==== Front Virchows Arch Virchows Arch Virchows Archiv 0945-6317 1432-2307 Springer Berlin Heidelberg Berlin/Heidelberg 23925389 1406-50-6775/s00428-013-1444-y Abstracts ESP Abstracts 2013 8 8 2013 8 8 2013 2013 463 2 101352 © Springer-Verlag Berlin Heidelberg 2013 Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. issue-copyright-statement© Springer-Verlag Berlin Heidelberg 2013 ==== Body Oral Free Paper Sessions Sunday, 1 September 2013, 08.30–12.00, Room 5C OFP-01 Oral Free Paper Session Breast Pathology OFP-01-001 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. OFP-01-002 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. OFP-01-003 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. OFP-01-004 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. OFP-01-006 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 OFP-01-007 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. OFP-01-008 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. OFP-01-009 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. OFP-01-010 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. OFP-01-011 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. OFP-01-012 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 OFP-02-001 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. OFP-02-002 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. OFP-02-003 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. OFP-02-004 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. OFP-02-005 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. OFP-02-006 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. OFP-02-007 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. OFP-02-008 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. OFP-02-009 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). OFP-02-010 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 OFP-03-001 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. OFP-03-002 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. OFP-03-003 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. OFP-03-005 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. OFP-03-006 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. OFP-03-007 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. OFP-03-009 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. OFP-03-010 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 OFP-04-001 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. OFP-04-002 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. OFP-04-003 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. OFP-04-004 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. OFP-04-005 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
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