Adriatic Society of Pathology, 23rd Meeting, June 27 – 29 ... · PDF fileAdriatic...

Adriatic Society of Pathology, 23rd Meeting, June 27 – 29, 2008, Dubrovnik, Croatia

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BOOK OF ABSTRACTS

23rd Meeting of the Adriatic Society of Pathology

June 27-29, 2008

Dubrovnik


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Scientific Committee:

Mladen Belicza (President of the Congress) Božo Krušlin (Vice President of the Congress)

Vladimiro Mambelli (President Elect of the Adriatic Society of Pathology)

Carlo Alberto Beltrami (Udine), Gianni Bussolati (Torino), Vincenzo Eusebi (Bologna), Guidalberto Fabbris (Ancona), Snježana Grazio

(Ljubljana), Sigurd Lax (Graz), Lucio Palombini (Napoli), Valdi Pešutić-Pisac (Split), Giorgio Stanta (Trieste).

Organizing Committee:

Mladen Belicza, Božo Krušlin, Zlatko Marušić, Tanja Leniček, Igor Borić,

Alma Demirović, Ilija Jurković, Zdenko Njirić.

E-mail address: [email protected] Phone: 00 385 1 37 87 907

Fax: 00 385 1 37 87 244


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PROGRAMME

Adriatic Society of Pathology 23rd Meeting

June 27 – 29, 2008, Dubrovnik, Croatia

Friday, 27th of June, 2008 06.00 pm Registration 06.30 pm Opening and Welcome Ceremony Mladen Belicza, President of the Organizing Committee 07.00 pm Keynote Lecture AGOSTINO FARAVELLI (MILAN, ITALY): PATHOLOGISTS OVER FRONTIERS

Saturday, 28th of June, 2008

08.30-10.00 am Gastrointestinal pathology Chairpersons: Božo Krušlin & Vincenzo Eusebi VINCENZO VILLANACCI (BRESCIA, ITALY): POLYPS OF THE GASTROINTESTINAL TRACT FELIX ALBERT OFFNER (FELDKIRCH, AUSTRIA): BARRETT'S OESOPHAGUS 10.00-10.30 am Break 10.30-12.00 am Proffered papers (Oral presentations) Chairpersons: Matej Bračko & Bettina Zelger 12.00-02.00 pm Lunch break 02.00-03.30 pm Head & neck pathology Chairpersons: Valdi Pešutić-Pisac & Sven Seiwerth MARIA PIA FOSCHINI (BOLOGNA, ITALY): IMPACT OF RESECTION MARGINS IN THE FOLLOW UP OF SQUAMOUS CELL CARCINOMA OF THE ORAL CAVITY NINA GALE (LJUBLJANA, SLOVENIA): CLEAR CELL SALIVARY GLAND TUMORS: A DIFFERENTIAL DIAGNOSTIC CHALLENGE


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03.30-04.00 pm Break 04.00-05.30 pm Proffered papers (Oral presentations) Chairpersons: Sigurd Lax & Lucio Palombini 06.00 pm Starting point - Reception of Hotel Excelsior Social dinner at the Karaka boat; a cruise of the Old harbour (Free of charge for Lecturers, Chairmen and Presenters of oral presentations)

Sunday, 29th of June, 2008

09.00-10.30 am Technology applied to pathology Chairpersons: Snježana Grazio & Giorgio Stanta IRMGARD VERDORFER (INNSBRUCK, AUSTRIA): FISH TECHNOLOGY GIORGIO STANTA (TRIESTE, ITALY): ARCHIVE TISSUES: A SHORTCUT FOR MOLECULAR MEDICINE 10.30-11.00 am Break 11.00-12.30 pm Proffered papers (Oral presentations) Chairpersons: Nives Jonjić & Carlo Alberto Beltrami 12.30 pm Closing ceremony The meeting participation, in the tradition of ASP, is free of charge. Official language is "Bad English" (Continental English). So speak slowly and don't worry, be happy ! Abstracts have been published in their original form, as received, and the authors are exclusively responsible for the content of the abstracts.

Our special thanks for the financial support provided goes to the Croatian Academy of Sciences and Arts


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PROFFERED PAPERS

June 28, 2008 (10.30-12.00 am)

GENETIC SIMILARITIES AND DIFFERENCES IN DUCT CARCINOMA IN SITU OF THE BREAST F. Flamminio, L. Morandi, M.P. Foschini, V. Eusebi (Italy)..................................................................... 9 ENDOMETRIAL INTRAEPITHELIAL NEOPLASIA – HISTOPATHOLOGICAL, MORPHOMETRICAL AND IMMUNOHISTOCHEMICAL FEATURES Cornelia Amalinei, Raluca Balan, Corina Cianga, Petru Cianga, Irina Draga Caruntu (Romania)........10 RELATIONSHIP BETWEEN RENAL ARTERY CHANGES AND EXPRESSION OF VEGF AND HIF-1α IN RENAL CELL CARCINOMA Alma Demirović, Karla Tomić, Davor Tomas, Mladen Belicza, Božo Krušlin (Croatia)………….……...11 HEPATIC STEATOSIS, NECROINFLAMMATORY ACTIVITY AND FIBROSIS IN PATIENTS WITH CHRONIC HEPATITIS C Maria Comanescu, Violeta Comanescu, Corina Dochit, Carmen Popescu(Romania)..........................12 CORRELATION BETWEEN NOS2 EXPRESSION AND APOPTOSIS IN CHRONIC HEPATITIS C – AN IMMUNOHISTOCHEMICAL STUDY Camelia Stanciulescu, Catalina Pisoschi, Monica Banita, Oana Purcaru, B.Stanoiu, Niculina Mitrea (Romania)..............................................................................................................................................13 E-CADHERIN AND β-CATENIN EXPRESSION IN SOLID PSEUDOPAPILLARY TUMOR OF PANCREAS S. Gašparov, A. Borovečki, A. Škrtić, B. Kocman, K. Horvat, M. Dominis (Croatia)……………..……. 14 DIAGNOSIS OF CONGENITAL AGANGLIONIC MEGACOLON IN CHILDREN TREATED IN “ST MARY’S” EMERGENCY HOSPITAL, IASI, ON THE LAST 5 YEARS (2003-2008) Doina Mihaila, P. Plamadeala, S.G.Aprodu, Adana Varna, Mioara Trandafirescu (Romania)……..… 15 TWO DIFFERENT INTESTINAL METASTATIC SITES FROM LOBULAR CARCINOMA OF THE BREAST – REPORT OF A CASE Irina Florea, Dan Ferariu, Niculina Florea, Camelia Chifu, L.Ionescu, C. Radulescu, E. Patrascanu (Romania)…………………………………………………………………………………………………..….. 16 HISTOPATHOLOGY OF COLORECTAL TUMORS IN OUR MATERIAL Manxhuka-Kerliu S, Baruti A, Ahmetaj H, Loxha S, Ceku S, Kerliu A, Shahini L, Goneta Gashi, Podrimaj A, Hashani M (Kosovo)……………………………………………………………………………. 17


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PROFFERED PAPERS

June 28, 2008 (04.00-05.30 pm)

PODOPLANIN EXPRESSION IN SQUAMOUS CELL CARCINOMA OF THE ORAL CAVITY AS PREDICTOR OF AGGRESSIVE BEHAVIOUR Anna L. Tosi, Roberto Cocchi, Maria G. Pennesi, Maria P. Foschini (Italy)......................................... 18 THE p53 EXPRESSION IN ORAL SQUAMOUS CELL CARCINOMAS Raluca Ciurea, Cristiana Simionescu, Claudiu Margaritescu, Marius Ciurea (Romania)……………... 19 NIS EXPRESSION IN NORMAL AND NEOPLASTIC SALIVARY GLAND TISSUE A. Farnedi, S. Lega, K. J. Rhoden, R. Cocchi, G. Farneti, C. Marchetti, M.P. Foschini (Italy)............. 20 EXPRESSION OF EXTRACELLULAR MATRIX COMPONENTS IN MELANOCYTIC NEVI AND MELANOMA Vesna Jurčić, Tanja Perković, Boris Vodopivec (Slovenia)………………………………………………. 21 PURE RED CELL APLASIA ASSOCIATED TO MALIGNANT THYMOMA Amelia Gaman, Camelia Dobrea, G.Gaman (Romania)....................................................................... 22 PRIMARY MEDIASTINAL LARGE B CELL LYMPHOMA INITIALLY PRESENTED WITH UPPER CAVA SYNDROME – CASE REPORT Doina Butcovan, Doina Murarescu, Maria Sultana Mihailovici (Romania).......................................... 23 HISTOLOGICAL CRITERIA IN DIFFERENTIATION OF MALIGNANT LYMPHOMAS FROM REACTIVE CHANGES OF LYMPH NODE Maria-Sultana Mihailovici, M.Danciu, D. Ferariu, Doina Murarescu, Delia Ciobanu (Romania)........... 24 FETUS IN FETU: DIFFERENTIAL DIAGNOSIS OF AN OVARIAN MASS OF A PREGNANT WOMAN C.I. Paulon, D.Graziani, G.P.Bulfamante (Italy).................................................................................... 25


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PROFFERED PAPERS

June 29, 2008 (11.00-12.30 am)

NEW APPLICATIONS OF TISSUE MICROARRAY TECHNIQUE Szasz AM, Tokes AM, Lotz G, Nemeth ZS, Kulka J (Hungary)………………………………………..… 26 EFFECTS OF FIXATIVES AND FIXATION TIME ON TISSUE HISTOMORPHOLOGY AND mRNA PRESERVATION Isabella Dotti, Serena Bonin, Ermanno Nardon, Giorgio Stanta (Italy)................................................. 27 A NEW METHOD TO ISOLATE AND IN VITRO EXPAND CANCER STEM CELLS FROM HUMAN GLIOMAS Musiello Daniela, Gallelli Annarita, Puppato Elisa, Toffoletto Barbara, Ius Tamara, Vindigni Marco, Laura Mariuzzi, Nicoletta Finato, Beltrami Antonio Paolo, Cesselli Daniela, Skrap Miran, Beltrami Carlo Alberto (Italy)............................................................................................................................... 28 PROTEOMICS OF STEM CELL LINES OBTAINED FROM LOW- AND HIGH-GRADE HUMAN ASTROCYTOMAS Gallelli Annarita, Odreman Federico, Musiello Daniela, Beltrami Antonio Paolo, Cesselli Daniela, Ius Tamara, Vindigni Marco, Nicoletta Finato, Laura Mariuzzi, Skrap Miran, Vindigni Alessandro, Beltrami Carlo Alberto (Italy)............................................................................................................................... 29 HIGH THROUGH-PUT MOLECULAR ANALYSIS OF MUTATIONAL STATUS OF EGFR AND K-RAS IN ALCOHOL FIXED FINE-NEEDLE ASPIRATE BIOPSY Lorenzo Daniele, Sofia Asioli, Sara Mariani, Donatella Pacchioni, Gianni Bussolati (Italy)................. 30 CRITICALS IN THE PRE-ANALYTIC STEPS OF HISTOPATHOLOGICAL EXAMINATION IN HUMAN NEOPLASIAS Giuseppe D’Armento, Luca Molinaro, Sara Mariani, Gianni Bussolati (Italy)....................................... 31 VALUE OF FLUORESCENCE IN SITU HYBRIDIZATION IN TISSUE MICROARRAYS Rosa Noguera, Isidro Machado, Marta Piqueras, Eva Villamón, Antonio Llombart-Bosch, Samuel Navarro (Spain)……………………………………………………………………………………….……….. 32 ANALYSIS OF GENETIC MARKERS IN PEDIATRIC SOLID TUMORS USING FISH IN TMA Marta Piqueras, Isidro Machado, Eva Villamón, Antonio Llombart-Bosch, Samuel Navarro, Rosa Noguera (Spain)……………………………………………………………………………………………..… 33


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OTHER PAPERS SIMULTANEOUS CARCINOMAS OF THE OVARY AND ENDOMETRIUM Cornelia Amalinei, Raluca Balan, Laurette Cozma, Ioana Buda, Irina Draga Caruntu (Romania)....... 34 INTERFERON EFFECTS ABOUT MORPHOLOGICAL RED BLOOD CELLS IN PATIENTS WITH CHRONIC HEPATITIS C Adriana Bold, Gabriela Iliescu, Garofita Mateescu, Viorel Biciusca (Romania)................................... 35 EBV VIRUS DETECTION IN GASTROESOPHAGEAL JUNCTION ADENOCARCINOMAS – AN IMMUNOHISTOCHEMICAL AND IN SITU HYBRIDIZATION STUDY Cristina Iosif, Alina Georgescu, Alina Nicolae, Maria Neagu, Rodica Birla, Narcis Copca, Silviu Constantinoiu, Carmen Ardeleanu (Romania)...................................................................................... 36 TUMOROUS CALCINOSIS - CASE REPORT Košuta Iva, Gamulin Ozren, Bogdanović Iva, Bilić Ranko, Kavur Lovro, Batelja Lovorka (Croatia)..... 37 IMMUNOHISTOLOGICAL STUDY OF THE SECRETING-EXCRETING PANCREATIC COMPONENT IN THE CHRONIC PANCREATITIS Garofiţa Mateescu, Adriana Bold, Liliana Stanca, Maria Manolea, Eugen Petcu (Romania, Australia)38 P53 EXPRESSION IN BASAL CELL CARCINOMAS FROM PHOTOEXPOSED AREAS OF HEAD AND NECK REGION Claudia Mateoiu, Maria Comanescu, Claudia Georgescu, Florin Bogdan (Romania)…………..…….. 40 PRELIMINARY STUDY OF BIPOLAR HIP PROSTHESIS INFULENCE ON ACETABULAR ROOF MORPHOLOGY Iancu Emil Pleşea, Mirela Ghiluşi, Dan Nelu Anuşca, Oltin Tiberiu Pop, Valentin Dascălu, Claudia Valentina Georgescu (Romania)…………………………………………………………………………….. 41 QUNATITATIVE ASSESSEMENT OF INTRALOBULAR COMPONENTS IN AGEING TESTIS Iancu Emil Plesea, Stelian Danut Enache, Petrica Badea, Oltin Tiberiu Pop, Mirela Ghilusi, Carmen Florina Popescu, Bogdan Stanoiu, Raluca Ciurea (Romania)……………………..…………………….. 42 CORRELATIONS BETWEEN CLINICAL AND MORPHOLOGICAL ASPECTS IN HYPERTENSIVE PATIENS DEAD WITH PRIMARY INTRACEREBRAL NON LOBAR HAEMORRHAGE Iancu Emil Plesea, Stelian Danut Enache, Mirela Ghilusi, Oltin Tiberiu Pop, Simona Bondari, Corneliu Cristian Georgescu, Dan Cioroianu (Romania).................................................................................... 43 ISOLATION AND CHARACTERIZATION OF CANCER STEM CELLS PRESENTS IN HUMAN LIVER HEPATOCELLULAR CARCINOMA Poz Alessandra, Marzinotto Stefania, Baccarani Umberto, Avellini Claudio, Laura Mariuzzi, Nicoletta Finato, Toffoletto Barbara, Puppato Elisa, Rigo Silvia, Beltrami Antonio Paolo, Cesselli Daniela, Beltrami Carlo Alberto (Italy)................................................................................................................. 44


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GENETIC SIMILARITIES AND DIFFERENCES IN DUCT CARCINOMA IN SITU OF THE BREAST

F. Flamminio, L. Morandi, M.P. Foschini, V. Eusebi

Servizio di Anatomia Patologica, Ospedale Bellaria, Bologna, Italy

It has been recently suggested that ductal carcinoma in situ (DCIS) develops

within a single lobe. This view has been challenged by a study of DCIS in large (macro) sections. It was found that well differentiated (grade 1) DCIS/DIN1 are multicentric in 76.9% of the cases. Multiple foci range from 1 to over 100 (mean 35.08) and are located at significant distance one another (range 12-55 mm, mean 35.42 mm), in 61.5% multiple foci are more than 20 (1).

Aim of the study: to find out genetic similarities (clonality) or differences among the widely spread foci of DCIS.

Materials and methods: ten randomly selected cases of DCIS were studied with large (macro) sections. Multiple DCIS foci from different cases were microdissected as well as areas of invasive duct carcinoma (DCI) when present. All cases were studied for mtDNA D-loop sequence. In addition, all cases were studied for oligonucleotides with CGH microarrays.

Results: Five cases of the ten DCIS randomly selected were constituted by G1 DCIS/DIN 1, five cases were G2 DCIS/DIN 2. Patients were all female ranging from 47 to 87 (mean 68.9). All cases were unilateral (five cases in the right and five case were located in the left breast). The mtDNA D-loop sequencing analysis indicated that four cases of multiple DCIS G1 showed same mtDNA features when their foci where located within 30 mm one other (range 10-30 mm, mean 19.87). Remarkable differences were seen in two cases of G1DCIS were foci were distant 70 and 55 mm respectively. On the contrary DCIS G2, appeared clonal and grouped all within 35 mm at the most, with the exception of one case that showed foci distant 35mm. Some results were obtained with CGH array analysis. DCI foci, with the exception of three case, were considerably different from the closest DCIS.

Conclusions: 1) DCIS when located close together appear to be clonal. On the contrary, those located at a distance superior to 35 mm are genetically different, with only one exception. This indicates that they probably arise in different lobes.

2) DCI are often genetically different from the closest focus of DCIS. This result is difficult to explain and might be due to the higher genetic instability of the invasive tumours. References: Foschini MP et al. The impact of large sections on the study of in situ and invasive duct carcinoma of the breast. Human Pathology (2007) 38, 1736-43.


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ENDOMETRIAL INTRAEPITHELIAL NEOPLASIA – HISTOPATHOLOGICAL, MORPHOMETRICAL AND

IMMUNOHISTOCHEMICAL FEATURES Cornelia Amalinei, Raluca Balan, Corina Cianga, Petru Cianga, Irina Draga Caruntu

Normal and Pathological Morphology Department, University of Medicine and Pharmacy “Gr. T. Popa”

Iasi, Romania

Background. Identification of endometrial precancers has implemented EIN (endometrial intraepithelial neoplasia), as a descriptive term for monoclonal endometrial precancers, defined on a combined molecular, image analysis, and clinical outcome diagnostic schema.

Design. Our research was designed to evaluate architectural, cytological, morphometrical criteria, and immunohistochemical profile, using routine histological methods, computerized morphometry (KS.400 updated system), and hormonal receptors (ER, PR), proliferation marker (PCNA), apoptosis (Bcl-2), and matrix enzymes (MMP-2, MMP-9) on 17 cases of EIN.

Results. EIN was diagnosed in lesions that exceeded 1-2 mm as diameter, with a stromal volume less than that of the glands, and abnormal cytology when compared to background, after exclusion of mimics. For each case the D-score was calculated, using volume percentage stroma (VPS), standard deviation of shortest nuclear axis (SDSNA), and gland outer surface density (OUTSD), and then classified as EIN (D≤1), based on the outcome-predictive formula. ER and PR showed a concordant high expression, both in glandular and stromal components, and occasionally endothelial staining. PCNA index was variable but high, in glandular, stromal, and endothelial cells, correlated with ER and PR expression. Epithelial cells showed an increased Bcl-2 expression. MMPs expression was increased both in glandular and stromal components. Furthermore, MMP-9 expression was higher than that of MMP-2 and supplementary noticed in migrated cells. Five cases (19%) associated concurrent or subsequent endometrial carcinoma.

Conclusion. EIN is a valuable diagnosis in patient management. Overexpression of steroid receptors in EIN demonstrates the role of hormonal stimulation in carcinoma pathogenesis. PCNA, ER and PR coordinated increased expression suggests a correlation between hormonal status and cellular proliferation. Deregulated apoptosis enhances endometrial proliferation in EIN. As markers of extracellular matrix degradation, MMP-2 and MMP-9 demonstrate the role of epithelio-stromal interactions in the development and progression of endometrial neoplasia.


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RELATIONSHIP BETWEEN RENAL ARTERY CHANGES AND

EXPRESSION OF VEGF AND HIF-1α IN RENAL CELL CARCINOMA

Alma Demirović1, Karla Tomić2, Davor Tomas1, Mladen Belicza1, Božo Krušlin1

1Ljudevit Jurak University Department of Pathology, Sestre milosrdnice University Hospital, Zagreb, Croatia 2Department of Pathology, Dr. Josip Benčević General Hospital, Slavonski Brod, Croatia

Background: Renal cell carcinoma (RCC) represents 1% to 3% of all human

visceral malignancies and more than 90% of all malignancies of the kidney in adults. Main renal artery may show different lesions such as atherosclerosis or fibromuscular dysplasia (FMD). Some recent studies showed that necrosis of tumor tissue seem to be an independent prognostic factor. The aim of this study is to correlate expression of vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1α (HIF-1α) with renal artery changes and necrosis in RCC.

Patients and methods: We analyzed consecutive series of 55 patients (M:F=35:20) with RCC who underwent nephrectomy. Patients were aging from 35 to 79 years (mean 59.8) and the tumor size was 2.5 to 16 cm (mean 7.1). Specimens were routinely processed and stained with hematoxylin and eosin, Mallory trichrome method and orcein. Immunohistochemical analysis was performed using primary antibodies to VEGF and HIF-1α. The area of tumor that showed strongest expression (hot spot) and two adjacent areas have been identified by scanning entire tumor at low power field (X40). Expression of VEGF and HIF-1α was assessed under high power field (X400) and classified as: 0–no positive tumor cells; 1–up to 10% positive tumor cells; 2- >10-50% positive tumor cells; 3- >50% positive tumor cells. Necrosis was observed as: no necrosis, < 50%, and > 50% necrotic tumor tissue. Statistical analysis was performed using χ2 test and Pearson correlation test. The level of significance was set at p <0.05.

Results: Renal arteries of 23 (41.8%) patients showed no changes, FMD was found in 25 (45.5%) and atherosclerosis was observed in 7 (12.7%). VEGF expression was negative in 14 (25.5%) cases, 19 (34.5%) cases had less than 10% positive tumor cells, 13 (23.6%) cases had 10-50% positive tumor cells and 9 (16.4%) tumors expressed VEGF in more that 50% tumor cells. HIF-1α expression was negative in 15 (27.3%) cases, 19 (34.5%) cases had less than 10% positive tumor cells, 10 (18.2%) cases had 10-50% positive tumor cells and 11 (20%) tumors expressed HIF-1α in more that 50% tumor cells. 12 (21.8%) tumors had no necrosis, 32 (58.2%) had <50% and 11 (20%) had >50 % necrotic tissue.

Conclusion: Renal artery changes, especially FMD, are very common in patients with RCC. Striking correlation between VEGF and HIF-1α expression was observed. More necrotic tumors showed higher expression of HIF-1α and VEGF. There was significant relationship between FMD and expression of HIF-1α, but a larger number of patients should be analyzed to determine the cause of renal artery changes and their relationship to the tumor behavior and morphology.


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HEPATIC STEATOSIS, NECROINFLAMMATORY ACTIVITY AND FIBROSIS IN PATIENTS WITH CHRONIC HEPATITIS C

Maria Comanescu1, Violeta Comanescu2, Corina Dochit2, Carmen Popescu2

1Departament of Pathology, Clinical Emergency Hospital Bucharest, Romania,

2Departament of Pathology,Clinical County Hospital of Emergency Craiova, Romania

Infection with hepatitis C virus is a major cause of chronic liver disease. A common histological feature of chronic hepatitis C is steatosis and its association with fibrosis has been reported. The aim of this study was to elucidate the association between the markers of inflammation, fibrosis and steatosis and the histopathological changes.

371 patients with chronic hepatitis C who had undergone liver biopsy were included in this study . At the time of biopsy, patients were aged between 21 and 65 years old. Biopsies were routinely processed and special stainings were used with antibodies directed against different subsets of T cells, B cells and macrophages, and growth factors. The immunohistochemical markers included UCHL1, CD20, CD4, CD8, CD68 and TGF-beta. We assessed the activity and fibrosis using the HAI score. The degree of steatosis was assessed subjectively.

In the piecemeal necrosis the predominant cells were CD4 helper T cells, whereas those seen in the lobule were predominantly CD8 suppressor/cytotoxic T cells. CD20 positive B cells were seen mainly in the portal areas. In the more aggressive forms there was an increased number of CD68 positive cells. The TGF-beta was increased in the foci of necrosis In our study, steatosis was confirmed as significantly and was associated with fibrosis independently of necroinflammation in CHC. There was a significant association between stage of fibrosis and patient age. In our study, steatosis was invariably associated with fibrosis progression and was dependent on a simultaneous association between steatosis and hepatic necroinflammatory activity scores.


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CORRELATION BETWEEN NOS2 EXPRESSION AND APOPTOSIS IN CHRONIC HEPATITIS C – AN IMMUNOHISTOCHEMICAL STUDY

Camelia Stanciulescu1, Catalina Pisoschi1, Monica Banita1, Oana Purcaru1,

B.Stanoiu1, Niculina Mitrea2

1University of Medicine and Pharmacy, Craiova, Romania 2University of Medicine and Pharmacy „Carol Davila”, Bucharest, Romania

Introduction: The purpose of the present study was to assess the expression

of the inducible nitric oxide synthase protein (iNOS or NOS2) and its correlation with apoptosis in liver tissues of patients with chronic hepatitis C. In order to evaluate apoptosis we used TUNEL method and we analyzed the expression of Bcl-2 protein.

Methods: Liver biopsies from patients with chronic hepatitis C were used for the immunohistochemical study. Immunohistochemical reactions were performed on 3 μm rehydrated sections with the following primary antibodies: rabbit policlonal anti-NOS2, diluted 1: 100 (SantaCruz Biotechnology) and mouse monoclonal anti-Bcl2, diluted 1:75 (Dako Cytomation). Streptavidin-biotin-peroxidase complex was used to amplify the immune reactions and diaminobenzidine tetrahydrochloride and H2O2 were used as staining revelators. As a final step nuclear counterstaining with hematoxylin was performed. Apoptosis was assessed using DeadEnd Colorimetric TUNEL system (Promega).

Results: We observed NOS2 immunoreactivity in hepatocytes with predominant cytoplasmic staining. The cells from the inflammatory infiltrate were negative for NOS2. The reaction for Bcl-2 was negative in hepatocytes. Instead, many lymphocytes infiltrating portal triads and liver lobules were positive for this protein. TUNEL positive nuclei were observed in hepatocytes all around the liver lobules mainly in those surrounding the inflammatory infiltrate.

Conclusions: In our study we observed that the expression of NOS2 was directly correlated with apoptosis and inversely correlated with the expression of the antiapoptotic protein Bcl-2. These data suggest that NO could inhibit the expression of Bcl-2 proteins but the precise mechanism by which NO modulate apoptosis is still unclear.


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E-CADHERIN AND β-CATENIN EXPRESSION IN SOLID PSEUDOPAPILLARY TUMOR OF PANCREAS

S. Gašparov1, A. Borovečki1, A. Škrtić1, B. Kocman2, K. Horvat1, M. Dominis1

1Department of Clinical Pathology and Cytology, Merkur University Hospital, Zagreb, Croatia

2University Department of Surgery, Merkur University Hospital, Zagreb, Croatia

Solid pseudopapillary tumor of pancreas (SPT) is an extremely rare neoplasm of unknown origin that constitutes only about 1% of pancreatic neoplasms. SPT occurs most frequently in young females and has favorable prognosis. E-cadherin is a cell adhesion molecule that binds to catenins. Together they form cell adhesion junctions, which are associated with the cytoskeleton formation.

Four female patients, with age ranging from 17 to 28, underwent pancreatic surgical resection due to tumors, at the Merkur University Hospital between 1994 and 2007. The tumors were solitary, measured 6.5 – 18 cm at the time of resection. One patient underwent living donor liver transplantation due to multiple liver metastases three years after diagnosis. All four patients are alive and well.

Grossly, tumors were encapsulated, on cut sections solid with cystic, necrotic and hemorrhagic areas. Histologically, all SPT showed a solid monomorphous growth in peripheral parts, whereas in the centre tumor cells were dyscohesive and formed pseudopapillae surrounded by empty gap-like spaces.

Wide immunohistochemistry was performed. The tumor cells were vimentin+, NSE+, progesteron+, CD10+, CD56+, chromogranin-/+ (one case completely negative), synaptophysin- (one case focally positive), CK-, HMB45-, estrogen-, Ki67-.

In addition to the nuclear localization of β-catenin all tumors showed complete absence of E-cadherin expression. This may help to explain the discohesive nature of cells and cystic changes in these tumors. Nuclear expression of β-catenin and loss of E-cadherin with negative CK could also be used in definite diagnosis of SPT especially on small biopsy specimens.


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DIAGNOSIS OF CONGENITAL AGANGLIONIC MEGACOLON IN CHILDREN TREATED IN “ST MARY’S” EMERGENCY HOSPITAL,

IASI, ON THE LAST 5 YEARS (2003-2008)

Doina Mihaila1, P. Plamadeala1, S.G.Aprodu2, Adana Varna3, Mioara Trandafirescu4

1Department of Pathology of “St Mary’s” Emergency Hospital for children, Iasi, 2Pediatric Surgery Clinic, University of medicine “Gr.T.Popa” Iasi, 3Pathology Laboratory, Tg. Frumos City Hospital 4Histology Department, University of medicine “Gr.T.Popa” Iasi, Romania

Objectives: Congenital Aganglionic Megacolon or Hirschprung Disease (HD) is

an anatomic anomaly characterized by the absence of ganglion cells in both Meissner’s submucosal and Auerbach’s myenteric plexuses. The diagnosis is based on the microscopic examination of the intestinal wall. With this study we propose to highlight the diagnosis stages of our cases of HD.

Methods and materials: In the time span from January 2003 to April 2008 (5 years) we had 36 cases with Hirschprung Disease in our hospital, age 2 days to 14 years. Thirty of them were males, so the M:F ratio was of 5:1. We divided the age intervals into categories, resulting in 14 cases under 1 year, 13 between 1 and 3 years, and 9 cases over 4 years.

The microscopic exam was made on biopsies from the colonic or/with ileal muscular wall, on frozen sections stained with Toluidine Blue, and on routinely processed, paraffin-embedded tissue sections, stained H&E. We performed 17 frozen sections examinations, and most of them (16 cases) were in agreement with H&E sections. On the last 6 cases we also performed immunohistochimic stains (Synaptophysin).

Results: In half of the cases (18 patients), having the classic form of Hirschprung Disease (recto-sygmoidian), only one biopsy was enough to allow diagnostic and surgical treatment.

The remainder cases needed 2 or more (to 8) surgical interventions, all completed with wall biopsies, some of them due to infectious complications, followed by ulcerations and stenoses, or due to small dimensions of the biopsy, not enough to evaluate the plexuses. One case raised diagnostic difficulties, because it involved the entire intestines, with atypical spread of ganglion cells.

The immunohistochimic stains (Synaptophysin) allowed us to assess correctly the presence of ganglion cells where the muscular wall was markedly inflamed or/and with post-hypoxic degenerative changes of the plexuses.

Conclusions: The histopathologic diagnosis of Hirschprung Disease in the classic form was quickly done on frozen sections, in only one intervention, in half of our cases. In complicated cases, with infections, or in cases involving all the intestinal length, were helpful IHC investigations (Synaptophysin), for the right evaluation of the myenteric plexuses.


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TWO DIFFERENT INTESTINAL METASTATIC SITES FROM LOBULAR CARCINOMA OF THE BREAST – REPORT OF A CASE

Irina Florea1, Dan Ferariu1, Niculina Florea1, Camelia Chifu2, L.Ionescu2, C.

Radulescu2, E. Patrascanu2

1“Sf. Spiridon” University Hospital, Pathology Department, Iasi, Romania 2“Sf. Spiridon” University Hospital, Surgery Department, Iasi, Romania

It was previously reported that lobular carcinoma has a propensity to

metastasize to unusual sites such as the gastrointestinal tract, peritoneum and gynecologic organs. However, metastatic involvement of the intestinal tract secondary to breast carcinoma is a rare event.

We report here a particular case of a 50- year- old patient who was initially treated for a small bowel obstructive tumor whose primary site was revealed two years later and who subsequently developed a second rectal metastasis. The female patient presented in 2004 with an obstructive tumor of the ileum for which she underwent an emergent enterectomy. The pathology report revealed a small cell carcinoma the result coming out after ruling out the diagnostic of intestinal lymphoma. The patient underwent six cycles of adjuvant chemotherapy (Cisplatin and Etoposide). After two years the patient was readmitted in the hospital for a right breast tumor for which a modified Madden mastectomy was performed. Pathologic report revealed the diagnostic of lobular carcinoma (pT2 N2a Mx –G2) with morphologic modifications due to the chemotherapy. At this moment the question was if the previous ileal tumor was related to the breast malignancy. Indeed, the reassessment of ileal tumor revealed the diagnostic of carcinoma of nonintestinal origin (cytokeratin cocktail positive, CK20 negative). The diagnostic of small cell carcinoma (poorly differentiated endocrine neoplasm) was invalidated through immunohistochemical analysis (chromogranin and NSE negative). More than that, immunohistochemical results for hormone receptors and Her2 neu were identical with those achieved for the breast tumor (ER positive, PR negative, Her2 neu negative). In addition, E-cadherin was negative for both ileal and breast tumors. Retrospective, the diagnostic was lobular carcinoma with ileal metastasis. The patient underwent adjuvant chemotherapy for breast carcinoma. In spite of this, two years later she developed a rectal tumor synchronous with the recurrence of the ileal tumor. Morphological and immunohistochemical features confirmed that these tumors represented metastases of the breast lobular carcinoma.

Even though distant metastases of clinically occult breast cancers were previously reported these still represent a diagnostic trap for both the clinician and the pathologist. Awareness of this condition may lead to accurate and prompt diagnosis and early initiation of the appropriate treatment, thus avoiding the recurrence of the disease.


17

HISTOPATHOLOGY OF COLORECTAL TUMORS IN OUR MATERIAL

Manxhuka-Kerliu S, Baruti A, Ahmetaj H, Loxha S, Ceku S, Kerliu A, Shahini L, Goneta Gashi, Podrimaj A, Hashani M.

Institute of Pathology, Faculty of Medicine, University of Prishtina, Kosovo

Colorectal cancer is the second after lung cancer as a cause of cancer-related

death. The purpose of this research was to analyze the colorectal cancer cases in our material with respect to all prognostic values such as histological type and grade, vascular invasion, perineural invasion, and tumor border features. There were investigated 149 cases of resection specimens with colorectal cancer, which were fixed in buffered neutral formalin and embedded in paraffin. Tissue sections (4μm thick) were cut and stained with H&E. Adenocarcinoma was the most frequent histological type in 85.90% of cases, with 60.94% males and 39.06% females; squamous cell carcinoma in 7.38%, with 63.63% males and 36.36% females; mucinous carcinoma in 4.68%, with 57.15% males and 42.85% females; while adenosquamous carcinoma, undifferentiated carcinoma and carcinoma in situ in 0.71% of cases each. Dukes’ classification was used in order to define the depth of invasion. Dukes B was found in 80% of cases whereas in 20% of cases was found Dukes C. As far as histological grading is concerned colorectal cancer in our material was mostly with moderate differentiation and without vascular nor perineural invasion. Resection margins were in all cases free of tumor. Our date indicate that the pathologic features of the resection specimen constitute the most powerful predictors of postoperative outcome in colorectal cancer. Dukes' stage and degree of differentiation provide independent prognostic information in colorectal cancer. However, differentiation should be assessed by the worst pattern.

Keywords: Circumferential margin, Colon cancer, Grade, Pathology, Prognostic factors, Dukes’ system


18

PODOPLANIN EXPRESSION IN SQUAMOUS CELL CARCINOMA OF THE ORAL CAVITY AS PREDICTOR OF AGGRESSIVE BEHAVIOUR

Anna L. Tosi, Roberto Cocchi1, Maria G. Pennesi1, Maria P. Foschini

Anatomic Pathology, University of Bologna, at Bellaria Hospital, Bologna, Italy 1Department of Maxillo-Facial Surgery, Bellaria Hospital, Bologna, Italy

Podoplanin is a mucin-type transmembrane glycoprotein recognized by the

D2-40 monoclonal antibody. It is specifically expressed by lymphatic but not blood vascular endothelial cells. In fact podoplanin is used as a specific marker for recognizing lymphatic vessels. This new marker is also found in peritoneal mesothelial cells, osteocytes, glandular myoepithelial cells, ependymal cells, stromal reticular cells and follicular dendritic cells of lymphoid organs. Occasionally detected weak focal podoplanin expression in basal epidermal keratinocytes of normal human skin, although the majority of keratinocytes was podoplanin- negative but its expression is strongly in squamous cell carcinomas and expression of podoplanin is restricted to the invasive front of tumours.

Podoplanin promotes the formation of cell-surface extensions and diminishes cell-cell adhesiveness when expressed in cultured human keratinocytes.

E-cadherin is a member of the cadherin super family known as the main mediator of the cell-cell calcium dependent adhesion interactions and have an important role in carcinogenesis and tumour progression.

Purpose of the present study is to evaluate D2-40 and E-Cadherin expression in 35 cases of oral squamous cell carcinoma (T1-T2, N0-N1), and to correlate the results with clinical outcome.

All cases were formalin fixed and paraffin embedded. From selected block sections were cut and immunostained with anti D2-40 (Signet, diluted 1:50) and with anti E-cadherin (Dako, clone NCH38, diluted 1:50) antibodies. Semiquantitative evaluation was performed counting the percentage of positive neoplastic cells. Immunohistochemical values, below or equal to 50% of positive cells were considered “low expression” (LE), values higher than 50% were considered “high expression” (HE) for E-cadherin. Cut off value between LE and HE was 20% of positive cells for D2-40. Follow-up varied from 8 to 48 months.

Ten cases showed the contemporary loss of E-cadherin expression (LE) and the HE of D2-40. Five of these cases developed lymph-node metastases during follow-up. Among the remaining 25 cases only one developed metastases during follow-up.

In conclusion these data demonstrate that these markers are important to predict oral carcinoma aggressiveness and its prognosis.

Podoplanin is involved in oral tumorigenesis and may serve as a predictor for lymph node metastasis and poor clinical outcome.


19

THE p53 EXPRESSION IN ORAL SQUAMOUS CELL CARCINOMAS

Raluca Ciurea1, Cristiana Simionescu1, Claudiu Margaritescu1, Marius Ciurea2

1Pathology Department, University of Medicine and Pharmacy, Craiova, Romania 2Plastic Surgery Department, University of Medicine and Pharmacy, Craiova, Romania

ABSTRACT: Oral carcinogenesis is a process with many stages consisting in

an accumulation of some genetic disturbances, because of the action of carcinogenic factors which modify the regulatory mechanisms of the basic cellular functions. We proposed to analyze the immunochistochemical expression for p53, with the purpose to identify their implications in oral carcinogenesis. We used te following score: 0 degree – under 10% positive cells; 1 degree – between 10 – 25 % positive cells; 2 degrees – between 25 – 50 % positive cells; 3 degrees – between 50 – 75 % positive cells; 4 degrees – over 75 % positive cells. We have studied 62 cases of OSCC selected in 2007, with various sites, with different histopathological types, also with various grades of differentiation, 9 of those cases with dysplasia noticed to the surgical margins. We performed the LSAB/HRP immunohistochemical study using markers to appreciate the tumor progression (p53, DO-7)). The histopatological study of the 62 cases of OSC indicated: 24 well-differentiated cases, 18 moderately differentiated cases and 20 poorly differentiated cases. p 53 was positive in 92,8 % cases, grade 2 in 15 cases, grade 3 in 9 cases, grade 1 in 9 cases and grade 4 in 5 cases. To the surgical margins p53 was positive corresponding to grade 1 and 2 in 9 cases with dysplasia and in situ carcinoma.

Conclusions: p53 did not correlate with the histopathological type or differentiation grade, but it can be a indicator of tumoral progression in early stages, but not a prediction factor of the malignant potential.


20

NIS EXPRESSION IN NORMAL AND NEOPLASTIC SALIVARY GLAND TISSUE

A. Farnedi, S. Lega, K. J. Rhoden1, R. Cocchi2, G. Farneti3, C. Marchetti4,

M.P. Foschini

Section of Anatomic Pathology, University of Bologna at Bellaria Hospital, Bologna, Italy 1Department of Medical Genetics, University of Bologna, Bologna, Italy 2Unit of Maxillo-Facial Surgery, Bellaria hospital, Bologna, Italy 3Unit of Otorhinolaryngology, Budrio hospital, Budrio (BO), Italy 4Department of Oral Sciences, Maxillo-Facial Surgery, University of Bologna, Italy

Introduction: The “Sodium/Iodide Symporter” (NIS) is a trans-membrane

glycoprotein that drives iodide (I-) transport into cells. Its expression in both normal and neoplastic thyroid tissues is well established. Recently, it has been demonstrated that NIS is also expressed in other tissues including the salivary glands.

The aim of the present work is to study NIS expression in normal and neoplastic salivary glands tissues.

Materials and methods: Forty-two cases of benign and malignant salivary gland lesions and the relative normal adjacent tissue, randomly selected from the files of the Section of Anatomic Pathology of University of Bologna at Bellaria hospital constituted the basis of the study. Tissues were formalin-fixed and paraffin-embedded. From selected blocks sections were obtained for immunohistochemical reactions with an anti-hNIS antibody (hNIS Ab-1-Clone FP5A, NeoMarkers-LabVision Corporation, Fremont, Ca. dilution 1:200).

Results: Normal tissue: NIS is expressed in the basolateral membrane of luminal cells in the striated ducts, whereas it is negative in acinar, myoepithelial and basal cells. NIS is expressed in almost 100% of ductal cells in the parotid gland; in contrast, NIS expression in the submandibular gland and in minor salivary glands is limited to 60 to 80% of ductal luminal cells.

Neoplasia: NIS positivity was found in Warthin tumours (7/7), mucoepidermoid carcinoma (2\9), and oncocytic adenoma (2\2). In addition, occasional positive cells were detected in pleomorphic adenoma (1\8). NIS is expressed in the plasma-membrane of cells lining Warthin tumours cystic cavities and in oncocytomas. Furthermore, rare cells showed positivity in a case of pleomorphic adenoma. Among malignant neoplasia only mucoepidermoidal carcinoma has shown positive cells. Positivity was localized to the plasma-membrane of epidermoidal and intermediate cells. NIS was not expressed in all the other remaining malignant lesions included in the present study (adenoido-cystic, myoepithelial and squamous carcinomas) (16 cases).

Conclusions: The present study confirms that in salivary gland tissues NIS is expressed in striated ducts and shows that expression is stronger in parotid than in other salivary gland types.

Furthermore NIS expression is maintained in tumours showing differentiation towards striated ducts such as Warthin tumours, oncocytomas and mucoepidermoid carcinomas.


21

EXPRESSION OF EXTRACELLULAR MATRIX COMPONENTS IN MELANOCYTIC NEVI AND MELANOMA

Vesna Jurčić, Tanja Perković, Boris Vodopivec

Institute of Pathology, Medical Faculty, University of Ljubljana, Ljubljana, Slovenia

In the past decade the interaction between tumor cells and the surrounding

extracellular matrix in the process of tumor development, invasion and metastasis has been a focus of interest.

Aim: to analyse the composition of extracellular matrix and immunohistochemical phenotype of stromal cells in melanocytic nevi and in melanoma.

Methods: Immunohistochemistry was performed on bioptic samples of 15 melanomas (5 in situ and 10 invasive) and 5 melanocytic nevi using antibodies against collagen IV, laminin, fibronectin, tenascin, CD34, desmin and α-smooth muscle actin (SMA).

Results: 1) In nevi, melanocytic cell nests were surrounded by basal lamina positive for collagen IV and laminin, which focally dissapeared in melanomas; 2) tenascin was weakly and focally positive in nevi, while in melanomas stroma and also peritumoral zone showed strong positivity; 3) CD34 positive cells were found in normal skin around nevi, but not in the stroma and peritumoural zone of melanomas. All described changes were found only in invasive melanomas and were the most prominent at invasion fronts. With the exception of focally thinning of collagen IV and laminin positivity of basal lamina, there was no altered extracellular matrix expression in melanomas in situ.

Conclusions: Our results demonstrated that the stromal changes in melanomas were specific and not present in nevi. Enhanced expression of tenascin may be related to proliferation and invasive growth in melanomas. Reduced expression of basal lamina components colagen IV and laminin around tumor cells may also be important in invasion. Stromal remodeling induced by melanoma was also characterized by a loss od CD34 positive fibrocytes. Considering that CD34 positive fibrocytes are antigen-presenting cells, their elimination may enable an invasive tumor to escape immune-surveillance.


22

PURE RED CELL APLASIA ASSOCIATED TO MALIGNANT THYMOMA

Amelia Gaman1, Camelia Dobrea2, G.Gaman1

1University of Medicine and Pharmacy Craiova, Romania 2Hemopathology Laboratory, Fundeni Clinic Institute Bucharest, Romania

We are presenting the case of a female, 61 years old, hospitalized in Clinic of

Hematology Craiova (Romania) with severe anemic syndrome(Hb=3,5g%)and crisis of angor pectoris. The patient presented sinusale bradycardia and deafening cardiac noises. Bone marrow smear showed normal granulocyte and megacaryocyte series and almost absence for erythroid serie. Bone marrow biopsy confirmed diagnosis of pure red cell aplasia showed a reduced erythroid serie (fig1). Virusologic exam were negative (absence of atc anti Parvovirus B19);Nezelof and Blackfan-Diamond syndromes were excluded. Thoracic X-ray and computer tomograph exam showed a large fore mediastinum with a good delimited tumour. Surgical intervention was done and macroscopic has been seen an ovoid tumour(10/6/6cm) whitish-pink, high consistency, alternance between whitish areas (adequately tumorale proliferation) and blackish-grey areas, with more callous zone adequate cholesterolic granulomas on section. Histopatology and immunochemistry established diagnosis of a high differentiated malignant thymoma. At the mediastinal tumour, there has been seen an epithelial tissue of a malignant proliferation, with lobular pattern, delimited by a thick fibroconnective bag (fig.2,3,4). Tumour cells present pan-citokeratina (fig.5) and Ki67/MIB1 nuclear factor of cell proliferation in almost 40% from tumour cells (fig,6). After six day from surgical intervention hemoglobin value was normal and lossed transfusional necessities. At this hour the patient receives chemotherapy for malignant thymoma. Particularity of the case was old age of apparition for malignant thymoma associated with pure red cell aplasia.


23

PRIMARY MEDIASTINAL LARGE B CELL LYMPHOMA INITIALLY PRESENTED WITH UPPER CAVA SYNDROME – CASE REPORT

Doina Butcovan, Doina Murarescu, Maria Sultana Mihailovici

Department of Pathology UMF „Gr T Popa” Iasi, Romania

A 21-year-old young woman was admitted in our hospital with left arm pain and parestesia, and left chest pain and progressive dyspnea. She was presented a significant weight loss, as well. A chest X ray and a chest computed tomography revealed a mediastinal tumor mass. She was succesfully treated by surgical removing of the tumoral mass and chemotherapy. The histological diagnosis of diffuse B cell lymphoma was established on the tumor specimen obtained through surgical biopsy and immunohistochemical analysis. This is a rare case of primary mediastinal large B cell lymphoma, showing abundant stromal sclerosis forming thick bands separating tumor clear cells into lobules of varying size, with initial presentation of symptoms related to superior vena caval syndrome.


24

HISTOLOGICAL CRITERIA IN DIFFERENTIATION OF MALIGNANT LYMPHOMAS FROM REACTIVE CHANGES OF LYMPH NODE

Maria-Sultana Mihailovici, M.Danciu, D. Ferariu, Doina Murarescu, Delia Ciobanu

Pathology Department, University of Medicine and Pharmacy “Gr. T. Popa”, Iasi, Romania

Reactive changes might be a problem, mimicking malignant lymphomas. In

these conditions, immunohistochemistry (IHC) is of real help for a correct diagnosis. Aims: The purpose of this study was to evaluate the importance of different

histological and IHC criteria in distinguishing reactive changes from incipient malignant lymphomas in lymph nodes.

Material and methods: The study group analyzed 794 lymph nodes biopsies. On paraffin-included tissues sections we performed current staining (HE, Gordon-Sweets) and IHC tests (CD45, CD45RO, CD20, CD1-4, FVIII, p53, BCL2, Ki67, CK cocktail).

Results: Following complete histological diagnosis and IHC characterization upon WHO 2001 classification, we found: 309 (39%) malignant lymphomas, 189 (24%) carcinomatous metastasis, 95 (12%) specific lymphadenitis, 189 (23.5%) reactive lymphadenopathy and 12 (1.5%) lymph node infarcts.

Conclusions: The main criterion in differentiation of non-specific follicular hyperplasia from follicular lymphoma is the bcl2 pattern. Differential diagnosis between follicular hyperplasia, follicular lymphoma and interfollicular Hodgkin lymphoma is a challenge, IHC being mandatory. Non-specific reactive hyperplasia in children can be a result of an immune response following infectious diseases. In necessary that clinical and serological data are integrated with histological aspects.


25

FETUS IN FETU: DIFFERENTIAL DIAGNOSIS OF AN OVARIAN MASS OF A PREGNANT WOMAN

C.I. Paulon, D.Graziani, G.P.Bulfamante1

1Unit of Human Pathology, D.M.C.O.-Medical School, University of Milan, Italy

Background: Fetus in fetu is a malformed parasitic monozygotic diamniotic twin that installs and grows inside the body of its partner. This pathology is rare and the incidence is 1 per 500 000 births. To date 11 cases of living children and 3 cases of living adults with fetus in fetu were published. Case Report: An ovarian mass was diagnosed by ultrasonography on first trimester in a 33-year-old at its first pregnancy. At term, a cesarean section was performed and the ovarian mass was removed. On the macroscopic pathologic examination, the partially encapsulated solid-cystic mass measured 7,5x5,5x5 and was composed of an elementary trunk partially covered with hair and two rudimentary limbs corresponding to an incompletely developed fetus. On cut sections, a soft vertebral axis, insufficiently calcified, was found. Microscopically, there were intestinal, cartilaginous, hemopoietic, endocrine and nervous tissues. Most of these cells were well-differentiated. The differential diagnosis of fetus in fetu should include teratoma (an accumulation of pluripotential cells in which there is neither organogenesis nor vertebral segmentation), acardic twin fetus (abnormal karyotype whereas the karyotype of fetus in fetu is normal and similar to his host’s), extra-uterine pregnancy (no evidence of amniotic sac, neither placenta in our case). At the moment molecular study of DNA profiling is expected to confirm the diagnosis. Conclusion: The diagnosis of fetus in fetu was made by pathology and attends the DNA-analysis confirmation.


26

NEW APPLICATIONS OF TISSUE MICROARRAY TECHNIQUE

A.M. Szasz, A.M.Tokes, G. Lotz, Z.S. Nemeth, J. Kulka

Semmelweis University, 2nd Department of Pathology, Budapest, Hungary

Introduction: There have always been approaches to improve our understanding of carcinomas when considering tumor heterogeneity. Targeted analysis of tumors may be performed with tissue microarrays (TMA). Still, the main resources of research are formalin-fixed, paraffin-embedded tissue blocks in pathological archives. TMA technique is a high-throughput method to investigate a large number of tissue samples cost-effectively and in a short time. Therefore, the use of tissue microarray in research is rapidly increasing.

Methods: In our department TMAs were used in the last 4 years to investigate protein expression characteristics of different types of breast carcinomas. Recently, we developed new applications which are based on TMAs.

Results: Not only immunohistochemistry can be performed on TMAs, but we adapted the fluorescent in situ hibridization technique to evaluate gene amplification in a safe manner. Targeted nucleic acid purification from a certain part of tumor could be performed with the use of laser capture microdissection. We may also use TMA technique to isolate RNAs or DNAs from punched cores of tumors. We compared our final data to those purified from whole tissue sections, and our results validate the use of the new technique provided the selection was carefully done.

Conclusion: Tissue microarray technique is a versatile and safe method to investigte tumors. It may be used to investigate protein expression, and additionally, to evaluate transcriptomes, and furthermore, to uncover information encoded in the DNA.


27

EFFECTS OF FIXATIVES AND FIXATION TIME ON TISSUE HISTOMORPHOLOGY AND mRNA PRESERVATION

Isabella Dotti1,2, Serena Bonin1,2, Ermanno Nardon1,2, Giorgio Stanta1,2

1Department of Clinical, Morphological and Technological Sciences, University of Trieste 2International Centre for Genetic Engineering and Biotechnology, Area Science Park, Trieste, Italy

Currently, formalin-fixed and paraffin-embedded tissues provide the most

abundant supply of archival material for morphological and molecular analyses. Although formalin is adapted to morphological examination of tissues, it induces chemical modifications and fragmentation on the RNA, affecting the quantification of gene expression. Recently, new less toxic fixation procedures have been introduced to overcome limitations of formalin fixation.

In the present study we compared the conventional formalin fixation with that performed with two alcohol-based fixatives, the toxic home-made methacarnoy, and the non-toxic commercial FineFIX. We evaluated the effects of fixation time of these fixatives on both tissue morphology and mRNA preservation using unfixed and 1-24h-fixed colon cancer cultured cells and unfixed and 24-48h-fixed colon cancer surgical specimens. Morphology was observed by conventional haematoxylin and eosin staining, while mRNA preservation in the different solvents was assessed by evaluating total RNA quantity, integrity and target mRNA reliability for subsequent transcriptional analysis.

We found that FineFIX fixation ensures the best combination of tissue component preservation and mRNA accessibility for expression studies. Tissue morphology obtained using Finefix fixation was the most similar to that observed with formalin fixation, while methacarnoy fixation gave no comparable results with conventional formalin treatment. When fixed and unfixed cells were compared, RNA quantity in FineFIX-fixed cells was the most similar to that recovered from unfixed ones. Moreover, RNA integrity in cells and tissues fixed with both alcoholic solvents was comparable to that found in unfixed ones and the expression levels of differently transcribed target genes were kept constant during all the fixation times.

We concluded that FineFIX fixative could represent in the future an ideal substitute of formalin and methacarnoy in routinary tissue fixation for its promising applicability in both morphologic and gene expression studies.


28

A NEW METHOD TO ISOLATE AND IN VITRO EXPAND CANCER STEM CELLS FROM HUMAN GLIOMAS

Musiello Daniela1, Gallelli Annarita1, Puppato Elisa1, Toffoletto Barbara1, Ius

Tamara2, Vindigni Marco2, Laura Mariuzzi1, Nicoletta Finato1, Beltrami Antonio Paolo1, Cesselli Daniela1, Skrap Miran2, Beltrami Carlo Alberto1

1University of Udine, Udine, Italy 2Neurosurgery department, Azienda Ospedaliero-Universitaria , Udine, Italy

Background: The possibility to isolate and expand in vitro cancer cell with

stem cell properties from human gliomas could open new perspectives in “patient tailored” therapies aimed at targeting specifically this rare population of cells, possibly responsible for recurrences and drug resistance.

Aims: to develop a methodology alternative to in vitro neurosphere formation or CD133 selection, capable of isolating and rapidly expanding a population of cells with stem cell features, both from low- (I, II) and high- grade (glioblastoma multiforme, GBM) human glial tumors.

Methods and results: 0,2-2 g in weight, non necrotic fragments of low- (n=20) and high- (n=21) grade lesions were obtained by surgical resection at the Neurosurgical Unit of the hospital of Udine (Italy). Neoplasms were mechanically-enzimatically dissociated and cells were cultured in a medium optimized in our laboratory for the growth of Multipotent Adult Stem Cells. Cell lines were tested for growth kinetics, stemness and in vitro tumorigenicity.

Results: Cell lines were obtained from all the samples with high efficiency and reproducibility. Cell population doubling time of glioma cell lines (calculated at passage 3) was 25 hours, allowing obtaining 106 cells in less than 20 days. About stemness, CSCs expressed a typical mesenchymal immunophenotype, being usually CD13high/CD59high/CD49bhigh/CD90high/CD73low/CD44low/HLA-ABClow/CD29low/CD105low/KDRlow/CD49alow/CD49dlow/CD14neg/CD45neg/CD38neg/HLA-DRneg/CD133neg/CD117neg/CD34neg. Further, the average percentages of Oct-4 and Nanog, pluripotent state-specific transcription factors, were 86% and 91%, respectively, and cell lines displayed in vitro multipotency.

Regarding tumorigenicity, the majority of CSCs displayed aberrant growth features such as loss of contact inhibition (transformation foci assay) and adhesion independent growth (soft agar assay). Importantly, CSCs at passage 3 demonstrated a 160 fold enrichment in soft agar colony formation capability with respect to uncultured cells.

Conclusions: We developed a methodology, alternative to in vitro neurosphere formation or CD133 selection, for the rapid expansion, from low- and high-grade gliomas, of cells that retain stem cell features and aberrant growth properties. The high reproducibility and yield of the method would allow to obtain a sufficient number of cells, even for high throughput approaches and drug screening in a short period of time.


29

PROTEOMICS OF STEM CELL LINES OBTAINED FROM LOW- AND HIGH-GRADE HUMAN ASTROCYTOMAS

Gallelli Annarita1, Odreman Federico2, Musiello Daniela1, Beltrami Antonio Paolo1, Cesselli Daniela1, Ius Tamara3, Vindigni Marco3, Nicoletta Finato1, Laura Mariuzzi1,

Skrap Miran3, Vindigni Alessandro2, Beltrami Carlo Alberto1

1Univeristy of Udine, Udine, Italy 2International Centre for Genetic Engineering and Biotechnology, Trieste, Italy 3Neurosurgery department, Azienda Ospedaliero-Universitaria, Udine, Italy

In a previous study, the protein expression profiles of low- and high-grade

human brain astrocytoma tissues was compared by two-dimensional gel electrophoresis (Odreman et al., J. Proteome Res. 2005). In this way, a number of proteins differentially expressed between the low- and high-grade tumors were identified. These proteins could be potentially used as novel moleculars biomarkers for an accurate classification of the grade of astrocytomas. In this study, we extended the same analysis to cell lines obtained from the low- and high-grade tumor samples.

For this reason, main objectives of our study were: 1. Optimize a method to isolate and expand in vitro cancer stem cell (CSC) population from low-grade and high-grade human brain astrocytomas (named, respectively, l-CSCs and h-CSCs); 2. Demonstrate in vitro l-CSC and h-CSC stemness and tumorigenicity; 3. Compare the protein expression profiles of l-CSCs and hCSCs.

Low- (n=17) and high- (n=19) grade astrocytomas were obtained by surgical resection at the Neurosurgical unit of the Azienda Ospedaliero-Universitaria of Udine. Neoplastic tissues were mechanically-enzimatically dissociated and the single cell suspension cultured in a medium selective for the growth of Multipotent Adult Stem Cells. Cultured cell lines showed a mesenchymal immunophenotype, being usually CD13high CD59high CD49bhigh CD90high CD73int CD44int HLA-ABCint CD29int CD105int KDRint CD49aint CD49dint CD14neg CD45neg CD38neg HLA-DRneg CD133neg CD117neg CD34neg, and expressed Oct-4, REX1 and NANOG, pluripotent state-specific transcription factors. Tumorigenicity of expanded cell lines was confirmed by the demonstration of anchorage independence growth (soft agar assay) and loss of contact inhibition (sphere formation assay, transformation foci assay, and growth curve analysis).

The proteomic studies performed to compare h-CSCs and l-CSCs allowed us to identify a number of proteins that are more abundant either in the low- or in the high-grade tumors and were not detected in the previous study using the brain tissues.


30

HIGH THROUGH-PUT MOLECULAR ANALYSIS OF MUTATIONAL STATUS OF EGFR AND K-RAS IN ALCOHOL FIXED FINE-NEEDLE

ASPIRATE BIOPSY

Lorenzo Daniele, Sofia Asioli, Sara Mariani, Donatella Pacchioni, Gianni Bussolati

Department of Biomedical Sciences and Human Oncology, University of Turin, Italy

Introduction: In the majority of the cases, non-small cell lung cancer (NSCLC) and pancreatic neoplasms present in advanced stage and EUS or CT-guided fine-needle aspirate biopsy (FNAB) represents the gold standard method for diagnosis. Ethanol fixation, followed by paraffin embedding, is the currently processing to fix cytological samples. Besides, ethanol fixation seems to preserve the integrity of bio-molecules as proteins and nucleic acids. In fact, it is extremely important to test the feasibility and reliability of specific DNA analyses on ethanol-fixed cytological specimens to allow “tailor therapy” in these advanced neoplastic lesions. For this purpose, we decided to analyse the mutational status of both EGFR and K-Ras on cytological FNA samples of both NSCLC and pancreatic neoplasms.

Material and methods: We investigated FNA specimens from 6 cases of NSCLC and 2 cases pancreatic tumours. All samples were ethanol-fixed and paraffin-embedded. Six sections 7 ��deep were submitted to DNA extraction after conventional steps of paraffin removing. DNA obtained from lung and pancreatic lesions were PCR amplified for EGFR and K-Ras, respectively. EGFR mutational status was obtained after PCR by nucleotide sequencing, while specific gene amplifications were checked by FISH. K-Ras mutational status was investigated on codon 12 by restriction endonuclease-mediated selective PCR (REMS-PCR), and confirmed by nucleotide sequencing.

Results: DNA was successfully extracted in all cytological specimens analysed. Mutational analyses identified specific EGFR mutations in 2 out of 6 NSCLS cases. FISH analyses demonstrated that those mutated cases were also polysomic for EGFR. Lastly, K-Ras were found mutated in 2 out of 2 cases of pancreatic tumours.

Conclusion: We found that ethanol fixation of cytological specimens is a useful method to preserve nucleic acids for delay DNA analyses, i.e. PCR, sequencing, endonuclease restrictions and FISH. In particular, early mutational status assessment of genes as EGFR and K-Ras is of extremely interest for diagnosis, prognosis and prediction of recurrence/response to therapy.


31

CRITICALS IN THE PRE-ANALYTIC STEPS OF HISTOPATHOLOGICAL EXAMINATION IN HUMAN NEOPLASIAS

Giuseppe D’Armento, Luca Molinaro, Sara Mariani, Gianni Bussolati

Department of Biomedical Sciences and Human Oncology, University of Turin, Italy

Introduction: Pre-analytical treatment of the samples explanted by surgery is

a still debated question, in particular it is difficult to obtain information about: pre-fixation time, expectation in the fixative and temperature of conservation. Therefore, pre-analytical time interval (PATI) is pivotal in the standardization of the histo-pathological examination for different points of view as well as molecular (RT-PCR), morphological (H&E) and immunophenotipic (IHC). It is needful to consider: a) In vivo step: specimen remains into non-permissive conditions for a variable time (at least 30 mins). Thus molecular targets can be rapidly modified. b) Transfert step: transport time can vary from few minutes to several hours. c) Fixation before embedding step: prolonged fixation, could invalidate IHC and molecular tests. Preservation and integrity of nucleic acids (expecially RNA) directly correlates with the efficacy of previous steps.

Materials and methods: Samples from surgery (colon, thyroid, lung, breast) were stored under vacuum (U.V.) or ambient pressure (A.P.) and under different temperature conditions [room temperature(RT/18-22°C), 4°C, -20°C] for different times (1 day-7days). These specimens were processed in parallel for morphology (H&E), epitope retrieval (IHC) and molecular approach (RNA analysis). Further we performed the same test on laboratory rats.

Results: We observed that both integrity of nucleic acids and morphology were compromised in the specimens stored U.V. conditions at RT within 2 days. On the other hand, both H&E and IHC staining were adequate and well preserved in samples stored U.V. at 4°C (colon, breast, lung). Further, rRNA resulted well preserved in the specimens stored U.V. at -20°C and 4°C within 7 days (colon, thyroid). Reverted RNA produced amplicons until 716 nt (CK20 in colon). In rat specimens rRNA showed a partial degradation within 24h U.V. at RT, while it was full preserved in the specimen kept U.V. at 4°C.

Conclusions: U.V. condition make feasible prolonged conservation of RNA/DNA in surgical specimens, promoting their cooling, avoiding de-idratation. Transfer and preservation of surgical specimens, plays a central role in the pathological examination of the tissue. In conclusion, the application of standard criteria in the management of surgical specimens could contribute to an improvement in diagnostic and research responses.


32

VALUE OF FLUORESCENCE IN SITU HYBRIDIZATION IN TISSUE MICROARRAYS

Rosa Noguera, Isidro Machado, Marta Piqueras, Eva Villamón, Antonio Llombart-

Bosch, Samuel Navarro

Department of Pathology, Medical School, University of Valencia, Spain

The application of Fluorescence in Situ Hybridization (FISH) in Tissue MicroArrays (TMA) has been shown to provide an effective genetic evaluation tool. This high-throughput, TMA-based, dual-color breakapart interphase FISH assay for the detection of gene rearrangements, gains and/or deletion as well as for screening the gene amplifications is of great value in our pathology department. Interphase FISH on paraffin sections permits direct correlation of the genetic event with the morphologic features. Inclusion of normal control tissue samples with tumor samples in a TMA format serves as an ideal control for establishing cutoff percentages for deletion or gain of a chromosomal locus in tumor samples. Furthermore, it is is a useful method for determining the clinical value of genetic markers in large groups of samples and for the analysis of tumor cell heterogeneity, since it allows genetic alterations present in a low percentage of tumour cells to be found. It can be used for validation of phenotypic/genotypic studies and genetic stability related to gene aberrations in primary and xenografted tumors. The elaboration of TMA and its use in FISH techniques optimizes the teaching of our students in practical investigation within the field of Molecular Pathology.

However, it is limited by the inability to optimize tissue preparation and hybridization conditions to suit individual tissue cores and problems associated with truncated nuclei in paraffin sections. Such problems can be minimized by the application of an efficient pretreatment protocol and by optimizing the enzyme concentrations and digestion times to achieve improved FISH signals and high hybridization rate. In addition, our simplified scoring system enables us to group the cells, based on their pattern of FISH signals, into distinct categories in normal tissues and tumor samples, and differentiate true chromosomal aberrations from false positives. We established a scoring scheme that can be used for standardization of genetic studies.

Studies by FISH assays in TMA of paraffin embedded tumors samples are an interesting tool to continue completing our molecular knowledge about complex cancer disease and to use them for validation studies. Supported with grant from EU IMPACTS 037211 and from Instituto Carlos III (Acción transversal del Cancer 20080143, RETICS RD06/2010102)


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ANALYSIS OF GENETIC MARKERS IN PEDIATRIC SOLID TUMORS USING FISH IN TMA

Marta Piqueras, Isidro Machado, Eva Villamón, Antonio Llombart-Bosch, Samuel

Navarro, Rosa Noguera

Department of Pathology, Medical School, University of Valencia, Spain

Our main purpose in the present study is to analyse specific genetic markers of different formalin-fixed paraffin-embedded tissue of pediatric solid tumours included in TMAs to confirm diagnosis, to compare the genetic characteristics between original and xenografted tumours and to determine the presence and prognostic value of some genetic markers.

We used interphase FISH assay in TMA for the detection of SYT disruption in 97 small round/spindle cell tumours with a presumptive diagnosis of synovial sarcoma. True positive SYT rearrangement was found in 41 cases and heterozygous deletion of 9p21 region (p16) in 28 samples. In addition, the translocation t(X;18) was identified in the 8 primary tumours and in their xenografted (XT) passages, which were followed for several generations, included in 2 TMAs. No significant differences were detected among them despite wide variations in xenotransplantation time.

The phenotypic and genetic heterogeneity of neuroblastic tumors is the most important characteristic of these neoplasms. 139 samples were included in different TMAs and the status of MYCN gene, the genetic prognostic factor of excellence, and two frequent structural alterations (1p36 loss and 17q gain) were analyzed by FISH. Amplification of MYCN gene was detected in 49 cases while 12 showed MYCN gain. 65 samples displayed deletion in 1p36, while 6 showed imbalance. Gain of 17q was present in 81 cases. For the validation of TMA, the results were compared with those obtained previously by FISH on imprints or by PCR/LOH. The diagnosis was concordant in all cases.

The use of FISH in formalin-fixed paraffin-embedded tissue assembled in TMAs saves time, probes and facilities the study and genetic characterization tumour cells. The application of this method could prove useful in the high-throughput analysis of cell heterogeneity in other genetic aberrations associated to neoplasms. Supported with grant from EC IMPACTS 037211 and Instituto Carlos III: Acción transversal del Cáncer 20080143 and RETICS RD06/2010102.


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SIMULTANEOUS CARCINOMAS OF THE OVARY AND ENDOMETRIUM

Cornelia Amalinei, Raluca Balan, Laurette Cozma, Ioana Buda, Irina Draga Caruntu

Normal and Pathological Morphology Department, University of Medicine and Pharmacy “Gr. T. Popa”

Iasi, Romania

Background: Simultaneous tumors of the ovaries and endometrium are relatively frequent. Cancers developing concomitantly in these locations may represent metastasis from the endometrium to the ovary, metastasis from the ovary to the endometrium or independent primary tumors, as the surface epithelium of the ovary has the same embryologic derivation as the Müllerian duct and therefore a given carcinogenic stimulus may produce similar epithelial proliferations in two separate sites of a single anatomic region. DNA flow cytometric indexes, LOH, gene mutations and clonal X-inactivation analyses are useful in the distinction of independent primaries from metastatic carcinomas when the diagnosis does not rely exclusively on a single molecular result, considering tumor progression and tumor heterogeneity.

Design: We analyzed 21 cases from our files, diagnosed with simultaneous endometrial and ovarian carcinomas. Routine histological technique was followed, in selected cases, by PAS, Alcian blue, and immunohistochemistry for Ck 7, EMA, vimentin, ER, PR, beta-catenin, PTEN, Cyclin D1, Bcl-2, and HER-2.

Results: The following criteria were used in order to discriminate primary versus metastasis: histologic pattern, size, myometrial invasion, direct extension into adnexa or from the ovary predominantly into the outer wall of the uterus, endometrial hyperplasia, vascular space invasion, unilateral/ bilateral and/or multinodular ovarian tumors, hilar/parenchymal ovarian location, surface implants, ovarian endometriosis, and spread to another organ. In difficult cases, comparative analysis of the immunhistochemical profiles of the two neoplasms and ultimately patient follow-up were useful.

Conclusion: The distinction between primary and metastasis in simultaneous ovarian and endometrial carcinomas is important because prognosis and treatment is different. Due to tumor progression and heterogeneity, gene mutations have to be interpreted in the light of conventional clinicopathological criteria. The overall survival of patients diagnosed with simultaneous carcinomas of the ovary and endometrium suggested multifocal rather than metastatic disease in about a third of cases, and metastases from the endometrium to the ovary occurred more often than the reverse.


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INTERFERON EFFECTS ABOUT MORPHOLOGICAL RED BLOOD CELLS IN PATIENTS WITH CHRONIC HEPATITIS C

Adriana Bold1, Gabriela Iliescu2, Garofita Mateescu1, Viorel Biciusca2

1University of Medicine and Pharmacy Craiova, Romania 2Emergency County Hospital Craiova, Romania

Introduction: Best current treatment for chronic hepatitis C (CHC), the

combination between peginterferon (PegIFN) and ribavirin (RBV), is potentially dose-limited by hematological side-effects. Mixed anemia is the most common hematological disorder. Hematological disorders are reversible after dose reduction or discontinuation of treatment.

Objectives: In our study we tried to achieve data about anemia mechanisms in patients with CHC under antiviral interferon-based treatment in order to assess new supportive therapeutic options for the hematological disorders.

Methods: For 22 patients with CHC treated with standard doses of combination between PegIFN and RBV we performed complete blood counts and morphological exam of peripheric blood cells, reticulocytes count, Coombs test.

Results: In 17 patients we recorded various degrees of anemia and hemoglobin decreased with a mean of 3,5 g/dl from the baseline. Over 93% of patients presented morphological disorders of the erythrocytes: anizocytosis, poikylocytosis from the first month of treatment. In 1 patient we noted positive test Coombs and microcytosis. 6 patients had microcytosis and 3 had macrocytosis. The reticulocytes count showed low values in 10 patients.

Conclusions: The most common and frequent complication of interferon-based treatment in patients with CHC is anemia. Investigating the red blood cells morphology it is possible to recognize the mechanisms in order to use efficient support therapies for maintaining standard doses and to ensure the treatment outcomes.


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EBV VIRUS DETECTION IN GASTROESOPHAGEAL JUNCTION ADENOCARCINOMAS – AN IMMUNOHISTOCHEMICAL AND IN SITU

HYBRIDIZATION STUDY Cristina Iosif1, Alina Georgescu1, Alina Nicolae1, Maria Neagu1, Rodica Birla2, Narcis

Copca2, Silviu Constantinoiu2, Carmen Ardeleanu1

1“Victor Babes” National Institute of Pathology, Bucharest, Romania 2Esophageal Surgery Department, “St. Mary” Hospital, Bucharest, Romania

Background: Ebstein-Barr Virus is a human herpes-virus which has a well-

established association with endemic Burkitt lymphoma, nasopharyngeal carcinoma, some B lymphomas in immunodeficiency, and high proportion of lymhoepithelioma-like gastric carcinoma. There are few dates about its implication in the pathogenesis of other digestive carcinomas like gastroesophageal junction (GEJ) adenocarcinomas. The aim of our study was to evaluate the association of EBV infection with GEJ adenocarcinomas.

Methods: We studied tumor specimens from 55 patients with GEJ adenocarcinomas (41 surgically resected and 14 unresectable); patient characteristics were median age (66 years), sex (85.5% male and 16% female), stage disease (1 patient with stage I disease, 1 patient with stage II disease, 38 patients with stage III disease and 15 patients with stage IV disease). These specimens were tested for EBV using immunohistochemistry (IHC) and in situ hybridization (ISH). The IHC bistadial indirect technique was performed using producer’s development kit for EBV latent membrane protein-1 (LMP-1) (Novocastra). ISH has been done using a detection kit from Novocastra for EBER transcripts of EBV. These cases were also IHC tested for EGFR (Sigma), and Ki67 (Dako).

Results: IHC reaction for LMP-1 was positive in tumoral cells in 10/55 cases (18,18%). Expression of EBERs in GEJ adenocarcinomas was further confirmed with ISH in 5/10 tested cases (50%). None of the benign portion of the cases was positive for EBERs. EGFR was expressed in 18/55 adenocarcinomas (32,72%) and was correlated with advanced stage of the disease (16/38 stage III disease, p=0.02 and 2/15 stage IV disease, p=0.053) and with presence of visceral metastasis 2/15, p=0.02). EGFR was expressed only in 3/5 ISH positive for EBV cases; all of them were stage III. The proliferation index Ki67 <10% was correlated with resectable status.

Conclusions: Our results indicate that EBV could be associated with GEJ adenocarcinomas, but the clinical role of EBV in these neoplasias remains to be determinate. EGFR could be considered an independent prognostic factor associated with poor outcome in GEJ adenocarcinoma and also a molecular target for therapy. To determine the correlations between EBV infection and tumor grading of GEJ adenocarcinomas, considering EBV as a possible poor prognosis factor, further studies on larger series will be necessary.


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TUMOROUS CALCINOSIS - CASE REPORT

Košuta Iva, Gamulin Ozren, Bogdanović Iva, Bilić Ranko, Kavur Lovro, Batelja Lovorka

Institute of Pathology Medical Faculty University of Zagreb, Croatia

Tumorous calcinosis is a rare disorder of unknown etiology in which nodular masses of calcium-containing material are deposited in the proximity of a joint in otherwise healthy patients. Soft tissues around the hip and elbow are most commonly affected, and multiple appearances are not uncommon. The masses show a tendency towards rapid growth, with no cutaneous or visceral involvement. The disorder occurs most frequently in females in the first and second decade of life and a familial tendency is often observed. Local trauma, or a metabolic disorder are possibly the causes. The masses are usually surrounded by a dense fibrous capsule with fibrous trebeculae transversing their bulk. The cystic spaces filled with calcium-containing material are lined with two main cell types, mononuclear and multinuclear. Non-specific inflammatory reaction may be present in the stroma. Case report: A 32 year old female presented with swelling of the proximal interphalangeal joint of the right index finger. She reported a history of more than one year of swelling, followed by mild pain. Radiologically opacities and calcifications in the joint capsule were noted. Laboratory data (e.g. BSR, BCC) were in normal range. A biopsy revealed a highly cellular synovial membrane with a diffuse lymphocytic infiltration and surface fibrin deposits. Multiple foci of cristalyne deposits were present both in the stromal tissue and in the vacuoles of multinuclear cells. Standard HE staining failed to show birefringence. Von Kosa stain demonstrated strong calcium presence in deposits. According to clinical presentation and histological features the lesion was diagnosed as Tumoral calcinosis. The differential diagnosis of arthropathies with crystalline material deposition primarily includes pseudogout and gout. The main crystalline form in gout is monosodium urate, and pseudogout is characterized by calcium pyrophosphate dihydrate (CPPD) deposits, in contrast to basic calcium phosphate depositions in tumorous calcinosis. In order to elucidate the content of deposits, further analysis of the tissue was performed using in situ methods. Proton induced X-ray emission (PIXE) revealed a calcium to phosphate ratio indicative of calcium phosphate, not CPPD. The crystalline structure was further evaluated using Raman spectroscopy, confirming it to be basic calcium phosphate. The application of additional in situ methods for deposit identification can foster their correct characterization on routinely processed tissue, enabling introduction of adequate therapy.


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IMMUNOHISTOLOGICAL STUDY OF THE SECRETING-EXCRETING PANCREATIC COMPONENT IN THE CHRONIC PANCREATITIS

Garofiţa Mateescu1, Adriana Bold1, Liliana Stanca1, Maria Manolea1, Eugen

Petcu2

1University of Medicine and Pharmacy Craiova, Romania 2Griffith University, Australia

Introduction: Important problem of public health by its spontaneous evolution,

often unfavourable, and by its many complications the chronic pancreatitis, and particularly the installation, in its context, of the modifications of the components of the exocrine pancreas, by the simultaneous pathological implication (functional and/or morphological), represented the object of this study.

Materials and method: The study was carried out on 35 parts of pancreas taken after the necropsies carried out in the laboratory of Pathological Anatomy and Cytological Diagnosis of the Municipal Hospital “Filantropia” of Craiova. The treatment of these parts was carried out in the Laboratory of Immunohistochemistry of the University of Medicine and Pharmacy Craiova. The immunohistochemical method used in paper was the complex method avidin-biotin (ABC); the markers used were: the nestine, collagen IV, the laminine, the TGF beta (Transforming Growth Factor beta), the actine.

Results and discussions: The examination of the preparations showed a positivity of the cells with the CK8 in the cells in the small pancreatic ducts and also in those of the large ducts into six of the studied cases. In exchange, in the endocrine pancreas, we met in all the cases CK8-positive cells isolated or in small cores, in the majority of the islands.

The Collagen IV was positive in all the cases, prevailing the distribution in narrow bands intra-acinar with more of dense collagen fibres (57%), followed by the dense collagen fibres distribution with rarer narrow fibres intra-acinar (10%).

The majority of the chronic cases of pancreatitis (11 cases) presented a pattern of continuous distribution for the collagen IV in the basal membrane around the residual ducts that is allowing the realization of a differential diagnostic with the pancreatic adenocarcinoma.

The laminine presents in the stroma (15,67%), in the acinar cells (44,33%) but also in the spindle-shaped cells (60%), was positive only in six cases, which can be due to a formol fixing some more prolonged or to the pancreatic modifications postmortem. It was positive only in five cases.

The actine was positive in the vascular walls and in the stroma in all the cases of chronic pancreatitis and in the vascular walls in three cases of acute pancreatitis. TGF-beta was positive in 9 cases in the spangled cells. On our sections, it was not very positive in the adjacent acinar cells with the surfaces of fibrosis, suggesting a paracrine secretion of TGF-beta. The immunomarking with TGF-beta was positive in the cytoplasm of the cells of the canaliculaire epithelium and not very positive in the acinar epithelium. Conclusions: In conclusion of the study carried out we can say that:

• The transformer growth factor beta (TGF-beta) achieved the function of regulating key of the genesis of the extracellular matrix and the myofibroblastic


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proliferation; TGF-beta played a big role in the fibrogenesis by the stimulation of the formation of the collagen deposits as much as by the inhibition of the metalloproteinase MMP3 and MMP9, being known that those have a role in the degradation of collagen;

• The immunomarquage with laminine did not give us satisfactions because it is an antibody more sensitive to fixing and that’s why we used collagen IV more;

• The immunomarquage with collagen IV was positive in all the cases, the majority of the cases presenting a uniform distribution of collagen IV in the basal membrane around the residual ducts;

• The expression of the CK8 in the cells of the ductal epithelium suggests a possible role in the neogenesis of the insular cells, because it is believed that the birth of new endocrine cells in the mature pancreas has place by the interlude of the ductal cells.

We consider that the immunohistochemical study we carried out was important in the decoding of the lawsuit of fibrogenesis, of the “behaviour” of the pancreatic exocrine cellularity, in the context of the chronic pancreatitis.


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P53 EXPRESSION IN BASAL CELL CARCINOMAS FROM PHOTOEXPOSED AREAS OF HEAD AND NECK REGION

Claudia Mateoiu1, Maria Comanescu2, Claudia Georgescu1, Florin Bogdan3

1Departament of Pathology, Clinical County Hospital of Emergency, Craiova, Romania 2Departament of Pathology, Clinical Emergency Hospital Bucharest, Romania 3Departament of Research, University of Medicine and Pharmacy, Craiova, Romania BASAL CELL carcinomas (BCCs) are the most common cancer in humans.

Genetic and environmental factors contribute to their development. The major causative factor is UV radiation (UVR), which probably explains why the incidence of BCC in Queensland, Australia, is the highest reported for any cancer. Such epidermal p53 clones are common in chronically sun-exposed skin and have been suggested to play a role in skin cancer development.As point mutations in the coding sequence of the p53 tumor suppressor gene have been implicated in the progression of many human tumors, we studied the expression of p53 protein on this neoplasia.

Purpose: The purpose is to investigate whether aggressive basal cell carcinoma (BCC) differ from nonaggressive BCC with respect to the p53 mutation

spectrum and whether specific mutations can serve as prognostic indicators of tumor aggressiveness.

Experimental Design: An immunohistochemical study for p53 was performed in a series of 47 basal cell carcinomas (BCC) from photoexposed areas of head and neck region, comparing the findings with follow-up.We tested immunohistochemically the positivity for p53 protein on 23 cases of morphologically ″non aggressive″ BCC (BCC1) and on 24 ″aggressive″ BCC (BCC2), all with one or more relapses.

Results: Results were related to clinico-pathological and follow-up data. All but one BCC2 were found positive for p53 protein. Conversely, only 3 cases of BCC1 exhibited low immunoreactivity for p53 protein, with high statistical differences between the two groups. No correlation was found between the immunoreactivity, age of patients, and site of the lesions. The availability of immunohistochemistry and the relatively easy interpretation of the results make screening for p53 protein a possibly useful tool in the prognostic evaluation of BCC.

Conclusions: Our results indicate that UV radiation is responsible for the induction of p53 mutations and perhaps for the initiation of both aggressive and nonaggressive BCCs. Although some differences in p53 mutation frequency, types of mutation, and hot spots were seen between aggressive and nonaggressive BCCs these factors do not constitute as clear-cut diagnostic or prognostic indicators of tumor aggressiveness. Tumor aggressiveness may be attributable to other genetic changes or events that occur during tumor progression.


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PRELIMINARY STUDY OF BIPOLAR HIP PROSTHESIS INFULENCE ON ACETABULAR ROOF MORPHOLOGY

Iancu Emil Pleşea1, Mirela Ghiluşi3, Dan Nelu Anuşca2, Oltin Tiberiu Pop1, Valentin

Dascălu4, Claudia Valentina Georgescu3

1Department of Pathology 2Department of Orthopaedics and Traumatology, University of Medicine and Pharmacy 3Department of Pathology and Cytology 4Department of Orthopaedics and Traumatology, Emergency County Hospital, Craiova, Romania

Introduction. Aims: The status of the periprosthetic bone capital is an important element in the assessment of the total hip prosthesis revision. Periprosthetic bone changes following hip arthroplasty are yet to be completely described.The authors made a preliminary assessment of morphological changes of acetabular roof (AR) induced by the interaction with the bipolar hip prosthesis (BHP). Material and Method: The material consisted of acetabular bone tissue sampled from 3 cases with revision of a BHP. The finite element method (FEM) on the plane models was used to determine AR stress state and pressure area (PA). Two calculus models were developed considering a frontal section (FS) and a sagital one (SS) through the upper part of a bipolar prosthesis and the surrounding zone of the AR. Numerical simulations were performed for both one (OLSP) and two legs (TLSP) standing positions for both models. Main stress and equivalent stress fields distribution in the AR were obtained after processing of calculus results and the corresponding curves were plotted using a special post-processing program. Then, for each case were sampled: one bone fragment from the central zone of the maximal PA of the AR and one bone fragment from each of the medial, anterior and posterior aspects outside the maximal PA, at a distance of 1 cm. Bone tissue samples were fixed and decalcified in Duboscq-Brazil solution, embedded in paraffin wax, then stained with hematoxylin-eosin, van Gieson thrichrome and Masson thrichrome. Results: PA developed on AR by BHP components varied from an an ovoid shape, with the greater diameter in the SS shifted towards the anterior aspect of the AR and with values decreasing from lateral to medial, in OLSP to an antero-posterior narrow strip, near the acetabular rim with decreasing values from posterior to anterior in TLSP. Morphological aspects observed in maximal PA consisted of areas of bone necrosis in direct aposition with the prosthetic elements, mineralised lamellae alternating with poorly mineralized ones and focal presence of bone lacunae occupied by loose vascular connective tissue in the cortical layer and areas with thin bone lamellae circumscribing large lacunar spaces occupied by a small amount of bone marrow in the trabecular area. A large cellular population consisting of osteoblasts and osteoclasts, areas with a collagen network in which fibers gradually showed a lamellar disposition around wide channels filled with vascular connective tissue and lined by osteoblasts producing this osteoid material, as well as areas with gradual mineralisation of collagen lamellae, simultaneous with the narrowing of the vascular connective channels and shaping of new haversian systems were also observed. Outside the maximal PA, the bone structure showed characteristics of normal bone in both compact and trabecular components. Conclusions: Acetabular bone is hosting degenerative and regenerative changes seen in both compact and trabecular components but only inside the maximal PA of the AR. Our preliminary morphological study revealed the existence of an adaptation effort to the mechanical stress materialised through a dinamic process of bone remodelation in the maximal PA.


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QUANTITATIVE ASSESSEMENT OF INTRALOBULAR COMPONENTS IN AGEING TESTIS

Iancu Emil Plesea1, Stelian Danut Enache4, Petrica Badea2, Oltin Tiberiu Pop1, Mirela

Ghilusi4, Carmen Florina Popescu4, Bogdan Stanoiu3, Raluca Ciurea1

1Department of Pathology 2Department of Informatics 3Department of Cellular Biology, University of Medicine and Pharmacy 4Department of Pathology, Emergency County Universitary Hospital; Craiova, Romania

Background, Aims: The authors made a preliminary study of possible correlations between the percentage occupied by intralobullar stromal component, mean diameter of seminiferous tubules (ST-MD) and ageing. Material and Methods: The studied material consisted of surgical samples of testicular tissue from 192 cases with orchiectomy for prostate adenocarcinoma. Tissue samples were processed by the classical histological technique (neutral buffered formalin fixing and paraffin embedding) and stained, on serial slides, with haematoxilin eosin, trichrome Goldner silver staining following Gömöri technique and immunomarking for Collagen IV (CIV). Images were acquired and measurements were performed with a specialized software for image analysis after previous calibration. The assessed parameters were: intralobullar stroma/parenchyma ratio (IL-S/P-R) and ST-MD. For IL-S/P-R determinations, 10 rectangular areas were randomly selected with X20 objective for each case. For ST-MD determinations, 30 tubules were randomly selected, with X40 objective, for each case and greatest diameter (D) and smallest diameter (d) were determined for each tubule. Mean IL-S/P-R (M-IL-S/P-R) and Mean ST-MD were calculated for each case and for each age group. Regression line (RL), Slope (m) and Significance test for Slope ("p") were calculated in order to assess the correlation of each of two parameters with ageing. Results: Changes of intralobular septa (IS) varied from simple linear fibrillary assemblings to marked condensations which obviously enlarged the septa, dispersed especially toward peripheral lobular areas. Mean IL-S/P-R determined for the entire group, was 26,7% with limits of mean values/age group between 11% and 43% and individual variations between 2% (- 24,7) and 49% (+ 22,3), values situated in the range described in the literature for healthy adult testis [Trainer 1997]. RL showed a discrete decreasing trend with ageing (m = - 0,01) with an statistical correlation (SC) (“p” = 0,018).Mean ST-MD determined for the entire group, was 108,8 µm with limits of mean values/age group between 96,4 µm and 120,4 µm and individual variations between 78,9 µm (- 29,9) and 137,2 µm (+ 28,4), value representing 54% of mean value described in the literature but for young adults [Obafunwa et al 1993]. RL showed a discrete increasing trend with ageing (m = + 0,82) but without an obvious statistical correlation (SC) (“p” = 0,24). Conclusions: Our preliminary measurements and observations showed on one hand that changes of IS have, firstly, a widely variable intensity both individually and in each age group and, secondly, they have no significant dispersion, as quantitative determinations showed, and extensive trend which could be related to ageing. On the other hand, the somehow stationary trend and wide variability of ST-MD values are, probably, the consequence of unequal involution of seminal epithelium both individually and in each age group. References: Obafunwa JO, Elesha SO, Odunjo EO - Testicular morphometric studies in Nigerian males. Afr J Med Med Sci; 22 (1): 39-44, 1993 Trainer T.D. - Testis and excretory duct system. In: Histology for Pathologists, second edition, Sternberg S.S., (ed.), Lippincot-Raven Publishers, Philadelphia, p.p. 1019-35, 1997


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CORRELATIONS BETWEEN CLINICAL AND MORPHOLOGICAL ASPECTS IN HYPERTENSIVE PATIENS DEAD WITH PRIMARY

INTRACEREBRAL NON LOBAR HAEMORRHAGE

Iancu Emil Plesea1, Stelian Danut Enache2, Mirela Ghilusi2, Oltin Tiberiu Pop2, Simona Bondari2, Corneliu Cristian Georgescu1, Dan Cioroianu1

1University of Medicine and Pharmacy 2Emergency County Hospital, Craiova, Romania

Aims: The study is a retrospective integrated assesment of clinical, imagistic and morphological parameters in non lobar intracerebral hemorrhages (NLICH). Method: The material consisted of patient's medical records (clinical charts, CT scans, autopsy protocols and histopathology records) from 63 cases with NLICH hospitalised in the Emergency County Hospital of Craiova. The evaluated parameters were clinical (season relation, age, sex, arterial blood presure - HT, motor deficit - MD, Glasgow score at admission - GS) and morphological (ICH sites, size, perilesional edema - pE, microhemorrhages - mH, mass effect - ME, presence of intraventricular extension (IVE) and subarachnoid effusion (SE). Results: NLICH showed a season determinism, most probably related to changes in atmospheric pressure, with predilection for autumn and spring. Still, there are season related differences between the two genders, with higher incidence of NLICH in men during transition seasons, whereas in women, aparently there was no correlation between NLICH incidence and season. Gender distribution revealed no changes as compared to data from the literature, showing a slight predominance in men. The age-related NLICH incidence was somehow different in the two genders. In men, the most frequently involved age group was the fifth decade with a decreasing trend afterwards, while women showed an attenuated decreasing tendency after 40 years of age but with a peack in the seventh decade. Usualy severe HT as well as MD, in most cases of plegic type or decerebration-like reaction, were almost always present. An altered state of consciousness was also present at admission but not as frequently as HT and MD. The studied hemorrhagic foci following CT and autopsy were usualy associated with lobar hemorrhagic ones and more often located in the brainstem. Hemorrhagic foci were usualy large, regardless of location. The hemorrhagic strokes in our study were extremely severe, which is also sustained by the extent of the cerebal impairement up to the ventricular spaces in more than half of all cases as well as the SE in a significant number of cases. An altered state of consciousness is more frequent in women, between 40 and 60 years of age and in strokes occuring in summer and winter. MD is also more frequent in women, with a higher incidence with age and more often associated with strokes occuring in winter and spring. IVE is also more frequent in women and, has a decreasing trend with age and is more often associated with strokes occuring in autumn. Although less frequent, SE was slightly more frequent in women, less frequent between 60 and 70 years of age and more often associated with strokes occuring in autumn. Conclusion: LICH associated with one or more clinical and morphological poor outcome predictors (HT, MD, low GS, pE, mH and ME IVE and/or SE) result in patient's death, despite any sustained therapeutical intervention.


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ISOLATION AND CHARACTERIZATION OF CANCER STEM CELLS PRESENTS IN HUMAN LIVER HEPATOCELLULAR CARCINOMA

Poz Alessandra1, Marzinotto Stefania1, Baccarani Umberto1, Avellini Claudio2, Laura

Mariuzzi2, Nicoletta Finato2, Toffoletto Barbara1, Puppato Elisa1, Rigo Silvia1, Beltrami Antonio Paolo1, Cesselli Daniela1, Beltrami Carlo Alberto1

1University of Udine, Udine, Italy

2Azienda Ospedaliero-Universitaria, Udine, Italy Background: Hepatocellular carcinoma (HCC) represents the ultimate event of viral- or metabolic- related chronic liver processes, characterized by a gradual accumulation of genetic mutations. Recently, these alterations have been shown to occur not only in the mature parenchymal cells, but also in the resident stem cell compartment. Histologically, the multistep process of hepatocarcinogenesis is a continuum of lesions, in which dysplastic nodules have been suggested to behave as pre-neoplastic lesions. Aims: Purpose of this project was to isolate, expand in vitro, characterize, and compare stem cells obtained from normal livers (n=17) and from three different regions of neoplastic cirrhotic livers (n=12): the neoplastic area (N), the region bordering the neoplasia (C1) and a remote, macroscopically non-neoplastic region (C2). Methods and results: Utilizing a method optimized in our lab to grow multipotent-adult-human stem cells, we obtained cell lines from every normal and pathological specimen. Human liver stem cells (hLSCs) and human liver cancer stem cells (hLCSCs) shared a similar mesenchymal immunophenotype, expressing high levels of CD90 and CD105 and being mainly negative for CD34, CD45 and CD117. Interestingly, FACS analysis showed an increased expression of CD29, CD49a and CD49b in hLCSCs than in hLSCs. Regarding pluripotent state-specific transcription factor expression, every evaluated cell line showed high levels of OCT-4 (97,9±1,6%), SOX-2 (97,28±2,1%) and Nanog (99,28±0,9%). In contrast they were almost negative for hepato-specific markers such as cytokeratins (0,5±0,4%). hLCSCs were characterized, with respect to hLSCs, by a more restricted multipotency, failing to differentiate into glial or neural derivatives. Importantly, every tested hLCSC line possessed in vitro tumorigenicity, being able to grow in soft agar. Moreover, even cell lines obtained from regions distant from the neoplasia were able to grow embedded in soft agar. These areas were morphologically characterized by the presence of dysplastic nodules in the absence of overt neoplasia. In conclusion, we have isolated hLCSCs cells from livers affected by HCC; they possess both stemness and in vitro tumorigenic characteristics. The tumorigenicity of C1/C2-derived cell lines could be envisioned as supporting evidence for the presence, within dysplastic lesions, of cells already showing properties typical of cancer stem cells. Keywords: liver cancer stem cells, HCC, tumorigenicity, pluripotency.

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