillary endothelial proliferations in lymphangiomas have been described for long time. To our knowledge, this feature in the setting of splenic lymphangiomas is rare.

Method: We present a case of a 62-year-old woman with gastric cancer and a splenic nodule detected during staging CT, suspicious of metastasis.

Results: Total gastrectomy and splenectomy were performed. A 2 cm ulcerated tumour was found in the lesser curvature of the antrum and a 1,5 cm well-defined, grey-yellowish, hard nodule was found in the spleen. Histologically the gastric tumour was a moderately differentiated tubular adenocarcinoma invading the submucosa without lymph node metastasis. The spleen nodule consisted of luminal structures, without red blood cells, lined by a flat cell layer without atypia, with multifocal papillary proliferations into the lumen. These cells were CD34 and CD31 positive, as well as CAM5.2 negative, pointing towards an endothelial differentiation.

Conclusion: Splenic lymphangioma with papillary proliferations are rare lesions. This finding does not seem to represent a different subtype of lymphangioma nor have worse prognosis, and should not to be mistaken for a low-grade or malignant neoplasia.


A clinico-morphological review of tumors of heart

K. Dikarev *, L. Mitrofanova, I. Antonova

*Almazov Federal Heart, Blood, and Endocrinology Center, St. Petersburg, Russia

Objective: 200 cases benign and malignant tumors of heart have been investigated for 25 years (from 1988 to 2013).

Method: Conducted the statistical processing of the main clinical symptoms, microscopic examination of tumors with standard histological staining and immunohistochemical stain in some cases.

Results: The most number of tumors were benign (140 cases), predominantly myxoma (80 %). Other tumors were lipoma, papillary fibroelastoma, teratoma. The main symptoms were exertional dyspnea, edemas, arrhythmia, periodic pain of the chest. Sometimes tumors were identified by accident on clinical examination or autopsy. The surgical treatment benign tumors were effective. 2 patients without operation died from heart failure an arrhythmia. Among malignant tumors prevailed metastatic tumors (40 cases), among primary malignant tumors were different types of sarcomas. Prognosis is grave: the patients died during the term of 2 weeks to 1 year from metastases, progressive heart failure, pericardial tamponade. One patient who has received chemotherapy is alive for more than 1 year. Surgical intervention may reduce clinical symptoms and increase a term of life.

Conclusion: Prognosis cardiac tumors is difficult to predict, irrespect of their biological potential. There is not any TNM stading for malignant tumors yet. It is necessary to develop clinic-morphological criteria for optimal treatment and prognosis.


A single centre survey of cardiac tumours over a 16 year period from Singapore

S. H. Lai *

*Singapore General Hospital, Dept. of Pathology, Singapore

Objective: This study reviewed the pathology of cardiac tumours in a single tertiary institution in Singapore from 1997 to 2012.

Method: The departmental database was searched for cases of cardiac tumours during the period from January 1997 to December 2012 inclusive. Reports and slides of returned cases were reviewed. Demographical and pathological data was analysed and literature review was performed.

Results: here were a total of 61 cases, 53 were benign. The incidence was 3.8 per year. Age distribution ranged from 18 to 79 years, with a mean age of 55 years. The overall female to male ratio was 2.8:1. The eight malignant neoplasms included seven metastatic lesions and one primary lymphoma. Malignant lesions were lymphomas (2), renal cell carcinoma (1), sarcoma (1), hepatocellular carcinoma (1), mucoepidermoid carcinoma (1) and liposarcoma (1). Of the benign neoplasms, 48 were myxomas with annual incidence of 3 cases; a female to male ratio of 2.9:1; and occurred in the left atrium in 77 % of cases.

Conclusion: This is possibly the largest series of cardiac neoplasms reported from Singapore to date. The results provided an epidemiological and pathological description of cardiac neoplasms and are comparable to those described in the literature.


Metastasis of squamous cell carcinoma of the penis into a heart with pacemaker mimicking an endocardial vegetation

C. Tomikawa *, S. A. C. Siqueira

*FMUSP, Dept. of Pathology, Sao Paulo, Brazil

Objective: Metastasis of penile cancer to the heart is extremely rare and has been scarcely reported. Presented is an autopsy case of metastasis of squamous cell carcinoma of the penis to a heart with pacemaker mimicking an endocardial vegetation.

Method: Standardized autopsy examination was performed.

Results: A 79-year- old male patient with a permanent pacemaker underwent partial amputation of the penis due to a squamous cell carcinoma of the penis. Less than 1 year later, due to recurrence, emasculation was realized. After the surgery, the patient presented clinical features of sepsis and latter sudden discomfort, evolving to cardiac arrest. Autopsy was performed to elucidate the cause of death, being endocarditis the main hypothesis. The heart was augmented and on sectioning the right chambers a tumor was easily visible, from the atrium to the ventricle through the tricuspid valve, enclosing the pacemaker catheter and resembling an infectious endocardial vegetation. Histological examination showed extensively necrotic tumor, although some preserved areas displayed squamous cell carcinoma.

Conclusion: Penile cancer is an uncommon malignancy and his metastasis to the heart is even more. This case is presented to alert the physicians that metastatic tumor, albeit rare, may be a differential diagnosis of endocarditis.

Metastasis of squamous cell carcinoma of the penis to the heart:

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Intracardiac teratoma in a 7 year old boy: Case report

A. Rodrigues-Pereira *, E. Pinto, M. Ferraz de Oliveira

*Porto, Portugal

A 7-year-old male from Guine Bissau was diagnosed at birth with severe valvular stenosis and had two surgeries both at neonatal period and at 14 months old. He was followed by pediatric cardiology and the only symptom reported was fatigue after physical activity. The last transthoracic ultrasound (US) showed a multicystic mass measuring 6 × 3 cm in the right ventricle chamber adherent to the interventricular septum with US features suggestive of hydatid cyst. The magnetic ressonance confirmed these findings. The child had a third cardiac intervention to remove this mass. Macroscopically, two irregular white fragments were received measuring 4.5 × 3.5 cm and 3.5 × 2.5 cm. On section multiloculated cysts with a mucinous filling and intervening solid areas showing calcification were noted. Microscopically, they consisted in multicystic tumor lined by ciliated columnar epithelium and including structures such as bronchial glands, mature cartilaginous tissue, bone with bone marrow, adipose tissue, skeletal muscle and retina pigment cells. Since endodermal, mesodermal and ectodermal structures were identified, this lesion is classified as an intracardiac mature cystic teratoma. This is a benign rare primary tumor of the heart amenable to surgical cure.


Solitary fibrous tumor of the heart: An extremly rare neoplasm of the right atrium

D. Galler *, S. Reinold, D. Hoefer, M. Grimm, H. Popper, C. Ensinger

*Innsbruck, Austria

Objective: Cardiac solitary fibrous tumor (SFT) is a very rare entity with only 8 cases described in literature. We report on a case of a 68-year old man with a primary SFT developed in the right atrium.

Method: The tumor location was confirmed by CT and MRI after clinical symptoms of right atrial masses. Consecutively the well circumscribed 10 × 9 × 9 cm tumor was surgically resected and evaluated by intraoperative frozen sections wich revealed a partly myxoid, mostly fibrous tumor without signs of malignancy and tumor free resection margins. Formalin fixed histological slides showed variable cellularity with dense areas of spindle cells as well as pericytoma like vessels. Other parts revealed collagen bundles that are partly hyalinized.

Results: Tumor cells were reacting with CD34 as well as CD99 antibodies and were negative for S-100, desmin, actin, keratins and ALK. This reactivity excluded an inflammatory myofibroblastic tumor as well as neurofibroma from the differential diagnosis. In addition the tumor cells were strongly positive for IGF1R, typical for SFT and partly responsible for hypoglycemia of these patients.

Conclusion: Moreover, we found a PDGFR α and β reactivity in a large number of tumor cells, leading to an additional therapeutic option with imatinib in these tumors, especially important for the minority of malignant SFTs (5–10 %).


Expression of p53, p63 and Ki-67 in cardiac myxomas

T. Canpolat *, F. Kayaselcuk, A. Tünel

*Baskent University, Pathology, Adana, Turkey

Objective: Cardiac myxomas are the most common primary tumors of the heart, Although little is known about their etiology.

Method: We retrospectively evaluated 27 cases of myxomas and determined the role of p53, p63, Ki-67 in histogenesis.

Results: Nine patients (33.3 %) had immune staining in the nucleus for p53 in a limited number of cells. Similarly, nine patients (33.3 %) had immune staining for Ki-67. Especially the areas rich in cells were stained for Ki-67. P63 exhibited positive cytoplasmic and nuclear staining in the perivascular area or individual spindle- or star-like cells scattered on the myxoid stroma. Eight patients (29.6 %) had immune staining for P63 (+) and 12 patients (44.4 %) had immuhistochemical staining for p53 (++). Seven patients (25.9 %) were negative for P63. Tumor size, age, gender and presence of calcification were not correlated with immune staining for Ki-67 and p53 and p63 (p>0.05). Staining for P53 was significantly correlated with staining for Ki67 (p = 0.016).

Conclusion: We found immunoreactivity to p53 in few cells in 29.7 % of the cases of cardiac myxomas, which is consistent with the benign nature of cardiac myxomas. Extensive p53 and Ki-67 positivity especially in areas rich in cells was suggestive of cellular proliferation.


Valves damage in amyloidosis

M. Rybakova *, I. Kuznetsova, A. Goodkova, E. Semernin

*State Medical University, Dept. of Pathology, St. Petersburg, Russia

Objective: To identify the frequency of valve damage in amyloidosis.