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contents sadržaj Pozdravna reč 472 Welcome address Kongresni Odbori 473 Congress Commitees Organizator 475 Organizer HEMATOPATOLGIJA-Kratki kurs 1 477 HEMATHOPATHOLOGY-Short Course 1 Reaktivne limfadenopatije u svakodnevnoj praksi Vesna Čemerikić-Martinović Difuzni B krupnoćelijski limfom:varijante, podgrupe i podtipovi Maja Peruničić Jovanović HEMATOLOGIJA-Kratki kurs 2 Reactive lymphadenopathy in daily practice Vesna Cemerikic-Martinovic 489 494 Problematične „Low grade” lezije u limfoproliferativnoj patologiji Stefan Dojčinov Problematuične “High grade” lezije u limfoproliferativnoj patologiji Snježana dotlić Diffuse large B cell lymphoma: variants, subgroups and subtypes/entities Maja Perunicic Jovanovic HEMATHOPATHOLOGY-Short Course 2 Problematic “Low Grade” Lesions in Lymphoproliferative Pathology Stefan Dojcinov 498 Problematic “High Grade” Lesions in Lymphoproliferative Pathology Snjezana Dotlic Klasičan Hočkin limfom - diferencijalna dijagnoza i tumotska mikrosredina Slavko Gašparov 501 Classical Hodgkin lymphoma – differential diagnosis and tumour microenvironment Slavko Gasparov HEMATOPATOLGIJA-Kratki kurs 3 504 HEMATHOPATHOLOGY-Short Course 3 Proliferacije plazma ćelija Tatjana Terzić GINEKOLOŠKA PATOLOGIJA Kratki kurs 1 Plasma cell proliferations Tatjana Terzic 513 Patologija vulve – izabrane teme Vukomanović Đurđević Biserka Tumori uterusa Svetlana Milenković Važan napredak u ginekološkoj patologiji Sanjiv menek GYNECOLOGICAL PATHOLOGY Short Course 1 Vulvar Pathology - Selected Topisc Vukomanovic Djurdjevic Biserka 521 528 Tumors of the uterus Svetlana Milenkovic Important Recent Advances in Gynaecological Pathology Sanjiv Manek contents sadržaj GINEKOLOŠKA PATOLOGIJA Kratki kurs 1 533 Placenta-nemi svedok (klinički i medicinskopravni značaj patohistološkog ispitivanja) Marina Kos Novine u dijagnostici trofoblastnihbolesti Mihaela Mocko Kaćanski NOVINE U PATOLOGIJI Placenta – a silent witness: clinical and forensic importance of placental examination Marina Kos 540 Recent advances in diagnostics of trophoblastic disease Mihaela Mocko Kacanski 548 NEWS IN PATHOLOGY Endomiokardna biopsija: juče, danas i sutra Jovan D. Vasiljević Neuromišićne biopsije – tri godine nacionalnog iskustva Sanja M. Milenković Uvodno predavanje 3 Endomyocardial biopsy: yesterday, today and tomorrow Jovan D. Vasiljević 564 Neuromuscular biopsy - a review of 3 years nacional experience Sanja M. Milenkovic 573 Keynote Lecture 3 Cervikalna citologija i histologija u kontekstu skrining programa Sanjiv Menek Uvodno predavanje 4 Cervical cytology and histology in the context of a screening programme Sanjiv Manek 577 Profilaksa i rana detekcija HPV poveyanih neoplayija Hans Ikenberg USMENE PREZENTACIJE Keynote Lecture 4 Prophylaxis and Early Detection of HPVRelated Neoplasia Hans Ikenberg 586 Uloga eksfolijativne citologije u dijagnostici tumora donjeg urinarnog trakta Mirjana Ćuk, Slavica Knežević Ušaj, Radoslav Gajanin, Aleksandar Supić, Radmil Marić Ekspresija survivina kod bolesnika sa novootkrivenim nodalnim difuznim B krupnoćelijskim limfomom Olivera Marković, Dragomir Marisavljević, Vesna Čemerikić, Biljana Mihaljević, Branka Filipović GYNECOLOGICAL PATHOLOGY Short Course 1 ORAL FREE PAPER SESSIONS The role of exfoliative cytology in diagnosis of lower urinary tract tumor Mirjana Cuk, Slavica Knezevic Usaj, Radoslav Gajanin, Aleksandar Supic, Maric Radmil 587 Survivin expression in patients with newly diagnosed nodal diffuse large B cell lymphoma Olivera Markovic, Dragomir Marisavljevic, Vesna Cemerikic, Biljana Mihaljevic, Filipovic Branka contents sadržaj Prognostički značaj CD 68 pozitivnih makrofaga pridruženih tumoru u uznapredovalom stadijumu klasičnog Hočkinovog limfoma Ljubomir Jaković, Biljana Mihaljević, Maja Peruničić-Jovanović, Andrija Bogdanović, Boško Andelić, Vladimir Bumbaširević 588 The prognostic significance of CD 68 positive tumor associated macrophages in advanced stage classical Hodgkin lymphoma Ljubomir Jakovic, Biljana Mihaljevic, Maja Perunicic-Jovanovic, Andrija Bogdanovic, Boško Andelic, Vladimir Bumbasirevic Mogućnosti i ograničenja “imprint” citološke tehnike u dijagnostici koštanih i mekotkivnih tumora Jelena Sopta, Zoran Vučinić, Jelena Bokun , Dušan Ristić , Vesna Mijučić 589 Possibilities and limitations of „imprint” cytological techniques in diagnostics of bone and soft tissue tumors Jelena Sopta , Zoran Vucinic, Jelena Bokun, Dusan Ristic , Vesna Mijucic Imunoekspresija katepsina D kod meningioma Zorana Vukašinovic-Bokun, Lidija Prijić-Plećević, Milica Lavrnić, Nenad Miladinović, Marija Nikolić, Sanja M. Milenković 590 Immunoexpression of Cathepsin D in meningiomas Zorana Vukasinovic Bokun, Lidija Prijic-Plecevic, Milica Lavrnic, Nenad Miladinovic, Marija Nikolic, Sanja M. Milenkovic Nanog kao potencijalni marker kancerskih matičnih ćelija kod pacijenata sa gliomima visokog gradusa Irena Dimov, Sladana Ugrenović, Slavica Stojnev, Miloš Kostić, Tasic Desanka, Vladislav Stefanović Imunohistohemijska ekspresija p53 u Wilms-ovom tumoru Sanja Radojevic-Škodrić, Dimitrije Brašanac, Ljiljana Bogdanović, Milena Jovanović, Ivana Baralić, Gordana Basta-Jovanović 591 592 Nanog as the potential marker of the cancer stem cells in the high grade glioma patients Irena DimoV, Sladana Ugrenovic, Slavica Stojnev, Milos Kostic, Desanka Tasic, Vladislav Stefanovic Immunohistochemical expression of p53 in Wilms tumor Sanja Radojevic-Skodric, Dimitrije Brasanac, Ljiljana Bogdanovic, Milena Jovanovic, Ivana Baralic, Gordana Basta-Jovanovic Ispoljavanje FGFR1 i NCAM molekula u karcinomima bubrega Jasmina Marković-Lipkovski, Sanja Ćirović, Dragan Mitrović, Duško Dunđerović, Cane Tulić 593 Expression of FGFR1 and NCAM in renal tumors Jasmina Markovic-Lipkovski, Sanja Cirovic, Dragan Mitrovic, Dusko Dunderovic, Cane Tulic POSTER PREZENTACIJE 1 594 POSTER PRESENTATIONS 1 POSTER PREZENTACIJE 2 623 POSTER PRESENTATIONS 2 Beograd Belgrade Beograd Belgrade Beograd Belgrade Beograd Belgrade Welcome address Pozdravna reč Poštovane koleginice i kolege, Veliko nam je zadovoljstvo i čast da Vas pozdravimo u ime Udruženja patologa i citologa Srbije i da Vam poželimo uspešno učešće u radu 14. Kongresa Udruženja patologa i citologa Srbije koji se održava od 14.-16. juna 2012. godine, ponovo u Beogradu, kao i pre 37 godina, kada je održan II Kongres patologa Jugoslavije. 14. Kongres UPCS je naš prvi kongres koji se održava pod pokroviteljstvom Evropskog udruženja patologije. Naše najveće bogatstvo su naši doktori, naši članovi, zahvaljujući čijem radu, često i pored skromnih uslova i opreme za rad, uspevamo da održimo zavidan nivo stručnog i naučnog rada. U poslednjih nekoliko godina, uspeli smo da organizujemo niz značajnih aktivnosti, a posebno ističemo pet ESCOP škola (2009 - 2012) i kolektivno članstvo u ESP što je, uvereni smo, samo početak, puta kojim patologija Srbije vraća ugled i prepoznatljivost u svetu Organizovanje Evropskog kongresa patologa u Beogradu 2015. godine je naš najveći uspeh, ponos i velika šansa da mlade kolege uvedemo u svet razvijenih patologija. Nadamo se da će 14. Kongres UPCS biti zanimljiv i sadržajan. Služiće, ne samo razmeni stručnih i naučnih iskustava, nego i započinjanju novih projekata, obnavljanju starih i stvaranju novih prijateljstava. Ponosni smo što se u Udruženju oseća duh i atmosfera preovladavanja znanja i ideja, a ne zvanja, te u tom smislu učesnici će biti ne samo akademici ili profesori, već i mladi eksperti, asistenti, docenti ili vodeći patolozi u našim regionalnim zdravstvenim ustanovama Dobro došli! Welcome! Sanja M. Milenković Predsednik Izvršnog odbora Vesna Čemerikić-Martinović Predsednik Organizacionog odbora Jovan D. Vasiljević Predsednik Naučnog odbora 472 Dear colleagues, It is our great pleasure and honor to greet you on behalf of the Serbia Pathologists and Cytologists Association and wish you a successful participation in the work of the Congress which is to be held in June 2012 in Belgrade, where the 2nd Congress of Pathologists and Cytologists of Yugoslavia was held 37 years ago. 14th Congress of the SPCA is our first Congress held under the auspices of the European Society of Pathology. Our most valuable asset are our doctors, our members, and thanks to their hard work we manage to maintain an enviable level of professional and scientific work, despite our modest working conditions. In the recent years we have managed to organize a number of significant activities. Amongst them, I would like to mention five ESCOP schools (2009 2012) and collective membership in the ESP, which is just the beginning of our efforts to regain reputation and recognition of pathology of Serbia in the world. The organization of the European Congress of Pathology in 2015 is our biggest success, pride and a great chance to introduce our young colleagues to the world of developed pathologies We hope that the 14th Congress of SPCA will be interesting and educational. Not only it will serve for exchanging scientific experience, but also for starting new joint projects, renewal of old and beginning of new friendships. We are proud that the atmosphere of knowledge and ideas rather than people’s titles prevails in the Association and, in this sense, the participants will not only be academics or professors, but also young experts, assistants and leading pathologists of our regional institutions. Sanja M. Milenkovic President of the Executive Committe Vesna Cemerikic-Martinovic President of the Organising Committee Jovan D. Vasiljevic President of the Scientific Committee 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 Kongresni Odbori/Congress Committees Izvršni odbor/Executive Committee Organizacioni odbor/Organising Committee Predsednik/President Sanja M. Milenković Predsednik/President Vesna Čemerikić-Martinović Sekretar/Secretary Milos Mihajilović Sekretar/Secretary Svetlana Milenković Članovi/Members Dragan Mihailović Goran Stanojević Marjan Micev Slaviša Djuričić Svetislav Tatić Živka Eri Članovi/Members Biserka Vukomanović-Đurđević Mihaela Mocko-Kaćanski Maja Peruničić-Jovanović Snežana Vatrićević Naučni odbor/ Scientific Committee Počasni odbor/Honorary Committee Predsednik/President Jovan D. Vasiljević Ministar zdravlja Republike Srbije Prof. dr Zoran Stanković Predstavnik Srpske Akademije nauka i umetnosto Akademik Prof. dr Vladimir Kanjuh Rektor Univerziteta U Beogradu Akademik Prof. dr Vladimir Bumbaširević Predsednika SLD Prof. dr Radoje Čolović Predsednik Lekarske komore Srbije Prim. dr Tatjana Radosavljević Gradski sekretar za zdravstvo Prof. dr Zoran Blagojević Direktori Instituta za patologiju Prof. dr Gordana Basta-Jovanović, Beograd Prof. dr Ratko Ilić, Niš Prof. dr Milan Knežević, Kragujevac Sekretar/Secretary Jelena Sopta Članovi/Members Gordan Vujanić, GB Hans Ikenberg, Nemačka J. Han van Krieken, Holandija Jasmina Marković-Lipkovski, Srbija Marco Santucci, Italija Miroslav Đokić, SAD Radoslav Gajanin, BH Sanjiv Manek, GB Sigurd Lax, Austrija Slavica Knežević-Ušaj, Srbija Slavko Gašparov, Hrvatska Snežana Jančić, Srbija Stefan Dojčinov, GB Tatjana Terzić, Srbija 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 473 474 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 Organizator/Organizer 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 475 Beograd Belgrade Beograd Belgrade Beograd Belgrade Beograd Belgrade About Belgrade O Beogradu Beograd je središte kulture i umetnosti Srbije. U Beogradu stvaraju naši najznačajniji umetnici , a godišnje se održi više od 11.000 pozorišnih predstava, izložbi, koncerata, performansa i drugih umetničkih programa, gostuju brojni eminentni stvaraoci iz sveta umetnosti. Beograd je sedište najviših državnih i nacionalnih institucija kulture i umetnosti: Srpske akademije nauka i umetnosti, Narodne biblioteke Srbije, Narodnog muzeja, Narodnog pozorišta i Univerziteta umetnosti. Grad Beograd je osnivač i pokrovitelj 11 manifestacija u oblasti kulture (FEST, BITEF, BEMUS, BELEF, Međunarodno takmičenje muzičke omladine, Festival dokumentarnog i kratkometražnog filma, Oktobarski salon, Radost Evrope, Beogradski sajam knjiga, Filmski festival u Sopotu i Beogradski džez festival) Uopsteno govoreci, Beogradjani su prijatni i gostoljubivi, a moze se reci da su im ironija, crni humor i pozitivan stav pomogli da prevazicu teska vremena. Beogradjanke su lepe zene sa osecajem za modu, a Beogradjani visoki i zgodni. Iskreni su ljubitelji sporta, posebno vole kosarku, fudbal i tennis. Tokom dana ,za vreme kratke pauze ili za vreme poslovnog sastanka, uzivaju u kaficima dok je vece rezervisano za klubove i diskoteke.. Ko je imao sreće, da se jutros probudi u Beogradu, može se smatrati da je za danas dovoljno postigao u životu. Svako dalje insistiranje na još nečemu, bilo bi neskromno.“ Belgrade is the most flourishing heart of culture in Serbia! It offers an assorted selection of arts and cultural attractions: from art exhibitions to music performances, museums, architecture, theatres, photography and much more. Every year about 11.000 national and International cultural-artistic actvities take place in Belgrade: festivals such as FEST (Film Festival), BITEF (Theatre Festival), BELEF (Summer Festival) and BEMUS (Music Festival) along with the Book Fair and others. Generally speaking Belgraders are outgoing and friendly people: irony, self-criticism and black humor are the attitudes that helped Serbs overcome difficult times. Among its visitors Belgrade women are beautiful and with a sense for fashion and style while men are tall and handsome. Belgraders are big sports fans, they love particularly basketball, soccer and tennis. During the day Belgraders enjoy cafès both for a short break and for business meetings, meanwhile during the evening clubs and discos are always crowded: the vibrant and ever-changing nightlife is something not to be miss when visiting Belgrade Who was lucky this morning, to wake up in Belgrade, can be considered achieved enough in life for today. Any further insisting on something more it would be immodest“ Dusko Radovic Duško Radović 476 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 HEMATOPATOLGIJA- kratki kurs 1/HEMATHOPATOLOGY Short Course 1 Reaktivne limfadenopatije u svakodnevnoj praksi Reactive lymphadenopathy in daily practice Vesna Čemerikić-Martinović Vesna Cemerikic-Martinovic Beo-lab, Beograd, Srbija Beo-lab, Belgrade, Serbia Apstrakt Limfadenopatije su čest klinički nalaz u pacijenata svih uzrasta. Predstavljaju reakciju limfnog tkiva na različite spoljašnje i unutrašnje stimulanse. Najveći broj limfadenopatija su benigne promene koje mogu da liče na limfome i različite druge tumore tako da su njihovo poznavanje i diferencijalna dijagnoza bitni za isključivanje maligne bolesti. Limfni čvorovi veći od 1,5cm u najvećem prečniku, supraklavikularna lokalizacija i generalizovana limfadenopatija pobuđuju sumnju na malignu etiologiju. Lokalizovana limfadenopatija u starijih osoba je uvek sumnjiva na metastatski tumor. U slučaju generalizovane limfadenopatije limfomi, metastaze solidnih tumora i različite promene benigne etiologije se moraju razmatrati u diferencijalnoj dijagnozi. Benigne limfadenopatije se mogu podeliti na folikularno-nodularne, sinusne, interfolikularne ili mešovite i difuzne u zavisnosti od izgleda promena u različitim delovima limfnog čvora. Promene su dinamične tako da njihov izgled zavisi od momenta uzimanja biopsije. Najčešće benigne limfadenopatije su folikularna hiperplazija i limfadenitis u toksoplazmozi. U novije doba sve više viđamo limfadenopatije koje su posledica ugrađivanja implantata i proteza (silikonska limfadenopatija) ili su posledica davanja različitih kontrastnih sredstava (lipidna limfadenopatija). Specifična dijagnoza i diferencijacija od malignih bolesti zahteva korelaciju između kliničkih nalaza, morfologije limfnog tkiva, seroloških analiza i rezultata imunohistohemijskih, molekularnih i genetskih analiza. Ključne reči: limfadenopatije, klasifikacija, dijagnostika, FISH Abstract Lymphadenopathy is a common clinical finding, affecting patients of all ages. The majority of lymphadenopathies are reactive processes of lymph nodes in response to a variety of exogenous and endogenous stimulants. They are non-neoplastic conditions that can mimic lymphoma and other malignant tumors. Therefore their recognition and differential diagnosis is of great importance in order to rule-out the neoplastic disease. Signs of malignant etiology include lymph nodes >1,5cm in diameter, supraclavicular localization and generalized lymphadenopathy. A metastatic carcinoma is always in the differential diagnosis of localized lympadenophaty in older individuals. In case of generalized lymphadenopathy lymphomas, metastatic solid tumors and various benign etiologies need to be considered. The reactive lymphadenopathies are grouped into four major categories according to their predominant architectural histologic pattern: follicular-nodular, sinus, interfollicular or mixed, and diffuse. As reactive conditions of the lymph nodes are dynamic processes the predominant pattern may differ depending on when during the course of the disease the biopsy is performed. The most common reactive lymphadenopaties are follicular hyperplasia and toxoplasmic lymphadenitis. Nowdays some lymphadenopathies with foreign-body reaction are side effects of silicone prostheses (silicone lymphadenopathy) and various contrast media (lipid lymphadenopathy). A specific diagnosis and differentiation from neoplastic disease often requires correlation among the morphologic features, the clinical history, serologic studies, immunohistochemistry and molecular genetic analysis. Key words: lymphadenopathy, classification, diagnosis, FISH Uvod U ljudskom telu postoji oko 600 limfnih čvorova. Promene u njihovoj veličini, broju i konzistenciji se označavaju kao limfadenopatija. Čest je klinički nalaz u svim uzrastima. Preporuka je da se pacijenti sa limfadenopatijom većom od 1cm koja perzistira šest nedelja obavezno podvrgnu detaljnoj dijagnostičkoj proceduri, uključujući i biopsiju (1). Limfni čvorovi čiji je prečnik manji od 1cm se smatraju normalnim, mada to 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 477 HEMATOPATOLGIJA- kratki kurs 1/HEMATHOPATOLOGY Short Course 1 zavisi od njihove lokalizacije. Ingvinalni čvorovi veći od 1,5cm se smatraju izmenjenim dok je kod epitrohlearnih kriterijum 0,5cm. Godišnja incidenca limfadenopatije je oko 0,6%-0,7% u opštoj populaciji (1). Stoga tkivo limfnih čvorova predstavlja jedan od najčešćih biopsijskih materijala u patohistološkim laboratorijama. Osnovni problem koji se postavlja pred patologa je da li je limfadenopatija benigna ili maligna. Precizna dijagnoza zahteva korelaciju između kliničkih nalaza, morfologije izmenjenog limfnog tkiva, seroloških analiza, histohemijskih bojenja, uključujući i ona za identifikaciju mikroorganizama, imunohistohemijskih, molekularnih i genetskih analiza. U oko 50%-60% slučajeva postavi se pozitivna dijagnoza specifične limfadenopatije, različitih infektivnih limfadenitisa, metastaskih tumora i limfoma. Najveći broj uvećanih limfnih čvorova (40%-55%) je benigan i predstavlja nespecifičnu reakciju na različite antigene, bilo strane, bilo sopstvene. U opštim bolnicama neoplazme se nađu u oko 1%-2% biopsija limfnih čvorova, dok se u specijalizovanim ustanovama taj broj penje na 60%. U velikoj studiji autora iz Velike Britanije (2) koja je obuhvatila 550 bolesnika sa limfadenopatijom, 95 (17,3%) je imalo malignu bolest, od kojih 62 limfoproliferativnu bolest (19 Hodkinov limfom, 18 difuzni B-krupnoćelijski non-Hodgkinov limfom, a ostatak su bili različiti NHL). U 168 bolesnika je dijagnostikovana nespecifična, reaktivna limfadenopatija, u 139 benigne limfadenopatije različitih uzroka a u 21 bolesnika benigni tumori. Prvi korak u dijagnostici limfadenopatija je detaljno poznavanje kliničkih simptoma i karakteristika bolesti (3): postoje li znaci ili simptomi koji ukazuju na infekciju ili neoplazmu; postoje li simptomi kao što su bol, groznica, temperatura, gubitak težine, znojenje, osip po koži i sl.; epidemiološki podaci koji se odnose na ishranu, seksualne navike, profesionalno izlaganje štetnim materijama, uzimanje lekova i supstanci koji mogu da izazovu limfadenopatiju, postojanje proteza i implantata itd. Uzrast bolesnika je bitan. U dečijem uzrastu najveći broj limfadenopatija je benigan dok se u odraslih verovatnoća maligne limfadenopatije povećava sa starošću. Bolno uvećanje čvorova je obično benigno. Pokretni limfni čvorovi koji se brzo uvećavaju su verovatno ne-neoplastični za razliku od fiksiranih koji se sporo uvećavaju. Lokalizovana limfadenopatija (75%) je češća od generalizovane (25%). Limfni čvorovi glave i vrata su najčešće uvećani (55%), zatim slede ingvinalni (14%), aksilarni (5%), dok su supraklavikularni uvećani u 1% slučajeva. Lokalizacija limfadenopatije može biti značajna jer često ukazuje na njen uzrok. Limfni čvorovi na vratu su najpre uvećani u infektivnoj mononukleozi, toksoplazmozi i drugim infekcijama ali i u limfoproliferativnim bolestima. Supraklavikularni limfni čvorovi su često udruženi sa malignom bolešću (25% pacijenata mlađih od 40 godina i u 90% starijih od 40 godina). Generalizovana limfadenopatija koja predstavlja uvećane limfne čvorove u više od tri regije, više ukazuje na malignitet. Uzroci limfadenopatija su različiti (4, 5): • Infekcije (streptokok, stafilokok, tuberkuloza, atipične mikobakterije, toksoplazmoza, bolest mačije ogrebotine, sifilis, gljivice, paraziti, Epstein-Barrov virus, HIV, citomegalovirus, herpesvirus itd.) • Autoimune bolesti (reumatoidni artritis, SLE, Sjögrenov sindrom) • Lekovi (dilantin, antikonvulzanti, antibiotici, aspirin, alopurinol) • Sarkoidoza • Amiloidoza • Whippleova bolest • Vakcine • Silikonski implantati, kontrastna sredstva, proteze • Bolesti nakupljanja (Guacherova bolest) • Benigne limfoproliferativne bolesti (Kikuchieva bolest, Kawasakieva bolest, Kimurina bolest, RosaiDorfmanova bolest, progresivna transformacija germinativnih centara, vaskularna transformacija sinusa, inflamatorni pseudotumor, Castlemanova bolest, dermatopatska limfadenopatija) • Maligne limfoproliferativne bolesti (non-Hodgkinovi limfomi, Hodgkinovi limfomi, akutne i hronične mijeloidne leukemije) • Metastaski tumori 478 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 HEMATOPATOLGIJA- kratki kurs 1/HEMATHOPATOLOGY Short Course 1 Diferencijalna dijagnoza limfadenopatije je jako široka, slična diferencijalnoj dijagnozi nejasnih febrilnih stanja ili povećane sedimentacije eritrocita. Diferencijalne dijagnoze se mogu grupisati prema akronimu CHICAGO (Cancers, Hypersensitivity syndromes, Infections, Connective tissue diseases, Atypical lymphoproliferative disorders, Granulomatous lesions, Other unusual causes of lymphadenopathy) (6). Benigne limfadenopatije se mogu podeliti na folikularno-nodularne, sinusne, interfolikularne ili mešovite i difuzne u zavisnosti od izgleda promena u različitim delovima limfnog čvora (tabela 1). Ova podela nije striktna jer se često vide kombinacije promena u različitim odeljcima limfnog čvora. S druge strane promene su dinamične tako da njihov izgled zavisi od momenta uzimanja biopsije. Folikularno-nodularne Nespecifična, reaktivna folikularna hiperplazija Autoimune bolesti (reumatoidni artritis) HIV limfadenopatija Sifilis Castlemanova bolest, hijalino-vaskularni tip Progresivna transformacija germinativnih centara Hiperplazija mantle zone Mikobakterijski pseudotumor Sinusne Sinus histiocitoza Kontrastna sredstva, proteze, silikonski implantati Whippleova bolest Vaskularna transformacija sinusa Rosai-Dorfmanova bolest Sinus histiocitoza sa masivnom limfadenopatijom Hemofagocitni sindrom Interfolikularne ili mešovite Parakortikalna hiperplazija i dermatopatska reakcija Granulomatozni limfadenitis Nenekrotizirajući granulomi Nekrotizirajući granulomi Tuberkuloza Gljivične infekcije Bolest mačije ogrebotine Kimurina bolest Limfadenitis u toksoplazmozi Sistemski lupus Kikuchieva bolest Kawasakieva bolest Inflamatorni pseudotumor Bacilarna angiomatoza Limfadenopatije u sklopu imunodeficijencije Difuzne Infektivna mononukleoza Citomegalovirus infekcija Herpes simplex limfadenitis Dilantinska limfadenopatija Tabela 1. Morfološka klasifikacija reaktivnih limfadenopatija 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 479 HEMATOPATOLGIJA- kratki kurs 1/HEMATHOPATOLOGY Short Course 1 U radu će biti detaljno prikazane reaktivna folikularna hiperplazija i limfadenitis u sklopu toksoplazmoze kao najčešće benigne limfadenopatije koje srećemo u našem podneblju. Ponekad, floridna reakcija limfnog tkiva može imati atipičan morfološki izgled te se može zameniti za limfom (7). Uz to će biti prikazane neke limfadenopatije koje sve više viđamo, a mogu predstavljati diferencijalno-dijagnostički problem: silikonske i lipidne limfadenopatije. Reaktivna folikularna hiperplazija Folikularna hiperplazija (FH) je najčešća reaktivna limfadenopatija. Predstavlja umnožavanje sekundarnih limfoidnih folikula ili germinativnih centara po jedinici površine limfnog čvora. Jedan od kriterijuma za postavljanje dijagnoze je da >90% limfoidnih folikula sadrži aktivne germinativne centre. Folikuli su uvećani, hipertrofični, nepravilnog oblika. Česta je u dečijem uzrastu a ređa u starijem; retko se vidi posle 60-te godine kada je obično u sklopu reumatoidnog artritisa ili neke druge autoimune bolesti (8). Osnovni problem u dijagnostici reaktivne folikularne hiperplazije je diferencijacija od folikularnog NHL. FH je posledica antigene aktivacije B-limfocita (izazvane najčešće stranim, bakterijskim antigenima, ali ne-retko i sopstvenim). Ova aktivacija dovodi do somatskih mutacija, sazrevanja, klonalne ekspanzije i selekcije limfoidnih ćelija čiji je rezultat stvaranje plazma ćelija i produkcija antitela. Proces se može i morfološki pratiti u limfnom tkivu. Prisustvo brojnih apoptotskih tela i „tingible body“ makrofaga je morfološki znak klonalne selekcije, a brojne mitoze ukazuju na proliferativnu prirodu procesa. Citološke promene od malog, okruglog limfocita preko krupnih centroblasta i imunoblasta do zrelih limfocita sa potpunom antigenom selekcijom koje vidimo kao centrocite, su histološki dokaz aktivacije imunog sistema. Makroskopski, limfni čvorovi su sjajni, na preseku granulirane površine, a ponekad se mogu videti i lako uzdignuti noduli. Nažalost, isto ovako izgledaju i NHL koji pokazuju folikularni tip rasta. Mikroskopski, slikom dominiraju umnoženi limfoidni folikuli različite veličine i oblika, raspoređeni po svim odeljcima limfnog čvora (korteks, parakorteks, medula). Građa limfnog čvora je u osnovi očuvana, uz prisutan sinusni sistem. Reaktivni folikuli se obično ne nalaze van kapsule i u perinodalnom masnom i vezivnom tkivu za razliku od neoplastičnih folikula u folikularnom NHL. Na reaktivnu prirodu procesa ukazuju polarizacija folikula, očuvana mantle zona i prisustvo „tingible body“ makrofaga koji germinativnim centrima daju izgled zvezdanog neba (starry sky). Germinativni centri su upadljivi. Unutar njih postoji polimorfna ćelijska populacija centrocita, centroblasta i par imunoblasta. Krupne ćelije preovlađuju tako da germinativni centri deluju svetlo. Retikularna osnova je očuvana ili lako potisnuta ka periferiji, za razliku od folikularnog NHL gde je značajno zgusnuta i potisnuta ka periferiji. U folikularnom limfomu, folikuli su obično sitniji, mnogobrojniji, zgusnutiji, međusobno relativno jednaki, bez mantle zone u „back-to-back“ položaju, bez polarizacije. Ćelijski sastav germinativnih centara je monomorfan tako da deluju „mirnije“ od FH, bez „tingible body“ makrofaga. Dominiraju sitnije ćelije, centrociti pa germinativni centri izgledaju tamnije (8). U starijih bolesnika germinativni centri pokazuju manji stepen aktivacije nego kod mlađih tako da se folikularna hiperplazija lako zameni za folikularni NHL. Imunohistohemijski, germinativni centri se sastoje uglavnom od CD20+ B-ćelija, izmešanih sa različitim brojem CD4+, CD57+ T-limfocita kao i PD-1+ intrafolikularnih T-ćelija. Germinativni centri su BCL2 negativni za razliku od neoplastičnih folikula u folikularnom limfomu koji su BCL2 pozitivni. Ispitivanje sa BCL2 se uvek mora raditi zajedno sa reakcijama na CD20 i CD3 jer brojni BCL2+ T-limfociti mogu biti prisutni i u reaktivnim folikulima što se može pogrešno protumačiti kao BCL2+ NHL. Ekspresija BCL2 je obično jača u ćelijama folikularnog limfoma nego u okolnoj mantle ili interfolikularnoj zoni. Oko 5%-25% folikularnih NHL je BCL2 negativno. Pozitivnost opada sa gradusom; u folikularnim limfomima gradusa 3 se kreće od 50% do 75%. U slučaju sumnje na BCL2 negativan folikularni NHL obavezno uraditi FISH ili PCR analizu jer postoji BCL2 rearanžman, bez obzira na imunohistohemijsku negativnost (sl.1). I benigni i neoplastični folikuli eksprimiraju CD10 i BCL6, mada je ekspresija CD10 nešto slabija u folikularnoj hiperplaziji. BCL6 može biti pozitivan samo u delu ćelija folikularnog limfoma, za razliku od folikularne hiperplazije u kojoj su sve ćelije pozitivne. Proliferativni marker Ki-67 može biti od velike koristi u 480 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 HEMATOPATOLGIJA- kratki kurs 1/HEMATHOPATOLOGY Short Course 1 diferencijaciji FH od folikularnog limfoma: u reaktivnoj hiperplaziji skoro sve ćelije su Ki-67 pozitivne dok je u folikularnom limfomu ta pozitivnost daleko manja, obično 10-20%, retko do 60% i to u GR3. U folikularnoj hiperplaziji ćelije u proliferaciji su često skoncentrisane u jednoj polovini germinativnog centra što odražava zonalnu organizaciju proliferacije i selekciju ćelija za apoptozu u reaktivnom germinativnom centru. U folikularnom NHL malobrojne Ki-67 pozitivne ćelije su obično razbacane po celoj površini germinativnog centra. Niska proliferacija u folikularnim limfoidnim lezijama je uvek visoko suspektna na NHL. Imunohistohemijski dokaz monoklonalnosti, odnosno dokaz restrikcije lakih lanaca imunoglobulina (κ, λ) na smrznutim presecima ili putem protočne cytometrije je relativno pouzdana metoda za razlikovanje folikularnog NHL od FH. Lawrence i saradnici su prikazali pouzdane rezultate (64 pozitivnih od 67 slučajeva FH) primenom monoklonskih antitela (κ, λ) proizvođača Silver Lake Research Corporation (Monrovia, CA) (9) na parafinskim presecima limfnih čvorova. Nažalost, ovi markeri nisu registrovani u našoj zemlji pa su nam za sada nedostupni. Komercijalna antitela koja se kod nas mogu naći za parafinske preseke ne daju pouzdane rezultate. Slika 1. Karakteristike reaktivne folikularne hiperplazije (FH) i folikularnog NHL (FL) Folikularna hiperplazija se odlikuje polarizacijom folikula, očuvanom mantle zonom i prisustvom „tingible body“ makrofaga u germinativnim centrima. Unutar germinativnih centara postoji polimorfna ćelijska populacija centrocita, centroblasta i par imunoblasta (detalj). U folikularnom limfomu, folikuli su bez polariteta i mantle zone. Nema makrofaga a ćelijski sastav germinativnih centara je monomorfan tako da dominiraju centrociti (detalj). Imunohistohemijski, germinativni centri U FH su BCL2 negativni a u FL pozitivni. U reaktivnoj hiperplaziji skoro sve ćelije su Ki67 pozitivne dok je u folikularnom limfomu ta pozitivnost daleko manja. U FH BCL2 gen nije rearanžiran, dok je u FL rearanžiran, t(14;18). FiISH analiza pokazuje normalan BCL2 signal u jedrima ćelija FH i translokaciju BCL2 u FL 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 481 HEMATOPATOLGIJA- kratki kurs 1/HEMATHOPATOLOGY Short Course 1 Prema različitim literaturnim podacima, u 5-15% slučajeva diferencijalna dijagnoza između reaktivne limfoidne proliferacije i malignog limfoma zahteva molekularne genetske analize. Reaktivne limfoproliferacije imaju poliklonski rearanžman imunoglobulinskih (Ig) ili T-ćelijskih receptorskih (TCR) gena, za razliku od malignih koje imaju monoklonski rearanžman. Molekularne genetske analize (Southern blot i PCR) su najpouzdanije metode za detekciju imunoglobulinskih, TCR i BCL2 rearanžmana u FH. Za razliku od imunofenotipizacije, njima se može utvrditi prisustvo male klonalne populacije ćelija i može se utvrditi monoklonalnost u imunohistohemijski Ig i BCL2 negativnim tumorima. Southern blot je preciznija metoda, ali je problem što zahteva sveže, nefiksirano tkivo. PCR analiza je manje senzitivna, mada se sa pojavom novih standardizovanih reagenasa i metoda za PCR-baziranu klonalnost (BIOMED-2 Concerted Action BMH4 CT98-3936) senzitivnost značajno poboljšala (99% u B-ćelijskim NHL i 94% u T-ćelijskim) (10). Histološki reaktivne proliferacije predstavljaju široki spektar lezija sastavljenih od heterogene populacije poliklonskih limfocita preko lezija koje sadrže (oligo)klonalne aktivirane limfoidne ćelije ili, ponekad, monoklonsku komponentu. U studiji Van Kriekena i saradnika (11) urađena je ekstenzivna PCR analiza svih mogućih Ig/TCR genskih rearanžmana i translokacija t(11;14) i t(14;18) u 109 reaktivnih limfoidnih lezija, od kojih su 74 bile FH. U 75% slučajeva utvrđena je jasna poliklonalnost za Ig/TCR. U 10% (11/106) su uočeni jasni monoklonski produkti za jedan ili više Ig/TCR rearanžmana. U 2 od ovih 11, ponovnom patohistološkom analizom je utvrđeno delimično zahvatanje limfnog tkiva limfomom. Nalaz klonalnosti u reaktivnim lezijama zahteva tesnu saradnju patologa i molekularnog biologa u tumačenju rezultata da bi se izbegle potencijalne greške. Treba isključiti moguće tehničke poteškoće, imunobiološke karakteristike (EBV infekciju i sl.) i ako je rezultat suspektan klinički pratiti bolesnika, uz eventualne ponovljene biopsije sa ponovnim molekularnim analizama da bi se došlo do tačne dijagnoze. Različiti spektar hromozomskih aberacija (strukturnih i numeričkih) je utvrđen u 17% reaktivnih limfadenopatija (116 pacijenata), među kojima najveći broj pripada FH (12). Kariotipske anomalije su češće u generalizovanoj nego u lokalizovanoj limfadenopatiji. U 40% promene su bile na hromozomu 14, pre svega translokacije koje zahvataju IGH lokus. Rearanžman BCL6 je detektovan u 20%. Pacijenti su praćeni 5 godina i samo kod jednog se pojavio NHL. Najveći problem u diferencijalnoj dijagnozi FH je folikularni limfom. U najvećem broju slučajeva diferencijacija se može izvršiti na osnovu arhitekturalnih i citomorfoloških karakteristika. U komplikovanim i nejasnim slučajevima neophodna je detaljna imunohistohemijska i citogenetska analiza (sl. 1). Morfološki, imunohistohemijski i molekularni parametri za razlikovanje FH od folikularnog NHL su prikazani na tabeli 2. Poseban problem predstavlja BCL2 negativan folikularni limfom (10-15%) kada se dijagnoza može postaviti samo po utvrđivanju klonalnosti B-ćelijske populacije i citogenetskoj analizi. Različiti NHL koji mogu da rastu folikularno (NHL marginalne zone, mantle-ćelijski NHL i sitnoćelijski difuzni NHL) kao i Hodgkinov limfom tipa nodularne limfocitne predominacije mogu predstavljati problem u diferencijalnoj dijagnozi. Za diferencijaciju je neophodna pažljiva morfološka evaluacija, uz detaljno imunohistohemijsko i, eventualno, citogenetsko ispitivanje (FISH, PCR). U diskriminaciji između reaktivnih proliferacija i NHL ekspresije gena (mRNA microarray), može biti od velike koristi. Pokazano je da u reaktivnoj limfadenopatiji postoji niža ekspresija gena vezanih za imuni odgovor u odnosu na NHL (13). Postoji i redukovana ekspresija TAF3 i LDB2 gena. 482 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 HEMATOPATOLGIJA- kratki kurs 1/HEMATHOPATOLOGY Short Course 1 KarakteristikaFHFL Arhitektura čvora očuvana narušena Retikularna/FDRC osnova očuvana kondenzovana, iskidana Germinativni centri granica jasna nejasna,”back to back” veličina i oblik nejednaki relativno jednaki mantle zona prisutna odsutna ili uska polarnost folikula često prisutna nedostaje perinodalno tkivo odsutni folikuli prisutni folikuli uz fibrozu mitoze brojne retke Citologija folikula centrociti malobrojni mnogobrojni centroblasti mnogobrojni malobrojni ”starry sky” upadljiv nema Interfolikularna zona mešovita ćelijska populacija sitne ćelije (centorc.) plazmociti često (poliklonski) ponekad (monoklonski) imunoblasti nema ponekad inflamatorni infiltrat nema prisutan Imunohistohemija CD20 + + CD10 + (slabo) + BCL2 - + Ki-67 visok nizak Fascin u FDRC + Genetika klonalnost poliklonska monoklonska rearanžman BCL2 nema ima, t(14;18) Tabela 2. Histološke, imunohistohemijske i genetske karakteristike korisne za razlikovanje FH i folikularnog NHL Toxoplasma limfadenitis Toxoplasma gondii je jedan od najuspešnijih parazita na našoj planeti jer je njime inficirano više od trećine čovečanstva. Otkrivena je 1908. godine u Tunisu (Nicolle i Manceaux). Uzročnik je oko 15%-20% svih limfadenopatija. Prenosioci toksoplazme najčešće su mačke i, znatno ređe, psi. Jedan od čestih puteva infekcije je konzumiranje termički nedovoljno obradjenog mesa i povrća. Ređi putevi prenošenja obuhvataju laboratorijske infekcije, infekcije preko transfuzije krvi i transplantiranih organa i infekciju nerođenog ploda preko majke (14). Životni ciklus toksoplazme je osoben utoliko što se seksualni razvoj odigrava samo kod životinja iz porodice mačaka, a aseksualni kod svih ostalih domaćina. Kod mačke se razvoj parazita završava stvaranjem neinfektivnih oocista, koje inficirana mačka stolicom izbacuje u spoljnu sredinu. Posle 48 sati na spoljnoj temperaturi, ove oociste prelaze u spore i postaju visoko infektivne i veoma izdržljive: mogu da prežive u zemljištu do 18 meseci. Otporne su na uobičajene dezinficijense, a uništavaju ih isušivanje i temperatura iznad 60oC. Kod svih ostalih prelaznih domaćina, uključujući i čoveka, odvija se životni ciklus u kome se pojavljuju tahizoiti (2-6 μm veliki polumesečasti organizmi) koji se brzo dele, i bradizoiti (unutar intracelularne ciste) koji se sporo dele. Čovek se najčešće inficira preko usta (digestivnog trakta), unošenjem bilo tkivnih cista bilo oocista. Posle unošenja bilo kog od ovih infektivnih oblika, pod dejstvom želudačnog soka dolazi do brzog razlaganja opne ciste i oslobađanja parazita, koji prelaze u tahizoite i prodiru u sve vrste ćelija organizma. Međutim, infekcija 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 483 HEMATOPATOLGIJA- kratki kurs 1/HEMATHOPATOLOGY Short Course 1 podstiče imuni odgovor domaćina, pa paraziti da bi se zaštitili (ponovo) prelaze u oblik bradizoita koji se okružuju opnom (cista). Ciste se uglavnom smeštaju u tkivima slabije dostupnim imunom sistemu (centralni nervni sistem, oko, skeletni mišić, srce). Vrlo dugo preživljavaju, praktično doživotno, izazivajući trajnu latentnu odnosno hroničnu infekciju, i ova sposobnost konverzije u manje zahtevni životni oblik i predstavlja suštinu parazitizma Toxoplasme gondii. Infekcija toksoplazmom je veoma rasprostranjena i zastupljena na svim krajevima Zemljine kugle. U SAD varira od 13%-68%, zavisno od regije, pri čemu se 30% uzima kao prosek. U Evropi se zapažaju velike razlike u prokuženosti toksoplazmom: od 23-33% u Velikoj Britaniji, preko 62% u Austriji, do 67% u Francuskoj. U velikoj studiji Đuraković-Đaković i saradnika seroepidemiološkim ispitivanjem 2936 žena u periodu 1988. do 1997.godine utvrđena je prevalenca infekcije u 69% sa tendencijom pada tokom godina (od 88% do 39%), sa najvećom incidencom u Vojvodini (15). Toksoplazmoza je neobična infektivna bolest. Pored činjenice da je jedna od najčešćih infektivnih bolesti ona je u većini slučajeva asimptomatska. Kod 10-20% inficiranih može doći do uvećanja limfnih čvorova (najčešće zadnjih vratnih, ređe aksilarnih i ingvinalnih), uz lako povišenu temperaturu, malaksalost, bol u mišićima, znojenje, kožnu ospu, pa i uvećanje jetre i slezine. Kako tegobe posle nekoliko meseci i spontano iščezavaju, nije potrebna terapija. Sistemska toksoplazmoza je jedna od najznačajnijih oportunističkih infekcija u sklopu AIDS i jatrogene imunosupresije. Toksoplazmoza je najbitnija fetalna transplacentarna infekcija koja može da ošteti plod. Karakteristična trijada histoloških nalaza u limfnom čvoru upućuje na toksoplazmozu: folikularna hiperplazija, sitni klasteri epiteloidnih histiocita i hiperplazija monocitoidnih B-ćelija unutar sinusoida (16). Ova trijada ima specifičnost od 91-97% u dijagnozi toksoplazmoze što je potvrđeno i PCR analizom. Folikuli su uvećani, krupnih, aktivnih germinativnih centara u kojima postoji povećan broj centroblasta i veliki broj „tingible body“ makrofaga. Pojedinačne, krupne epiteloidne ćelije se vide u korteksu i parakorteksu. Prisutni su i brojni sitni klasteri epiteloidnih histiocita u parakorteksu ali i unutar germinativnih centra, što je karakteristično za toksoplazmozu. Germinativni centri stoga imaju nepravilnu, „nazubljenu“ ivicu. Ponekad se vide i veći klasteri epiteloidnih ćelija, tzv. „makrogranulomi“ koji sadrže više od 25 epiteloidnih histiocita. Pravih granuloma i multijedarnih džinovskih ćelija nema. Monocitoidne B-ćelije su krupne ćelije, oštrih granica, obilne, svetle citoplazme, relativno sitnog, tamnog jedra, neupadljivog nukleolusa. Raspoređene su u vidu plaža i ostrva oko i unutar sinusa limfnog čvora. Ponekad se nađu i perivaskularno (sl.2). Nekroza nema. Ciste parazita unutar makrofaga se retko nađu (u 1% slučajeva u AFIP-ovoj seriji). U obolelih od AIDS brojni slobodni tahizoiti se mogu naći u raznim tkivima. Najbolje se vide u Giemsom obojenim razmazima i inprintima. Mogu se identifikovati i imunohistohemijski. Pozitivan serološki nalaz je najbitniji za potvrdu dijagnoze. Dijagnoza se može potvrditi i imunohistohemijski anti-Toxoplasmosa antitelom ili PCR analizom (pozitivan rezultat u 83% u slučaju postojanja karakteristične dijagnostičke trijade) (17). Slika 2. Toksoplazma limfadenopatija 484 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 HEMATOPATOLGIJA- kratki kurs 1/HEMATHOPATOLOGY Short Course 1 Toksoplazma limfadenitis sa folikularnom hiperplazijom (FH), klasterima epiteloidnih histiocita (EH) u parakorteksu i u germinativnim centrima i hiperplazijom monocitoidnih B-ćelija (HMC), H&E. Diferencijalno dijagnostički na prvom mestu je limfadenitis u sklopu HIV infekcije i lajšmanijaze. Infektivna mononukleoza, bolest mačije ogrebotine i citomegalovirusni limfadenitis u ranoj fazi morfološki liče na toksoplazmozni limfadenitis. Sarkoidoza može predstavljati problem ali u njoj obično postoje jasno formirani granulomi i džinovske ćelije se uvek nađu. Dermatopatska limfadenopatija dolazi u obzir, ali nju odlikuje prisustvo melanina i lipida. Ponekad se sinus histiocitoza sa masivnom limfadenopatijom (Rosai-Dorfmanova bolest) zameni za toksoplazmozu ali se u ovoj bolesti histiociti nalaze unutar proširenih sinusa, a ne u tkivu. Neke forme NHL (Lennertov T-ćelijski NHL) mogu imati klastere epiteloidnih histiocita kao i Hodgkinov limfom. Limfadenopatije u XXI veku Silikonska limfadenopatija Medicinski silikon se sastoji od polimera dimetilsiloksana. Postoji u čvrstoj formi (sličnoj gumi), kao gel i kao ulje. Sve tri forme se široko upotrebljavaju kao implantati u kozmetičke, ortopedske ili rekonstruktivne svrhe (naročito dojke po mastektomiji). Silikonsko ulje, gel ili guma su potencijalni bioaktivni agensi koji mogu da izazovu reakciju tkiva domaćina. Silikonski implantati mogu da rupturiraju (što su stariji mogućnost rupture je veća), a ubrizgani gel ili ulje mogu da se rasprše po tkivima i da dospeju u limfotok i krvotok kojima se šire na udaljena mesta, uključujući i limfne čvorove (20). Jedna od čestih komplikacija ovih ruptura je silikonska limfadenopatija sve više dolazi kao diferencijalno dijagnostički problem i u klinici i u patologiji. Silikonska limfadenopatija može nastati u regionalnim limfnim čvorovima kao reakcija na silikonske implantate u dojkama i na drugim mestima, usled direktnog ubrizgavanja silikona ili posle artroplastike malih zglobova. Prvi put je opisana 1978. godine (18). Najčešće se pojavljuje u aksilarnim limfnim čvorovima kada se klinički često zameni za metastazu karcinoma dojke. Incidenca silikonske limfadenopatije kod artroplastike malih zglobova se kreće od 1,8% do 13% (19). Može se javiti i nekoliko godina po ugrađivanju proteze. Silikon se, ako nije nestao prilkom obrade tkiva, lako prepoznaje u tkivu. U vidu je sitnih, providnih kapi amorfnog materijal koji ne polarizuje. Preseci deblji od 4μm (najbolje 10-30μm) su najbolji za njegovu identifikaciju jer se onda silikon ne gubi pri procesiranju tkiva. Morfološki, normalna nodalna arhitektura je zamenjena sitnim i krupnim vakuolama koje deluju ili kao „prazne“ (jer je silikon ispran prilikom obrade tkiva) ili sadrže zaostale silikonske depozite (20). Džinovske ćelije tipa stranog tela i granulomi (silikonomi) se mogu naći u sinusima i parenhimu (sl.3). Slika 3. Silikonska limfadenopatija 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 485 HEMATOPATOLGIJA- kratki kurs 1/HEMATHOPATOLOGY Short Course 1 U limfnom tkivu se vide brojne sitne i krupne vakuole koje deluju ili kao „prazne“ (jer je silikon ispran prilikom obrade tkiva) ili sadrže zaostale silikonske depozite u vidu sitnih, providnih kapi amorfnog materijal. Detalj: džinovske ćelije tipa stranog tela sa silikonskim kapima u citoplazmi, H&E. U citoplazmi džinovskih ćelija se može naći silikon i inkluziona telašca. Mogu se videti i makrofagi penušave citoplazme, uz upadljivu sinus histiocitozu. Silkon se može identifikovati infracrvenom spektroskopijom ili bojenjem na Oil red O (ne uspeva uvek). Klinički podaci su presudni za postavljanje dijagnoze. Diferencijalno-dijagnostički u obzir dolaze razna nakupljanja lipidnih materija (lipidna limfadenopatija), Whippleova bolest i atipične mikobakterioze. U poslednje dve se diferencijacija lako izvrši bojenjem na PAS ili prikazivanjem acido-rezistentnih bacila. Lipidna limfadenopatija Granulomatozna reakcija po tipu stranog tela na lipide u obliku lipogranuloma se može videti u limfnim čvorovima. Lipidna limfadenopatija je česta u starijih i gojaznih, kod dugotrajne parenteralne ishrane, kod raznih lezija žučne kese i žučnih puteva, u blizini hematoma, nekroza i nekih tumora. Uz to se može pojaviti kao posledica davanja kontrasta na bazi ulja. Nekada su se ovakvi kontrasti davali u limfografiji (21), a danas se ponekad daju u angiografiji. Depo lekovi koji se daju u vidu lipidnih rastvora mogu dovesti do ove limfadenopatije. Mi smo imali dva slučaja neobične lipidne limfadenopatije: u submandibularnom limfnom čvoru po ubrizgavanju Botoxa radi zatezanja lica (sl.4A); u aksilarnom limfnom čvoru kao posledicu akumpukture. Sinusni sistem je proširen i ispunjen makrofagima koji u citoplazmi sadrže masne kapljice različite veličine koje imponuju kao prazne vakuole. Uz njih se vide epiteloidne i džinovske ćelije tipa stranog tela, ali nema pravih granuloma (sl.4B). Mogu se naći limfociti i plazmociti, a ponekad i eozinofili. Lipidne kapljice se mogu videti i unutar atrofičnih germinativnih centara. Diferencijalno dijagnostički, pored silikonske limfadenopatije u obzir dolaze i infekcije atipičnim mikobakterijama i gljivicama kao i Whippleova bolest. Slika 4. A. Lipidna limfadenopatija kao posledica ubrizgavanja Botoxa. Narušena građa limfnog tkiva usled prisustva brojnih krupnih masnih kapi bez ikakve reakcije u tkivu, H&E. B. Lipidna limfadenopatija.po ubrizgavanju kontrasta za angiografiju. Narušena građa limfnog tkiva usled prisustva brojnih masnih kapi i nakupljanja džinovskih ćelija tipa stranog tela, H&E. Detalj: Džinovske želije tipa stranog tela koje okružuju lipidne kapi. U citoplazmi se vide masne kapi, H&E. 486 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 HEMATOPATOLGIJA- kratki kurs 1/HEMATHOPATOLOGY Short Course 1 Imati u vidu: Reaktivne limfadenopatije mogu imati dramatičnu i „divlju“ histološku sliku tako da se lako mogu pogrešno dijagnostikovati kao limfom što može imati katastrofalne posledice po pacijenta. U razlikovanju folikularne hiperplazije od folikularnog NHL, prisustvo folikula van kapsule, pogotovo uz fibrozu, govori u prilog limfoma. Interfolikularno prisustvo centrocita je odlika folikularnog NHL. Germinativni centri u folikularnoj hiperplaziji su imunohistohemijski BCL2 negativni. Uvek imati na umu BCL2 negativan folikularni NHL. U slučaju sumnje na BCL2 negativan folikularni NHL obavezno uraditi FISH ili PCR jer postoji BCL2 rearanžman. Manje monoklonske B- i T-ćelijske populacije se mogu naći u reaktivnoj hiperplaziji. Ovakav nalaz uvek tumačiti u kontekstu kliničke i morfološke slike. Ako posle svih dostupnih ispitivanja niste sigurni da li je reč o FH ili nekom NHL bolje se izjasniti da je benigno, uz savet da se bolesnik klinički prati (watch and wait). Toksoplazmozni limfadenitis odlikuje trijada: folikularna hiperplazija, sitni klasteri epiteloidnih histiocita i hiperplazija monocitoidnih B-ćelija unutar sinusoida. Ovaj morfološki nalaz se mora potvrditi serološki i/ili imunohistohemijski i PCR reakcijom u tkivu. Klinički podaci o kozmetičkim, rekonstruktivnim ili ortopedskim protezama kao i o davanju kontrasta i lekova na bazi ulja su presudni za postavljanje dijagnoze silikonske ili lipidne limfadenopatije. Literatura: 1. Fijten GH, Blijham GH. Unexplained lymphadenopathy in family practice. An evaluation of the probability of malignant causes and the effectiveness of physicians’ workup. J Fam Pract. 1988;27(4):373-6. 2. Chau I, Kelleher MT, Cunningham D, Norman AR, Wotherspoon A, Trott P, Rhys-Evans P, Querci Della Rovere G, Brown G, Allen M, Waters JS, Haque S, Murray T, Bishop L. Rapid access multidisciplinary lymph node diagnostic clinic: analysis of 550 patients. Br J Cancer. 2003;10;88(3):354-61. 3. Ferrer R. Lymphadenopathy: differential diagnosis and evaluation. Am Fam Physician. 1998;58(6):1313-20. 4. Brown JR, Skarin AT. Clinical mimics of lymphoma. Oncologist. 2004;9(4):406-16. 5. Peric Lj, Zidovec-Lepej S, Jeren T, Kozic S, Peric N. Multifaktorijalno istrazivanje nemalignih limfadenopatija. Croatian J of Infection. 2005:25(3):1005-110. 6. Good DJ, Gascoyne RD. Atypical lymphoid hyperplasia mimicking lymphoma. Hematol Oncol Clin North Am. 2009;23(4):729-45. 7. Habermann T, Steensma DP. Lymphadenopathy. Mayo Clin Proc. 2000;75(7):723-732. 8. Wilkins BS. Pitfalls in lymphoma pathology: avoiding errors in diagnosis of lymphoid tissues. J Clin Pathol. 2011;64(6):466-76. 9. Weiss L, Loera S, Bacchi CE. Immunoglobulin light chain immunohistochemistry revisited, with emphasis on reactive follicular hyperplasia vs. Follicular lymphoma. Appl Immunohistochem Mol Morphol. 2010;18(3):199–205. 10.JH van Krieken, AW Langerak, EA Macintyre, M Kneba, E Hodges, RG Sanz, GJ Morgan, A Parreira, TJ Molina, J Cabecadas, P Gaulard, B Jasani, JF Garcia, M Ott, ML Hannsmann, F Berger, M Hummel, F Davi, M Bruggemann, FL Lavender, E Schuuring, PA Evans, H White, G Salles, PJ Groenen, P Gameiro, Ch Pott, and JJ Dongen. Improved reliability of lymphoma diagnostics via PCR-based clonality testing: report of the BIOMED-2 Concerted Action BHM4-CT98-3936. Leukemia, 2007;21(2):201-6. 11.Langerak AW, Molina TJ, Lavender FL, Pearson D, Flohr T, Sambade C, Schuuring E, Al Saati T, van Dongen JJ, van Krieken JH. Polymerase chain reaction-based clonality testing in tissue samples with reactive lymphoproliferations: usefulness and pitfalls. A report of the BIOMED-2 Concerted Action MH4-CT98-3936. Leukemia. 2007;21(2):222-9. 12.Sevilla DW, Murty VV, Sun XL, Nandula SV, Mansukhani MM, Alobeid B, Bhagat G. Cytogenetic abnormalities in reactive lymphoid hyperplasia: byproducts of the germinal centre reaction or indicators of lymphoma? Hematol Oncol. 2011;29(2):81-90. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 487 HEMATOPATOLGIJA- kratki kurs 1/HEMATHOPATOLOGY Short Course 1 13.Loi TH, Campain A, Bryant A, Molloy TJ, Lutherborrow M, Turner J, Yang YH, Ma DD. Discriminating lymphomas and reactive lymphadenopathy in lymph node biopsies by gene expression profiling. BMC Med Genomics. 2011;4:27. 14.Djurković-Djaković O, Bobić B, Klun I. Toxoplasmosis in Serbia: time for an action plan. Parasite. 2010 Sep;17(3):187-92. 15.Bobić B, Nikolić A, Đurković-Đaković O. Identifikacija faktora rizika za infekciju parazitom toxoplasma gondii u Srbiji kao osnov programa prevencije kongenitalne toksoplazmoze. Srp Arh Celok Lek. 2003, 31(3-4):162-167. 16.Eapen M, Mathew CF, Aravindan KP. Evidence based criteria for the histopathological diagnosis of toxoplasmic lymphadenopathy. J Clin Pathol. 2005;58(11):1143-6. 17.Lin MH, Kuo TT. Specificity of the histopathological triad for the diagnosis of toxoplasmic lymphadenitis: polymerase chain reaction study. Pathol Int. 2001;51(8):619-23. 18.Wintsh W, Smahel J, Clodius L. Local and regional lymph node response to ruptured gel-filled mammary protheses. Br J Plast Surg. 1978;31:349-352. 19.Péoc’h M, Duprez D, Grice G, Fabre-Bocquentin B, Gressin R, Pasquier B. Silicone lymphadenopathy mimicking a lymphoma in a patient with a metatarsophalangeal joint prosthesis. J Clin Pathol. 2000;53(7):549-51. 20.Van Diest PJ, Beekman WH, Hage JJ. Pathology of silicone leakage from breast implants. J Clin Pathol. 1998;51(7):493-7. 21.Smith T. Fatty replacment of lymph nodes mimicking lymphoma relapse. Cancer. 1986;58:2686-2688. 488 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 HEMATOPATOLGIJA- kratki kurs 1/HEMATHOPATOLOGY Short Course 1 Difuzni B krupnoćelijski limfom: varijante, podgrupe i podtipovi Diffuse large B cell lymphoma: variants, subgroups and subtypes/entities Maja Peruničić Jovanović Maja Perunicic Jovanovic Služba za patohistologiju, Klinički centar Srbije, Beograd, Srbija Department of Pathology, Clinical Center of Serbia, Belgrade, Serbia Apstrakt Difuzni B krupnoćelijski limfom (DBKL) je najčešći limfoidni tumor i predstavlja klinički, patološki i biološki veoma heterogenu grupu tumora. Novije studije su podelile difuzne B krupnoćelijske limfome u različite morfološke varijante, molekularne i imunofenotipske podgrupe i entitete.Imunofenotipska podela DBKL, na tip porekla germinativnog centra (GCB) i negerminativnih (non-GCB), koristeći kombinaciju antitela CD10, BCL6 i MUM1, ne korelira u potpunosti sa profilom genske ekspresije, GCB tipa i aktiviranih B ćelija (ABC). Neke studije su pokazale da kombinacija ekspresije CD10, BCL6 i MUM1 može podeliti pacijente sa DBKL u one sa dužim i kraćim preživljavanjem.Klasifikacija Svetske Zdravstvene Organizacije (SZO) iz 2008. godine prepoznaje grupu agresivnih B ćelijskih limfoma koja se ne može klasifikovati ni kao Burkitt-ov limfom ni DBKL, i kategoriju B ćelijskih neoplazmi sa karakteristikama između DBKL i klasičnog Hodgkin-ovog limfoma.Takođe, nova klasifikacija prepoznaje starosno doba pacijenta, mesto nastanka tumora i kliničke faktore u definisanju varijanti DBKL. Klasifikacija SZO iz 2008. je rezultat uspešne međunarodne saradnje između patologa, biologa i kliničara, ali heterogena grupa DBKL će i dalje biti predmet istraživanja. Ključne reči: difuzan veliki B ćelijski limfom, WHO klasifikacija, genski profil Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma worldwide, and represents a clinically, pathologically and biologicaly very heterogeneous group of tumours. Recent studies have subdivided diffuse large B cell lymphomas into morphological variants, molecular and immunophenotypical subgroups and distinct entities. An immunophenotypical subdivision of DLBCL, into germinal centre-like (GCB) and non-germinal centre-like (non-GCB) subgroups, using a combination of antibodies to CD10, BCL6 and MUM1, does not correlate exactly with gene expresion profile of GCB and activated peripheral B-cells (ABC). Some studies reported that combination of CD10, BCL6 and MUM1 expression could subdivide DLBCL patients into long- and short-time survivors.The WHO classification of 2008. recognizes a group of aggressive B-cell lymphomas that are not readily classified as either Burkitt lymphoma (BL) or DLBCL, and provisional category of B-cell neoplasms with features intermidiate between DLBCL and classical Hodgkin lymphoma. Furthermore, the new classification recognizes the patient age, site-specific categories, and clinical factors in defining variants of DLBCL.The WHO classification of 2008. is the result of successful international collaboration among pathologists, biologists and clinicians, but heterogeneous group of DLBCL will be the subject of further investigation. Key words: diffuse large B cell lymphoma, WHO classification, gene expresion profil Uvod Difuzni B krupnoćelijski limfom (DBKL) je najčešći limfoidni tumor i čini oko 30-40% svih non-Hodgkin limfoma kod odraslih, sa petogodišnjim preživljavanjem oko 50%1. Predstavlja grupu tumora koja je heterogena po svom kliničkom ishodu, morfologiji, imunofenotipu, molekularnoj genetici i citogenetici 1,2. Morfološka, biološka i klinička heterogenost je prepoznata od strane Svetske Zdravstvene Organizacije (SZO), tako da u Klasifikaciji tumora hematopoetskog i limfoidnog tkiva SZO iz 2008. postoje različite morfološke varijante DBKL, molekularne i imunohistohemijske podgrupe DBKL, kao i različiti klinički entiteti. DBKL koji nisu drugačije specifikovani (not otherwise specified, NOS) obuhvataju one tipove DBKL koji ne pripadaju specifičnim podtipovima ili entitetima1. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 489 HEMATOPATOLGIJA- kratki kurs 1/HEMATHOPATOLOGY Short Course 1 Promene u klasifikaciji limfoma, koje su rezultat novih saznanja nastalih na osnovu kliničkih i laboratorijskih istraživanja, su pokušaj da se bolje definiše heterogenost ovih bolesti. Klasifikacija limfoma iz 2008. godine, predviđa prepoznavanje ranih i in situ lezija, prepoznavanje starosne dobi kao karakteristike određenih limfomskih entiteta, kako kod starijih osoba tako i kod dece, kao i prepoznavanje „border-line“ kategorija2. Takođe, u Četvrto izdanje klasifikacije Tumora Hematopoetskog i Limfoidnog tkiva, uključeni su i neki provizionalni entiteti 1,2 (Tabela 1.). Tip Difuzni B krupnoćelijski limfom, (DBKL) NOS Difuzni B krupnoćelijski limfom, podtipovi Ostali limfomi krupnih B ćelija “Borderline” limfomi B-ćelijski limfom, neklasifikovan, sa karakteristikama između DBKL i Burkitt-ovog limfoma B-ćelijski limfom, neklasifikovan, sa karakteristikama između DBKL i Hodgkin-ovog limfoma Morfološke varijante Centroblastni Imunoblastni Anaplastični Retke morfološke varijante Molekularne podgrupe GCB tip ABC tip Imunohistohemijske podgrupe CD5 + DBKL GCB Non-GCB DBKL T-cell/histiocyte-rich tip Primarni DBKL centralnog nervnog sistema Primarni kutani DBKL, leg type EBV pozitivni DBKL starijih Primarni medijastinalni B krupnoćelijski limfom Intravaskularni B krupnoćelijski limfom DBKL udružen sa hroničnom inflamacijom Limfomatoidna granulomatoza Intravaskularni B krupnoćelijski limfom ALK+ B krupnoćelijski limfom Plazmablastni limfom Krupnoćelijski limfom udružen sa HHV8+ Castleman-ovom bolešću Primarni efuzioni limfom Tabela 1. Klasifikacija tumora hematopoetskog i limfoidnog tkiva Difuzni B krupnoćelijski limfom, NOS (Diffuse large B cell lymphoma, not otherwise specified; DLBCL, NOS) Difuzni B krupnoćelijski limfom je neoplazma difuznog tipa rasta, sastavljena od krupnih limfoidnih ćelija koje eksprimiraju B ćelijske antigene (CD20, CD79α, ili Pax-5)1. Obično nastaju de novo, kao primarni, ali mogu nastati i transformacijom manje agresivnih limfoma kao što su hronična limfocitna leukemija, folikularni limfom, limfom marginalne zone ili limfoplazmocitni limfom. Najčešće se javlja u sedmoj deceniji života, ali se može videti i kod dece. Nešto je češći kod muškaraca u odnosu na žene. Bolest se prezentuje kao brzorastuća nodalna ili ekstranodalna tumorska masa, kod imunokompetentnih, ali i kod pacijenata sa različitim oblicima imunosupresije 1,2. 490 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 HEMATOPATOLGIJA- kratki kurs 1/HEMATHOPATOLOGY Short Course 1 Imunohistohemijska analiza je postala integralni deo dijagnostičke hematopatologije i neophodna je u dijagnostici limfoma3,4. Ograničen panel antitela (CD20, CD79α, CD10, BCL-6, MUM-1/IRF-4 i CD138) može mnogo pomoći u svrstavanju različitih tipova limfoma u skladu sa morfološkim izgledom i stepenom diferencijacije 4. Određeni markeri korespondiraju sa setom ključnih proteina uključenih u ćelijski ciklus, apoptozu i B ćelijsku diferencijaciju 4,5. U poslednjih 10 godina, na osnovu istraživanja u oblasti molekularne genetike i imunohistohemije, napravljen je veliki napredak u subklasifikaciji DBKL u klinički relevantne grupe: porekla germinativnog centra (GCB) i non-GCB tip ili aktiviranih B ćelija (ABC). Stadijum diferencijacije B limfocita na kome se dešava neoplastična transformacija može definisati biološko ponašanje i ishod bolesti kod pacijenata sa DBKL. Mnoge studije su pokazale da podela DBKL na grupu tumora koji imaju profil B ćelija porekla germinativnog centra (GCB) i na grupu tumora koji imaju profil aktiviranih B ćelija (ABC odnosno “non GCB”) ima klinički značaj, odnosno da pacijenti iz GCB grupe imaju značajno bolje preživljavanje. Odkako su prepoznati biološki podtipovi DBKL na bazi „gene expression profiling”, istraživanje kliničkog značaja ovakve podele je postalo predmet mnogih studija6,7,8. Međutim, ovakve analize nisu lako primenljive u svakodnevnoj praksi, jer zavise od dostupnosti zamrznutih uzoraka, kao i sofisticiranih laboratorijskih metoda. Hans i saradnici su predložili algoritam koji je baziran na imunohistohemijskoj ekspresiji sledećih markera: CD10, bcl-6 i MUM-1/IRF4, a pomoću koga se mogu razlikovati raziličite grupe DBKL 8. Kombinovanom analizom ekspresije ovih antitela u skladu sa predloženim algoritmom može biti surogat za “gene expression” potpis prognostički različitih tipova8. Difuzni B krupnoćelijski limfomi porekla germinativnog centra (GCB) su imunohistohemijski CD10+ (>30% ćelija) ili CD10-, BCL-6+ i IRF4/MUM-1-1. Svi ostali slučajevi se svrstavaju u non-GCB tip (Slika 1.). dBKL, centroblastni GCB tip CD10 bcl-6 MUM-1 CD10 bcl-6 MUM-1 dBKL, centroblastni non GCB tip Slika 1. Imunohistohemijski profil DBKL DBKL koji imaju profil B ćelija porekla germinativnog centra (GCB) imaju bolju prognozu od onih koji su non-GCB tip (imaju profil aktiviranih B ćelija (ABC)8, iako neke studije ne potvrđuju ovu hipotezu9. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 491 HEMATOPATOLGIJA- kratki kurs 1/HEMATHOPATOLOGY Short Course 1 Novi entiteti U definisanju nekoliko novih entiteta, starosno doba i mesto nastanka tumora igraju veliku ulogu. Promene u klasifikaciji B ćelijskih limfoma su nastale kao rezultat prepoznavanja važnosti mesta nastanka tumora kao i određenih kliničkih karakteristika10. EBV+ DBKL starijih osoba je klinički agresivan tumor, koji najverovatnije nastaje zbog oslabljenog imunološkog statusa. Češće se javlja na ekstranodalnim lokalizacijama. Tumorske ćelije su polimorfne, mogu podsećati na Hodgkin-ove i Reed-Strenberg-ove, uz čestu nekrozu i inflamatorni infiltrat. DBKL udružen sa hroničnom inflamacijom je drugi EBV+ tumor sa određenim kliničkim karakteristikama, koji nastaje u specifičnim situacijama, najčešće na terenu prolongirane hronične inflamacije. Druge specifične kategorije DBKL vezane za mesto nastanka su DBKL centralnog nervnog sistema i primarni kutani DBKL, „leg type“. Primarni kutani DBKL, „leg type“ pokazuje profil aktiviranih B ćelija (ABC) u većini slučajeva. Takođe, primarni CNS DBKL pokazuje specifičan genski potpis, na osnovu kojeg se izdvaja kao poseban entitet. “Borderline” limfomi Savremene studije su skrenule pažnju na biološko, morfološko i imunohistohemijsko preklapanje između klasičnog Hodgkin-ovog limfoma i nekih B krupnoćelijskih limfoma, posebno primarnog medijastinalnog B krupnoćelijskog limfoma (PMBL) i medijastinalnog klasičnog Hodgkinovog limfoma, (cHL) tip nodularne skleroze. „gene expression profiling”, analize su potvrdile biološku povezanost ovih tumora. Obe neoplazme nastaju u medijastinumu, kod mlađih osoba1,2,10. Klasifikacija SZO iz 2008. prepoznaje kategoriju tumora nazvanu B ćelijska neoplazma, sa karakteristikama između DBKL i Hodgkin-ovog limfoma. Ovi tumori nastaju najčešće kod mlađih muškaraca i ponašaju se agresivnije od PMBL i cHL. Takođe, prepoznata je grupa limfoma koju nije moguće klasifikovati ni kao Burkitt ni kao DBKL.Ova kategorija je nazvana B-ćelijski limfom, sa karakteristikama između DBKL i Burkitt-ovog limfoma1,2,10. Ovi limfomi se javljaju kod odraslih osoba i pokazuju imunofenotip germinativnog centra i podsećaju na Burkittov limfom ali nemaju citomorfološke karakteristike Burkitt-ovog limfoma.Ovaj entitet takođe uključuje slučejeve sa translokacijom i myc i bcl-2 („double hit“). Finalnu dijagnozu je moguće postaviti kao skup morfoloških, imunofenotipskih i molekularnih karakteristika1,2,10. Terapija Standardni terapijski pristup za pacijente sa DBKL je CHOP (cyclophosphamide, doxorubicine, vincristine, prednisone) ili u kombinaciji sa rituximab-om (R-CHOP)1,6,9,10,11. Pri donošenju odluke koji terapijski protokol primeniti, rukovodimo se mnogobrojnim parametrima u cilju tzv. individualizacije terapije. Svakako da najveći značaj imaju prognozni indeksi, odnosno, Internacionalni Prognostički Indeks (IPI) za agresivne limfome, a na osnovu kojih se bolesnici dele u one sa niskim, srednjim ili visokim rizikom za nastanak progresije bolesti11. Internacionalni Prognostički Indeks (IPI), se bazira na pet nezavisnih prognostičkih faktora, uključujući starosnu dob, Ann Arbor tumorski stadijum, nivo serumske laktat dehidrogenaze (LDH), performans status i broj ekstranodalnih lokalizacija. Zaključak U svim kliničkim modelima, uključujući IPI indeks, postoji značajna heterogenost u ishodu, koja se ogleda u različitom preživljavanju pacijenata sa identičnim prognostičkim skorom2,7,11. Iako značajan procenat pacijenata sa DBKL može biti uspešno lečen kombinacijom različitih terapijskih procedura, ne postoji dostupan ni biološki ni klinički skor koji bi razlikovao pacijente koji mogu biti lečeni standardnom terapijom i one pacijente koji zahtevaju novi terapijski pristup7,11. 492 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 HEMATOPATOLGIJA- kratki kurs 1/HEMATHOPATOLOGY Short Course 1 Mnoge studije su pokazale da podela DBKL na grupu tumora koji imaju profil B ćelija porekla germinativnog centra (GCB) i na grupu tumora koji imaju profil aktiviranih B ćelija (ABC odnosno “non GCB”) ima klinički značaj, odnosno da pacijenti iz GCB grupe imaju značajno bolje preživljavanje. Umesto skupe i teško dostupne tehnologije “cDNA microarray gene expression profiling” može se koristiti imunohistohemijska analiza, pomoću koje je moguće klasifikovati DBKL u molekularno i prognostički različite grupe. Ovakav pristup omogućava široku praktičnu primenu u svakodnevnoj kliničkoj praksi. Od izuzetnog značaja je identifikovati, u vreme postavljanja dijagnoze, one pacijente koji mogu imati koristi od agresivnijeg terapijskog pristupa. Promene u klasifikaciji limfoma iz 2008. su nastale kao rezultat saradnje između patologa, kliničara, biologa u pokušaju da se poboljša preciznost dijagnostikovanja i terapijski pristup1,2,10. Dati su odgovori na moga pitanja ali istovremeno otvorena nova koja će biti predmet istraživanja u budućnosti, posebno u heterogenoj grupi DBKL, NOS. Literatura: 1. Swerdlow S.H., Campo E., Harris N.L., Jaffe E.S., Pileri S.A., Stein H., Thiele J., Vardiman J.W. (Eds): WHO Classification of Tumours of Haematopoietic and lymphoid Tissues. IARC: Lyon 2008 2. Jaffe E.S.:The 2008 WHO classification of lymphomas: implication for clinical practice and translational research. Hematology Am Soc Hematol Educ Program. 2009:523-31. 3. Pileri S A, Dirnhofer S, Went Ph, Ascani S, Sabattini E, Marafioti T, Tzankov A, Leoncini L, Falini B & Zinzani P L. Diffuse large B-cell lymphoma: one or more entities? Present controversies and possible tools for its subklassification. Histopathology 2002;41: 482-509 4. Garcia F. C, MD; Swerdlow H.S, MD. Best practices in Contemporary Diagnostic immunohistochemistry. Panel Approach to Hematolymphoid Proliferations. Arch Pathol Lab Med 2009; 133:756-765. 5. Russell A. Higgins, MD; Jennofer E. Blankenship, MD; Marsha C. Kinney, MD: Application of Immunohistochemistry in the Diagnosis of Non-Hodgkin and Hodgkin Lymphoma. Arch Pathol Lab Med 2008;132:441-461. 6. Lossos IS, Morgensztern D. Prognostic biomarkers in diffuse large B-cell lymphoma. J Clin Oncol. 2006;24:995-1007. 7. De Yong D, Rosenwald A, Chhanabhai M, Gaulard P, Klapper W, Lee A, et al. Immunohistochemical prognostic markers in diffuse large B-cell lymphoma: validation of tissue microarray as perequisite for broad clinical applications- A study from Lunenburg Lymphoma Biomarker Consortium. J Clin Oncol. 2007; 25:805-812. 8. Hans CP, Weisenburger DD, Greiner TC, Gascoyne RD, Delabie J, Ott G et al. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood. 2004;103:275-282. 9. Gustaaf W. van Imhoff et al. Prognostic Impact of Germinal Center-Associated Proteins and Chromosomal Breakpoints in poor-Risk Diffuse Large B-Cell Lymphoma. Journal of Clinical Oncology, 2006;25:4135-4142. 10.Jaffe S. E. and Pittaluga S.:Aggressive B-Cell Lymphomas: A Review of New and Old Entities in the WHO Classification. Hematology Am Soc Hematol Educ Program. 2011;2011:506-14. 11.Biasoli I, Morais JC, Scheliga A, Milito CB, Romano S, Land M et al. CD10 and Bcl-2 expression combined with the International Prognostic index can identify subgroups of patients with diffuse large-cell lymphoma with very good or very poor prognoses. Histopathology. 2005; 46(3):328-33. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 493 HEMATOPATOLGIJA- kratki kurs 2/HEMATHOPATOLOGY Short Course 2 Problematic “Low Grade” Lesions in Lymphoproliferative Pathology Stefan Dojcinov All Wales Lymphoma Panel University Hospital of Wales, Cardiff, United Kingdom Abstract Pathological diagnosis of lymphoproliferative processes has been associated with a high error rate of 1735%, compared to a low diagnostic error incidence of 1-3% in general histopathology. In lymphoma diagnosis, one half of the diagnostic errors result in significant clinical consequences such as delayed or inappropriate therapy, unnecessary treatment, avoidable morbidity and compromised survival.1 Inherent pathological ambiguity of lymphoproliferative processes, interpretational subjectivity, unfamiliarity with diagnostic criteria and novel entities, lack of expert opinion, inappropriate laboratory support and poor clinico-pathological correlation are the main reasons behind most pitfalls in this subspecialty. Over the past 20 years, advances in classification have aided pathologists to better characterise lymphoid proliferation. The WHO lymphoma classification has imposed additional requirements for genetic and molecular interrogations. As a result, it has now been widely advocated that diagnosis of lymphoproliferative processes is centralised and conducted in dedicated, highly specialised laboratories.2 However, even in the context of central pathology review of lymphoid proliferations, the initial steps in selecting cases for referral depend on the diagnostic skills of generalist pathologists. Their familiarity with reactive conditions many of which closely mimic neoplasms, is essential. Reactive lymphoproliferations represent 10-20% of the cases referred to subspecialist diagnostic services. This high referral rate outlines the pathological ambiguity of these conditions and their difficult differential diagnosis with lymphoma. In many instances, this is complicated by a dramatic clinical presentation resulting in high concern and “pressure” from clinicians, which may further compromise diagnostic confidence. Such conditions comprise a variety of morphological patterns including follicular proliferations, paracortical expansions, intrasinusoidal and necrotizing processes. In the recent update of the WHO lymphoma classification there is a “plethora” of new entities which further widen the differential diagnosis of reactive lesions and “low grade” lymphomas. This particularly refers to a spectrum of early lesions which sit at the interface between the reactive and neoplastic. In the context of this review, “low grade” lesions are not considered to represent a homogeneous group of entities. This colloquial term is here used for diverse both reactive and neoplastic processes the clinical course of which is mostly non-aggressive, even though the morphological features may sometimes imply otherwise. Examples of difficult differential diagnoses and newly recognised entities with their clinical implications are discussed with emphasis on the importance of clinico-pathological correlation. Reactive lymphoproliferations with nodular architecture may mimic lymphoma (and vice versa): Progressive transformation of germinal centres is a rare reactive process of uncertain aetiology which may result in bulky lymphadenopathy. The morphological features mimic nodular lymphocyte predominant Hodgkin lymphoma or follicular lymphoma. Diagnosis relies on the identification of disrupted but retained components of the lymphoid follicles, without L&H cells. Other nodular/follicular proliferations including florid non-specific follicular hyperplasia and hyaline vascular variant of Castleman’s disease pose a differential diagnosis with lymphomas exerting a nodular growth pattern. This relates primarily to variants of follicular lymphoma (FL)(Grade 3A/B, “floral variant”, FL with hyaline vascular changes). Appreciation of retained lymph node architecture is essential for diagnosis. Difficult cases may need clonality and genetic studies.3 “In situ” and early neoplastic lesions in lymphoproliferative pathology do exist: Follicular lymphoma in situ is a recently recognised entity representing an early phase of neoplastic transformation with uncertain 494 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 HEMATOPATOLGIJA- kratki kurs 2/HEMATHOPATOLOGY Short Course 2 potential for progression.4,5 The typical morphological and immunophenotypic features distinguish it from partial involvement of lymph nodes by systemic lymphoma. Recognition of this condition prevents unnecessary treatment and patient anxiety. Early and “in situ” forms of other lymphomas include monoclonal B-cell lymphocytosis 6, 7, “in situ” mantle cell lymphoma8, intestinal FL9, intraepithelial monoclonal T-cell lymphoproliferation associated with refractory celiac disease10 and early lesions of EBV positive age related lymphoproliferations.11, 12 Accurate recognition of these entities has important clinical implications. Lymphomas may display “reactive” architectural and immunocytochemical patterns; “Aggressive” looking morphology does not always indicate an aggressive clinical course: Follicular lymphoma grade 3B is part of the morphological spectrum of FL but appears to be different from grades 1, 2 and 3A. The genetic makeup of grade 3B is closer to the clinically aggressive diffuse large B-cell lymphoma.13, 14 Due to high proliferation, abundance of tingible body macrophages and immunohistochemical negativity for Bcl2 FL grade 3B could be misinterpreted as reactive follicular hyperplasia. This is particularly problematic in cases of paediatric FL.15 In adults, grade 3B is currently managed as most other aggressive large B-cell lymphoma, so accurate diagnosis of this subgroup is of high clinical relevance. However, childhood cases pursue an indolent course, despite grade 3 morphology. Florid, atypical blastic lymphoproliferations could be reactive: Infectious mononucleosis affects young individuals often presenting as an alarming, rapidly developing lymphadenopathy with a worrying histological appearance which may suggest lymphoma. Diagnosis relies on the identification of the polymorphous nature of the infiltrate, retention of lymph node architecture and EBV positivity. The differential diagnosis usually includes classical Hodgkin lymphoma or T-cell rich B-cell lymphoma. In very young patients a consideration should also be given to EBV positive T-cell lymphoproliferation of childhood. Similar problems may be seen with Kikuchi lymphadenitis, drug induced or post-vaccinal reactions further expanding the differential diagnosis. Clinico-pathological correlation is essential but early laboratory investigations (“monospot”) may be misleading.16, 17 Dramatic clinical presentation with massive or generalised lymphadenopathy is not always due to lymphoma: Rosai – Dorfman disease (sinus histiocytosis with massive lymphadenopathy) presents with massive localised or generalised lymph node enlargement, occasionally with extranodal involvement. Severe constitutional symptoms are common. Diagnosis relies on the identification of an intrasinusoidal proliferation of histiocytes with a specific S100/CD68 positive immunophenotype. The course is self limiting but administration of steroids may be beneficial.16 The differential diagnosis includes non-specific sinus histiocytosis and infection but also a sinusoidal spread of tumours such as carcinoma, melanoma, angiosarcoma, anaplastic large cell lymphoma or “villous/sinusoidal” large B-cell lymphoma. Examples of other reactive conditions resulting in a dramatic clinical presentation where the pathological diagnosis may be problematic include plasma cell variant of Castleman’s disease18 and infectious mononucleosis. Clinical history and correlation are essential for accurate diagnosis, prognostication and choice of management: EBV positive mucocutaneous ulcer (EBVMCU) is a localised immunosuppression associated B-cell lymphoproliferation involving skin and a range of mucosal sites, most commonly oropharynx. It displays Hodgkin-like morphological features and is frequently misdiagnosed as such. Despite its aggressive appearance, EBVMCU is an “early lesion” and follows an indolent course upon reduction of immunosuppression. However, with sustained iatrogenic immunosuppression this process is locally destructive and patients may receive unnecessary aggressive treatment.11, 12 This new entity is used to highlight a range of lymphoproliferations associated with various immunosuppressive aetiologies.11, 12, 14 History of immunosuppression is often omitted from the information available to the pathologist and some clinicians may not be aware of its importance in the context of B-cell lymphoproliferations.14, 16, 17 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 495 HEMATOPATOLGIJA- kratki kurs 2/HEMATHOPATOLOGY Short Course 2 Formalised communication between pathologists and clinicians in the form of Multidisciplinary Meetings (MDM) would prevent most misdiagnosis due to lack of relevant clinical information. It hugely aids diagnosis and management and has become essential. Professional regulatory and advisory bodies highlight the mandatory requirement for MDMs as part of the patient management pathway.19 Key learning points: All the common patterns of reactive lymphoid hyperplasias morphologically overlap with a range of lymphomas. Recognition of architecture retention by simple stains such as CD20, CD3 and bcl2 is often very helpful. Difficult cases may require additional clonality or cytogenetic studies; “In situ” and early lymphoms are being increasingly characterised. Awareness of these conditions is needed to prevent unnecessary treatment and to distinguish them from partial involvement by systemic disease; Reactive lymphoproliferations may result in a worrying histological picture as seen in infectious mononucleosis. This is one of the most misdiagnosed reactive lymphadenitidies with potentially catastrophic clinical consequences. Clinicopathological correlation is essential but laboratory tests may be misleading; Other reactive lymphadenitidies may have dramatic clinical presentation as seen in Rosai-Dorfman disease, Kikuchi disease and plasma cell variant of Castleman’s disease. In all these conditions conservative therapy is beneficial. While the clinical context is always important, the alarming clinical presentation and increased pressure from clinicians should not impact upon the pathological diagnosis; Immunosuppression associated B-cell lymphoproliferations represent a wide biological and pathological spectrum including the new entity of EBV positive mucocutaneous ulcer. Information on immunosuppressive management is frequently lacking from pathology requests. Information on immunosuppression should be specifically sought; Referral of reactive conditions to specialist diagnostic services is not unjustified as their precise characterisation may require complex ancillary studies which are routinely unavailable; MDM has become an essential and mandatory principle in the management of patients. Literature 1. Lester JF, Dojcinov SD, Attanoos RL, et al. The clinical impact of expert pathological review on lymphoma management: a regional experience. Br J Haematol. 2003;123:463-8. 2. Jaffe ES. Centralized review offers promise for the clinician, the pathologist, and the patient with newly diagnosed lymphoma. J Clin Oncol. 2011;29:1398-9. 3. Dojcinov SD. Self-Assessment: Lymph node pathology. Current Diagnostic Pathology. 2004;10:500-10. 4. Cong P, Raffeld M, Teruya-Feldstein J, et al. In situ localization of follicular lymphoma: description and analysis by laser capture microdissection. Blood. 2002;99:3376-82. 5. Jegalian AG, Eberle FC, Pack SD, et al. Follicular lymphoma in situ: clinical implications and comparisons with partial involvement by follicular lymphoma. Blood. 2011;118:2976-84. 6. Dagklis A, Fazi C, Sala C, et al. The immunoglobulin gene repertoire of low-count chronic lymphocytic leukemia (CLL)-like monoclonal B lymphocytosis is different from CLL: diagnostic implications for clinical monitoring. Blood. 2009;114:26-32. 7. Gibson SE, Swerdlow SH, Ferry JA, et al. Reassessment of small lymphocytic lymphoma in the era of monoclonal B-cell lymphocytosis. Haematologica. 2011;96:1144-52. 8. Carvajal-Cuenca A, Sua LF, Silva NM, et al. In situ mantle cell lymphoma: clinical implications of an incidental finding with indolent clinical behavior. Haematologica. 2012;97:270-8. 9. Schmatz AI, Streubel B, Kretschmer-Chott E, et al. Primary follicular lymphoma of the duodenum is a distinct mucosal/submucosal variant of follicular lymphoma: a retrospective study of 63 cases. J Clin Oncol. 2011;29:1445-51. 496 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 HEMATOPATOLGIJA- kratki kurs 2/HEMATHOPATOLOGY Short Course 2 10.Bagdi E, Diss TC, Munson P, et al. Mucosal intra-epithelial lymphocytes in enteropathy-associated T-cell lymphoma, ulcerative jejunitis, and refractory celiac disease constitute a neoplastic population. Blood. 1999;94:260-4. 11.Dojcinov SD, Venkataraman G, Raffeld M, et al. EBV Positive Mucocutaneous Ulcer-A Study of 26 Cases Associated With Various Sources of Immunosuppression. Am J Surg Pathol. 2010. 12.Dojcinov SD, Venkataraman G, Pittaluga S, et al. Age-related EBV-associated lymphoproliferative disorders in the Western population: a spectrum of reactive lymphoid hyperplasia and lymphoma. Blood. 2011;117:4726-35. 13.Ott G, Katzenberger T, Lohr A, et al. Cytomorphologic, immunohistochemical, and cytogenetic profiles of follicular lymphoma: 2 types of follicular lymphoma grade 3. Blood. 2002;99:3806-12. 14.Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue. Lyon: IARC; 2008. 15.Agrawal R, Wang J. Pediatric follicular lymphoma: a rare clinicopathologic entity. Arch Pathol Lab Med. 2009;133:142-6. 16.Ferry J.A., Harris N.L. Reactive Lymphoidd Hyperplasia. In: Atlas of Lymphoid Hyperplasia and Lymphoma W.B. Saunders Company; 1997:9-62. 17.Chan JK, Kwong YL. Common misdiagnoses in lymphomas and avoidance strategies. Lancet Oncol. 2010;11:579-88. 18.Dupin N, Diss TL, Kellam P, et al. HHV-8 is associated with a plasmablastic variant of Castleman disease that is linked to HHV-8-positive plasmablastic lymphoma. Blood. 2000;95:1406-12. 19.National Institute forClinical Excellence. Guidance on Cancer Services: Improving Outcomes in Haematological Cancers; The Manual: National Institute for Clinical Excellence, UK; 2003. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 497 HEMATOPATOLGIJA- kratki kurs 2/HEMATHOPATOLOGY Short Course 2 Problematic “High Grade” Lesions in Lymphoproliferative Pathology Snjezana Dotlic Department of Pathology and Cytology University Hospital Centre Zagreb, Croatia High error rate in primary lymphoma diagnosis by generalist pathologists has imposed a requirement for a redesign of diagnostic services in this subspecialty. 1 In all developed countries and in most of the countries in transition, diagnosis of lymphoid proliferations has become centralised, relying on regional panels of experts and dedicated specialised laboratories. However, the initial steps in the management and subspecialist referral of patients with suspected lymphoma still rely on diagnostic skills of general histopathologists. Their detailed awareness of classification changes, diagnostic requirements and standards is essential for the success of the diagnostic pathway. Here a range of scenarios are highlighted where the outcome of the initial pathological assessment, before any specialist investigations have been carried out, could “sidetrack” the referral process and adversely affect management. The term “High Grade” in this context is used for lesions histologically characterised by high pleomorphism and “blastic” appearance and also for processes with an aggressive clinical behaviour. Over the past two decades the wealth of accumulated knowledge on the biology of lymphoid cells and lymphomas culminated in a series of classifications which emphasised the need for extensive immunophenotypic and genetic interrogation of lymphoid proliferation in the course of pathological diagnosis. 2 As a consequence gone past are the days when treatment of lymphomas could commence after morphological assessment alone. On morphological grounds so many different aggressive lymphomas may show striking similarity. Burkitt lymphoma (BL), blastoid variant of mantle cell lymphoma, lymphomas of the “grey zone” between BL and diffuse large B-cell lymphoma (DLBCL), lymphoblastic lymphoma, plasmacytoid dendritic cell neoplasm and many others may show very similar morphology. This morphological mimicry is further complicated by similarities aggressive lymphoid malignancies may in certain circumstances show with non-haematological malignancies and reactive, inflammatory conditions. Examples of this contentious spectrum are provided together with an update of the most recent classification changes and the impact this has made on the practicalities of pathological diagnosis and management. Abundant reactive lymphoid infiltrate is seen in a range of different tumours: Follicular dendritic cell tumour / sarcoma is an example of this category. This is an uncommon entity displaying a spectrum of biological behaviour involving lymph nodes and a range of extranodal sites. The tumour cells amongst the abundant reactive lymphoid infiltrate could show spindle cell morphology, epithelioid or Reed-Sternberg-cell features. Accurate diagnosis relies on the recognition of the specific immunophenotype (CD21, CD23, CD35, clusterin). 2-4 This is of benefit only if this tumour is included in the initial differential diagnosis which should also consider tumours such as classical Hodgkin lymphoma, T-cell rich B-cell lymphoma, “lymphoepithelioma-like” carcinoma, inflammatory myofibroblastic tumour, metastatic germ cell tumour, medullary carcinoma of breast, “B-type” thymomas, inflammatory pleomorphic sarcoma or interdigitating dendritic cell sarcoma. Aggressive lymphomas may be negative for commonly used lymphoid lineage markers: ALK positive large B-cell lymphoma is a rare entity characterised by a high degree of pleomorphism and epithelioid morphology. 5 This aggressive lymphoma in addition displays an aberrant phenotype, lacking expression of CD45 and other lineage markers. The initial use of broad immunocytochemical screens may classify this lymphoma as undifferentiated malignancy. A range of haematolymphoid neoplasms may display loss of expression or are by definition characterised by the absence of markers generally considered to be robust lineage discriminators. Such tumours are myeloma, plasmablastic lymphomas, anaplastic large cell lymphoma and classical Hodgkin lymphoma which may all pose a difficult differential diagnosis with non-haematological malignancies. Plasma cell myeloma as well as other haematological malignancies may also aberrantly express cytokeratins, which in the context of paucity of expression of other B-cell lineage markers could be highly confusing.6 In addition, a common tumour such as small cell carcinoma of lung on occasions expresses 498 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 HEMATOPATOLGIJA- kratki kurs 2/HEMATHOPATOLOGY Short Course 2 lymphoid lineage marker PAX5 and lacks most other lineage markers, thus morphologically and immunophenotypically closely resembling lymphoblastic lymphoma. 7 Marked histiocytic infiltrate and granulomatous reaction may morphologically obscure aggressive lymphomas: Lymphoepithelioid (Lennert) lymphoma is a morphological variant of peripheral T-cell lymphoma of unspecified type. Due to its rich histiocytic infiltrate with formation of granulomas, Lennert lymphoma is frequently misinterpreted as an inflammatory process. 2;8 Granulomatous inflammation and rich histiocytic infiltrate could be diagnostically misleading in a range of lymphoma types such as BL lymphoma 9, classical Hodgkin lymphoma, nodular lymphocyte predominant Hodgkin lymphoma, T-cell rich B-cell lymphoma, mediastinal large B-cell lymphoma and lymphomatoid granulomatosis.10 Classification changes and how they affect practice: The update of the WHO classification (2008) introduced several new entities.2 It focused on resolving contentious differential diagnoses and providing pathologists and clinicians with therapeutically meaningful classification categories. To resolve sometimes difficult differential diagnosis between BL and DLBCL, a new category of “B-cell lymphoma, unclassifiable, with features intermediate between BL lymphoma and DLBCL” was introduced. This “grey zone” category encompasses lymphomas with atypical, BL-like morphological features but also with atypical immunophenotypes and genotypes, including the clinically aggressive “double hit” tumours. 11-13 The practical consequence of this attempt at more stringent classification is that pathological diagnosis of BL and the “grey zone” overlap with DLBCL requires mandatory genetic testing for MYC gene rearrangements but also for other most commonly encountered genetic abnormalities which include rearrangements of the BCL2 and BCL6 genes. In addition, patterns of MYC rearrangements in BL and those lymphomas which would fall into the “grey zone” category are different requiring specific testing for both immunoglobulin and non-immunoglobulin gene partners. The controversy of the differential diagnosis between BL and DLBCL has not been eliminated with the introduction of this category. The strongest criticism of this category is that it does not concern “typical” DLBCLs based on subjective assessment of morphology alone. There is accumulating evidence for the need of systematic genetic testing of morphologically typical DLBCLs which may also show a range of genotypes including those similar to BL and with much more diversity as seen in the “double hit” cases. This could only be resolved by a consensus on systematic genetic testing of all or most of DLBCLs and with additional clinical agreement how to treat this cases which are currently still being classified using diversely different approaches. 14 This will inevitably change practice of pathologists in the near future, requiring extensive routine genetic diagnosis in the context of highly specialised laboratories. Key learning points: Generalist pathologists play an essential initial role in the process of centralised, sub specialist diagnosis of lymphoid malignancies; Their awareness of non-haematological mimics of lymphoid tumours, and vice versa, is highly important for the process of case selection for the specialist referral pathway; A range of lymphomas and non-haematological malignancies are characterised by a dense reactive lymphoid infiltrate. In the differential diagnosis even rare entities such as follicular dendritic cell sarcoma should be considered to enable specific immunocytochemical diagnosis; A range of high grade lymphomas harbour abundant histiocytic infiltrate and granulomatous reaction which may obscure the underlying neoplastic process; Lack of expression of lineage markers such as CD45, CD3 and CD20 does not exclude diagnosis of aggressive lymphomas; Other tumours may in addition mimic lymphomas, expressing markers conventionally associated to lymphoid lineage; New classification changes introduced even more controversy and require further consensus amongst the experts regarding genetic classification of BL and DLBCL. Further consensus on specific treatments is also needed in this context; Diagnosis of haematological malignancies has become dependant on systematic genetic interrogation of the biopsy materials; This is increasingly becoming mandatory for more and more entities and forms basis for entity specific therapies. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 499 HEMATOPATOLGIJA- kratki kurs 2/HEMATHOPATOLOGY Short Course 2 Literature 1. Lester JF, Dojcinov SD, Attanoos RL et al. The clinical impact of expert pathological review on lymphoma management: a regional experience. Br.J.Haematol. 2003;123:463-468. 2. Swerdlow SH, Campo E, Harris NL et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue. Lyon: IARC; 2008. 3. Chan JK, Fletcher CD, Nayler SJ, Cooper K. Follicular dendritic cell sarcoma. Clinicopathologic analysis of 17 cases suggesting a malignant potential higher than currently recognized. Cancer 1997;79:294-313. 4. Pileri SA, Grogan TM, Harris NL et al. Tumours of histiocytes and accessory dendritic cells: an immunohistochemical approach to classification from the International Lymphoma Study Group based on 61 cases. Histopathology 2002;41:1-29. 5. Laurent C, Do C, Gascoyne RD et al. Anaplastic lymphoma kinase-positive diffuse large B-cell lymphoma: a rare clinicopathologic entity with poor prognosis. J.Clin.Oncol. 2009;27:4211-4216. 6. Adams H, Schmid P, Dirnhofer S, Tzankov A. Cytokeratin expression in hematological neoplasms: a tissue microarray study on 866 lymphoma and leukemia cases. Pathol.Res.Pract. 2008;204:569-573. 7. Mhawech-Fauceglia P, Saxena R, Zhang S et al. Pax-5 immunoexpression in various types of benign and malignant tumours: a high-throughput tissue microarray analysis. J.Clin.Pathol. 2007;60:709-714. 8. Spier CM, Lippman SM, Miller TP, Grogan TM. Lennert’s lymphoma. A clinicopathologic study with emphasis on phenotype and its relationship to survival. Cancer 1988;61:517-524. 9. Schrager JA, Pittaluga S, Raffeld M, Jaffe ES. Granulomatous reaction in Burkitt lymphoma: correlation with EBV positivity and clinical outcome. Am.J.Surg.Pathol. 2005;29:1115-1116. 10.Jaffe ES, Harris NL, Vardiman JW, Campo E, Arber DA. Hematopathology.: Elsevier, Saunders; 2011. 11.Schrader A, Bentink S, Spang R et al. High myc activity is an independent negative prognostic factor for diffuse large B cell lymphomas. Int.J.Cancer 2011 12.Hummel M, Bentink S, Berger H et al. A biologic definition of Burkitt’s lymphoma from transcriptional and genomic profiling. N.Engl.J.Med. 2006;354:2419-2430. 13.Jaffe ES, Pittaluga S. Aggressive B-cell lymphomas: a review of new and old entities in the WHO classification. Hematology.Am.Soc.Hematol.Educ.Program. 2011;2011:506-514. 14.Barrans S, Crouch S, Smith A et al. Rearrangement of MYC is associated with poor prognosis in patients with diffuse large B-cell lymphoma treated in the era of rituximab. J.Clin.Oncol. 2010;28:3360-3365. 500 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 HEMATOPATOLGIJA- kratki kurs 2/HEMATHOPATOLOGY Short Course 2 Classical Hodgkin lymphoma – differential diagnosis and tumour microenvironment Slavko Gasparov Medical School, University of Zagreb Institute of Pathology and Cytology, University Hospital Merkur, Zagreb, Croatia Introduction Hodgkin lymphoma (HL) accounts for approximately 15% to 30% of all malignant lymphomas. According to current diagnostic criteria, approximately 90% to 95% of HLs fall into the Classical Hodgkin lymphoma (CHL) category; the remaining cases are nodular lymphocyte-predominant subtype of Hodgkin’s lymphoma (NLPHL) which is recognized as a separate entity in the World Health Organization (WHO) classification1. WHO classification is based on the fact that there are clear and consistent histologic, epidemiologic, immunologic, and genetic differences between NLPHL and CHL. NLPHL is an indolent germinal center (GC) B-cell malignancy, that represents a nodular proliferation comprised of a minority of large neoplastic centroblasts with multilobated nuclei, the so-called popcorn or lymphocyte-predominant (LP) cells. Immunohistochemically LP cells are CD20+, PAX5+, BCL6+, EBV-LMP1-, CD30- and CD15-. Background inflammatory infiltrate represent mixture of small B and T lymphocytes1. This type of tumour is characterised clinically by a relatively indolent course and a very good response to standard therapy in cases with low stage disease. Unfortunately, the prognosis is unfavourable for advanced stages2. CHL is also a clonal, malignant lymphoproliferation originating from germinal center B cells3. CHL has a bimodal age curve in western countries, showing a peak at 15-35 years of age and a second peak later in life at 45-60 years1. A histopathologic diagnosis of CHL is based on the identification of diagnostic Reed-Sternberg (RS) cells in an appropriate inflammatory background of mixed infiltrate by histiocytes, small lymphocytes, eosinophils, neutrophils, plasma cells, fibroblasts and colagen. Based on characteristics of the reactive infiltrate and the specific features of neoplastic cells, cases may be subclassified into one of four subtypes: nodular sclerosis (NSCHL), lymphocyte-rich (LRCHL), mixed cellularity (MCCHL) and lymphocyte-depleted classical Hodgkin lymphoma (LDCHL)1,4. Although most cases can be diagnosed on the basis of morphology alone, diagnostic criteria include the characteristic immunophenotype of the neoplastic population. RS cells and variants express the CD30 and CD15 antigens in the majority of cases and lack the common leukocyte antigen CD455,6. The LMP-1 protein of EBV is expressed in approximately 25% to 50% of CHLs depending on the histologic subtype and patient age7. The staining is membranous and cytoplasmic, and usually most neoplastic cells are positive. Etiology of CHL is still questionable, but due to the unique epidemiologic and clinical features of the disease, an infectious cause has long been suspected. Currently, immunohistochemistry for the EBV latent membrane protein-1 (LMP-1) and nonradioactive in situ hybridization for EBV-encoded early RNAs (EBERs) are the methods of choice for the detection of EBV in routinely fixed, paraffin-embedded tissues8. Recent data suggest that the EBV status of tumour cells in classical HL could have prognostic significance for patients with this heterogenous disease9. Differential diagnosis of Hodgkin lymphoma Although most cases of CHL can now be safely classified on the basis of morphology and immunohistochemical features, distinction from some subtypes of NHL, reactive disorders, or even nonhematopoietic neoplasms can in some cases be difficult. Furthermore, the differential diagnosis between NLPHL and LRCHL can be particularly challenging. The morphology of the neoplastic cells is of limited value because LP cells can occur in both entities, so immunophenotyping and architecture are of paramount importance for the differential diagnosis (CD20, CD30, CD15, CD45, CD3, PD-1, CD57, EBV-LMP, EMA)10,11. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 501 HEMATOPATOLGIJA- kratki kurs 2/HEMATHOPATOLOGY Short Course 2 There are some subtypes of DLBCL that exhibit morphologic and phenotypic overlap with CHL. Those include primary mediastinal large B-cell lymphoma, THRLBCL, and EBV+ DLBCL of the elderly. There is also „grey zone“ lymphoma group, the new category of B-cell unclassifiable lymphoma, with features intermediate between DLBCL and CHL that sometimes has to be included in the differential diagnosis1,11. Anaplastic large cell lymphoma (ALCL) shows some morphologic and phenotypic similarities with CHL. ALCL tumour cells may resemble RS cells or mononuclear variants, but they are usually smaller than the neoplastic cells of CHL and often show bean-shaped or horseshoe-shaped nuclei (“hallmark” cells) rather than the round nuclei of Hodgkin cells. Peripheral T-cell lymphomas frequently show a polymorphic inflammatory background with eosinophils, neutrophils, plasma cells, and histiocytes and may contain RS-like giant cells. For this reasons ALCL as well as the peripheral T-cell lymphoma not otherwise specified (NOS) or angioimmunoblastic T-cell lymphoma (AITL) should sometimes be included in differental diagnosis. Microenvironment of Hodgkin lymphoma CHL is the example of a formerly lethal lymphoma that became curable mainly due to the considerable advances in therapeutic regimens12,13. These therapeutic improvements have transformed CHL into a curable disease in more than 85% of cases. However, a considerable percentage of patients still fail to respond to current standard therapies requiring more intensive treatments14. Identifying subgroups of patients with poor prognostic parameters has become the main objective of clinical and biological research. Studies on Hodgkin and Reed-Sternberg cells- related prognostic biomarkers have been unsuccessful but the microenvironmental composition seems to be of prognostic importance. Using immunohistochemical analysis some authors found that an increased number of tumour-associated macrophages was strongly associated with shortened survival in patients with classical Hodgkin lymphoma and provided a new biomarker for risk stratification15. Some other reports also now support the value of enumerating tumor-associated macrophages in pretreatment biopsies for outcome prediction in classical HL16,17,18. Their abundant signals could explain the deregulation of a critical apoptotic pathway in Reed-Sternberg cells that inhibits death in response to cytotoxic agents19. In HL several biomarkers other than CD68 have been reported to be associated with treatment outcome, in particular markers expressed by certain T-cell subsets20. A subset of regulatory T-cells (Treg) in the tumour microenvironment characterized by a CD4+CD25+ phenotype, is also in focus of interest, given the critical role of these cells in the modification of immune responses. FOXP3 is a master regulator of Treg cells. According to some autors FOXP3 density can contribute to the prediction of oucome in classical Hodgkin lymphoma. The important question is whether these findings will have a notable impact on general practice in the management of HL patients. As some authors point out, it is of pivotal importance that a personalized treatment strategy is developed in the future treatment of patients with HL, identifying at the time of a diagnosis those individuals with increased resistance to chemotherapy and radiotherapy21. Literature 1. Swerdlow SH. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC; 4th edition, 2008. 2. Jackson C, Sirohi B, Cunningham D, Horwich A, Thomas K, Wotherspoon A. Lymphocite-predominant Hodgkin lymphoma – clinical features and treatment outcomes from a 30-year experience. Annals of Oncology 2010;10:2061-2028 3. Kanzler H, Küppers R, Hansmann ML, Rajewsky K. Hodgkin and Reed-Sternberg cells in Hodgkin’s disease represent the outgrowth of a dominant tumor clone derived from (crippled) germinal center B cells. J Exp Med 1996;184:1495-1505. 4. Harris NL. Hodgkin’s disease: classification and differential diagnosis. Mod Pathol 1999; 12:159-176 502 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 HEMATOPATOLGIJA- kratki kurs 2/HEMATHOPATOLOGY Short Course 2 5. von Wasielewski R, Mengel M, Fischer R, Hansmann ML, Hübner K, Franklin J, Tesch H, Paulus U, Werner M, Diehl V, Georgii A. Classical Hodgkin’s disease: clinical impact of the immunophenotype. Am J Pathol 1997; 151:1123-1130. 6. Said JW. The immunohistochemistry of Hodgkin’s disease. Semin Diagn Pathol 1992; 9:265-271. 7. Herbst H, Steinbrecher E, Niedobitek G, Young LS, Brooks L, Müller-Lantzsch N, Stein H. Distribution and phenotype of Epstein-Barr virus-harboring cells in Hodgkin’s disease. Blood 1992; 80:484-491. 8. Gulley ML, Glaser SL, Craig FE, Borowitz M, Mann RB, Shema SJ, Ambinder RF. Guidelines for interpreting EBER in situ hybridization and LMP1 immunohistochemical tests for detecting Epstein-Barr virus in Hodgkin lymphoma. Am J Clin Pathol 2002; 117:259-267. 9. Keegan THM, Glaser SL, Clarke CA, Gulley ML, Craig FE, Digiuseppe JA, Dorfman RF, Mann RB, Ambinder RF. Epstein-Barr virus as a marker of survival after Hodgkin’s lymphoma: A population –based study. J Clin Oncol, 2005: 23;7604-7613 10.Kamel OW, Gelb AB, Shibuya RB, Warnke RA. Leu 7 (CD57) reactivity distinguishes nodular lymphocyte predominance Hodgkin’s disease from nodular sclerosing Hodgkin’s disease, T-cell-rich-B-cell lymphoma and follicular lymphoma. Am J Pathol 1993; 142:541-546. 11.Jaffe ES. Hematopathology. Philadelphia, PA Elsevier Saunders; 1st edition, 2011. 12.Connors JM. State of the art therapeutics: Hodgkin’s lymphoma. J Clin Oncology, 2005:23;6400-6408. 13.Diehl V, Engert A, Re D. . New strategies for the treatment of advanced stage Hodgkin’s lymphoma. Hematology/ Oncology. 2007:21;897-914. 14.David KA, Mauro L, Evens AM. . Relapsed and refractory Hodgkin lymphoma: transplantation strategies and novel therapeutic options. Curr Treat Options Oncol 2007:8;352-374. 15.Steidl C, Lee T, Shah SP, Farinha P, Han G, Nayar T, Delaney A, et al. Tumor-Associated Macrophages and Survival in Classic Hodgkin’s Lymphoma. N Engl J Med. 2010:362(10):875-885. 16.Tzankov A, Matter MS, Dirnhofer S. Refined prognostic role of CD68-positive tumor macrophages in the context of the cellular micromilieu of classical Hodgkin lymphoma. Pathobiology. 2010:77:301-308. 17.Farinha P. Lymphoma associated macrophages predict survival in uniformly treated patients with classical Hl. USCAP Annual Meeting Abstracts 2011 (1257). 18.Jakovic LR, Mihaljevic BS, Perunicic Jovanovic MD, Bogdanovic AD, Andjelic BM, Bumbasirevic VZ. The prognostic relevance of tumor associated macrophages in advanced stage classical Hodgkin lymphoma. Leuk Lymphoma. 2011:10;1913-19. 19.Tzankov A, Meier C, Hirschmann P, Went P, Pileri SA, Dirnhofer S. Correlation of high numbers of intratumoral FOXP3+ regulatory T cells with improved survival in germinal center-like diffuse large B-cell lymphoma, follicular lymphoma and classical Hodgkin lymphoma. Haematologica. 2008:93:193-200. 20.Kelley TW, Pohlman B, Elson P, Hsi ED. The ratio of FOXP3+ regulatory T cells to granzyme B+ cytotoxic T/NK cells predicts prognosis in classical Hodgkin lymphoma and is independet of bcl-2 and MAL expression. Am J Clin Pathol. 2007;128:958-65. 21.DeVita VT Jr, Costa J. Toward a personalized treatment of Hodgkin’s Disease. N Engl J Med. 2010;362:942-943. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 503 HEMATOPATOLGIJA- kratki kurs 3/HEMATHOPATOLOGY Short Course 3 Proliferacije plazma ćelija Plasma cell proliferations Tatjana Terzić Tatjana Terzic Institut za patologiju, Medicinski fakultet Univerziteta u Beogradu, Srbija Institute of Pathology, School of Medicine University of Belgrade, Serbia Apstrakt Abstract Plazmociti su terminalno diferentovane efektorne ćelije B-ćelijske linije. Cilj ovog rada je sveobuhvatni prikaz relevantnih podataka o fenotipu plazmocita, reaktivnih i neoplastičnih. Savremena klasifikacija limfoproliferativnih bolesti po Svetskoj zdravstvenoj organizaciji se zasniva na koreliranju njihovih morfoloških, histoloških, imunofenotipskih, citogenetskih i kliničkih karakteristika. Primena imunohistohemije je neophodna ne samo u dijagnostici plazmocitnih neoplazmi, već i u određivanju prognoze, kao i evaluaciji rezidualne bolesti i relapsa.U ovom radu je istaknut značaj imunohistohemijske analize neneoplastičnih i neoplastičnih proliferacija plazmocita u cilju lakše dijagnostike, sa posebnim osvrtom na savremene pristupe u njihovoj subklasifikaciji. Ključne reči: plazma ćelije, proliferacije, klasifikacija Plasma cells (PC) are the terminally differentiated effector cells of the B-cell lineage. The aim of this review is to integrate relevant data on the phenotype of plasma cells, including reactive and malignant PC. The current World Health Organisation classification of lymphoid neoplasm is based on correlation of their morphologic, histologic, immunophenotypic, genetic and clinical features. The extensive application of imunohistochemistry is necessary in diagnosis of plasma cell neoplasm, but also in determining prognosis as well as in evaluating residual/ relapsing disease. This review focuses on immunohistochemical analysis non-neoplastic and neoplastic plasma cell proliferation in facilitating the diagnosis and highlights the recent advances that have been made with regard to their stratifying. Key words: plasma cell proliferation, classification Plasma cells: a new light at the end of B cell development As the final mediators of a humoral response, plasma cells play a critical role in adaptive immunity. However, the mechanisms that control the differentiation of germinal center (GC) B cells toward the plasma cell or memory B cell pathway are not known. Both cell types derive from antigen-activated B cells that have undergone the “GC reaction”, in which they specifically modify their immunoglobulin through somatic hypermutation and class-switch recombination.1 Klein et al2 report that the transcription factor Interferon Regulatory Factor 4 (IRF4) is required for the generation of plasma cells. Transgenic mice with conditional deletion of IRF4 in germinal center B cells lacked post–germinal center plasma cells and were unable to differentiate memory B cells into plasma cells. In addition, IRF4-deficient B cells had impaired expression of activation-induced deaminase and lacked classswitch recombination, suggesting an independent function for IRF4 in this process.2 These results identify IRF4 as a crucial transcriptional “switch” in the generation of functionally competent plasma cells. IRF4, also called MUM1, originally identified as the product of a proto-oncogene involved in chromosomal translocations in multiple myeloma, is capable of transforming cells in vitro and is often abnormally expressed in B cell lymphomas.3 504 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 HEMATOPATOLGIJA- kratki kurs 3/HEMATHOPATOLOGY Short Course 3 Figure 1. Termination of the GC transcriptional programme and post-GC B-cell development1 The GC transcriptional programme culminates in the differentiation of a centroblast into a centrocyte through the stimulation of CD40 mediated by GC T cells that leads to repression of B-cell lymphoma 6 (BCL-6) by IRF4 (Figure 1).1 This centrocyte stage could represent the common precursor of memory B cells and plasma cells. Inactivation of PAX5 (paired box protein 5) by an unknown stimulus and mechanism appears to be the first step towards plasma-cell differentiation. The pre-plasmablast stage is characterized by low-levels of immunoglobulin secretion, which results from the release of the repression of the genes encoding XBP1 (X-box binding protein 1) and J (joining) chain. The subsequent upregulation of BLIMP1 (B-lymphocyteinduced maturation protein 1) and IRF4, during a plasmablast stage, then establishes the characteristic plasma-cell phenotype. BCL-6 and BLIMP1 establish a mutual suppression loop between the centroblast and the plasmablast and/or plasma cell, respectively. BLIMP1 represses PAX5 in plasmablasts and plasma cells; evidence suggests that PAX5 might also repress positive-regulatory-domain-containing 1 (Prdm1), which encodes BLIMP1.4 Continued signalling through CD40 may be crucial in driving the centrocyte towards memory B-cell differentiation, and continued PAX5 expression maintains B-cell identity in memory B cells.1 Because they are terminally differentiated, end-stage cells, plasma cells do not divide. However, “plasmablasts” in extrafollicular regions or exiting GCs do undergo cell division just before they become plasma cells.1, 5 The lifespan of nondividing plasma cells varies from a few days to many months. Numerous B cell–specific surface proteins are down-regulated upon plasma cell differentiation, including major histocompatibility complex (MHC) class II, B220, CD19, CD21 and CD22.6 The proteoglycan syndecan-1 (CD138), which recognizes extracellular matrix and growth factors, is induced on antibody-secreting B cells and is often used as a marker of plasma cells.7 Most normal plasma cells express CD138, CD38, VS38, cIg, CD19, CD10, EMA, CD27, CD31, hTPD52, MUM1, CD20v, CD45v and HLA-DR antigens, although there is extensive antigenic heterogeneity reflecting a spectrum of differentiation that ranges from the immature plasmablast to the mature plasma cell. Among integrin family proteins, which mediate cell-matrix and cell-cell adhesion functions, VLA-4 (very late antigen 4) is most predominant on plasma cells. Perhaps most important for differential homing of plasma cells, the chemokine receptors CXCR5 and CCR7 are decreased, which reduces responsiveness to the B and T cell zone chemokines CXCL13, CCL19 and CCL21.8 In contrast, expression of CXCR4, which recognizes CXCL12 present in splenic red pulp, lymph node medullary cords and in bone marrow, remains high.8 These changes mediate movement of plasma cells from the follicles to other locations, including the bone marrow. The signaling pathways that normal B cells utilize to sense antigens are frequently derailed in B cell malignancies, leading to constitutive activation of prosurvival pathways.9 A better understanding of these issues will provide major insights into the mechanisms that regulate humoral immunity, as well as in those that lead to its pathological manifestations such as B‑cell lymphomas. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 505 HEMATOPATOLGIJA- kratki kurs 3/HEMATHOPATOLOGY Short Course 3 Immunoglobulin Stains for Clonality Assessment Staining for kappa and lambda light chains may help to identify the presence of an abnormal clonal population, particularly plasma cells. Immunohistochemistry is less sensitive than flow cytometry for the detection of immunoglobulin expression, as it does not detect surface immunoglobulin. Immunohistochemistry in fixed tissue relies on some degree of cytoplasmic immunoglobulin production. Plasma cells and immunoblasts strongly express cytoplasmic immunoglobulin, whereas only a small subset of other B-cell proliferations has detectable immunoglobulin expression by paraffin immunoperoxidase techniques.10 Non-neoplastic plasma cell proliferation Benign polyclonal plasmacytosis (BPP) was first described in 1988 by Peterson et al. as benign polyclonal immunoblast proliferation.11 The most common clinical presentations have been fever with leukocytosis and skin rash. Other presenting signs include lymphadenopathy, dyspnea, hepatosplenomegaly, jaundice, and autoantibodies. The BPP cases showed an association with bacterial sepsis (Staphylococcus aureus, Pseudomonas aeruginosa), viral infection (hepatitis C, infectious mononucleosis), serum sickness-like syndrome (streptokinase therapy) and immunological disorders and they can be interpreted as a dysregulated hyperactive immune response.12 A reactive increase of polyclonal plasma cells is common and is associated with a variety of conditions including HIV and other infections, chronic inflammatory diseases, haemopoietic and non-haemopoietic malignant disease, angioimmunoblastic lymphadenopathy, systemic Castleman’s disease, cirrhosis, diabetes mellitus, iron deficiency, megaloblastic anaemia and haemolytic anaemia.13 Pathologically, the plasma cells increased in number and accounted for 20–40% of nucleated cells of bone marrow. Cytological features must be assessed as well as plasma cell number. The presence of plasmablasts and marked plasma cell dysplasia, e.g. giant forms, striking nuclear lobulation and prominent nucleoli, are strongly suggestive of multiple myeloma. Reactive plasma cells showed mature cytomorphology. Immunoperoxidase studies of light chain determinants for plasma cells and their precursors demonstrated a polyclonal pattern.14 In reactive plasmacytosis immunocytochemistry shows that κ- and λ-expressing plasma cells are present in a ratio of approximately 2:1. About half the plasma cells express γ heavy chain, about a third α and the remainder µ.13 The plasma cell type of Castleman’s disease (CD) is often associated with polyclonal gammaglobulinemia and increased serum levels of IL-6. Anemia and elevated erythrocyte sedimentation rate are frequent findings. The most frequent site of involvement is the abdomen, particularly in the small bowel mesentery.15 Lymph node sections show follicular hyperplasia with a well-defined mantle zone, surrounded by sheets of mature plasma cells and scattered immunoblasts. Vascular proliferation or hyalinization is usually absent. In approximately 40% of the cases the plasma cells are monotypic and express Ig lambda light chain. The plasma cell type resembles other follicular hyperplasias, such as those associated with rheumatoid arthritis, or other autoimmune disorders. Plasma cell variant of CD should be considered in the differential diagnosis of plasma cell neoplasms (primary lymph node plasmacytoma), polyclonal plasma cell-rich lymphoproliferative disorder (marginal zone lymphoma, lymphoplasmacytic lymphoma, Burkitt’s lymphoma with plasmacytoid differentiation, plasmablastic lymphoma, angioimmunoblastic lymphadenopathy with dysproteinemia, Hodgkin’s lymphoma), plasma cell granuloma, syphilitic lymphadenitis, etc. Neoplastic plasma cell proliferation Plasma cell neoplasms encompass a group of diseases with varying clinical manifestations but with at least one common feature, the production by neoplastic plasma cells with a monoclonal immunoglobulin protein (M-protein, or paraprotein). These diseases include monoclonal gammopathy of undetermined significance (MGUS), plasma cell myeloma (PCM) (and its clinical variants), plasmacytoma, the monoclonal 506 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 HEMATOPATOLGIJA- kratki kurs 3/HEMATHOPATOLOGY Short Course 3 immunoglobulin deposition diseases (primary amyloidosis and monoclonal light and heavy chain deposition diseases) and osteosclerotic myeloma (POEMS syndrome).16 The diagnosis of a plasma cell neoplasm rests on the correlation of clinical and pathological findings. At one end of the spectrum of plasma cell dyscrasia is monoclonal gammopathy of undetermined significance, which is the most common and relatively indolent form of plasma cell dyscrasia; this contrasts with plasma cell myeloma, which is aggressive and requires treatment. Patients presenting with MGUS demonstrate no B-symptoms (e.g. weight loss, night sweats), no bone lesions, and no symptoms that indicate organ or tissue impairment. A low level of M-protein of <30 g/L in serum, and clonal plasma cells accounting for <10% of total marrow cellularity, are characteristic features in MGUS.17 Recent studies indicate that the diagnosis of symptomatic multiple myeloma is always preceded by monoclonal gammopathy for 2 or more years. Plasma cell myelomas are more advanced neoplasms and classified as either asymptomatic (smouldering) myeloma or symptomatic myeloma. In the former, the patients have either an increase in M-protein (>30 g/L) or an increase in clonal plasma cells (>10%) in the bone marrow without end organ impairment, and lack symptoms and signs. Symptomatic myeloma is diagnosed when there is end-organ or tissue impairment (most classically defined as the “CRAB” findings: hypercalcemia, renal insufficiency, anemia and bone lesions) with the presence of M-protein in serum or urine and bone marrow clonal plasma cells or plasmacytoma17. When there are multiple bone lesions or the disease is systemic, the clinical term is multiple myeloma (MM), as opposed to plasma cell myeloma (plasmacytoma). The risk that MGUS may evolve to a malignant state (MM, primary amyloidosis, macroglobulinaemia, chronic lymphocytic leukaemia or plasmacytoma) is about 1–2% of cases per year, and patients are at risk of progression even after 25 years or more of stable MGUS.18The differences in follow-up, treatment, and survival between MM and MGUS necessitate discrimination between these two entities. Most normal plasma cells express CD138, CD38, CD19, CD10, CD27, CD45 and HLA-DR antigens19, although there is extensive antigenic heterogeneity reflecting a spectrum of differentiation that ranges from the immature plasmablast to the mature plasma cell. Benign plasma cells are typically negative for CD117 and CD56. Malignant plasma cells are usually positive for CD27dim, CD28, CD56, and negative for CD19, CD20, and CD45, or dimly positive for CD45.20 However, CD45 and CD20 can be expressed at moderate or higher intensity in 10% and 20% of cases, respectively. CD56 is negative in about 40% to 45% of MM, as it has in association with progressive disease.21 CD117 is aberrantly expressed in 30% of cases and is a useful marker for malignancy. Myeloid markers CD13 and CD33 or CD10 can also be identified in a small subset of cases. Myelomatous plasma cells may express other antigens associated with different haematopoietic lineages such as CD45R, CD25, CD2, CD420. The monoclonal antibody CD56⁄NCAM (clone 1B6) seemed to have potential for discriminating between myelomatous, MGUS and reactive plasmacytosis cells in bone marrow sections and aspirates, especially in those cases with low infiltration. 20 Of the MM samples 78% were CD56+ in smears and 92% positive in biopsies. Martín et al. did not find strong CD56 expression in MGUS samples.22 One of five samples of polyclonal plasmacytosis was CD56+ (a case was considered to be positive for CD56 expression if >50% of the CD138+ plasma cells expressed NCAM with an intensity on a par with that of the osteoblasts). 22 All normal and malignant PC express CD38 and CD138. However, the level of expression is different and allows normal PC to be distinguished from malignant PC: myeloma cells express more CD138 but less CD38 than do normal PC23. Bataille et al. have never observed viable CD138– myeloma cells whereas CD38– myeloma cells.23 The normal ratio of plasma cells is 2-4 kappa-expressing cells to each lambda positive cell; a ratio of 8 (or more) kappa to 1 lambda strongly suggests a monoclonal kappa-positive population and can be readily visualised by ISH with a plasmacytosis of approximately 5% in trephine sections. Conversely, a ratio of even 4 lambda-expressing cells to 1 kappa-positive one indicates the presence of a monoclonal lambda-positive plasma cell population. Cytologic features of plasma cells in the bone marrow smears may vary from normal-appearing mature plasma cells to immature and anaplastic forms. Plasmablasts show a high nuclear:cytoplasmic ratio, deep blue cytoplasm, with or without perinuclear hof, round or irregular nucleus, fine chromatin, and one or several prominent nucleoli. Multinucleated or multilobated plasma cells may be present. Cells with cherry-red, 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 507 HEMATOPATOLGIJA- kratki kurs 3/HEMATHOPATOLOGY Short Course 3 round cytoplasmic (Russell bodies) or nuclear (Dutcher bodies) inclusions, as well as cytoplasmic crystals may be present. Some plasma cells may appear like grapes and demonstrate numerous, round, Ig-containing cytoplasmic structures (Mott and Morula cells). Blood smears may show rouleaux formation of the red blood cells or the presence of plasma cells in various proportions. In some patients with multiple myeloma, the neoplastic cells are so immature or atypical that they are cytologically indistinguishable from large cell lymphoma or other anaplastic tumours, including carcinoma, melanoma and acute leukaemias. Plasmablastic myeloma and large cell lymphoma with cells having the features of immunoblasts are particularly likely to be confused. Cytogenetics and molecular genetics are not essential for diagnosis of PCM. Conventional cytogenetic analysis is frequently unsuccessful but it appears that the detection of deletions of 13q by metaphase analysis may have prognostic value, along with t(4;14) and deletion of TP53 by FISH24. Translocations involving chromosomes 4, 14, and 16 as well as del17p13 (TP53) have been associated with a poor prognosis.24 DNA aneuploidy is observed in more than 90%; these are predominantly hyperdiploid, with less than 10% being hypodiploid, and the latter carries a poor prognosis. The t(11;14) abnormality is found in 10-15% of cases, resulting in over-expression of cyclin D1. Different surface molecules could be targeted as individual therapies for either well-defined MM entities i.e., CD19, CD20, CD27 or CD117, or subpopulations of MM i.e., CD33, CD52.23 Half of MM retain CD27 and its expression is associated with a better prognosis. CD27 expression is lost with myeloma progression.23 The expression of CD117 seems to be restricted to patients with indolent MM.23 Clinical grade monoclonal antibodies exist for CD20, CD33 and CD52 and clinical trials are ongoing for some of them. Non-secretory myeloma, which accounts for 1% to 5% of all myelomas, is characterized by the absence of detectable M-protein in serum and urine. The presenting features of nonsecretory myeloma are similar to those in patients with a detectable M-protein, except for the absence of renal function impairment and hypercalcemia. The response to therapy and survival of patients with nonsecretory myeloma are similar to those of patients with measurable M-protein. Plasma cell leukaemia (PCL) is also a rare form of plasma cell dyscrasia (2% to 4% of all myelomas). It is a variant of multiple myeloma characterized by greater than or equal to 2 x 109 circulating plasma cells in one litre of peripheral blood. Pateints can present with primary PCL, or it can evolve from previously recognized multiple myeloma (secondary PCL). The primary form accounts for 60% of the cases. In primary PCL, the constellation of adverse biologic prognostic factors in patients with advanced aggressive myeloma is already present at diagnosis. Immunohistochemically, PCL tends to be positive for CD20 (50%) but negative for CD117 and CD56 compared to myeloma. Approximately 90% of secondary PCL cases express CD28 as compared with 30% in primary PCL. The plasma cells express cytoplasmic but not surface Ig light chain. Primary PCL has a more aggressive clinical presentation than MM, with a higher frequency of extramedullary involvement, anemia, thrombocytopenia, hypercalcemia, and renal failure. Plasmacytoma Solitary bone plasmacytomas (SBP) are tumors of neoplastic plasma cells localized to a single bone and represent 3% of plasma cell neoplasms. Some patients with SBP present with a single painful bone lesion due to a monoclonal plasma cell infiltrate, and further studies show no evidence of myeloma elsewhere. In other cases, SBP may be discovered during roentgenographic studies for another condition or the patient presents with a painless swelling of the sternum, rib, or other bone. Electrophoresis of serum and urine samples reveals monoclonal protein in 24% to 72% of patients with SBP, although levels of the protein are much lower than those patients with MM. In true solitary or multifocal osseous plasmacytomas, trephine biopsy show no plasmacytosis and have no demonstrable clonal population of plasma cells. Most patients (70%) eventually develop systemic disease at a median of 2 to 4 years. Solitary extramedullary (extraosseous) plasmacytomas tend to be localized to the head and neck regions, where 80% of cases occur, although they can occur in many other parts of the body such as the gastrointestinal tract, central nervous system, and skin. The majority of the tumors do not produce detectable serum 508 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 HEMATOPATOLGIJA- kratki kurs 3/HEMATHOPATOLOGY Short Course 3 paraprotein (less than 25%) and the tumors rarely spread (less than 30%). Primary lymph node plasmacytomas (PLNPs) represent 2% of all extramedullary plasmacytomas and 0.5% of lymph node malignant neoplasms. PLNPs can be diagnosed only after exclusion of metastatic multiple myeloma (which metastasizes to lymph nodes in up to 40% of cases of advanced-stage disease) and metastatic upper respiratory tract plasmacytomas (which represent 76% of extramedullary plasmacytomas and infiltrate cervical lymph nodes in approximately 15% of cases).25 A total of 33 PLNPs have been described, primarily as single case reports, 7 of them arising in Castleman disease, plasma cell type. Because most PLNPs and other types of extramedullary plasmacytomas show mature plasma cell morphologic features relatively often and because reactive plasmacytoses form dense tumefactive plasma cell infiltrates simulating plasmacytoma in multiple body sites, as previously reported in the upper respiratory tract.25 Monoclonal immunoglobulin deposition diseases (MIDD) Primary amyloidosis In this condition there is deposition of a fibrillary protein in the liver, kidneys, heart, gastrointestinal tract, peripheral nerves and other tissues resulting in organ impairment. Diagnosis is dependent on demonstration of amyloid protein and exclusion of secondary (non-immunoglobulin) amyloidosis. The protein binds Congo Red dyes with apple-green birefringence when viewed under polarised light. The light chain type in primary amyloidosis is usually lambda, presumably reflecting easier conversion of this protein, compared with kappa, into beta-pleated sheet structures. Primary amyloidosis is always the result of a clonal plasma cell neoplasm, but symptoms because of the amyloid deposition usually become clinically evident at a time when the plasma cell tumor burden is relatively low; most cases have < 10% bone marrow plasma cells with low M-protein levels (< 30 g/L), similar to that seen in MGUS.16 If the plasma cell count and M-protein level are consistent with MGUS and the patient’s symptoms are entirely attributable to organ damage from amyloid deposition, the resulting organ failure does not constitute a criterion for the diagnosis of symptomatic PCM, and the diagnosis remains primary amyloidosis. Amyloidosis may also occur in the presence of a larger plasma cell burden and other symptoms of myeloma (≤ 10% of patients with myeloma); if the level of M-protein or the plasma cell count is sufficient for a diagnosis of asymptomatic myeloma, the diagnosis is amyloidosis and PCM.26 Light and heavy chain deposition disorders These may present with features resembling primary amyloidosis but the protein is non-fibrillary as seen by electron microscopy and does not bind Congo Red. Abnormal protein comprising heavy or light chains (or both) is deposited in tissues such as the heart and liver leading to organ dysfunction. As with primary amyloidosis, the associated tumor burden is usually low. In light chain deposition disease, the light chain type is usually kappa and neoplastic cells, if identifiable, are usually plasma cells. Heavy chain diseases (mu and gamma) usually affect older individuals and involve lymph nodes, marrow, spleen and peripheral blood, with varying lymphocytic, lymphoplasmacytoid and mature plasma cell morphology in cellular components. Occasional examples appear to be variants of diffuse large B-cell lymphoma. Alpha chain disease differs in presenting typically with small intestine and mesenteric lymph node involvement causing malabsorption in younger individuals. The small bowel in alpha chain disease has histological features resembling those seen in extranodal marginal zone B-cell lymphomas of MALT type. Osteosclerotic myeloma is a rare form of plasma cell neoplasm (less than 1%) usually seen in POEMS (polyneuropathy, organomegaly, endocrinopathy, M component, and skin changes) syndrome. The bone changes are characterized by osteosclerosis rather than lytic lesions. The monoclonal protein is usually IgG or IgA type (with a marked predominance of lambda light chain) and generally less than 3 g/dL. Lymph nodes may show Castleman disease. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 509 HEMATOPATOLGIJA- kratki kurs 3/HEMATHOPATOLOGY Short Course 3 Other plasma cell proliferations Lymphoplasmacytic lymphoma (LPL) is a slowly progressive, clonal disorder of mature B cells, with features of plasma cell differentiation. In some patients this can be associated with peripheral neuropathy. Splenomegaly is frequent but not usually massive and lymphadenopathy, when present, is not usually prominent. Waldenströmľs macroglobulinemia (WM) is the term used to describe cases of LPL in which there is an IgM paraprotein, which may be associated with hyperviscosity. Trephine biopsy histology shows irregular nodular and paratrabecular infiltrates, with or without additional diffuse interstitial infiltration. Intrasinusoidal infiltration is uncommon, in contrast with splenic marginal zone and mantle cell lymphomas. Plasma cells may contain PAS-positive inclusions of immunoglobulin, which may appear in the cytoplasm (Russell bodies) or indenting the nucleus (Dutcher bodies). The proportions of lymphocytes, lymphoplasmacytoid cells and plasma cells vary widely. There may also be scattered larger blast cells but no true para-immunoblasts or proliferation centres. Accompanying reactive mast cells are often abundant.27 The lymph nodes contain diffuse or vaguely nodular infiltrates of mixed lymphoid cells encompassing the spectrum described above. Absence of neoplastic follicles, expanded marginal zones or infiltrates of monocytoid B cells is important in differentiating lymphoplasmacytic lymphoma from other types of small B-cell lymphoma. Immunophenotype of LPL is Sm IgM+ CD5- CD10- CD19+ CD20+ CD22+ CD23- CD25+ CD27+ FMC7+ CD103- CD138-. Cases with little evidence of plasma cell differentiation may be confused with other small B-cell lymphomas; immunophenotyping can exclude chronic lymphocytic leukaemia, mantle cell lymphoma and follicular lymphoma, but not splenic or extranodal marginal zone lymphomas.28 Plasmablastic lymphomas or lymphomas with plasmacytic differentiation Plasmablastic tumors are composed of large cells with abundant, often eosinophilic cytoplasm and immunoblastic, anaplastic, or plasmacytoid morphology. The classification of tumors with plasmablastic morphology has become increasingly complex.29 Important features to subclassify these neoplasms include the clinical site (oral cavity, body cavity, etc), morphologic spectrum (pure immunoblasts vs mixture of immunoblasts, plasmacytoid cells, and plasma cells), differential antigen expression (CD20, CD138, immunoglobulin, CD30, and CD56), and association with viruses10. Tumors with plasmablastic morphology typically occur in patients with an abnormal immune state (HIV positive, posttransplantation, or the elderly). These tumors often arise in the oral cavity or other mucosal sites of the head and neck, or body cavity (PEL), or in association with multicentric Castleman disease.30 In addition, a Kaposi sarcoma-associated herpesvirus–positive solid lymphoma/ extracavitary PEL/human herpesvirus 8–associated DLBCL has been described, predominantly in HIV-positive patients and shows coexpression of EBV. Atypical BL with plasmacytoid differentiation is seen in HIV-positive patients, representing approximately 20% of AIDS-related NHL. Other tumors with plasmablastic morphology and less association with an immunocompromised state include PBL with plasmacytic differentiation defined by Colomo et al31 as prominent immunoblasts or plasmablasts but with some admixed smaller cells with plasmacytic differentiationand by little or weak expression of CD20, DLBCL with prominent plasmablastic/secretory differentiation, pyothorax-associated lymphoma, and PCM with a dysplastic, plasmablastic appearance. Morphologically, the PBL of the oral cavity and the rare ALK-positive DLBCL are composed of a very monomorphic, sheetlike proliferation of immunoblasts. PBL with plasmacytic differentiation and DLBCL with secretory differentiation (immunoblasts and plasmacytoid cells) are distinguished by the presence of centroblasts in the latter.10 CD138 and MUM-1, markers of post germinal center/terminal B-cell/plasmacytic differentiation, are useful in identifying the B-cell origin of these tumors that show variable or negative expression of CD20 and CD79a. 10 Expression of Pax-5 has not been investigated in a significant number of cases to be informative at the present. Non HIV patients with plasmablastic lymphoma may present with nodal, soft tissue or bone marrow disease. 31 The distinction from myeloma may be problematic. The immunophenotype can be difficult due to lack of the B antigens with weak/ absent CD19 and 510 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 HEMATOPATOLGIJA- kratki kurs 3/HEMATHOPATOLOGY Short Course 3 CD20, PAX5 negative, CD138 positive, CD79 variable, MUM1 positive, CD56 positive (most), CD45 negative (usually) with a few cases expressing ALK1.32 Open questions and future challenges The 2008 WHO classification of lymphoid neoplasms has been a major consensus effort in updating new knowledge, concepts, and criteria in the taxonomy of plasma cell neoplasms. However, many questions still remain. Literature 1. Klein U, Dalla-Favera R. Germinal centres: role in B-cell physiology and malignancy. Nat Rev Immunol. 2008;8(1): 22-33. 2. Klein U, Casola S, Cattoretti G, Shen Q, Lia M, Mo T, Ludwig T, Rajewsky K, Dalla-Favera R. Transcription factor IRF4 controls plasma cell differentiation and class-switch recombination. Nat Immunol. 2006;7(7): 773-782. 3. Iida S, Rao PH, Butler M, Corradini P, Boccadoro M, Klein B, Chaganti RSK, Dalla-Favera R. Deregulation of MUM1/ IRF4 by chromosomal translocation in multiple myeloma. Nat Genet. 1997;17: 226-230. 4. Garcia JF, Roncador G, Garcia JF, Sanz AI, Maestre L, Lucas E, Montes-Moreno S,Fernandez Victoria R, MartinezTorrecuadrara JL, Marafioti T, Mason DY, Piris MA. PRDM1/BLIMP-1 expression in multiple B and T-cell lymphoma. Haematologica 2006; 91: 467-474. 5. Sze DMY, Toellner KM, de Vinuesa CG, Taylor DR, MacLennan ICM. Intrinsic constraint on plasmablast growth and extrinsic limits of plasma cell survival. J Exp Med. 2000;192(6): 813-822. 6. Calame K. Immunology: end game for B cells. Nature 2001;412: 289-290. 7. Sanderson RD, Lalor P, Bernfield M. B lymphocytes express and lose syndecan at specific stages of differentiation. Cell Reg. 1989;1: 27–35. 8. Hargreaves, D. C. Hyman PL, Lu TT, Ngo VN, Bidgol A, Suzuki G, Zou YR, Littman DR, Cyster JG. A coordinated change in chemokine responsiveness guides plasma cell movements. J Exp Med. 2001;194: 45–56. 9. Klein U, Pasqualucci L. B-cell receptor signaling derailed in lymphomas. Immunol Cell Biol. 2010;88(4):346-347. 10.Higgins RA, Blankenship JE, Kinney MC. Application of immunohistochemistry in the diagnosis of non-Hodgkin and Hodgkin lymphoma. Arch Pathol Lab Med. 2008;132: 441–461. 11.Peterson LC, Kueck B, Arthur DC, Dedeker K, Brunning RD. Systemic polyclonal immunoblastic proliferations. Cancer 1988;61:1350-1358. 12.Li L, Hsu P, Patel K, Saffari Y, Ashley I, Brody J. Polyclonal plasma cell proliferation with marked hypergammaglobulinemia and multiple autoantibodies. Ann Clin Lab Sci. 2006;36(4): 479-484. 13.Bain BJ, Clark DM, Lampert IA, Wilkins BS. Infections and reactive changes. In: Bone marrow pathology (3th ed) Blackwell Science Ltd, Oxford, 2001. 14.Kojima M, Murayama K, Igarashi T, Masawa N, Shimano S, Nakamura S. Bone marrow plasmacytosis in idiopathic plasmacytic lymphadenopathy with polyclonal hyperimmunoglobulinemia: a report of four cases. Pathol Res Pract. 2007;203(11): 789-794. 15.Dham A, Peterson BA. Castleman disease. Curr Opin Hematol 2007;14: 354–359. 16.Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW., eds. WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon, France: IARC;2008, pp. 200–213. 17.International Myeloma Working Group. Criteria for the classification of monoclonal gammopathies, multiple myeloma and related disorders: a report of the International MyelomaWorking Group. Br J Haemotol. 2003;121: 749-757. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 511 HEMATHOPATHOLOGY –Short Course 3 18.Kyle RA, Therneau TM, Rajkumer SV, Offord JR, Larson DR, Plevak MF, L. Joseph Melton LJ. A long-term study of prognosis in monoclonal gammopathy of undetermined significance. N Engl J Med. 2002;346: 564–569. 19.Ruiz Argüelles GJ, San Miguel JF. Cell surface markers in multiplemyeloma. Mayo Clin Proc. 1994; 69: 684–690. 20.Ely SA, Knowles DM. Expression of CD56 ⁄ neural adhesion molecule correlates with the presence of lytic bone lesions in multiple myeloma and distinguishes myeloma from monoclonal gammopathy of undetermined significance and lymphomas with plasmacytoid differentiation. Am J Pathol. 2002; 160; 1293–1299. 21.Ashcroft JA, Rawstron AC, Owen RG, Morgan GJ. Phenotyping in myeloma: identification of a rare CD19+ CD56– subgroup.Blood 2000;96: 156a (Abstract). 22.Martín P, Santón A, Bellas C. Neural cell adhesion molecule expression in plasma cells in bone marrow biopsies and aspirates allows discrimination between multiple myeloma, monoclonal gammopathy of uncertain significance and polyclonal plasmacytosis. Histopathology 2004; 44(4): 375-380. 23.Bataille R,Jégo G, Robillard N, Barillé-Nion S, Harousseau JL, Moreau P, Amiot M, Pellat-Deceunynck C. The phenotype of normal, reactive and malignant plasma cells. Identification of “many and multiple myelomas” and of new targets for myeloma therapy. Haematologica 2006; 91:1234-1240. 24.Stewart AK, Fonseca R. Prognostic and therapeutic significance of myeloma genetics and gene expression profiling. J Clin Oncol. 2005; 23: 6339-6344. 25.Menke DM, Horny HP, Griesser H, Tiemann M, Katzmann JA, E. Kaiserling E, Parwaresch R, Kyle RA. Primary lymph node plasmacytomas (Plasmacytic lymphomas). Am J Clin Pathol. 2001;115:119-126. 26.Campo E, Swerdlow SH, Harris NL, Pileri S, Stein H, Jaffe ES. The 2008 WHO classification of lymphoid neoplasms and beyond: evolving concepts and practical applications. Blood 2011;117: 5019-5032. 27.Wilkins B, Buchan S, Webster J, Jones D. Tryptase-positive mast cells accompany lymphocytic as well as lymphoplasmacytic lymphoma infiltrates in bone marrow trephine biopsies. Histopathology 2001; 39: 150-155. 28.Berger F, Traverse-Glehen A, Felman P, Callet-Bauchu E, Baseggio L, Gazzo S, Thieblemont C, Ffrench M, Magaud JP, Salles G, Coiffer B. Clinicopathologic features of Waldenstrom’s macroglobulinemia and marginal zone lymphoma: are they distinct or the same entity? Clin Lymphoma 2005; 5: 220-224. 29.Teruya-Feldstein J. Diffuse large B-cell lymphomas with plasmablastic differentiation. Curr Oncol Rep. 2005;7: 357–363. 30.Delecluse HJ, Anagnostopoulos I, Dallenbach F, Hummel M, Marafioti T, Schneider U, Huhn D, Schmidt-Westhausen A, Reichart PA, Gross U, Stein H. Plasmablastic lymphomas of the oral cavity: a new entity associated with the human immunodeficiency virus infection. Blood 1997; 89:1413- 1420. 31.Colomo L, Loong F, Rives S, Pittaluga S, Martínez A, López-Guillermo A, Ojanguren J, Romagosa V, Jaffe ES, Campo E. Diffuse large B-cell lymphomas with plasmablastic differentiation represent a heterogeneous group of disease entities. Am J Surg Pathol. 2004;28: 736–747. 32.Vega F, Chang CC, Medeiros LJ, Udden MM, Cho-Vega JH, Lau CC, Finch CJ, Vilchez RA, McGregor D, Jorgensen JL. Plasmablastic lymphomas and plasmablastic plasma cell myelomas have nearly identical immunophenotypic profiles. Mod Pathol. 2005;18: 806-815. 512 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 1/GYNECOLOGICAL PATHOLOGY - Short Course 1 Patologija vulve/izabrane teme Vulvar Pathology - Selected Topisc Vukomanović Đurđević Biserka, Vukomanovic Djurdjevic Biserka Vojnomedicinska akademija Beograd, Srbija Military Medical Academy, Belgrade, Serbia Apstrakt Koža vulve se razlikuje od koze drugih regija po različitoj bakterijskoj flori I uslovima trenja. Dermatološke bolesti vulvarne kože i mukoze se dele u dve grupe. Prvu grupu čine dermatoze slične ekstravulvarnim dermatozama, a drugu čine dermatoze koje zahvataju predominantno vulvarnu regiju. Spečificne dermatoze vulve su kontaktni dermatitis, lichen planus, lichen sclerosus. Infektivne bolesti vulve se mogu preneti direktnim kontaktom. Prepoznavanje tipičnih mikroskopskih karakteristika pomaze u dijagnostici scabiesa, herpes virusne infekcije i molluscum contagiosuma. Humani papilloma virusi (HPV) su epiteliotropni virusi i mogu uzrokovati premalgnu i malignu transformatciju epitelnih ćelija. Sledeći kriterijumi kao što su virusne promene, klinički parametri i mikroskopski nalaz upućuju na postojanje dva puta u karcinogenezi skvamoznog karcinoma vulve i to HPV-zavisni put udružen sa Vulvarnom intraepitelialnom neoplazijom/VIN/ klasičnog tipa i ne -HPV sa VIN diferentovanog (simlex) tipa često udruženim sa lichen sclerosusom i/ili vulvarnom hiperplazijom.Invazivni planocelularni karcinom je najčešći karcinom vulve. Procena prognostičkih faktora je neophodna komponenta patohistoloskog izveštaja. Visoki gradus tumora, vaskularna invazija, veće dimenzije tumora i dubina invazije zaslužuju imunohistohemijsku analizu limfnih čvorova radi nalazenja metastaskih ćelija karcinoma. Extramamarna Pagetova bolest je retka. Može biti primarnog kožnog porekla ili udružena sa nekožnim tumorima gastrointestinalnog ili porekla mokraćne bešike. Imunohistohemijskla analiza je važna za odredjivanje primarnog porekla tumora. Ključne reči: vulve, dermatoza, herpers virus, šuga, Molluscum contagiosum, humanim papiloma virusima (HPV), Vulvar intraepitelna neoplazija / VIN, planocelularni karcinom, ektramamarna Pagets bolesti Abstract Skin of the vulva differs from the other sites from the different bacterial and friction features. Dermatologic diseases of vulvar skin and mucosa can be divided in two groups. The first includes dermatoses similar extravulvar sites,and second includes dermatoses affected predominantly vulvar region.Specific dermatoses of vulva are contact dermatitis, seborrhoic dermatitis, lichen planus, lichen sclerosus. Infectios deseases of vulva can be transmited by direct contact. Recognition of typical microscopic findings can helped for dignosis of scabies,herpes virus,molluscum contagiosum diseases of vulva. Human papilloma viruses (HPV) are epithelitrophic viruses and cause premalgnant and malignant transformation of epithelial cells.According criteria like viral associated changes,clinical paramethars, microscopic features there are two pathways in carcinogenesissquamous cell carcinoma HPVrelated pathway associated with Vulvar intraepithelial neoplasia/VIN of the classic type, and the nonHPV related with VIN of the differentiated (simlex) type frequently associated with lichen sclerosus and/or vulvar hyperplasia. Invasive squamous carcinoma is the most common carcinoma of vulva. Prognostic factors like stage, extracapsular nodal spread,infiltrative margins, vascular invasion, degree of differentiation, status of adjacent skin, stromal response, p53 overexpression are necessary for report. High tumor grade, capillary lymphatics invasion, biger tumor size and depth invasion deserve imunohistochemical analysis lymph nodes for metastatic cells of carcinoma. Extramammary Pagets desease is rare neoplasm. Paget desease can be primary cutaneus vulvar disease,or associated with noncutaneous carcinoma gastrointenstinal origin or bladder carcinoma. Immunohistochemical analysis is very helpful to determine primary origin. Key words: vulva, dermatoses, herpers virus, scabies, molluscum contagiosum, Human papilloma viruses (HPV), Vulvar intraepithelial neoplasia/VIN, squamous carcinoma, extramammary Pagets desease 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 513 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 1/GYNECOLOGICAL PATHOLOGY - Short Course 1 Introduction Skin of the vulva differs from the other sites from the different bacterial and friction features. Dermatologic diseases of vulvar skin and mucosa can be divided in two groups. The first includes dermatoses similar extravulvar sites,and second includes dermatoses affected predominantly vulvar region1. Specific dermatoses of vulva Contact dermatitis Agents like hygienic, synthetic materials may cause inflammatory response of the squamous epithelium. Histologic features are not specific and include intracellular and intercellular edema of squamous epithelim, with edema of dermis and dillatation of blood vessels. Inflammatory infiltrtation consist lymphocytes and histiocytes, and eosinophiles may be or not prominent. Figure 1. Dermal inflammatory infiltration Figure 2. Fungal organisms on surface vulvar skin (HEx40) (PASx40) Seborrhoic dermatitis Vulvar erruptions of in obese woman with, frictin cause, exematous apperance can have nonspecific histologic changes. Microscopic features may be akantosis, spongiosis and parakeratosis, with elongation of rete pegs of epidermis.Mild chronic dermal inflammation persist, and finding fungal and other infectious agents are according for the diagnosis sebborhoic dermatitis2. Psoriasis Vulvar psoriasis may be part of generalize disease or affect vulvar region.Erruptions have sharp dermacated areas with red surface covered with white plaques. The clinical course can be persistent with progress in chronic and generalized disease, or have remission. Histologic findings are hypekeratosis, parakeratosis,uniform elongation of rete pegs, lack of granular layer, thinning of stratum malpighii. Munro microabscesses are accumulation neutrofils in the epidermis.Dermal capillares are dilated, and dermis is with edema,with minimal inflammatory cells1. 514 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 1/GYNECOLOGICAL PATHOLOGY - Short Course 1 Lichen planus Lichen planus may present like vulvar white plaques or lines with pruritus in the womans after 30 years . It can be localized or generalized and can spontanous regress with exacerbations. Histologic features this interface dermatitis are akantosis, parakeratosis, hypekeratosis and hypergranulosis with elongation of rete pegs. Band-like lymphocytic infiltration next the epidermis is typically present and infiltrate extend in epidermis bassal cells.. Liquefaction degeneration of basal epithelial cells is present. Cytoid bodies present degenerated keratinocytes and dysceratotic cells, and they are localized in the epidermis or superficial dermis. Lichen sclerosus (et atrophicus) Lichen sclerosus affected predominantly postmenopausal women, but and young womens may be affected.Desease can affect any or all areas of vulva, and may extend perianal skin, or extragenital sites (neck, extremimites). Microscopic features can vary related to stage of desease. In the advanced the epithelium is atrophic, hyperkeratotic in some cases, with flattened rete pegs.In basal layers of epidermis are present edema and hydropic degeneration what sometimes separate basal cells from the basment membrane in some cases. Infiltration of lymphocytes in the basal and parabasal layers are visibly. Homogenisation, edema, collagenisation of the dermis are in association with mild chronic inflammation3,4. Infectious disease of vulva- selected topics Scabies Infestation of scabies is related with sexual contact.Pruritic disease cause excoriation, secondary infection. Microscopic features are epidermal irregular acantosis, focal spongiosis, psoriasiform epidermal hyperplasia with exoskeleton on the lower stratum corneum, dermal inflammation with perivascular infiltrate of lymphocites and variable numbers of eosinophils5. Figure 3. Exosceleton of scabies on the surfaceof Figure 4.Vulvar herpes simplex (PASx40) vulvar skin(HEx10) Herpesvirus infection Herpes simplex virus hominis type 2 can be transmited by direct contact.Fever, inguinal lymphadenopathy, dysuria, urinary retention and vesicles, pustules and painful ulcers are typical clinical findings. Microscopic 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 515 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 1/GYNECOLOGICAL PATHOLOGY - Short Course 1 features of herpes simpex virus infected epithelial cell is nuclear „ground glass“ appearence and typical eosinophilic intranuclear inclusion body predominantly in the margins of lesion, and finally lysis of cells. Imunohistohemistry for HSV is helpful for diagnosis of viral presence6. Molluscum contagiosum Poxyviral disease transmited with direct contact.Vulvar an perianal pruritic papules with central umbilication or punctum are present, and can be single or multiple. Microscopic findings are acantosis, spongiosis, ballonooning degeneration of epithelial cells which contain intracytoplasmatic eosinophilic inclusion bodies (Henderson-Paterson ) which push the nucleus on the periphery of cells.Edema and perivascular inflammation with endothelial vascular proliferation are present in the derm. Recognition of typical microscopic findings can be helped with electron mycroscopy of the virus, but wery rare analisis7. Figure 5. Molluscum contagiosum of vulvar skin (HEx40) Human papilloma viruses HPV Human papilloma viruses (HPV) are epithelitrophic viruses and cause premalgnant and malignant transformation of epithelial cells8. One-half of womens have other deaseases of genital tract cause HPV. Like other sexually trasmited deseases, direct contact is way for infection. Low-Risk HPV types for genital premalignant and malignant deseases are 6, 11, 42, 43, 44, IntermediateRisk HPV types are 33, 35, 39, 51, 52 and High-Risk HPV are types 16, 18, 31, 45, 56. HPV type 6,11,16,18,33 are most commonly in vulva8,9. In the geometric style HPV started with disorders in the lowest third of epithelium where HPV include in cell nuclei, and transform and deregulate cell cycle and inhibition of apopthosis. Condyloma are verrucous, papilary lesions of skin or mucous membrane.This benign neoplasms may be solitary, but may involve cervix, vagina, urethra, peranal skin and anal canal, predominantly in immunosuppresed patients.Central fibrovascular cores covered with squamous epithelium with acantosis, hyperkeratosis, parakeratosis with or without koilocytic atypia in epithelium are microscopic features . Hyperplasia an enlarging of the parabasal cells,with accentation of itracellular bridges and granular layer may be also present. Perinucler „halos“, with picnotic or enlarged nuclei are present in the superfitial cells, and multinucleated cells too, but they may be focal or absent.Abnormal mitosis are absent. Viral cytopathic changes are associated with HPV 6 and 11, but lack of koilocytic atypia does not exclude diagnosis of condyloma,an HPV infection8. Vulvar intraepithelial neoplasia ( VIN) are precancerous changes of the squamous type. International Society for Study of Vulvovaginal Disease and the International Society of Gineclogical Pathologist recommanded the use of term VIN. 516 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 1/GYNECOLOGICAL PATHOLOGY - Short Course 1 According criteria like viral associated changes,clinical paramethars, microscopic features there are two pathways in carcinogenesis sqimous cell carcinoma HPV-related pathway associated with VIN of the classic type, and the non-HPV related with VIN of the differentiated (simlex) type frequently associated with lichen sclerosus and/or vulvar hyperplasia9,10. Vulvar intaepithelial neoplasia, classic type are present like focal or multifocal lesions in womens with HPV infection,and they have concomitant cervical lesion in 50%(). Risk factors are cigarete smokong, immunodeficiency. Microscoping features of VIN are high nucleo-cytoplasmatic ratio, lack of cytoplasmatic maturation of basal and parabasal layers with crowding and cellular disarray,hyperchromasia and nuclear pleomorphism, mitotic figures, parakeratosis, hyperkeratosis,individual-cell keratinisation. Koilocytosis and binucleated and multinucleated cells are present too.Skin appendages are involved often. Imunohistohemichal analisis p16, p63, survivin are very helpful for diagnostic HPV infection in VIN11,12. Figure 6. p16 immunoreactivity in cells of VIN Figure 7. p63 immunoreactivity in cells of VIN (x40) (x40) VIN differentiated (simplex) type affected predominantly older patients, andoften associated with vulvar inflammatory desease or lichen sclerosus. Lesion is predimninatly solitary without assotiation with cervical desease, and HPV infection.The keratinocytes of differetiated VIN are large and pleomorphic, with eosinophilic cytoplasm in the basal and parabasal keratinocytes in the base of rete ridges.Prominent nucleoli in the enlarged nuclei are present predominantly in basal and parabasal keratinocytes.Elongation and anastomosis of rete pegs, and keratin pearl within rete may be present. Parakeratosis is often present10. Some patients have “mixed” VIN lesion, and the report of microscoping findings must have predominant type of VIN. Vulvar malignant epithelial tumors - selected topics Vulvar squamous cell carcinoma Invasive squamous carcinoma is the most common carcinoma of vulva. Carcinomas associated with HPV infection affected younger patients in risk groups of smokers, immunosupresion9,13. Morphologic subtypes of invasive squamous cell carcinoma include keratinizing, nonkeratinizing, basaloid, warty, spindled, verrucous.Prognostic factors like stage, extracapsular nodal spread,infiltrative margins, vascular invasion, degree of differentiation, status of adjacent skin, stromal response, p53 overexpression are necessary for report14,15,16. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 517 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 1/GYNECOLOGICAL PATHOLOGY - Short Course 1 Some authors sugest to detect dendritic cells by the Cd1a immunohistochemical analysis liike prognostic factor17. Figure 8.Immunoreactivity CD1a in squamous Figure 9. Metastatic cells of squamous cell carcinoma cell carcinoma of vulva (x5) in lymph node(HEx10) High tumor grade, capillary lymphatics invasion, biger tumor size and depth invasion deserve imunohistochemical analysis lymph nodes for metastatic cells of carcinoma. Figure 10. Pancitokeratin immunoreaktivity of Figure 10. CK7 immunoreactivity in cells ov metastatic cells of carcinoma in lymph node (x10) vulvar Paget desease (x5) MALIGNANT GLANDULAR TUMOR OF VULVA- selected topic Vulvar Pagets disease Extramammary Pagets desease is rare neoplasm, predominantly in the seventh decade. Desease can be focal or extensive. Microscopic features are typical with intraepidermal proliferation of large, atypical gladular-type cells, with granular or vacuolated cytoplasm and round nuclei with prominent nucleoli. Localisation of this cells is predominantly in the parabasal area, singly or in clusters, but may be in diferent layers of epithelium with “Pagetoid spread”. Mitotic figures are present, but not very frequent. 518 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 1/GYNECOLOGICAL PATHOLOGY - Short Course 1 Paget desease can be primary cutaneus vulvar disease,or associated with noncutaneous carcinoma gastrointenstinal origin or bladder carcinoma. Immunohistochemical analysis is very helpful to determine primary origin. Immunohistochemical finding in primary vulvar cutaneus Paget desease are CK7+, EMA+, CEA+, S100, MelanA-, HMB45-. Immunohistochemical finding in pagetoid rectal adenocarcinoma are CK7-, CK20+, CEA+. Immunohistochemical finding in pagetoid transitional cell carcinoma are CK7+/-,CK20+/-, CEA+, Uroplakin+.18 Some author suggest that survivin is useful prognostic marker for vulvar Paget desease19. Figure 11. Survivin immunoreactivity in cells of vulvar Paget desease (x5) Literature 1. Barchino-Ortiz L, Suárez-Fernández R, Lázaro-Ochaita P.Vulvar Inflammatory Dermatoses. 2. Actas Dermosifiliogr. 2012;103(4):260-275. 3. Schwartz JL, Clinton TS. Darier’s disease misdiagnosed as severe seborrheic dermatitis.Mil Med 2011;176(12):1457-9. 4. Yasar S, Mumcuoglu CT, Serdar ZA, Gunes P. A case of lichen sclerosus et atrophicus accompanying bullous morphea. Ann Dermatol. 2011;23(Suppl 3):S354-9. 5. Sander BB, Damsgaard K. Lichen sclerosus--a neglected disease.Ugeskr Laeger. 2005;173(46):2951-4. 6. Gaspard L, Laffitte E, Michaud M, Eicher N, Lacour O, Toutous-Trellu L. Scabies.Rev Med Suisse.2006;8(335):718-22, 7. Amaral RL, Giraldo PC, Cursino K, Gonçalves AK, Eleutério J Jr, Giraldo H. Nodular vulvar herpes in an HIVpositive woman.Int J Gynaecol Obstet. 2009;107(3):255. 8. Lin HY, Linn G, Liu CB, Chen CJ, Yu KJ. 4. An immunocompromised woman with severe molluscum contagiosum that responded well to topical imiquimod: a case report and literature review.J Low Genit Tract Dis. 2010;14(2):134-5. 9. Horn LC, Klostermann K, Hautmann S, Höhn AK, Beckmann MW, Mehlhorn G. HPV-associated alterations of the vulva and vagina. Morphology and molecular pathology.Pathologe. 201 ;32(6):467-75. 10.Alonso I, Fusté V, del Pino M, Castillo P, Torné A, Fusté Pet al. Does human papillomavirus infection imply a different prognosis in vulvar squamous cell carcinoma?Gynecol Oncol. 2011;122(3):509-14. 11.Ordi J, Alejo M, Fusté V, Lloveras B, Del Pino M, Alonso I et al. HPV-negative vulvar intraepithelial neoplasia (VIN) with basaloid histologic pattern: an unrecognized variant of simplex (differentiated) VIN.Am J Surg Pathol. 2009;33(11):1659-65. 12.Immunohistochemical staining for p16 and p53 in premalignant and malignant epithelial lesions of the vulva.Santos M, Montagut C, Mellado B, García A, Ramón y Cajal S, et al. Immunohistochemical staining for p16 and p53 in premalignant and malignant epithelial lesions of the vulva.Int J Gynecol Pathol. 2004;23(3):206-14. 13.Brustmann H, Hinterholzer S, Brunner A.Iimmunohistochemical expression of survivin and γ-H2AX in vulvar intraepithelial neoplasia and low-stage squamous cell carcinoma.Int J Gynecol Pathol. 2011;30(6):583-90. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 519 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 1/GYNECOLOGICAL PATHOLOGY - Short Course 1 14.Petignat P, Achtari C. Gynecology.Rev Med Suisse. 2010;6(232):105-8. 15.Woelber L, Choschzick M, Eulenburg C, Hager M, Jaenicke F, Gieseking F et al. Prognostic value of pathological resection margin distance in squamous cell cancer of the vulva.Ann Surg Oncol. 2011;18(13):3811-8. 16.Konidaris S, Bakas P, Gregoriou O, Kalampokas T, Kondi-Pafiti A. Surgical management of invasive carcinoma of the vulva. A retrospective analysis and review.Eur J Gynaecol Oncol. 2011;32(5):505-8. 17.Balega J, Butler J, Jeyarajah A, Oram D, Shepherd J, Faruqi A et al. Vulval cancer: what is an adequate surgical margin? Eur J Gynaecol Oncol. 2008;29(5):455-8. 18.Brustmann H. Galectin-3 and CD1a-positive dendritic cells are involved in the development of an invasive phenotype in vulvar squamous lesions.Int J Gynecol Pathol. 2006;25(1):30-7. 19.Shiomi T, Yoshida Y, Shomori K, Yamamoto O, Ito H. Extramammary Paget’s disease: evaluation of the histopathological patterns of Paget cell proliferation in the epidermis.J Dermatol. 2011;38(11):1054-7. 20.Shan SJ, Zhang N, Geng SL, Zhou Z, Chen X, Nie T, Guo Zet al Expression of survivin and human telomerase reverse transcriptase in extramammary Paget’s disease..J Cutan Pathol. 2010;37(6):635-40.. 520 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 1/GYNECOLOGICAL PATHOLOGY - Short Course 1 Tumori uterusa Tumors of the uterus Svetlana Milenković Svetlana Milenkovic Ginekološko akušerska klinika Višegradska, Klinički centar Srbije, Beograd, Srbija Gynecology and Obstetrics Clinic Visegradska, Clinical Center of Serbia, Belgrade, Serbia Apstrakt Poslednja revizija klasifikacije tumora uterusa uradjena je 2003.godine. Prema WHO, karcinomi endometrijuma podeljeni su u dve grupe, nazvane tip I i tip II. Tip I - grupa endometrioidnih adenokarcinoma, ima veću učestalost. Serozni i svetloćelijski adenokarcinom su u grupi II, po definiciji su karcinomi visokog gradusa, loše prognoze, bez jasno definisanih prekursora i predisponirajućih faktora rizika. U tipu I postoji PTEN mutacija, a u tipu II prekomerna ekspresija p53, pa se ova dva biomarkera koriste u njihovoj diferencijalnoj dijagnozi.WHO je definisala trostepeni sistem u odredjivanju gradusa endometrioidnih adenokarcinoma u kome skvamozna komponenta tumora nema uticaja. Postoje i drugi, dvostepeni, načini gradiranja ovih tumora, ali nisu opšteprihvaćeni. U odredjivanju stadijuma tumora važeća je FIGO klasifikacija 2009. u kome se tumori ograničeni na telo uterusa dele na stadijum IA i IB.U grupi mezenhimalnih tumora izdvojen je nediferentovani sarkom, koji se svojom morfologijom i imunohistohemijski razlikuje od leiomiosarkoma i endometrijalnog stromalnog sarkoma. Prihvaćeni su i novi termini kao što je UTROSCT, ali i nova teorija o nastanku karcinosarkoma. Smatra se da ovi tumori imaju epitelno poreklo, zbog čega sarkomska komponenta pokazuje pozitivnost i na epitelne i na mezenhimne markere.Otkriven je i veliki broj novih antitela, koja u dijagnostici i diferencijalnoj dijagnostici tumora uterusa imaju značaja samo ako se koriste u korelaciji sa morfološkom slikom i kliničkim podacima (karcinom endometrijuma vs karcinom jajnika, adenosarkom vs adenofibrom). Abstract The latest version of uterine tumors classification was done in 2003. According to WHO classification, endometrial cancers are divided into two groups, so called Type I and Type II. Endometrioid adenocarcinoma has a higher frequency and belongs to Type I. Serous and clear-cell adenocarcinoma, are forming Type II and by definition, they are high grade cancers with poor prognosis and no clearly defined precursors and predisposing risk factors. In Type I, there is a PTEN mutation and in Type II overexpression of p53. These two biomarkers are used in their differential diagnosis.WHO has defined a three-step system in determining the grade of endometrioid adenocarcinoma in which squamous component has no significance. There are other, two-step, grading systems which are not generally accepted.2009 FIGO classification is still in use for tumor staging and according to that tumors confined to the uterus body are divided into stages IA and IB. In the group of mesenchyme tumors, undifferentiated sarcoma was separated, due to different morphology and immunohistochemical profile compared to leiomyoma and endometrial stromal sarcoma. New terms, like UTROSCT, are accepted as well as new theory about the origin of carcinosarcoma. Common believe is that these tumors have epithelial origin and therefore sarcoma component showed positivity on epithelial and mesenchymal markers too. Large number of new antibodies was discovered which in the diagnosis and differential diagnosis of uterine tumors are only meaningful when used in correlation with the clinical picture and morphological data (endometrial carcinoma vs. ovarian carcinoma, adenosarcoma vs. adenofibroma). Uvod Endometrijalni karcinom je najčešći invazivni tumor ženskog genitalnog trakta u razvijenim zemljama sveta, a peti po učestalosti maligni tumor žena uopšte. Dva puta češće oboljevaju pripadnice bele rase, ali je smrtnost veća kod Crnkinja verovatno kao posledica lošeg kvaliteta zdravstvene zaštite i/ili genetskih razloga. Na osnovu kliničkopatološke slike i molekularno genetskih promena, karcinomi endometrijuma su podeljeni u dva tipa, jednostavno nazvanih Tip I i Tip II. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 521 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 1/GYNECOLOGICAL PATHOLOGY - Short Course 1 Tip I: 90% slučajeva endometrijalnih karcinoma pripada ovoj grupi. Javlja se kod premenopauzalnih i perimenopauzalnih žena, uslovljen je hroničnom estrogenom stimulacijom, a vodeći rizični faktori za njegov nastanak su: ovarijalna disfunkcija, gojaznost, dijabetes i nuliparitet. Najčešće su dobro diferentovani, simulirajući u histološkoj slici normalne endometrijalne žlezde, pa se zbog toga nazivaju endometrioidni adenokarcinomi. Imaju dobru prognozu, a prihvaćeni patogenetski put njihovog nastanka je sledeći: povećani nivo estrogena → anovulatorni endometrijum → atipična hiperplazija (EIN) → adenokarcinom. Pretpostavka je da je potrebno da protekne 3 do 5 godina za transformaciju od atipične hiperplazije do adenokarcinoma. Tip II: Manje učestali tip karcinoma, od koga oboljevaju žene u sedmoj i osmoj deceniji života. Prognostički su lošiji od prve grupe tumora, nisu uslovljeni hiperestrinizmom, dijabetesom niti gojaznošću, a razvijaju se na terenu atrofičnog endometrijuma. Ovoj grupi pripadaju serozni i svetlo ćelijski tip adenokarcinoma. Parametar Tip I Tip II Starost 50-60 godina 70-80 godina Gojaznost/hiperestrinizam Česta neuobičajena Endometrijum Anovulatorni Atrofični Prekursorna promena Atipična hiperplazija (EIN) Nepoznat (EIS?) Transformacija Spora Brza Tip karcinoma Endometrioidni Serozni ili svetloćelijski Molekularna promena MSI, PTEN mutacija p53 mutacija Familijarni sindrom HNPCC Način širenja Limfni nodusi peritoneum Zahvatanje jajnika Često Retko Prognoza Dobra Loša Tabela 1. Sumirane karakteristike karcinoma endometrijuma tipa I i II Tip I - Endometrioidni adenokarcinom¹ Mikroskopski nalaz: maligne žlezdane strukture koje odgovaraju normalnom proliferativnom endometrijumu, ali se od njega razlikuju kribriformnim rastom, viloglandularnim strukturama, većom nuklearnom atipijom nego u slučaju atipične hiperplazije, kao i oskudnom ili čak otsutnom stromom izmedju. Epitel je najčešće pseudostratifikovan, bazalno orjentisanih uniformnih jedara.Retko, i žlezdane strukture mogu biti izrazito ujednačene, tubularne, stvarajući sliku koja odgovara Sertoli ćelijskom tumoru jajnika. Često su prisutni i penušavi histiociti u stromi, što po nekim autorima ukazuje na primarno endometrijalno pre nego na cervikalno poreklo tumora, ukoliko postoji diferencijalno dijagnostička dilema. Vrlo bitna karakteristika ove grupe karcinoma je i skvamozna diferencijacija, koja stepenom atipije može varirati: a) diskretna gnezda – morule uniformnih nekeratinizirajućih skvamoznih ćelija koja se obično vide kod EIN ili metaplazije, b)manja polja čak i bizarne skvamozne diferencijacije sa prominentnom keratinizacijom i nuklearnom atipijom obično udružena sa slabo diferentovanim tumorom i c) manje upadljiva područja skvamozne diferencijacije sa fokalnom keratinizacijom i većim nuklearnim gradusom koja se teško mogu razlikovati od skvamocelularnog karcinoma i obično su udružena sa slabo diferentovanim endometrioidnim adenokarcinom. Prognostički značaj skvamozne diferencijacije nekada je bio predmet rasprave, ali se danas smatra da na prognozu utiče samo stepen diferencijacije žlezdane komponente, pa su termini „adenoakantom“ i „adenoskvamozni“ karcinom napušteni, a prihvaćen je termin „endometrioidni adenokarcinoma sa skvamoznom diferencijacijom“. Pored ovog tipa, u grupi endometrioidnog adenokarcinoma svrstane sudrugi, redji podtipovi²sa: 1) mucinoznom 2) tubalnom (cilijarnom) 3) sekretornom 4)skvamoznom(tranziciocelularnom) diferencijacijom, kao i 5)viloglandularni tip. Mucinozna diferencijacija: često je prisutna kao minor komponenta uobičajenog tipa endometrioidnog adenokarcinoma i definiše se prisustvom veće količine intracelularnog mucina. Svojom histološkom gradjom odgovara endocervikalnom tipu žlezda, ali se mnogo češće sreće u endometrijalnim nego endocervikalnim karcinomima. U retkim slučajevima može se uočiti gastrična ili intestinalna diferencijacija karcinomskih ćelija. 522 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 1/GYNECOLOGICAL PATHOLOGY - Short Course 1 Tubalna diferencijacija: tubalna diferencijacija je uobičajena i u benignom i u maligno izmenjenom endometrijumu. Termin se koristi u slučajevima veoma izraženog prisustva cilija na površini karcinomskih ćelija i po definiciji, ovaj karcinom pripada grupi dobro diferentovanih tumora. Sekretorna diferencijacija: retka je i karakteriše se prisustvom bazalnih ili supranuklearnih vakuola slično rano sekretornoj fazi, ali su žlezde veće, nepravilnije, sa pregradjivanjem lumena. Viloglandularni karcinom: dominantna histološka slika su brojne razgranate papile diskretne vezivno tkivne strome, obložene pseudostratifikovanim endometrioidnim epitelom. U većem broju endometrioidnih adenokarcinoma (čak i do 30%) može se uočiti izvestan stepen viloglandularne gradje, ali je u samo manje od 5%slučajeva dominantan mikroskopski nalaz. Postoje izvesna neslaganja oko prognostičkog značaja ove karcinomske forme u zavisnosti od studije, ali se misli da nema bitne razlike u odnosu na uobičajeni tip diferencijacije. FIGO sistem odredjivanja histološkog gradusa Gradus 1 Manje od 5% tumora su područja solidne gradje Gradus 2 Područja solidne gradje obuhvataju 5 – 50% tumora Gradus 3 Područja solidne gradje obuhvataju preko 50% tumora Napomena: izražena nuklearna atipija povećava gradus tumora za jedan stepen, a prisustvo skvamozne diferencijacije ne utiče na gradus tumora Tabela 2. Histološki gradus endometrioidnih adenokarcinoma Postoje i drugi predloženi načini odredjivanja stepena diferencijacije, jer se kao najveći nedostatak FIGO sistema ističe problem pri odredjivanju donje predložene granice -5% solidnog rasta izmedju gradusa 1 i 2, kao i mala razlika u prognozi izmedju njih (92% vs 88% 5-godišnjeg preživljavanja). Taylor i saradnici predlažu binarni sistem u kojima kod gradusa 2 tumor pokazuje više od 20% solidnog rasta, što daje veću reproducibilnost u odnosu na trostepeni sistem (κ 0,97 vs 0,53 ). Lax i saradnici³ takodjesugerišu binarni sistem u kojima o gradusu 2 tumora odlučuju prisutna dva ili više parametra od sledećih predloženih: (1) više od 50% tumora je solidnog načina rasta (nema uticaja da li se o radi o skvamoznim ili neskvamoznim poljima), (2)difuzno infiltrativna vs ekspanzivna miometrijalna invazija (3) tumorska nekroza. Za tumore koji su ograničenina endometrijum, 50% ili više solidnih polja i tumorska nekroza odredjuju visoki gradus tumora.U njihovoj analizi postignuta je visoka korelacija u odnosu na 5-godišnje preživljavanje, ali se kao osnovni nedostatak ističe primenjivost samo na već operisane pacijente, jer je važan kriterijum način miometrijalne invazije. Endometrioidni adenokarcinom Serozni adenokarcinom Slika 1. Histološki tipovi carcinoma endometrijuma 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 523 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 1/GYNECOLOGICAL PATHOLOGY - Short Course 1 Imunohistohemijski profil: Endometrioidni adenokarcinomi pokazuju ekspresiju pan-citokeratina, EMA, CA125, dok je ekspresija CEA neuobičajena. Skoro svi su CK7 pozitivni i CK 20 negativni, a mucinozna varijanta je nekada CDX2 pozitivna. Za razliku od ostalih karcinoma, endometrioidni su izrazito vimentin pozitivni. FIGO gradus I i II tumora ispoljavaju pozitivnost za ER i PR kao i oko polovine slučajeva gradusa 3.p53 ekspresija nije uobičajena u gradusu 1 i samo je minimalno izražena u gradusu 2, ali je prisutna u velikom broju slučajeva gradusa 3. Manji panel imunohistohemijskih markera može biti koristan u diferencijalnoj dijagnozi izmedju primarnih endometrijalnih i endocervikalnih karcinoma: p16fokalno+/ER+/PR+ je profil endometrijalnih, dok se p16difuzno+/ER-/PR- sreće u endocervikalnim tumorima. Tip II Ovoj grupi pripadaju dva tipa endometrijalnog karcinoma: serozni (sinonim serozni papilarni adenokarcinom) i svetloćelijski (clear cell) adenokarcinom. Po definiciji, oba tipa tumora pripadaju karcinomima visokog stepena maligniteta i ne gradiraju se. Serozni (papilarni) adenokarcinom Entitet koji se izdvaja u poslednjih 20-tak godina, javlja se sa učestalošću manjom od 10% (u nekim studijama oko 1,1%),prepoznat u nekoj od sledećih formi, koje se uzajamno ne isključuju: (1) mešoviti serozni i endometrioidni adenokarcinom (2)serozni adenokarcinom u polipu (3)serozni karcinom ograničen na endometrijalnu površinu (tzv.intraepitelni karcinom). Mikroskopska slika: papilarne strukture koje mogu biti velike, široke, nepravilne, obložene tumorskim ćelijama sa visokim jedarno/citoplazmatskim odnosom su jedna od karakterističnih nalaza. Drugi tip promene je udružen sa prethodnim i odnosi se na eksfolijaciju karcinomskih ćelija sa površine papila (mikropapilarni rast). Treći način rasta je prisustvo mikropapila unutar žlezdanih struktura. U 30-40% slučajeva prisutna su psamoma tela. Imunohistohemijskiprofil⁴: oko 75% seroznih adenokarcinoma pokazuju p53 ekspresiju, Ki-67 indeks je izrazito visok (čak 50-75%), a difuzno jaka pozitivnost postoji i za p16. Istovremeno postoji difuzni gubitak ekspresije za ER i PR, ali kao endometrioidni adenokarcinomi obično pokazuju pozitivnost za pan citokeratin, EMA, CA 125, CK 7 i vimentin i gubitak ekspresije CEA. WT1 pozitivnost je prisutna u najviše 2030% slučajeva, što je bitno razlikuje od primarno ovarijalne, tubalne ili peritonealne lokalizacije gde je WT1 pozitivnost prisutna u najmanje 70-80% slučajeva. Svetloćelijski (clear cell) adenokarcinom Mikroskopska slika: solidna polja, tubulocistične ili papilarne strukture sagradjene od svetlih, glikogenom bogatih, ili „hobnail“ ćelija, izrazito atipičnih, bizarnih jedara, sa često prisutnim PAS pozitivnim, dijastaza rezistentnim eozinofilnim intracelularnim i ekstracelularnim hijalinim globulama. Mešoviti adenokarcinomi Mešavina tipa I (endometrioidni/mucinozni) i tipa II (serozni/svetloćelijski) karcinoma u kome je manja komponenta zastupljena sa najmanje 10% ukupnog volumena tumora naziva se mešovitim adenokarcinom. Nediferentovani karcinom Ovaj tumor se izdvaja kao poseban entitet po svojoj histološkoj slici u kojoj postoji potpuni gubitak bilo kakvog tipa diferencijacije karcinomskih ćelija. Treba ga razlikovati odendometrioidnog adenokarcinoma gradusa 3 u kojem postoje očuvani bar minimalni delovi žlezdane diferencijacije, skvamozne metaplazije ili eventualne trabekularne gradje. MEZENHIMALNI TUMORI Endometrijalni stromalni nodul Definicija: Jasno ograničeni tumor sagradjen od stromalnih ćelija koje odgovaraju stromalnim endometrijalnim ćelijama proliferativne faze. Izmedju njih se uočavaju brojni sitni krvni sudovi arteriolarnog tipa. Retko se mogu uočiti prstolike projekcije u miometrijum, ali ne smeju bitidubljeod 3mm⁵. Diferencijalna dijagnoza: (1)celularni leiomiom (jako pozitivna reakcija za desmin, h-caldesmon i negativna reakcija za CD 10) i (2) endometrijalni stromalni sarkom niskog stepena maligniteta (bazira se na evaluaciji miometrijalne infiltracije i zato se ne može postaviti definitivna dijagnoza na kiretaži!) Endometrijalni stromalni sarkom (ESS) 524 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 1/GYNECOLOGICAL PATHOLOGY - Short Course 1 Učestalost ovog tipa tumora je oko 0,2% od svih genitalnih malignih tumora žene. Mikroskopska slika: hipercelularni tumor sagradjen od uniformnih ćelija tipa endometrijalnih stromalnih ćelija sa brojnim malim arteriolama koje odgovaraju spiralnim u kasno sekretornom endometrijumu. Izražena je invazija limfatika, mitotska aktivnost obično je niska (manje od 10 mitoza na 10HPF), a nekroza otsutna.U nekim slučajevima naglašena je i perivaskularna hijalinizacija. Endometrijalni stromalni tumori mogu dodatno pokazivati i druge, različite tipove diferencijacije kao što su: glatko mišićna, obično fokalna i manja od 30%. Ako je prisustvo ove druge komponente veće od 30% tumor se naziva mešovitim endometrijalnim stromalnim i glatkomišićnimtumorom⁶. Takodje, mogu biti prisutni i elementipolnihtrakajajnika, najčešće u formi anastomozirajućih trabekula ili traka, retko tubula. Ukoliko predominiraju (pozitivnost na Inhibin!) tumor se naziva „uterine tumor resembling ovarian sex cord stromal tumor“. Treća varijanta je prisustvo endometrijalnih žlezda koje se sreću u 10-40% slučajeva. Napomena: Drugi tipovi diferencijacije mogu se javiti i kod endometrijalnih stromalnih nodula kao i kod endometrijalnih stromalnih sarkoma, niskog stepena maligniteta, pa se kao diferencijalno dijagnostički kriterijum uzima miometrijalna infiltracija. Diferencijalna dijagnoza: pored stromalnog nodula, uključuje i celularni leiomiom, intravensku leiomiomatozu kao i adenomiozu. Nediferentovani endometrijalni sarkom Uterini mezenhimalni tumor viskog stepena maligniteta koji ne pokazuje specifičnu diferencijaciju, ćelijska i nuklearna atipija su veoma izražene, gubi se karakterističan način rasta i vaskularnost ESS niskog stepena maligniteta, izraženo je krvavljenje i nekroza a tumorske ćelije jako liče na sarkomsku komponentu karcinosarkoma, koga je neophodno diferencijalno dijagnostički isključiti. Uopšteno, dijagnoza ovog tipa tumora uglavnom se zasniva na sekvencijalnoj eliminaciji slabo diferentovanog karcinoma, leiomiosarkoma i karcinosarkoma (od formiranja većeg broja uzoraka za analizu do korišćenja imunohistohemijskih metoda). Leiomioma Mikroskopska slika: Anastomozirajuće trake uniformnih, fuziformnih – vretenastih ćelija, nejasnih granica, izduženih jedara, uobičajeno retkih mitoza. Kod trudnica ili žena koje koriste progesteronsku terapiju krvavljenje, nekroza, edem, miksoidne promene i hipercelularnost se mogu javiti i kod leiomioma. Patološka dijagnoza Karakteristična histološka slika Leiomioma sa povećanom mitotskom aktivnošću 5-10 mitoza/10HPF Atipični leiomioma Pleomorfne džinovske tumorske naglašenom nuklearnom atipijom Celularni leiomiom Značajno veća celularnost nego uobičajeno Epitelioidni leiomiom Okruglaste/ovalne/poligonalne odgovaraju epitelnim Lipoleiomioma Veći procenat zrelog masnog tkiva Neurilemmoma-like leiomioma Naglašeno nuklearno palisadanje koje odgovara benignim tumorima perifernih nervnih omotača Intravaskularna leiomiomatoza „Crvoliki“ intravenski benigni glatkomišićni tumori Diseminovana peritonealna leiomiomatoza Histološki benigne, tumorolike glatkomišćne lezije na peritoneumu ili omentumu Benigni metastazirajući leiomiom Histološki benigne glatkomišićne mase u plućima koje se tumače kao metastaze primarno uterinih leiomioma ćelije ćelije sa koje Tabela br.3. Histološke varijante leiomioma⁷ 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 525 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 1/GYNECOLOGICAL PATHOLOGY - Short Course 1 Atipični leiomiom Epiteloidni leiomiom Slika 2. Histološke varijante lejomioma Leiomiosarkoma Mikroskopska slika: fascikule vretenatastih mezenhimalnih ćelija sa hiperhromatičnim jedrima, zgrudvanim hromatinom, prominentnim nukleolusima, naglašenom tumorskom nekrozom (koja nije uvek prisutna). Celularnost visoka, mitotske figure brojne, najčešće više od 15/10HPF, a vaskularna invazija prisutna u 25% slučajeva. Kriterijumi za dijagnozu leiomiosarkoma (WHO 2003.) 1.koagulativna ćelijska nekroza 2.U odsustvu nekroze mora postojati difuzna, umerena do naglašena citološka atipija, a broj mitoza najmanje 10/10HPF. Epiteloidna varijanta leiomiosarkoma Mikroskopski nalaz: Hipercelularan tumor, „epiteloidnog“ fenotipa tumorskih ćelija, sa naglašenom citološkom atipijom, tumorskom nekrozom i visokim mitotskim indeksom⁸. Ukoliko nema nekroze, dijagnoza epiteloidnog leiomiosarkoma zahteva difuznu, umerenu do veoma izraženu citološku atipiju i 5 ili više mitoza na 10HPF. Miksoidna varijanta leiomiosarkoma Mikroskopski nalaz: Tumorske ćelije široko razdvojene obilnim miksoidnim materijalom. Niska celularnost i niska mitotska aktivnost se sreće u najvećem broju slučajeva.Medjutim, većina tumora pokazuje izrazit celularni pleomorfizam. Ukoliko nema nekroze, dijagnoza miksoidnog leiomiosarkoma zahteva difuznu, umerenu do veoma izraženu citološku atipiju i 5 ili više mitoza na 10HPF. Tumorska nekroza Prisutna prisutna prisutna Nije prisutna Nije prisutna Atipija Difuzna, umerena do izražena Nema/laka Nema/laka Difuzna, umerena do izražena Difuzna, umerena do izražena MF/10HPF Bilo kog stepena ≥10 <10 ≥10 <5 Nije prisutna Nije prisutna Nije prisutna Nije prisutna Nije prisutna Difuzna, umerena do izražena Nema/laka Nema/laka Fokalna, umerena do izražena Fokalna, umerena do izražena 5-9 ili atipične mitotske figure <5 ≥5 ≥5 <5 Dijagnoza leiomiosarkoma Leiomiosarkoma STUMP Leiomiosarkoma Atipični leiomiom sa niskim stepen rizika ponovnog javljanja STUMP leiomioma Mitotski aktivan leiomiom STUMP Atipični leiomiom Tabela br.4 Histološki kriterijumi za dijagnozu glatko mišićnih tumora 526 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 1/GYNECOLOGICAL PATHOLOGY - Short Course 1 MEŠOVITI EPITELNI I MEZENHIMNI TUMORI Maligni mešoviti Milerov tumor (carcinosarcoma) Po definiciji ovaj tumor predstavlja kombinaciju epitelijalne (karcinomske) i mezenhimalne (sarkomske) komponente, sa učestalošću do 10% svihendometrijalnihmaligniteta⁹. Iako se danas misli da je poreklo obe komponente epitelno (pa zbog toga sarkomski delovi tumora pokazuju pozitivnost i za epitelne i za mezenhimne biomarkere) preduslov za dijagnozu je histološki aspekt mezenhimalne diferencijacije. Tradicionalno se dele na homologi i heterologi tip, što se odredjuje na osnovu diferencijacije sarkomske komponente. Adenosarkoma Mikroskopska slika¹⁰: bifazični tumor sa benignom epitelnom i sarkomatoznom – malignom komponentom. Karakteriše se postojanjem kleftova na površini, stromalnog kondenzacijom koja okružuje žlezde i kleftove, a u kojoj se vidi citološka atipija i mitotske figure (više od 1/10HPF). Sarkomska komponenta je najčešće homologi stromalni sarkom niskog stepena maligniteta, ali se ponekad mogu javiti ielementi heterologe diferencijacije na pr. rabdomiomske, hrskavičave... Diferencijalna dijagnoza uključuje adenofibrom i botrioidni rabdomiosarkom. Adenofibroma Mikroskopska slika: Veoma redak bifazični tumor u kome su benigne i epitelna i mezenhimna komponenta. Površina tumora može biti papilarna, sa kleftovima, ali nema stromalne atipije, mitoza niti periglandularne kondenzacije. Literatura 1. Albertini AF, Devouassoux-Shisheboran M, Genestie C. Pathology of endometrioid carcinoma.Bull Cancer. 2012 Jan;99(1):7-12. 2. Silvereberg SG.. Tumors of the uterine corpus:epithelialtumours and related conditions.In: Tavassoli FA, DevileePI,eds. Tumors of the breast and female genital tract. Lyon: WHO; 2003; 2002:218. 3. Lax SF, Kurman RJ, Pizer ES, Wu L, Ronnett BM. A binary archictural grading system for uterin endometrial endometrioid carcinoma has superior reproducibility compared FIGO grading and identifies subsets of advanced –stage tumors with favourable and unfavourable prognosis. Am J SurgPathol 2000; 24: 1201-1208. 4. Lax SF. Precursor lesions of endometrial carcinoma: diagnostic approach and molecular pathology. Pathologe. 2011;32Suppl 2:p.255-64 5. Dionigi A, Oliva E, Clement PB, Young RH. Endometrial stromal nodules and endometrial stromal tumors with limited infiltration:aclinicopathologicstady of 50 cases. Am J surgPathol 2002; 26:567-581. 6. ClementPB.The pathology of the uterine smooth muscle tumors and mixed endometrial stromal-smooth muscle tumors: a selective review with emphasis on recent advances. Int J GynecolPathol 2000; 19:39-55. 7. Hendrickson MR, Tavassoli FA, Kempson RL, et al. Mesenchymal tumorous and related lesions. In: Tavassoli FA, DevileePI,eds.Tumors of the breast and female genital tract. Lyon: IARC Press; 2003; 233-244. 8. Atkins K, Bell S, Kempson RL, Hendrickson MR. Epitheloid smooth muscle tumors of uterus. Mod Pathol 2002; 14(1):132A. 9. RaniKanthan and Jenna-Lynn Senger.Uterine Carcinosarcomas (Malignant Mixed MüllerianTumours): A Review with Special Emphasis on the Controversies in Management. ObstetGynecol Int. 2011; 2011: 470795. 10.Zaloudek CJ, Norris HJ. Adenofibroma and adenosarcoma oh the uterus: a clinicopathologic study of 35 cases. Cancer 1981; 48: 354-366. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 527 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 1/GYNECOLOGICAL PATHOLOGY - Short Course 1 Important Recent Advances in Gynaecological Pathology Sanjiv Manek Consultant Gynaecological Pathologist Oxford, UK In recent years there have been a significant number of changes in gynaecological pathology practice. Many of these deal with the molecular basis of common and rare gynaecological tumours but there are also many that deal with morphology, terminology and new perspectives. This talk will focus on a few chosen entities and concepts that have been recently introduced and have reached everyday practice but which can cause problems or diagnostic difficulties and can require alteration in protocols. The areas to be covered are: The concept of endometrial intraepithelial neoplasia (EIN) The concepts of endometrial intraepithelial carcinoma (EIC) and minimal serous carcinoma Undifferentiated endometrial carcinoma Inherited endometrial tumour syndromes The relevance of tubal carcinoma in situ and the preceding dysplastic lesion in the pathogenesis of pelvic serous carcinoma The concept of differentiated VIN and its association with squamous carcinoma Microinvasion in borderline serous ovarian tumours The implications of the 2009 FIGO staging system for endometrial and vulval malignancies and uterine sarcomas Endometrial intraepithelial neoplasia (EIN) The 2003 WHO and ISGP review of endometrial hyperplasia has retained the following classification. Hyperplasias (typical) Atypical Hyperplasia Simple without atypia Simple atypical Complex without atypia Complex atypical The atypia refers to cytological rather than architectural atypia and is commonly diagnosed when the nuclear/cytoplasmic ratio is increased and there is loss of axial polarity with nuclei becoming large and rounded with prominent nucleoli. Mitoses and apoptotic bodies are frequently found. Because of the common problem of reproducibility in identifying genuine cytological atypia, an alternative classification, the endometrial intraepithelial neoplasia (EIN) system has been proposed and is being increasingly used. In this system, there is a spectrum from benign hyperplasias through EIN to adenocarcinoma. The EIN approach is supported by morphometric, molecular and genetic data. There is a concept of latency with PTEN mutation. Some latent glands may involute and others may progress to EIN. From the molecular point of view, EIN is a monoclonal proliferation with PTEN mutation and microsatellite instability. Histologically, EIN is identified by: Foci of atypical hyperplasia at least 1 mm and greater in linear dimension (usually >5-10 gland clusters). Gland to stroma ratio >1. Cytological features that are different from those of background glands. Exclusion of potential mimics such as disordered proliferation, polyps and adenocarcinomas. Most complex atypical hyperplasias could be reclassified as EIN. In carcinoma cases with background atypical hyperplasia/EIN, PTEN mutations, b-Catenin, MLH 1 and K-ras oncogene mutations and microsatellite instability are seen in both processes, lending support to the fact that atypical hyperplasia/EIN is pre-cancerous. Many studies have shown an increased risk of associated 528 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 1/GYNECOLOGICAL PATHOLOGY - Short Course 1 carcinoma when an endometrial biopsy shows atypical hyperplasia. With EIN this risk is up to 46x more than without EIN. It may be difficult to distinguish between atypical hyperplasia/EIN and well-differentiated (FIGO Grade 1) endometrial adenocarcinoma. Features which help to identify adenocarcinoma are: prominent nuclear pleomorphism a confluent, fused glandular pattern a cribriform architecture of glands a confluent papillary pattern desmoplastic stroma replacement of stroma by squamous epithelium The confluent or papillary pattern or squamous epithelial predominance has to occupy at least half of a low-power field. EIC and minimal serous carcinoma Endometrial intraepithelial carcinoma (EIC) or serous EIC is the precursor lesion of serous endometrial adenocarcinoma and occurs typically in post-menopausal women on a background of atrophy. It comprises highly pleomorphic cells on the surface and in glands without invasion but with frequent mitoses and prominent nucleoli in atypical nuclei. Mib 1 (Ki67) staining shows a high proliferation index and there is strong p53 staining indicating p53 gene mutation. Rarely, there may be microinvasion associated with EIC. It is well known that EICs can be associated with a high incidence of extrauterine metastatic disease despite lack of invasion in the uterus. Hence, the concept of ‘minimal uterine serous carcinoma’ has developed. This is diagnosed when there is EIC with focal superficial/surface serous carcinoma. These lesions are WT1 and ER negative. Recently, it has been shown that some serous carcinomas are WT1+ and p53 – (?dedifferentiation of type I cancers). Another lesion, endometrial glandular dysplasia (EmGD) has been described and this appears to be a precursor of EIC. Undifferentiated endometrial carcinoma This is a new entity which is now increasingly used but it is poorly recognised because of its ‘weak definition’ in the literature. It is generally included in FIGO G3 endometrioid adenocarcinomas in textbooks. The WHO definition is ‘no evidence of glandular or squamoid differentiation ‘. The tumour is composed of medium or large cells with a complete absence of glandular differentiation and absent or minimal ( < 10 % ) neuroendocrine differentiation. There are no specific gross features and microscopically there are solid sheets of these medium or large cells with marked pleomorphism, rhabdoid cells, foci of necrosis and many mitoses (>10/10hpf). Immunohistochemically, they are CK focally positive, EMA positive, Vimentin positive and <10% cells show 1 positive neuroendocrine marker. Its significance is in that it has a poor prognosis (75 % cases died of disease), as compared to the excellent prognosis of G2 and intermediate prognosis of G3 endometrioid carcinomas (30% cases died of disease) Inherited endometrial tumour syndromes Lynch Syndrome or Hereditary Nonpolyposis Colon Cancer Syndrome (HNPCC) patients are vulnerable to developing endometrioid adenocarcinomas and this may be the initial presentation in more than 50% of cases. They usually occur in young pre-menopausal patients and are more common than colorectal cancers in female patients with this syndrome. The genetic defect is in DNA mismatch repair genes (MLH-1, MSH6 and 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 529 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 1/GYNECOLOGICAL PATHOLOGY - Short Course 1 MSH2). Women with HNPCC have a 20% chance of developing endometrial cancer compared to 3% without HNPCC. HNPCC/Lynch Syndrome endometrial cancers have the following characteristics: predilection for isthmic and cornual areas poor differentiation peritumoral, Crohn’s-like lymphoid reaction intratumoral lymphocytic infiltration higher incidence of lymphatic permeation higher incidence of mixed carcinomas and carcinosarcomas Tubal carcinoma in-situ (serous tubal intraepithelial carcinoma – STIC) and its precursor lesions and the link to pelvic serous carcinoma There is a vast amount of new data showing the increasing importance of STIC. In a significant number of cases of endometrial serous carcinoma, including EIC, there is co-existent STIC with similar p53 signatures. In some cases, the STIC is WT1 negative suggesting spread from the endometrium or at least a field change. In other cases the STIC is WT1 positive indicating a synchronous lesion when the endometrial primary is WT1 negative. STIC or tubal invasive serous carcinoma may also be the origin of some endometrial serous carcinomas which have the reversed immunoprofile of WT1 positivity and p53 negativity. The STIC is also thought to be the origin of many peritoneal and ovarian serous carcinomas particularly in cases of high grade serous histology. Until recently it was suggested that the p53 signature (synonymous with tubal dysplasia and the tubal intraepithelial lesions in transition - TILTs) was only limited to BRCA+ cases but new evidence shows that its importance goes beyond that. Vulval intraepithelial neoplasia (VIN) and squamous carcinoma (SCC) There are two distinct types of VIN; each with a related SCC: Differentiated (Simplex) Classical (Bowenoid) 1. Undifferentiated appearance Basaloid ↔ Warty 2. Associated with high risk HPV (16,18) 3. Multifocal 4. ± CIN, VAIN, warts 5. Young women 6. Lower risk for SCC 7. p16 positive 8. p53 negative 9. Associated with basaloid/warty/ verrucous SCC 10. Rare mucinous differentiation 1. Difficult to diagnose in isolation (i.e. without adjacent SCC) 2. Hyperplastic epithelium with basal atypia 3. Arises in vulval dystrophy (e.g hyperplasia) or lichen sclerosus 4. Older women 5. p16 negative 6. p53 positive 7. Associated with keratinising SCC 8. Basaloid variant recognised Recently, there has been a proposal to reclassify VIN. Essentially it has been suggested that in classic VIN, the VIN 1 category be abandoned because of its rarity and instead be referred to as flat condyloma or HPV effect and VIN 2 and VIN 3 be lumped together as classic VIN. This change is to reflect the lack of evidence of progression from VIN 1 to VIN 3 (as in the cervix). However, some cases of VIN 1 with high-risk HPV are known, suggesting VIN 1 could be pre-malignant in nature. Thus, there has also been a proposal to have a two-tier clarification with low-grade VIN encompassing condylomas and VIN 1 and high-grade VIN encompassing VIN 2 and 3 and also differentiated VIN. 530 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 1/GYNECOLOGICAL PATHOLOGY - Short Course 1 Microinvasion in borderline serous ovarian tumours Is this a risk lesion? The conventional wisdom suggests that it is not but recent data has shown that microinvasion is an adverse prognostic factor independent of stage of disease and implant status in non-pregnant patients (71% v 97% 5 year survival) The implications of the 2009 FIGO staging system for endometrial and vulval malignancies The introduction of lymph node status in endometrial malignancies has meant additional specimens to handle but at the same time the problem of false positive peritoneal fluid cytology has been resolved. Assessment of cervical involvement by the endometrial malignancy has become easier. In vulval cancer, there is more detailed analysis of lymph node involvement required with the introduction of substages in stage 3. One now needs to asses how many lymph nodes are involved and what the size of the metastasis is. For the first time there are now FIGO stages available for the uterine sarcomas rendering it easier to assign the appropriate stages based on size, etc. Literature 1. Broaddus RR, Lynch HT, Chen LM, et al. Pathological features of endometrial carcinoma associated with HNPCC: A comparison with sporadic endometrial carcinoma. Cancer 2006; 106(1): 87-94. 2. Meyer LA, Broaddus RR, Lu LH. Endometrial cancer and Lynch Syndrome: Clinical and pathologic considerations cancer control, 2009; 16(1): 14-22. 3. WHO classification of tumours: Pathology and Genetics of Tumours of the Breast and Female Genital Organs: Tumours of the uterine corpus. IARC Press; 2003: 228-233. 4. Bergeron C, Nogales F, Masseroli M, et al. A multicentric European study testing the reproducibility of the WHO classification of endometrial hyperplasia with a proposal of a simplified working classification for biopsy and curettage specimens. Am J Surg Path 1999; 23: 1102-1108. 5. Mutter GL. Diagnosis of premalignant endometrial disease. J Clin Path, 2002; 55: 326-331. 6. FIGO Committee on Gynaecologic Oncology. Int J Gynaecol & Obs 2009; 105: 103-104. 7. Carlson JW & Mutter GL. Endometrial intraepithelial neoplasia is associated with polyps and frequently has metaplastic change. Histopathology, 2008; 53: 325-332. 8. Zhang W, Liong SX, Yu X, et al. Occurrence of endometrial glandular dysplasia precedes uterine papillary serous carcinoma. Int J Gynaecol Pathol 2007; 26: 38-52. 9. Miltel K & Da Costa D. Endometrial hyperplasia and carcinoma in endometrial polyps: Clinicopathologic and follow-up findings. Int J Gynaecol Pathol 2008; 27: 45-48. 10.Hui P et al. Minimal uterine serous carcinoma: a clinicopathological study of 40 cases. Mod Pathol 2005; 18: 75-82 11.Sherman ME, Ronnett BM, Ioffe OB, et al. Reproducibility of biopsy diagnosis of endometrial hyperplasia: Evidence supporting a simplified classification. Int J Gynaecol Pathol 2008; 27: 318-325. 12.Mutter, MD. www.endometrium.org 13.Hirschowitz L, Ganesan R and McCluggage WG. WT1, p53 and ER receptor expression in uterine serous carcinoma. Histopathol 2009; 55: 478-82 14.Rabban JT and Zaloudek CJ. Minimal uterine serous carcinoma: current concepts in diagnosis and prognosis. Pathology 2007; 39: 125-33 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 531 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 1/GYNECOLOGICAL PATHOLOGY - Short Course 1 15.Longacre TA, McKenney JK, Tazelaar HD et al. Ovarian serous tumours of low malignant potential (borderline tumours): outcome-based study of 276 patients with long-term (> or =5-year) followup. Am J Surg Pathol 2005; 29: 707-723 16.McKenney JK, Balzer BL, Longacre TA. Patterns of stromal invasion in ovarian serous tumours of low malignant potential (borderline tumours): a reevaluation of the concept of stromal micro-invasion. Am J Surg Pathol 2006; 30: 1209-21 17.Carcangiu ML, Peissel B, Pasini B et al. Incidental carcinomas in prophylactic specimens in BRCA1 and BRCA2 germi-line mutation carriers, with emphasis on fallopian tube lesions: report of 6 cases and review of the literature. Am J Surg Pathol 2006; 30: 1222-30 18.Jarboe EA, Folins AK, Drapkin R et al. Tubal and ovarian pathways to pelvic epithelial cancer: a pathological perspective. Histopathology 2008; 53: 127-38 532 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 2/GYNECOLOGICAL PATHOLOGY - Short Course 2 Placenta – a silent witness: clinical and forensic importance of placental examination Marina Kos Clinical Department of Pathology “Ljudevit Jurak” Clinical Hospital Center “Sestre milosrdnice”, Zagreb, Croatia University of Zagreb Medical School. Zagreb, Croatia Introduction There is no doubt that obstetrics carries high medical liability risk. In many countries, gynecologists-obstetricians who attend childbirths and perform complex obstetric procedures are faced with increasing malpractice insurance premiums and litigation risk. The American College of Obstetricians and Gynecologists (ACOG) publishes its Survey of Professional Liability since 1983, with the objective to analyze the effect that malpractice litigation has had on the practice of obstetrics and gynecology in the United States1. According to the 2003 ACOG survey, 76.3% of the members who answered the questionnaire have been involved in a lawsuit at least once in their professional career; gynecologists/obstetricians have been sued a total of 2.64 times per individual over the course of their careers1,2. In the 2006 ACOG Survey, 89% of respondents indicated that they had been sued during their careers. The average number of claims per obstetrician was 2.6 (3). The ACOG’s 2009 Survey on Professional Liability showed that nearly 91% of gynecologists/obstetricians had experienced at least one liability claim filed against them during their professional careers, with an average of 2.69 claims per physician. In 2009, 62% percent of the total reported claims were for obstetric care as opposed to gynecology, the same as in the 2006 Survey3,4. In the 2003 ACOG survey, fetal monitoring, neurologically impaired children, neonatal death, shoulder dystocia, uterine rupture, and “decision-to-incision” time were identified as clinical factors frequently present in obstetric malpractice cases1. In both 2006 and 2009 Surveys the reasons for claim were neurologically impaired infant (in 31% of cases in both Surveys), stillbirth/neonatal death (with 16 % of cases in both Surveys), and delay or failure in diagnosis (in 11% of cases in 2009 vs. 14% in 2006)3,4. In all the Surveys, neurological impair is the leading cause of the reasons for liability claim, with the cerebral palsy being the most serious damage. The possible etiologies have been discussed for years, and although the damage to neural tissue is undebatable, there is still no agreement upon the timing of the damage. Some authors think that 90% of the cases of cerebral palsy are not due to intrapartum events, while in the opinion of others most of the devastating events occurred in the perinatal period5,6. It is still impossible to firmly determine in each single case whether the hypoxic insult has developed during delivery, in the first few hours after birth, or was already present before the labor began, as a consequence of long lasting hypoxia during pregnancy. Careful gross and histopathological examination of the placenta in chosen cases can elucidate the events that occurred some time before labor, and help to connect and reconstruct the course of disease7,8. Importance of pathological examination of the placenta Today, it is undisputable that all the samples of diagnostic value removed from the human body should be histologically examined, with only a few exceptions, one of them being the healthy human placenta. The placenta forms a functional unit between the mother and the fetus and any pathological event that concerns one ore both of them will influence the normal function of the placenta, resulting sometimes in morphological gross and/or histological change(s). It is the most important fetal organ because it is responsible for exchange of all nutrients, oxygen, and fluid from mother to fetus and removal of fetal waste products. It has also been called the ‘‘diary of gestational life’’ 9. The placenta provides important information’s on the timing and 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 533 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 2/GYNECOLOGICAL PATHOLOGY - Short Course 2 etiology of many adverse events, including neurologic injury, fetal distress, infections, growth restriction, demise and many other fetal conditions. It also reflects the intrauterine environment, helps in identification of unsuspected maternal disorders, such as lupus or maternal vascular disease, and primary placental disorders, such as maternal floor infarction or chronic villitis. Furthermore, the placenta, being a fetal organ, expresses the fetal genotype and thus may provide diagnostic information on various genetic, chromosomal, congenital metabolic, or hematologic disorders10. Severe abnormalities of the placenta may lead to adverse fetal outcome. Fortunately, the vast majority of pregnancies and newborns are normal, so only a subset of placentas requires submission to the pathology department for gross and histological examination. However, the clinical indications for placental examination have no gold standards. Some organizations have offered more or less similar guidelines, but the choice whether the placenta will be sent for histopathological examination is still left to the attending obstetrician. Indications for pathological examination of the placenta According to the guidelines issued by College of American Pathologists at their XIX Conference dedicated exclusively to the examination of the placenta and some other considerations, the main indications for placental examination are shown in Table 1.11,12 Maternal conditions Fetal conditions Placental conditions Systemic disorders (e.g. diabetes mellitus, hypertensive disorders, collagen vascular disease) Stillbirth/neonatal death Abnormalities of the placental shape Unexplained third-trimester bleeding Multiple pregnancy Retroplacental hematoma Severe oligohydramnios Congenital malformations, dysmorphic phenotype or abnormal karyotype Small or large placental size or weight for gestational age Peripartum fever and/or infection Intrauterine growth restriction Abnormalities of the umbilical cord (length, appearence) Premature delivery Prematurity Abnormalities of the fetal membranes (e.g. amnion nodosum, meconium staining, etc) Drug addiction/alcochol abuse Hydrops Invasive procedures with suspected placental injury Meconium Placental abruption Admittance to neonatal intensive care unit Apgar <=3 in 5. minute Neurological problems (seizures) Suspected infection Table 1. The main indications for placental examination (modified from ref. 11, 12) 534 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 2/GYNECOLOGICAL PATHOLOGY - Short Course 2 Others recommend that placentas from pregnancy complicated with cholestasis, hepatitis B, human immunodeficiency infection, other maternal diseases with normal pregnancy outcome, placenta previa and postpartum hemorrhage should not be sent for pathological examination in spite of moderate cost of this examination13. These and other guidelines are only recommendations, and each institutions may accept all or only some of them, to meet the needs of the population they serve. Even with valid indications the human placenta is one of the most under examined specimens. However, all placentas should be examined grossly, immediately after delivery, by the attending physician or a nurse. To conclude whether the placenta is normal, or should be sent for pathological examination, the person examining the placenta should have at least a basic knowledge of placental anatomy and pathology. The description of the placental shape, the color of the fetal membranes, the insertion and length of the umbilical cord and placental weight must be recorded in the clinical history. Even though the placenta should be left suspended on the umbilical cord for at least an hour after delivery, this practice is never followed, so the placental weight measured immediately after delivery is about 10 to 20% greater than the real weight because of the blood it contains8. No matter the indications that are followed, the chosen placentas are sent to the pathology department. Placentas submitted to pathological examination should be accompanied by a specimen requisition form containing clinical information. The importance of providing the clinical information cannot be overemphasized, because the absence of clinical informations prevents the appropriate evaluation and hampers the conclusions that are drawn from it. The informations that must be included are gravidity and parity, obstetric history, obstetric estimate of gestational age, route of delivery, fetal birth weight, gender, Apgar scores, maternal and fetal complications of pregnancy, labor, delivery, and total umbilical cord length. Some institutions even have a dedicated specimen requisition form for the placenta that facilitates the provision of these informations to the pathologist. The clinician should also state the indication(s) for which the placenta is being submitted14. The obstetricians should bear in mind that in case bacterial or/and viral cultures, cytogenetic and metabolic studies are needed the samples must be taken from the fresh placental tissue and in sterile conditions, immediately after delivery, in the delivery room. Depending on a pathologist that is going to examine the placenta, it can be submitted fresh, without fixative, or in the appropriate amount (10 times placental volume) of fixative, usually 10% buffered formalin. If there is no possibility of the pathological examination of the placenta in the near future, or a delay is anticipated between delivery and receipt at the pathology department the placenta can be stored in a refrigerator at 4oC for a week. It would be ideal, but is practically impossible in most institutions, for all placentas not requiring pathological examination to be stored in a refrigerator for a week in case the clinical status of the newborn changes, so that the placenta can still be pathologically examined. The placenta should never be deep frozen, because the ice crystals distort the villi, and the fine pathological changes are impossible to appreciate on histological examination8. Pathological evaluation and reporting The fact of life is that in most pathological departments, other biopsies have precedence over placentas, so the obstetrician often receives the report on a placenta several weeks after submission. The report usually contains only placental measures and gross description of the placenta and the umbilical cord (frequently not even that) and the description of the histological appearance is „immature placenta“ or „mature placenta“, or „placenta without pathological changes“. After reading such reports, many obstetricians that are not convinced in the usefulness of the placental pathological examination in the first place, are discouraged to continue seeking pathological consultation. Placental lesions associated with adverse perinatal outcome can be roughly divided into those with abnormal blood flow in the maternal circulation, abnormal blood flow in the fetal circulation, inflammatory processes, and primary placental lesions8,10. Each of these categories is associated with identifiable pathologic lesions. Intrauterine demise or neurologic injury can occur by sudden and possibly devastating events, by chronic processes that lead to decreased placental and fetal reserves, or by a combination of both. It is helpful to distinguish between pathologic placental lesions that result in acute and chronic intrauterine compromise (Table 2.)15 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 535 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 2/GYNECOLOGICAL PATHOLOGY - Short Course 2 Acute Normal placental weight or weight appropriate for fetal weight Acute villous edema Intravillous hemorrhage Acute retroplacental hemorrhage Acute meconium staining Chronic Abnormal placental weight in relation to fetal weight Chorangiosis Fetal normoblastemia Chronic meconium staining Meconium associated myonecrosis of cord vessel(s) Acute or necrotising funisitis Significant chronic villitis Amnion nodosum Significant placental ischemia or infarction Decidual vasculopathy Maternal floor infarction/massive perivillous fibrinoid deposition Fetal thrombotic vasculopathy Table 2. Placental findings indicating acute and chronic in utero compromise15 The quality of reports on the investigation of the placenta also varies greatly from institution to institution. One study showed that general surgical pathologists have a higher rate of under diagnosis of placental lesions compared to pediatric pathologists16. These findings underline the fact that the reporting pathologist should be adequately trained and experienced in placental pathology. Templates and checklists for the reporting of placentas might help to improve the completeness and uniformity of reporting17. The pathological report on placenta is usually sent only to the attending obstetrician, while the neonatologists or pediatricians caring for a newborn are not aware of the findings. In the opinion of the author, the copy of the placental pathology report should be filed in both the mother’s and newborn’s clinical history. Discussion Of the ACOG Survey respondents who reported making changes to their obstetric practice as a result of the risk or fear of professional liability claims or litigation, 30% decreased the number of high-risk obstetric patients that they accepted. Performing more cesarean sections was reported by 29% of respondents that changed their obstetric practices, and 25.9% stopped offering/performing vaginal births after cesarean (VBACs). An additional 13.9% decreased the number of total deliveries. About 8% of survey respondents reported that they had stopped practicing obstetrics altogether4. Other studies addressing this problem showed that malpractice claims led to a small reduction in physician delivery volume, but they did not have a significant impact on cesarean section rates 18,19. Litigation risk 536 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 2/GYNECOLOGICAL PATHOLOGY - Short Course 2 and high malpractice premiums certainly affect negatively the gynecologist-obstetricians’ career satisfaction. The result of a relatively recent study revealed that 43.7% of gynecologist-obstetricians had become less satisfied over the last 5 years and 34.0% would not recommend obstetrics/gynecology to students seeking career advice20. Gynecologists/obstetricians are not the only doctors involved in delivery that are sued very frequently. Anesthesiologists, and pediatricians/neonatologists are also often accused of malpractice in cases of unfavorable outcome of pregnancy. Before 1990 maternal death and newborn death/brain damage were the most common complications in obstetric anesthesia malpractice claims. Newborn death/brain damage has decreased, yet it still remains also a leading cause of obstetric anesthesia malpractice claims21. The basis of litigation claims against obstetricians, anesthesiologists and neonatologists is the notion that fetal death or neurological disabilities are the result of failure or delay in intervention or inappropriate management of injuries believed to have occurred during the process of delivery. The intense fetal monitoring and changes in methods of delivery have decreased the incidence of cerebral palsy, but not substantially22-24. One of the reasons for this is that most of the fetal brain injuries occur before hospital admission and the beginning of labor, many of them being the result of intrauterine infection and inflammation, or reduced or interrupted placental vascular perfusion25). The majority of cases of cerebral palsy, particularly in term infants, are now considered to be due to ante partum events26,27. In the context of stillbirth or neurological impairment, examination of the placenta may be helpful in several ways. Firstly, the placenta itself may be abnormal and contribute directly to the adverse outcome, (e.g. when there is a tight umbilical cord knot, maternal floor infarction or a large chorangioma). The category of primary placental lesions also contains massive perivillous fibrinoid deposition, decidual vasculopathy leading to placental ischemia and/or infarction. Placenta itself may sometimes function normally, but the pathologic findings reflect abnormal intrauterine environment (e.g. intervillous thrombosis in a pale and hydropic placenta whose villous capillaries contain nucleated fetal red blood cells that reflects fetomaternal hemorrhage). Some findings (such as chorangiosis) reflect an adaptation to adverse intrauterine conditions (hypoxia). The adverse outcome may be due to pathologic processes that are not placental in origin but that lead to abnormal placental function such as maternal under perfusion and fetal thrombotic vasculopathy. .Many placental lesions develop during the prenatal period, long before labor and delivery. They cannot be prevented even by the best of obstetrical care, but they can be identified and documented by pathological examination of the placenta28. In case of adverse pregnancy outcome, the normal placental findings on pathological examination are also very important, because in such a case certain conditions may be ruled out and the attention should be directed elsewhere to look for the cause of injury. During legal proceedings, the pathologist functioning as an expert witness may be asked to specify a time frame for a placental lesion. It is often impossible to provide answers accurate to days or hours, but the rough distinction and framework of placental pathologies that result in acute or chronic compromise can be used for determining the timing of the fetal insult. Pathologic examination provides more information on chronic than on acute events. However, many acute events can also be diagnosed or confirmed on placental examination. Acute lesions may be associated with sudden catastrophic events whereas chronic lesions develop over a period of time leading to decreased placental reserves at a minimum. Markedly depleted reserves will render the infant susceptible to stresses of labor and to more acute events and therefore may also be associated with significant injury or death. In stillborn or neurologically impaired infants, multiple placental lesions are often present. By the timing of all lesions found, a sequence of events can be reconstructed in the development of an adverse intrauterine environment. The decrease of placental reserves and function is usually the result of synergistic action of multiple lesions of different etiologies that involve different aspects of placental function (fetal or maternal blood circulation). Acute events also frequently occur in combination with chronic processes29. Multiple placental lesions greatly increase the susceptibility of the fetus to neurologic damage29-31. The type and significance of placental lesions determine the extent to which placental pathology can be helpful in understanding adverse antenatal and perinatal events. Interpretation of these lesions is complex and requires experience and insight into clinicopathologic correlation with outcome. However, the most important part of placental examination is ensuring that it is performed, because the slides and paraffin blocks of 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 537 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 2/GYNECOLOGICAL PATHOLOGY - Short Course 2 placental tissue remain in the archives and can be retrieved even after a couple of years, should the need for revision or expertise arise. A photographic record of gross pathologic findings is also very useful. The gross and histological pathologic report of the placenta may not completely explain the etiology and timing but is an important and essential witness in understanding adverse pregnancy outcome. One also has to bear in mind that placental lesions are not necessarily the cause of unfavorable pregnancy outcome, and some structural changes may be the consequences of poor fetal condition. The placenta is an easily available specimen and the costs of a routine pathological examination are moderate, so in all doubtful cases, the clinicians should not hesitate to ask for a pathological analysis and opinion. References 1. Strunk AL, Esser L. Overview of the 2003 ACOG Survey of Professional Liability. ACOG Clinical Review. 2004; 9:1; 13-6. 2. American College of Obstetricians and Gynecologists. Survey of professional liability. Washington, DC: American College of Obstetricians and Gynecologists; 2003;. 3. Chervenak JL. Overview of professional liability. Clin Perinatol 2007; 34:227-32 4. http://www.theunnecesarean.com/blog/2009/9/11/acog-releases-survey-results-ob-gyns-ultimately-hurt-patient.html 5. Maclennan A. A template for defining a causal relation between acute intrapartum events and cerebral palsy: international consensus statement. BMJ 1999; 319:1054–9 6. Cowan R, Rutherford M, Groenendaal F, et al. Origin and timing of brain lesions in term infants with neonatal encephalopathy. Lancet 2003; 361:736–42. 7. d’Aloja E, Müller M, Paribello F, Demontis R, Faa A. Neonatal asphyxia and forensic medicine. J Matern Fetal Neonatal Med. 2009; 22:54-6. 8. Kos M, Leniček T. Osnove patologije posteljice. Medicinska Naklada, Zagreb, 2011. 9. Altshuler G. Some placental considerations in alleged obstetrical and neonatology malpractice. In: Wecht CH, editor. Legal medicine. Salem (NH): Butterworth Legal Publishers; 1994. p.27–47. 10.Baergen RN. The Placenta as Witness. Clin Perinatol 2007; 34:393–407. 11.Altshuler G, Deppisch LM .College of American Pathologists Conference XIX on the Examination of the Placenta: report of the Working Group on Indications for Placental Examination. Arch Pathol Lab Med. 1991; 115:701-3. 12.Langston C, Kaplan C, Macpherson T, et al. Practice guideline for examination of the placenta: developed by the Placental Pathology Practice Guideline Development Task Force of the College of American Pathologists. Arch Pathol Lab Med 1997; 121:449-76 13.B Hargitai, T Marton, P M Cox. Examination of the human placenta. J Clin Pathol 2004; 57:785–792 14.Bull AD, Cross SS, James DS, Silcocks PB. Do pathologists have extrasensory perception? BMJ 1991; 303:1604-5. 15.Chang KTE. Pathological examination of the placenta: Raison d’être, clinical relevance and medicolegal utility. Singapore Med J 2009; 50:1123-33. 16.Sun CC, Revell VO, Belli AJ, Viscardi RM. Discrepancy in pathologic diagnosis of placental lesions. Arch Pathol Lab Med 2002; 126:706-9. 17.Khong TY, Gordijn SJ. Quality of placental pathology reports. Pediatr Dev Pathol 2003; 6:54-8. 18.Gimm GW. The impact of malpractice liability claims on obstetrical practice patterns. Health Serv Res 2010; 45:195-211. 538 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 2/GYNECOLOGICAL PATHOLOGY - Short Course 2 19.Grant D, McInnes MM. Malpractice experience and the incidence of cesarean delivery: a physician-level longitudinal analysis. Inquiry 2004; 41:170-8. 20.Xu X, Siefert KA, Jacobson PD, Lori JR, Ransom SB The impact of malpractice burden on Michigan obstetriciangynecologists’ career satisfaction. Womens Health Issues 2008; 18:229-37. 21.Davies JM, Posner KL, Lee LA, Cheney FW, Domino KB. Liability associated with obstetric anesthesia: a closed claims analysis. Anesthesiology 2009; 110:131-9. 22.Scheller JM, Nelson KB. Does cesarean delivery prevent cerebral palsy or other neurologic problems of childhood? Obstet Gynecol 1994; 83:624-30. 23.Winter S, Autry A, Boyle C, Yeargin-Allsopp M. Trends in the prevalence of cerebral palsy in a population-based study. Pediatrics 2002; 110:1220-5. 24.Nelson KB. Can we prevent cerebral palsy? N Engl J Med 2003; 349:1765-9. 25.Nelson KB, Grether JK. Causes of cerebral palsy. Curr Opin Pediatr 1999; 11:487-91. 26.Kuban KC, Leviton A. Cerebral palsy. N Engl J Med 1994; 330:188-95. 27.Cowan R, Rutherford M, Groenendaal F, et al. Origin and timing of brain lesions in term infants with neonatal encephalopathy. Lancet 2003; 361:736–42. 28.Ward CJ. Analysis of 500 obstetric and gynecologic malpractice claims: causes and prevention. Am J Obstet Gynecol 1991; 165:298-304; discussion 304-6. 29.Redline RW, O’Riordan A. Placental lesions associated with cerebral palsy and neurologic impairment following term birth. Arch Pathol Lab Med 2000;124:1785–91. 30.Redline RW. Severe fetal placental vascular lesions in term infants with neurologic impairment. Am J Obstet Gynecol 2005;192:452–7 31.Viscardi RM, Sun CJ. Placental lesion multiplicity: risk factor for IUGR and neonatal cranial ultrasound abnormalities. Early Hum Dev 2001;62:1–10. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 539 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 2/GYNECOLOGICAL PATHOLOGY - Short Course 2 Novine u dijagnostici trofoblastnih bolesti Recent advances in diagnostics of trophoblastic disease Mihaela Mocko Kaćanski Mihaela Mocko Kacanski Odsek patologije, Klinički centar Vojvodine, Novi Sad, Srbija Department of Pathology, Clinical CenterVojvodina, Novi Sad, Serbia Apstrakt Abstract Gestacijske trofoblastne bolesti (GTB) su bolesti i tumorska stanja u vezi sa trudnoćom a koje se najčešće javljaju u generativnom periodu žene. Ovo su retki tumori a javljaju se kod neadekvatne proliferacije trofoblasta. U ovim stanjima uvek dolazi do povišenja vrednosti beta subjedinice humanog horionskog gonadotropina (βhCG). Hidatidne mole, parcijalne i kompletne, su u većini slučajeva benignog karaktera i karakterišu se edemom gotovo avaskularnih horionskih resica, nazubljenih ivica, pseudoinkluzijama hiperplastičnog trofoblasta i sincicijalnim izdancima. Invazivne mole su agresivne lezije trofoblasta sa miometrijalnom i/ili vaskularnom invazijom. Horiokarcinom, trofoblastni tumor posteljičnog ležišta i epiteloidni trofoblastni tumor su jasno maligni tumori proliferisanog intermedijernog trofoblasta, sa metastatskim potencijalom. Tumorolika stanja su placentalni nodul i naglašeno postaljično mesto koji predstavljaju proliferativne lezije i reaktivne procese i ne smatraju se pravim tumorima. U GTB najčešći simptom je vaginalno krvarenje koje je praćeno povišenjem serumskih vrednosti βhCG. GTB moraju biti potvrđene histološki na većem broju uzoraka. Kontrole su neophodne kod svih pacijentkinja sa dijagnostikovanom GTB a to se rutinski sprovodi merenjem nivoa βhCG-a. S obzirom da se kod GTB u svim entitetima radi o proliferaciji jednog ili više trofoblastnih tkiva, od izuzetne je važnosti dobro poznavati i uobičajene razvojne oblike tkiva normalne trudnoće da bi se adekvatno mogli primeniti poznati dijagnostičko histološki kriterijumi tumorskih bolesti ovog tipa. Ključne reči: gestacijske trofoblastne bolesti, hidatidna mola, horiokarcinom, humani horioni gonadotropin. Gestational trophoblastic disease (GTD) is a term used for a group of pregnancy-related tumours, overwhelmingly affecting women of childbearing age. These tumours are rare, and they appear when trophoblastic cells start to grow out of control. In these conditions production of beta subunit of human chorionic gonadotropin (hCG) is evident. Hydatidiform moles, partial or complete, are in most cases benign featured by villous hydrps, scalloping effect, hyperplastic trophoblastic pseudoinclusions and syncytiotrophoblastic sprouts. Invasive mole is an aggressive trophoblastic lesion with myometrial and/or vascular invasion. Choriocarcinoma, placental site trophoblastic tumor and epitheloid trophoblastic tumor are clearly malignant tumors with proliferation of intermediate trophoblast, with metastatic potential. Tumor-like trophoblastic conditions are placental site nodul and exaggerated placental site wich are proliferative lesions and reactive processes and are not considered as true tumor lesions.In all of these conditions vaginal bleeding is the most common symptom followed by elevation of serum beta hCG. GTD has to be confirmed histologically with extensive sampling of the material. Follow up is necessary in all women with GTD and it is rutinley done by measurment of serum levels of hCG. Since GTDs are proliferative conditions of diferent trophoblastic tisues, pathologists should be well histologicly educated about normal pregnancy stages and its abnormalities in order of adeqate diagnosing these rare conditions. Key words: gestational trophoblastic disease, hydatid mole, choriocarcinoma, human chorionic gonadotropin. Uvod Gestacijske trofoblastne bolesti (GTB) su lezije trofoblasta različitih proliferativnih kapaciteta i to u rasponu od neneoplastičnih hidatidnih mola (kompletne mole, parcijalne mole i invazivne mole) do jasno neoplastičnih stanja (gestacijski horiokarcinom, trofoblastni tumor posteljičnog ležišta i epiteloidni trofoblastni 540 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 2/GYNECOLOGICAL PATHOLOGY - Short Course 2 tumor). Svaki od ovih entiteta ima jasnu sliku ponašanja koja je u vezi sa proliferatinim kapacitetom konstitucionog trofoblasta. Ujedno u sklopu ovih stanja ističu se i dva entiteta i to naglašeno posteljično mesto i placentni nodul kao tumorolika stanja 1,2,3. Kategorija Hidatidne mole Trofoblastni tumori Tumorolika stanja Podtipovi Parcijalna mola Kompletna mola Invazivna mola Horiokarcinom Trofoblastni tumor posteljičnog ležišta (TTPL) Epiteloidni trofoblastni tumor (ETT) Naglašeno posteljično mesto Placentni nodul/plak Tabela 1. Vrste mola i tumorolika stanja U cilju adekvatnog razumevanja faza razvoja ranog trofoblasta neophodno je upoznavanje i shvatanje morfologije i embrionalnih stadijuma kroz koje ovo tkivo u normalnoj trudnoći prolazi. Posle oplodnje, blastocita se diferentuje u embrionalne i ekstraembrionalne ćelije a nešto kasnije nastaje trofoblast odnosno preteča posteljice. Trofoblastne ćelije se dalje diferentuju u vilozni i ekstravilozni trofoblast. Vilozni trofoblast se sastoji od viloznog citotrofoblasta i sinciciotrofoblasta. Ekstravilozni trofoblast se sastoji od horionske ploče, choriona leave, ćelijskih ostrvaca, septi, implantacionog mesta i bazalne ploče. Vilozni i ekstravilozni trofoblast proizilaze iz različite faze trofoblastne diferencijacije, a isto tako njihove lezije imaju drugačije morfološko, kliničko i biološko ponašanje 4,5,6. Nedavne studije biomarkerske ekspresije ukazuju na dualnu diferencijaciju ćelijskih populacija u različitim trofoblastnim bolestima. Hidatidne mole su proliferativne lezije viloznog trofoblasta. Horiokarcinom je jasno maligni tumor čiji trofoblast pokušava da oponaša primitivne ćelije previloznog stadijuma posteljice. Intermedijerni trofoblast na nivou implantacionog mesta daje ćelije koje daju trofoblastni tumor posteljičnog ležišta i naglašeno posteljično mesto. Dokazano je da intermedijerni trofoblast u horionu leve daje ćelije koje čine epitelni trofoblastni tumor i placentni nodul 7. Najveća incidenca hidatidne mole na 1000 trudnoća se registruje u jugo-istočnoj Aziji i to 13 u Indoneziji, 8 na Tajvanu, 5 na Filipinima i Kini a 3.8 u Japanu. Severna Amerika, Evropa i Okeanija imaju najnižu incidencu od oko 0.5 - 1.84 na 1000 trudnoća. Prijavljivanje gestacijskih trofoblastnih tumora značajno varira zbog dijagnostičkih definicija i individualizovanih kriterijuma patologa ali i od uključivanja kliničkih parametara te biohemijskih dijagnostičkih metoda. Najviša stopa gestacijskih trofoblastnih neoplazmi se beleži u Indoneziji i to 5.4 na 1000 trudnoća dok je najniža u severnoj Americi, Evropi i Okeaniji i to oko 0.05 na 1000 trudnoća 8,9. Velike svetske studije su pokušale da identifikuju rizične faktore za nastanak GTB i najčešće se nalaze starost majke (trudnoće preko 40 godine živora), ranije trudnoće (prethodna molarna trudnoća nosi rizik za kasniji nastanak iste), etnička pripadnost (češće kod malezijki, kineskinja i indonežanki), genetika (češće u pojedinim porodicama) i niski socio-ekonomski status 10,11. KOMPLETNA HIDATIDNA MOLA – nazvana još i “klasična” mola nosi naziv hidatidna s obzirom da se makroskopski opis odnosi na grozdaste mase koje su karakteristika ove bolesti. Kompletna mola je proliferativni poremećaj cito i sinciciotrofoblastnog bez razvoja embroina. Kompletna hidatidna mola čiji je celokupan genetski material očinskog porekla rezultat je oplodnje zrelim spermatozoidom jajne ćelije koja je izgubila jedro odnosno hromatin (“prazno jaje” engl. blighted ovum). Sledeće umnožavanje haploidnog spermatozidnog kompleta genetskog materijala vodi ka diploidnom genotipu. Stoga komletne mole su obično genetskog profila 46XX, a mali broj njih je 46XY. Triploidne i tetraploidne komletne mole se javljaju izuzetno retko i one takođe potiču jedino od očeve DNA 12. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 541 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 2/GYNECOLOGICAL PATHOLOGY - Short Course 2 Najčešće se kompletne mole kod pacijentkinja prepoznaju vaginalnim krvarenjem do 16 nedelje trudnoće, dok se ređe od simptoma mogu naći i hiperemeza, toksemija, hipertiroidizam ili plućna embolija. Kod kompletne mole je tipično izraženo povišenje serumskih vrednosti βGCG-a (i preko 100,000 mIU/mL) iznad normalnih vrednosti, dok se na ultrazvučnom pregledu plod ne nalazi ali je prisutan efekat “snežne mećave” (engl. snowstorm) 13. Kod oko 25% pacijentkinja nalaze se preeklampsijske ili eklampsijske tegobe dok oko trećina razvija ovarijalne teka luteinske ciste. Makroskopski materijal kompletne mole je po pravilu voluminozan, hemoragičan i grožđu sličan sa manjim ili većim vezikulama a što su edematozno izmenjene horionske resice, dok se plod po pravilu ne nalazi. Slika 1. Makroskopski i histološki izgled hidatidne mole. Histološki jasno formirana mola ima dve osnovne karakteristike i to difuzni edem horionskih resica i naglašena hiperplazija trofoblasta. Stromalni edem horionskih resica je često izražen sa formiranjem centralnih pukotina (cisterni) koje su acelularne ali sa trofoblastnim inkluzijama. Hiperplazija trofoblasta se karakteriše iregularnom ali difuznom proliferacijom koja je ujedno nepolarna i multifokalna i prostire se oko cele horionske resice. Plaže ili konfuentni agregati intermedijernog trofoblasta se mešaju sa cito ili sinciciotrofoblastom. Citološka atipija na nivou sinciciotrofoblasta i intermedijernog trofoblasta je ponekad izražena uz naglašeniju mitotsku aktivnost na nivou citotrofoblasta i intermedijernog trofoblasta. Iako je vilozna stroma hipocelularna, celularna područja se mogu naći pri vrhovima horionskih resica sa vretenastim ćelijama u miksoidnom matriksu sa brojnim apoptotičnim telima. U ovim horionskim resicama krvni sudovi nedostaju ili su svedeni na rudimentne strukture 14,15. S obzirom da u ranim kompletnim molama edem resica ili naglašenija proliferacija trofoblasta ne postoji histološka dijagnostika je izuzetno teška te se u tim situacijama mora posmatrati needematozna stoma koja pokazuje neadekvatnu bulboznu, polipoidnu ili filodesu sličnu konfiguraciju. Stroma je često u ovim slučajevima hipercelularna građena od zvezdastih fibroblasta u miksoidnoj stromi sa velikim brojem apoptotičnih tela i nešto malo rudinentnih krvnih sudova 15,16. Trofoblastna proliferacija je minimalna i samo fokalna, često slična uobičajenoj gestacjskoj proliferaciji. Po pravilu, kod kompletnih mola je prisutna jedarna negativnost na p57 antitelo u citotrofoblastu i stromalnim ćelijama dok je kod parcijalnih mola i spontanog abortusa pozitivno u citotrofoblastu, intermendijernom trofoblastu, stromalnim viloznim ćelijama i stromalnim decidualnim ćelijama 17. Diferencijalna dijagnoza kompletne hidatidne mole podrazumeva normalnu ranu trudnoću, hidropsni edem ne molarne trudnoće, ektopičnu trudnoću i parcijalnu hidatidnu molu. 542 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 2/GYNECOLOGICAL PATHOLOGY - Short Course 2 PARCIJALNA HIDATIDNA MOLA - Parcijalne mole su najčešće triploidne i nastaju iz jajne ćelije koja je oplođena sa dva spermatozoida, vođene od očevog ka majčinom hromozomu u odnosu 2:1. Uglavnom su 69XXX, 69XXY genetskog profila a retko se nalazi i 69XYY. Tetraploidne mole su isto opisane i imaju odnos očevih i majčinih hromozoma 3:1 18. Kod parcijalnih mola najčešći simptom je vaginalno krvarenje u kasnom prvom ili početkom drugog trimestra trudnoće. Kod ovih pacijentkinja veličina uterusa odgovara gestacijskoj starosti dok su serumske vrednosti βHCG-a uobičajene ili samo blago povišene a preeklampsija ili eklampsija se retko sreću. Ultrazvučnim pregledom prisutne su samo fokalne cistične promene na posteljici uz povišenje transverzalnog dijametra gestacijskog meška. Kod parcijalnih hidatidnih mola može se naći plod ali često smanjene veličine za gestacijsku dob te sa karakterističnim malformacijama kao što je sindaktilija, spina bifida, kriptorhizam i renalna hipoplazija 13,19. Makroskopski, po pravilu, količina kiretiranog materijala je manja nego kod kompletne mole ali je svakako veća nego kod hidropsnog abortusa, dok je izgled uobičajen za trudnoću sa samo fokalno prisutnim grozdastim formacijama. Histološki kod parcijalnih mola se nalaze dve vrste horionskih resica i to jedne uobičajenog izgleda a druge edematozne, veće i nepravilne. Ove veće nepravilne resice su izreckanih nazubljenih ivica dok su okruglaste trofoblastne pseudoinkluzije česte. Prisutna je ujedno i umerena cirkumskriptna nepolarna trofoblastna hiperplazija bez atipije, dok je formiranje centralnih stromalnih cisterni ređe u odnosu na kompletne mole. Sinciciotrofoblast može ponekad da bude prominentan sa formiranjem izraštaja ili invaginatima odnosno intracitoplazmatskim lakunama. Fetalni krvni sudovi i eritrociti u njima se često registruju 13,20. U citotrofoblastu i viloznim stromalnim ćelijama se nalazi pozitivna p57 nuklearna eksperesija 17. Diferencijalna dijagnostika parcijalnih mola podrazumeva kompletnu molu, hidropsni abortus, gestacije sa hromozomskim anomalijama, placentalnu mezenhimnu displaziju i blizanačku trudnoću sa kompletnom molom i jednim prisutnim fetusom. TROFOBLASTNI TUMOR POSTELJIČNOG LEŽIŠTA (TTPL) - je prava neoplastična proliferacija ćelija intermedijernog trofoblasta na mestu implantacije koja se zasniva na genetskoj nestabilnosti. Za ovakvu tumorsku proliferaciju ranije su korišćeni nazivi kao što su atipični horioepiteliom, atipični horiokarcinom, sinciciom i horioepitelioza ali su tek Scully i Young 1981 godine predložili naziv koji se i do danas koristi 21. TTPL se najčešće javlja u ranim tridesetim godinama života i preduslov je makar i jedna prethodna trudoća sa kojom ne mora da bude u bliskoj vremenskoj povezanosti. Kod ovih pacijentkinja kao simptom se javlja vaginalno vanmenstrualno krvarenje praćeno uvećanjem materice 22. U inicijalnoj dijagnostici u oko 80% slučajeva prisutno je blaže do umereno povišenje serumskih vrednosti βHCG-a koje ne prelazi 700 mIU/mL ali koje se duplira u metastatskoj varijante ove bolesti 22. Makroskopski TTPL se nalazi u endometrijumu u vidu jasno ograničenog čvora ili polipozne mase dok na poprečnom presku se nalazi žućkasto solidno tkivo mesnatog izgleda sa fokusima nekroze i krvarenja. Duboka ili transmuralna invazija miometrijuma je česta i nalazi se u oko 50% slučajeva. Histološki se registruje masivna proliferacija većih, okruglastih, mononuklearnih ili ređe multinuklearnih ćelija, eozinofilne obilne citoplazme intermedijernog trofoblasta posteljičnog implantacionog mesta. Jedarni pleomorfizam je povremeno izraženiji kao i povišenje broja mitoza. Tumorsko tkivo je aranžirano u manjim gnezdima ili trakama gde na periferiji tumora ćelije raslojavaju mišićna vlakna miometrijuma. U stromi se konstatuju ekstracelularni fibrinski depoziti dok tip vaskularne invazije pokušava da napravi sličanu sliku normalnog trofoblasta. Nekroze i krvarenja su često opsežni i mogu biti dominantni. Horionske resice ili delovi ploda se ne nalaze. Okolni endometrijum može pokazivati deciduiformne promene sa Arias-Stella reakcijom. Metastatski depoziti ovog tumora su građeni isključivo od mononuklearnih trofoblastnih ćelija. Pojedini TTPL mogu fokalno pokazivati histološku sliku drugih gestacijskih tumora i to najčešće epiteloidnog trofoblastnog tumora 23,24,25. U dijagnostici TTPL značajnu ulogu igra imunohistohemijska dijagnostika sa difuznom pozitivnom ekspresijom na hPL (humani placentalni laktogen), AE1/3 i CK 18 a fokalnom slabijom pozitivnosti na MUC-4, 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 543 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 2/GYNECOLOGICAL PATHOLOGY - Short Course 2 HSD3B1, CD10, HLA-G, CD146 i hCG dok je p63 negativne ekspresije. Faktor proliferacije Ki-67 je 10%30% 24,25. Dijagnoza ovog tumora se mora raditi isključivo na operativnom materijalu dok se samo sumnja sme postaviti na materijalu dobijenom kiretmanom. Diferencijalna dijagnoza TTPL je sa naglašenim posteljičnim mestom, epiteloidnim trofoblastnim tumorom, loše diferentovanim karcinom i epiteloidnim glatko mišićnim tumorima. Iako se TTPL terapijski rešava histerektomijom metastaze se beleže u literaturi i nalaze se najčešće u plućima, jetri i vagini. EPITELOIDNI TROFOBLASTNI TUMOR (ETT) – tumorska proliferacija porekla horionog tipa intermedijernog trofoblasta a u literaturi se opisuje tek stotinak slučajeva od 1989 godine kada je prvi put prepoznat kao zaseban entitet 26. Morfološki je izuzetno sličan planocelularnom karcinomu. EET se može naći zajedno i sa ostalim GTB i to najčešće sa TTPL i horiokarcinomom i tada se ovi tumori nazivaju mešanim trofoblastnim tumorima. EET se javlja u generativnom periodu i to 1 – 25 godina nakon trudnoće a praćen je blažim do umerenim povišenjem serumskih vrednosti βHCG-a (oko 2500 mIU/ml) 26,27. U oko polovine slučajeva se lokalizuje u telu dok je druga polovina sa mestom ishodišta u grliću materice te je upravo iz ovog razloga ovaj tumor neadekvatno dijagnostikovan kao planocelularni karcinom. Metastaze se verifikuju u četvrtine pacijentkinja i nalaze se u plućima, limfnim čvorovima, jetri, žučnoj kesi, bubrezima, pankreasu, kičmenom stubu i vagini 27. Makroskopski ETT je jasno ograničeni delom cistično-hemoragijski degenerativno izmenjeni čvor do 5cm koji infiltruje miometrijum u “gurajućem” maniru u širokom frontu. Histološki nalaze se mononuklearne ćelije horion leve tipa intermedijernog trofoblasta, umerene količine fino granulirane bledo eozinofilne citoplazme, većih ovalnih jedara sa uočljivim jedarcima a koje su aranžirane u gnezda, trake i solidne plaže. Nuklearni pleomorfizam je blaži do umeren a fokalno se nalaze i pojedinačne više jedarne ćelije. Mitotski indeks je mali (obično 2/10 HPF). Karakteristična osobina ETT je i formiranje gnezda koja su okružena nekrozom i hijalinim matriksom. Nekroze su geografskog tipa dok su mali krvni sudovi okruženi hijalinom nekrozom. Oko tumorskog tkiva nalazi se deciduiformno izmenjena stroma endometrijuma ili endocervikalna sluznica 27,28. Imunohistohemijski u ETT je prisutna pozitivna eksperesija trofoblastnih markera i to H3D3B1, HLA-G, hPL, Inhibin-alpha i Mel-CAM ali i CK18, AE1/3 te p63. Negativnost se registruje kod CK20, CK 5/6, TTF1, S100, CA-125 i calretininu. Diferencijalno dijagnostički problem ETT, s obzirom na čestu lokalizaciju u grliću materice, je sa planocelularnim karcinomom. Kod dijagnostike ovog tumora treba razmišljati i o TTPL, horiokarcinomu, nodulu posteljičnog mesta, epiteloidnom leiomiosarkomu te loše diferentovanom endometrioidnom adenokarcinomu. GESTACIJSKI HORIOKARCINOM – najagresivnija forma trofoblastne bolesti gde tumor pokušava da imitira trofoblast razvojne placente. Vaginalno krvarenje je najčešći simptom ali se povremeno nalaze prvo metastatski depoziti u plućima, jetri, centralnom nervnom sistemu ili gastrointestinalnom sistemu a kasnije i primarni tumor. Javlja se u generativnom periodu, i to u periodu od 1–23 godine nakon trudnoće. Dovodi se u tesnu vezu sa ostalim GTB ali najčešće sa hidatidnim molama iz kojih može nastati 29,30. Makroskopski horiokarcinom je veća, destruktivna tamnije mrko – hemoragična masa sa opsežnim nekrotičnim promenama koja masivno infiltruje miometrijum. Histološki se nalazi destruktivna agresivna nejasno ograničena lezija sa izraženim nekrotičnim i hemoragičnim promenama. Tipičan je bilamelarni tip rasta gde su u centralnim delovima smeštene mononuklearne ćelije dok je periferija okružena višejedarnim sinciciotrofoblastnim ćelijama naglašene citološke atipije i izraženo povišenog broja mitoza. Horionske resice se ne nalaze kod kompletno formiranih horiokarcinoma. Okolni endometrijum pokazuje deciduifomne promene dok se u oko polovine slučajeva nalaze na jajnicima teka-luteinske ciste 29,30. 544 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 2/GYNECOLOGICAL PATHOLOGY - Short Course 2 Slika 2. Histološki izgled horiokarcinoma IN SITU ILI INTRAPLACENTALNI HORIOKARCINOM nastaje u zreloj, razvijenoj, ročnoj placenti i ima sve karakteristike “klasičnog” horiokarcinoma 31. Imunohistohemijski sinciciotrofoblastne ćelije su pozitivne na hCG, hPL i HSD3B1 dok su ćelije neoplastičnog intermendijernog trofoblasta pozitivne na HLA-G, MUC-4, CD 146 i hPL. Ki 67 pokazuje proliferaciju oko 90% 32. Diferencijalna dijagnoza horiokarcinoma je sa kompletnom hidatidnom molom, ranim gestacijskim viloznim trofoblastom, naglašenim posteljičnim mestom, TTPL, ETT, negestacijskim horiokarcinomom te loše diferentovanim karcinomima uterusa 32. PERZISTENTNA TROFOBLASTNA NEOPLAZMA – kao termin je prvi put predložena od strane WHO 2002 godine i podrazumeva perzistentne mole, invazivnu molu, metastatsku molu, horiokarcinom ili drugu gestacjsku bolest kod kojih perzistiraju osnovni simptomi čak i nakon terapijskih protokola 33. Kod svih ovih pacijentkinja neophodno je sprovesti hemioterapijski modalitet dopunskog lečenja. Osnovni kriterijum u dijagnostici ovih stanja su perzistentno povišene serumske vrednosti βHCG-a. INVAZINA HIDATIDNA MOLA – molarna trudnoća ali sa naglašenim urastanjem horionskih resica u miometrijum. Kod ovih pacijentkinja prisutno je čak i nakon evakuacione kiretaže vaginalno krvarenje uz konstantno povišene vrednosti βHCG-a. Makroskopski i histološki se nalazi raslojavanje mišićnog tkiva molarno izmenjenim horionskim resicama i hemoragičnim masama 34. Slika 3. Histološki izgled invazivne mole u kiretmanu i operativnom materijalu. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 545 GINEKOLOŠKA PATOLOGIJA - Kratki kurs 2/GYNECOLOGICAL PATHOLOGY - Short Course 2 TUMORIMA SLIČNA TROFOBLASTNA STANJA – Izdvajaju se dva entiteta koja ne predstavljaju tumorske proliferate nego hiperplastične reakcije intermedijernog trofoblasta a to su naglašeno posteljično mesto (NPM) i posteljični nodul (PN). POSTELJIČNI NODUL (PN) – je nodularna hiperplazija intermedijernog trofoblasta na horionu leve maksimalnog prečnika 10mm. Javlja se kod pacijentkinja srednje životne dobi oko godinu do dve dana nakon trudnoće, a praćeno je vaginalnim krvarenjem 35. NAGLAŠENO POSTELJIČNO MESTO (NPM) – Ewing je ovaj entitet 1910 godine nazvao sincicijalni endometritis ali se danas naziv više ne koristi jer nije reč o upalnom procesu. Javlja se u generativnom periodu i predstavlja umnožavanje intermedijernog trofoblasta u endometrijumu i površnom miometrijumu na mestu implantacije 36. Nakon potvrđene dijagnoze GTB i tačne tipizacije lezije pacijentkinja dalje podleže dijagnostičko – terapijskim protokolima u cilju stejdžinga i proširenosti bolesti uz obavezno merenje vrednosti βHCG-a jer se na taj način obezbeđuje adekvatno kasnije kontrolisanje uklonjenosti ili perzistencije tumorskog tkiva. S obzirom da se kod GTB u svim entitetima radi o proliferaciji jednog ili više trofoblastnih tkiva od izuzetne je važnosti dobro poznavati i uobičajene razvojne oblike tkiva normalne trudnoće da bi se adekvatno mogli primeniti poznati dijagnostičko histološki kriterijumi tumorskih bolesti ovog tipa 37. Literatura 1. Tavassoli F, Peter D. World Health Organization: tumours of the breast and female genital organs. Lyon: IARC PRESS; 2004 2. Tham KF, Ratnam SS. 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Hum Pathol. 1999;30(6): 687–94 36.Chen X, Shi Y, Xie X. The clinical and pathological characteristics of exaggerated placental site. Zhonghua Fu Chan Ke Za Zhi. 1998;33(6):352–4 37.Buza N, Hui P. Gestational trophoblastic disease: histopathological diagnosis in the molecular era. Diagn Histopathol. 2010;16(11):526–37 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 547 novine u patologiji/NEWS IN PATHOLOGY Endomiokardna biopsija: juče, danas i sutra Endomyocardial biopsy: yesterday, today and tomorrow Jovan D. Vasiljević Institut za patologiju, Medicinski fakultet, Univerzitet u Beogradu, Beograd, Srbija Jovan D. Vasiljevic Abstract Apstrakt Glavni ciljevi u ovom radu su bili da se prikaže aktuelno znanje o primarnim bolestima srčanog mišića, kardiomiopatijama (KMP), da se da njihova klasifikacija, dijagnostičke mogućnosti endomiokardne biopsije (EMB), da se utvrdi vrednost EMB u izboru najbolje terapije. Uz mnoge kontroverze, EMB se danas smatra široko prihvaćenom metodom, sa malo komplikacija, i izvodi se rutinski u mnogim kardiološkim centrima. Iako smo mi otpoćeli analizu EMB još u osamdesetim godinama, danas se EMB u Beogradu relativno retko radi. Jako je teško dati odgovor – zašto? KMP se dele na dve osnovne grupe, prema funkcionalnim i strukturalnim, patološkim karakteristikama, na idiopatske i specifične, odnosno na primarne i sekundarne. Idiopatska grupa se zatim deli na 5 podgrupa: hipertrofičnu, dilatacionu, restriktivnu, aritmogenu KMP desne komore, i neklasifikovane KMP. Specifične KMP se dele na 8 podgrupa, baziranih na etiopatogenetskim karakteristikama. Na infektivne, sa virusnim miokarditisom (VMK) kao najčešćim entitetom, na metabolične, uključujući i endokrine poremećaje i bolesti nakupljanja. Defekti u deficitu oligoelemenata i vitamina, zahvaćenost srca u bolestima vezivnog tkiva, kao i granulomi i neoplazme takađe spadaju u ove grupe oboljenja. Poremećaji (pre)osetljivosti i toksične reakcije, sa dugim nizom substanci koje mogu da utiču na srce, su vrlo česte u današnjoj patologiji. Na kraju, u poslednju grupu bi spadala oštećenja srca nastala u sklopu različitih sistemskih sindroma, ipak relativno retkih. EMB je u svetu u znatnom porastu primene, posebno u dijagnostici akutnog odbacivanja kod srčane trnasplantacije, dijagnostici KMP, posebno VMK, sa malom stopom komplikacija, te se smatra, efikasnom, korisnom i sigurnom procedurom. Ključne reči: endomiokardna biopsija, primarna bolest srčanog mišića, kardiomiopatija, dijagnoze, lečenje, immunomodulation, ljudi 548 Institute of Pathology, Medical School, University of Belgrade, Belgrade, Serbia The major goals of this presentation are to give the updated knowledge of primary heart muscle disease, cardiomyopathies (CMP), their classification, the diagnostic possibilities using endomyocardial biopsy (EMB) and to estimate the value of EMB in the choice of the right therapeutic approach. Despite many controversies, EMB is today a widely accepted method, with low percentage of complications for analysing CMPs, and is considered to be a routine procedure in many cardiological centers. Inspite the fact that we started with the use of EMB in 80’s, it is not performed often today in Belgrade, like many yeras ago (2). It’s difficult to say why ? The CMPs may be subdivided, according to the functional and structural features, into two groups: idiopathic and specific, or primary and secundary. Idipathic group is consisted of 5 subgroups: hypertrophic, dilated, restrictive, arrithmogenic right ventricular CMP and unclassified. Specific CMPs may be subdivided into 8 groups mainly based on etiopathogenetic characteristics: infective, with viral myocarditis as the most common entity, metabolic, including endocrine disorders and infiltration and storage diseases. Deficiency disorders and heart involvement in connective tissue disorders are also included. Granulomas, neoplasms and neuromuscular disorders are also wery often presented with cardiac disfunction and structural abnormalities. Sensitivity and toxic reactions with long list of substances wich may affect the heart are probably the most present today. Finally, the last group represent miscellaneous systemic syndromes with heart affection. EMB has been increasingly used in the diagnosis of CMPs, with special influnce on diagnosis of heart transplant rejection, myocarditis, treatment modalities of different types of myocarditis, with low complication rate, considered effective, usefull and safe procedure. Key words: endomyocardial biopsy, primary heart muscle disease, cardiomyopathy, diagnosis, treatment, immunomodulation, human 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 novine u patologiji/NEWS IN PATHOLOGY Introduction The major goals of this presentation are to give the updated knowledge of primary heart muscle disease, of diagnostic possibilities using endomyocardial biopsy (EMB) and to estimate the value of EMB in the choice of the right therapeutic approach. Despite many controversies, EMB is today a widely accepted method, with low percentage of complications for analysing cardiomyopathies (CMPs), and is considered to be a routine procedure in many cardiological centers 1. Inspite the fact that we started with the use of EMB in 80’s, it is not performed often today in Belgrade, like many yeras ago 2. It’s difficult to say why? INDICATIONS OF EMB Although the technique of EMB was introduced in the early 1960`s for diagnosing myocarditis and primary myocardial diseases (Sakakibara and Konno 3, it became popular in the 1970 when “Stanford technique” was introduced for evaluation of cardiac rejection. Unlike King`s College bioptome technique 4, where left venricular biopsy is usually performed 5, Caves-Schultz or Stanford bioptome uses percutanous approach through the right internal jugular vein. Several biopsy specimens can be obtained, and possible frozen section for immunohistochemical analysis could be done. Controversial issues concerning EMB and its clinical indications could be found in the literature 6-8. It is universally agreed that the value of EMB in the diagnosis and management of cardiac allograft rejection is well established, in the assessment of anthracycline cardiotoxicity, and myocarditis and secondary myocardial diseases can be readily diagnosed by EMB. Today, there is little dispute about main indications and contraindications for EMB, listed in Tables 1 and 2. Evaluation of cardiac allograft rejection Monitoring anthracycline cardiotoxicity Diagnosis of inflammatory myocarditis Distinction between restrictive and constrictive heart disease Diagnosis of specific cadiomyopathies (storage diseases, etc.) Diagnosis of neoplasm (primary and metastatic) Idiopathic chest pain Idiopathic arrhythmia Idiopathic cardiomyopathies Table 1. Main indications for EMB The EMB may provide confirmatory morphologic data in many idiopathic or specific CMPs. While right ventricular biopsy is easier to perform and likely to make the diagnosis, there may be specific indications for left EMB. The contraindications to EMB are few. Bleeding disorders are the most common contraindication. The presence of intraventricular mural thrombus, if involving the left ventricle, may predispose to systemic embolisation, and intracardiac shunts may carry a risk of paradoxical systemic embolisation 9. Prior myocardial infarction and arrhythmogenic right ventricular CMP may also carry a risk. EMB is a technique that can be mastered by a cardiologist with basic cardiac catheterization training. The equipment is standard, and multiple 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 549 novine u patologiji/NEWS IN PATHOLOGY sequential biopsies can be obtained from a single patient, the technique is safer and has fewer serious complications than conventional percutaneous liver or kidney biopsies. EMB EVALUATION OF CARDIAC ALLOGRAFT REJECTION Two major problems arise following successful cardiac transplantation: acute rejection and infections, due to immunosuppression treatment. Untreated, of unnecessary overtreated, acute rejection in heart transplant patients will frequently result in patient dearth or his marked disability. Therefore, much attention has been paid to monitor the degree of cardiac allograft rejection and thereby to determine the most appropriate immuno-suppression regimen, especially if myocarditis was present before the transplant 10. Many different techniques have been used to evaluate the evidence of rejection, such as: electrocardiographic monitoring, echocardiography radioactively labelled different cells or isotopes, magnetic resonance imaging, etc, So far, none of these techniques have shown to be sensitive enough to replace endomyocardial biopsy (EMB). Histologic examination of cardiac tissue samples obtained by EMB remains the most effective method for surveillance of cardiac rejection in heart transplant patients. Electron microscopy is not very useful in the diagnosis of acute cardiac rejection, because longer processing time is required for this method. However, electron microscopy or immuno-electronmicroscopy may be useful in the research field, for better understanding the mechanisms of myocyte injury, and in identifying cells involved in the rejection process. Histologic grading systems for diagnosing cardiac allograft rejection Two major systems were in everyday use to measure cardiac allograft rejection: qualitative, or Stanford system 11, introduced by M. Billingham, and quantitative or THI (Texas Heart Institute), system uses the terms mild, moderate and severe, in order to describe the degree of acute rejection. The THI system is based on numerical scale, ranging from 0 to 10, and describe the degree of rejection by assigning to it a numerical value12. A comparison of the THI and Stanford grading systems is illustrated on Table 2. The other important difference between the two systems is that Stanford system uses, in addition to the amount of mononuclear cells, myocyte necrosis as a criterion for distinguishing between moderate and severe rejection. The THI system is based on findings of intensity of myocyte degeneration as the criterion to evaluate the degree of rejection. True necrosis is rarely observed in rejection process except in high grades of rejection and degeneration is usually reversible if the patient is appropriately treated. Table 2. Comparison of the THI and Stanford grading system of rejection A modification of the Stanford classification was proposed by Kemnitz and colleagues in so-called “Hannover classification”. The main difference is subdivision of the mild acute rejection and introduction of early and late resolution of rejection 13. However, this combination of quantitative and qualitative grading system has not been widely accepted. 550 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 novine u patologiji/NEWS IN PATHOLOGY Grade 0 1 2 3 4 New nomenclature No rejection A - Focal infiltrate without necrosis, Perivascular or intersticial B-Diffuse infiltrate without necrosis One focus with infiltration and/or focal Miocene damage A - Multifocal infiltration and/or myocyte damage B - Diffuse inflammatory process with necrosis Diffuse polimorphonuclear infiltrate with oedema, haemorrhage and necrosis of cardiomyocytes Old nomenclature No rejection mild rejection “Focal”, moderate rejection “mild” moderate “Border-line” severe Severe acute rejection Resolving - rejection in phase of diminution, presented with one smaller number Resolved - finished rejection, presented with grade 0. (Modified from: International Society for Heart Transplantation) Table 3. Standardisation of classification of cardiac allograft rejection Histologic findings corresponding to the numerical value of 0 (THI system) indicated that there is no evidence of cardiac rejection. A grade of 1 and 2 indicates only perivascular aggregates of mononuclear cells. Grade 3 indicates that mononuclear cells are extending into the interstitium. Grade 4 do 8 represent presence of interstitial mononuclear cells with cardiac myocyte degeneration of different, increasing severity cardiac myocyte degeneration of different, increasing severity. Grade 4 represents occasional myocyte degeneration; grades 5 and 6 – scattered myocyte degeneration and grades 7 and 8 signify multifocal degeneration (present in every piece and in all high-power fields). Maintenance of the cardiac transplant patients EMBs are performed in heart transplant patients with frequency depending on patient status, level of immunosuppressive agents, and time of previous biopsy. By correlation of the numerical values and date of the patients previous EMB, we can estimate: the degree of rejection, the direction of change (resolving or progressing), and the speed of change 14. Using the THI grading system 12, we can avoid extensive immunosuppression treatment of hear transplant patients when impressive cellular infiltrate is present with no evidence of marked degeneration. Or, we can introduce higher levels of immunosuppressive agents in patients with multifocal myocyte degeneration and clinical signs of rejection with relatively few mononuclear cells. This change of levels of immunosuppressive treatment is practically impossible under diagnosis of “moderate” rejection. Other factors of cardiac allograft damage can also be detected by EMB. These include: increased graft eosinophilia as sensitive indicator of severe graft rejection, eosinophilic coronary arteritis without classical lymphocytic allograft rejection, occlusive coronary arteritis as consequence of cyclosporine immunosuppression, and evidence of ischemic damage of myocyte in patients with longer survival and present coronary artery disease. Finally, by EMB is possible to identify toxoplasmosis, cytomegalic viral inclusions, fungi, leishmania, and coccidioidomycosis. It is difficult to clinically diagnose these affections of the myocardium. All these conditions are treatable, which support the use of EMB in maintenance of cardiac transplant patients. EMB DIAGNOSIS OF CARDIOMYOPATHIES Idiopathic cardiomyopathies are heart muscle diseases of unknown aetiology (unaccompanied by any other disease process in the body 15. Different classification have been proposed 16-19. The so-called “secondary 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 551 novine u patologiji/NEWS IN PATHOLOGY cardiomyopathies” of known cause are often accompanied or preceded by disease process elsewhere in the body. We are calling them today - specific cardiomyopathies 16-19, besides familial group, how are also idiopathic 20. Hypertrophic cardiomyopathy Hypertrophic cardiomyopathy (HCM) is another entity with many previous names, definitions, and clinical presentations 21, 22 . Hypertrophhic obstructive CMP, the so-called HOCM, implies that the obstructive element is an essential part of the manifestations. Typical clinical presentation is (1) abnormal stiffness of the hypertrophied ventricular muscle with impaired distensibility of the left ventricle; (2) a pressure gradient in systole between the outflow of the left ventricle and the aorta (usually over 60 mmHg and 70 –80 % of patients). Therefore, HCM without obstruction is well-recognised condition with signs confined to a powerful left ventricle and atria thrust and evidence of pulmonary hypertention. Asymmetrical apical hypertrophy is another condition recognised by ECG and echocardiography. In classical HCM, macroscopically, there is bulging, extensive hypertrophy of the left ventricle, especially in the septum of the outflow tract, giving rise to the pressure gradient between the left ventricle and aorta, usually exuding 60 mmHg. This is the most common picture of HCM, also called “subaortic muscular stenosis” and once classified as congenital heart disease. A genetic basis (autosomal dominant trait with almost complete penetrance) has been established, with sequelae on catecholamin function and abnormal muscle fibre alignment (disarray). Histological examination shows short myocardial fibres running in all directions (disarray), and often forming small whorls. Severe hypertrophy of individual myocardial fibres, which often measure 90 -100 m in diameter is striking (normal range is 5 -12 m, average cell diameter in hypertrophy is about 22-25m). Additional histological features include: large bizarre-shaped nuclei, each surrounded by a clear zone, the so-called “perinuclear halo”. Various amounts of interstitial fibrosis, often apparently interrupting short myocardial fibres, are seen. Only the combination of the abnormal features (detailed above) is highly characteristic of HCM and permit diagnosis 23. The so-called “histological HOCM index” is applied and values represent semiquantitative grading system (values 1-3 for each of the 5 histological features - hypertrophy, disarray, bizarre nuclei with perinuclear halo, short runs fibres and fibrosis). If the values over 50% of the maximal 15 are obtained, the PH diagnosis of HCM is confirmed 24, 25. HCM without obstruction has PH, histochemical and EM changes similar to those already presented, and on EMB findings it is impossible to distinguish these to conditions. In all, the use of EMB in diagnosis of HCM is restricted by non-pathognomonic findings, absence of EM or enzymatic differences (22), and should be used only in combination with clinical data. Probably, the main value of EMB in HCM is to rollout other possible conditions with similar clinical impairment (amyloidosis, glicogenosis, small vessel diseases, etc.). Dilated (congestive) cardiomyopathy This world wide distributed cardiac condition have as main clinical sign congestive heart failure which may rapidly progress to end-stage heart disease. Today, among different heart transplant candidates, post-viral DCM is the leading condition 27-29. Macroscopically non-specific features are found. The weight of the heart is around 600-700g with hypertrophy of myocardial wall, but all cardiac chambers are severely dilated and often masking the degree of hypertrophy. The parietal thrombus is often found. The myocardium is pale and flabby, and the coronary arteries are usually normal and patent 26. Histologicaly, EMB findings in DCM shows changes that are non-specific, reflecting changes of hypertrophy with various degrees of degeneration and fibroses. The EMB diagnosis can be made only by exclusion of other specific histiological features (MC), and of other causes that may lead to heart failure. Although 552 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 novine u patologiji/NEWS IN PATHOLOGY a multifactorial aetiology was estimated likely, many experimental, epidemiological and new sophisticated pathological methods has shown the strong evidence of linking idiopathic viral myocarditis to DCM. Familial DCM is also a separate entity, with proof fo different gene involvement 20 . Alcoholic CMP or alcohol induced heart disease, has no consistent features, and distinction from DCM is usually impossible on histologic level. Only enzymatic differences between two groups of patients have been observed by Richardson et al.30 They found that thining and atrophy of the cardiomyocytes, with fatty infiltration, EM characteristics of dilatation of the sarcoplasmic reticulum, lipids, mitochondriosis and increased number of lysosome, followed by decreases in mitochondrial enzyme activity studied by enzyme histochemistry, represent the changes specific for alcoholic CMP. Similarly, congestive heart failure associated with pregnancy, in so-called peripartum cardiomyopathy (PPCMP), cannot be distinguished pathologically from DCM 31. PPCMP is defined as congestive heart failure in the last trimester of pregnancy or within the first six months postpartum. Although positive etiological connection with different factors have been established (viruses, toxoplasmosis, malnutrition, hypertension, immunological mechanism, race, number of pregnancies, etc.), unknown multifactorial aetiology is the most likely 32. Higher incidence of MC in cases of PPCMP suggests that this condition is a separate entity, and similar relation with post-myocardial DCM 31,32. Restrictive cardiomyopathy Although a possible association between eosinophilia, endomyocardial disease and adherent thrombi had been suggested in the late 1800s, it was Loffler in 1936 who described two Swiss patients with hronic heart failure and marked eosinophilia 33. Latter, this entity was recognised under variety of names: Loffler`s endocarditis parietalis fibroplastica, hypereosinophilic syndrome, endomyocardial fibrosis with eosinophilia, disseminated eosinophilic collagen disease, etc. and similarities with tropical endomyocardial fibrosis (EMF) was recognised. These two conditions give very similar clinical picture that may resemble constrictive pericarditis or mitral insufficiency with pulmonary hypertension, but any hypothesis of common aetiology has to reconcile the following: myocardial damage has been reported in cases with increased eosinophilia other than Loffler (filariasis, trichinosis, acute leukaemias); some patients with long standing eosinophilia may have no evidence of EMF; a form of CMP similar to Loffler`s disease has been described in pts without eosinophilia. Brockington and Olsen have suggested that acquired eosinophilia (including the idiopathic hypereosinophilic syndrome and eosinophilic leukaemia) lead to Loffler’s endocarditis, and finally endomyocardial fibrosis 34. These two entities belong to the same disease process with evidence that a large proportion of circulating eosinophils are abnormal with reduced numbers of crystalloid granules. The degranulation of eosinophils is producing MC with nospecific fibrosis at the end of the process. The unitarian hypothesis that “the presence of an eosinophilic leucocytosis, in a susceptible person, by some not completely explained mechanism, causes endomyocardial damage”, is very likely. The cardiac damage is not dependent on the number of circulating eosinophils but on whether they are normal or they might be releasing their cationic proteins – potentially cardiotoxic agents. Degranulated eosinophils with individual immunological abnormalities are the basis for pathologycal changes in “eosinophilic heart disease” (EHD), clinically manifested as RCM. Finally, there are opinions that two form of RCM can be found: one without eosinophilia, with tipical findings of EMF and called idiopathic RCM, and the other form associated with eosinophilia and named EHD 35. Macroscopically, the distribution of the thickened endocardium may vary and may involve the left, right or both ventricles. The endocardium may be several mm thick, and thrombus is superimposed in about 50% of patients. Fibrous septa, extending for a short distance into the underlying myocardium, are common. The cardiovascular involvement are shown on Table 4. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 553 novine u patologiji/NEWS IN PATHOLOGY Table 4.Cardiovascular findings in EHD zone consisted of hyaline collagen. The middle layer is occupied by fibrous tissue and the deepest layer is the “granulation tissue layer”, with variable number of eosinophils. The myocardial fibres between the septa may show degenerative changes, like in other CMPs. The disease passes through 3 stages: first, acute, necrotic phase with inflammatory reaction and variable number of eosinophils. Interval between onset of symptoms and potential death are measured in weeks. The second stage is characterised by thrombus formation and represents the complication of eosinophilic endomyocarditis. There is prominent fibrous endocardial thickening, and arteritis may be still present. Interval between onset of symptoms and death are measured in months. The third, fibrotic stage represent the last phase with fibrosis and septa made of superficial layer of hyaline collagen, middle layer of fibrous tissue, and deepest layer of chronic inflammatory cell with varying number of eosinophils. These three pathological patterns of disease are one continious process connected with length of time between onset of disease and death. EMB diagnosis of EHD is of great importance, especially in children 36. Depending of the time of the biopsy, the possibility of founding degranulated eosinophils, thrombus, and MC, makes the EMB fundamental techniques in diagnosing this condition 37. Arrhythmogenic Right Ventricular Cardiomyopathy This entity is characterised by progressive fibrous-fatty replacement of right ventricular myocardium, mainly the free wall, followed by rhythm disturbances as major clinical problem. Initially, the changes are consistent with regional, and later global, right and some left ventricular involvement, and with relative sparing of the septum. Although familial occurence has been documented and a gene defect was recently localized on chromosome 14q23-q24, the etiopathogenesis of the disease is still obscure. Familial form of arrhythmogenic right ventricular cardiomyopathy (ARVC) disease is very common, with autosomal dominant inheritance and 554 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 novine u patologiji/NEWS IN PATHOLOGY incomplete penetrance, although a recessive form is also described. Presentation with arrhythmia and sudden death is also very common, particularly in the young and athletes, as indicated by Thiene et al 38. Contrary to the genetic form, some recent clinical and morphological findings demonstrated its acquired, progressive, frequently inflammatory character, and supports the notion of chronic, continuous process of injury and repair. Several hypotheses are made upon pathogenesis of ARVC, with idiopathic myocarditis as the most probable cause (Fontaine, Hofman, et al.). The findings of adipose tissue in the heart is not allways the indication of pathologic process, and frequency, extent, and distribution of endomyocardial adipose tissue was analysed by morphomety from EMBs in 241 patients, indicating the particularities of adipose replacement in ARVC. It should be noted that pathologic evidence of extensive left ventricular involvement in ARVC was demonstrated. Finally, as possible causes of ARVC apoptosis was implicated, showing how normal and abnormal consequences of apoptosis can lead from postnatal morphogenesis to paroxysmal arrhythmias39. The frequent findings of MC with myocyte death lead to the consideration of the disease as chronic MC, with some immune factors involved. The EMB diagnosis of ARVC should be performed with coution of perforation, and maneuver under echocardiographic guide is recommended. The usual site for EMB, the interventricular septum, is not the ideal location for biopsy. Unother problem is the fibrous-fatty atrophy and its pahtgnomonity for definitive diagnosis. Adipose and fibrous tissue in the myocardiun is not so specific findings, interstitial replacement is also often observed in many CMPs, so the issue is to quantify the components, rather then qualify them. As suggested by Angelini et al., the following diagnostic parameters should be obtained by morphometric analysis: myocardial atrophy with residual myocytes less then 45%; fobrous tissue less then 40%; and fatty tissue more then 3%. The sensitivity and specificity were 67% and )”%, respectively. This quantitative histologic criteria give better definitive diagnosis by tissue characterization then magnetic resonance imaging or other noninvasive procedures 38. Unclassified Cardiomyopathies Unclassified CMPs incised a few cases that do not fit readily into any group, like fibroelastosis, noncompacted myocardium, systolic dysfunction with minimal dilatation, mitochondrial disorders in myocardial involvement, etc. Some of CMPs may present with features of more than one type of CMP (eg. amyloidosis, systemic hypertention), and overlapping morphology is even more common (end-stage HCM with dilatation, peripartum CMP, alcohol induced heart disease, and other entities similar with DCM). It is recognised that arrhythmias and conduction tissue diseases may be primary myocardial disorders, but, at this time, they are not included as cardiomyopathies. They remain as one of the frequent indications for EMB (mainly to rule-out MC), but as many conditions may present with rrhythm disturbances, they are not a distinctive entity. 6. EMB DIAGNOSIS OF SPECIFIC CARDIOMYOPATHIES Specific cardiomyopathies (SCMP) may be defined as “secondary CMPs” or heart muscle disease of known cause, associated with recognised general disorders. These conditions have been classified by many authorities in this field (15-19), but our approach to the classification of SCMP is mainly based on EMB findings, indicating three main groups of diseases: first, in which the pathognomonic EMB features are seen, that allows the morpho diagnosis of “specific entity”; second group of diseases in which “specific” morphological features can not be demonstrated, and the diagnosis is made by exclusion 40 . In spite of this luck of “morphological specificity”, many general condition in which the heart can be affected are classified under SCMP. We strongly believe that division of these two groups, based on possibility of EMB diagnosis, is the most appropriate way to classify, diagnose and treat SCMP. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 555 novine u patologiji/NEWS IN PATHOLOGY Entities with pathognomonic PH findings 1. Infective diseases (MC) 2. Metabolic (familial storage and endocrine diseases) 3. Infiltrations, granulomas and neoplasia 4. Sensibility and toxic reactions (adriamycin) Entities with non-pathognomonic PH findings 5. Connective tissue disorders 6. Heredofamilial neuromuscular diseases 7. Deficiencies and avitaminoses 8. Rare syndromes Table 5. Classification of Specific CMPs (specific heart muscle diseases). Besides infective diseases (viral, bacterial, fungal, protozoal, etc.), with specific or pathognomonic findings, many storage diseases (glicogenoses, haemochromatosis, amyloidosis, etc.) can be diagnosed by EMB. All otter specific CMPs have less pathogno-monic findings and only clinical characteristics makes them “specific” heart disease 33. Glycogen storage disease Of the twelve types of glycogen storage disease, the heart is involved in three: type II (Pompe,s disease), deficiency of alpha-4,4-glucosidase (acid maltase); type III (Cori,s disease), a deficiency of the debranching enzyme amylo-1,6-glucosidae; and type IV (Andersen,s disease), caused by a deficiency of the branching enzyme alpha-1,4-glucan-6 glucosyltransferase. These diseases are transmitted as autosomal recessives and are manifest by accumulation of glycogen in various tissues 41. The diagnosis should be based not only on the demonstration of increased glycogen, but also by demonstration of the enzyme defect. Most cases of glycogen storage disease, causing cardiomegaly, are due to type II glycogenosis. The heart is enlarged, and all chambers have thickened walls and small cavities. Progressive impairment of myocardial function ensues, and Pompe,s disease is fatal within the first year of life. Death is due to cardiac failure or respiratory complications. Grossly, the heart appears rubbery and pale pink. There may be fibroelastotic thickening of the endocardium. In histologic sections there will be severe vacuolisation within the central areas of the myocytes, giving a lacework appearance to the tissue, due to massive deposits of glycogen which displace myofibrils to the periphery. Myofibrillar loss related to cardiac failure can also be demonstrated ultrastructurally. If a glycogen storage disease is suspected, the endomyocardial biopsy can be fixed in absolute alcohol tryed in preserving the glycogen, and electron microscopy is usually required as well (42). The characteristic ultrastructural alteration is large collections of glycogen (either free in the cytoplasm in Pompe,s disease, or within lysosomes). The glycogen may be in a morpholo-gical form, granules (type II and III) or in an abnormal form (fibriles, in type IV glycogenosis). Fabry,s Disease Fabry,s disease (angiokeratoma corporis diffusum universale) is an X-linked recessive disorder caused by deficiency of lysosomal alpha-galactosidase A, resulting in excessive deposits of ceramide trihexoside, particularly in the skin, cornea, kidneys, and heart. It is manifest by angiokeratomas, pain and paresthesias of the extremities, and progressive renal and cardiovascular disease 43. Symptoms relative to the heart include cardiac hypertrophy and dilatation, congestive heart failure, angina, and hypertension, all of which are due to 556 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 novine u patologiji/NEWS IN PATHOLOGY deposits of ceramide trihexoside in lysosomes in endothelial cells, smooth muscle and pericytes throughout the vascular system. Because of these deposits, patients may have myocardial infarction at an early age, microaneurysms, cystic medial necrosis of the aorta, and valvular lesions (mitral stenosis, aortic regurgitation, pulmonic regurgitation). Death is often from congestive heart failure. Endomyocardial biopsy from patients with Fabry,s disease will demonstrate a lacework appearance of the myocardial fibres on routine light microscopy, with marked perinuclear vacuolisation and displacement of the contractile elements to the periphery. In frozen sections, the deposits of ceramide trihexoside appear as vacuoles, which are sudanophilic and PAS-positive. By electron microscopy, these deposits appear as intralysosomal aggregates of concentric or parallel lamellae, which stain positively by ultrastructural techniques that demonstrate cardohydrates 44. Endomyocardial biopsy may be helpful in making the diagnosis of Fabry,s disease, particularly in cases not recognised in childhood, or in patients who lack the usual signs and symptoms (i.e., proteinuria, corneal apacities). Cardiac Amyloidosis Amyloidosis describes disease processes, which are characterised by extracellular deposits of proteins, which have a beta-pleated sheet conformation. Current classification is based on the biochemistry of the amyloid fibril, and who major groups are recognised: AL, in which fibrils consist of light chains of immunoglobulin, and AA, in which the fibrils consist of fragments of serum amyloid A protein. A third group, in which the fibrils are comprised predominantly of prealbumin and which occurs in senile cardiac amyloidosis and familial polyneuropathy. The AL form is associated with plasma cell dyscrasias or can occur as localised deposits without evidence of generalised involvement, while AA often occurs with inflammatory processes, such as rheumatoid arthritis or chronic infection. Amyloid deposits can be found in the heart in elderly people or as part of generalised amyloidosis. Senile cardiac amyloid has been reported in 30-69% of patients older than 60 years, while 54-90% of patients with generalised amyloidosis (usually AL associated with multiple myeloma, or less often AA) have cardiac involvement 45. Depending upon its origin and extent, cardiac amyloidosis may be asymptomatic, or it may cause progressive heart failure and refractory arrhythmia. Clinically significant cardiac amyloidosis must be differentiated from constrictive pericarditis, hypertrophic cardiomyopathy, storage diseases, or other infiltrative myocardial diseases. It can also manifest as ischemic heart disease, with typical or atypical angina and a “pseudoinfarct” pattern on ECG, and it may produce arrhythmias or conduction defects. Cardiac amyloidosis produces a stiff myocardium, which impedes diastolic ventricular filling and creates restrictive hemodynamics. In clinically significant cardiac amyloidosis, the heart may be heavy and the walls are thickened, firm, and have a pale colour and rubbery consistency. Amyloid deposits occur in the interstitium, conduction tissue, valves, endocardium, pericardium, and in small intramural arteries, veins and capillaries. In the myocardium, amyloid may be found surrounding individual myocytes, or in the form of focal nodular interstitial deposits that push aside and replace fibres, or both. Myocardial fibrosis may be assesed by different methods 46 In coronary vessels, it may involve all vascular layers and even cause luminal occlusion. The presence of amyloid can be confirmed by the apple green birefringence it gives under polarised light after Congo red staining, or by metachromasis with methyl violet, or ultraviolet fluorescence with thioflavin T (the latter may yield false positives). Amyloid fibrils can also be identified by their characteristic ultrastructural appearance as 7.5 nm diameter nonbranching fibrils. If clinically indicated, immunohistochemical typing of amyloid can be done using paraffin embedded sections with antisera against purified amyloid fibril protein and monoclonal antibodies against protein AA. In patients suspected of having cardiac amyloidosis, an endomyocardial biopsy cannot be substituted by biopsy of another site, such as the rectum, as only 60-80% of patients with idiopathic (primary) amyloidosis will have a positive rectal biopsy. At least four biopsy specimens should be obtained to minimise the possibility of a false negative result. While patients with known amyloidosis who have typical echoradiographic 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 557 novine u patologiji/NEWS IN PATHOLOGY features do not necessarily need to undergo endomyocardial biopsy, cardiac amyloidosis cannot be excluded on the basis of negative biopsies of extracardiac tissues. In patients who have clinically unexplained cardiac disease and suspected amyloidosis, endomyocardial biopsy is a reliable diagnostic procedure. Cardiac hemochromatosis Iron deposits occur in the myocardium in idiopathic (familial) hemo-chromatosis as well as in hemosiderosis secondary to iron overload (e.g., multiple transfusions, dietary intake). The clinical manifestations vary, depending on the extent of myocardial involvement, but patients usually have a dilated, or rarely restrictive, cardiomyopathy. Some patients are asymptomatic but may have echocardiographic evidence of myocardial infiltration (increased left ventricular wall thickness). Other abnormalities include ECG changes (ST-segment and T-wave changes), supraventricular arrhythmias, atrioventricular conduction disturbances, and ventricular arhythmias. The severity of myocardial dysfunction may be proportional to the amount of iron present, and extensive deposits are usually associated with congestive heart failure (occurring in one third of patients) which is usually the cause of death 47. The heart appears brown and may be hypertrophied and/or dilated. Normally, there is no stainable iron within the myocardium. In hemochromatosis/hemosiderosis, iron deposits are more extensive in the epicardial third, intermediate in the inner (subendocardial) third, and least extensive in the middle third of the ventricular wall. They are typically perinuclear in location initially, but eventually occupy most of the cells. Involvement of the conduction system, coronary arteries, and valves in limited. There may be associated fibrosis, in which case restrictive hemodynamics may be present. As hemochromatosis in usually associated with involvement of other organs, EMB is not required for diagnosis. If a biopsy is performed, however, multiple specimens should be obtained to minimise sampling error, since iron deposition may be focal 48. Cardiac Sarcoidosis Most patients with cardiac sarcoidosis have clinically apparent systemic sarcoidal involvement, but in some patients the heart may be the primary site, without clinical evidence of other organ involvement. The clinical manifestations are determined by the extent and location of process and may include atrioventricular conduction defects, ventricular arrhythmias, sudden death, congestive heart failure (due to widespread myocardial involvement, ventricular aneurysms, arrhythmias, or cor pulmonale due to pulmonary hypertension), chest pain with or without ECG changes of ischemia or dysfunction of papillary muscles and mitral regurgitation. Pericardial abnormalities (effusion, constrictive pericarditis, tamponade) may also occur. Cardiac sarcoidosis is a focal disease. Therefore, when endomyocardial biopsy is performed in patients with suspected cardiac involvement, multiple specimens from several sites should be obtained. The predominant sites of myocardial involvement are, in decreasing order of frequency, left ventricular free wall, base on the interventricular septum, right ventricular free wall, and atrial walls. A negative biopsy does not rule out the diagnosis of cardiac sarcoidosis 49. The lesions in the heart are identical to those described in the lungs, consisting of histiocytes, giant cells, lymphocytes and plasma cells. Patchy fibrosis and lymphocytic myocarditis can also be observed but are nonspecific. Other conditions associated with giant cells in the heart that may need to be distinguished from sarcoid include idiopathic giant cell myocarditis, infective endocarditis, rheumatoid arthritis, Takayasu,s arteritis, and Wegener,s granulomatosis 50. In summary, sarcoidosis should be suspected in young adults, especially in blacks, who have cardiomyopathy, conduction disturbances or other ECG abnormalities. Although sarcoid involves the heart in only 20% of autopsy-proven cases of sarcoidosis, endomyocardial biopsy may aid in the diagnosis. 558 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 novine u patologiji/NEWS IN PATHOLOGY Cardiac Tumors Primary cardiac tumors are rare, ranging in incidence at autopsy from 0.0017 to 0.28%, compared to 1.22% for metastatic cardiac tumors. The most common primary cardiac tumor is myxoma (40%), followed by angiosarcoma and rhabdomyosarcoma, with these three comprising over half of all cardiac tumors. Other primary cardiac tumors include papillary fibroelastoma, fibroma and fibrosarcoma, hemangioma, teratoma, and mesothelioma of the atrioventricular node. Metastatic tumors to the heart include lung carcinoma, hematopoietic-lymphoid neoplasms, and melanomas, with cardiac involvement occurring in about 20% of patients with malignancies. Endomyocardial biopsy has the potential to aid in the diagnosis of primary and secondary cardiac tumors, and there have been several reports in which the diagnosis of an intracardiac tumor was made by left or right ventricular transvenous biopsy 40. Some patients may benefit from the procedure, which has less discomfort, risk and expense than an open thoracotomy, particularly those in whom the benefit of surgery might be questionable. The risk of tumor embolization is unknown, but could be a potential problem, particularly if the tumor is friable (i.e., myxomas). Sensitivity and toxic reactions Anthracycline cardiotoxicity Anthracyclines, antineoplastic drugs that include doxorubicin (Adriamycin) and daunomycin, are effective chemotherapeutic agents for the treatment of numerous solid and hematopoietic malignancies. Cardiac toxicity is a well-recognised complication and is the doselimiting factor in the use of these drugs. While the usual practice is to administer these agents up to a maximum total dosage of 500-550 mg/m2, some patients may suffer cardiotoxic effects at lower cumulative doses, particularly if they have pre-existing cardiac disease (including hypertension), are greater than 70 years in age, or have received prior irradiation. Prior cyclophosphamide therapy may also potential the cardiotoxic effects of anthracyclines. There is significant individual variation in a patients susceptibility to anthracycline-induced cardiac damage, with the subsequent development of heart failure. Radionuclide ejection fraction is sensitive but cannot differentiate heart failure due to other causes from that due to anthracycline alone, and the drug may be withdrawn prematurely in some patients. For these reasons, serial EMBs have become a reliable method to evaluate anthracycline cardiotoxicity 51. The degree of myocardial injury can be estimated by histologic grading of biopsy specimens, and the EMB is an effective means to monitor patients. Anthracycline produces both early and late cardiotoxic effects. The early effects, which can occur after one dose, include a pericarditis-myocarditis syndrome, drug-induced cardiovascular dysfunction, and arrhythmias. Dose-related myocyte damage and heart failure is directly related to the amount of myocyte damage. The cardiomyopathy appears 1 to 6 weeks after the last dose is given and prognosis is poor, with death occurring in 79% of patients. It is not possible to accurately evaluate anthracycline cardiotoxicity by light microscopy. Therefore all EMBs must be submitted for EM 15,52 . Two characteristic lesions can be seen ultrastructurally: sarcotubular dilatation and loss of myofibrils. The extent of these changes are the basis for the grading system. Sarcotubular dilatation is due to coalescence of dilated sarcoplasmic reticulum and, when severe, can be seen by light microscopy, but this must be confirmed by electron microscopy. Myofibrillar loss may be detected by light microscopy by the appearance of small, shrunken cells with homogenous, pale cytoplasm but, again, this must be confirmed ultrastructurally. If cardiotoxicity is severe, the non-specific finding of interstitial fibrosis may also be seen.The pathogenesis underlying anthracycline cardiotoxicity is not clear. One postulated mechanism is via generation of free radicals, which has considerable corroborating experimental evidence. Other possibilities include inhibition of coenzyme Q 10 (ubiquinone) which is involved in oxidative phosphorylation. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 559 novine u patologiji/NEWS IN PATHOLOGY 7. SPECIAL VALUE OF EMB IN DIAGNOSIS AND CHOICE OF TREATMENT MODALITIES OF IDIOPATHIC MYOCARDITIS Among all SHMD, the most common and important is viral MC, which diagnosis is based mainly on “Dallas criteria”. By consensus, the authors defined acute MC as “a process characterised by an inflammatory infiltrate of the myocardium with necrosis and/or degeneration of adjacent myocytes which was not typical of the ischaemic damage associated with coronary artery disease”. Idiopathic (presumed viral) MC would be termed “primary MC”, whereas acute MC due to other causes (toxoplasmosis or Chagas disease) would be termed secondary MC. As mentioned previously, the Dallas criteria were defined for the MC trial, therefore, separate terminology was adopted for the first diagnostic biopsy and for the subsequent biopsies 53. On the first EMB the following diagnoses could be made: (1) active MC, with or without fibrosis: both, an infiltrate and damage of the adjacent myocytes were required for this diagnosis. (2) Borderline MC: (not diagnostic findings, requiring a repeat biopsy). This term implies that the inflammatory infiltrate is too sparse or that the myocyte damage is not seen on PH. Additional sections of the original biopsy might demonstrate diagnostic changes in which active MC can be diagnosed. (3) No evidence of MC: a biopsy in which there is no inflammatory infiltrate or myocyte damage. All subsequent biopsies were divided also into three categories. (1) Persistent or ongoing MC: this diagnosis is made when the degree of interstitial infiltrate is the same or worse than on the first biopsy. (2) A resolving (healing) MC: this diagnosis is made when the inflammatory infiltrate is less expressed than in the previous biopsy, and reparative changes are evident. (3) Resolved (healed) MC: where there is now no inflammatory infiltrate remaining and no evidence of ongoing cellular necrosis. Scar tissue ( fibrous replacement or collagen fibrosis) may be present with adjacent compensatory hypertrophy. It is becoming increasingly apparent that following an acute or subacute episode of MC due to a virus infection, the virus might persist in the cardiac tissues causing a DCM. It is not clear how RNA viruses switch from acute to persistent infections, but it seems that viral transcription and translation are reduced. The treatment of viral MC may be different and depend on therapeutic approach of the clinician and can be defined as 3 main modalities: conventional therapy, immunosuppressive and immunomodulatory treatment 53-56. The conventional therapy is mainly based on bed rest, aspirin, and all other necessary cardiac medicaments (including diuretics, ACE-inhibitors, b-blockers, etc). There is no attempt to eliminate the virus or to interfere with patients’ immune system. From the very “popular” immunosuppressive treatment there was too many expectations, and unfortunately not very promising results. Introduced and recommended by many, the final trial had no definitive proof for recommendation of these medicaments. Finally, the immunomodulatory therapy was introduced. If the viruses are involved in MC (and consequently with DCM), therapeutic use of interferons or its inducers seems to be appropriate, either due to its direct action on the target cell or indirect effects (activation of cytotoxycity, modulation of immune response, and interaction with other mediators of immune response). Presuming that heart in MC and DCM may represent a site of low-grade persistent infection, intermediate doses of interferon (IFN) should be considered as a treatment schedule. Administration of IFN in doses over a certain level does not increase antiviral activity, and too frequent administration may prolong the refractory state of the cells. We enrolled 180 patients (clinical study by Dr M.Mirić at “Dedinje” Institute) to receive a-interferon (IFN) with doses of 3-5 million units per day, for 3 months, and thymomodulin 10 mg 3 times per week, for 2 months. Some patients received conventional therapy alone (depending on EMB analysis). Patients were followed up for 7 years after the end of treatment. Left ventricular function, exercise tolerance and survival rate were significantly better at long-term follow-up in patients treated with IFN or thymomodulin, than in conventionally treated patients. These results implicate that immune modulating therapy might represent important contribution for treatment of MC and DCM, and suggest that adding immunomodulators to the conventional therapy improved the functional capacity and survival of these patients 55. To improve the treatment, Figulla et al. presented a new classification of MC/DCM patients21. The term idiopathic DCM should be used if myocardial dysfunction of unknown cause is detectable by hemodynamic 560 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 novine u patologiji/NEWS IN PATHOLOGY measurements. According to EMB findings, 4 subsets of MC/DCM can be differentiated: patients without any infectious agent present on EMB and without inflammatory infiltrate; patients without infectious agent but with cellular infiltrate; the third group are patients with an infectious agent and with an infiltrate; and the last group are patients with infectious agent without infiltrate. Those patients without any infectious agents and without cellular infiltrates should receive unspecific heart failure therapy (conventional). If a virus is detectable, heart failure therapy in combination with a virus-suppressive agent may be more appropriate. If a cellular infiltrate is present in combination with an infectious agent and severe left ventricular dysfunction is found, unspecific (conventional) therapy is recommended in combination with a virus-suppressive agent and cytokine blockers. In the case of an infectious agent without cellular infiltrates, a combination of unspecific therapy and virus-suppressive agent should be used. OVERALL DIAGNOSTIC VALUE OF ENDOMYOCARDIAL BIOPSY During the past three decades, the technique for performing EMBs has been improved substantially. However, this alone does not explain the increasing clinical use of this procedure. Probably, the combination of experience, diagnostic accuracy, new technical aspects 57, and sophisticated technique in analysing fresh myocardial tissue revealed such interest for enlarged EMB applications. The main value of EMB in diagnosis and treatment of primary heart diseases is the possibility of giving the precise diagnosis (with severity and extent of the pathologic process), followed by the choice of treatment modalities 55,56). EMB analyses not only aid diagnosis, but also help in the differential diagnosis of endomyocardial fibrosis from other causes of heart failure57. Further, it has helped to evaluate prognosis, particularly in patients with congestive CMP. The personal experience of the author extends to biopsies obtained from over 1100 patients. It has been found that in 82 % of patients, morphological information that is helpful to the referring physician can be obtained. Additional 13% of analysed material present non-specific findings with no PH diagnosis. Only the remaining 5% of EMB were to small or inadequate material for appropriate diagnosis. If we consider that in non-specific findings we did not found any other new entity or changed the clinical diagnosis, by elimination these results can also be considered as helpful. In that case, we can estimate clinical merit in over 80% of the cases. With special references to the choice of the treatment, EMB will play even more important role in the future, for the management of patients with CMPs and SHMD. Literature 1. Aretz HT: The endomyocardial biopsy revisited (Editorial). Mayo Clin Proc 1990, 65 : 1506 -1509. 2. Vasiljevic JD: Endomiokardna biopsija – patohistološka, histohemijska i elektronskomikroskopska ispitivanja uzorak. Kardiologija, Medicinski Fakultet Beograd, NIRO Zrenjanin (izd.), 1986, 289 -99. 3. Sakakibara S, Konno S: Endomyocardial biopsy. Jpn Heart J 1962, 3:537 - 41. 4. Richardson PJ: King’s endomyocardial biopsy. Lancet 1974, i :660. 5. Mason J: Left ventricular biopsy. In: Fenoglio J.J. (ed.) Endomyocardial biopsy: techniques and applications. CRC Press, Bocca Raton 1983: 15-23. 6. Mills AS, Hastillo A, Thompson JA, Hess ML: Expectations and limitaions of endomyocardial biopsy in cardiomyopathy. Can J Cardiol 1985, 1 : 358 -62. 7. Starling RC, Van Fossen DB, et al.: Morbidity of endomyocardial biopsy in cardiomyopathy. Am J Cardiol 1991, 68 : 133 -36. 8. Vasiljević JD: The role of endomyocardial biopsy in diagnosis and choice of treatment modalities in idiopathic and specific cardiomyopathies. Review article. Arch Balk Med Union, 2000, 35(2) : 95-106. 9. Seferović P, Vasiljević JD, Ostojić M, et al.: Komplikacije endomiokardne biopsije. Kardiologija (Beograd), 1992, 13 : 175 -81. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 561 novine u patologiji/NEWS IN PATHOLOGY 10.Vasiljevic JD, Radovancevic R, McAllister HA, et al.: Influence of pre-transplant myocarditis on post-transplant cardiac rejection episodes. XIII European Congress of Pathology, Ljubljana, 1991. Path Research Pract, 1991; 187(6): 778. 11.Billingham ME: Dilema of variety of hitopathologic grading systems for acute cardiac allograft rejection by endomyocardial biopsy. J Heart Transplant 1890, 9 . 272 -6. 12.McAllister HA Jr: Histologing grading of cardiac allograft rejection: a quantitative approach. 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Arch Anat Cytol Path Clin Exp Path, 1998, 46 : 328 (Abst. 212). 25.Vasiljević JD, Tucaković G, Mirić M, et al. : The role of endomyocardial biopsy in patients with hypertrophic cardiomyopathy. Virchow’s Archive, 1999, 435: 343. 26.Figulla HR : Idiopathic Dilated Cardiomyopathy: Current Concepts in Clinical Research. In : Figulla et al. (eds.): Idiopathic Dilated Cardiomyopathy, Springer Verlag, Berlin-Heilderberg, 1993 : 3 - 9. 27.Kawai C., Matsumori A., Kitaura Y., Takatsu T.: Viruses and the heart - viral myocarditis and cardiomyopathy. Prog in Cardiol 1978, 7: 141 - 9. 28.Hirshman S.Z., Hammer G.S.: Coxsackie virus myocarditis. A microbiological and clinical review. Am J Cardiol 1974, 34: 224 - 30. 29.Martino TA, Liu P, Petric M, et al.: Enteroviral myocarditis and dilated cardiomyopathy: a review of clinical and experimental studies. In: Rotbart HA, ed. Human enterovirus infections. Washington, DC: American Society for Microbiology; 1995, 291 - 351. 30.Richardson PJ, Wodak AA: Alcohol-induced heart muscle disease. In : Symons C., Evans T, Mitchell AG (ed): Specific Heart Muscle Disease, Wright PSG, 1983 : 99 -122. 31.Vasiljević JD, Kanjuh V, Seferović P, Šesto M, Ostojić D, Olsen E: The Incidence of Myocarditis in Endomyocardial Biopsy Samples from Patients with Conjestive Heart Failure. Am Heart J, 1990; 120:1370-1377. 32.Vasiljević JD, Mirić M: Virusni miokarditis i dilataciona kardiomiopatija. Tersit (Izd.), Beograd, 1995: 1 - 395. (Oktobarska nagrada Beograda za medicinu za 1996. godinu) 33.Brockington IF, Olsen EGJ, Goodwin JF: Endomyocardial fibrosis in Europeans resident in tropical Africa. Lancet 1967, i : 583. 34.Oakley CM, Olsen EGJ: Eosinophilia and heart disease. (Editorial). Br Heart J 1977, 39 : 223. 562 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 novine u patologiji/NEWS IN PATHOLOGY 35.Webb-Peploe MM: Eosinophilic heart disease. In: Symons C, Evans T, Mitchell AG (ed): Specific Heart Muscle Disease. Wright PSG (Publ.), 1983 : 24 - 32. 36.Vasiljević JD, Nedeljković V, Seferović P : Eosinophilic herat disease in children confirmed by endomyocardial biopsy. Second Balkan Meeting of pediatric cardiology and cardiac surgery. Sofia, 1994, Abstracts, 55. 37.Vasiljević JD, Stojšić ĐS, Cvejin B, et al.: Morphologic diagnosis of eosinophilic heart disease - biopsy and pathologic findings. 10th Anniversary International Symposium on Cardiovascular Diseases, Novi Sad, 1994, Abstracts, 245. 38.Thiene G, Corrado G, Basso C: Arrhythmogenic right ventricular cardiomyopathy / dysplasia. Orphanet J Rare Dis 2007, 2 : 45. 39.Thiene G, Basso C: Arrhythmogenic right ventricular cardiomyopathy: An update. Cardiovasc Pathol 2001,10 :109 -117. 40.Vasiljević JD: Seminar iz endomiokardne biopsije. VII Kongres patologa Jugoslavije, Budva, 1996. Institut za Patologiju, Medicinski fakultet u Beogradu (Izd.), 1996: 1-22. 41.Wenger NK: Specific heart muscle disease. In: Goodwin J.F. (ed.): Heart Muscle Disease, MTP Press 1985 : 108 - 15. 42.Howell RR: The glycogen storage diseases. In: Stanbury J.B., Wyngaarden J.B., Frederickson D.S. (ed.): The Metabolic Basis of Inherited Disease, 1982, 149 - 152. 43.Blieden LC, Moller JH: Cardiac involvement in inherited disorders of metabolism. Prog Cardiovasc Dis 1974, 16: 615 - 19. 44.Ferrans VJ, Hibbs RG, Burda CD: The heart in Fabry, s disease. A histochemical and electron microscopic study. Am J Cardiol 1969, 24 : 95 - 103. 45.Mirić M, Vasiljević JD: Imunske bolesti srca i krvnih sudova. Cicero (Izdavač), Beograd, 2000:1-222. 46.Vasiljević JD, Popović Z, Vidaković M, et al.: Myocardial fibrosis assesment from semiquantitative, point-counting and computer-based methods: a comparative study. Histopatology 2001, 38 : 338-343. 47.Buja LM, Roberts WC: Iron in the heart. Etiology and clinical significance. Am J Med, 1971, 51 : 209 - 15. 48.Vasiljevic JD, Kanjuh V, Tucakovic G et al.: Myocardial lesion in idiopathic hema-chromatosis : Findings in 2 autopsied cases and 2 endomyocardial biopsy specimens. Path Res Pract 1989, 185 (1) : 168. 49.Roberts WC, McAllister HA, Ferrans VJ: Sarcoidosis of the heart. A clinicopathologic study of 35 necropsy patients and review of 78 previously described necropsy patients. Am J Med 1977, 63 : 86 - 98. 50.Fauci AS, Haynes BF, Katz P: The spectrum of vasculitis : Clinical, pathologic, immunologic and therapeutic considerations. Ann Intern Med 1978, 89 660 - 69. 51.Friedman MA, Bozdech MJ, Billingham ME, Rider AK: Doxorubicin cardiotoxicity. Serial endomyocardial biopsies and systolic time intervals. J Am Med Assoc 1978, 240 :1603 - 1611. 52.Vasiljević JD: Uloga endomiokardne biopsije srca u dijagnostici primarnih bolesti srčanog mišića. MAG Medic (Beograd), 1995, 1(2):9. 53.Aretz HT, Billingham ME, Edwards WD et al.: Myocarditis / a histopathologic definition and classification. Am J Cardiovasc Pathol, 1986,1 : 3 -14. 54.Maisch B, Herzum M, Schonian U: Immunomodulating factors and immunosuppressive drugs in the therapy of myocarditis. Scand J Infect Dis . Suppl 1993, 88 : 149 – 62. 55.Mirić M, Mišković A, Brkić S, Vasiljević JD et al. : Long-term follow-up of patients with myocarditis and idiopathic dilated cardiomyopathy after immunomodulatory therapy. FEMS Immunology and Medical Microbiology 1994, 10 : 65 -74. 56.Mirić M, Vasiljević JD, Bojić M, et al. : Long term follow-up of patients with dilated heart muscle disease treated with human leucocytic interferon-alpha or thymic hormones. Initial results. Heart 1996, 75 : 596 – 601. 57.Vasiljević JD, Otasevic P, Popovic Z, et al.: Semi-quantitaitve Histomorphometric analysis of myocardium following partial left ventriculectomy: One-year follow-up. Europ J Heart Failure 2005, 7: 763-767. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 563 novine u patologiji/NEWS IN PATHOLOGY Neuromišićne biopsije – tri godine nacionalnog iskustva Neuromuscular biopsy - a review of 3 years nacional experience Sanja M. Milenković Sanja M. Milenkovic Služba kliničke patologije, Kliničko bolnički centar Zemun, Beograd, Srbija Department of Clinical Pathology, Clinical Hospital Centeer Zemun, Belgrade, Serbia Apstrakt Abstract U ovom radu prikazujemo iskustvo sa 162 konsekutivne neuromišićne biopsije analizirane u period od 2009 do 2012 godine. Neuromišićne bolesti predstavljaju veliku grupu naslednih i stečenih bolesti koje se karakterišu gubljenjem mišićne mase i slabošću mišića. Razlikovanje miopatija od perifernih neuropatija, bolesti ćelija prednjih rogova kičmene moždine (i bolesti neuromišićne spojnice zahteva pažljivu kliničku evaluaciju, laboratorijska, neurofiziološka i elektromiografska ispitivanja, radiološka ispitivanja (npr.magnetna rezonanca), mišićnu biopsiju i genetsko ispitivanje. Rezultati mišićne biopsije se isključivo mogu interpretirati u kontekstu prethodno navedenih ispitivanja. U Republici Srbiji je 2009.god. u Službi kliničke patologije Kliničko bolničkog centra Zemun, a po odluci Ministarstva zdravlja Republike Srbije, počela rutinska dijagnostika NMB na biopsijskim uzorcima mišiča, nerava i kože Ključne reči: biopsija mišića, biopsija nervaa, imunohistohemija, populaciona studija In this paper we present the experience with 162 consecutive neuromuscular biopsies analyzed in the period from 2009 to 2012. Neuromuscular diseases are a large group of inherited and acquired diseases that are characterized by loss of muscle mass and muscle weakness. Distinguishing myopathies from peripheral neuropathy, diseases of the anterior horn cells of the spinal cord and the diseases of the neuromuscular junction requires careful clinical evaluation, laboratory, neurophysiological and electromyographical examination, radiological tests, muscle biopsy and genetic testing. Muscle biopsy results can solely be interpreted in the context of the above tests. In Serbia, 2009. in Department of Clinical Pathology Clinical Hospital Center Zemun, a decision by the Serbian Ministry of Health, began a routine diagnostic biopsy specimens in the NMB muscles, nerves and skin. Key words: muscle biopsy, nervaa biopsy, immunohistochemistry, population studies Uvod Neuromišićne bolesti predstavljaju veliku grupu naslednih i stečenih bolesti koje se karakterišu gubljenjem mišićne mase i slabošću mišića. Razlikovanje miopatija od perifernih neuropatija, bolesti ćelija prednjih rogova kičmene moždine (npr. bolest motornog neurona) i bolesti neuromišićne spojnice (mijastenija gravis) zahteva pažljivu kliničku evaluaciju, laboratorijska, neurofiziološka i elektromiografska ispitivanja, radiološka ispitivanja (npr.magnetna rezonanca), mišićnu biopsiju i genetsko ispitivanje. Rezultati mišićne biopsije se isključivo mogu interpretirati u kontekstu prethodno navedenih ispitivanja1, 2, 3. Ni u jednoj grani patologije neuromišićni patolog ne pokazuje toliku sklonost da podržava uputnu kliničku dijagnozu. Prilikom analiziranja neuromišićne biopsije neuromišićni patolog uporno i neprekidno traga za morfološkim detaljima koji bi potvrdili uputnu dijagnozu4. U tom dugotrajnom procesu, nizu ponovljenih bojenja i velikom broju preseka on odustaje i počinje da traži alternativne morfološke pokazatelje tek kada je duboko uveren da nema patognomoničnih promena za predloženu dijagnozu. Takođe, neuropatolog pri analizi neuromišićne biopsije, pokazuje duboko nepoverenje u morfološke promene koje vidi u kontinuirano ih upoređuje sa naučnim usvojenim standardima i sopstvenim kontrolnim bojenjima. Mišićne biopsije su još, daleke 1860. godine postale oruđe u dijagnostici neuromišićnih bolesti kada je Duchenne izveo prvu biopsiju kod pacijenta sa simptomima miopatije 5. Uvođenje enzimohistohemijskih metoda od strane Victora Dubowitz 1970 –tih godina je napravilo revolucionarni napredak u dijagnostici različitih primarnih i sekundarnih bolesti mišića3, 5. 564 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 novine u patologiji/NEWS IN PATHOLOGY Novi progress nastaje 90-tih godina dvadesetog veka sa uvođenjem imunohistohemije, a početak 21 veka je obeležen spektakularnim progresom molekularnih metoda. Terapija neuromuskularnih bolesti takođe pokazuje snažan napredak sa uvođenjem genetskog terapijskog pristupa. Napredak postignut u proteklih 25 godina je omogućio otkrivanje novih uzročnika genetskih defekata sa mnogo novih proteina uključenih u neuromišićne bolesti (ažurirani spisak MDS i odgovornih gena mogu se naći na http://vvv.musclegenetable.org). Danas, mišićna biopsija uz primenu svih savremenih metoda obrade predstavlja zlatni standard u dijagnostici neuromišićnih bolesti6,3. Neuromuskularne bolesti (NMB) se javljaju kod ljudi svih uzrasta od rođenja, preko adolescentnog i odraslog doba do duboke starosti. Mogu biti u vidu blagih formi, ali i u vidu teških formi sa smrtnim ishodom. Ne postoji zemlja ni region na svetu u kome se ove bolesti ne javljaju. U opštoj populaciji incidenca iznosi 1 na 1000 novorođenčadi, muskularne distrofije javljaju sa incidencom 1 na 2000 novorođene dece a, posebno teška, ali i najčešća Duchenne-ova muskularna distrofija je sa incidence 1 na 3.500 muške novorođene dece7. Veliki, vekovni, problem pacijenat sa NMB je njihova kulturalna i socijalna marginalizacija u većini zemalja sveta, a posebno u nerazvijenim zemljama i zemljama u razvoju. Pored navedenog problema, dodatni problem zemalja u razvoju i nerazvijenih zemalja je nepostojanje adekvatne dijagnostike i formiranje nacionalnih baza podataka koje bi omogućile uvid u distribuciju i familijarne rizike oboljevanja. U Republici Srbiji je 2009.god. u Službi kliničke patologije Kliničko bolničkog centra Zemun, a po odluci Ministarstva zdravlja Republike Srbije, počela rutinska dijagnostika NMB na biopsijskim uzorcima mišiča, nerava i kože. Većina pacijenata su pacijenti iz pet centara: Klinike za neurologiju Kliničkog centra Srbije, Instituta za dečiju neurologiju i psihijatriju, Vojnomedicinske akademije Beograd, Univerzitetske dečije klinike Tiršova Beograd i Kliničkog centra Vojvodine. Takođe, sporadično se upućuju pacijenti iz drugih ustanova u Srbiji, susednih država, ali i iz inostranstva. U cilju formiranja Nacionalne baze za mišićne distrofije Ministrstvo nauke je odobrilo petogodišnji naučni projekat pod nazivom “Ispitivanje molekularno genetskih, patohistoloških i biohemijskih karakteristika neuromišićnih bolesti”8 (br. 175083). U ovom radu prikazujemo naše iskustvo sa 162 konsekutivne neuromišićne biopsije analizirane u period od 2009 do 2012 godine. Materijal i metode Za protekle tri godine je analizirano 162 neuro-mišična biopsijska uzorka dobijena procedurom otvorene hirurške biopsije4. Kriterijumi na osnovu kojih su pacijenti uključeni u studiju su: a) da su bili upućeni iz primarnih i sekundarnih zdravstvenih institucija u referentne centre za adultnu i dečiju neurologiju b) da su detaljno, svim raspoloživim metodama pregledani od strane neurologa u referentnim centrima c) da su biopsije otvorenim putem uzete od strane posebno edukovanih hirurga u Kliničko bolničkom centru Zemun za adultne pacijente, a za dečiji uzrast u Univerzitetskoj dečijoj klinici Tiršova c) da su sveži reprezentativno uzeti i pripremljeni biopsijski uzorci u vremenskom period ne dužem od 30 minuta dopremljeni u Službu kliničke patologije KBC Zemun d) Pojedinačni pacijenti, sporadično upućeni iz drugih ustanova ili parafinski blokovi doneti na konsultaciju nisu uključeni u ovu studiju (ukupno 32). Uzorci biopsija mišića i nerava su obrađeni u Službi kliničke patologije KBC Zemun Beograd. Pravilnom orjentacijom uzorka pod stereomikroskopom Nikon SMZ1500 i brzim smrzavanje uzorka u tečnom azotu (ili izopentanu ohlađenom u tečnom azotu) odmah po dostavljanju uzorka iz hirurške sale smo započinjali proces obrade. Pripremano je 20-30 smrznutih preseka na super frost pločicama za histohemiju, enzimohistohemiju ili imunohistohemiju. Obrada se nastavlja procesiranjem uzorka kroz formalinsku fiksaciju do parafinskog kalup, a deo tkiva za elektronsku mikroskopiju smo procesirati kroz 4% glutaraldehid. Rutinski smo preseke neuromišićnih biopsija bojili hematoksilin eozin (H&E) i modifikovano Gomori trichrom bojenja na osnovu koji smo procenjivali: arhitekturu mišićnih vlakana (oblik, veličinu, atrofiju i hipertrofiju, poziciju jedara) 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 565 novine u patologiji/NEWS IN PATHOLOGY inflamatorne infiltrate intersticijum i krvne sudove patognomonične histološke promene za mitohondrijalnie miopatija, nemalinske miopatije, tubularnih agregata i “rimmed” vakuole Smrznuti preseci tkiva su obrađivani histohemijskim i enzimohistohemijskim metodama bojenja na osnovu standardno preporučenih paleta3 (Tabela 1.) Spectrin Dystrophin (N term) Dystrophin (rod domain) Dystrophin (C-term) Utrophin nNOS Myotilin Lamin A/C Dysferlin Ham1 Dysferlin Ham2 a-Sarcoglycan b-Sarcoglycan g-Sarcoglycan δ-Sarcoglycan b-Dystroglycan Collagen VI Emerin Desmin MHC class I Fast Myosin Slow Myosin Develpmental Myosin Neonatal Myosin g-Sarcoglycan Tabela 1. Lista sadrži deo palete antitela za otkrivanje primarnih ili sekundarnih oštećenja, kao i za procenu kvaliteta i očuvanja uzoraka Imunohistohemijsko bojenje je rađeno automatski u imunostejneru Labvision sa automatskim predtretmanom u PT modulu. Svi uzorci mišićnih biopsija su rutinski bojeni na izoforme miozina Tip 1 (slow), Tip 2 (fast) i neonatalni miozina (Slika ). Kompjuterskim softverom LAS V37 programom je merena veličina vlakana kod svakog uzorka na 100 preseka (50 obojenih brzim miozinom, 50 vlakana obojenih sporim miozinom) po najužem prečniku. Očuvanost mišićne sarkoleme je procenjivana imunohistohemijskim bojenjima ili na laminin α2 ili collagen VI. Za procenu statusa mnogih protein kod različitih miopatija je bila korišćena široka paleta komercijalnih antitela dostupnih na tržištu (Tabela 1.). Kompjuterska baza analiziranih uzoraka je formirana za period 2009-2012 godine. Patohistološki izveštaj je sadržao detaljnu analizu svih parametara sa reprezentativnim fotografijama, dijagnozom ukoliko je bilo moguće postaviti, ili predlogom za eventualna dodatana imunohistohemijska, molekularna ili elektronsko mikroskopska ispitivanja. Rezultati Analizirano je ukupno 162 uzorka neuromišićne biopsije koje su upućene iz referentnih centara i 31 sporadične biopsije (Dijagram 1.). Dijagram 1. Broj pacijenata po godinama koji su upućeni iz referentnih centara 566 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 novine u patologiji/NEWS IN PATHOLOGY Od ukupnog broja pacijeneta 34 (21,11%) su bili pacijenti starosti do 10.godina, a najveći broj pacijenata je bio u starosnim grupama posle 30 godine (Dijagram 2. ) Dijagram 2. Starosna struktura pacijenata U odnosu na regione republike Srbije nije postojala posebna učestalost pacijenata iz nekog dela Srbije, i uglavnom su bili pacijenti iz centralnih delova Srbije. (Dijagram 3.). Dijagram 3. Distribucija pacijenata prema regionima Srbije U odnosu na grupu bolesti najviše dijagnostikovanuh oboljenja je bilo iz grupe miopatija i miozitisa, a znatan broj je bio i pacijenata sa neurogenim lezijama mišića (Dijagram 4.) Dijagram 4. Broj pacijenata u odnosu na tip bolesti 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 567 novine u patologiji/NEWS IN PATHOLOGY Prilikom analize biopsija postavljene su precizne dijagnoze za mnoge bolest, ali je jedan broj sadržavao samo opisnu formu uočenih abnormalnosti sa preporukama za dalja uglavnom molekularno genetska ispitivanja. Na HE obojenim preparatima su utvrđivane grupe promene arhitekture ali su postavljene i neke decidne dijagnoze kao npr. (Slika 1.): polyglucosan body disease (Slika 1a.), granulomatozni miozitis (Slika 1b,) ili Inclusion Body Myositis (Slika 1c.). Slika 1. Patognomonične histološke promene za određene bolesti mišića i nerva Takođe, pojedine promene ukazuju na određene proteinske poremećaje koji se mogu potvrditi imunohistohemijskim metodama, kao što je utvrđeno u slučaju dezmonopatije (Slika 2.) Slika 2. Vakuolarna izmenjenost vlakana sa iregularnom distribucijom dezmina u slučaju dezminopatije 568 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 novine u patologiji/NEWS IN PATHOLOGY Primenjena enzimohistohemijska bojenja su omogućila analizu intermiofibrilarne mreže koju grade mitohondrije i sarkoplazmatični retikulum. Abnormalnosti unutrašnje fibrilarne arhitekture su veoma važni pokazatelji mnogih mišićnih bolesti i zahvaljujući ovim bojenjima smo utvrdili postojanje ciljnih tzv.”target” vlakana taman, svetao ili prazan centar bez bojenja mitohondrij i pojavu tzv. “lobuliranih” vlakana je opšta karakteristika ali je česta kod LGMD 2A, Ullrich Congenital Muscular Dystrophy, Bethlem myopathyi sl. (Slika 3). Slika 3. Abnormalnosti unutrašnje fibrilarne arhitekture mišićaa. Abnormalnosti u bojenju čistih mitohondrijalnih enzima, kao na primer subsarkolemalnaa akumulacija succinic dehydrogenase (SDHkoje smo utvrdili u slučajevima dijagnostikovanih mitohondrijalnih miopatija (Slika 3c). Modifikovano Gomori trichrom bojenje (Engel trichrome uvedeno od strane W. King Engel 1963.god.) je omogućilo dijagnostiku patognomoničnih abnormalnosti kod: mitohondrijalnih miopatija, nemalinske miopatije, tubularnih agregata i “rimmed” vakuola (Slika 4.). Slika 4. Patognomonične abnormalnosti kod mitohondrijalne i nemalinske miopatije 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 569 novine u patologiji/NEWS IN PATHOLOGY Ova metoda bojenja je bila veoma uspešna kod nervnih biopsija za procenu različitih patoloških stanja, a posebno demijelinizacija (Slika 5.), Slika 5 . Uspešnost Modifikovanog Gomori Trichrome bojenja na biosijskim uzorcima nerva U laboratoriji smo rutinski koristili na parafinskim presecima imunohistohemijsko bojenje na brzi miozin Tip 2 (Myosin Heavy Chain, Fast, Clone Wb-MHCf) a, na kriostatskim presecima naspori T2 miozin (Myosin Heavy Chain, Slow, Clone WB-MHCs) (Slika 6.). Pored dva osnovna tipa smo posebno u proceni kongenitalnih miopatija i utvrđivanju da li su atrofična vlakna regeneratorna ili stvarno atrofilčna primeniti i drufe izoforme miozina: neonatalni i razvojni. (Slika 6.). Slika 6. Ekspresija različitih izoformi miozina. Za neke distrofija (udno pojasnih i fascioscapulohumeralnih) su detektovani proteinski defekti i moguće ih je utvrditi imunohistohemijski što smo primenjivali u dijagnostici korišćenjem svih raspoloživih klonova (Slika 7 i 8). 570 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 novine u patologiji/NEWS IN PATHOLOGY Slika 7. Detektovani proteinski defekti7 Slika 8. Detektovani proteinski defekti Diskusija Skeletni mišić je najveći organ u telu i posebno je teško reprezentativno odrediti mesto biopsije. Biceps, deltoideus i kvadriceps su najčešće birani mišići, ali je uvek optimalno izabrati umereno zahvaćen mišić. Druga važna tačka uspešne dijagnosstike je reeprezentativno uzorkovanje i sledstvene laboratorijske metode obrade tkiva koje mogu imati značajan uticaj na rezultat biopsije. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 571 novine u patologiji/NEWS IN PATHOLOGY Zlatni standard u dijagnostici neuromuskularnih bolesti je histološka analiza. U novije vreme je dijagnostika znatno unapređena metodama imunohistohemije, pre svega u sferi utvrđivanja tipa vlakana i potiskuje 50 godina korišćenu tehniku ATP-aza koja je veoma zahtevna i kapriciozna 8,9, 10. Drugi veoma važan doprinos imunohistohemije je mogućnost utrđivanja statusa pojedinih specifičnih proteinskih defekata što vodi ili decidnom postavljanju dijagnoze ili usmeravanju molekularno genetske analize. Utvrđivanje populacione distribucije pojedinih naslednih bolesti i familija u kojima se one javljaju predstavlja glavni zadatak ozbiljnog vođenja brige o zdravlju ljudi jedne zemlje. No, u nerazvijenim zemljama i zemljama u razvoju finansijska nestabilnost i nedovoljna opremljenost laboratorija u tehničkom i kadrovskom smislu usporava taj process. Čak, i u mnogim razvijenim zemljama process stvaranja nacionalnih baza je predstavljao problem i rešavan je višedecenijski. Walton i Nattrass12 su 1954 godine publikovali originalan rad u kojem su opisali 105 slučajeva od Northumberland do Durham u Severnoj Engleskoj. Oni su tada i predložili novu klasifikaciju mišićnih bolesti baziranu na detaljnoj kliničkoj opservaciji. Posle 50 godina, urađena je detaljna populaciona studija12 sa genetskim analizama u istom region na 1100 pacijenata kod koji su utvrđivane molekularne karakteristike za 31 entitet. Ukupno 75.7% pacijenata je svrstano u taj 31 entitet. Navedena studija takođe ilustruje ogroman dijagnostički napredak od prvog regionalnog istraživanja pre više od 50 godina. Literatura 1. Anderson JR. Recommendations for the biopsy procedure and assessment of skeletal muscle biopsies. Virchows Arch 1997;431(4):227-33. 2. Edwards R, Young A, Wiles M. Needle biopsy of skeletal muscle in the diagnosis of myopathy and the clinical study of muscle function and repair. N Engl J Med 1980;302(5):261-71. 3. Dubowitz V, Sewry C A. Muscle Biopsy: A Practical Approach. 3rd ed ed. China: Saunders Elsevier; 2007. 4. Milenkovic S, Rakočevic-Stojanovic V. Biopsije mišića: Zašto, ko i kako? Zbornik sažetaka II kongres patologa Bosne i Hercegovine sa međunaarodnim učešćem, Banja Luka 2012, str.215-227 (predavanje po pozivu) 5. Sundaram C, Uppin SM. Approach to the Interpretation of Muscle Biopsy. In Muscle biopsy. Ed. Sundaram C. Croatia, InTech 2011:15-33. 6. Rakocević-Stojanović V. Mišićne distrofije. Medicinski fakultet Beograd, I izd., CIBID, 2011, Beograd 7. Nešić S, Andrić N, Jovanović M, Milenković S, Aleksić Kovačević S. Heritable myopathy in a Labrador retriever. Journal of Comparative pathology 2012;146(1):74 (Meeting Abstract) 8. Padykula HA, Herman E. The specificity of the histochemical method for adenosine triphosphatase. J Histochem Cytochem 1955;3:170–95. 9. Padykula HA, Herman E. Factors affecting the activity of adenosine triphosphatase and other phosphates as measured by histochemical techniques. J Histochem Cytochem 1955;3:161–9. 10.Brooke MH, Kaiser KK. Some comments on the histochemical characterisation of muscle adenosine triphosphates. J Histochem Cytochem 1961;17:431–2. 11.Meola G, Bugiardini E, Cardani R. Muscle biopsy.J Neurol. 2012;259(4):601-10. 12.Fiona L. M. Norwood FLM, Harling C, Chinnery FP, Eagle M, Bushby K, Straub V. Prevalence of genetic muscle isease in NorthernEngland: in-depth analysis of a muscle clinic population. Brain 2009: 132; 3175–3186 572 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 Uvodno predavanje 3/Keynote Lecture 3 Cervical cytology and histology in the context of a screening programme Sanjiv Manek Gynaecological Pathologist Oxford UK In the UK, the national cervical cancer screening programme is probably the most established, robust and rigorous of all screening programmes that has evolved over 25 years. It has seen a multitude of changes based on evidence obtained from a vast amount of research and observations, as well as from advances in technology. This success has led to a definite impact on cervical cancer incidence with reduced mortality and morbidity related to cervical cancer. More recently, the changes introduced to the programme have been exponential and in the UK we are at the brink of a new direction and a new style of provision of the screening programme. Throughout the years, there has been a continued effort to understand morphological aspects of the cytology and histology in cervical neoplasia and many new entities have been recognised or variations of known entities realised. The intricate measures to ensure quality in the screening programme have led to close working relationships between cytologists, histopathologists, colposcopists, and now, molecular biologists. The UK cervical cancer screening programme (CCSP) is likely to change significantly in the next few years, and although reduced in quantity, cytology will still remain an important aspect of it. The importance of histopathology is unlikely to change, but more onus will be placed on the molecular biologists and colposcopists. In order to discuss and understand the importance of cytology and histology in the cervical screening programme and the absolute requirement of correlation between the two, it is important first to appreciate how the UK CCSP has evolved and how it has taken with it the evolution of new technologies in cytology leading to improved correlation between histology and cytology and quality assurance with obvious benefits to the patient. Milestones in the UK CCSP 1987/8 - Launch of formal programme. 1989 - Introduction of 3-tier system of reporting (Code 7 – moderate dyskaryosis). 1994 - (Code 8). Formal introduction and definition of the borderline category 1996 - Formal introduction of review of screened slides. 1998 - Introduction of quality assurance measures including external quality assurance tests. 2000 -Achievable Benchmark Criteria (ABC) I. 2002 - Terminology refinement. 2003 -Achievable Benchmark Criteria (ABC) II 2005 - Introduction of the invasive cancer audit 2006 - Changes in age ranges for screening and follow-up protocols. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 573 Uvodno predavanje 3/Keynote Lecture 3 2007 - Liquid-based cytology (± HPV vaccine). 2011 - Targets for quick turnaround. 2012 - Introduction of HPV testing as triage and test of cure Future - Networks, automation in screening and HPV testing as a primary screening tool. Of these milestones, the major ones have been: 1) the ABC I and II documents which have introduced not only the finer details of morphology in cervical cytology and histology, but also guidelines for reporting within certain parameters; 2) the terminology refinement; 3) the change in screening age ranges; 4) LBC; and 5) HPV testing. Each time such changes were introduced, there were changes in reporting practice also seen and considered necessary and the management of patients better defined and tailored. As a result, there has been a vast amount of literature on morphology in conventional cytology and LBC, as well as literature on outcomes. Cytology The main categories of reporting currently are: Unsatisfactory Negative Mild dyskaryosis Severe dyskaryosis Severe dyskaryosis ?invasive Glandular dyskaryosis Moderate dyskaryosis Borderline changes – NOS Warty ?High grade Glandular These categories were very well defined in conventional cytology and for many, particularly for severe dyskaryosis, many patterns have been recognised through careful observation and regular audits. These categories are equally reproducible in LBC and, in fact, are somewhat easier to diagnose. With the advent of HPV testing, some of these categories will be refined in due course. Histology The UK NHS CSP national office has published multiple documents on guidelines and amongst these, there are two on the histopathology of cervical lesions. The main categories to recognise are: HPV changes Cervical Intraepithelial Neoplasia (CIN) 1, 2 and 3 CIN 3 with crypt involvement Low and high grade cervical glandular intraepithelial neoplasia (CGIN) Early stromal invasion >Stage 1A2 carcinomas and all the subtypes Stratified mucinous intraepithelial lesion (SMILE) Tubo-endometrioid metaplasia 574 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 Uvodno predavanje 3/Keynote Lecture 3 In the UK, the retention of the 3-tier system of reporting in cytology and histology has allowed detailed correlation between the two. In everyday practice, correlation plays a very important role in providing the best management to a patient and it helps to enhance failsafe procedures and to improve quality assurance measures. Correlation in cervical pathology Much of cervical biopsy reporting is dependent on correlation with referral or recent cytology. An adequate request form should state the expected degree of abnormality by mentioning the referral cytology findings. This can be accompanied by the colposcopic impression which is useful but less important where correlation is concerned. If clinical details are inadequate or variable, it is good practice to have the past history readily available at the time of reporting both histology and cytology. This allows appropriate and safe prospective reporting. The typical cervical histology report should have a comment on correlation, and where possible, all non-correlation cases should be worked up prospectively prior to issuing the final report. This would involve examining multiple levels of the biopsy, immunohistochemistry (p16, bcl2, etc) and reviewing the cytology. If the cytology is confirmed, then the appropriate management could be advised in the histology report and this includes repeat colposcopy and biopsy. Where relevant, cases should be discussed at dedicated colposcopy MDT meetings. The review of the cytology could be used as a learning exercise when there is overcall and undercall of abnormalities. Examining levels could mean cutting through the block if necessary, particularly for high grade lesions. If the cytology is high grade and confirmed by more than one person, and the subsequent histology is repeatedly lower grade or negative, follow up should be according to cytology in case of vaginal or high grade canal disease. Problems arise when cytology and histology are divorced and correlation exercises are carried out retrospectively, often with significant delays. Patients may slip through failsafe, which can lead to delayed diagnosis. The commonest situation of non-correlation is where the cytology has been reported as high grade dyskaryosis and the subsequent biopsy is negative or only low grade. If levels do not reveal any correlating lesions, the possible reasons to consider are high canal disease, vault disease, tiny lesions and inadequate colposcopy. In a significant proportion of cases, the expected lesion is discovered in subsequent biopsies. Sometimes a loop excision is performed as treatment after a biopsy diagnosis of a high grade lesion and there is no correlating lesion. Once again, the possibility of a tiny lesion (treated by the biopsy) needs to be considered if multiple levels still do not reveal the expected histology. Undercall in cytology is usually due to misinterpretation of crowded groups or missing single dyskaryotic cells that may be small or keratinised, dark or pale. Occasionally immature metaplastic cells can be overlooked. Overcall in cytology is usually due to mistaking immature or reactive metaplastic cells as high grade dyskaryosis and crowded groups as microbiopsies of CIN 3. Correlation with cytology reported as borderline is interesting. Borderline NOS or borderline reactive usually correlates with inflammatory changes or low grade CIN. Occasionally, a high grade lesion is seen and review of the cytology may show high grade dyskaryotic features. Borderline glandular changes may yield reactive features or CGIN on biopsy. It is a matter of experience and constant review of such cases to gain the confidence to report the cytology as either negative or glandular dyskaryosis. When LBC was introduced, there was a high incidence of reporting borderline ?high grade dyskaryosis. Most of these turn out to be CIN 2/3 on histology. With time and with learning from consistent reviews, most of these can be reclassified as high grade dyskaryosis. With HPV testing, a new type of correlation challenge is being encountered. What is to be expected when the cytology is negative and high risk HPV testing is positive? Many biopsies are negative (correlate with cytology) but some are high grade. Review of cytology in such high grade cases tends to show negative features in most and abnormalities in only a few. In negative biopsy cases, follow up would be return to normal recall unless the colposcopist decides otherwise. The other conundrum that has emerged is where the first cytology 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 575 Uvodno predavanje 3/Keynote Lecture 3 after a treatment is negative but HPV testing is positive rendering it necessary to colposcope. In many such cases, the colposcopy is also negative. The type of further management is then decided by the colposcopist. One more aspect of correlation in cervical pathology is in cervical cancer staging. Beyond stage 1A2 tumours, which depend on measurements, the more reliable modality for staging is imaging (MRI) and clinical, partly because of the three dimensional nature of lesions. There are a significant number of cases where staging by pathology is much lower than that shown by imaging; hence, final staging has to be left to multidisciplinary team meetings. Litigations Over the years, there have been numerous litigation cases in cervical cancer development. Some are related to surgical issues but most are for possible errors in cytology reporting and occasionally for histology reporting. In the UK there have been famous instances of litigation and these have led to some of the changes in the CCSP mentioned above. In particular, the invasive cancer audit was introduced as an educational tool to understand why cancers develop despite women adhering to the screening programme. One has to appreciate that with human interpretation there will be a small percentage of false negative cytology but what is really important in this situation is to know what leads to false negative cytology. The audit is a very useful tool for this and with its use, the UK cytopathologists have seen some common patterns and variations in morphology accountable for false negative reporting. These include the categories of scanty dyskaryosis, small keratinised cells, small cell (dark) dyskaryosis, pale dyskaryosis and microbiopsies. The ‘litigation’ cell is often the small dyskaryotic cell! The future There will be a reduction in the amount of cytology required as a result of HPV testing, to the point where HPV testing may become the primary screening tool and cytology, the triage test. Much of the cytology screening will be automated, leaving only a small amount for human interpretation. All the processing and screening is likely to be carried out in hyper-laboratories. There will be changes in correlation processes because histology will need to be matched with the HPV test result rather than with the cytology in low grade lesions. Despite all the major overhauls, the UK CCSP is likely to remain cost-effective with major benefits which, when the effects of the HPV vaccine are felt, should see a dramatic reduction in the cervical cancer incidence in the UK whilst avoiding major surgical interventions and hence other complications. References 1. www.cancerscreening.nhs.uk/cervical 2. www.britishcytology.org.uk 576 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 Uvodno predavanje 4/Keynote Lecture 4 Prophylaxis and Early Detection of HPV-Related Neoplasia Herbert Pfister, Cologne, Editor Monographs in Virology. S. Karger Detection of HPV DNA and RNA Hans Ikenberg, Germany Abstract The balance between analytical (low) and clinical (high) sensitivity is crucial for the specificity of a routine HPV test as limited specificity will be harmful due to unnecessary treatment of healthy women. Up to now HPV diagnostics is mainly based on DNA detection for which target and signal amplification methods are available. PCR techniques can be divided into type-specific and consensus PCRs. Due to its high clinical sensitivity and its relatively high specificity the HC2 test is still regarded as the gold standard in routine HPV testing. It hybridizes 13 (near) full-length stabilized RNA probes of high-risk HPV types to denatured target DNA followed by detection via antibodies and chemiluminescence. To avoid costly validation studies for each new HPV test standards for evaluation have been defined. Recently several new HPV detection assays have been commercialized. They all show promising data in first published studies but still await full validation according to the criteria mentioned above. Among them only the cobas HPV test has already been fully validated for use in triage and as adjunct to cytology. HPV 16 and 18 confer a much higher risk for development of a CIN 2+ compared to the other HPV high-risk types. It is therefore appropriate to test for these HPV types independently. Apart from that testing for individual HPV types is of very limited clinical value up to now.HPV RNA testing is a promising option with potentially higher specificity. As a first system, the APTIMA test has now received an FDA approval. Background While cytology fails to detect prevalent CIN 2+ in a first approach in half of the cases (McCrory D 1999) molecular HPV diagnostics is able to achieve this goal in up to 99%. Unlike morphology it also predicts the risk of developing CIN 2+. Otherwise the presence of HPV DNA often is transient if not even contamination (Schiffman, Wentzensen et al. 2011). Therefore standardized methods of HPV detection are of utmost importance to achieve clinically relevant results. Except in some rare conditions only HPV high-risk testing is regarded of clinical value. In this chapter HPV testing means identification of HPV high-risk types which are defined by their association with cancer. In the future HPV diagnostics may become helpful also for diagnostics of oral, laryngeal and pharyngeal (D’Souza, Kreimer et al. 2007) as well as anal (Frisch, Glimelius et al. 1997) neoplasias. To date it is clinically almost solely used in cervical lesions. Today HPV diagnostics is a routine tool in three areas. First, triage of cytologically borderline (Wright, Cox et al. 2002) or low grade (Wright, Massad et al. 2007) abnormalities. Second, as a test of cure after therapy of CIN (Zielinski, Bais et al. 2004).Third, HPV testing as an adjunct to cytology is recommended in major guidelines (Saslow, Runowicz et al. 2002) and meanwhile the replacement of cytology as primary screening method by HPV is intensively discussed (Katki, Kinney et al. 2011). The detection of HPV is not always indicative of the disease resulting from the infection. A certain amount of virus has to be present for a certain time in order to induce cervical neoplasia (Josefsson, Magnusson et al. 2000; Snijders, van den Brule et al. 2003). Hence transient infections with HPV and low amounts of virus are clinically irrelevant. Routine HPV testing requires a clinically defined cut-off value of a detection system to 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 577 Uvodno predavanje 4/Keynote Lecture 4 avoid HPV positivities which compromise specificity and cause unnecessary further diagnostic procedures and treatments. It is rare that a 12 year old diagnostic test like HC2 is regarded as gold-standard and the reference of a major guideline. The fact that this holds in clinical HPV testing points to a special situation with distinct technical requirements not easy to meet. Methods of HPV detection Although in-situ-hybridization is able to locate the viral DNA its low sensitivity and lacking ability for highthroughput preclude a routine use (Hesselink, van den Brule et al. 2004). Any routine diagnostic use of HPV protein detection is excluded by the fact that the HPV oncogenes E6 and E7 are expressed in very differential levels and no validated tests for their detection are available. The inconsistent seroconversion rate even in persons with high grade cervical lesions precludes any routine serology tests (Wang, Schiffman et al. 2003). Therefore only HPV DNA and (to a lesser extent) RNA assays are currently available for clinical applications. Because HPV DNA is present in productive as well as in transforming infections but also potentially as a contamination its detection can primarily not inform about the clinical importance of this finding. Technically for HPV DNA testing target and signal amplification methods are available. PCR techniques can be divided into type-specific and consensus (general primer) PCRs. The products of consensus PCRs can also be used for genotyping, eg by hybridizing them to multiple HPV types fixed on a membrane strip (Linear Array) or to a DNA chip (Papillocheck). PCR Several PCRs for HPV detection are commercially available. The most widely distributed is meanwhile the cobas assay, a realtime consensus PCR which covers 14 HPV high-risk types and additionally differentiates HPV 16 and 18 (Roche Molecular Diagnostics, Pleasanton, CA; see below). It replaced the Amplicor test which never met all criteria for routine use. The linear array assay is a type-specific method detecting 37 high- and low-risk types (Roche) while the SPF10 assay (Kleter, van Doorn et al. 1999) (Innogenetics, Gent, Belgium), the most sensitive HPV assay available, identifies 28 types. The most common PCR techniques only available for scientific applications are the GP5+/6+ (Jacobs, Snijders et al. 1997) and the PGMY (Gravitt, Peyton et al. 2000) general primer sets. An interesting multiplex variant of GP5+/6+ with Luminex detection with more even coverage of up to 100 different HPV types has been recently presented (Schmitt, Bravo et al. 2006). All these tests target the L1 region of the HPV genome with resulting amplicons from 65 to 450 bp length. Enzyme-immuno assays or reverse line blot assays are used as detection systems. The analytical sensitivity varies from less than 10 copies with SPF10 primers with purified DNA to around 1000 copies with the GP5+/ 6+ primers in crude extracts (Snijders, van den Brule et al. 2003). Numerous commercial labs have made up their own PCRs which are at best internally validated, generally have not been investigated in scientific studies and are therefore less suitable for clinical use. The analytical sensitivity of PCR can go down to some copies of HPV DNA. Surprisingly that is not reflected in a similar clinical sensitivity. Only a few larger studies showed an ability to identify more than 95% of CIN 2+ lesions and the median sensitivity of HPV PCR was reported to be 82% in 16 studies (Lorincz and Smith 2006). This can be explained by several factors. Primers usually targeting the L1 region of the HPV genome may lack to bind in up to 7% of high grade lesions and cancers that have lost this region (Karlsen, Kalantari et al. 1996). Other reasons are random partial inhibition (which is not necessarily indicated by internal controls) and competition effects if several HPV types are present (Qu, Jiang et al. 1997). One challenge with consensus HPV PCRs is to obtain an equal and regular level of detection of different HPV types as shown by the range of sensitivity of the GP5+/6+ assay depending on the HPV type (Jacobs, Snijders et al. 1997). Another challenge is the adjustment of a reliable cut-off, which is even a major point in ultra-sensitive type-specific PCRs. 578 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 Uvodno predavanje 4/Keynote Lecture 4 Signal amplification Two signal amplification technologies (HC2 test, Qiagen, Gaithersburg, MD; Cervista, Hologic, Bedford, MA) are on the market. The HC2 test hybridizes 13 (near) full-length stabilized RNA probes of high-risk HPV types to denatured HPV target DNA followed by detection via antibodies and chemiluminescence. The threshold value of the system is 1 pg/ml which corresponds to around 5.000 copies of HPV DNA per test (Lorincz and Smith 2006). It is very robust in the pre-analytical phase, has a simple sample preparation and requires no separate rooms for different work steps. Up to now only partial automation is available. The inter-laboratory variation of the HC2 test is very low and its results are highly reproducible (Castle, Wheeler et al. 2004). Respective data on PCR are less consistent (Quint, Pagliusi et al. 2006). One theoretical drawback of the HC2 test is the lack of a control for the presence of human cellular material. In routine application this seems to be of minor importance as it shows a sensitivity of up to 99% for CIN 3+ measured on a histology gold standard (Bohmer, van den Brule et al. 2003) (Castle, Cox et al. 2008) and the low rate of specimens (~1%) without amplification of a house-keeping gene in major studies (Castle, Gutierrez et al. 2011; Youens, Hosler et al. 2011). Additionally risk estimates were hardly affected by adjusting for the amount of a housekeeping gene in a PCR system (van Duin, Snijders et al. 2002). Another potential problem may be a cross reaction with a couple of low-risk HPV types which was determined to be 7.8% of all positive HC2 results (Castle, Solomon et al. 2008). Because some of them are regarded in fact as being of intermediate risk, this may even contribute to the high clinical sensitivity of the test (Castle, Solomon et al. 2008). While the HC2 test out of the vial of the ThinPrep liquidbased cytology has been approved by the FDA there are hints that the performance of the system under these conditions (which requires an elaborate transformation process) is somewhat poorer than from the standard medium (Carozzi, Del Mistro et al. 2005). The clinical sensitivity of the HC2 test is very high, in 26 trials worldwide its median was 94% (Lorincz and Smith 2006). More than 200.000 women have been included in screening studies with this assay published in peer reviewed journals. This figure exceeds by far the data available for any single PCR method. Significant differences in performance were observed between HPV screening studies conducted in Europe and Northern America reaching up to 99% sensitivity compared to investigations in developing countries. Here regularly the sensitivity was 20-30% lower which might be explained by weaknesses of regional colposcopy and histology leading to deficits in gold standard definition (Sankaranarayanan, Chatterji et al. 2004). Though in “real life” the clinical sensitivity even under optimized conditions will not exceed 95-98% due to sampling errors or minor technical variations (Lorincz and Smith 2006). Qiagen developed careHPV, a basic version of HC2 without need for running water and main electricity with a turn-around time of only 2.5 hours. That allows further diagnostic or therapeutic procedures in the same visit. A study in China showed 90% sensitivity for CIN 2+ (Qiao, Sellors et al. 2008). Further trials are ongoing and a broad roll-out in developing countries is intended. A second signal amplification test (Cervista, Hologic, Bedford, MA) gained FDA approval in 2009. It uses two isothermal reactions with a proprietary technology (Invader) which detects 14 high-risk HPV types as a group and in addition HPV 16 and 18 together. First data from the package insert pointed to a much lower specificity than eg of HC2 and were negatively discussed (Kinney, Stoler et al. 2010). Newly published data showed a similar sensitivity and specificity as with HC2 in a NILM (Quigley, Potter et al. 2011); (Youens, Hosler et al. 2011) and ASC-US population (Einstein, Martens et al. 2010; Quigley, Potter et al. 2011). The central role of specificity The major problem of routine HPV diagnostics is its relatively low specificity which is limited even with non-DNA-amplifying methods. This restricts its use in a screening approach to women over 30 years (Saslow, Runowicz et al. 2002). Below this age testing is regarded as useful in case of cytological and/or colposcopic abnormality (triage) and after therapy of CIN as a test of cure (Wright, Cox et al. 2002). But even in this triage setting the revised ASCCP guidelines in the US discourage HPV testing in the below 20s due to very 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 579 Uvodno predavanje 4/Keynote Lecture 4 high remission rates in this age group (Wright, Massad et al. 2007). Currently HPV testing is regarded more useful i.e. predictive the older the tested women are. If however the appropriate test is used in the appropriate age group screening by HPV DNA testing is able to reach the specificity of cytology as demonstrated by 5 of 26 studies reviewed in a HTA (Mittendorf, Nocon et al. 2007). The specificity can be increased by raising the viral load cut-off. This can be achieved more easily with signal-amplifying methods than with PCR. Studies here show a slightly lower (Lorincz and Smith 2006) or unchanged (Guyot, Karim et al. 2003; Hesselink, Bulkmans et al. 2006) sensitivity, while in either case the specificity was raised. A recent systematic review underlined these findings (Rebolj, Bonde et al. 2011). Even though the importance of a minimal viral load for the development of HPV induced disease is obvious there seems to be no clear-cut association between quantitative (real-time PCR) or semiquantitative (HC2) measurements of viral load and prognostic potential at a level above the detection limit of the HC2 test (Bory, Cucherousset et al. 2002; Lorincz, Castle et al. 2002; Snijders, van den Brule et al. 2003) A trade-off between sensitivity and specificity also limits an extension of the number of types covered by clinical HPV tests. The inclusion of very rarely occurring types beyond the 13-14 included in most tests increases sensitivity minimally, while leading to an unproportional decrease in specificity (Schiffman, Khan et al. 2005). The main issue when using HPV testing as an adjunct to cytological screening is the rate of women positive for high-risk HPV in the absence of cytological abnormalities. This rate is usually very low for women of 30 years and older. Among more than 800.000 tests in nearly 600.000 women in California tested with the HC2 system the rate equalled 3.99% with only about one third of them remaining positive over one year, thus leaving less than 1.5% for colposcopy (Castle, Fetterman et al. 2009). An even lower rate (1.9%) was observed among cytologically normal women screened with liquid-based cytology and computer-assistance (Bansal, Austin et al. 2009). That means that with even an ASC-US rate of just 3% (where at maximum half of the cases will be HPV positive) the total number of women scheduled for colposcopy will not increase whereas this group now comprises also all cytologically false-negatives. The crucial criterion for the clinical value of a routine HPV test is not its analytical but its clinical sensitivity. Because limited specificity will be harmful via unnecessary treatment of healthy women also clinical sensitivity has to be traded off with specificity (Kinney, Stoler et al. 2010). The observance of a well defined diagnostic threshold must be guaranteed. Up to now this has only been proven in large-scale studies for the HC2 and the cobas test. For several reasons as stated above this objective is rather complicated to achieve for PCR techniques. A guideline for new HPV tests It is indispensable that new tests for routine HPV testing prove a balance between clinical sensitivity and specificity. To avoid costly validation studies for each new HPV test standards for evaluation have been defined by leading experts on the field (Meijer, Berkhof et al. 2009). These standards of noninferiority are based on the results of numerous large screening studies with HC2 and the GP5+/6+ PCR which set the standards for cut-off, sensitivity and specificity in clinical use. A new HPV test among women above 30 years should reach a relative sensitivity of 90% and a relative specificity of 98% of that of the HC2 test. To test for that around 60 HPV-positive probes of CIN2+ and around 800 HPV-negative specimens are needed. Additionally an intra- and interlaboratory concordance of at least 87% in 500 specimens is requested. New commercial HPV tests 2011 data from the largest clinical HPV study conducted until now have been published assessing the cobas HPV test (Roche). The ATHENA trial, comprising over 47.000 women proved performance comparable to the HC2 test in triage of women with ASC-US cytology (Stoler, Wright et al. 2011) and high sensitivity for CIN2+ in cytologically normal women (Castle, Stoler et al. 2011). The test has further met the criteria of the 580 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 Uvodno predavanje 4/Keynote Lecture 4 guideline for new HPV tests (Heideman, Hesselink et al. 2011). Meanwhile the cobas test has received FDA approval for triage and co-testing with cytology (each from the Preservcyt LBC vial). Recently also other new HPV detection assays have been commercialized. The Abbott Real Time High Risk HPV Test (Abbott Molecular, Wiesbaden, Germany) is a PCR DNA assay which detects 14 high-risk HPV types as a group-specific test and separately HPV 16 and 18. In a first comparison it showed similar performance as HC2 and Linear Array (Cuzick, Ambroisine et al. 2010). As did the B&D Viper HPV realtime PCR which detects 14 high-risk HPV types and discriminates 7 of them (Castle, Gutierrez et al. 2011). A DNA chip test for the type-specific detection of 18 high-risk and 6 low-risk HPV types which is based on the detection of E1 sequences (Papillocheck, greiner bio-one, Frickenhausen, Germany) was found clinically compatible with the GP5+/6+ assay (Hesselink, Heideman et al. 2010). HPV Genotyping Several studies showed a significantly higher risk for development of a CIN 2+ among women positive for HPV 16, 18 and 45 (which is closely related to HPV 18) compared to positivity for other HPV high-risk types. This was valid for cytologically normal women (Khan, Castle et al. 2005; Castle, Rodriguez et al. 2009) as well as with borderline cytologic findings (Wheeler, Hunt et al. 2006). It is therefore appropriate to test for these HPV types independently. This makes only sense after or in parallel with a HPV high-risk basic test because clinically relevant cases positive for high-risk types beside 16,18 and 45 would otherwise be missed. It is generally accepted that a diagnosis of individual HPV types is of scientific interest but its clinical value is currently very limited. First, the risk potential of other HPV types than 16, 18 and 45 is not well defined and seems to be similar (Khan, Castle et al. 2005), second, even if this potential would be better defined it might be difficult to implement the routine application of complex type-specific algorithms. Already at present the follow-up of HPV positivity in clinical practice is rather challenging. Another problem with routine full HPV typing is the high analytical sensitivity and consecutively lower specificity when using the PCR assays which are only available for that purpose. On the other hand detection of HPV 16, 18 and 45 can be achieved by a variant of the HC2 test (which is currently under FDA review) with the same cut-off as the basic test as well as HPV 16 and 18 can be detected separately with some newer tests like cobas or Cervista. HPV RNA Testing A logic approach to overcome some limitations in HPV diagnostics is testing for HPV RNA because elevated expression of viral oncogenes E6 and E7 is associated with malignant transformation by high-risk HPV. Due to non-transforming activities of HPV oncogenes and subsequent transcription also in non-neoplastic lesions and the general instability of RNA HPV mRNA analytics is more complicated than initially expected. First promising results (Molden, Kraus et al. 2005) of a commercial assay (pretectprooferTM, norchip, Klokkarstua/Norway now NuclisenseTM, Biomérieux, Marcy L´Étoile/France) have not be confirmed. In the PREDICTORS study (Szarewski, Ambroisine et al. 2008) the sensitivity of this test for CIN 2+ was only 73,6%, beside other reasons probably due to the fact that it targets only five HPV types. However, it is not finally determined whether lacking HPV mRNA expression might also indicate high grade disease without the potential for progression. Another HPV mRNA test, the APTIMA HPV test (Genprobe, San Diego, CA) detects 14 high-risk HPV types as a group and is run on a fully automated system. Data point to a similar sensitivity and a higher specificity of the APTIMA test as for DNA detection assays (Castle, Dockter et al. 2007; Szarewski, Ambroisine et al. 2008). A promising aspect of the system is that no RNA isolation is required and sealed vials are used. Late in 2011 it received an FDA approval, joining the HC2, Cervista, and cobas tests. Very recently a new approach applying several marker transcripts showed high sensitivity for grading HPV 16 positive cervical lesions (Schmitt, Dalstein et al. 2010). A first systematic review attested HPV RNA testing diagnostic potential but saw the need for further studies (Burger, Kornor et al. 2011). 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 581 Uvodno predavanje 4/Keynote Lecture 4 Tab. 1. Test systems for the detection of HPV DNA and RNA Name HPV DNA Amplicor Producer Technology Target sequence HPV types Genotyping Approval Roche PCR 13 ca no careHPV Qiagen Signal amp 14 ca no Cervista cobas GP 5+/6+ Hologic Roche non commercial Signal amp Realtime PCR PCR 14 ca 14 ca 14 ca HPV 16 + 18 HPV 16 + 18 no CE WHO prequalification FDA + CE FDA + CE no HC2 Qiagen Signal amp 13 ca HPV 16 + 18 FDA + CE Inno-LiPA Linear Array Multiplex HPV Genotyping Kit Papillocheck RealTime HR HPV Innogenetics Roche PCR PCR L1 (part) full genome L1 L1 L1 (part) full genome L1 L1 13 ca + 15 nca 14 ca + 23 nca yes yes CE CE Multimetrix PCR L1 13 ca + 11 nca yes Greiner bioone PCR E1 13 ca + 11 nca yes CE Abbott Realtime PCR L1 14 ca HPV 16 + 18 CE B&D Realtime PCR L1 14 ca HPV 16, 18, 31, 45, 51, 52, 59 Genprobe TMA E6/E7 mRNA 14 ca no HPV 16 + 18 planned CE + FDA Biomérieux NASBA E6/E7 mRNA HPV 16,18,31,33,45 yes CE non commercial mRNA analysis E1/4/6 + L1 mRNA 16 yes BD Viper HPV HPV RNA APTIMA Nuclisens EasyQ HPV HPV 16 transcriptome Ca: carcinogenic; CE: EU-registration; FDA: Food and Drug Administration approval; nac: non carcinogenic; NASBA: Nucleic acid sequence-based amplification; Signal amp: signal amplification; TMA: Transcription-mediated amplification; WHO: World Health Organization References Bansal, M., R. 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(2007). “2006 consensus guidelines for the management of women with abnormal cervical cancer screening tests.” Am J Obstet Gynecol 197(4): 346-355. Youens, K. E., G. A. Hosler, et al. (2011). “Clinical experience with the Cervista HPV HR assay: correlation of cytology and HPV status from 56,501 specimens.” J Mol Diagn 13(2): 160-166. Zielinski, G. D., A. G. Bais, et al. (2004). “HPV testing and monitoring of women after treatment of CIN 3: review of the literature and meta-analysis.” Obstet Gynecol Surv 59(7): 543-553. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 585 usmene prezentacije/ORAL FREE PAPER SESSIONS CITOPATOLOGIJA CITOPATHOLOGY UP1 Uloga eksfolijativne citologije u dijagnostici tumora donjeg urinarnog trakta OP1 The role of exfoliative cytology in diagnosis of lower urinary tract tumor Mirjana Ćuk1, Slavica Knežević Ušaj2, Radoslav Gajanin3, Aleksandar Supić4, Radmil Marić4 Mirjana Cuk1, Slavica Knezevic Usaj2, Radoslav Gajanin3, Supic Aleksandar4, Radmil Maric4 1 Služba za patologiju i citodijagnostiku, Univerzitetska bolnica u Foči, Foča, Bosna i Hercegovina 2 Institut za onkologiju Vojvodine, Sremska Kamenica, Srbija 3 Klinički centar Banja Luka, Zavod za patologiju, Banja Luka, Bosna i Hercegovina 4II hirurška klinika, Univerzitetska bolnica u Foči, Foča, Bosna i Hercegovina Cilj: Analiza parametara uspješnosti primjene citologije urina u dijagnostici malignih tumora donjeg urinarnog trakta. Uvod: Citologija urinarnog trakta je jedna od najznačajnijih dijagnostickih metoda u urološkoj onkologiji, ali pod jasno definisanim uslovima. Materijal i metode: U studiju je uključeno 30 pacijenata sa kliničkom dijagnozom hematurije i 5 pacijenata kod kojih je prethodno urađena hirurška resekcija papilarnih tumora mokraćne bešike. Citološki materijali su bojeni May Grinwald Giemsa metodom i analizirani svjetlosnim mikroskopom. Rezultati: Odnos prema polu bio je muškarci:žene = 6:1. 94,28% pacijenata je bilo starije od 50 godina. Kod svih pacijenata sa malignitetom bio je prisutan urotelni karcinom, a samo kod 3 pacijenta ravni infiltrativni tip. Na ukupnom uzorku apsolutna senzitivnost je bila 42,85 %, a specifičnost 100%. U grupi pacijenata sa low- grade lezijama apsolutna senzitivnost je bila 0%. Papilarne karcinome visokog histološkog gradusa smo podijelili u dvije grupe prema stepenu diferentovanosti: u grupi karcinoma sa HG2 apsolutna senzitivnost je iznosila 62,5%, a u grupi HG3 apsolutna senzitivnost je iznosila 85,71 %. Zaključak: Citologija urina je lako primjenjiva i uspješna metoda za identifikaciju novih i rekurentnih high- grade papilarnih i ravnih karcinoma donjeg urinarnog trakta. Citologija urina nije adekvatna metoda u otkrivanju low-grade tumora donjeg urinarnog trakta. Ključne reči: citologija, urin, urotelni karcinom 586 University Hospital in Foca, Pathology and Cytodiagnostics Department, Foca, BiH 2Oncology Institute of Vojvodina, Sremska Kamenica, Serbia, 3Clinical Centre Banja Luka, Pathology Institute, Banja Luka, BiH, 4University Hospital in Foca, II Surgery Clinic, Foca, BiH, 1 Aim: The analysis of success parameters of urine cytology application in diagnosis of malignant tumors of lower urinary tract. Introduction: Cytology of urinary tract is one of the most significant diagnostic methods in urologic oncology, but under clearly defined conditions. Material and methods: 30 patients with clinical diagnosis of hematuria were included in this study and 5 patients with previously performed surgical resection of papillary tumors of urinary bladders. Cytological material was coloured by May Grinwald Giemsa method and analysed by light microscope. Results: Sex ratio was male: female = 6:1. 94.28% of patients were older than 50. In all patients with malignant tumors, urotel cancer was present, and, only in 3 patients, there was a plane infiltrative type. In all samples absolute sensitivity was 42.85%, and specificity was 100%. In the group of patients with lowgrade lesions, absolute sensitivity was 0%. Papillar carcinoma with high histological grade was divided into two groups according to the level of differentiation: in the group of carcinoma with HG2, absolute sensitivity was 62.5%, and in the HG3 group, absolute sensitivity was 85.71%. Conclusion: Cytology of urine is an easily applied and successful method for identification of new and recurrent high-grade papillar and plane carcinoma of lower urinary tract. Urine cytology is not an adequate method in diagnosing low-grade tumors of lower urinary tract. Key words: cytology, urine, urothelial carcinoma 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 usmene prezentacije/ORAL FREE PAPER SESSIONS HEMATOPATOLOGIJA HAEMATOPATHOLOGY UP2 Ekspresija survivina kod bolesnika sa novootkrivenim nodalnim difuznim B krupnoćelijskim limfomom OP2 Survivin expression in patients with newly diagnosed nodal diffuse large B cell lymphoma Olivera Marković1, Dragomir Marisavljević1, Vesna Čemerikić2, Biljana Mihaljević3, Branka Filipović1 Olivera Markovic1, Dragomir Marisavljevic1, Vesna Cemerikic2, Biljana Mihaljevic3, Branka Filipovic1 2Beolab, 2Beolab, 1Kliničko bolnički centar Bežanijska kosa, Beograd, Srbija Beograd, Srbija 3Institut za hematologiju, Klinički centar Srbije, Beograd, Srbija Cilj: Ova studija je urađena u cilju utvrđivanja kliničkog i prognostičkog značaja survivina kod bolesnika sa nodalnim DBKL. Uvod: Survivin je jedan od članova porodice inhibitora apoptoze (IAP) koji ima značajnu ulogu u patogenezi difuznog B krupnoćelijskog limfoma (DBKL). Materijal i metode: Mi smo analizirali uzorke tkiva limfnog čvora dobijene od 56 bolesnika sa novootkrivenim nodalnim DBKL, lečenih imunohemioterapijom(R-CHOPprotokolom). Ekspresija survivina je analizirana standardnom imunohistohemijskom metodom na parafinskim isečima tkiva limfnog čvora i procenjivana semikvantitativno kao procenat tumorskih ćelija. Rezultati: Imunoekspresija survivina (>45% pozitivnih tumorskih ćelija) je registrovana kod 22(39.28%) bolesnika, u vidu citoplazmatske pozitivnosti kod 15 bolesnika ili mešovite (citoplazmatske i jedarne) pozitivnosti kod 15 bolesnika. Utvrđena je statistički značajna razlika u ekspresiji survivina između GCB i non-GCB podtipa DBKL(p=0.031). Imunoekspresija survivina nije u korelaciji sa IPI bulky bolešću, ekstranodalnom lokalizacijom, vrednošću hemoglobina, ekspresijom Ki-67 i drugim kliničko-patološkim parameterima. Univarijantna analiza je pokazala da je bolesnici sa ekspresijom survivina imaju značajno lošiji terapijski odgovor i krace preživljavanje u odnosu na bolesnike koji su survivin negativni (p=0.048 and p=0.034, respektivno). Bolesnici sa ekspresijom survivina relapsiraju češće nego bolesnici koji ga ne eksprimiraju (27.3% vs. 11.8%), ali ova razlika nije dostigla nivo statističke značajnosti (p=0.131). Zaključak: Rezultati ove studije pokazuju da ekspresija survivina ima značajnu ulogu u određivanju toka i prognoze bolesnika sa DBKL lečenih R-CHOP protokolom. Prema tome, survivin predstavlja potencijalni cilj za terapijske intervencije u DBKL. Ključne reči: difuzni B krupnoćelijski limfom, apoptoza, survivin, imunohistohemija, prognoza. 1Clinical Hospital Center Bezanijska Kosa, Belgrade, Serbia Belgrade, Serbia 3Institut of Hematology, Clinical Center of Serbia, Belgrade, Serbia Aim: The present study was designed to investigate the clinical and prognostic significance of survivin expression in nodal DLBCL. Introduction: Survivin is one of the inhibitors of apoptosis proteins (IAP) that might play an important role in the pathogenesis of diffuse large B cell lymphoma (DLBCL). Material and methods: We analyzed lymph node biopsy specimens obtained from 56 patients with newly diagnosed nodal DLBCL, treated with immunochemotherapy (R-CHOP). The expression of survivin was analyzed using the standard immunohistochemical method on formalin-fixed and routinely processed paraffin-embedded lymph node specimens, and evaluated semiquantitatively as a percentage of tumor cells. Results: Survivin immunoexpression (>45% positive tumor cells) was found in 22 (39.28%), and observed as cytoplasmic staining in 15 patients, or mixed staining in 7 patients. A significant difference in survivin immunoexpression was noticed between the GCB and the non-GCB subtypes of DLBCL (p=0.031). A univariate analysis showed that survivin positivity was an unfavorable factor for therapy response and a predictor of shorter survival in patients with DLBCL (p=0.048 and p=0.034, respectively). Patients with survivin overexpression experienced a relapse more often than patients without expression of this apoptotic protein (27.3% vs. 11.8%), but this difference did not reach statistical significance (p=0.131). Conclusion: The results of this study showed that disregulation of survivin expression had an important role in determination of the course of the disease in patients with nodal DLBCL treated with R-CHOP. Therefore, survivin represents a potential target for therapeutic intervention in DLBCL Key words: diffuse large B cell lymphoma, apoptosis, survivin, immunohistochemistry, prognosis. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 587 usmene prezentacije/ORAL FREE PAPER SESSIONS UP3 Prognostički značaj CD 68 pozitivnih makrofaga pridruženih tumoru u uznapredovalom stadijumu klasičnog Hočkinovog limfoma OP3 The prognostic significance of CD 68 positive tumor associated macrophages in advanced stage classical Hodgkin lymphoma Ljubomir Jaković1, Biljana Mihaljević1, Maja PeruničićJovanović2, Andrija Bogdanović1, Boško Andelić1, Vladimir Bumbaširević3 Ljubomir Jakovic1, Biljana Mihaljevic1, Maja Perunicic Jovanovic2, Andrija Bogdanovic1, Vladimir Bumbasirevic3 1Klinika Srbija 2Odeljenje za Histopatologiju, Klinicki Centar Srbije,Beograd, Srbija 3Institut za Histologiju i Embriologiju, Medicinski fakultet, Univerzitet u Beogradu, Beograd, Srbija Clinic for Hematology, Clinical Center of Serbia, Belgrade, Serbia 2Department for Histopathology, Clinical Center of Serbia, Belgrade, Serbia 3 Institute for Histology and Embryology, Medical Faculty, University of Belgrade, Belgrade, Serbia Cilj: Analizirati prognostički značaj makrofaga pridruženih tumoru (MPT) i standardnih kliničkih parametara u uznapredovalom HL u cilju određivanja inicijalnog prognoznog modela. Uvod: Rezultati lečenja Hockinovog limfoma (HL) su danas bolji nego u prošlosti. Doprinos daljem poboljš anju rezultata lečenja može dati otkrivanje pouzdanih prognostičkih biomarkera. Materijal i metode: Prognostički značaj MPT analizirali smo u grupi od 52 bolesnika sa uznapredovalim HL, lečenih ABVD protokolom u periodu od 1997-2005. Imunohistohemijska ispitivanja primenom CD68 antitela su obavljena na tkivnim isečcima limfnih čvorova u trenutku postavljanja dijagnoze. Broj CD68 MPT je analiziran na 10 polja velikog uveličanja i podeljen u dve grupe (CD68 manje od 25% i CD68 više od 25%). Medijana praćenja je 7 godina, a značaj parametara je određivan u odnosu na vreme do neželjenog događaja i sveukupno preživljavanje u korelaciji sa standardnim kliničkim parametrima. Rezultati: Bolesnici sa >25% CD68 MPT u poređenju sa bolesnicima sa =25% CD68 MPT su imali kraće petogodišnje preživljavanje (45% vs. 77% log rank p=0.019) i trend kraćeg vremena do neželjenog događaja (51 vs. 71% log rank p=0.19). Nije nađena značajna korelacija sa praćenim kliničkim parametrima. Značajno kraće preživljavanje je bilo udruženo sa IPS>2, voluminoznom tumorskom masom i povišenom sedimentacijom eritrocita (log rank test, p=0.003, p=0.049, p=0.007). U multivarijantnoj analizi >25% CD68 MPT, IPS>2 i voluminozna tumorska masa su pokazale značaj nezavisnih prognostičkih faktora za sveukupno preživljavanje (Cox multivarijantni model p=0.006, p=0.007, p=0.013). Zaključak: Makrofagi koji su pridruženi tumoru predstavljaju potencijalni prognostički biomarker koji bi mogao doprineti boljoj stratifikaciji rizika bolesnika sa klasičnim HL. Ključne reči: Makrofagi, uznapredovali Hočkinov limfom Aim: To analyze the prognostic value of tumor associated macrophages (TAM) and standard clinical parameters in advanced HL patients in order to determine optimal initial prognostic model. Introduction: Although the treatment of Hodgkin lymphoma (HL) has been improved, distinguishing reliable prognostic biomarkers could better stratify patients for more effective treatment. Material and methods: In a group of 52 advanced HL patients treated with ABVD regimen from 19972005, we analyzed the prognostic relevance of TAM using a CD68 antibody in the immunohistochemical analysis, on lymph nodes tissue sections, at diagnosis. The number of CD68 positive TAM was analyzed on 10 different high power microscopy fields (HPF, x400) and scored (CD68 less than 25% and CD68 grater than 25%). The median follow-up was 7 years (yrs). Their significance was evaluated according to the response to treatment (EFS) and survival period (OS) and correlated with standard clinical parametres. Results: Patients with >25% CD68 TAM compared to those with =25% had worse 5-yrs OS (45% vs. 77% log rank p=0.019) and showed trend towards shorter 5-yrs EFS (51 vs. 71% log rank p=0.19). Additionally no significant correlation with selected clinical features were found. Significantly shorter OS was associated with IPS>2, bulky disease, elevated sedimentation rate (log rank test, p=0.003, p=0.049, p=0.007, respectively). In multivariate analysis increased CD68 TAM, IPS>2 and bulky disease were identified as independent prognostic factors for overall survival (Cox multivariate model, p=0.006, p=0.007, p=0.013, respectively). Conclusion: Tumor associated macrophages represent potential prognostic biomarker which could contribute to better risk stratification of cHL patients. Key words: Macrophages, advanced Hodgkin’s 588 za Hematologiju, Klinički Centar Srbije, Beograd, 1 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 usmene prezentacije/ORAL FREE PAPER SESSIONS BONE AND SOFT TISSUE PATHOLOGY KOŠTANA I MEKOTKIVNA PATOLOGIJA UP4 Mogućnosti i ograničenja “imprint” citološke tehnike u dijagnostici koštanih i mekotkivnih tumora OP4 Possibilities and limitations of „imprint” cytological techniques in diagnostics of bone and soft tissue tumors Jelena Sopta1, Zoran Vučinić2, Jelena Bokun 3, Dušan Ristić 3, Vesna Mijučić 3 Jelena Sopta 1, Zoran Vucinic 2, Jelena Bokun 3, Dusan Ristic 3, Vesna Mijucic 3 Cilj: Cilj ovog rada je da utvrditi stepen korelacije citoloških i definitivnih patohistoloških dijagnoza primenom imprint citološke tehnike u biopsijama tumora kostiju i mekih tkiva. Uvod: Koštani i mekotkivnu tumori predstavljaju veliki izazov u standardnoj analizi bioptičkog ili operatvnog uzoraka. Od svih citoloških tehnika, otisak bioptičkog uzorka („imprint” citološka tehnika) najbliži i je patohistološkom. Materijali i metode: Na Institutu za patologiju Medicinskog fakulteta u Beogradu u period od 5 godina (2007-2011.) uradjeno je 286 imprint citoloških analiza tumora kostiju i mekih tkiva. Rezultati: Poredeći rezultate citološke imprint analize sa definitivnom patohistološkom dijagnozom utvrdili smo da je stepen sigurnosti u našem uzorku iznosio 89%. Zaključak: Mesto citološke analize je sve zapaženije u sklopu multidisciplinarne i sve složenije interperetacije bioptičkog materijala. Ključne reči: imprint citologija, tumori, kosti, meka tkiva Aim: The aim of thys study is to determine the correlation of cytologic and histologic diagnosis of definite application of imprint cytology techniques in tumor biopsies of bone and soft tissue. Introduction: Bone and soft tissue tumors represent a major challenge in the standard analysis of biopsy samples. Of all cytological techniques, fingerprint biopsy specimen („imprint” cytological techniques) is closest to histopathology. Materials and Methods: At the Institute of Pathology, University of Belgrade in the period of 5 years (2007-2011.) was done 286 imprint cytological analysis of bone tumors and soft tissue. Results: Comparing the results of imprint cytology analysis with definitive histopathological diagnosis we found that the level of security in our sample was 89%. Conclusion: Place of cytology is noticeable in the multidisciplinary and more complex biopsy interpretation. Key words: imprint cytology, bone, soft tissue, tumor 1Institut za patologiju, Medicinski fakultet, Univerzitet u Beogradu, Srbija 2Institut za ortopedsko hirurške bolesti Banjica, Beograd, Srbija 3Institut za onkologiju i radiologiju Srbije, Beograd, Srbija 1Medical Faculty Belgrade,University Belgrade,Institute of Pathology, Serbia 2Institute for orthopoedic surgery „Banjica”,Belgrade, Serbia 3Institute for Oncology and Radiology, Belgrade, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 589 usmene prezentacije/ORAL FREE PAPER SESSIONS NEUROPATOLOGIJA NEUROPATHOLOGY UP5 Imunoekspresija katepsina D kod meningioma OP5 Immunoexpression of Cathepsin D in meningiomas Zorana Vukašinovic-Bokun, Lidija Prijić-Plećević, Milica Lavrnić, Nenad Miladinović, Marija Nikolić, Sanja M. Milenković Zorana Vukasinovic Bokun, Lidija Prijic-Plecevic, Milica Lavrnic, Nenad Miladinovic, Marija Nikolic, Sanja M. Milenkovic Cilj: Ciljevi ove studije bili su definisanje osnovnih socioepidemioloških karakteristika (starost, polna distribucija pacijenata), prevalenca različitih histoloških tipova meningioma u grupi primarnih i recidivantnih tumora u odnosu na imunoekspresiju katepsina D. Uvod: Meningiomi su većinom benigni neglijalni tumori koji nastaju iz ćelija arahnoidalne kape ovojnica mozga i kičmene moždine. Ovi tumori čine 30% svih primarnih tumora mozga sa godišnjom incidencom od 4,5/100000. Sposobnost recidiviranja je tipična za meningiome. Katepsin D je lizozomalna cisteinska peptidaza koja može imati ulogu u ponašanju agresivnih meningioma. Materijali i metode: Analizirano je 50 pacijenata sa primarnim i recidivirajucim meningiomima, operisanih u klinicko-bolničkom centru Zemun tokom 2004 koji su praćeni tokom dve godine. Svi uzorci su histološki klasifikovani prema WHO klasifikaciji, a reprezentativni isečci su imunohistohemijski obojeni sa anti-katepsinom D. U grupama primarnih i recidivirajućih meningioma brojane su mitoze na 10 i 20 polja velikog uveličanja, kao faktor tumorske agresivnosti i rezultati su statisticki obrađeni. Rezultati: Ukupno je analizirano 64 uzorka tumorskog tkiva primarnih i recidivirajućih meningioma. Najveći broj meningioma klasifikovano je kao WHO gradus I (57 slučajeva). Ostali su pripadali WHO gradusu II (5) i WHO gradusu III (2). Utvrđena je statistički značajna razlika u ekspresiji katepsina D u odnosu na broj mitoza i histološki gradus tumora. Zaključak: Imunoekspresija katepsina D značajna je kod benignih meningioma te ne može biti upotrebljena kao prognostički faktor maligniteta i agresivnosti tumora. Gubitak pozitivnosti meningioma za katepsin D može biti signal za potencijalno agresivno biološko ponašanje tumora. Ključne reči: meningiomi, broj mitoza, katepsin D Aim: This study was performed to establish the socioepidemiological caracteristics, the prevalence of histological types of meningiomas in the groups of primary and recidivant tumors and Cathepsin D immunoexpression. Introduction: Meningiomas are mostly benign nonglial tumors arising from arachnoidal cap cells of the brain. They rappresent 30% of all primary brain tumors with an annual incidence of 4,5/100000. The ability to recurre is a feature typical of meningiomas. Cathepsin D, a lysosomal cistein pepridase could play a role in the biological behaviour of agressive meningiomas. Material and methods: We analysed 50 patients with meningiomas who underwent surgery in KBC Zemun during the year 2004. All surgical specimen were evaluated for histologic type of tumor according to WHO classification and representative slides were immunostained with the anti-Cathepsin D antibody. In primary and recidivant meningioma groups the number of mitosis was counted on 10 and 20 HPF and those results were statistically analyzed. Results: A total of 64 meningioma specimen have been submitted to histological analysis.Most of the analysed tumors were graded as the WHO I group (57 cases). The remainder were classified as WHO II (5) and WHO III (2). A considerable difference was found when the number of mitosis and the histologic tumor grade have been compared with the Cathepsin D immunoexpression. Conclusion: Since Cathepsin D exibits a strong expression in benign meningiomas, it could not be used as a prognostic factor of malignancy and invasivness. On the contrary, the loss of positive staining could be an alert for a potentialy agressive biological behaviour. Key words: meningiomas, mitosis, Cathepsin D Služba kliničke patologije, Kliničko-bolnički centar Zemun, Beograd, Srbija 590 Department of Pathology, Clinical Hospital Center Zemun, Belgrade, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 ORAL FREE PAPER SESSIONS UP6 Nanog kao potencijalni marker kancerskih matičnih ćelija kod pacijenata sa gliomima visokog gradusa OP6 Nanog as the potential marker of the cancer stem cells in the high grade glioma patients Irena Dimov1, Sladana Ugrenović2, Slavica Stojnev3, Miloš Kostić1, Tasic Desanka3, Vladislav Stefanović2 Irena Dimov1, Sladana Ugrenovic2, Slavica Stojnev3, Milos Kostic1, Desanka Tasic3, Vladislav Stefanovic2 2Medicinski 2Faculty 1Institut za imunologiju, Medicinski fakultet, Niš, Srbija fakultet, Niš, Srbija 3Institut za patologiju, Medicinski fakultet, Niš, Srbija Cilj: Reevaluirati Nanog kao potencijalni marker kancerskih matičnih ćelija i molekularnu terapijsku metu u gliomima visokog gradusa Uvod: Visokogradusni gliomi su najsmrtonosniji tumori mozga, otporni na sve forme terapije. Hipoteza o kancerskim matičnim ćelijama je koja je u fokusu novih istraživanja je odraz novih aspekata razumevanja patogeneze malignih tumora uključujući i visokogradusne gliome i pruža mogućnosti za nove terapijske pristupe. Nanog je transkripcioni faktor dokazano povezan sa kapacitetom za samoobnavljanje embrionalnih i neuralnih stem ćelija. Stoga bi mogao biti koristan marker matičnih ćelija visokogradusnih glioma i potencijalna molekularna meta za terapiju. Materijal i metode: Patohistološki uzorci 79 visokogradusnih glioma su imunohistohemijski bojeni i kmvantifikovani za pozitivnost Nanog nuklearnog antigena. Rezultati: Nuklearna ekspresija Nanog je pokazala ekstremno visoku ili ekstremno nisku pozitivnost u različitim tumorima. Isti nivo ekspresije pokazan je u recidivima. Zaključak: Nanog se čini interesantim kao potencijalni genetski marker i može se pokazati kao značajan događaj u patogenezi određenih visoko gradusnih glioma. Ključne reči: gliomi, kancerske matične ćelije, Nanog 1Institute for Immunology, Faculty of Medicine, Nis, Serbia of Medicine, Nis, Serbia 3Institute for Pathology, Faculty of Medicine, Nis, Serbia Aim: Reevaluation of Nanog as the potential cancer stem cell (CSC) marker and therapeutic molecular target in the high grade gliomas Introduction: High grade gliomas are the most deadly malignant brain tumors, persistant to all forms of threatment.The rapidly emerging focus on cancer stem cell (CSC) hypothesis embodies a paradigm shift in our understanding of the pathogenesis of malignomas incuding high grade gliomas, and gives new hope for more effective therapeutic approach.Nanog is a transcriptonal factor critically involved with self-renewal of undifferentiated embryonic and neural stem cells. Therefore, it might be useful marker of the CSCs in the high grade gliomas and potential molecular target for therapy. Material and methods: Pathohistological samples 79 high grade gliomas were immunochistocemically stained and quantified for Nanog nuclear positivity. Results: The Nanog expression expression in some tumors was extremely high, while in other extremely low or negative. Recurrenbt tumors showed same pattern of expression. Conclusion: Nanog may be interesting as an genetic marker and might show to be another important genetic point in the development of high grade gliomas. Key words: gliomas, cancer stem cells, Nanog 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 591 usmene prezentacije/ORAL FREE PAPER SESSIONS PERINATALNA I PEDIJATRIJSKA PATOLOGIJA PERINATAL AND PEDIATRIC PATHOLOGY UP7 Imunohistohemijska ekspresija p53 u Wilms-ovom tumoru OP7 Immunohistochemical expression of p53 in Wilms tumor Sanja Radojevic-Škodrić1, Dimitrije Brašanac1, Ljiljana Bogdanović1, Milena Jovanović2, Ivana Baralić3, Gordana Basta-Jovanović1 Sanja Radojevic-Skodric1, Dimitrije Brasanac1, Ljiljana Bogdanovic1, Milena Jovanovic2, Ivana Baralic3, Gordana Basta-Jovanovic1 Cilj: Da se analizira ekspresija p53 imunohistohemijskom metodom u WT kao i odnos ekspresije sa stadijumom tumora, histološkim tipom i prognostičkom grupom. Uvod: Brojne studije su pokazale prognosticki značaj p53 ekspresije u različitim malignim tumorima. Materijal i metode: Imunohistohemijska ekspresija p53 je analizirana u 59 WT i 5 slučajeva normalnog tkiva bubrega koje nije zahvaćeno tumorom. Rezultati: 41 od 59 slucajeva WT (61,5%) su pokazali smanjenje ekspresije p53 u poređenju sa nivoom ekspresije u normalnom tkivu bubrega. Sve tri komponente WT su pokazale smanjenje ekspresije u približno istom procentu. Smanjenje ekspresije p57 je bilo cešće u višim stadijumima (stadijumi III i IV) u poređenju sa nižim stadijumima WT (stadijum I ai II), u tumorima sa difuznom anaplazijom u odnosu na ostale histološke tipove i u slučajevima visokog rizika u odnosu na tumore srednjeg rizika, ali bez statistički znacajne razlike. Zaključak: Smanjena ekspresija p53 je mnogo cešćeuočena u odmaklim stadijumima, u difuznoj anaplaziji i u tumorima visokog rizika ukazujući da je smanjena ekspresija p53 povezana sa nepovoljnom prognozom. Ključne reči: WT, p53, ćelijski ciklus, imunohistohemija Aim: To determine p53 immunohistochemical expression patterns in WTs and to analyze it in relation to tumor stage, histological type and prognostic category. Introduction: A number of studies have indicated that the expression of cell cycle inhibitor p57 is of prognostic importance in a variety of cancers. Material and methods: Imunohistostochemical expression of p53 was analyzed in 59 cases of WT and in five kidney specimens uninvolved by the tumor. Results: 41 of 59 WTs (61.5%) showed decreased expression of p53 compared to the expression level in normal kidney tissue. All components of WTs showed decreased expressed with similar frequency. Decreased p53 expression was found more often in high-stage tumors (stage III and IV) compared to lowstage WTs (stages I and II), in diffuse anaplastic WTs compared to other histological types, and in cases of high-risk compared to intermediate-risk tumors, but without significant difference. Conclusion: Decreased p53 expression was observed more often in advanced stages, in diffuse anaplasia and in high-risk group of WTs, suggesting that it may be associated with the unfavorable prognosis. Key words: WT, p53, cell cycle, immunohistochemical 1Institut za patologiju, Medicinski fakultet, Univerzitet u Beogradu, Beograd, Srbija 2Institut za mikrobiologiju, Stomatolo ki fakultet, Univerzitet u Beogradu, Beograd, Srbija 3Institut za bromatologiju, Farmaceutski fakultet, Univerzitet u Beograd, Beograd, Srbija 592 1Institute of pathology, Medical School, University of Belgrade, Serbia 2Institute of microbiology, Dental School, University of Belgrade, Serbia 3Institute of bromatology, Faculty of Pharmacy, University of Belgrade, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 usmene prezentacije/ORAL FREE PAPER SESSIONS UROLOŠKA PATOLOGIJA UROPATHOLOGY UP8 Ispoljavanje FGFR1 i NCAM molekula u karcinomima bubrega OP8 Expression of FGFR1 and NCAM in renal tumors Jasmina Marković-Lipkovski1, Sanja Ćirović1, Dragan Mitrović1, Duško Dunđerović1, Cane Tulić2 Jasmina Markovic-Lipkovski1, Sanja Cirovic1, Dragan Mitrovic1, Dusko Dunderovic1, Cane Tulic2 2Urološka 2Clinic 1Institut za patologiju, Medicinski fakultet, Beograd, Srbija klinika KCS, Beograd Cilj: Detekcija koekspresije receptora fbroblast faktora rasta 1 (engl.-FGFR1) i neuralnog ćelijskog adhezionog molekula (engl.-NCAM) u karcinomu bubreznih ćelija (KBĆ). Uvod: KBĆ potiču od proksimalnih ili distalnih tubula koji vode embrionalno poreklo od mesonefrosa i tokom fetalnog razvoja nastaju putem mezenhimalno-epitelijalne transformacije. S obzirom da mezonefros snažno eksprimira NCAM nije iznenađujuće da tokom tumorske transformacije tubulskih ćelija bubrega dolazi do ponovne ekspresije NCAM-a. Fibroblast faktori rasta i njihovi receptori (engl.-FGFR1-4) neophodni su za ćelijski rast i diferencijaciju, pa se mogu ispoljiti i u KBĆ. Materijali i metode: RT-PCR analizom (engl. – Reverse Transcriptase Polymerase Chain Reaction) izvršena je detekcija 3 specifične NCAM izoforme, dok je ispoljavanje FGFR1 i NCAM antigena u tumorskom tkivu detektovano imunohistohemijskom i duplom imunofluorecsentnom metodom. Analizirano je ukupno 35 tumora bubrega. Rezultati: RT-PCR rezultati tumora pokazali su prisustvo NCAM-140 kDa izoforme kod svih testiranih tumora, dok su NCAM-120 i NCAM-180 kDa izoforme detektovane samo u 3 tumora. Intenzitet ispoljavanja FGFR1 i NCAM-a nije konstantan u KBĆ. Koekspresija FGFR1/NCAM detektovana je u polovini analiziranih uzoraka. Tipovi KBĆ koji eksprimiraju FGFR1/NCAM su svetloćelijski, papilarni, hromofobni, kao i KBĆ sa sarkomatoidnom varijantom. Broj FGFR1/NCAM ćelija je u korelaciji se stepenom maligniteta tumora (veći nuklearni gradus - jača ekspresija FGFR1/NCAM). Ipak 20 % analiziranih KBC bilo je negativno na oba markera, dok je 20 % imalo pozitivnost samo na jedan marker. Zaključak: FGFR1/NCAM koekspresija nije specifična za određeni tip KBC. Ipak jači intenzitet FGFR1/ NCAM koekspresije kod KBC je u korelaciji sa povećanjem nuklearnog gradusa tumora. S tim u vezi koekspresija FGFR1/NCAM može predstavljati važan pokazatelj agresivnosti KBC. Ključne reči: FGFR1, NCAM, karcinom bubrežnih ćelija (KBĆ), nuklearni gradus 1Institute for Pathology, Medical Faculty, Belgrade, Serbia of Urology, Clinical Center of Serbia, Belgrade Aim: Co-expression of FGFR1 and NCAM in tumor samples was analyzed in this study. Introduction: Fibroblast growth factor receptor (FGFR1-4) is critical for cell growth and differentiation, and could be over expressed in renal cell carcinoma (RCC). Most of RCC develop from tubular cells, which arise from mesenchyme after mesenchymal-epithelial transformation, these mesenchymal cells widely express neural cell adhesion molecule (NCAM), so NCAM re-expression is expected in RCC Material and methods: 35 RCC samples were analyzed. Antibodies for FGFR1 and NCAM were used in immunohistochemisrty and double immunofluorescent analysis and specific primers for each NCAM isoform were used in RT-PCR. Results: RT-PCR analysis showed that NCAM-140 kDa isoform is predominantly expressed in all samples, while isoforms of 120 and 180 kDa were present in only 3 samples. Expression of FGFR1 and different NCAM molecules slightly varied in renal tumour cells. In half of the analyzed samples co-expression FGFR1/NCAM was detected. Co-expression of FGFR1/NCAM-was detected in clear cell, papillary, sarcomatoid, and chromophobe RCC. The number of cancer cells which co-expressed FGFR1 and NCAM seems to correlate with tumour malignancy. However, 20% of all analyzed samples were negative for both markers and 20% were negative for either NCAM or FGFR1. Conclusion: FGFR1 and NCAM are mainly coexpressed on the same RCC cells Co-expression of FGFR1/NCAM-180 seems to be present on RCC tissue regardless of histological cell type. RCC with high nuclear grade (III/IV) usually has higher number of FGFR1/NCAM cells. Interaction between FGFR1 and NCAM may be relevant for cell migration, growth and malignancy in RCC. Key words: FGFR1, NCAM, RCC, nucl 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 593 POSTER PRESENTATIONS 1/poster prezentacije 1 CITOPATOLOGIJA CITOPATHOLOGY P01 Tačnost citološke dijagnostike metastatske bolesti glave i vrata na materijalu dobijenom aspiracijom tankom iglom The accuracy of fine needle aspiration cytology in the diagnosis head and neck metastatic disease Željka Tatomirović, Vesna Škuletić, Jovana Trimčev, Lidija Zolotarevski Military Academy Belgrade, Belgrade, Serbia Vojnomedicinska akademija, Beograd, Srbija Cilj: Procena tačnosti citološke dijagnostike metastatske bolesti glave i vrata (GiV) na materijlalu dobijenom aspiracijom tankom iglom (ATI). Uvod: ATI je dobro poznata tehnika karakterizirana minimalnim komplikacijama, tako da je veoma pogodna za ovu regiju sa vitalnim organima i različitim tkivima na relativno malom prostoru. Materijal i metode: Aspiracija je urađena u 54 bolesnika sa metastatskom bolešćuti GiV, iglama od 25 G. Aspiraciju je radio citolog, a razmazi su bojeni May-Grunwald-Giemsom. Rezultati: Postavljena je citiološka dijagnoza 13 skvamoznih, 8 adeno, 13 nemikrocelularnih, 13 mikrocelularnih i 6 slabo diferencirani metastatskih karcinoma.Korelacijom sa histologijom, potvrdena je dijagnoza svih skvamoznih i mikrocelularnih karcinoma, adenokarcinoma u 6 slučajeva dok je jedan bio adenoskvamozni, a jedan je citološki pogrešno dijagnosticiran kao skvamozni karcinom. Od 13 citolški nemikrocelularnih karcinoma, 4 su bila skvamozna, 3 adenokarcinoma, 5 adenoskvamozna, a jedan je ostao nemikrocelularni karcinom. Od 6 citolo ki slabo diferenciranih karcinoma 4 su bila skvamozna, jedan adenoskvamozni, a jedan je ostao sa istom dijagnozom. Tri metastaze nisu prepoznate: Jedan liomfoepiteliom (dijagnostkovan kao Hodgkinov limfom), i dva slučaja metastaze papilarnog karcinoma štitaste žlezde (dijadnostikovane kao nodularna struma i adenoid cistični karcionom). Tačnost citološke dijagnostike za metastatsku bolest GiV u ovoj grupi bolesnika je iznosila 94,5%. Zaključak: Veoma dobra korelacija citoloških, sa histološkim dijagnozama, pokazala je da je ATI pogodna kao prva dijagnostička linija kod metastatske bolesti GiV. Ključne reči: citologija, histologija, aspiracija tankom iglom, glava i vrat, metastaza 594 Zeljka Tatomirovic, Vesna Skuletic, Jovana Trimcev, Lidija Zolotarevski Aim: The aim of this study was to assess the diagnostic accuracy of fine needle aspiration (FNA) cytology of head and neck (H&N) metastatic disease. Introduction: FNA cytology is technique associated with minimal complications, very suitable for obtaining material in the head and neck region. Material and methods: Aspirations were performed in 54 patients with H&N metastatic disease by cytologist, using 25 G needles and smears were stained with May-Grunwald-Giemsa. Results: Cytological diagnosis of 13 squamous, 8 adeno, 13 non small cell, 13 small cell and 6 poorly differentiated metastatic carcinoma were established. Histological correlation confirmed FNA diagnosis of all squamous, and small cell carcinoma, adenocarcinoma in 6 cases, while one was adenoaquamous carcinoma and one was cytologically misdiagnosed as squamous cell carcinoma. Out of 13 cases cytologically diagnosed as non small cell carcinoma, 4 were squamous cell carcinoma, 3 adenocarcinoma, 5 adenosquamous carcinoma and one remained non small cell carcinoma. Out of 6 cases cytologically diagnosed as poorly differentiated carcinoma 4 were squamous cell carcinoma, one adenosquamous carcinoma and one remained with the same diagnosis. Three metastases were not recognized: One lymphoepithelioma (diagnosed as Hodgkin lymphoma), and 2 cases of metastases of papillary thyroid carcinoma (diagnosed as nodular gotier and adenoid cystic carcinoma). The accuracy FNA cytology for metastatic H&N disease was 94,5%. Conclusion: A good concordance between cytological and histological diagnoses, showed that FNA cytology is suitable as a first line of investigation of metastatic H&N disease. Key words: fine needle aspiration, cytology, histology, head, neck, metastasis 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 1/poster prezentacije 1 DERMATOPATOLOGIJA DERMATOPATHOLOGY P02 Ekstraokularni sebacealni karcinom kože Exraocular sebaceous carcinoma of the skin Nada Vučković, Bojana Andrejić2, Aleksandra Levakov1, Mirjana Živojinov1, Dejan Vučković3 Nada Vuckovic1, Bojana Andrejic2, Aleksandra Levakov1, Mirjana Zivojinov1, Dejan Vuckovic3 Cilj: Cilj rada je prikaz retke lokalizacije sebacealnog karcinoma kože. Uvod: Sebacealni karcinomi čine 1% malignih tumora kože, i po lokalizaciji se dele na okularne i ekstraokularne. Ekstraokularni sebacealni karcinomi su kod četvrtine ovog broja (0.25% svih karcinoma kože). Klinički izgled ukazuje na brojne benigne i inflamatorne lezije, to utiče na produženje vremena do postavljenja dijagnoze i adekvatne terapije. Materijal i metode: Ekscizioni biopsijski uzorak kože sa tumorom, sa levog ramena, pacijentkinje stare 56 godina. Tumorski nodul je slobodno pokretan, u odnosu na kožu i niže ležeća tkiva strukture ramena.Rezultati: Makroskopski promena je 2 cm, jasno ograničena, lobulirana, žute boje, srednje čvrstine i homogenog izgleda na preseku. Mikroskopski tumorsko tkivo je jasno ograničeno, bez kapsule, bez kontakta sa epidermisom. Tumorski lobuli su sa bazaloidnim ćelijama u perifernom rubu i centralno smeštenim ćelijama, vakuolizovane citoplazme sebacealnog izgleda. Vidljive su pojedinačne mitoze, nakupine keratina i fokalne nekroze. Tumorske ćelije su CEA negativne i EMA pozitivne. Postavljena je dijagnoza dobro diferentovanog sebacealnog karcinoma kože. Zaključak: Ekstraokularni sebacealni karcinom čini manje od 4% svih sebacealnih karcinoma kože. Usled dobre ograničenosti, obično se dijagnostikuju kao benigna lezije. Iako su ovi tumori veoma retki, neophodno ih je uvrstiti u diferencijalnu dijagnozu tumora kože Ključne reči: sebacealni adenokarcinom, koža, ekstraokularni Aim: The aim of the paper is to present a rare localisation of sebaceous skin carcinoma. Introduction: Sebaceous carcinoma makes 1% of skin cancer, and it can be ocular and extraocular. Extraocular sebaceous carcinoma makes a quarter of this number (0.25% of skin cancer). The clinical appearance of sebaceous carcinoma may resemble many benign and inflammatory changes, which contributes to the longer time required to set the correct diagnosis and initiate appropriate treatment. Material and Methods: The excision skin biopsy of the tumor from a 56-year-old female patient located on left shoulder. The nodule was freely movable to the skin surface and appeared not to be firmly attached to deeper structures of the shoulder. Results: Gross examination revealed clearly demarcated, lobular, yellow, medium firm node, 2 cm in size with homogeneous appearance on cut section. Histologically, tumour tissue was clearly demarcated, without capsule, without any contact with the epidermis. Tissue consisted of basaloid cells on the periphery of tumour lobules, as well as of large centrally localized cells with vacuolated cytoplasm showing sebaceous differentiation. A small number of mitosis, keratin masses and focal necrosis were noted. Tumour tissue showed CEA negativity and EMA positivity. The diagnosis of well differentiated sebaceous carcinoma was made. Conclusion: Extraocular sebaceous carcinoma is found in less than 4% of all skin sebaceous carcinomas. Usually, due to well demarcation, they are clinically diagnosed as benign changes. Regardless of the rare occurrence, it is necessary to include this tumour in the differential diagnosis. Key words: sebaceous adenocarcinoma, skin, extraocular 1 Centar za patologiju i histologiju, Klinicki centar Vojvodine, Novi Sad, Srbija 2 Odseka za za histologiju i embriologiju, Medicinski fakultet, Novi Sad, Srbija 3 Centar za patologiju, Institut za plućne bolesti Vojvodine, Sremska Kamenica, Srbija 1 Center for Pathology and Histology, Clinical Center of Vojvodina, Novi Sad, Serbia 2 Department of Histology and Embryology, Faculty of Medicine, Novi Sad, Serbia 3 Center for Pathology, Institute for Lung Diseases of Vojvodina, Sremska Kamenica, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 595 POSTER PRESENTATIONS 1/poster prezentacije 1 P 03 Imunoekspresija protein regulatora ćelijskog ciklusa (p53 i p16INK4a) i BCL-2 onkoproteina kod primarnog melanoma kože Immunoexpression of cell cycle regulatory proteina (p53 and p16INK4a) and BCL-2 oncoproteina in primary cutaneus melanoma Miloš Kostov1, Žaklina Mijović2, Dragan Mihailović2, Snežana Cerović3, Milorad Pavlović4 Milos Kostov1, Zaklina Mijovic2, Dragan Mihailovic2, Snezana Cerovic3, Milorad Pavlovic4 2Instutut 2Institute 1Odsek za patologiju, Vojna bolnica Niš, Srbija za patologiju, Medicinski fakultet Niš, Srbija 3Institut za patologiju i sudsku medicinu, Vojnomedicinska akademija Beograd, Srbija 4Opšta bolnica Leskovac, Služba za patologiju, Leskovac, Srbija Cilj: Cilj studije je da utvrdi učestalost ekspresije p53 i p16INK4a proteina i bcl-2 onkoproteina kod primarnog melanoma kože. Uvod: Kontrola ćelijskog ciklusa je ključni događaj u regulaciji normalnog funkcionisanja humanog tkiva, i smatra se da abnormalnosti gena regulatora ćelijskog ciklusa doprinose nastanku nekih humanih malignoma. Melanom nije izuzetak u biologiji tumora i njegovo poreklo predominatno je rezultat akumulacije mutacija gena. Materijal i metode: Ispitivanje je obuhvatilo 53 uzoraka primarnog melanoma kože. Imunohistohemijska analiza uradena je primenom monoklonskih antitela za p53, p16INK4a i bcl-2 i visoko senzitivne i specifične obeležene streptavidin-biotin kompleksne imunohistohemijske metode DAKO LSAB TM HRP kit. Rezultati: Ekspresija mutirane forme p53 proteina utvrđena je kod 33/33 (100%) nodularnih melanoma (NM), dok je kod superficijalno šireceg melanoma (SSM) nađena kod 13/20 (65%) slučajeva. Statistički značajne razlike izmedu NM i SSM nađene su za p53 i p16INK4a imunoekspresiju (p < 0,01). Ekspresija bcl-2 onkoproteina utvrđena je kod 50/54 (94%) melanoma kože. Nisu nađene statistički značajne razlike izmedu NM i SSM za bcl-2 imunoekspresiju. Zaključak: Prekomerna ekspresija mutiranog p53 proteina i bcl-2 onkoproteina, uz gubitak ekspresije p16INK4a proteina kod primarnog mealnoma potvrđuje čest gubitak funkcije ovih tumor supresorskih gena i onkogena. Prekomerna ekspresija mutiranog p53 proteina i gubitak ekspresije p16INK4a proteina kod NM ukazuje na vertikalnu fazu rasta tumora. Ključne reči: Melanom, koža, regulatori ćelijskog ciklusa, Bcl-2, progresija melanoma 596 1Department Serbia of Pathology, Military hospital of Nis, Nis,Serbia of Pathology, Faculty of Medicine, University of Nis, 3Institute for Pathology and Forensic Medicine, Military Medical of Academy Belgrade, Serbia 4General hospital of Leskovac, Department of Pathology, Leskovac, Serbia Aim: The aim of the study was to determine the frequency of expression p53 and p16INK4a proteins and bcl-2 oncoprotein in primary skin melanoma. Introduction: Cell cycle control is a crucial event in the regulations of human normal tissues functions, and abnormalities of regulatory cell cycle genes are considered to contribute to the development of several human malignancies. Melanoma is not an exception in the tumor biology and its occurrence is predominantly the result of the gene mutations accumulation. Material and methods: The study involved 53 samples of the primary melanoma of the skin. Immunohistochemical analysis was performed using monoclonal antibodies antihuman for p53, p16INK4a and Bcl-2 and highly sensitive and specifically marked streptavidin-biotin complex immunoassays DAKO LSABTM Kit/HRP. Results: The p53 protein mutated form expression was found in 33/33 (100%) nodular melanoma (NM), while in the superficially spreading melanoma (SSM) was found in 13/20 (65%) cases. Statistically relevant differences between NM and SSM were found for the p53 and p16INK4a immunoexpresion (p < 0.01). The bcl-2 oncoprotein expression was found in 50/53 (94%) melanoma of the skin. There were no significant statistical differences between the NM and SSM for the bcl-2 immunoexpression. Conclusion: Overexpression p53 protein and bcl2 oncoprotein, with the loss p16INK4a protein of expression in the primary melanoma, confirms a frequent loss of function of these tumor suppressor gene and oncogene. Overexpression of mutated p53 protein and loss p16INK4a protein in NM indicate a vertical tumor growth phase. Key words: Melanoma, skin, cell cycle regulators, bcl-2, melanoma progression 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 1/poster prezentacije 1 P04 Hibridni kožni švanom - miksom omotača nerva Hybrid schwannoma-nerve sheath myxoma of the skin Dimitrije Brašanac, Martina Stojanović Dimitrije Brasanac, Martina Stojanovic Cilj: Histopatološka i imunohistohemijska analiza hibridnog tumora omotača perifernog nerva. Uvod: Hibridni tumori omotača perifernog nerva, koji pokazuju nagli ili postepeni prelaz između dve komponente, se retko opisuju u literaturi. Najčešće kombinacije su neurofibrom-švanom, švanom-perineuriom i neurofibrom-perineuriom. Materijal i metode: Analiza pločica bojenih hematoksilin-eozinom i imunohistohemijskom strepatvidin-biotin tehnikom. Rezultati: Pacijentkinji starosti 55 godina je dijagnostikovan kožni čvor, dimenzija 13x11x7 mm, na potiljačnom delu glave. Posle hirurške ekscizije uočen je potkožni nastavak (20x15x13mm) u kontinuitetu sa kožnim tumorom. Mikroskopskom analizom u površnom delu uočeni su multipli lobulusi, u celosti ili delimićno okruženi vezivnim trakama, građeni od vretenastih, ovalnih ili zvezdastih ćelija rasutih po miksoidnoj supstanci. U dubljim delovima uzorka nodularnu građu su postupno zamenjivala celularnija polja, ponegde sa palisadnim ređanjem jedara razdvojenih kolagenom stromom (Verocay-eva telašca). U površnim delovima jedra su bila okrugla-ovalna, bez atipije i mitotske aktivnost, dok su se u dubljim, celularnim delovima mogle naći do 2 mitoze/10polja velikog uveličanja. Imunohistohemijsko bojenje pokazalo je S-100 (difuzno) i D2-40 (dublji celularni delovi) pozitivnost. Periferni delovi miksoidnih lobulusa su bili pozitivni na epitelni membranski antigen i Glut-1. Proliferativni indeks (KI-67) je bio 1% u miksoidnim, a 5% u celularnim delovima. Postavljena je dijagnoza hibridnog švanoma-miksoma omotača nerva. Zaključak: Miksom omotača nerva (NSM) do sada nije opisan kao deo hibridnih tumora omotača nerva. Rezultati imunohistohemijske analize ukazuju da su švanom i NSM bliski ali zasebni patološki entiteti. Ključne reči: švanom, miksom omotača nerva, hibridni tumor, koža, imunohistohemija Aim: Histopathological and immunohistochemical analysis of hybrid peripheral nerve sheath tumor. Introduction: Hybrid peripheral nerve sheath tumors showing abrupt or indistinct transition of two components have been reported uncommonly in the literature. Most frequent combinations described are neurofibroma-schwannoma, schwannoma-perineurioma and neurofibroma-perineurioma. Material and methods: Analysis of slides stained with hematoxylin-eosin and streptavidin-biotin immunohistochemical method. Results: A 55-year-old female presented with a cutaneous node on the occipital region, measuring 13x11x7 mm. Surgical excision revealed subcutaneous extension (20x15x13 mm) continuous with the skin tumor. Microscopic analysis showed superficial area with multiple lobules, partially or completely surrounded by fibrous bands, with spindled, oval, or stellate-shape cells dispersed in myxoid matrix. In deeper portions of the specimen nodular architecture gradually disappeared, being replaced with cellular areas, somewhere showing palisaded arrangement of nuclei separated by an acellular collagen-rich matrix (Verocay bodies). In superficial parts, cells displayed small, round to oval, cytologically bland nucleus and no mitotic figures, whereas in deep cellular areas up to 2 mitoses/10 high-power fields were detected. Immunohistochemically, tumor was positive with S-100 (diffusely), and with D2-40 (deep cellular parts). Epithelial membrane atigen and Glut-1 positive cells were identified at the periphery of myxoid lobules. Ki-67 labelling index was 1% and 5% in myxoid and cellular areas, respectively. Diagnosis of hybrid schwannoma-nerve sheath myxoma has been made. Conclusion: Nerve sheath myxoma (NSM) has not been previously identified as a part of hybrid nerve sheath tumors. Results of immunohistochemical analysis suggest that NSM and schwannoma are closely related but distinct entities. Key words: schwannoma, nerve sheath myxoma, hybrid tumor, skin, immunohistochemistry Institut za patologiju, Medicinski fakultet Univerziteta u Beogradu, Srbija Institute of pathology, School of Medičine, University of Belgrade, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 597 POSTER PRESENTATIONS 1/poster prezentacije 1 P05 Ožiljna endometrioza i endosalpingioza kože ingvinuma Cutaneous inguinal scar endometriosis and endosalpingiosis Martina Stojanović1, Dimitrije Brašanac1, Milan Stojičić2 Martina Stojanovic1, Dimitrije Brasanac1, Milan Stojicic2 Cilj: Histopatološka i imunohistohemijska analiza ožiljne endometrioze i endosalipingioze kože. Uvod: Endosalpingioza i endometrioza predstavljaju ektopičan rast epitela jajovoda i endometrijalnih žlezda i strome. Endometrioza kože je poznat entitet sa učestalošću od 1.1% u operisanih pacijentkinja. Endosalpingioza kože je redak entitet sa samo pet do sada opisanih slučajeva. Materijal i metode: Analiza preparata bojenih hematoksilin-eozin i streptavidin-biotin imunohistohemijskom metodom. Rezultati: U ovom radu prikazujemo slučaj žene starosti 40 godina koja se javila zbog potkožnpg čvora u desnom ingvinumu, koji je sporo sporo rastao tokom dve godine i boleo tokom menstruacije. Pacijentkinja je pre 10 godina imala miomektomiju materice uz duže (trajanja sedam dana) prisustvo hirurškog drena u desnom ingvinumu. Uradena je hirurška ekscizija potkožnog čvora, čvrste konzistencije, dimenzija 40x25x20 mm. Histopatološkom analizom uočene su razbačane žlezde i ciste sa papilarnim produžecima u potkožnom masnom tkivu i umereno celularnom ožiljnom vezivu. Žlezde su bile obložene kockastim i cilindricnim epitelom i okružene stromom nalik endometrijalnoj. Papile su bile prekrivene cilndričnim epitelom sa cilijama i bazalnim ćelijama. Promene su bile fokalno prisutne na linijama resekcije. Nalaz je odgovarao endometriozi i endosalpingiozi u ožiljku. Imunohistohemijska analiza je potvrdila dijagnozu uz citokeratin 7, estrogen- i progesteron-receptor pozitivne fokuse endometrioze i endosalpingioze, CD10 pozitivnu endometrijalnu stromu i WT-1 i kalretinin pozitivan tubalni epitel. Zaključak: Ovo je prvi slucaj kombinovane endosalpingioze i endometrioze kože, kao i prvi slučaj endosalpingioze u ožiljku i van pupka. Ključne reči: endosalpingioza, endometrioza, koža, imunohistohemija Aim: Histopathological and immunohistochemical analysis of cutaneous scar endometriosis and endosalpingiosis. Introduction: Endosalpingiosis and endometriosis are an ectopic growth of the Fallopian tube epithelium and endometrial glands and stroma. Cutaneous endometriosis is well known entity with an incidence of 1.1% in surgically proven cases. Cutaneous endosalpingiosis is a rare entity, with only 5 cases reported up today. Material and Methods: Analysis of slides stained with hematoxylin-eosin and streptavidin-biotin immunohistochemical method. Results: A 40-year-old Caucasian woman presented with a 2-year history of a slowly growing subcutaneous nodule in right inguinum associated with cyclical pain during menses. Her past surgical history revealed myomectomy with prolonged placement of surgical drain in right inguinum. An excision under general anesthesia was made. A firm subcutaneous nodule measuring 40x25x20 mm was removed. Routine histological examination revealed scattered glands and cystic spaces with papillary structures within subcutaneous fat tissue and in the moderately cellular fibrous scar. Glands were covered with cubical to cylindrical epithelium and surrounded with endometrium-like stroma, and papillae were covered with cylindrical epithelium with cilia and peg cells. These structures were focally present on surgical margins. Diagnosis of scar endometriosis and endosalpingiosis has been made. Immunohistochemical analysis confirmed the diagnosis with cytokeratin 7, estrogen- and progesterone- receptors positive foci of endometriosis and endosalpingiosis, CD10 positive endometrial stroma and WT-1 and calretinin positive tubal epithelium. Conclusion: This is the first case of combined endosalpingiosis and endometriosis of the skin, and the first with endosalpingiosis occurring on scar and outside umbilicus. Key words: endosalpingiosis, endometriosis, skin, immunohistochemistry 1Institut za patologiju, Medicinski fakultet, Univerzitet u Beogradu, Beograd, Srbija 2Klinika za opekotine,plastičnu i rekonstruktivnu hirurgiju, Klinički centar Srbije, Beograd, Srbija 598 1Institute of Pathology, Faculty of Medicine, University of Belgrade, Belgrade, Serbia 2Clinic for Burns, Plastic and Reconstructive Surgery, Clinical Centre of Serbia, Belgrade, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 1/poster prezentacije 1 P06 Diferencijalna dijagnostika mastocitoma i histiocitoma pasa Differential diagnosis of mastocytoma and histiocytoma in dogs Ivana Vučičević, Sladjan Nešić, Sanja Aleksić-Kovačević Ivana Vucicevic, Sladjan Nesic, Sanja Aleksic-Kovacevic Cilj: Utvrdivanje ekspresije molekula značajnih za diferencijalnu dijagnozu i kontrolu rasta mastocitoma. Uvod: Mastocitomi su neoplazme poreklom iz kostne srži, iz linije opredeljene za mastocite. Kod pasa čine 7 - 21% od ukupnog broja tumora i mnogi imaju vrlo agresivan klinički tok sa metastazama. Diferencijalno dijagnosticki ih je teško razlikovati od histiocitoma samo na osnovu patohistološkog pregleda preparata bojenih hematoksilin-eozinom. Imunohistohemijsko ispitivanje mastocitoma pored diferencijalno dijagnostickog značaja, bitno je i sa aspekta klasifikacije mastocitoma. Materijal i metode: Ispitivanjem su obuhvaćeni isečci tumora kože ukupno 30 pasa različitog uzrasta, rase i pola. Uzorci kože su fiksirani u 10% neutralnom formalinu, a zatim su standarno procesovani do parafinskih blokova. Parafinski isečci bojeni su hematoksilin-eozinom i specifičnim bojenjem toluidine blue (TB). Kod isecaka tumora kože pozitivnih na toluidine blue bojenje, uradeno je imunohistohemijsko ispitivanje za CD117 (c-kit) i CD45RA. Rezultati: Od 30 uzoraka tumora kože, nakon patohistološkog ispitivanja, utvrdeno je 8 histiocitoma, a specificnim TB bojenjem potvrdeno je 16 mastocitoma sa intenzivno ljubičasto obojenim granulama histamina. 12 od 16 uzoraka mastocitoma bilo je pozitivno na CD117, to odgovara mastocitomima II gradusa. U njima je takode intenzivna ekspresija CD45RA. Zaključak: Mastocitomi pasa II gradusa eksprimiraju CD117 i CD45RA. Ovi receptori predstavljaju molekule značajne za ćelijsku proliferaciju, brzinu rasta tumora i metastatski potencijal. Za razliku od njih, histiocitomi su negativni na TB, brzo regresiraju i benignog su toka. Ključne reči: CD117, CD45RA, mastocitom, histiocitom, pas Aim: Determination of molecular expression important for differential diagnosis and growth control of mastocytomas. Introduction: The mast cell tumors originating from bone marrow, the line determined to mast cells. Mastocitomas represent 7 - 21% of all canine tumors and many show an aggressive clinical course with metastases. Differential diagnosis of mastocytoma and histiocytoma based on histopathological examination of samples stained with hematoxylin-eosin is very difficult. Immunohistochemical examination of mastocytomas has not only the differential diagnostic value, but is also important for the mast cell tumors classification. Material and Methods: The skin tumor samples included in this study were from 30 dogs of different ages, breed and sex. After the fixation in 10% neutral formalin, the skin tumor samples were cut into slices and processed by routine method to paraffin blocks. For paraffin embedded slices staining were used hematoxylin-eosin and specific toluidine blue (TB) staining. TB positive tumor samples were immunohistochemically analyzed for CD117 and CD45RA expression. Results: After histopathological analysis of the 30 skin tumor samples, 8 samples were histiocytomas, and using TB specific staining was confirmed that 16 samples were mastocytomas with purple colored granules of histamine. 12 of 16 mastocytomas were CD117 positive, which correspond to the grade II mast cell tumors. They also had intense CD45RA expression. Conclusion: Grade II mast cell tumors in dogs express CD117 and CD45RA. These receptors are important molecules for cell proliferation, tumor growth rate and metastatic potential. Unlike mastocitomas, histiocytomas were negative for TB, rapidly regresses and has benign course. Key words: CD117, CD45RA, dog, histiocytoma, mastocytoma Fakultet veterinarske medicine, Univerzitet u Beogradu, Beograd, Srbija Faculty of Veterinary Medičine, University of Belgrade, Belgrade, Republic of Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 599 POSTER PRESENTATIONS 1/poster prezentacije 1 P07 Elektronska mikroskopija kao pomoćna metoda u dijagnostici Histiocitoze - prikaz tri slučaja Electron microscopy as a supporting method in a diagnosis of Histiocytosis - report of tree cases Tamara Kravić Stevović1, Tamara Martinović1, Jelena Sopta2, Vesna Čemerikic Martinović3, Slaviša Djurišić4, Vesna Lačković1 Tamara Kravic Stevovic1, Tamara Martinovic1, Sopta Jelena2, Vesna Cemerikic Martinovic3, Djuricic Slavisa4, Lackovic Vesna1 Cilj: Cilj ovog rada je da se prikažu tri bolesnika sa histiocitozom kod kojih je kao pomoćna dijagnostička metoda uradjena ultrastrukturna analiza biopsijskog materijala elektronskim mikroskopom. Uvod: Histiocitoza Langerhansovih ćelija je klonalna proliferativna bolest ovih antigen prezentujućih dendritičnih ćelija. Materijali i metode: Uzorak tkiva jednog bolesnika fiksiran je u glutaraldehidu, dok su uzorci dva bolesnika bili prethodno fiksirani u formalinu, kalupljeni u parafinu, a kasnije deparafinizirani i fiksirani u glutaraldehidu. Uzorci fiksirani u glutaraldehidu prvo su ispirani u kakodilatnom puferu, fiksirani u osmijum tetroksidu, dehidrirani u alkoholima rastuće koncentracije i propilenoksidu, i kalupljeni u EPON-u. Na polutankim isečcima bojenim toluidin plavim procenjivano je prisustvo Langerhansovih ćelija, a na ultratankim isečcima, kontrastiranim uranil acetatom i olovo citratom, elektronskim mikroskopom analizirana je ultrastuktura Langerhansovih celija i detektovano prisustvo Birbekovih granula. Rezultati: Birbekove granule detektovane su u Langerhansovim ćelijama dva od tri bolesnika. Karakterističan izgled i mnoštvo Birbekovih granula vidjeno je u uzorku koji je prethodno bio fiksiran u glutaraldehidu. U uzorku koji prethodno nije bio adekvatno fiksiran, tj. koji je bio fiksiran u formalinu, membranske strukture, pa samim tim i Birbekove granule nisu bile u potpunosti očuvane, pa su vidjene retke membranske tubularne strukture karakterističnog periodičnog izgleda za Birbekove granule. Zaključak: Elektronska mikroskopija ima značaja kao pomoćna dijagnostička metoda u dijagnostici Histiocitoze Langerhansovih ćelija. Iako se Birbekove granule mogu detektovati i iz parafinskih blokova, u slučaju kada je moguće uzorak tkiva uzeti ponovo, sa adekvatnom fiksacijom i obradom za elektronsku mikroskopiju, povećava se verovatnoća i sigurnost detekcije Birbekovih granula. Ključne reci: histiocitoza, Langerhansove ćelije, Birbekove granule, ultrastruktura Aim: The aim of this study is to report tree cases with histiocytosis in which the electron microscopy as a supporting method in diagnosis was performed. Introduction: Langerhans cell histiocytosis is a clonal proliferative disorder of these antigen presenting dendritic cells. Material and methods: Biopsy material of one patient was fixed in gluaraldehyde, while other two were fixed in formalin, embedded in paraffin, and later deparaffinized and fixed in glutaraldehyde. Specimens fixed in glutaraldehyde, were washed in cacodylate buffer, fixed in osmium tetroxide, dehydrated in alcohols and propylene oxide, and embedded in EPON. Semi thin sections, stained with toluidin blue were used to asses the presence of Langerhans cells, while ultra thin sections, contrasted with uranyl acetate and lead citrate, were used for ultrastructural analysis and detection of Birbeck granules. Results: Birbeck granules were detected in Langerhans cells of two out of tree patients. Characteristic appearance and abundance of Birbeck granules were seen in a specimen that was adequately fixed in glutaraldehyde. In the other specimen that was first fixed in formalin, membrane structures, including Birbeck granule, were not completely preserved, due to the inadequate fixation. Rare membrane tubular structures, with characteristical periodicity for Birbeck granules were seen in this patient. Conclusion: Electron microscopy is a valuable supporting method in a diagnosis of Langerhans cell histiocytosis. Although detection of Birberck granules is possible from paraffin blocks, when it is possible to obtain tissue with adequate fixation, the possibility and specificity of the detection of Birbeck granules is increasing. Key words: histiocytosis, Langerhans cells, Birbeck granules, ultrastructure 1Institut za histologiju i embriologiju, Medicinski Fakultet Beograd, Beograd, Srbija 2Institut za patologiju, Medicinski Fakultet Beograd, Beograd, Srbija 3Beo-lab, Beograd, Srbija 4Institut za majku i dete Dr Vukan Čupić, Beograd, Srbija 600 1Institute for histology and embryology, Medical Faculty Belgrade, Belgrade, Serbia 2Institute for pathology, Medical Faculty Belgrade, Belgrade, Serbia 3Beolab, Belgrade, Serbia 4Mother and Child Health Care Institute of Serbia Dr Vukan Cupic, Belgrade, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 1/poster prezentacije 1 DIGESTIVNA PATOLOGIJA P08 Solidni pseudopapilarni tumor pankreasa GASTROENTHEROHEPATO PATHOLOGY Solid Pseudopapillary Neoplasm of the Pancreas Mirjana Živojinov1, Jelena Ilić2, Sandra Trivunić3, Mihaela Mocko-Kaćanski4, Aleksandra Levakov4, Srđan Živojinov5 Mirjana Živojinov1, Jelena Ilić2, Sandra Trivunić3, Mihaela Mocko-Kaćanski4, Aleksandra Levakov4, Srđan Živojinov5 2Univerzitet 2University 1KCV, Centar za histologiju i patologiju, Novi Sad,R.Srbija u Novom Sadu, Medicinski fakultet, Katedra za histologiju, Novi Sad, Srbija 3KCV, Centar za patologiju i histologiju, Katedra za patologiju, Novi Sad, Srbija 4KCV, Centar za patologiju i histologiju, Katedra za histologiju, Novi Sad, Srbija 5KCV, Institut za hirurgiju, Novi Sad, Srbija Cilj: Prikazati slučaj pacijenta sa pseudopapilarnim tumorom pankreasa. Uvod: Solidni pseudopapilarni tumor pankreasa je veoma retka neoplazma opisana od strane Frantz-a 1956. god. Pretežno je benignog karaktera sa učestalošcu od 1-2% svih egzokrinih tumora pankreasa. Najčešće se javlja kod adolescentkinja i žena starosti oko 35 godina. Materijal i metode: Odrađen je imunohistohemijski profil na Hromogranin, Vimentin, CD56 i S100. Postavljena je dijagnoza solidnog pseudopapilarnog (cističnog) tumora pankreasa sa neuroendokrinom diferencijacijom. Rezultati: U radu je prikazan slučaj pacijentkinje starosti 22 godine, kojoj je odstranjen deo pankreasa. Makroskopskim pregledom opisan je inkapsulisan fragment velicine 10x6 cm, sjajne i glatke površine, podlivene krvi. Na preseku se uočava meko tkivo, jasno ograničeno od okolnog tkiva pankreasa u vidu cistične formacije. Na serijskim rezovima se nalaze solidna područja tkiva i manje cistične strukture ispunjene koagulisanom krvi. Patohistološkom analizom tumorsko tkivo je sagrađeno od solidnih plaža koje čine okrugle tumorske ćelije, čija su jedra ovalna, pravilna, bez uočljivih jedaraca,oskudne acidofilne citoplazme. Mitoze su retke ( manje od 2 na 10 HPF ). Mestimično se uočavaju papile čija je stroma sagrađena iz vezivno-vaskularnog, delimično hijalinizovanog tkiva, a koje su obložene opisanim tumorskim ćelijama. Prisutni su holesterolski kristali, makrofagi penušave citoplazme, džinovske ćelije tipa oko stranog tela kao i okruglasta hijalina područja amorfnog izgleda. Prisutno je krvarenje u vezivnoj hijalinoj kapsuli koja tumorsko tkivo ograđuje od okolnog tkiva pankreasa, kao i znaci starog krvarenja. Zaključak: Pravilna dijagnostika ovih tumora je bitna, s obzirom da se pravovremenom i kompletnom resekcijom mož e postići potpuno izlečenje ove neoplazme. Ključne reči: pseudopapilarni tumor, pankreas, imunohistohemija 1CCV, Center for histology and pathology, Novi Sad, Serbia of Novi Sad, Medical faculty, Department for histology, Novi Sad, Serbia 3CCV, Center for pathology and histology, Department for pathology, Novi Sad, Serbia 4CCV, Center For pathology and histology, Department for histology, Novi Sad, Serbia 5CCV, Institut for surgery, Novi Sad, Serbia Aim: To show case report of pacient with solid pseudopapillary neoplasm of the pancreas. Introduction: Solid pseudopapillary tumor of the pancreas is rare neoplasm, described in 1959 by Frantz. It is mostly of low malignant potential. It accounts for approximately 1 2% of all exocrine pancreatic tumors. It occurs predominantly in adolescent girls and young women (mean 35 years). Material and methods: Positive immunoreactivity was for Chromogranin, Vimentin, CD56 and S100. After histological examination, diagnosed of solid pseudopapillary tumor of the pancreas with neuroendocrine differentiation was done. Results: Our paper showed a case in 22-year-old female with tumor of pancreas. After removing a part of pancreas, pathohistological analysis was done. By macroscopic examination, it was described encapsulated fragment, size 10x6 cm, shiny and smooth surface with haemorrhage zones. There was found soft tissue, well demarcated from the surrounding pancreas. It was in a form of cystic formation. On serial sections there were solid areas and cystic structures filled with coagulated blood. Histologicaly tumor tissue was built of round cells with ovoid nuclei and clear cytoplasm. Nucleoli were inconspicuous. Mitotic figures were rare (less than 2 on 10 HPF). In some places papillae were observed, composed from partially hyalinised connective tissue. Groups of foamy macrophages were accumulated, and there were clusters of lipid crystals surrounded by foreign body giant cells. There was a haemorrhage in connective hyalinised capsule and signs of old haemorrhages. Conclusion: Proper diagnosis of these tumors is important. Considering well-timed diagnosis and complete resection, healing is possible. Key words: pseudopapillary neoplasm, pancreas, immunohistochemistry 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 601 POSTER PRESENTATIONS 1/poster prezentacije 1 P09 Prevalenca i tipovi kongenitalnih anomalija gastrointestinalnog trakta Prevalence and types of the congenital anomalies of the gastrointestinal tract Radmila Janković1, Martina Stojanović1, Branislav Lekić1, Biljana Parapid2, Srđan Aleksandrić2, Zorica Stojšić1 Radmila Jankovic1, Martina Stojanovic1, Branislav Lekić1, Biljana Parapid2, Srdjan Aleksandric2, Zorica Stojšić1 Cilj: Cilj rada je da se odrede prevalenca i kategorizacija kongenitalnih malformacija gastrointestinalnog trakta u pedijatrijskoj populaciji. Uvod: Kongenitalne anomalije gastrointestinalnog trakta su retke. Neke od njih su letalne po rodjenju, dok neke anomalije treba razlikovati od drugih cističnih lezija abdomena. Materijal i metode: Retrospektivna analiza učestalosti različitih gastrointestinalnih anomalija operisanih u Dečjoj Univerzitetskoj Klinici u Beogradu u desetogodišnjem periodu (2002-2011). Podaci o makro- i mikromorfologiji prikupljani su iz patoloških izveštaja arhive Instituta za patologiju u Beogradu. Rezultati: Registrovano je 113 slučajeva kongenitalnih malformacija gastrointestinalnog trakta: 61 Mekelov divertikulum (54%), 25 intestinalnih atrezija/stenoza (28%) i 14 mezenterijalnih cista crevnog porekla (12%). Retke anomalije su bile: 4 umbilikoilealne fistula i ciste (3,5%) i divertikulumi želuca, duodenuma i jejunuma (po 0,8%). Najčešći tip atrezije je bio tip I (11), zatim tip IIIa (5), tip II (4) sa retkom naslednom formom i tip IIIb (1). Mezenterijalne ciste crevnog porekla su uključivale cistične duplikature u zidu ileuma (9), ileuma i cekuma (2) i u torakoabdominalnoj šupljini (1) i izolovane enterične ciste u mezenterijumu ileuma (1) i okoline pankreasa (1). Zaključak: Cistične kongenitalne anomalije gastrointestinalnog trakta neophodno je ispravno diganostikovati i razlikovati od drugih oblika mezenterijalnih cista. Ključne reči: malformacija gastrointestinalnog trakta, intestinalna duplikatura, intestinalna atrezija, mezenterijalna cista Aim: The aim of this study was to determine the prevalence and categorize the variants of congenital malformations of the gastrointestinal tract in the pediatric age. Introduction: Congenital anomalies of the gastrointestinal tract are relatively rare. Some forms cause inevitably neonatal mortality, and the others are included in the differential diagnosis of the cystic lesions of the abdominal cavity. Material and methods: The retrospective analysis of a 10-year (2002-2011) prevalence of various gastrointestional congenital malformations operated at the University Children’s Hospital Belgrade. Gross and histological findings were recorded from the pathology reports of the Institute of Pathology archival files. Results: 113 cases of congenital malformations of the gastrointestinal tract were disclosed as follows: Meckel s diverticulum, 61 cases (54%), intestinal atresia/stenosis, 25 (28%) and mesenterial cysts of enteric origin, 14 (12%). Rare anomalies included umbilicoileal fistula and vitelline cysts, 4 (3,5%) and congenital diverticulum of the stomach, duodenum and jejunum, one case each (0.8%). The most frequent type of intestinal atresia was type I (11), followed by type IIIa (5), type II (4), type IV (2) including a rare form of hereditary seieve-like atresia and type IIIb (1). Mesenteric cysts of enteric origin included duplication cyst that were situated within ileum (9), ileum and cekum (2) and thoracoabdominal cavity (1), as well as isolated enteric cysts positioned within ileal mesenterium (1) and immediate to the pancreas (1). Conclusion: Appropriate diagnosis of cystic congenital anomalies of the gastrointestinal tract is essential regarding the other forms of mesenteric cysts. Key words: gastrointestinal malformation, intestinal duplication, intestinal atresia, mesenteric cysts 1Institut za patologiju, Medicinski fakultet Univerziteta u Beogradu, Beograd, Srbija 2Klinika za kardiologiju, Klinički centar Srbije, Beograd, Srbija 602 1Institute of pathology, Faculty of medicine, University of Belgrade, Belgrade, Serbia 2Division of Cardiology, Clinical Center of Serbia, Belgrade, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 1/poster prezentacije 1 P10 Imunohistohemijsko proučavanje ekspresije mucina u želudacnom karcinomu Immunohistochemical study of mucins expression in gastric carcinoma Miljan Krstić, Dragan Mihailović, Žaklina Mijović, Ljubinka Veličković, Slavica Stojnev, Ana Ristić, Nikola Živković Krstic Miljan, Dragan Mihailovic, Zaklina Mijovic, Ljubinka Velickovic, Slavica Stojnev, Ana Ristic, Nikola Zivkovic Cilj: Imunohistohemijsko ispitivanje ekspresije MUC2, MUC5AC, MUC6 kod intestinalnih i difuznih karcinoma želuca sa ciljem utvrdjivanja razlika u ekspresiji i uticaju na diferencijaciju, progresiju i prognozu. Uvod: Karcinom želuca je, kao jedan od najčešcih malignih tumora na globalnom svetskom nivou, još uvek je veliki zdravstveni problem i izazov za kliničare i patologe. Histogeneza nije u potpunosti razjaš -njena, a saznanja o prekanceroznim lezijama su nekompletirana. Klasifikacione šeme su često komplikovane, ali je najprihvaćenija podela na dva osnovna tipa, intestinalni i difuzni karcinom želuca. Za svaki od njih je pretpostavljena drugacija etiologija, karcinogeneza i prognoza. Materijali i metode: Korišćen je operativni materijal 62 pacijenta sa resekovanim želucem (29 intestinalnih i 33 difuzna). Materijal je obradjivan klasično, bojen hematoxylin i eosin (HE) i imunohistohemijski (EnVisionZ Dual Link System-HRP (DAB ). Korišćena su antitela: 1. MUC2 (Clone Ccp58), 2. MUC5AC (Clone CLH2) i 3. MUC6 (Clone CLH5) - Novocastra, Newcastle, UK. Bodovanje imunohistohemijske ekspresije je bazirano na German ImmunoReactive Score. Rezultati: Postoji statistički značajna razlika u ekspresiji MUC5AC (t=0.006) i MUC2 (t=0.004) izmedju intestinalnih i difuznih karcinoma želuca. Ekspresija MUC2 opada sa opadanjem diferencijacije. Ekspresija MUC6 ne pokazuje statistički signifikantnu razliku izmedju 2 ispitivane grupe. Zaključak: Istraživanje je pokazalo da je mucinska ekspresija koristan dijagnostički parametar za ispravnu histogenetsku dijagnozu i klasifikaciju želudacnih karcinoma.MUC5AC je prediktor za difuzni karcinom želuca, a MUC2 ekspresija je značajno učestalija kod intestinalnog tipa karcinoma želuca i signifikantniji marker stepena diferencijacije. Ključne reči: karcinom želuca, imunohistohemija, mucini Aim: Immunohistochemical study of expression of mucins, that included tissue specimens of the intestinal and diffuse gastric cancer, has been carried out in order to determine differences in the expression between the two cancer types, influence on differentiation, progression and their possible significance for prognosis. Introduction: Gastric carcinoma still represents a major health problem and remains enigma and a challenge for both clinicians and pathologists. The histogenesis has not been fully elucidated so far and the knowledge of precancerous lesions is incomplete. Classification schemes are often complicated, but the most accepted is the division into intestinal and diffuse gastric cancer. Each of these two cancer types has different and specific etiology, carcinogenesis and prognosis. Material and methods: The operative material obtained from 62 patients with stomach carcinoma (29 intestinal and 33 diffuse) was used. The samples were processed conventionally and HE stained The immunohistochemical analysis was performed using EnVisionŽ Dual Link System-HRP (DAB ) with MUC2 (Ccp58), MUC5AC (CLH2) and MUC6 (CLH5) - Novocastra, Newcastle, UK. Scoring of immunohistochemical expression was based on German ImmunoReactive Score. Results: There was statistically significant difference in expression of MUC5AC and MUC2 between intestinal and diffuse gastric cancer. Expression of MUC2 decreases with decreasing of differentiation. Expression of MUC6 showed no statistically significant difference. Conclusion: The study shows that mucin expression represents a useful diagnostic tool for the correct histogenetic diagnosis and classification of gastric carcinoma, with MUC5AC being a predictor of diffuse type, and MUC2 a significant marker for degree of differentiation. Key words: Stomach carcinoma, immunohistochemistry, mucins Institut za patologiju, Medicinski fakultet Niš, Centar za patologiju, Klinički centar Niš Department of Pathology, Medical Faculty Nis and Center for Pathology of Clinical Center Nis 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 603 POSTER PRESENTATIONS 1/poster prezentacije 1 ENDOKRINA PATOLOGIJA P11 Plazmocitni granulom štitaste žlezde udružen sa Hašimotovim tireoiditisom Duško Dunđerović1, Tatjana Terzić1, Svetislav Tatić1, Jasmina Marković- Lipkovski1, Vesna Božić2, Vladan Živaljević2, Marija Havelka Đuković1 1Institut za patologiju, Medicinski fakultet, Beograd, Srbija 2Klinički centar Srbije, Beograd, Srbija Cilj: Presentovati slučaj pacijenta sa plazmocitnim granulom, retkim uzrokom strume i hipotireoze. Uvod: Plazmocitni granulom (plasma cell granulom-PCG) je retka tumoru-slična lezija, sa obimnim plazmocitnim infiltratom u fibrotičnoj stromi. Pojava PCG u štitastoj lezdi je veoma neuobičajena i retka. Do sada je u literaturi opisano 19 slucajeva PCG tireoidne ž lezde od kojih su četiri udružena sa Hašimotovim tireoiditisom. Materijal i metode: Dijagnoza je postavljena na osnovu analize hematoksilin-eozin obojenih preseka parafinskih uzoraka, kao i imunohistohemijske analize (CD20, CD3, CD38, CD138, kappa, lambda, IgG, IgA, IgM, CD56). Rezultati: Žena starosti 57 godina je primljena radi operativnog lečenja recidivantne strume. Pacijentkinji je urađena subtotalna tiroidektomija u 27 godini života. Sa navršenih 55 godina starosti, primećen je ponovni rast štitaste lezde, zbog čega je bila godinu dana na supstitucionoj terapiji tiroksinom. Preoperativno, biopsijom tankom iglom (FNAB) postavljena je dijagnoza papilarnog karcinoma. Uradena je totalna leva lobektomija i parcijalna desna lobektomija. Patohistološki, tkivo štitaste žlezde je bilo obimno difuzno prožeto zapaljenskim infiltratom između atrofičnih tireoidnih folikula, sa naglašenom fibrozom strome. Zapaljenski infiltrat je predstavljen retkim sekundarnim limfnim folikulima (CD20 ) i brojnim plazmocitima (CD38, CD138). Imunohistohemijski, plazmociti su poliklonski (odnos kappa/lambda oko 3:1, dominantno IgG, CD56-). Fokalno se uočava skvamozna metaplazija rezidualnih tireoidnih folikularnih ćelija. Biohemijska testiranja funkcije štitaste žlezde i postojanje antitela na tireoidnu peroksidazu su u saglasnosti sa postojanjem Hašhimotovog tireoiditisa. Morfološki i imunohistohemijski nalaz odgovara PCG sa morfološ kim i kliničkim znacima Hašimotovog tireoiditisa. Zaključak: Dijagnoza PCG može predstavljati diferencijalno dijagnostički problem u odnosu na papilarni karcinom (posebno u FNAB-u), ekstramedularni plazmocitom, kao i infektivne i autoimune bolesti. Ključne reči: struma, Hashimoto thyreoiditis, plazmocitni granulom 604 ENDOCRINE PATHOLOGY Plasma cell granuloma of the thyroid gland associated with Hashimoto thyroiditis Dusko Dunderovic1, Tatjana Terzic1, Svetislav Tatic1, Jasmina Markovic- Lipkovski1, Vesna Bozic2, Vladan Zivaljevic2, Marija Havelka Djukovic1 1Institute 2Clinical of pathology, Faculty of medicine, Belgrade, Serbia center of Serbia, Belgrade, Serbia Aim: To report case of a patient with a plasma cell granuloma of the thyroid,a rare cause of goiter and hypothyroidism. Introduction: Plasma cell granuloma (PCG) is rare tumor-like lesion, with numerous polyclonal plasma cells and stromal fibrosis. Thyroid gland involvement by PCG is rare and unusual. There are only 19 cases in literature until now, including 4 cases associated with Hashimoto thyroiditis Material and methods: The diagnosis was obtained on review of hematoxylin and eosin stained paraffin- embedded slides and immunohistochemical data (CD20, CD3, CD38, CD138, kappa, lambda, IgG, IgA, IgM, CD56). Results: A 57-year-old female, was admitted to hospital for operative treatment of relapsing goiter. Patient has had subtotal thyroidectomy in the age of 27. Twenty eight years later, increase of the size of thyroid gland has been observed. Patient was treated by thyroid hormones for one year. Preoperatively, fine needle aspiration biopsy (FNAB) was performed and diagnosis of papillary carcinoma has been made. Total left lobectomy and partial right lobectomy has been done. Histological examination revealed an abundant inflammatory infiltrate between atrophic thyroid follicles and prominent stromal fibrosis. Inflammatory infiltrate was composed of rare secondary lymphoid follicles (CD20 ) and numerous plasma cells (CD38,CD138 ). Immunohistochemically, plasma cells were polyclonal (kappa/lambda approximately 3:1, predominantly IgG, CD56-). Focally, squamous metaplasia of residual thyroid follicles was present. Biochemical functional tests and presence of thyroid peroxidase antibodies were consistent with Hashimoto thyroiditis. Morphological, immunohistochemical and clinical findings correspond to PCG associated with Hashimoto thyroiditis. Conclusion: Diagnosis of PCG could be diagnostic problem, and differential diagnosis with papillary carcinoma (especially in FNAB), extramedullary plasmacytoma, infectious and autoimmune diseases should be taken into consideration. Key words: Goiter, Hashimoto thyreoiditis, plasma cell granuloma 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 1/poster prezentacije 1 P12 Medularni karcinom i dobro diferentovani karcinomi štitaste žlezde kao komponente kolizionih tumora: prikaz tri slučaja Božidar Kovačević, Snežana Cerović, Mile Ignjatović, Sanja Nikolajević, Petar Noack, Snežana Kuzmić Janković Institut za patologiju i sudsku medicinu Vojnomedicinska akdemija, Beograd, Srbija Cilj: Prikaz tri slučaja kao kolizionih tumora štitaste žlezde: dva slučaja predstavljaju istovremeno prisustvo medularnog karcinoma sa papilarnim karcinomom, u trećem slučaju prijavljujemo udruženost papilarnog karcinoma sa oksifiolnim karcinomom štitaste žlezde. Uvod: Primarni kolizioni tumori štitaste ž lezde su retke neoplazme (manje od 1%), sačinjene od najmanje dve različite histomorfološke komponente koje mogu biti u kontinuitetu ili odvojene normalnim tkivom. Materijal i metode: Kod tri bolesnika ženskog pola učinjena je totalna tireoidektomija zbog preoperativno citološki i klinički utvrđjene sumnje na postojanje medularnog karcinoma i folikularne lezije nejasnog malignog potencijala nalik oksifilnoj leziji. U svim slučajevima primenjena je intraopreativna dijagnostika sa dva odgovora u smislu medularnog karcinoma i jednim odgovorom da se radi o benignoj leziji. Rezultati: Kod jedne bolesnice utvrdili smo izdvojene tumore lokalizovane u odvojenim lobusima- medularni i papilarni karcinom, dok je u drugom slučaju u okolini medularnog karcinoma u istom lobusu, kao i usuprotnom, detektovano nekoliko mikrofokusa papilarnog karcinoma. U trećem slučaju dominanti supstrat predstavlja nodus sa karakteristikama oksifilnog karcinoma i papilarni karcinom sa multiplim poljima rasta u oba lobusa. Imunohistohemijski profil korišćenjem antitela za neuroendokrine markere, uz CK19, HBME, CEA i tireoglobulin pokazao je dvojne antigene strukture u različitim tipovima karcinoma. Zaključak: Medularni i dobro diferentovani karcinomi folikularne diferencijacije kao komponente kolizionih tumora imaju različiti maligni potencijal i biolo ko ponašanje. Njihova dijagnoza i terapija predstavljaju izazov. Tretman kolizionih tumora zavisi od kombinacije primarnih tumora. Svaka komponenta se tretira kao nezavisni primarni tumor. Ključne reči: karcinomi štitaste žlezde, kolizioni tumori Medullary and well differented thyroid carcinomas occurring as a collision tumors: report of three cases Bozidar Kovačevic, Snezana Cerovic, Mile Ignjatovic, Sanja Nikolajevic, Petar Noack, Snezana Kuzmic Jankovic Institute of pathology and forensic medicine Military medical Academy, Belgrade Aim: We present three cases as a collision tumors of the thyroid gland: two cases represent simultaneous occurrence of medullary carcinoma (MTC) and papillary carcinoma (PTC) and the thrid case represents coexistence of an oxyphylic carcinoma and a PTC of the thyreoid gland. Introduction: Primary collision tumors of the thyroid gland are rare neoplasms, composed of at least two different hystomorfologicall components which can be in continuitiy or separated with normal tissue. Material and methods: A total thyroidectomy was performed in the three female patients due to a preoperatively cytologically and clinically confirmed suspicion of the existence of a MTC and a follicular lesion of an uncertain malignant potential, alike to an oxyphylic lesion. In both cases an intraoperative diagnosis was performed, in two cases with a MTC and in one case with a benign lesion being diagnosed. Results: In two patients we detected MTC and PTC. In the third case we detected oxyphylic carcinoma and a PTC with multiple regions of growth in both lobes. The immunohistochemical profile (neuroendocrine markers, calcitonine, CEA, thyreoglobuline, CK19, HBME-1) showed antigene structures in different tumors, which corresponded with two different types of carcinomas in each single case. Conclusion: MTC and well differented carcinomas of follicular differentiation as components of the collision tumors have different malignant potential as well as biological behavior. Tretmant of collision tumors depend on the combinantion of primary tumors. Each component is treated as an independent primary tumor. Key words: thyroid gland carcinoma, collision tumors 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 605 POSTER PRESENTATIONS 1/poster prezentacije 1 GLAVA I VRAT PATOLOGIJA HEAD AND NECK PATHOLOGY P13 Mukozni melanom - prikaz slučaja Mucosal melanoma - case report Jordan Radojčić, Dragan Živojinovic, Petar Noack, Milo Zarić, Jovanka Trifunović, Saša Ristic, Jelena Vrtikapa, Biserka Vukomanović - Đurdević Jordan Radojcic, Dragan Zivojinovic, Petar Noack, Milo Zaric, Jovanka Trifunovic, Sasa Ristic, Jelena Vrtikapa, Biserka Vukomanovic Djurdevic Cilj: Prikaz slučaja primarnog malignog melanoma respiratorne sluznice kao ekstremno retkog tumora. Uvod: Pacijent u straosti 73 godine se javio sa nespecifičnom simptomatologijom od strane sinonazalnog trakta, znacima nazalne opstrukcije i epistaksom. Klinickim pregledom je utvrđeno prisustvo ekspanzivne tumorske mase koja je zahvatala sinonazalni trakt. Učijena je operacija. Materijal i metode: Imunohistohemijske analize tumorskog tkiva su neophodna dijagnostička procedura. Za definitivni staging bolesti je neophodna kliničko-patološka korelacija. Rezultati: Patohistološkim pregledom je pokazano prisustvo loše diferentovanog malignog tumora koga su činile pleomorfne tumorske ćelije epiteloidnog izgleda. Imunohistohemijsko analizom tumorske ćelije su bile Pancitokeratin-, Vimentin, GFAP-, S100, MelanA, HMB45 . Daljom, detaljnom kliničkom analizom je isključeno postojanje melanoma van sinonazalnog trakta, što je u korelaciji sa mikroskopskom slikom upućivalo sa se radi o primarnom malignom melanomu respiratorne sluznice. Zaključak: Dijagnostika malignog melanoma sinonazalnog trakta zahteva pored standradnog HE bojenja i dopunske imunohistohemijske analize, a za definitivni staging bolesti je neophodna klinička korelacija koja upućuje na primarno ishodište. Ključne reči: melanom, imunohistohemija, epistaksa, sinonazalni trakt Aim: Case report of a primary malignant melanoma of the respiratory mucosa as an example of an extremely rare tumor. Introduction: A patient aged 73 years was submitted with nonspecific symptomatology of the sinonasal cavity, nasal obstruction and epistaxis. Clinical examination revealed an expansive tumor mass in the sinonasal cavity. Surgery was performed. Material and methods: Immunohistochemical analyses are essential diagnostic tools. For a definitive staging of the disease, a clinico-pathological correlation is needed. Results: Pathohistological examination revealed the presence of a poorly differentiated malignant tumor which consisted of pleomorphic epithelial cells. Immunohistochemically, tumor cells were Pancitokeratin-, Vimentin, GFAP-, S100, MelanA, HMB45 . Detailed clinical examinations excluded the presence of melanoma of other localizations, which confirmed in correlation with the microscopic findings the presence of a primary malignant melanoma of the respiratory mucosa. Conclusion: The diagnosis of the malignant melanoma of the sinonasal cavity requires the use of standard HE and additional immunohistochemical staining and analysis. For definitive staging of the disease requires clinical correlation suggests that the primary outcome. Key words: melanoma, immunohistochemistry, epistaxis, sinonasal mucosa Vojnomedicinska akademija, Beograd, Srbija 606 Military Medical Academy, Belgrade, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 1/poster prezentacije 1 P14 Jugulotimpanični paraganglion Jugulotympanic paraganglioma Desanka Tasić-Dimov1, Irena Dimov2, Milan Stanković3, Aleksandar Stojanov4, Petar Stanković4, Dragan Dimov1 Desanka Tasic Dimov1, Irena Dimov2, Milan Stankovic3, Aleksandar Stojanov4, Petar Stankovic4, Dragan Dimov1 2Institut 2Institute 1Institut za Patologiju, Medicinski fakultet, Niš, Srbija za imunologiju, Medicinski fakultet, Niš, Srbija, 3Klinika za otorinolaringologiju, Klinički centar Niš, Srbija, 4Medicinski fakultet, Niš, Srbija, Cilj: Prikaz slučaja jugulotimpaničnog paraganglioma sa ranim recidivom, neuobičajenog za pol i uzrast pacijenta. Uvod: Jugulotimpanični paragangliomi su retki neuroendokrini tumori, koji pokazuju izrazitu predilekciju za ženski pol i javljaju se većinom u srednjoj životnoj dobi. Obično se prezentuju u srednjem uhu. Materijal i metode: Prikazujemo slučaj jugulotimpaničnog paraganglioma kod muškarca životne dobi od 28 godina sa poremećajem sluha. Tumor je hirurški uklonjen u delovima. Na osnovu histopatološke slike i imunohistohemijskih analiza utvrđeno je da se radi o paragangliomu. Posle godinu dana javio se lokalni recidiv, zbog lokalizacije i invazivne prirode lezije. Metastaze u limfnim nodusima i u udaljenim organima nisu bile prisutne. Rezultati: Mikroskopski, tumor je bio građen od solidnih gnezda ćelija (Zellballen) okruženih fibroznim trakama sa brojnim krvnim sudovima tipa kapilara. Jedra tumorskih ćelija bila su uglavnom mala, okrugla, sa neupadljivim jedarcima, međutim, nađene su i ćelije sa uvećanim, iregularnim i hiperhromnim jedrima. Količina stromalnog vezivnog tkiva je varirala. Polja sa širokim trakama vezivnog tkiva i hijalinizovanim masama bila su takođe zastupljena i dominirala su u recidivantnom tumoru. U recidivantnom tumoru uočena je i infiltracija okolnih struktura. Glavne ćelije su bile imunopozitivne na neuron specificnu enolazu (NSE) i hromogranin A. Tanke, izdužene sustentakularne ćelije identifikovane su na bazi imunopozitivnosti na S-100 protein, ali su bile relativno retke. Zaključak: Prikazani slučaj jugulotimpaničnog paraganglioma kod mladog odraslog muškarca sa pojavom ranog recidiva ukazuje na nepohodnost daljeg praćenja pacijenta i sugeriše potrebu za genetskom analizom. Ključne reči: jugulotimpanični paragangliom, vrat, mlađi muškarac 1Institute for Pathology, Faculty of Medicine, Nis, Serbia for Immunology, Faculty of Medicine, Nis, Serbia 3Clinic for Othorinolaringology, Clinical Center of Nis, Serbia, 4Faculty of Medicine, Nis, Serbia Faculty of Medicine, Nis,Serbija, Aim: Presentation of jugulotympanic paragangioma with early recurrence unusual for age and sex of the patient. Introduction: Jugulotympanic paragangliomas are uncommon neuroendocrine tumors, show a strong female predilection and occur mainly in the middle age group. They usually arise laterally and are present in the middle ear. Material and methods: The authors present a case of jugulotympanic paragangioma in a 28-year old man with hearing impairment. The tumor was surgically removed into fragments. The histopathological feature and immunohistochemical analysis revealed that the tumor was paraganglioma. Because of the invasiveness and location of lesion, local recurrence occurred after one year. The lymph node involvement and distant metastases were not present. Results: Microscopically, the tumor was composed of solid cell nests (Zellballen) surrounded by fibrous trabeculae containing numerous capillary blood vessels. The tumor cell nuclei were mainly small and round with inconspicuous nucleoli, however, cells with enlarged, irregular and hyperchromatic nuclei could be easily found. Stromal connective tissue varied throughout the tumor, and areas with fibrous bands and densely hyalinized masses were present as well, and dominated in recurred tumor mass. In the recurrent tumor infiltration of surrounding structures were readily seen. The chief cells were immunopositive for neuron specific enolase (NSE) and chromogranin A. The thin, elongated sustentacular cells were identified by immunopositivity to S-100 protein, but were relatively sparse. Conclusion: Presented case of jugulotympanic paraganglioma with early recurrence in young adult man indicates that extended follow-up is required and suggests need for genetic analysis. Key words: jugulotympanic paraganglioma, neck, younger man 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 607 POSTER PRESENTATIONS 1/poster prezentacije 1 P15 Imunohistohemijska analiza Spindle - cell carcinoma larinksa - prikaz slučaja Immunohistochemical analysis Spindle-cell carcinoma of the larynx Case report Biserka Vukomanović-Djurdjević, Jordan Radojičić, Jovanka Trifunović, Petar Noack, Jelena Ristić, Jelena Vrtikapa Biserka Vukomanovic-Djurdjevic, Jordan Radojicic, Jovanka Trifunovic, Petar Noack, Jelena Ristic, Jelena Vrtikapa Cilj: Prikaz značaja imunohistohemije u dijagnozi spindle cell karcinoma Uvod: Spindle-cell karcinomi su predmet kontoverze u literaturi zbog svoje bifazične strukture lezije sa skvamocelularnim fokusima i učestalom proliferacijom vretenastih ćelija sarkomskog izgleda. Ovakva slika u literaturi se pominje kao skvamozni karcinom sa sarkomatozno nalik stromom, polipoidni karcinom, sarkomatoidni karcinom. U analizi tumora je neophodna dijagnostička procedura kojom se identifikuju komponente tumora. Materijal i metode: Pacijent starosti 80 godina sa nespecifičnim simptomima od strane larinksa. Endoskopski dijagnostifikovan tumor je bio lociran u glotičnoj regiji, polipoidnog izgleda najvećeg prečnika 8mm sa ulceracijom i fibrinskim naslagama. Rezultati: Mikroskopski u celini je bio sačinjen od vretenastih kao i atipičnih, bizarnih, dzinovskih ćelija iregularno organizovanih sa vaskularnom invazijom tumorskim tkivom. Površni epitel je pokazivao displaziju visokog stepena. Imunohistohemijski profil pokazan na tumorskim ćelijama je bio Vimentinpozitivan u svim ćelijama, a markeri HMWCK, PanCK, CK7 su bili pozitivni u delu tumora, a CK5/6, CK17, CK18, CK20 i S-100 su bili negativni. Naša dijagnoza je bila spindle-cell karcinom. Zaključak: Imunohistohemijske analize su neophodne za dijagnostiku spindle cell karcinom larinksa kojom se dokazuje epitelna komponenta tumorskih ćelija. Ključne reči: larinks, imunohistohemija, spindle cell karcinom Aim: To show the imortance of immunohistochemistry in the diagnosis of the spindle cell carcinoma Introduction: Spindle cell carcinomas are an issue of controversies in the literature, which has its origin in the characteristic biphasic structure of the lesion with squamocellular foci and frequent spindle sarcoma-like cell proliferation. This appearance is mentioned in the literature as a squamocellular carcinoma with a sarkomatoid stroma, as a polypoid carcinoma, as a sarkomatoid carcinoma. For the analysis, a diagnostical procedure is required which allows the identification of the tumor component. Material and methods: A patient aged 80 years was submitted with nonspecific symptoms of the larynx. The endoscopically diagnosed tumor was located in the glottic region, was of a polypoid shape with a diameter of 8mm with an ulcer and fibrinous deposits. Results: Microscopically, the tumor consisted of spindle-like as well as atypical, bizarre, giant cells of an irregular organization and with vascular infiltration. The superficial epithelium showed a high-grade displasy. The immunohistochemical profile within the tumor cells was Vimentin-positive in all cells, whereas the markers HMWCK, Pan-CK, CK7 were positive in a part of the neoplasm. CK5/6, CK17, CK18, CK20 and S-100 were negative. Our diagnosis was spindle cell carcinoma. Conclusion: Immunohistochemical analyses are necessary for the diagnosis of the spindle cell carcinoma of the larynx, with which aid the epithelial component of tumor cells can be shown. Key words: larynx, immunohistochemistry, spindle cell carcinoma Vojnomedicinska Akademija, Beograd, Srbija 608 Military Medical Academy, Belgrade, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 1/poster prezentacije 1 HEMATOPATOLOGIJA HAEMATOPATHOLOGY P16 Morfološke karakteristike leukemijskih limfocita bolesnika sa hroničnom limfocitnom leukemijom posle inkubacije sa lovastatinom Morphological characteristics of leukemic cells of patients with chronic lymphocytic leukemia treated with lovastatin Tamara Kravić-Stevović1, Andrija Bogdanović2, Darinka Bošković2, Vladimir Bumbaširević1 Tamara Kravic-Stevovic1, Andrija Bogdanovic2, Darinka Boskovic2, Vladimir Bumbasirevic1 Cilj: Cilj rada bio je da se ispita uticaj lovastatina na pojavu ćelijske smrti leukemijskih limfocita bolesnika sa hroničnom limfocitnom leukemijom (HLL). Uvod: Lovastatin inhibi e3-hidroksi-3 metil glutaril koenzim A reduktaza, enzim vazan u mevalonatnom putu sinteze holesterola, ciji su produkti neophodni za aktivaciju proteina uključenih u procese ćelijske smrti. Materijali i metode: Mononuklearne ćelije izdvojene iz periferne krvi 30 bolesnika sa HLL inkubirane su u RPMI-1640 medijumu, sa 10% seruma, i lovastatinom (10 ľM, 20 ľM, 50 ľM, 100 ľM). Posle 24h inkubacije (370C, 5% CO2, 98% vlažnosti) uzorci ćelija su bojeni akridin oranžom i Mitotrackerom, pravljeni su razmazi ćelija, dok je ostatak ćelija fiksiran u glutaraldehidu i dalje obradjivan radi dobijanja ultratankih preparata. Rezultati: Elektronskim mikroskopom je utvrdjeno da značajan broj leukemijskih ćelija tretiranih lovastatinom pokazuje karakteristike apoptoze i autofagije, kao i da ćelije imaju različite promene na mitohondrijama. Procenti apoptoze ćelija inkubiranih sa lovastatinom, odredjivani na razmazima bojenim TUNEL tehnikom, bili su veći od vrednosti dobijenih posle inkubacije bez leka, za sve korišćene koncentracije lovastatina (p<0,05, Exact Wilcoxon test). Indukcija autofagije u leukemijskim ćelijama tretiranim lovastatinom pokazana je akumulacijom crvenih vezikula obojenih akridin oranžom u citoplazmi ovih ćelija. Primenom konfokalne mikroskopije zaključeno je da Mcl-1 protein u ćelijama tretiranim lovastatinom prelazi iz citoplazme u mitohondrije, dok je Bim protein u leukemijskim ćelijama lokalizovan u mitohondrijama, a primena lovastatina ne dovodi do promene lokalizacije ovog proteina. Zaključak: Rezultati ovog istraživanja pokazuju da lovastatin dovodi do indukcije ćelijske smrti u leukemijskim ćelijama bolesnika sa HLL. Ključne reči: lovastatin, HLL, apoptoza, autofagija, ultrastruktura Aim: The aim of this study was to investigate the effect of the lovastatin on the induction of the cell death of leukemic lymphocytes of patients with chronic lymphocytic leukemia (CLL). Introduction: Lovastatin inhibits 3-hydroxy-3-methil glutaryl coenzyme A reductase, important enzyme in mevanolate pathway of cholesterol synthesis, whose products are necessary for activation of proteins involved in cell death. Material and methods: Mononuclear cells isolated from the peripheral blood of 30 patients with CLL were incubated in RPMI-1640 medium, with 10% serum, and lovastatin (10 ľM, 20 ľM, 50 ľM, 100 ľM). After 24h of incubation (370C, 5% CO2, 98% humidity) cells were stained with acridin orange and MitoTracker, cell smears were made, and the rest of cells were fixed in glutaraldehyde and processed for electron microscopy. Results: Significant number of cells treated with lovastatin had ultrastructural characteristics of apoptosis and autophagy, and changes of mitochondria. Percentage of apoptotic cells incubated with lovastatin, estimated on cell smears stained with TUNEL technique, were higher than the control, for all of the used concentration (p<0,05, Exact Wilcoxon test). Accumulation of red vesicles stained with acridin orange in lovastatin treated cells confirmed autophagy. Confocal microscope analysis discovered that Mcl-1 protein changes its position from cytoplasm to mitochondria after treatment with lovastatin, while Bim protein that is localized in mitochondria of leukemic cells, do not change its localization after treatment with lovastatin. Conclusion: The results of this study show that lovastatin induces cell death in leukemic cells of patients with CLL. Key words: lovastatin, CLL, apoptosis, autophagy, ultrastructure 1Institut za histologiju i embriologiju, Medicinski Fakultet Beograd, Beograd, Srbija 2Klinika za hematologiju, Klinički Centar Srbije, Medicinski Fakultet Beograd 1Institute for histology and embryology, Medical Faculty Belgrade, Belgrade, Serbia 2Clinic for Haematology, Clinical Centre of Serbia, Faculty of Medicine, University of Belgrade 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 609 POSTER PRESENTATIONS 1/poster prezentacije 1 P17 Ekstranodalni krupnoćelijski NonHodginovi limfomi diferencijalno dijagnostički problem Extranodal Diffuse large cell NonHodgin Lymphoma problem in differentiall diagnosis Vesna Čemerikić-Martinović1, Olivera Marković2, Dragomir Marisavljević2, Tamara Martinović3, Vesna Božić4, Iva Paunović4, Katarina Marković5 Vesna Cemerikic-Martinovic1, Olivera Markovic2, Dragomir Marisavljevic2, Tamara Martinovic3, Vesna Bozic4, Iva Paunovic4, Katarina Markovic5 2KBC 2KBC 1Beo-lab, Beograd, Srbija Bežanijska kosa, Beograd, Srbija 3Institut za histologiju i embriologiju, Medicinski fakultet Univerziteta u Beogradu, Beograd, Srbija 4Klinički centar Srbije, Beograd, Srbija 5KBC Zvezdara, Beograd, Srbija Cilj: Prikazujemo tri slučaja primarnih krupnoćelijskih NHL ekstranodalne lokalizacije. Uvod: Primarni ekstranodalni non-Hodkinovi limfomi (NHL) čine oko 20-30% limfoidnih tumora. Najčešće nastaju u digestivnom traktu. Materijal i metode: Prvi slučaj je muškarac, star 46 godina, kome je gastroskopski vidjen veliki tumor u želucu. Dijagnoza endoskopske biopsije, bez imunohistohemije, je bila anaplastični karcinom želuca. Po uradjenoj gastrektomiji nadjen je veliki ulcerovegetativni tumor, precnika 7cm, uz uvecane limfne čvorove uz malu i veliku krivinu. U drugog bolesnika, starog 73 godine, kompjuterska tomografija je otkrila veliki tumor leve nadbubrežne železde, velicine 9x7cm. Uradjena je leva adrenalektomija uz splenektomiju. U treceg bolesnika, starog 42, godine vidjen je veliki tumor u predelu mišica ramena sa aksilarnom limfadenopatijom. Uradena je biopsija tumora i limfnog cvora. Rezultati: U sva tri slučaja histološki nalaz je odgovarao slabo diferentovanoj krupnoćelijskoj neoplazmi. U prvog bolesnika postojala je i infiltracija limfnih čvorova slična metastazi karcinoma, dok je u treceg histološki nalaz u limfnom čvoru odgovarao reaktivnoj hiperplaziji. Posle detaljne imunohistohemijske obrade u kojoj je korišćen širok panel antitela u prva dva bolesnika je postavljena dijagnoza difuznog B-krupnoćelijskog NHL, porekla aktivirane B-celije (non-GCB). FISH analiza limfoma nadbubrega je pokazala da postoji rearan man MYC i BCL2 gena. U trećeg bolesnika je postavljena dijagnoza anaplastičnog krupnoćelijskog NHL, ALK (anaplastic large cell lymphoma kinase). Imunohistohemijska analiza je pokazala da se i u limfnom čvoru nalaze pojedine ALK tumorske ćelije. Zaključak: Uvek treba misliti na NHL u diferencijalnoj dijagnozi slabo diferentovanih krupnoćelijskih tumora. Ključne reči: Ekstranodalni limfomi, krupnoćelijski NHL, NHL nadbubrežne želzde, NHL mišica, NHL želuca 610 1Beo-lab, Belgrade, Serbia Bezanijska kosa, Belgrade, Serbia 3Institute of Histology, Faculty of Medicine, University of Belgrade, Belgrade, Serbia 4Clinical Center of Serbia, Belgrade, Serbia, 5KBC Zvezdara, Belgrade, Serbia Aim: We report three cases of the primary extranodal large-cell NHL. Introduction: Primary extranodal non-Hodgkin lymphomas are uncommon and represent 20-30% of all NHL. Gastrointestinal localization represent the most common form of extranodal NHL. Matherial and methods: The first patient, 46-yearold man, had a huge tumor in his stomach that was seen on endoscopy. Biopsy, without immunohistochemistry revealed anaplastic carcinoma. After the operation the large exophytic, ulcerated lesion measuring 7cm was found with enlarged lymph nodes in omentum. In second patient, 73-year-old man CT tomography of abdomen demonstrated 9x7cm mass of the left adrenal gland. Laparatomy with left adrenalectomy and splenectomy was done. Third was an 42-year-old man who underwent biopsy of a shoulder muscle mass and enlarged axillary lymph node. Results: Histological examination revealed poorly differentiated large cell tumor in all three cases. In the first patient the lymph nodes were involved by tumor in a manner of cancer metastases. In third patients the lymph node showed only reactive changes. Immunohistochemical studies showed that tumors in first two patient were diffuse large B-cell NHL with non-GCB phenotype. FISH analysis show CMYC and BCL2 rerrangement in adrenal NHL. Immunohistochemical features of the tumor in shoulder muscle revealed a primary anaplastic large cell NHL with positive staining for anaplastic large cell lymphoma kinase (ALK). Immunophenotyping showed few ALK positive cells in histologicaly reactive lymph node. Conclusion: Primary lymphoma should be kept in mind in the differential diagnosis of poorly differentiated extranodal tumors. Key words: Ekstranodal lymphoma, large-cell NHL, NHL of adrenal gland, NHL of muscle, NHL of stomach 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 1/poster prezentacije 1 P18 Uticaj polimorfizama FCGR3A I FCGR2A gena na kliničke karakteristike i ishod bolesti kod pacijenata sa difuznim B krupnoćelijskim limfomom Impact of FCGR3A and FCGR2A gene polymorphisms on clinical characteristics and outcome in patients with diffuse large B-cell lymphoma Trimčev Jovana1, Cikota-Aleksić Bojana2, Tarabar Olivera3, Tukić Ljiljana3, Magić Zvonko2 Trimcev Jovana1, Cikota-Aleksic Bojana2, Tarabar Olivera3, Tukic Ljiljana3, Magic Zvonko2 1Institut za patologiju, Vojnomedicinska akademija, Beograd, Srbija 2Institut za medicinska istrazivanja, Vojnomedicinska akademija, Beograd, Srbija 3Klinika za hematologiju, Vojnomedicinska akademija, Beograd, Srbija Cilj: Cilj ove studije je da proceni da li polimorfizmi FCGR3A i FCGR2A gena mogu uticati na klinički tok i ishod bolesti kod pacijenata sa DLBCL koji su lečeni R-CHOP terapijom Uvod: Difuzni B krupnoćelijski limfomi (DLBCL), predstavljaju heterogenu grupu limfoproliferativnih oboljenja sa veoma različitim kliničkim tokom i ishodom bolesti. Tačan mehanizam dejstva R-CHOP terapije nije u potpunosti objašnjen. Pokazano je da polimorfizmi FCGR3A i FCGR2A gena imaju uticaj na afinitet za vezivanje monoklonalnog antitela za FC receptor na efektornoj ćeliji. Materijal i metode: Studija je uključila 64 pacijenta obolelih od DLBCL, lečenih R-CHOP terapijom. Genske analize su rađene PCR-RFLP metodom Rezultati: Nije nađena značajna statistička veza izmedu polimorfizama FCGR3A i FCGR2A gena, i kliničkih karakteristika kod DLBCL ( klinički stadijum, tumorski oblik bolesti, IPI, odgovor na terapiju, učestalost relapsa i ishoda bolesti), osim kod FCGR2A HR genotipa koji je bio češći kod pacijenata sa poodmaklim stadijumom bolesti (stadijum III i IV). (OR 6.1111, 95% CI 1.1842-31.5364, p=0.0091). Zapaženo je da je bolji DFS ( preživljavanje bez bolesti), kod pacijenata sa FCGR2A HR i RR genotipima u poredenju sa pacijentima koji imaju FCGR2A HH genotip. (log rank test, p=0.0595). FCGR3A genotip nije uticao na DFS. Nije pokazana značajna veza izmedju FCGR3A i FCGR2A polimorfizama/genotipa i EFS (preživljavanje bez događaja) I OS (ukupno prež ivljavanje) Zaključak: Imajući u vidu kliničke karakteristike i tok bolesti kod pacijenata sa DLBCL, FCGR2A HR genotip je udružen sa višim kliničkim stadijumom bolesti, dok nosioci FCGR2A HR I RR genotipa imaju bolji DFS. Ključne reči: DLBCL, R-CHOP,polimorfizmi gena, FCGR2A, FCGR3A, PCR-RFLP 1Institute of pathology and forensic medicine, Military medical academy, Belgrade, Serbia 2Institute of medical research, Military medical academy, Belgrade, Serbia 3Clinic for hematology, Military medical academy, Belgrade, Serbia Aim: The aim of this study was to assess whether FCGR3A and FCGR2A gene polymorphisms may affect the clinical course and outcome in DLBCL patients treated with R-CHOP therapy Introduction: Diffuse large B-cell lymphomas (DLBCL) represent a heterogeneous group of lymphoproliferative disorders with highly variable clinical course and outcome. The precise mechanism of R-CHOP therapy in DLBCL is not fully elucidated. It has been shown that polymorphisms of FCGR3A and FCGR2A genes influence the afinity of monoclonal antibodies to the FCreceptor on the effector cells. Material and methods: The study included 64 DLBCL patients treated with R-CHOP. Genotype analyses were performed by PCR-RFLP methodology. Results: Significant statistical association between different FCGR3A and FCGR2A gene polymorphisms/ genotypes and clinical characteristics of DLBCL (clinical stadium, bulky disease, IPI, response to therapy, incidence of relapse and outcome) was not observed, except for FCGR2A HR genotype, which was more frequent in patients with advanced clinical stage (III and IV) (OR 6.1111, 95% CI 1.1842-31.5364p: 0.0091). However, we observed a trend of better disease free survival(DFS) in patients with FCGR2A HR and RR genotypes than in those with FCGR2A HH genotype (log rank test, p=0.0595). FCGR3A genotypes haven t influenced DFS. There was no observed association between FCGR3A and FCGR2A gene polymorphisms/genotypes and event free survival (EFS) and overall survival(OS) . Conclusion: Considering clinical features and course of DLBCL, FCGR2A HR genotype is associated with advanced clinical stage of DLBCL, while the carriers of FCGR2A HR and RR genotypes have better DFS. Key words: DLBCL,R-CHOP, gene polymorphism, FCGR3A, FCGR2A,PCR-RFLP 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 611 POSTER PRESENTATIONS 1/poster prezentacije 1 P19 Nesvakidašnji tumor limfnog čvora, prikaz slučaja Unusual tumor of lymph node - a case report Zoran Nikin1, Tatjana Kapicl1, Slavica Knežević Ušaj1, Bratislav Stojiljković1, Milana Panjković, Dragana Tegeltija2, Aleksandra Lovrenski2 Zoran Nikin, 1, Tatjana Kapicl1, Slavica Knežević Ušaj1, Bratislav Stojiljković1, Milana Panjković2, Dragana Tegeltija2, Aleksandra Lovrenski2 1Institut 2Institut za onkologiju Vojvodine, Sremska Kamenica, Srbija za plucne bolesti Vojvodine, Sremska Kamenica, Srbija Cilj: Prikaz slučaja. Uvod: Sarkom dendritinih ćelija je retka hematološka bolest. Materijal i metode: Prikaz slučaja. Rezultati: Bolesnik starosti 78 godina se javio lekaru u novembru 2011. godine sa brzorastućim bolnim čvorom na vratu od septembra, afebrilan, bez gubitka težine, sa glavoboljom koja prolazi na Diklofen. CT snimak prikazuje tumor dimenzija 65mm na vratu. Klinička dijagnoza je bila anaplastični karcinom štitaste žlezde. Na bioptatu patolog se izjašnjava da je tumor CD 20 negativni difuzni krupno-celijski B limfom. Hematolozi su zatražili reviziju zbog nekompatibilnosti sa kliničkom slikom. U donetim parafinskim kalupima se nalaze isečci iz tumorom razorenog limfnog čvora sa invazijom okolnog mišicnog tkiva. Tumorske ćelije su pleomorfne atipične srednje krupne i krupne, obilne acidofilne ili svetle citoplazme, često nejasnih granica, mehurastih jedara sa vidljivim jedarcima. Prisutne su i “Reed Sternberg like”, vretenaste i potkovičaste celije. U pratećem zapaljenskom infiltratu se nalaze limfociti, plazmaciti, granulociti i makrofagi. Nakon panela dostupnih imunohistohemijskih analiza je zaključeno da nalaz prvenstveno može odgovarati sarkomu dendritičnih ćelija (Vimentin, S-100, CD10, CD43, CD4, CD68, CD23 -, CD30 -, CD20 -, CD79a -, CD3 -, CD5 -, CD8 -, Bcl-6 -, Bcl-2 -, CD56 -, CD138 -, CK ae1/ae3 -, Thyreoglobulin -, MelanA -, HMB-45 -). Bolesnik je primio do sada 3 ciklusa terapije po protokolu CHOP, koje je dobro podneo. Zaključak: Potrebno je uvek sistematično dijagnostikovati pacijente i misliti i na ređe entitete jer savremena terapija može produ iti ž ivot i povećati njegov kvalitet. Ključne reči: dendritične ćelije, sarkom 612 1Institut for oncology of Vojvodina, Sremska Kamenica, Serbia 2Institut for pulmonary diseases of Vojvodina, Sremska Kamenica, Serbia Aim: Case report. Introduction: Dendritic cell sarcoma is a rare hematologic disease. Material and methods: Case report Results: A 78 years old patient came to doctor in November 2011. with fast growing and dolorous cervical node since September. He was afebrile, without losing weight, with headaches relieving on Diclofen. CT scan showed cervical tumor dimensioned 65mm. Clinical diagnosis was anaplastic carcinoma of thyroid gland. After biopsy pathologist diagnosed CD 20 negative diffuse large B-cell lymphoma. Hematologists asked for revision due to incompatibility with clinical presentation. We obtained paraffin embedded tissue samples with lymph node destructed by tumor, invading nearby muscles. Tumor cells were pleomorphic, middle large and large, with abundant acidophilic or clear cytoplasm, often with unclear borders, with vesicular nuclei and visible nucleoli. “Reed Sternberg like” cells, fusiform and “horse shoe” cells were present too. In the background there were lymphocytes, plasmacytes, granulocytes and macrophages. After performing a panel of attainable immunohistochemical analyses it was concluded that the tumor is probably dendritic cell sarcoma (Vimentin, S-100, CD10, CD43, CD4, CD68, CD23 -, CD30 -, CD20 -, CD79a -, CD3 -, CD5 -, CD8 -, Bcl-6 -, Bcl-2 -, CD56 -, CD138 -, CK ae1/ ae3 -, Thyreoglobulin -, MelanA -, HMB-45 -). The patient was treated with 3 CHOP regimens up to date and is in a good condition. Conclusion: Doctors should always systematically perform diagnostic procedures and think about rare entities because nowadays therapy can prolong life and improve its quality. Key words: dendritic cell, sarcoma 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 1/poster prezentacije 1 P20 Korelacija između KI67 indeksa i kariometrijskih varijabli kod neHočkinovih limfoma od B- limfocita Correlation between Ki-67 index and karyometric variables in B-cell nonHodgkin lymphomas Dragan Mihailović, Žaklina Mijović, Nikola Živković, Miloš Kostov, Miljan Krstić Dragan Mihailovic, Zaklina Mijovic, Nikola Zivkovic, Milos Kostov, Miljan Krstic Cilj: Cilj ovog rada je određivanje korelacije izmedu Ki67 indeksa i kariometrijskih varijabli kod B-ćelijskih ne-Hočkinovih limfoma. Uvod: Ki67 je protein koji se sintetiše u aktivnim fazama ćelijskog ciklusa. Poznato je da je kinetika ćelijskog ciklusa od vitalnog znacaja za ponašanje tumora. Kariometrija je objektivni metod koji se koristi za određivanje veličine, oblika i optičke gustine jedara. Materijal i metode: U centru za patologiju Kliničkog centra u Nišu, od januara 2008. godine do januara 2012. godine imunohistohemijski i kariometrijski ispitano je 20 ne-Hočkinovih limfoma od B-limfocita (11 difuznih B-krupnoćelijskih limfoma (DLBCL), 3 folikularna limfoma, 3 mantle-ćelijska limfoma, 2 medijastinalna difuzna B-krupnoćelijska limfoma i 1 Burkitt-ov limfom), kao i 7 reaktivnih hiperplazija. Odredivani su Ki67 indeks i 7 jedarnih parametara (površina, optička gustina, Feret-ov dijametar, obim, cirkularnost, okruglost i intregrisana optička gustina). Korišćene su digitalne slike rezolucije 1280x1024 piksela, dobijene na mikroskopu NU-2 (Carl Zeiss, Jena, Nemačka) sa objektivom x63. Korišćen je program ImageJ, za određivanje kariometrijskih varijabli i Ki67 indeksa (plagin Cell Counter), na najmanje 100 jedara tumorskih ćelija kod svakog pacijenta. Rezultati: Najveće prosečne vrednosti Ki67 indeksa nadene su kod Burkitt limfoma (98%) i DLBCL (43%) u odnosu na ostale histološke tipove ne-Hočkinovih limfoma od B-limfocita. Statistički značajna pozitivna korelacija nađena je između površine, Feret-ovog dijametra i Ki67 indeksa (p<0.05). Cirkularnost jedara koja su Ki67 pozitivna je u statistički značajnoj negativnoj korelaciji sa optičkom gustinom (p<0.05). Zaključak: Kod B-ćelijskih ne-Holkinovih limfoma Ki67 indeks je u pozitivnoj korelaciji sa veličinom jedara. Ključne reči: Ćelijski ciklus, Ki67 indeks. neHočkinovi limfomi Aim: Aim of this study was to estimate correlation between Ki67 index and karyometric variables in B-cell non-Hodgkin lymphomas. Introduction: KI67 antigen is a nuclear protein synthesized in the active phase of the cell cycle. Cell cycle kinetics plays a vital role in tumor behaviors. Karyometry in an objective method to estimate nuclear size, shape and optical density. Material and methods: At Centre of Pathology, Clinical Centre of Niš, from January 2008 to January 2012, 20 cases of non-Hodgkin lymphomas (11 diffuse large B-cell lymphomas (DLBCL), 3 follicular lymphomas, 3 mantle cell lymphomas, 2 mediastinal diffuse large B-cell lymphomas, 1 Burkitt lymphoma) and 7 cases with reactive hyperplasia were immunohistochemically and karyometricaly analyzed to Ki67 index and 7 karyometric variables (nuclear area, optical density, Feret diameter, perimeter, circularity, roundness and integrated optical density). Using digital pictures of 1280x1024 pixels resolution, obtained on NU-2 microscope (Carl Zeiss, Jena, Germany) at objective x63, and ImageJ software (plugin Cell Counter), Ki67 index and karyometric variables were estimated on at least a 100 of tumor cell nuclei per case. Results: Higher mean values of Ki-67 index were found in Burkitt lymphoma (98%) and DLBCL (43%) compared to other histological types of B-cell nonHodgkin lymphomas. Statistically significant positive correlation was found between area, Feret diameter and Ki67 index (p<0.05). Circularity of Ki67 stained nuclei was in statistically negative correlation with optical density (p<0.05). Conclusion: In B-cell non-Hodgkin lymphomas, Ki-67 index is in positive correlation with nuclear size. Key words: cell cycle, Ki67 index, nonHodgkin lymphomas. Centar za patologiju, Klinički Centar Niš, Niš, Srbija Centre of Pathology, Clinical Centre of Nis, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 613 POSTER PRESENTATIONS 1/poster prezentacije 1 UROLOŠKA PATOLOGIJA UROPATHOLOGY P21 Periacinarne retrakcione pukotine u adenokarcinomu prostate u korelaciji sa Gleason scorom i kliničkim stadijumom Periacinar retraction clefting in prostatic adenocarcinoma in correlation with Gleason score and clinical stage Milica Mijović1, Boris Dobrojević2, Danica Vukičević1, Nebojša Mitić1, Branislav Đerković1, Vladica Nedeljković 1 1Institut za patologiju, Medicinski fakultet Priština-Kosovska Mitrovica, Srbija 2Odeljenje patologije, Opšta bolnica Brčko, Brčko Distrikt, BIH Cilj: Ispitati značaj PRP kao pomoćnog kriterijuma u dijagnozi AP i korelirati ih sa Gleason scorom i kliničkim stadijumom Uvod: Periacinarne retrakcione pukotine (PRP) predstavljaju prazne prostore oko žlezda adenokarcinoma prostate (AP) i smatraju se za pomoćni dijagnostički kriterijum ako su prisutne u >1/2 žlezda i ako pri tome zahvataju najmanje 1/2 do 2/3 cirkumferencije žlezde Materijali i metode: Analizirano je 70 AP. Materijal za HP analizu dobijen je needle core biopsijom ili radikalnom prostatektomijom. PRP su analizirane na deset neoplastižnih i deset normalnih žlezda na tri različita polja VMU (400x). Gleason score je odreden na standardnim HE preparatima, klinički stadijum prema TNM klasifikaciji Rezultati: Nađene su žlezde sa PRP u >50% žlezdane cirkumferencije u <50% prisutnih žlezda (grupa 2) u 34(48,6%) AP ili u =50% prisutnih žlezda (grupa 3) u 31 (44,3%) AP. Žlezde bez PRP su bile redak nalaz (grupa 1) u 5 (7,1%) AP. ROC kriva pokazaje senzitivnost od 92,9% i specifičnost od 73,3% (AUC =0.831 ?<0,001). Najčešći Gleason score bio je 7 i nađen je u 33 (47,14%) AP. Najveći broj AP dijagnostikovan je u stadijumu IV, kod 31 (44,29%). Korelacionom neparametarskom analizom navedenih parametara utvrđena je da su PRP sa zadovoljavajućim koeficijentom korelacije od -0,268 i -0,247, praćeni manje uznapredovalim kliničkim stadijumom i nižim Gleason scorom Zaključak: PRP mogu se svrstati u pomoćne kriterijume za dijagnozu AP. Najčešće su kod srednje diferentovanih AP i u neuznapredovalim kliničkim stadijumima. Ključne reči: Prostata, karcinom, periacinarne retrakcione pukotine 614 Milica Mijovic1, Boris Dobrojevic2, Danica Vukicevic1, Nebojsa Mitic1, Branislav Đerkovic1, Vladica Nedeljkovic1 1Institut of pathology, Medical faculty Pristina-Kosovska Mitrovica, Serbia 2Department of pathology, General hospital Brcko, Brcko District, BIH Aim: Importance of PRC in diagnosis of PA and their correlation with Gleason score and clinical stage Introduction: Periacinar retraction clefting (PRC) are “empty spaces” around glands of prostatic adenocarcinoma (PA) and are supportive criteria if they are present in >1/2 glands and are around 1/2 to 2/3 circumference of gland. Material and methods. We analyzed 70 PA. Material for analysis was obtained by needle core biopsy or radical prostatectomy. PRC were analyzed on ten neoplastic and ten normal glands in three different high power fields (400x). Gleason score was determined on standard HE preparations, clinical stage were determined by TNM classification. Results: We found glands with PRC in >50% of the glandular circumference in <50% of all examined glands (group 2) in 34 (48,6%) PA or in ≥50% of all examined glands (group 3) in 31 (44,3%) PA. We found only 5 (7,1%) PA with glands without PRC (group 1). ROC curves showed sensitivity of 92.9% and specificity of 73.3% (AUC = 0831; р<0,001). The most common Gleason score was 7 and was found in 33 (47.14%) PA. The greatest number of PA was diagnosed in stage IV, in 31 (44.29%). According to nonparametric correlation analysis of these parameters it was found that the PRC, with satisfactory correlation coefficient of -0.268 and -0.247, are followed by less advanced clinical stage and lower Gleason score. Conclusion: PRC can be classified as supportive criteria for diagnosis of PA. They are commonly found in intermediate grade AP and at not-advanced clinical stages. Key words: Prostate, cancer, periacinar retraction clefting. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 1/poster prezentacije 1 P22 Mucinozni adenokarcinom prostate: histopatološke i imunohistohemijske karakteristike Mucinous Adenocarcinoma of the Prostate: Histopathological and Immunohistochemical Characteristics Sandra Trivunic Dajko1, Matilda Djolai1, Nenad Šolajic1, Jelena Amidžić1, Mirjana Živojinov1, Bojana Andrejić2, Snežana Božanić1 Sandra Trivunic Dajko1, Matilda Djolai1, Nenad Solajic1, Jelena Amidzic1, Mirjana Zivojinov1, Bojana Andrejic2, Snezana Bozanic1 Cilj: Odrediti histopatološke i imunohistohemijske karakteristike mucinoznog adenokarcinoma prostate. Uvod: Mucinozni (koloidni) adenokarcinom prostate je subtip prostatičnog karcinoma koji se karakteriše velikim jezerima ekstracelularnog mucina, prema definiciji najmanje 25% tumorskog volumena i čini 0,4% svih adenokarcinoma prostate. Tradicionalno je mucinozni adenokarcinom prostate smatran mnogo agresivnijim nego drugi češći tipovi nemucinoznih prostatičnih adenokarcinoma, medutim ova tvrdnja je i osporavana. Imajući u vidu ovu tvrdnju Internacionalno udruženje uroloških patologa je na Konsenzus konferenciji o Gleason gradiranju prostatičnog karcinoma 2005. godine sugerisalo da bi ovaj subtip tumora trebalo klasifikovati kao Gleason score 8(4 4). Materijal i metode: Studija je bila retrospektivna i sprovedena je u Centru za patologiju i histologiju Kliničkog Centra Vojvodine. U periodu od januara 2007. godine do marta 2012. godine je analizirana mucinozna komponenta u adenokarcinomu prostate, nakon totalne prostatektomije. Rezultati: U petogodšnjem periodu ukupno je dijagnostikovano 302 adenokarcinoma prostate, njih 4 je imalo mucinoznu komponentu, ali je ona bila manja od 25% tumorskog volumena kod 3 slučaja. Kod samo jednog pacijenta starosti 69 godina, mucinozna komponenta je činila preko 30% tumorskog tkiva, te je tumor klasifikovan kao mucinozni (kololidni) tip adenokarcinoma prostate, Gleason score 4 4(8), pT3aN1. Imunohistohemijski profil je bio sledeci: PSA+/-, Androgeni receptori-, CEA -, CK7 -, CK20 i 34ßE12 -. Zaključak: Zastupljenost mucinoznog adenokarcinoma prostate je u korelaciji sa literaturnim navodima, kao i njegov imunohistohemijski profil, čime ga jasno razlikujemo od veoma retkog i agresivnog mucinoznog adenokarcinoma prostatične uretre - urinary bladder type . Ključne reči: prostata adenokarcinom, mucinozni. Aim: Determine histopathological and immunohistochemical characteristics of the mucinous adenocarcinoma of the prostate. Introduction: Mucinous (or colloid) adenocarcinoma of the prostate is a subtype of prostate cancer that is characterized by the large pools of extracellular mucin that by definition compose at least 25% of the tumor volumen. Mucinous adenocarcinoma represent approximately 0.4% of all prostate adenocarcinoma. Traditionally, mucinous adenocarcinoma of the prostate has been considered to be more agressive than the more common nonmuconous prostate adenocarcinoma, althougt this notion has been challenged. Material and methods: Retrospective study was conducted in the Clinical Center of Vojvodina. From January 2007 to March 2012 mucinous component was analyzed in prostate adenocarcinoma and the material used was from the radical prostatectomy. Results: Within a five-year period the total of 302 adenocarcinoma of the prostate was diagnosed, only 4 of them had a mucinous component, but it was less than 25% tumor volume in 3 cases. In only one patient aged 69 years, mucinous component accounted for over 30% of tumor tissue and the tumor was classified as a mucinous (colloid) type of the prostate adenocarcinoma, Gleason score 4 4 (8), pT3aN1. Immunohistochemical profile of the tumor was as follows: PSA + /-, Androgen Receptors + /-, CEA -, CK7 -, CK20 - and 34ßE12 -. Conclusion: Presence of the mucinous adenocarcinoma of the prostate is correlated with the literature claims, as well as its immunohistochemical profile, which clearly distinguishes it from a very rare and aggressive urinary bladder type. Key words: prostat, adenocarcinoma, mucinous. Centar za patologiju i histologiju, Klinički centar Vojvodine, Novi Sad, Srbija. 2Katedra za histologiju i embriologiju, Medicinski fakultet, Novi Sad, Srbija. 1 1Centar of pathology and histology, Clinical centar of Vojvodina, Novi Sad, Serbia. 2Dipartment of histology and embriology, Medical faculty of Novi Sad, Serbia. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 615 POSTER PRESENTATIONS 1/poster prezentacije 1 P23 Značaj stromalne eozinofilije u neinvazivnim (pTa) I invazivnim (pT1) papilarnim urotelnim karcinomima Ljiljana Bogdanović1, Sanja Radojević- Škodrić1, Miodrag Lazić 2, Cane Tulić3, Zoran Džamić3, Gordana Basta- Jovanović1 1Institut za Patologiju, Medicinski fakultet, Univerzitet u Beogradu, Beograd, Srbija 2Klinika za urologiju, KBC”Dr Dragisa Mišović”,Beograd, Srbija 3Institut za urologiju i nefrologiju, Medicinski fakultet, Univerzitet u Beogradu, Beograd, Srbija Cilj: Cilj rada bio je da se uporedi broj eozinofila, koji su identifikovani na rutinskim hematoksilin eozin preparatima, u urotelnom papilarnom karcinomu mokraćne besike sa invazijom lamine proprie. Uvod: Tumorska eozinofilija je retka, ali je značajan fenomen koji je pronaden u mnogim tumorima. Mnogi autori navode da prisustvo nekog zapaljenskog odgovora u okviru tumora predvida napredovanje mnogih tumora, uključujuci i urotelni karcinom mokraćne bešike. Materijali i metode: Izmedu 2007. i 2011., 40 bolesnika sa primarnim urotelnim karcinomom mokraćne bešike, koji su imali neku hiruršku intervenciju, su uključeni u ovu studiju. Od njih, 20 je imalo neinvazivni papilarni UC mokraćne bešike, a 20 invazivni papularni UC. U svim slučajevima odreden je stadijum i histološki gradus i prisustvo eozinofila u stromi. Eozinofili su brojani na optičkom mikroskopu na 10 uzastopnih polja velikog uveličanja (uveličanje 400x). Rezultati: Nadena je statisticki značajna razlika izmedu broja eozinofila u neinvazivnom papilarnom urotelnom karcinomu i invazivnom papilarnom urotelnom karcinomu mokraćne bešike. Maksimalan broj eozinofila na jednom i deset vidnih polja velikog uveličanja statistički je značajno visi u invazivnom papilarnom urotelnom karcinomu mokraćne bešike. Zaključak: Ovi rezultati potvrduju da prisustvo eozinofilne reakcije u stromi ima prognostički i dijagnostički značaj u UC. Razlika u intenzitetu eozinofilne reakcije može se koristiti kao dodatan dijagnostički kriterijum u dijagnozi invazije karcinoma u laminu propriju. Ključne reči: stromalna eozinofilija, urotelni karcinom 616 Significance of stromal eosinophilia in non-invasive (pTa) and invasive (pT1) papillary urothelial carcinoma Ljiljana Bogdanovic1, Sanja Radojevic Skodric1, Miodrag Lazic 2, Cane Tulic3, Zoran Dzamic3, Gordana Basta Jovanovic1 1Institute of Pathology, School of Medicine, University of Belgrade, Belgrade, Serbia 2Clinical Center Dr Dragisa Misovic, Department of Urology, Belgrade, Serbia 3Institute of Urology and Nephrology, School of Medicine, University of Belgrade, Belgrade, Serbia Aim: The aim of this study was to compare the number of eosinophils, identified routinely with hematoxylin and eosin stain, in papillary urothelial carcinoma of the urinary bladder with invasion of lamina propria. Introduction: Tumor eosinophilia is an uncommon, but striking phenomenon which has been found in many tumors. Many authors have indicated that the presence of an inflammatory response within the tumor may predict progression in many tumors, including urothelial carcinoma (UC) of urinary bladder. Material and methods: Between 2007. and 2011., 40 patients with primary UC of bladder, who had undergone surgical treatment, were selected for the study. Of these, 20 had non-invasive papillary UC of the urinary bladder and 20 had invasive papillary UC. All cases were reviewed to assess stage and grade of the tumor and presence stromal eosinophilia. The number of eosinophils in tumor stroma was counted. Eosinophils was assessed in 10 consecutive fields of vision, under the optical microscope at 400 x magnification. Results: A statistically significant association was shown between the number of eosinophils in non-invasive papillary urothelial carcinoma of the urinary bladder and invasive carcinoma. The maximun number of eosinophils per one as well as per ten high power fields was significantly higher in invasive papillary urothelial carcinoma. Conclusion: These results confirm that the presence of eosinophils reaction in tumor stroma has prognostic and diagnostic value in UC. The difference in the intensity of eosinophilic reactions may be used as a helpful additional diagnostic tool in diagnosis of lamina propria invasion. Key words: stromal eosinophilia, urothelial carcinoma 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 1/poster prezentacije 1 P24 Ekspresija Estrogenog Receptora B u nodularnoj hiperplaziji prostate The Estrogen Receptor B expression in prostate nodular hyperplasia Aleksandra Levakov, Mihaela Mocko-Kaćanski, Mirjana Živojinov, Nada Vučković, Pavle Budakov Aleksandra Levakov, Mihaela Mocko Kacanski, Mirjana Zivojinov, Nada Vuckovic, Pavle Budakov Cilj: Ustanoviti stepen ekspresije i lokalizaciju ERB u BHP Uvod: Nakon 50. godine dolazi do proliferacije žlezdanih struktura i strome prostate. Hiperplazija prostate je povezana sa pomeranjem ravnoteže izmedu androgena i estrogena. Estrogeni uticu na rast, diferencijaciju i funkciju muškog reproduktivnog sistema. ERB je steroidni receptor lokalizovan u jedru bazalnih ćelija acinusa i manjim delom u stromalnim ćelijama. Bazalne ćelije su androgen nezavisne i predstavljaju matične ćelije. Sekretorne ćelije sa visokom ekspresijom ERB su diferentovane, androgen zavisne, bez proliferativnog kapaciteta. Materijal i metode: Sekstant biopsije sa BHP su imunohistohemijski bojene na ERB (liofilizirano mišije monoklonalno antitelo razblaženo 1:50 Novocastra). Ocenjena je lokalizacija i stepen ekspresije ERB na uvećanju 40x. Proporcionalni score predstavlja odnos pozitivnih ćelija u acinusima: 0 nula 1 : 1% 1% 5 >66%. Posmatran je u odnosu na pozitivno obojene fibroblaste i endotelne ćelije. Rezultati: Najveći stepen ekspresije ERB je u bazalnim ćelijama acinusa (score 4,33), ne to manji u sekretornim luminalnim ćelijama (score 4), a prisutan je i u stromalnim ćelijama (score 3). Visoka ekspresija ERB je nadena u slučajevima BHP sa PSA: 10 ng/ml (srednji 9,52 ng/ml). Ovakav nalaz se slaze sa većinom aktuelnih radova. Zaključak: Uloga ERB je u zastitnom dejstvu estrogena na epitel prostate od nepredviđene proliferacije, neoplastične transformacije i oksidativnog oštećenja. ERA ima prolifertivnu ulogu koja se ograničava dejstvom ERB. Gubitak ERB vodi u nekontrolisanu ćelijsku proliferaciju. Lokalizacija ERB u sekretornim ćelijama pokazuje da je diferentovani odeljak glavna meta estrogenog delovanja u prostati. Ključne reči: ERB, hiperplazija prostate, bazalne ćelije, sekretorne ćelije, imunohistohemija Aim: Expression intensity and localization of ERB in BHP Introduction: Prostate acinar and stromal proliferation develops after fifties. Prostatic hyperplasia is related with disturbed balance between androgens and estrogens. Estrogens take influence on growth, differentiation and function of male reproductive system. ERB is steroid receptor located in nuclei of acinar basal cell and partially in stromal cells. Androgen independent basal cells are stem cells. Secretory cells are differentiated androgen dependent, show high ERB expression, without proliferating capacity. Material and methods: Sextant biopsies with BHP are imumnohistochemistry treated for ERB (lyophilized mouse monoclonal antibody diluted 1:50 Novocastra). Localization and intensity of ERB?expression is determined in 40x magnification. Proportional score presents relation of positive acinar cells: 0 zero 5 >66%. For comparison are used positive fibroblasts and endothelial cells. Results: ERB expression is the greatest in acinar basal cells (score 4,33), lesser in secretory luminal cells (score 4) and also noticed in stromal cells (score 3). High expression of ERB is registered in prostate hyperplasia with PSA: 10 ng/ml (median 9,52 ng/ml). This finding is in consistency with majority of current reports. Conclusion: ERB enables the protection role of estrogen on prostate epithelium from unpredicted proliferation, neoplastic transformation and oxidative damage. ERA has a proliferative role determined with ERB action. Losing of ERB leads to uncontrolled cell proliferation. ERB localization in secretory cell indicate that differentiated section is target of estrogen action in prostate. Key words: ERB, prostate hyperplasia, basal cells, secretory cells, immunohistochemistry Klinički centar Vojvodine, Novi Sad, Srbija Clinical Center of Vojvodina, Novi Sad, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 617 POSTER PRESENTATIONS 1/poster prezentacije 1 P25 Angiomyolipom bubrega Renal angiomyolipoma - case report Nikola Živkovic1, Dragan Mihailovic1, Sladana Petrovic1, Zaklina Mijovic1, Aleksandra Radovanovic1, Simonida Stojanovic1, Ana Ristic Petrovic1, Slavica Stojnev1 Nikola Zivkovic, Dragan Mihailovic, Sladana Petrovic, Zaklina Mijovic, Aleksandra Radovanovic, Simonida Stojanovic, Ana Ristic Petrovic, Slavica Stojnev Cilj: Prikaz morfoloških i imunohistohemijskih karakteristika angiomiolipoma u bubregu. Uvod: Prikaz morfoloških i imunohistohemijskih karakteristika angiomiolipoma u bubregu. Materijali i metode: Melan-A, HMB45. CD68, CD117, CK, CEA Rezultati: Prikaz pacijenta. Prikazujemo 65 godina starog muškarca sa spontanom rupturom angiomiolipoma, koji je lečen parcijalnom nefrektomijom. Makroskopski, lezija je žute boje, u delovima sa mišićnom komponentom tamno braon boje. Mikroskopski, lezija je izgrađena od jednakog odnosa masnog, glatkomišićnog tkiva i krvnih sudova. Masno tkivo čine zrele masne ćelije sa svetlom vakuolizovanom citoplazmom sa malim periferno postavljenim jedrom. Glatke mišićne ćelije su vretenastog oblika, mestimično epiteloidne sa svetlom eozinofilnom citoplazmom. Vaskularnu komponentu čine veliki krvni sudovi tankih zidova. Imunohistohemijski, tumorske ćelije su pozitivne na Melan-A, HMB 45, CD117, CD68. Nasuprot, tumorske ćelije su negativne na S-100 protein, epitelne markere citokeratin i EMA. Slučajan nalaz je bio mali kortikalni adenom. Zaključak: Angiomiolipomi su obično benigni tumori, izuzev epiteloidnog angiomiolipoma, koji se javlja u 3% slučajeva i može imati agresivan tok Ključne reči: angiomiolipomi, bubreg, imunohistohemija Aim: To present histological and immunohistochemical findings in renal angyomyolipoma Introduction: Angiomyolipomas are benign neoplasm of the kidney that are composed of fat, smooth muscle, and thick-walled blood vessels in varying proportions. Tumor is usually present in adults and rarely in children, with an overall median age 50 years. Material and methods: Melan-A, HMB45. CD68, CD117, CK, CEA Results: Case report. We report the case of a 65-year-old man with spontaneous rupture of an angiomyolipoma, who was treated with a partial nephrectomy. Grossly, lesion was predominantly pale yellow, whereas tumors with an extensive smooth muscle component are brownish. Microscopically, the lesions have the relative proportions of fat, smooth muscle, and blood vessels. The adipose tissue was composed of uniform fat cells with large cytoplasmic vacuoles and small peripheral nucleus. The smooth muscle cells were typically spindle shaped but occasionally they are epithelioid and have abundant eosinophilic cytoplasm. The vascular components consisting of large thick walled tortuous blood vessels. According to immunohistochemistry, tumor cells were positive for Melan A, HMB-45, CD117, CD68. Moreover, tumor cells were negative for S-100 protein and epithelial markers such as cytokeratin and epithelial membrane antigen. We were incidentally found small renal adenoma. The cells have round to oval nuclei with chromatin that ranges from stippled to clumped, as well as inconspicuous nucleoli. Conclusion: The true nature of these lesions is unclear, but they are usually classified as hamartomas. Angiomyolipomas are generally benign lesions, although the epithelioid angiomyolipoma, a subtype that occurs in about 3% of cases, can have an aggressive behavior. Key words: angiomyolipoma, kidney, immunohistochemistry 1Medicinski 618 fakultet u Nisu, Institut za patologiju, Niš, Srbija University of Nis, Faculty of Medicine, Institute of Patology, Nis, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 1/poster prezentacije 1 P26 Non Hodgkin limfom bubrega Non Hodgkin lymphoma in kidney Jelena Vještica1, Milica Čekerevac2, Tatjana Terzić1, Radmila Janković1,Sanja Ćirović1, Vesna Čemerikic3, Jasmina Marković-Lipkovski1 Jelena Vjestica1, Milica Cekerevac2, Tatjana Terzic1, Radmila Jankovic1,Sanja Cirovic1, Vesna Cemerikic3, Jasmina Markovic-Lipkovski1 Institut za patologiju,Medicinski fakultet,Beograd, Srbija za urologiju, Klinički centar Srbije, Beograd, Srbija 3Laboratorija za patologiju Beolab, Beograd, Srbija 1Institute 2Klinika 2Urology Cilj: Ovde smo prikazali 3 slučaja non Hodgkin limfoma bubrega dijagnostikovanih u Kliničkom centru Srbije. Uvod: Limfomi bubrega su veoma retki i mogu da se jave u sklopu sistemskih bolesti limfnog tkiva. Mnogo redje se javljaju kao izolovana forma limfoma bubrega. Najčešci histološki tip ekstranodalnog limfoma bubrega je difuzni non Hodgkin limfom krupnih B- ćelija (diffuse large B-cell lymphoma- DLBCL). Materijal i metode: Analiziran je materijal Urološke klinike, Kliničkog centra Srbije, dobijen perkutanom biopsijom bubrega i nefrektomijom. Materijal je obrađjen i imunohistohemijski analiziran na Institutu za patologiju, Medicinskog fakulteta u Beogradu, upotrebom markera za tumore bubrđnih ćelija (RCC, CD10, CK7, Pax-2, vimentin, AMACR, HMWCK, sinaptofizin) i limfome (bcl-2, bcl-6, Pax2, LCA, CD3, CD5, CD20, CD23, CD30, Cd45Ro, CD45, CD79a). Rezultati: Non Hodgkin limfom bubrega dijagnostikovan je kod 3 bolesnice na osnovu morfološkog izgleda tumorskih ćelija i imunohistohemijskog bojenja. Bolesnice su bile stare 53, 56 i 73 godina. Kod najstarije bolesnice dijagnoza DLBCL postavljena je posle nefrektomije. Perkutanom biopsijom bubrega je kod mlađe bolesnice postavljena dijagnoza DLBCL bubrega u sklopu sistemskog limfoma, dok je kod druge bolesnice na osnovu perkutane biopsije bubrega postavljena dijagnoza izolovanog B ćelijskoglimfoma malih limfocita (B-cell small lymphocytic lymphoma- B-SLL) bubrega. Zaključak: Non-Hodgkin limfom bubrega dijagnostikovan je kod žena, prosečne starosti 60 godina, nasuprot navedenim podacima iz literature da limfomi bubrega pogadaju mahom mušku populaciju. Zahvaljujuci imunohistohemijskoj analizi danas smo u mogućnosti da postavimo dijagnozu limfoma i na biopsijskom materijalu bubrega. Ključne reči: Non Hodgkin limfom, bubreg, imunohistohemija Aim: We reported 3 cases of non Hodgkin lymphoma in kidney, diagnosed at the Clinical Center of Serba. Introduction: Renal lymphomas are very rare and they can be associated with lymphoid tissue systemic dissease. Rare thay can be diagnossed as primary renal lymphoma. The most common histological subtype encountered is diffuse large B cell lymphoma (DLBCL). Material and methods: Tissue samples from kidney biopsies and nepherctomy were collected from Urology Clinic, Clinical center of Serbia. Samples were analyzed at the Institute for Pathology, Medical Faculty in Belgrade. Markers for renal carcinoma cells (RCC, CD10, HMWCK, vimentin, AMACR, synaptophysin) and for lymphoma (bcl-2, bcl-6, Pax2, LCA, CD3, CD5, CD20, CD23, CD30, CD45Ro, CD79a) were used and the material was analyzed by using immunohistochemisty staining. Results: We diagnosed Non Hodgkin lymphoma in 3 female patients according to morphology of tumor cells and immunohistochemistry analyses. Patients were 53, 56, and 73 year old. After nephrectomy the oldest patient was diagnosed with diffuse large B cells non Hodgkin lymphoma (DLBCL). After kidney biopsies, in one younger patient we diagnosed systemic DLBCL, while the second patient had primary B-cell small lymphocytic lymphoma (B-SLL). Conclusion: In our study all 3 cases of non Hodgkin lymphoma in kidney were detected in female patients, in contradictory to previously shown data that kidney lymphoma are predominant in male population. Using immunohistochemistry analysis these days we are able to diagnose kidney lymphoma in kidney biopsy samples. Key words: Non Hodgkin lymphoma, kidney, immunohistochemistry 1 for Pathology, School of Medicine, Belgrade,Serbia clinic, Clinical center of Serbia, Belgrade, Serbia 3Laboratory for pathology,Belgrade, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 619 POSTER PRESENTATIONS 1/poster prezentacije 1 P27 Primarni maligni mezoteliom tunike vaginalis testis Primary malignant tunica mesothelioma of testicular vaginalis Milorad Pavlović1, Ljubinka Veličković2, Žaklina Miović2, Desanka Dimov Tasić2, Irena Dimov3, Miloš Kostov4 Milorad Pavlovic1, Ljubinka Velickovic2, Jacqueline Miovic2, Desanka Dimov Tasic2, Irena Dimov3, Milos Kostov4 2Institut 2Institute 1Služba patologije, Opšta bolnica Leskovac, Srbija za patologiju, Medicinski fakultet Niš, Srbija 3Institut za imunologiju, Medicinski fakultet, Niš, Srbija 4Služba za patologiju, Vojna bolnica, Niš, Srbija Cilj: Prikaz pacijenta sa retkom lokalizacijom malignog mezotelioma. Uvod: Maligni mezoteliom je retka neoplazma koja se najčešće javlja na pleuri, peritoneumu i perikardu, a vrlo retko na tunici vaginalis testisa. Materijal i metode: Resekat tumorske promene i operativni materijal. Korišćena je klasična obrada materijala i bojenje na HE i imunohistohemijska bojenja. Rezultati: Prikazujemo redak slučaj malignog mezotelioma tubulopapilarni epiteloidni tip kod muškarca životne dobi od 72 godine sa hidrokelom i bolovima u predelu levog testisa. Izvršena je hirurška ekscizija cistične promene na granici testisa i funikulusa. U zidu ciste nađena je tumorska promena prečnika 4mm. Mikroskopski, tumorska lezija je građena od tubulopapilarnih struktura sa oskudnom vezivno tkivnom stromom. Tumorske ćelije epiteloidnog tipa sadržavale su ovalna jedra sa upadljivim jedarcima. Jedra su pokazivala umereni atipizam i hiperhromaziju, a mitoze su relativno česte. Imunohistohemijski tumorske ćelije su bile pozitivne na CK 5/6, EMA, calretinin i mezothelin-1. Na osnovu kliničke, histološke i imunohistohemijske slike postavljena je dijagnoza malignog mezotelioma. CT je isključio prisustvo tumora u maloj karlici i abdomenu. Na operativnom materijalu nadena je tumorska formacija koja infiltruje funikulus spermatikus i testis, a prisutna je i na gornjoj ivici resekcije. Nakon četiri meseca pacijent je egzitirao. Zaključak: Prikazan je slučaj retkog primarnog malignog mezotelioma tunike vaginalis testisa zbog izrazito agresivne prirode. Ključne reči: Maligni mezoteliom, tubulopapilarni epiteloidni tip, testis 620 1Pathology Department, General Hospital Leskovac, Serbia of Pathology, School of Medicine, Nis, Serbia 3Institute of Immunology, Faculty of Medicine, Nis, Serbia 4Department of Pathology, Military Hospital Nis, Serbia Aim: Review of the patient with a rare localization of malignant mesothelioma. Introduction: Malignant mesothelioma is a rare neoplasm that occurs most frequently in the pleura, peritoneum and pericardium, and rarely on the tunic of vaginalis testis. Material and methods: The resect of tumor changes and operational material. We used a classical treatment of the material and the HE staining as well as immunohistochemical staining. Results: We present a rare case of malignant mesothelioma of tubulopapilar epitheloid type in man 72 years with hydrocell and pain in the left testicle. Surgical excision of cystic change was performed on the border of the testicles and the funiculus. The tumor change of 4 mm in diameter was found in the cyst wall. Microscopically, the tumor lesion was built from tubulopapilar structures with sparse connective tissue of stroma. Tumor cells of epitheloid type contained oval nuclei with distinctive nucleus. Nucleus showed moderate atypicality and hyper chromaticity, while mitosis were relatively frequent. Immunohistochemical, tumor cells were positive on CK 5/6, EMA, calretinin and mesothelin-1. The diagnosis of malignant mesothelioma was set on the basis of clinical, histological and immunohistochemical images. CT excluded the presence of tumor in the pelvis and abdomen. Tumor formation that infiltrates funiculus spermaticus and testicle, was found in the operational material. It was also present on the upper edge of the resection. After four months the patient has died. Conclusion: The case of a rare primary malignant mesothelioma of testicle tunica vaginalis, due to highly aggressive nature, has been presented. Key words: malignant mesothelioma, tubulopapilar epitheloid type, testicle 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 1/poster prezentacije 1 P28 Metastaze sitnoćelijskog karcinoma pluća u svetloćelijski karcinom bubrežnih ćelija - prikaz slučaja Small cell lung carcinoma metastasized to clear cell renal carcinoma: a case report Ivana D. Savić1, Martina M. Stojanović1, Snežana V. Raljević2, Radmila M. Janković1, Jovan D. Vasiljević1 Ivana D. Savic1, Martina M. Stojanovic1, Snezana V. Raljevic2, Radmila M. Jankovic1, Jovan D. Vasiljevic1 Cilj: Histopatološka i imunohistohemijska analiza sitnoćelijskog karcinoma pluća (SCKP) u svetloćelijski karcinom bubrežnih ćelija (SCKBC). Uvod: Prisustvo dva ili više primarnih tumora kod pacijenta, nije neuobičajeno, ali metastaze jednog tumora u drugi (tumor-u-tumor metastaze)jesu retka pojava. Najčešći tumori donori metastaza u okviru ovog fenomena su karcinomi pluća, prostate i štitne ž lezde, a najčešći tumor primalac ovih metastaza je karcinom bubrežnih ćelija. Materijal i metode: Analiza preparata uzoraka tkiva uzetih na autopsiji, bojenih hematoksilin-eozin i streptavidin-biotin imunohistohemijskom metodom. Rezultati: Muškarac, starosti 53 godine je primljen u Kliniku za pulmologiju Klinickog Centra Srbije, zbog dispneje, parapereze, gubitka svesti i prisustva tumorske mase u medijastinumu (prethodno CT-om verifikovane u okruženoj bolnici). Pacijent je preminuo jedan dan nakon prijema. Na autopsiji je nađen infiltrativan tumor u donjem desnom lobarnom bronhu sa metastazama u kontralateralnom plućnom krilu, desnoj nadbubrežnoj žlezdi, epikardu i više limfnih čvorova (hilusnim, medijastinalnim i vratnim). Histopatološka slika je bila tipična za SCKP. Takode, na gornjem polu desnog bubrega uočen je jasno ograničen, žućkast tumor, dimenzija 2.5 x 2 cm. Histopatološkom analizom nađen je SCKBC, nuklearni gradus II po Fuhrman-u, sa metastatskim gnezdima SCKP. Imunohistohemijskom analizom tumora bubrega dijagnoza je potvrđena. U gnezdima SCKP uočena je TTF-1, hromogranin A, sinaptofizin, i CD56 pozitivnost, dok je u SCKBC uočena vimentin, citokeratin, CD10 i RCC pozitivnost. Zaključak: Ovo je do sada prvi objavljen slučaj metastaza sitnoćelijskog karcinoma pluća u svetloćelijski karcinom bubrežnih ćelija. Ključne reči: sitnoćelijski karcinom pluća, svetloćelijski karcinoma bubrežnih ćelija, tumor-u-tumor metastaze Aim: Histopathological and immunohistochemical analysis of small cell lung carcinoma (SCLC) metastasized to clear cell renal cell carcinoma (ccRCC). Introduction: Presence of two or more malignancies in a single patient is not uncommon, but tumor-to-tumor metastases are considered rare. The most frequent donor tumors are the lung, prostate and thyroid gland tumors, whereas renal cell carcinoma is the most common recipient. Material and methods: Analysis of slides, from tissue samples collected at the autopsy, stained with hematoxylin-eosin and streptavidin-biotin immunohistochemical method. Results: A 53-year old male with dyspnea, paraparesis and loss of consciousness was admitted at the Clinic of Pulmology, Clinical Center of Serbia. Thoracal CT scan, performed previously in community hospital, showed mediastinal tumorous mass. He died a day after admission. On autopsy, infiltrative tumor in the right inferior lobar bronchus was found with metastases in contralateral lung, right adrenal gland and epicardium and in multiple lymph nodes (hilar, mediastinal and neck). Histology was typical for SCLC. Also, in the upper pole of right kidney sharply circumscribed, yellowish tumor measuring 2.5 x 2 cm was noted. Histopathological analysis showed ccRCC, Fuhrman nuclear grade II, with metastatic nests of small cell lung carcinoma within it. Immunohistochemical analysis of kidney tumour confirmed the diagnosis with TTF-1, chromogranin A, synaptophysin and CD56 positivity in SCLC nests and vimentin, cytokeratin, CD10 and RCC positivity in ccRCC. Conclusion: According to our knowledge, this the first reported case of small cell lung carcinoma metastasized to clear cell renal cell carcinoma. Key words: small cell lung carcinoma, clear cell renal carcinoma, tumor-to-tumor metastases 1Institut za patologiju, Medicinski fakultet, Univerzitet u Beogradu, Beograd, Srbija 2Klinika za pulmologiju, Klinički Centar Srbije, Beograd, Srbija 1Institute of Pathology, Faculty of Medicine, University of Belgrade, Belgrade, Serbia 2Clinic of Pulmology, Clinical Center of Serbia, Belgrade, Serbia, 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 621 POSTER PRESENTATIONS 1/poster prezentacije 1 P29 Ispoljavanje razlićitih progenitorskih markera i njihova korelacija sa NCAM ekspresijom u fetalnom bubrežnom tkivu Sanja Ćirović1, Jelena Vještica1, Martina Stojanović1, Jasmina Tadić2, Svetislav Tatić1, Jasmina MarkovićLipkovski 1 1Institut za patologiju, Medicinski fakultet, Beograd, Srbija klinika „Višegradska”, Beograd, 2Ginekološko-akušerska Srbija Cilj: Korelacija NCAM ekspresije sa markerima stem celija TRA-1-60, CD24, CD133 i CD34 u fetalnom tkivu bubrega. Uvod: Interakcija izmedu ureteralnog pupoljka i mezenhimalnih ćelija dovodi to formiranja bubrega tokom embrionalnog razvoja. NCAM se eksprimira na svim mezenhimalnim ćelijama u ovom periodu i zato se smatra markerom progenitorskih ćelija bubrega. Novija istraživanja pokazala su postojanje adultnih renalnih progenitorskih ćelija koje eksprimiraju CD24 i CD133. Materijal i metode: Prisustvo NCAM izoformi u humanom fetalnom tkivu bubrega različite gestacione starosti analizirano je RT-PCR. Duplom imunofluorescencom detktovana je koekspresija NCAM-a sa markerima stem ćelija: TRA-1-60, CD24, CD133, CD34. Takode je analizirano i ispoljavanje PSA/NCAM, FGFR1 i Ki67 na istim uzorcima. Rezultati: RT-PCR analiza je pokazala postojanje sve 3 NCAM izoforme (120, 140, 180 kDa) u tkivu. Mezenhimalne ćelije oko ureteralnog pupoljka ispoljile su NCAM PSA/NCAM-CD24-CD133- fenotip. U ovoj regiji nadene su i FGFR1 NCAM-, kao i CD133 NCAM- i CD34 NCAM- mezenhimalne ćelije. Takode je primećeno da TRA-1-60, CD24, CD133 eksprimiraju ćelije ureteralnog pupoljka. S druge strane, mezenhimalne ćelije u nefrogenoj zoni (kondenzovanom mezenhimu i njegovim derivatima) imale su NCAM PSA/ NCAM CD24 Ki67 fenotip. PSA/NCAM prisutan je samo u nefrogenoj zoni, kao i retke NCAM/FGFR1 ćelije, u ovoj regiji nisu nađene mezenhimalne ćelije pozitivne na TRA-1-60, CD34, CD133. Zaključak: Različita koekspresija NCAM-a sa stem marketima TRA-1-60, CD24, CD133, CD34 ukazuje na postojanje bar 2 vrste renalnih fetalnih progenitor ćelija. PSA/NCAM učestvuje u ranom formiranju nefrona. Iznenađujuća je detekcija malog broja NCAM/ FGFR1 ćelija. Ključne reči: progenitorski-stem markeri, NCAM, CD24, CD133, TRA-1-60 622 Expression of different progenitor markers in correlation with NCAM in human fetal kidney Sanja Cirovic1, Jelena Vjestica1, Martina Stojanovic1, Jasmina Tadic2, Svetislav Tatic1, Serbia Jasmina Markovic-Lipkovski1 1Institute for Pathology, Medical Faculty, Belgrade, Serbia and Gynecology Clinic “Visegradska”, Belgrade, 2Obstetrics Serbia Aim: Co-expression of NCAM and other progenitor markers TRA-1-60, CD24, CD133 and CD34 in fetal tissue was investigated in this study. Introduction: Structures of adult kidney arise from interactions between metanephric mesenchyme and ureteric bud. The neural cell adhesion molecule (NCAM) is widely expressed during development. Recent study showed CD24 and CD133 positive adult renal progenitor cells. Material and methods: Human fetal kidney tissue on different stages of gestation was analyzed using double immunofluorescent staining of NCAM and stem cell markers: TRA-1-60, CD24, CD133, CD34, as well as PSA-NCAM, FGFR1 and Ki67. RTPCR was used to evaluate NCAM isoforms. Results: RT-PCR shows presence of 120, 140, and 180 kDa isoforms. Mesenchymal cells around ureteric bud were NCAM PSA/NCAM-CD24CD133-. FGFR1 NCAM- mesenchyme cells were found in this area, single cells CD133 NCAM- and CD34 NCAM- were also found here. TRA-1-60, CD24 and CD133 stain uretetic bud. PSA/NCAM NCAM, Ki67 NCAM, CD24 NCAM was detected in nephrogenic zone (cap mesenchyme, pretubular aggregates, renal vesicle, comma and S-shaped body). In these areas NCAM co-expression with TRA-1-60, CD34, CD133 and FGFR1 was not detected. Conclusion: Different co-expression of NCAM with CD24, CD133, and TRA-1-60 suggests that more than one type of renal stem cells exist. PSANCAM is important for early development of the nephron. Interestingly, only restricted number of cells coexpressed FGFR1 and NCAM. Key words: progenitors/stem markers,NCAM, CD24, CD133, TRA-1-60 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 2/poster prezentacije 2 P30 Sklerozirajući peritonitis kao posledica hroničnog lečenja peritoneumskom dijalizom - prikaz slučaja Ivana Tufegdzić1, 1Institut Dejan Pilčević2 za patologiju i sudsku medicinu, Vojnomedicinka akademija, Beograd, Srbija 2Klinika za nefrologiju, Vojnomedicinska akdemija, Beograd, Srbija Cilj: Ukazivanje na moguću posledicu dugotrajnog lečenja peritoneumskom dijalizom sa pojavim ozbiljne komplikacije sklerozirajućim peritonitisom. Uvod: Sklerozirajući peritonitis je retka, ali jedna od najozbiljnijih komplikacija kod pacijenata na hroničnoj peritoneumskoj dijalizi. Nejasne je patogeneze, dovodi do morfoloških promena peritoneumske membrane sa sklerozom, formiranjem adhezija i inkapsulacijom crevnih vijuga i posledičnim (sub)okluzijama i pojavom ultrafiltracione slabosti. Dijagnoza se zasniva na kliničkim simptomima subokluzije, laboratorijskim i radiološkim nalazima, makroskopskim i histopatološkim kriterijumima. Materijal i metode: Muškarac starosne dobi 52 godine, na programu lečenja peritoneumskom dijalizom oko 6 godina usled razvoja terminalne bubrežne insuficijencije koja se javila zbog hroničnog glomerulonefritisa. Primljen je zbog peritonitisa klinički manifestovanog bolovima u abdomenu, povraćanjem, razvojem subokluzija i laboratorijskim nalazom koji je pokazao upalni proces. Usled infekcije izlaznog mesta peritonealnog katetera, pristupilo se vadjenju istog, prilikom čega je identifikovan zadebljao visceralni peritoneum sa izraženom fibrozom koja fiksira crevne vijuge. Uzet je uzorak tkiva parijetalnog peritoneuma, sa kojeg su načinjeni hematoksilin-eozin bojeni preparati, kao i specijalnim histohemijskim bojenjem Lendrum s Picro Mallory i imunohistohemijskom metodom Ki67 i SMA. Rezultati: Nadjene histološke promene su: mezotelna denudacija, deponovanje fibrina uz infiltraciju sa mononuklearnim celijama i uvećanim aktivisanim fibroblastima, kao i kapilarnom angiogenezom i hijalinizacijom- opisani kriterijumi odgovaraju sklerozirajucem peritonitisu. Zaključak: Skerozirajuci peritonitis je ozbiljna komplikacija kod pacijenata na peritoneumskoj dijalizi sa visokim stepenom mortaliteta. Ukoliko postoji klinička sumnja za razvoj istoga, neophodna je ciljana dijagnostička obrada i histološka evaluacija radi rane detekcije i adekvatnog terapijskog pristupa. Ključne reči: Sklerozirajući peritonitis, peritonealna dijaliza, renalna insuficijencija Encapsulating peritoneal sclerosis in patient on peritoneal dialysis-a case report Ivana Tufegdzic, Dejan Pilcevic Institute for pathology and forensic medicine, Military Medical Academy, Belgrade, Serbia Clinic for nephrology, Military Medical Academy, Belgrade, Serbia Aim: Pointing out the possible consequence of long term peritoneal dialysis treatment with the most serious complication: encapsulating peritoneal sclerosis. Introduction: Eencapsulating peritoneal sclerosis is rare, but the most serious complication of chronic peritoneal dialysis treatment. Pathogenesis is not clear, disease is characterized by sclerotic thickening of the peritoneal membrane, formation of adhesions, accompanied by bowel encapsulation and subsequent bowel (sub)occlusion with ultra filtration weakness. The diagnosis is established by clinical, laboratory, radiographic and histological findings. Material and methods: Male, age 52, on going peritoneal dialysis treatment for last 6 years due to terminal renal failure caused by chronic glomerulonephritis. He was admitted due to peritonitis with severe abdominal pain, vomiting, developing suboclusions and laboratory findings which point out inflammatory process. Due to infection of peritoneal catheter, the same was removed and during that procedure the sclerotic thickening of the peritoneal membrane with bowel sclerosis and fixation had been seen. Histological examination of peritoneal membrane was performed along with histochemical (Lendrum s Picro Mallory) and immunohistochemical (Ki67 and SMA) analysis. Results: Histological analysis: mesothelial denudation, fibrin deposition, mononuclear cell infiltration, fibroblast enlargement, capillary angiogenesis and hyalinisation-poposed histological criteria for a diagnosis of encapsulating peritoneal sclerosis. Conclusion: Encapsulating peritoneal sclerosis is the most serious complication of peritoneal dialysis treatment with high mortality rate. If there is any clinical suspicion, it is a need to establish the possible diagnosis by clinical criteria and histological evaluation in order to early detection and adequate therapy. Key words: Encapsulating peritoneal sclerosis, peritoneal dialysis, renal failure 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 623 POSTER PRESENTATIONS 2/poster prezentacije 2 GINEKOLOŠKA PATOLOGIJA GINECOLOGICAL PATHOLOGY P31 Upotreba Wilms tumor 1 imunohistohemije u evaluaciji ovarijalnih karcinoma Use of Wilms tumor 1 immunohistochemistry in the evaluation of ovarian carcinomas Biljana Đordević1, Dragan Mihailović1, Simonida Stojanović1, Živković Nikola1, Danijela Živanović1, Milorad Pavlović2 Biljana Djordjevic1, Dragan Mihailovic1, Simonida Stojanovic1, Nikola Zivkovic1, Danijala Zivanovic1, Milorad Pavlovic2 1Institut za patologiju, Medicinski fakultet, Univerzitet u Nišu, Srbija 2Služba patologije, Opšta bolnica Leskovac, Srbija Cilj: Cilj ove studije bio je da proceni da li Wilms tumor 1 (WT1) imunohistohemija ima dvostruku upotrebu u evaluaciji tumorskih i endotelnih ćelija karcinoma jajnika. Uvod: Nedavno je pokazano da WT1 imunohistohemija može biti upotrebljena za analizu tumorskih i endotelnih ćelija karcinoma jajnika. Materijal i metode: Parafinski blokovi tkiva janika 40 bolesnica izabrani su iz arhiva Instituta za patologiju Medicinskog fakulteta Univerziteta u Nišu. Tri uzastopna preseka svakog slučaja bili su predmet imunohistohemijskih analiza mišijim monoklonskim antitelima protiv WT1 proteina, dobro definisanog ovarijalnog markera, CA-125, i fenotipskog markera endotelnih ćelija, CD34, redom. Rezultati: Različita WT1 imunoreaktivnosti bila je nađena u tumorskim i endotelnim ćelijama. Više od 90% WT1 pozitivnih tumorskih i endotelnih ćelija bilo je pozitivno za CA-125 (tumorske ćelije) i CD34 (endotelne ćelije). Slično, više od 90% CA-125 ili CD34 pozitivnih ćelija eksprimiralo je i WT1 u tumorskim ili endotelnim ćelijama, redom. Zaključak: Rezultati ove studije sugeri u da WT1 imunohistohemija može biti upotrebljena za procenu tumorskih ćelija i mikrovaskularne gustine kod karcinoma jajnika. Budući da je WT1 eksprimiran u tumorskim i endotelnim ćelijama, razvoj terapijskih sredstava protiv WT1 može da predstavlja efikasnu terapijsku opciju za karcinoma jajnika. Ključne reči: Karcinom jajnika, imunohistohemija, WT1 protein 624 1Institute of Pathology, Faculty of Medicine, University of Nis, Serbia 2Departmeny of Pathology, General Hospital, Leskovac, Serbia Aim: The aim of this study was to assess if Wilms tumor 1 (WT1) immunohistochemistry has dual usages in the evaluation of tumor and endothelial cells of ovarian carcinomas. Introduction: Previous studies revealed that a single WT1 immunohistochemistry can be used to elucidate both the cancer cells and blood vessels of EOCs. Material and methods: Paraffin-embedded ovarian tissue blocks from 40 patients were retrieved from the files of the Institute of Pathology, Faculty of Medicine, University of Nis. A set of three consecutive sections from each case were subjected to immunohistochemical analyses with a mouse monoclonal antibody against the human WT1 protein, a well defined ovarian tumor marker, CA-125, and a endothelial cell phenotypic marker, CD34, respectively. Results: Different WT1 immunoreactivity was found in both tumor and endothelial cells. Over 90% of WT1 positive tumor and endothelial cells were positive for CA-125 and CD34, respectively. Similarly, over 90% of CA-125 or CD34 positive cells co-express WT1 in tumor or endothelial cells, respectively. Conclusion: The results of this study suggest that WT1 immunohistochemistry can be used to assess both tumor cells and microvascular density in ovarian carcinomas. Since WT1 is expressed in both tumor and endothelial cells, the development of therapeutic agents to target WT1 may provide an effective therapeutic option for ovarian carcinomas. Key words: Ovarian carcinoma, immunohistochemistry, WT1 protein 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 2/poster prezentacije 2 P32 Prvi srpski imunohistohemijski dokazan limfoepitelioma-nalik karcinom u cerviksu First Serbian immunohistochemicaly detected Lymphoepithelioma-like carcinoma in cervix Ljiljana Tomić1, Miloš Zarić1, Vuk Milutinović2, Jovanka Trifunović1 Ljiljana Tomic1, Milos Zaric2, Vuk Milutinovic1, Jovanka Trifunovic1 2 Opšta 2General 1Vojnomedicinska Akademija Beograd, Srbija bolnica Vršac, Srbija Cilj: Kad god se susretnemo sa loše diferentovanim karcinomom, sa izraženim limfoplazmocitnim infiltratom, bez obzira na mesto nastanka, treba pomisliti na LELC. Uvod: Lymphoepithelioma-like carcinom(LELC) se detektuje van nazofarinksa u 0,7% slučajeva u cervixu, kao primarnom mestu pojave. Literaturno navodi se hipotetska sumnja u povezanost sa Epstajn Barovim virusom(EBV) na drugim anatomskim pojavnim mestima, osobito kod Azijatkinja a ne kod belkinja. Materijal i metode: Lymphoepithelioma-like carcinom je opisan na mnogim mestima, van nazofarinksa. Ova retka forma skvamocelularnog karcinoma opisana je u 0,7% slučajeva cervikalne primarne maligne forme. Smatra se da cervikalni LELC je u vezi sa Epstein-Barr virusnom (EBV) infekcijom, ali u našem slučaju, je u pitanju belkinja nasuprot Azijatkinji, koje literaturno, imaju, EBV genom. Rezultati: Mikroskopski pregled je potvrdio loše diferentovanu formu skvamoznog karcinoma sa prominentnim limfoplazmocitnim infiltratom. Nije dokazana glandularna komponenta. Ćelije su bile krupne, nejasnih margina-”syncytial”-ni izgled. Tumorske ćelije su bile jako pozitivne na Anti-Epitelni Membrane antigen(EMA). P63 protein kao homolog p53 proteina, marker skvamozne diferencijacije, bio je difuzno eksprimovan, sto je isključilo glandularnu ili neuroendokrinu diferencijaciju. Anti-p53 protein takođe pozitivan. Tumorske ćelije su jako eksprimirale visok proliferativni index Ki-67 (MIB1) u više od 80% pozitivnih tumorskih jedara. Inflamatorni infiltrat je bio Leucocyte Common Antigen (LCA) positivan. Imunohistohemijski EBV nije ubedljivo detektovan. Zaključak: LELC je retka forma skvamocelularnog karcinoma. Ovo je prvi imunohistohemijski detektovan slučaj lelca kod srpske žene. Ključne reči: cerviks, belkinja, limfoepiteliomanalik karcinom 1Military Medical Academy, Belgrade, Serbia Hospital Vrsac, Serbia Aim: We present LELC in order to bear in mind such possibility whenever confronted with poorly differentiated carcinoma rich in lymphoplasmocytic infiltration regardless of the anatomic site. Introduction: Lymphoepithelioma-like carcinomas have been reported outside the nasopharynx in many sites.This distinct neoplasm is a very rare variant of squamous cell carcinoma accounting for only 0,7% of all uterine cervix primary malignant neoplasms. It has been proposed that cervical LELC may be related to Epstein-Barr virus infection,likewise at other locations and it has been demonstrated in Asian women. Materialand methods: We report a case of LELC of the uterine cervix in a 64 year old woman from Serbia detected immunohistochemically, fungating tumor, occupied the posterior lip of the cervix. Results: Microscopic examination of the biopsy specimen disclosed a poorly differentiated nonkeratinizing carcinoma composed of cohesive nests surrounded by prominent lymphoplasmocytic infiltrate.Small foci of nonkeratinizing carcinoma appeared in the stroma, surrounded by marked inflammatory reaction. Cells were large and had indistinct cell margins (syncytial-like pattern). The tumor cells were strongly positive for Anti-Epithelial Membrane antigen. P63 protein as homologue of the p53 protein, being a powerful marker for squamous differentiation, was diffusely expressed, which excluded a glandular or neuroendocrine differentiation. Anti-p53 protein was expressed as well. Tumor cells also expressed a high proliferative rate i.e. Ki-67 of more than 80% positive tumor nuclei. Conclusion: LELC is a rare variant of squamous cell carcinoma of the uterine cervix. This is its first case in Caucasian woman from Serbia detected immunohistochemicaly. Key words: cervix, caucasian woman, lymphoepithelioma-like cancer 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 625 POSTER PRESENTATIONS 2/poster prezentacije 2 P32 Tumor granuloza ćelija ovarijumaprikaz slučaja Granulosa cell tumor ovary - case description Radmila Bajić1, Golubović Mileta2, Željko Lazović1 Radmila Bajic1, Mileta Golubovic2, Zeljko Lazovic1 1Opšta bolnica Bijelo Polje, Bijelo Polje, Crna Gora 2Klinicki Centar Crne Gore, Podgorica, Crna Gora Cilj: Prikaz slučaja Uvod: Tumori granuloza ćelija spadaju u grupu ovarijalnih neoplazmi, koje u potpunosti čine granuloza ćelije. Ove novotvorine čine 3% svih tumora jajnika. Mogu se javiti u svakom uzrastu, ali se dvije trećine javlja poslije menopauze. Tumori granuloza ćelija su obično unilateralni. Veličina i građa im variraju od mikroskopskih fokusa do velikih solidnih ili cističnih inkapsuliranih masa. Materijal i metode: Pacijentkinja u menopauzi starosti 56 godina primljena na odjeljenje radi operativnog liječenja miomatoznog uterusa. Nakon klasične histerektomije sa adneksektomijom tumefakcija na lijevom jajniku je bila dimenzija 5x 4x 3 cm, glatke spoljašnje površine, na presjeku solidne grade, žutonarandžaste boje, okružena vezivnom kapsulom. Materijal je u patohistološkoj obradi bojen metodama HE (hematoksilin eozin) i IHH (imunohistohemijsko bojenje). Histološki, tumorsko tkivo je komponovano dominantno u vidu solidnih i trabekularnih formacija. U centralnim pojedinim djelovima uocavaju se ovalni mali prostori koji sadrže eozinofilan materijal i celularni debris (Call Exnerbodies). Tumorske ćelije su srednje krupne, blijedoeozinofilne citplazme, nejasnih granica, okruglih hiperhromatičnih jedara, niske mitotske aktivnosti. Stroma dijelom srednje obilna. Rezultati: Imunohistohemijska verifikacija: VIMENTIN jako pozitivan, CK 18 negativan, EMA negativan i CEA negativan Zaključak: Patohistološki nalaz: Granulosa Cell Tumor ovarii (solid type). Ključne reči: tumor, granuloza ćelije, jajnik 626 1General 2Clinical Hospital Bijelo Polje, Bijelo Polje, Montenegro Center of Montenegro, Podgorica, Montenegro Aim: Case description Introduction: Granulosa cell tumors belong to the group of ovarian neoplasms, which entirely consists of granulosa cells. These neoplasms constitute 3% of all ovarian tumors. They can occur at any age, but two-thirds occurs after menopause. Granulosa cell tumors are usually unilateral. The size and structure are moving from microscopic forms to large solid or cystical encapsulated mass. Material and methods: The pacient in menopause at the age of 56 is recived on the department because of operational treatment miomatous uterus. After classic hysterectomy with adnexectomy tumefact on the left ovary was 5x 4x 3 cm, smooth exterior area, on the cut of solid structure, yellow-orange color, circled with a connective capsule. The material is in the pathohystological processing coloured with metods of HE (Hematoxylin eosin) and IHH (Immunohistochemical colour). Histologically, the tumor was composed predominantly in the form of solid and trabecular formation. In the central individual parts there are small oval spaces that contain eosinophilic material and cellular debris (Call Exner bodies). Tumor cells are medium large, pale eosinophilic cytoplasms, with unclear borders, with round hiperhromatic sails, with low mitotic activity. Stroma mostly medium dense. Results: Immunohystochemical verification: Vimentin strongly positive, CK 18 is negative, EMA is negative and CEA negative. Conclusion: Histopathological Findings: Granulosa Cell Tumor ovarii (solid type). Key words: tumor, granulosa cell, ovarii 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 2/poster prezentacije 2 P33 Distribucija histoloških tipova carcinoma jajnika na operativnom i biopsijskom materijalu- GAK Narodni front Beograd u 2011 Danica Vukičević1, Igor Kuzmanović2, Marijana Cimbaljević2, Milica Mijović1, Nebojša Mitić1 1Institut za patologiju, Medicinski fakultet, Priština, Kosovska Mitrovica, Srbija 2Ginekološko akušerska klinika Narodni front, Beograd, Srbija Cilj: Utvrđivanje zastupljenosti karcinioma jajnika u celokupnom analiziranom operativnom i biopsijskom materijalu, utvrdivanje distribucije histopatoloških tipova ovog karcinoma, kao i u kom stadijumu prema TNM klasifikaciji je bolest dijagnostikovana. Uvod: Od malignih tumora jajnika su najčešći karcinomi i njihova učestalost se kreće u svetu, prema Svetskoj zdravstvenoj organizaciji u rasponu od 2,0-15,3 a u Srbiji ta vrednost je od 9,11-11,1 na 100.000 žena. Materijali i metode: Korišcen je biopsijski i operativni materijal koji je obrađen i analiziran u laboratoriji na ginekološko akušerskoj klinici Narodni front u Beogradu za 2011 godinu. Rezultati: Od 9119 žena od kojih je uzet biopsijsjki materijal karcinom jajnika je potvrden u 108 (1,18%). Epitelnih malignih tumora je bilo u 75 (69,44%) slucajeva od kojih su borderline tumori zabeleženi u 17 (22,67%), tumora porekla polnih traka u 6 (5,56%), tumora porekla germinativnih celija u 4 slučaja (3,70%), mešovitih epitelno-mezenhimnih tumora u 3 slučaja (2,78%), metastatskih karcinoma jajnika u 20 slučajeva (18,52%). Prosecna životna dob pacijentkinja bila je 56,66ą13,23g. Bolest je najce ce dijagnostikovana u stadijumu FIGO IA u 32.95%, FIGO IIIC u 20.45% dok je u ostalim stadijumima ima značajno manje. Zaključak: Na analiziranom materijalu najučestaliji histološki tip karcinoma jajnika su epitelni maligni tumori, dok je bolest najčešće dijagnostikovana u stadijumu FIGO IA. Ključne reči: Karcinomi jajnika, TNM klasifikacija, Metastatski karcinomi jajnika. Distribution of histological types of ovarian cancer in operational and biopsy material in Gynecological and Obstretic Clinics Narodni FrontBelgrade during 2001. year Danica Vukicevic1, Igor Kuzmanovic2, Marijana Cimbaljevic2, Milica Mijovic1, Nebojsa Mitic1 1Institute of pathology, Medical school Pristina, Kosovska Mitrovica, Serbia 2Gynecologic and Obstetrics Clinic Narodni Front, Belgrade, Serbia Aim: Determine the presence of ovarian cancer in the overall analyzed operational and biopsy material, determine the distribution of histological types of cancer, as well as determine TNM stage in which the disease is diagnosed. Introduction: Cancers are the most common malignant ovarian tumors and according to the World Health Organization, their frequency ranges in the world, from 2.0 to 15.3. in Serbia, this value is from 9.11-11.1 per 100,000 women. Material and Methods: Biopsy and operational material that is processed and analyzed in the laboratory of Gynecologic and Obstetrics Clinic Narodni Front, Belgrade in 2011. Results: Of 9119 women of whom the biopsy material was taken ovarian cancer was confirmed in 108 cases (1.18%). We found 75 (69.44%) malignant epithelial tumors. Among them there were 17 (22.67%) borderline tumors, 6 cases (5.56%) sex cord tumors, 4 (3.70%) germ cell tumors, 3 (2.78%) mixed epithelial-mesenchymal tumors and 20 (18.52%) metastatic ovarian cancer. The average age of patients was 56.66 ą 13.23 g. The disease was usually diagnosed in FIGO stage IA at 32.95%, FIGO IIIC at 20.45%, while in the other stages is significantly less. Conclusion: The most common histological type of ovarian cancer in the analyzed material are epithelial malignant tumors, usually diagnosed in FIGO stage IA. Key words: Ovarian cancers, TNM classification, metastatic ovarian cancer. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 627 POSTER PRESENTATIONS 2/poster prezentacije 2 P34 Diferencijalna dijagnostika džinovskih mnogojedarnih ćelija u Papanikolau - brisu Differential Diagnostics of Multinucleated Giant Cells in the Papanicolau Smear Dušan Lalošević1, Milana Panjković2, Marko Maksimović3, Jovan Maksimović4 Dusan Lalosevic1, Milana Panjkovic2, Marko Maksimovic3, Jovan Maksimovic4 1Medicinski 2Institut fakultet i Pasterov zavod Novi Sad, Srbija za plućne bolesti Vojvodine, Sremska Kamenica, Srbija 3Klinika za ginekologiju i akušerstvo Vojvodine, Novi Sad, Srbija 4Ginekološka ambulanta ”Maksimović”, Novi Sad, Srbija Cilj: Cilj istraživanja je komparacija citološke i histološke slike džinovskih mnogojedarnih ćelija u Papanikolau (Papa) brisu i biopsiji grlića materice. Uvod: Nalaz mnogojedarnih džinovskih ćelija ukazuje na granulomatozno zapaljenje. Mnogojedarne džinovske ćelije mogu biti dva tipa, Langhansove i oko stranog tela. Međutim, naročito u citološkom razmazu je teško razlikovati ova dva tipa ćelija. U Papa-brisu grlića materice taj nalaz je vrlo redak, kao i granulomatozni cervicitis. Bethesda vodič pominje mnogojedarne džinovske ćelije kod starijih žena kao ćelijsku promenu povezanu sa starenjem. Brojna patološka stanja mogu biti povezana sa mnogojedarnim džinovskim ćelijama u Papa-brisu, ali takođe mogu da imitiraju karcinom ili da budu povezana sa karcinomom. Razmotreni su etiološki faktori granulomskog cervicitisa, tuberkuloza, talk-granulomi, ceroid granulomi, amebijaza, šistozomijaza, sarkoidoza, Crohn-ova bolest, spirala, kao i granulomi oko stranog tela. Materijal i metode:Analizirani su Papa-brisevi i komparirani sa biopsijskim uzorcima grlića materice. Od pregledanih 500 Papa-briseva, džinovske mnogojedarne ćelije su nađene samo u jednom slučaju. Rezultati: Prikaz slučaja.- Pacijentkinja stara 61 godinu, u menopauzi 6 godina, pregledana je u ginekološkoj ambulanti, kada je konvencionalni Papa-bris interpretiran kao diskariotičan. U ponovljenom razmazu vidjena je atrofija, brojni leukociti i džinovske mmnogojedarne ćelije. Nakon toga, urađena je biopsija cerviksa i nađen atrofični cervicitis sa granulomima oko hirurškog konca. Pacijentkinja je imala dve trudnoće od kojih je poslednja završena sa ginekološkom operacijom pre 32 godine. Zaključak: Granulomi na hirurški konac posle više od 30 godina od operacije su jako neobičan nalaz, te je za razjašnjenje pojave džinovskih mnogojedarnih ćelija u Papa-brisu bila potrebna biosija grlića materice. Ključne reči: Papa-bris, džinovske mnogojedarne ćelije, granulomi, konac 628 1Faculty of Medicine and Pasteur Institute of Novi Sad, Hajduk Veljkova 1, Serbia 2Institute for Pulmonary Diseases of Vojvodina, Sremska Kamenica, Serbia 3Clinic of Obstetrics and Gynecology of Vojvodina, Novi Sad, Serbia 4Gynecological clinic “Maksimovic,” Novi Sad, Serbia Aim:The goal of this study is comparison of cytological and histological pictures with multinucleated giant cells in Papanicolau (Pap) smear and cervical biopsy. Introduction: Finding of multinucleated giant cells strongly suggest a granulomatous inflammation. Multinucleated giant cells may be presented as two types, Langhans and foreign body giant cells. However, especially in smears, it is difficult to distinguish these two types of cells. In Pap smears of uterine cervix this finding is very rare, as well as granulomatous cervicitis. Bethesda manual mentioned multinucleated giant cells in older women as age-related cellular changes. Numerous pathological conditions may be associated with multinucleated giant cells, but also these cells can mimic cancerous changes in Pap smear and may be associated with carcinoma. Etiological factors of granulomatous cervicitis are discussed such as tuberculosis, talc-granuloma, ceroid granuloma, amoebiasis, schistosomiasis, sarcoidosis, Crohn disease, contraceptive intrauterine device, and foreign body granulomas. Material and Methods: Analysis of Pap smears was performed and compared with conventional biopsies of uterine cervix. Among 500 examined Pap smears, the giant multinuclear cells were found in one case. Results:Case report.-Female aged 61, 6 years in menopause, was seen in the outpatient gynecology clinics when conventional Pap smear was interpreted as abnormal with dyscaryosis. In repeated smear, atrophy, many leukocytes, and multinucleated giant cells were found. After that, cervical biopsy was performed and chronic atrophic cervicitis with surgical suture granulomas were found. Patient had two pregnancy and last one 32 years ago was finished with gynecologic operation. Conclusion:Postsurgical granulomas after more than 30 years are very unexpected finding and need biopsy to resolve Pap smear with giant cells. Key words: Pap smear, giant multinuclear cells, suture granulomas. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 2/poster prezentacije 2 P35 Kombinovani granulosa cell tumor i mucinozni cistadenom jajnika Danica Jovanović1, Neda Drndarević1, Popovic Marija2, Stojadinovic Vladimir2 1Poliklinika Beo-Lab Beograd,SR Srbija 2Zdravstveni centar Užice, Užice, Srbija Cilj: Želimo da prikažemo kombinovani tumor jajnika koji se retko javlja i za koga postoji veoma malo podataka u literaturi. Uvod: U literaturi je objavljen mali broj slucajeva kombinovanog tumora jajnika (adult granulosa cell tumor-AGCT et cystadenoma mucinosum ovarii). Teorije histogeneze uključuju kolizioni tumor i heterologu mucinoznu diferencijaciju unutar AGCT. Materijali i metode: Prikazujemo neuobičajeni tumor jajnika kod pacijentkinje stare 53 g. kod koje je ultrazvučnim pregledom utvrden multicistični tumor levog jajnika, prečnika 80mm.Hiruški odstranjen tumor bio je promera 90x80x70mm, glatke i sjajne površine, na reseku multicisticnog izgleda, mestimično ispunjen mucinoznim sadrž ajem. Sa različitih mesta uzeto je 7 isečaka za mikroskopsko ispitivanje koji su obojeni HE metodom. Reprezentativni isečak iz tumora obojen je imunohistohemijskom metodom za CK7, CK20, a-inhibin i calretinin. Rezultati: Mikroskopskim ispitivanjem ustanovljen je tumor koga grade dve komponente intimno povezane jedna sa drugom. Jednu komponentu gradi mešovit mucinozni epitel endocervikalno-intestinalnog tipa na unutrašnjoj strani cisticnih prostora, difuzno CK7 i fokalno CK20 . Druga komponenta, granuloza ćelijski tumor, se nalazi odmah ispod epitelne, pozitivna za a-inhibin i calretinin Zaključak: Intimna povezanost obe komponente ide u prilog teoriji da je mucinozna komponenta verovatno heterologa mucinozna diferencijacija unutar granulozo ćelijskog tumora. Ključne reči: Jajnik, cistadenom, granulozoćelijski tumor Combined adult granulosa cell tumor and mucinous cystadenoma of the ovary Danica Jovanovic1, Drndarevic Neda1, Marija Popovic2, Vladimir Stojadinovic2 1Polyclinic 2Health Beo-Lab Belgrade, Serbia Center Uzice, Uzice, Serbia Aim: We would like to describe the combined ovarian tumor that rarely occurs,and for wich there is a few data in the literature. Introduction: Only few cases of combined ovary tumor (adult granulosa cell tumor-AGCT et mucinosum cystadenoma ovarii) have been described and reported in the literature Theories of histogenesis include a collision tumor and heterologous mucinous differentiation within an AGCT. Material and Methods: We herein report a rare tumor of the ovary of the 53-year old woman. Ultrasound examination showed a multicystic tumor mass in the left ovary, 80 mm in diameter. Surgically removed tumor diameter measuring 90x80x70mm, with smooth and glossy surface. Cross-section revealed multicystic appearance, sporadically filled with mucinous content. Samples were taken from seven different sites, for microscopic examination, stained for routine histology (HE). Only representative specimens were immunostained for CK7, CK20, a-inhibin and calretinin. Results: Microscopically, the tumor was composed of two components that were intimately intermingled. One component was composed of mixed mucinous epithelium of endocervical-intestinal type that lined the inner side of cystic area, diffusely positive for CK7 and focally immunoreactive for CK20. The other was a granulosa cell tumor, just below the epithelial component, positive for a-inhibin and calretinin Conclusion: The intimate admixture of both components supports the theory that the mucinous component is probably heterologous mucinous differentiation within the adult granulosa cell tumor. Key words: Ovary, cystadenoma, granulosa cell tumor 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 629 POSTER PRESENTATIONS 2/poster prezentacije 2 P36 Prognostički značaj humanog horionskog gonadotropina i korelacija sa morfološkim parametrima kod gestacijskih trofoblastnih bolesti Prognostic significanse of human chorionic gonadotrophin and its correlation with morphology in gestational trophoblastic diseases Jelena Amidžić 1, Mihaela Mocko-Kaćanski 1, Matilda Đolai1, Aleksandra Levakov 1, Mileva Orelj- Popić, Jelena Amidzic1, Mihaela Mocko-Kacanski1, Matilda Đolai1, Aleksandra Levakov1, Mileva Orelj- Popic2 Cilj: Prikaz vrednosti humanog horionskog gonadotropina (ßHCG) u serumu i patohistoloških karakteristika kod gestacijskih trofobasnih bolesti, uz procenu njihove zastupljenosti. Uvod: Gestacijske trofoblastne bolesti, kao retke komplikacije trudnoce, obuhvataju oboljenja kod kojih je prisutna neadekvatna proliferacija tkiva trofoblasta. Mogu imati različito biološko ponašanje, od benignih stanja kao što su hidatidne mole (parcijalna i kompletna) i invazivna mola do ređih ali izuzetno agresivnih horiokarcinoma i tumora ležišta posteljice. Zajednička karakteristika ovih lezija je da dovode do porasta nivoa ßHCG-a u krvi, koji je bitan pokazatelj u dijagnostici, prognozi i praćenju učinka terapije. Materijal i metode: U istraživanje je uključeno 30 pacijentkinja, starosti od 16 do 49 godina, kod kojih je u Centru za patologiju i histologiju Kliničkog centra Vojvodine, u periodu od 2009 do 2011, na osnovu patohistološkog pregleda dijagnostikovana gestacijska trofoblasna bolest, a za koje postoje kompletni klinički i patohistološki podaci. Rezultati: Patohistološkim pregledom odabranih pacijentkinja dijagnostikovano je: 16 parcijalnih hidatidnih mola (53,33%), 10 kompletnih hidatidnih mola (33,33%), 2 invazivne hidatidne mole (6,67%), 1 horiokarcinom (3,33%) i 1 trofoblastni tumor posteljičnog ležišta (3,33%). Srednja vrednost ßHCG-a je kod parcijalnih mola bila 99212 mIU/ml, kod kompletnih mola 230070 mIU/ml, kod invazivnih mola 570150 mIU/ ml, a kod horiokarcinoma 290000 mIU/ml. Kod pacijentkinje sa trofoblastnim tumorom posteljičnog ležišta nisu nađene povišene vrednosti ovog hormona. Zaključak: Gestacijske trofoblastne bolesti se najčešće javljaju u reproduktivnom periodu žene, sa najvećom incidencom javljanja hidatidnih mola. Nivo ßHCG-a u krvi može ukazati na biološko ponšanje jer se naglašeno povišene vrednosti nalaze kod agresivnijih formi bolesti. Ključne reči: gestacijske trofoblastne bolesti, ßHCG Aim: Correlation of human chorionic gonadotrophin (hCG) values with histopathological features in gestational trophoblastic diseases (GTD). Introduction: GTD as uncommon complications of pregnancy, encompass group of diseases with abnormal proliferation of trophoblast. They include the benign condition such as hydatidiform mole (both partial and complete), invasive mole and malignant tumours such as choriocarcinoma and placental site trophoblastic tumour. A common feature of all of these trophoblastic lesions is production of hCG, which is used as an important marker for estimating presense, prognosis and adequate therapy of disease. Material and methods: The study population consists of 30 patients (16 to 49 years), with the GTD diagnosed on histological examination of endometrial curettage in the Pathology and Histology Centre of Clinical Centre of Vojvodina, from 2009 to 2011. Only patients with complete medical history were selected for the study. Results: Among patients, 16 partial hydatidiform moles (53.33%), 10 complete hydatidiform moles (33.33%), 2 invasive moles (6.67%), 1 choriocarcinoma (3.33%) and 1 placental site trophoblastic tumour (3.33%) were diagnosed. Following average serum hCG levels were found: 99212 mIU/ml in patients with partial hydatidiform moles, 230070 mIU/ml with complete hydatidiform moles, 570150 mIU/ml with invasive moles, 290000 mIU/ml with choriocarcinoma. Elevated values of this hormone were not found in patient with placental site trophoblastic tumour. Conclusion: The majority of GTD occur in generative period, with the highest incidence of mollar pregnaces. Biological behavior can be predicted by serum levels of hCG, as higher levels are assosiated with an aggressive forms of disease. Key words: gestational trophoblastic disease, hCG 1Centar za patologiju i histologiju, Klinički centar Vojvodine, Novi Sad, Srbija 2Klinika za ginekologiju i aku erstvo, Klinički centar Vojvodine, Novi Sad, Srbija 630 1Pathology and histology centre, Clinical Centre of Vojvodina, Novi Sad, Serbia 2Clinic of Gynecology and Obstetric,Clinical Centre of Vojvodina, Novi Sad, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 2/poster prezentacije 2 P37 Korelacija Papanicolau testa sa atipičnim skvamoznim ćelijama neodređjenog značaja (ASCUS) i patohistoloških nalaza Correaltion between PAP smear with atypical squamous cells of undetermined (ASCUS) and pathohystological findings Jasmina Atanacković1, Svetlana Milenković1, Biljana Kastratović- Kotlica2, Milena Mitrović2 Jasmina Atanackovic1, Svetlana Milenkovic1, Biljana Kastratovic- Kotlica2, Milena Mitrovic2 1Služba za patohistologiju, Klinički centar Srbije, Beograd, Srbija za ginekologiju i akušerstvo, Klinički centar Srbije, Beograd, Srbija 2Klinika Cilj: Sagledavanje primenjenih morfoloških kriterijuma u citološkoj analizi PAP testa sa atipičnim skvamoznim ćelijama i njihovo poređenje sa patohistološ kim nalazima, kao zlatnim standardom u dijagnostici Uvod: U interpretaciji cervikovaginalnih briseva se koristi numerička PAP klasifikacija, a od 1988. deskriptivna Bethesda klasifikacija, koja uvodi termin atipične skvamozne ćelije neodređenog značaja-ASCUS. ASCUS se karakteriše citološkim promenama koje upućuju na skvamozne lezije lakog stepena - L-SIL, ali su kvalitativno i kvantitativno nedovoljne za svrstavanje u L-SIL kategoriju. Zastupljenost ASCUS je do 5% u referentnim citološkim laboratorijama. Materijal i metode: U periodu 2008-2009. na Klinici za ginekologiju i akušerstvo KCS, ukupno je urađeno 13666 PAP testova, na konvencionalan način, a bojenih klasičnom Papanicolau metodom. Biopsije grlića materice sa kiretažom cervikalnog kanala su rađene u slučaju da se ASCUS ponavlja na kontrolnim pregledima više od dva puta ili ako je udružen sa atipičnom kolposkopijom. Biopsijski materijal je fiksiran u 4% puferisanom formalinu, klasično obrađen, sečen na 5 mikrona i bojeni HE metodom. Rezultati: Od ukupno analiziranih 13666 PAP testova 440 je interpretirano kao ASCUS (3,22%). Biopsije su urađenje kod 108 pacijenkinja sa ASCUS i dobijeni su sledeci patohistološki rezultati: 24 (22,22%) H-SIL, 19 (17,59%) L-SIL, 34 (31,48%) skvamozne metapalazije, 13 (12,03) hiper i parakeratoze, 10 (9,27%), polipa cerviksa i 8 (7,41%) cervicitisa. Zaključak: Histološka zastupljenost H-SIL od 22,22% nakon ASCUS u odnosu na literaturne podatke od 17,20% se može objasniti time što se kod nas citološka atipija tipa ASCUS ako je udružena sa promenama u kolposkopskoj slici odmah bioptira Ključne reči: Atipične ćelije, PAP test, ASCUS 1Department of Pathology, Clinical center Srbia, Belgrade, Serbia 2Clinic of Gynecology and Obstetric,Clinical Centre Serbia, Belgrade, Serbia Aim: Perceiving morphological criteria used in cytological analysis of PAP test with atypical squamous cells and correlation with patohystological findings as golden standard in diagnostic. Introduction: PAP classification is used as a numerical method in interpretation of cervicovaginal smears. Since 1988. descriptive Bethesda classification introduces term of atypical squamous cells of undetermined signifinace-ASCUS. ASCUS is based on cytological changes that direct to squamous cells of low-grade aquamous lesions (L-SIL), but their quality and quantity are insufficient to be classified as L-SIL category Material and methods: During one year period (2008. to 2009.) 13666 PAP smears were analysed in Clinic of gynecology and obstetrics in Belgrade, using conventional Papaniclolau method. Cervical biopsies with endocervical curettage were performed in cases when ASCUS was repeated more than two times or was connected with atypical colposcopic findings. Biopted material was fixed in 4% buffered formalin, classicly processed, cut in leyers of 5 microneand coloured with HE method Results: From total number of 13666 analiesed PAP smears 440 were interpreted as ASCUS (3.22%) with the following patohystological results: 24 (22.2%) H-SIL, 19 (17.59%) L-SIL, 34 (31.48%) squamous metaplasia, 13 (12.03%) hyperkeratosis and parakeratosis, 10 (9.27%) endocervical polyps and 8 (7.41%) cervical inflammation. Conclusion: There are more frequent results of H-SIL (22.22%) after ASCUS findings. It can be explained with doing immediate biopsy in all the cases when atypical colposcopic findings are connected with ASCUS Key words: Atipical cells, PAP test, ASCUS 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 631 POSTER PRESENTATIONS 2/poster prezentacije 2 P38 Morfološki sistem bodovanja u preoperativnoj diferencijaciji neoplazmi jajnika kod devojčica i adolescentkinja Slaviša Đuričić, Zoran B. Stanković, Dragana Stanković, Đorde Savić, Katarina Sedlecki – Akugin Institut za zdravstvenu zaštitu majke i deteta Srbije “Dr Vukan Čupić”, Beograd, Srbija Cilj: Procena kliničkog znacaja Uelandovog morfološkog sistema bodovanja u predvidanju osnovnih patohistoloških kategorija neoplazmi jajnika kod devojčica i adolescentkinja. Uvod: Korisnost Uelandovog morfološkog sistema bodovanja u predvidanju patohistoloških kategorija neoplazmi jajnika kod dece do danas je nedovoljno ispitana. Materijal i metode: Statističkim metodama analize procenjivana je povezanost Uelandovog indeksa i histoloških kategorija neoplazmi jajnika kod devojčica i adolescentkinja operisanih u periodu od januar 1997.-decembar 2006. godine. Evaluiran je i prediktivni značaj serumskih tumorskih markera. Rezultati: Kod 153 devojčice (uzrasta 6 meseci-19 godina) odstranjeno je 160 tumora jajnika, uključujući 7 bilateralnih. Od toga su 43 devojčice bile u premenarhalnom dobu. Prema patohistološ kom nalazu kod 62 devojčice radilo se o neneoplastičnim lezijama, kod 68 tumori su bili benigni, kod 30 maligni ili granične malignosti. Stadijumi maligne bolesti bili su: I stadijum 18, II 5, III 7 devojčica. Senzitivnost, pozitivna prediktivna vrednost i tačnost Uelandovog indeksa za benigne tumore (skor <7), iznosila je: 0,94, 0,84 i 0,93. Povišen nivo tumorskih markera nađen je kod 64 pacijentkinje, uključujuci 43 sa benignim tumorima. Kod 120 pacijetkinja sačuvan je deo neoštećenog tkiva jajnika. U odnosu na sistem bodovanja tkivo jajnika je sačuvano kod 103 od 109 jajnika sa Uelandovim skorom =6 i kod 17 jajnika sa skorom =7. Zaključak: Morfološki sistem bodovanja na osnovu ultrazvučne analize preporučen od Uelanda veoma je pouzdan metod za preoperativno razlikovanje benignih od malignih tumora jajnika devojčica i adeolescentkinja. Tumorski serumski markeri mogu biti korisni u preoperativnoj proceni tumorskog uvećanja jajnika. Ključne reči: jajnik, neoplazma, devojčice, adolescencija, sistem bodovanja 632 Morphological scoring system in preoperative differentiation of pediatric and adolescence ovarian tumors Slavisa Djuricic, Zoran B. Stankovic, Dragana Stankovic, Djordje Savic, Katarina Sedlecky - Akugin Mother and Child Health Institute of Serbia, Belgrade, Serbia Aim: To asses the clinical relevance of Uelands morphological scoring system in prediction of histopathological categories of ovarian neoplasms in childhood and adolescence. Introduction: The utility of morphological scoring system in differentiation of main histopathological categories of ovarian neoplasms in childhood is unquestioned by now. Material and methods: Morphological assessment using Uelands index were performed for all patients with histopathological confirmation of ovarian tumor, with evaluation of tumor markers, from January 1997.-Decembar 2006. Results: 153 girls (age range 6 months-19 years) were surgically treated for 160 ovarian tumors, including 7 bilateral. 43/153 girls were in premenarchal period. Histopathology report described 62 non-neoplasms, and 68 benign and 30 malignant neoplasms. Stages of malignant disease were distributed as follows: stage I, 18, stage II, 5, stage III, 7. Sensitivity, positive predictive value and accuracy by Ueland s index for benign tumors (score <7) was: 0.94, 0.84, 0.93, respectively. Elevated levels of tumor markers were observed in 64 patients, including 43 with benign tumors. In 120 girls some part of ovarian tissue were preserved during surgery. Depending on the Ueland s scor we preserved 103/109 ovaries with score =6 and 17/44 ovaries with score =7. Conclusion: Ultrasonographic assessment using morphological analysis and scoring system recommended by Ueland is a very useful procedure for differentiating benign from malignant ovarian tumors in children and adolescents. Tumor markers and endocrinological investigation may also be useful for preoperative evaluation. Key words: ovary, neoplasm, girls, adolescence, scoring system 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 2/poster prezentacije 2 P39 Tumori germinativnih ćelija jajnika u dečjem uzrastu: 35 - godišnji period Ovarian germ cell tumors of childhood: 35 years period Milena Đukić, Gordana Samardžija, Slaviša Đuričić Milena Djukic, Gordana Samardzija, Slavisa Đuricic Cilj: Utvrditi epidemiološke i patohistološke karakteristike TGC jajnika u dečijem uzrastu. Uvod: Tumori germinativnih ćelija jajnika (TGC) su retki u ranom dečijem uzrastu, ce ce se pojavljuju u pubertetskom periodu, to se objašnjava hormonalnim fiziološkim promenama. TGC čine više od 50% svih tumora jajnika i oko 25% svih teratoma u dečijem uzrastu, većinom su benigni teratomi sa dobrom prognozom. Najčešći maligni tumori u jajniku su disgerminom i tumor žumančane kese. Materijal i metode: Retrospektivna analiza epidemioloških i patohistoloških karakteristika TGC jajnika u dečjem uzrastu (0-18 godina) operisanih u Institutu za zdravstvenu za Institutu majke i deteta Srbije u 35.godišnjem periodu (1976-2011). Rezultati: U analizu je uključeno 88 bolesnika sa TGC jajnika. Uzrast pri operaciji bolesnika za benigne teratome je između 32-216 meseci, srednja vrednost (SD) 146,6±50,1, za nezrele teratome između 59-210, SD 149,2±50, a za maligne tumore između 12-216, SD 137,2ą50,4. U svim uzrasnim grupama dominiraju benigni teratomi 55 (62,5%), sa vrhom učestalosti oko puberteta, zatim slede maligni TGC 23 (26,1%) i nezreli teratomi 10 (11,4%), koji su cešći u prepubertalnom periodu. Najčešć maligni TGC su maligni mešoviti tumori 8 (34,8%) i disgerminomi 7 (30,4%). Zaključak: TGC jajnika u ranom dečijem uzrastu su retki, najviše je benignih teratoma, dok su ređi maligni tumori, sa predominacijom mešovitih malignih tumora i disgerminoma. Najređi su nezreli teratomi. Ključne reči: jajnik, tumori germinativnih celija, dečiji uzrast, epidemiologija, histopatologija Aim: The aim of the study was to determine the epidemiologic and histopathologic characteristics of ovarian GCT in childhood. Introduction: Ovarian germ cell tumors (GCT) are rare in early childhood. They appear more often in puberty, which is explained by physiological hormonal changes. GCT represent more than 50% of all ovarian tumors and approximately 25% of all teratomas in childhood, most of which are benign teratomas with a good prognosis. Most common malignant ovarian tumors are dysgerminoma and yolk sac tumor. Material and methods: Retrospective analysis of epidemiologic and histopathologic features of ovarian GCT in children (0-18 years) operated at the Mother and Child Health Care Institute of Serbia in the period of 35 years (1976-2011). Results: The analysis included 88 patients with ovarian GCT. Age at surgery for benign teratoma is 32-216 months, mean age (SD) 146.6 ą 50.1, for immature teratoma 59-210 months, SD 149.2 ą 50.1, for malignant tumors 12-216 months, SD 137.2 ą 50.4. In all age groups, benign teratomas dominate 55 (62.5%), with peak incidence around puberty, followed by malignant GCT 23 (26.1%) and immature teratomas 10 (11.4%), which are more common in prepuberty period. The most common malignant GCT are malignant mixed 8 (34.8%) and dysgerminoma 7 (30.4%). Conclusion: Ovarian GTC in early childhood are rare, being mostly benign teratomas. Malignant tumors are rare, with a predominance of mixed tumors and dysgerminoma. The rarest are immature teratomas. Key words: ovary, germ cell tumors, childhood, epidemiology, histopathology 1Institut za zdravstvenu za titu majke i deteta Srbije “Dr Vukan Čupić”, Beograd, Srbija Mother and Child Health Care Institute of Serbia “dr Vukan Cupic”, Belgrade, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 633 POSTER PRESENTATIONS 2/poster prezentacije 2 P40 Uzroci spontanog pobačaja na nivou placente i pupčane vrpce Causes of miscarriage at the level of placenta and umbilical cord Jovanka Trifunović, Petar Noack, Kristina Noack, Hristina Djordjević, Momčilo Ristanovic, Mladjen Veselinovic, Nebojsa Jovčić Jovanka Trifunovic, Petar Noack, Kristina Noack, Hristina Djordjevic, Momcilo Ristanovic, Mladjen Veselinovic, Nebojsa Jovcic Cilj : Ispitati učestalost patoloških promena na posteljici i pupčanoj vrpci koji su najčešci uzrok spontanog pobačaja. Uvod: Spontani pobačaj je neželjeni prekid trudnoće, pre nego što je plod sposoban za vanmaterični život. Uzroci mogu biti od strane majke ili na nivou fetusa. Promene placente mogu biti uzrokovane poremećajem cirkulacije majke, poremećajima fetalnog krvotoka i ostalim nevaskularnim lezijama. Najčešće malformacije pupčanika su vezane za dužinu. Materijal i metode: Naše ispitivanje predstavlja retrospektivnu studiju kojom su obuhvaćena oba pola fetusa u drugom i trećem trimestru trudnoće.Uzorak obuhvata 60 protokola koji su uzeti sa Instituta za Patologiju. Rezultati: Zastupljenost polova bila je 33 muških fetusa (55%) i 27 ženskih fetusa (45%). Razlika u starosti među polovima nije statistički značajna p=0.222 (p>0.05). Promene na placeti su bile prisutne u 52 slučaja a na pupčaniku u 8 slučajeva. Poremecaji placente kao uzrok spontanog pobačaja su češći jer se mogu javiti u drugom i trećem trimestru, dok su pobačaji uzrokovani patološkim promenama pupčanika uglavnom ograničeni na zadnji trimestar. Najčešće promene na placenti su bile: infekcije, infarkt placente, abrupcija placente, placenta previa. Od malformacija pupčanika najviše prisutni su čvorovi koji nastaju usled dugačkog pupčanika. Zaključak: U ovom ispitivanju smo pokazali da poremećaji na nivou placente u odnosu na poremećaje na pupčaniku češće dovode do pobačaja. Najčešći uzrok spontanog pobačaja na nivou placente je infekcija. Ključne reči: placenta,pupčanik,spontani pobačaj,učestalost Aim: Examine the frequency of pathological changes in the placenta and umbilical cord which are the most common cause of spontaneous abortion. Introduction: Spontaneous abortion is the unwanted termination of pregnancy, before the fetus is capable for extrauterine life. Spontaneous abortion may be caused by mother or fetus. Changes of placenta may be caused by the mother’s circulatory disorder, fetal circulatory disorders or other non-vascular malformations. The most frequent umbilical cord malformacion are connected to length. Material and methods: Our examination is retrospective study and it s covering both sexes of fetus in the second and third trimester of pregnancy . Sample includes 60 protocols. Results: Gender was 33 male fetuses (55%) and 27 female fetuses (45%). The difference in age between the sexes was not statistically significant p = 0,222 (p> 0.05). Changes in placenta were present in 52 cases and the umbilical cord in 8 cases. Disorders of the placenta as a cause of spontaneous miscarriages are more common because it may occur in the second and third trimester, while the pathological changes of umbilical cord usually are limited to the last trimester The most common changes on placentawere: infection, infarction of the placenta, abruption of placenta, placenta previa .The most common malformations of umbilical cord are nodes that result from long umbilical cord. Conclusion: In this study we showed that disturbances at the level of the placenta in relation to the umbilical cord disorders often leads to abortion. The most common cause of spontaneous abortions at the level of the placenta is an infection. Key words: placenta, umbilical cord, spontaneous abortion, fequency Vojnomedicinska Akademija, Beograd, Srbija 634 Medical Military Academy, Belgrade, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 2/poster prezentacije 2 PATOLOGIJA DOJKE BREAST PATHOLOGY P42 Primarni sitnoćelijski karcinom dojke: dijagnostička dilema Primary small cell breast carcinoma : diagnostic dilemma Nenad Rsovac, Snežana Vatričević, Zoran Babić Nenad Rsovac, Snzana Vatricevic, Zoran Babic Cilj: Predstavljanje našeg iskustva u dijagnostici primarninog sitnoćelijskog karcinomom dojke Uvod: Primarni sitnoćelijski karcinom dojke je redak tumor. Prikazujemo primarni tumor leve dojke kod žene stare 45 godina, klinički prezentovan kao palpabilna, brzo rastuća tumorska masa, sa pozitivnim limfnim čvorovima u aksili. Radiografski i ultrazvučno nisu viđeni primarni ili sekundarni tumori izvan dojke. Materijal i metode: HE, CD117,EMA, CD99, S-100, NSE, vimentin, HMB45, CD34, CD68, hromogranin A, LCA, CK, sinaptofizin Rezultati: Na H&E preparatima viđen je maligni epitelni tumor koji pokazuje visoku atipiju malih do srednjih ćelija, sa slabo granuliranom ili agranuliranom citoplazmom i brojnim mitozama, koji raste u formi gnezda, međusobno razdvojenih tankim vezivnim septama, a mestimično rasporedjenih u vidu traka, sa poljima opsežne nekroze i invazijom limfatika. Imunohistohemijski utvrđena je pozitivnost na CD117, EMA, CD99, kao i delimična pozitivnost na NSE. Negativni su bili vimentin, HMB45, CD34, CD68, hromogranin A, LCA, hormondki receptori ER i PR kao i HER2. CK i sinaptofizin su pokazali pozitivnost u različitim delovima tumora. Zaključak: Diferencijalno dijagnostički smo razmatrali invazivni lobularni karcinom dojke sa delovima koji pokazuju neuroendokrinu diferencijaciju, primarni sitnoćelijski neuroendokrini karcinom dojke ili mešoviti tumor (isprepletanost sitnoćelijske komponente sa lobularnim karcinomom), to se prema podacima iz literature sreće u oko 40% slučajeva. Ključne reči: sitnoćelijski karcinom dojke Aim: Presentation of our experience in diagnostics of primary small call breast carcinoma Introduction: Primary small cell breast carcinoma is a rare tumor. We present primary left breast tumor in 45 old woman clinically presented as rapidly growing tumor mass with positive axillary lymph nodes. Radiographic and ultrasound findings were negative for other primary or secondary tumor. Matherial and methods: HE, CD117,EMA, CD99, S-100, NSE, vimentin, HMB45, CD34, CD68, chromogranin A, LCA, CK, sinaptophyisin Results: HE revealed malignant epithelial tumor with high atypia of small to medium cells, with poorly granulated or agranulated cytoslasm and numerous mitotic figures, which grows in form of nests separated with thin fibrous septa, partly arranged in bundles with huge necrotic zones and lymphatic invasion. Immunohistochemicaly we found positivity for CD117, EMA, CD99, S-100 and partly NSE. Negativity was recorded for vimentin, HMB45, CD34, Cd68, chromogranih A, LCA, hormon receptors ER i Pr as well as HER2. CK and sinaptophysin was positive in different areas of tumor. Conclusion: In differential diagnostics we discussed invasive lobular breast carcinoma with neuroendocrine differentiation, neuroendocrine breast tumor or mixed tumor ( small cell carcinoma intermingled with lobular carcinoma) which are reported in about 40% of cases. Key words: small cell, breast carcinoma. Služba patologije, Zdravstveni centar “Studenica”, Kraljevo, Srbija Department of Pathology, Health Center “Studenica”, Kraljevo, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 635 POSTER PRESENTATIONS 2/poster prezentacije 2 P 43 Bilateralni karcinom dojke –prikaz slučaja Boris Dobrojević1, Gordana Mitrović2, Milica Mijović1 1Opšta bolnica, Brčko Distrikt, BiH za patologiju, Medicinski fakultet Priština-Kosovska Mitrovica, Srbija 2Institut Cilj: Prikazujemo slučaj BKD kod 86-o godišnje žene. Uvod: Bilateralni karcinom dojke (BKD) se retko javlja. Njegova incidenca je od 0,3 12%. Etiologija BKD je nepoznata, ali većina dokaza upućuje da su to nezavisni tumori, a ne metastaza primarnog karcinoma. Postoji velika neusaglašenost u vezi definicije kad je drugi tumor sinhroni, a kad metahroni. Materijal i metode: Pacijentkinja je primljena u bolnicu u januaru 2012. godine. Fizikalnim pregledom nađeni su egzulcerisani tumori na obe dojke, kao i uvećani limfni nodusi u levoj pazušnoj jami. Pacijentkinja u anamnezi navodi da je napipala tumor na levoj dojci pre 13 godina, a na desnoj pre 7 do 8 godina. Odlukom onkološkog konzilijuma urađena je obostrana simplex mastektomija sa uklanjanjem limfnih nodusa iz leve aksile. Rezultati: Makroskopski, tumor iz leve dojke egzulcerisan, loptasto nodularan, veličine 75x60x55mm, beličaste boje, tvrd. Patohistološki postavljena je dijagnoza invazivnog duktusnog karcinoma, gradusa II sa invazivnom mikropapilarnom komponentom i DCIS visokog gradusa. U svih šest limfnih nodusa nađene su metastaze i to mikropapilarne komponente. Tumor iz desne dojke egzulcerisan, loptasto nodularan, veličine 60x55x50mm, sivobeličaste boje, meki. Patohistološki postavljena je dijagnoza invazivnog mucinoznog karcinoma (tip A). Imunohistohemijski oba tumora su pozitivni za estrogene i progesteronske receptore, a HER2/neu negativni. O daljem tretmanu čeka se odluka onkolo kog konzilijuma. Zaključak: Iako je BKD redak, uvek treba misliti na mogućnost nastanka karcinoma u kontralateralnoj dojci. Ključne reči: bilateralni karcinom, dojka 636 Billateral brest cancer – case report Boris Dobrojevic1, Milica Mijovic2, Gordana Mitrovic1 1General Hospital, Brcko District, BiH of pathology, Medical faculty Priština-Kosovska Mitrovica, Serbia 2Institut Aim: We report a case of BBC in 86-year-old woman. Introduction: Bilateral breast cancer (BBC) is rare. Its incidence is from 0.3 to 12%. BBC etiology is unknown, but most evidence suggests that these are independent tumors, rather than metastasis of primary tumor. There is a large discrepancy regarding the definition of synchronous or metachronous appearing of second tumor. Matherial and methods: The patient was admitted to the hospital in January 2012. By physical examination, egzulcerans tumors in both breasts and enlarged left armpits lymph nodes were found. The patient was given information about existence of tumor in left breast during last 13 years and in right breast during last 7-8 years. By the decision of oncology council a bilateral simple mastectomy with removal of left armpits lymph nodes was made. Results: Grossly, the tumor from left breast was egzulcerans, globular nodular, size 75x60x55mm, whitish, solid. Histopathological diagnosis was invasive ductal carcinoma, grade II with invasive micropapillary component and high grade DCIS. Metastases, dominantly of micropapillary component, were found in all six axillary lymph nodes. Tumor from right breast was egzulcerans, globular nodular, size 60x55x50mm, greyishwhite color, softer. Histopathological diagnosis was invasive mucinous cancer (type A). Immunohistochemically both tumors were positive for estrogen and progesterone receptors and HER2/neu negative. Further treatment depends on the decisions of the council of oncology. Conclusion: Although BBC is a rare, we should always think of the possibility of cancer in the contralateral breast. Key words: Key words: bilateral carcinoma, breast 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 2/poster prezentacije 2 INFEKTIVNA PATOLOGIJA P44 Imunohistohemijska analiza aktiviranih stelatnih ćelija u akutnom oštećenju jetre INFECTIOUS DISEEASES PATHOLOGY Immunohistochemical analysis of activated stellate cells in acutely damaged liver Nada Tomanović1, Dragan Delić2, Dimitrije Brašanac1, Ivan Boričić1 Nada Tomanovic1, Dragan Delic2, Dimitrije Brasanac1, Ivan Boricic1 2Institut 2Institute 1Institut za patologiju, Medicinski fakultet, Beograd za infektivne i tropske bolesti, Medicinski fakultet, 1Institute of Pathology, Medical School, Belgrade of Infectious and Tropical Diseases, Medical School, Beograd Belgrade Cilj: U ovoj studiji ispitano je prisustvo aktiviranih stelatnih ćelija u akutnom oštećenju tkiva jetre. Uvod: Nakon oštećenja tkiva jetre, stelatne ćelije se aktiviraju i transdiferenciraju u miofibroblastni fenotip imunoreaktivan na α-glatkomišićni aktin. Materijali i metode: Retrospektivna studija obuhvatila je uzorke tkiva jetre 9 ispitanika (6 ženskog pola, prosečne starosti 43 godine i 3 muškog pola, prosečne starosti 38 godina) koji su umrli pod kliničkom dijagnozom fulminantnog hepatitisa na Institutu za infektivne i tropske bolesti (Medicinski fakultet Univerziteta u Beogradu) tokom perioda od 6 godina. Uzorci tkiva dobijeni su perkutanom iglenom (18-G) nekropsijom i obrađeni na Institutu za patologiju (Medicinski fakultet Univerziteta u Beogradu). Nakon standardnog hematoksilin-eozin bojenja, uzorci su bojeni imunohistohemijski na α-glatkomisicni aktin (DAKO, Carpenteria, Ca, USA, razblaženje 1:100). Prisustvo aktiviranih stelatnih ćelija pozitivnih na α-glatkomišićni aktin evaluirano je u portalnim prostorima i u zonama nekroze. U svrhu kontrole koristili smo 10 uzoraka tkiva jetre u kojima nije bilo patoloških promena. Rezultati: U kontrolnoj grupi, retke, pojedinačne aktivirane stelatne ćelije bile su prisutne u portalnim prostorima 7 uzoraka. U grupi ispitanika sa fulminantnim hepatitisom, kod 8 uzoraka je nađena panacinusna nekroza, a u jednom uzorku spojne nekroze hepatocita. U svim uzorcima uočen je povećan broj aktiviranih stelatnih celija u portalnim prostorima ali u zonama nekroze. Zaključak: U akutnom oštećenju jetre prisutan je povećan broj aktiviranih stelatnih ćelija u portalnim prostorima i u zonama nekroze, to se može objasniti ulogom ovih ćelija u remodeliranju ekstracelularnog matriksa i reparaciji. Ključne reči: stelatne celije, akutno oštećenje Aim: We investigated presence of activated stellate cells in acutely damaged liver tissue. Introduction: Upon liver injury, stellate cells become activated and transdifferentiate into extracellular-matrix producing myofibroblasts immunoreactive for a-smooth muscle actin. Material and methods: Liver tissue samples from 9 patients (6 female, mean age 43 years and 3 male, mean age 38 years) that died under clinical diagnosis of fulminant hepatitis at Institute for infectious and tropical diseases during a 6 year period were included in this retrospective study. Samples were obtained post mortem, percutaneously, using 18-G needle and evaluated at Institute of pathology (Medical Faculty, Belgrade University). After standard hematoxillin-eosin staining, samples were stained immunohistochemically for a-smooth muscle actin (DAKO, Carpenteria, Ca, USA, dilution 1: 100). Presence of activated stellate cells was evaluated in portal spaces and in areas of necrosis. As a control group we used 10 samples of liver tissue that showed no evidence of histopathological changes. Results: In control group, rare individual activated stellate cells were present in portal spaces of 7 samples. In fulminant hepatitis group, in 8 samples there was panacinar necrosis and in one sample there was bridging hepatocyte necrosis. In all examined samples there was an increased number of activated stellate cells in portal spaces as well as in areas of necrosis. Conclusion: In acutely damaged liver, increased number of activated stellate cells is present in portal spaces as well as in areas of necrosis Key words: stellate cells, acute damage, liver 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 637 POSTER PRESENTATIONS 2/poster prezentacije 2 P45 Retke parazitoze u biopsijskom i autopsijskom materijalu: Ehinokokoza, dirofilarijaza, enterobijaza Ljiljana Đurdev1, Dušan Lalošević2 1 2 Opšta bolnica ‘’Dr Djordje Joanovic’’ Zrenjanin, Srbija Pasterov zavod, Novi Sad, Srbija Cilj: Opis morfologije retkih parazitoza ili retke lokalizacije parazita. Uvod: Multipna ehinokokoza kao uzrok epilepsije, akutnog infarkta miokarda, te smrtnog ishoda otkrivena tek na obdukciji, dirofilarijaza, novoregistrovana zoonoza kod nas i enterobijaza u adenomu debelog creva, predstavljaju jako redak nalaz te je potrebno poznavanje morfologije parazita. Za razliku od hidatidnog ehinokoka poznatog po velikim cistama, multilokularni ehinokok daje sitne grozdaste ciste koje rastu kao maligni tumor i čak metastaziraju. Materijal i metode: Materijal predstavlja obdukcioni nalaz kod slučaja ehinokokoze, kao i bioptički materijal kod dirofilarijaze i enterobijaze. Rezultati: Pacijent star 50 godina dobio je epileptični napad, akutni infarkt miokarda i pretkomornu fibrilaciju, te je sutradan preminuo. Na obdukciji u predelu hilusa desnog pluća nalazi se više cisticnih formacija do najvećeg precnika 7 cm, koje su delom kalcifikovane, delom ispunjene kasastim sadržajem, sa beličastom kapsulom, naležu na perikard, vršeći spoljnu kompresiju srca. U desnoj hemisferi velikog mozga nalazi se cista od 5 cm, a na slezini od 4 cm. U drugom slučaju sa potkolenice u biopsji potkožnog čvora su nađeni preseci parazita ženke Dirofilaria spp. U adenomu debelog creva dvogodišnjeg deteta nađen je Enterobius vermicularis, to predstavlja anegdotsku lokalizaciju. Zaključak: Nas slučaj je dijagnostikovan kao hidatidni ehinokok nalazom kukica parazita u cistama, jer multilokularni kod čoveka ne razvija protoskolekse, pa prema tome ni kukice. Dirofilarija se identifikuje po karakteristicno naboranoj kutikuli kao i rasporedu unutra njih organa, kao i enterobijaza. Morfološka identifikacija parazita u bioptičkom i autopsijskom materijalu predstavlja i dalje standard u dijagnostici. Ključne reči: patologija, ehinokokoza, dirofilarijaza, enterobijaza 638 Rare parasitic infections in biopsies and autopsies: Echinococcosis, dirofilariosis, enterobiasis Ljiljana Djurdev1, Dusan Lalosevic2 1General 2Pasteur Hospital Dr Đordje Joannovic Zrenjanin Institute of Novi Sad, Serbia Aim: To describe morphology of rare parasitic infections and their unusual localizations. Introduction: Multiple echinococcosis as cause of epilepsy, acute myocarial infarction and letaly outcome diagnosed by autopsy, dirofilariasis, newly registered zoonosis in Serbia, and enterobiasis in colon adenoma, are very rare finding that required knowledge of their morphology. Unlike hydatide Echinococcus, multilocular gives a small cluster of cysts that grow like a malignant tumor, and even metastasize. Material and methods: Materials are autopsy case of echinococcosis, and biopsies from dirofilariasis and enterobiasis. Results: Case reports. - 50 years-old patient was given epileptic attack, myocardial infarction and atrial fibrillation, and died on the next day. At autopsy, the right lung hilus consist several cystic formations to the largest diameter of 7cm, which are partly calcified, partly filled mashy content, with white capsule. Cysts placed against the pericardium performing external compression of the heart. In the right hemisphere of the brain was a cyst of 5 cm and at the spleen of 4 cm. The second case had hypodermic node in the leg in biopsy identified as a female Dirofilaria spp. In adenoma of the colon of two years child was found Enterobius vermicularis, which is anecdotal localization. Conclusion: Our case was diagnosed as a hydatid Echinococcus by finding hooks in cysts, because multilocular in the man does not develop hooks. Dirofilaria is identified by its characteristic cuticular ridges and structure of internal organs, and enterobiasis too. Morphologically identification of parasites in the bioptic and autopsy material is still standard in the diagnosis. Key words: pathology, echinococcosis, dirofilariasis, enterobiasis. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 2/poster prezentacije 2 KARDIOVASKULARNA PATOLOGIJA CARDIOVASCULAR PATHOLOGY P46 Apoptoza u virusnom miokarditisu Apoptosis in viral myocarditis Sofija Glumac1, Radmila Janković1, Zorica Stojšić1, Milana Lazić1, Isidora Ranisavljević-Katuca2, Jelena Marković2, Jovan D. Vasiljević1 Sofija Glumac1, Radmila Jankovic1, Zorica Stojsic1, Milana Lazic1, Isidora Ranisavljevic-Katuca2, Jelena Markovic2, Jovan D. Vasiljevic1 2Klinicki 2Department Cilj: Cilj naučnog rada je da se odredi apoptotski indeks (AI) kod pacijenata obolelih od VMK i ispita odnos AI kod različitih morfoloških oblika VMK. Uvod: Virusni miokarditis (VMK) predstavlja zapaljensku infiltraciju srčanog mišića praćenu oštećenjem kardiomiocita neishemijske etiologije. Apoptoza je programirana smrt ćelije kojom se putem eliminacije neželjenih ćelijskih elemenata održava normalna gustina ćelijske populacije. Danas se smatra da apoptoza ima značajnu ulogu u patogenezi različitih kardiovaskularnih oboljenja, kao što su dilataciona kardiomiopatija, VMK, infarkt miokarda, srčana insuficijencija, itd. Materijal i metode: Ispitivanjem je obuhvaćeno 30 bolesnika oba pola sa kliničkom dijagnozom VMK, i to 13 žena (43.3%) i 17 muškaraca (56.7%). Za postavljanje patohistološke dijagnoze VMK korišćena je modifikacija Dallas-ovih kriterijuma, a za detekciju apoptoze TUNEL metoda. Rezultati: Prosečne vrednosti AI u grupi od 30 pacijenata sa MK bile su 4,23 I 12,16 (Med=0,005). U grupi ispitanika koji su imali fokalni VMK, AI je iznosio 10,99, to je bila i najveća izmerena vrednost. U grupi ispitanika sa healing VMK, AI je imao najnižu vrednost 1,55, a u grupi sa akutnim i borderline VMK, izmereni AI je iznosio 3,32, odnosno 3,40. Zaključak: Naše istraživanje je pokazalo da je najveća vrednost AI uočena kod fokalnog VMK, ali da nije bilo statistički značajne razlike izmedu njega i vrednosti AI kod ostalih stadijuma bolesti, kao ni kod međugrupnog poredenja vrednosti AI. Naši rezultati ukazuju na potrebu proširivanja istrazivanja na većem broju ispitanika, primenu dodatnih metoda, pored TUNEL metode, za detekciju apoptoze kod VMK. Ključne reči: apoptoza, virusni miokarditis, TUNEL metod Aim: The aim of this study was to determine apoptotic index (AI) in patients with (VMC) and to compare the rate of AI in groupes with different morphological stages of VMC. Introduction: Viral myocarditis (VMC) is an inflamatory disease of cardiac muscle that is associated with non-ischemic cardiomyocyte injury. Apoptosis is defined as programmed cell death which eliminates most of the damaged and non-functional cells, managing the normal density of cell population in human tissues. Apoptosis of cardiomyocytes has been reported to be involved in pathogenesis of cardiovascular diseases like infarction, VMC, heart failure. Material and methods: Research included 30 patients with clinical diagnosis VMC13 women (43.3%) and 17 men (56.7%). All 30 patients fullfilled modified histopathological Dallas criteria, while apoptosis was detected using TUNEL method in myocardial tissue sections Results: The group of 30 patients with VMC demonstrated an elevated rate of AI 4.23 12.16 (Med: 0,005). The group of patients diagnosed with focal VMC demonstrated the highest rate of AI (10.99). In patients diagnosed with healing VMC the rate of AI was 1.55. Increased rates of apoptosis were also find in borderline (3.40) and acute (3.32) VMC. Conclusion: Our research has indicated the highest rate of apoptosis in patients with focal VMC. The rate of AI in focal VMC was not significantly increased when compared to AI of other groups. The rate of AI was not significantly increased when we compared results amongst groups with different morphological stages of VMC. Key words: apoptosis, viral myocarditis, TUNEL method 1Institut za patologiju, Beograd, Srbija centar Srbije, Beograd 1Institute of Pathology, Medical School, Belgrade of Pathology, Clinical Centre of Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 639 POSTER PRESENTATIONS 2/poster prezentacije 2 P47 Značaj endomiokardne biopsije u hipertrofičnoj kardiomiopatiji The use of endomyocardial biopsy in hypertrophic cardiomyopathy Jelena Marković1, Ivana Ranisavljević-Katuca2, Sofija Glumac2, Radmila Janković2, Jovan D. Vasiljević2 Jelena Markovic1, Isidora Ranisavljevic-Katuca2, Sofija Glumac2, Radmila Jankovic2, Jovan D. Vasiljevic2 1Odeljenje torakopulmonalne patologije, Služba za patologiju, Klinički Centar Srbije, Beograd 2Institut za patologiju, Medicinski fakultet, Beograd Uvod: Endomiokardna biopsija (EMB) je metoda koja se koristi u dijagnostikovanju različitih tipova kardiomiopatija, u cilju praćenja odbacivanja transplantata, evaluaciji miokarditisa, srčane insuficinjencije nepoznatog uzroka, aritmija, toksičnosti lekova i srčanih neoplazmi. Cilj: ovog rada je da ukaže kako neinvazivne dijagnostičke metode nisu uvek dovoljne u diferencijalnoj dijagnozi hipertrofične opstruktivne kardiomiopatije i ostalih bolesti srca. Materijal i metode: Analizirali smo trideset jednu reprezentativnu endomiokardnu biopsiju sa uputnom dijagnozom hipertrofične opstruktivne kardiomiopatije (HOCM). Uzorci su obrađivani standardnom procedurom i bojeni hematoksilin-eozin i specijalnim bojenjem. Za procenu smo koristili pet parametara (neregularnost pružanja vlakana-dissaray i hipertrofija miokardnih vlakana, kratka vlakna, perinuklearni halo sa bizarnim jedrima i fibroza) koje smo bodovali od 0 do 3 i prikazali zbir histološkim HOCM indeksom (HHI), čija vrednost može biti u rasponu 0 do 15, sa pozitivnim nalazom preko 8. Rezultati: Histološki nalazi dobijeni endomiokardnom biopsijom trideset jednog pacijenta su pokazali da je kod 8 pacijenata (25,8%) potvrđena uputna dijagnoza HOCM, kod 6 pacijenata (19,35%) nije bila potvrđena dijagnoza HOCM, jer je HHI bio manji ili jednak 50%, amiloidoza je dokazana kod jednog pacijenta (3,22%), miokarditis kod 4 (12,9%), dilataciona kardiomiopatija kod 6 (19,35%) i nespecifične srčane promene kod 6 pacijenata (19,35%). Zaključak: Za definitivnu dijagnozu je potreban multidisciplinarni pristup, koji u određenim slučajevima zahteva primenu endomiokardne biopsije, jer je klinička dijagnoza bila potvrđena u manje od 50% slučajeva. Ključne reči: Hipertrofična kardiomiopatija, endomiokardna biopsija, patohistologija 640 1Department 2Institute of Pathology, Clinical Centre of Serbia, Belgrade of Pathology, Medical School, Belgrade Aim: Endomyocardial biopsy (EMB) may be used to diagnose different types of cardiomyopathies, to monitor transplant rejection, evaluation of myocarditis, heart failure of unknown origin, arrhythmias, drug toxicity and heart neoplasmas. The aim of this presentation is to show how noninvasive cardiac investigations are sometimes not sufficiently conclusive for distinguishing between hypertrophic obstructive cardiomyopathy (HOCM) and the other cardiac diseases. Material and methods: Thirty-one representative left ventricle biopsies were obtained following suspected HOCM. Samples underwent routine standard and special staining procedures. Five histologic parameters were used for assesment (disarray and hypertrophy of myofibers, myocardial short runs, perinuclear halo and bizarre nuclei and fibrosis) and graded from 0 to 3, presenting in summary Histological HOCM Index (HHI- ranging from 0-15) with positive findings over 8. Results: The histological findings in EMB of thirty-one patients, in 8 patients (25,8%) HOCM diagnosis was confirmed, in 6 (19,35%) HHI was under or equal to 50% of HOCM index (8) so HOCM diagnosis couldn’t be confirmed, amyloidosis in 1 (3,22%), myocarditis in 4 (12,9%), dilatative CMP in 6 (19,35%) and unspecific cardiac changes in 6 patients (19,35%). Conclusion: A definitive diagnosis could be obtained by means of a multidisciplinary approach including EMB findings, because in less than 50% of patients the clinical diagnosis was confirmed. Key Words: Hypertrophic cardiomypathy, endomyocardial biopsy, pathohistology 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 2/poster prezentacije 2 P48 Ruptura papilarmog mišica usled infarkta miokarda Papillary Muscle Rupture in Myocardial Infarction Ranisavljević-Katuca Isidora1, Jelena Marković2, Sofija Glumac1, Radmila Janković1, Jovan D. Vasiljević1 Ranisavljevic-Katuca Isidora1, Jelena Markovic2, Sofija Glumac1, Radmila Jankovic1, Jovan D. Vasiljevic1 1Institut za patologiju, Medicinski fakultet Univerzitet u Beogradu 2Klinicki centar Srbije, Beograd Cilj: Cilj rada je utvrditi da li okluzija dominantne koronarne arterije, anatomske varijacije i postojanje anomalija PM mogu da favorizuju nastanak postinfarktne rupture PM. Uvod: Ruptura papilarnog mišića (PM) je retka, ali često fatalna mehanička komplikacija akutnog infarkta miokarda(AIM). Ruptura na zadnjem PM je više uobičajena (u oko 75% slučajeva) nego na prednjem PM, jer se zadnji PM ishranjuje preko zadnje descedentne grane dominantne desne koronarne arterije i u vezi je sa infarktom zadnjeg zida leve komore. Prednji PM se ishranjuje preko dijagonalnih grana prednje descedentne grane leve koronarne arterije i preko marginalnih grana leve cirkumflekse arterije. Nekoliko faktora igra važnu ulogu u razvoju rupture PM: okluzija dominantne koronarne arterije, anatomske varijacije i postojanje anomalija PM. Materijal i metode: Kao material za istrazivanje koristili smo nalaze 12 obdukovanih slučajeva postinfarktne rupture PM. Rezultati: U 11 slučajeva se radilo o akutnom infarktu miokarda zadnjeg zida leve komore, a u jednom slučaju o AIM prednjeg zida leve komore. 10 pacijenata je imalo rupturu zadnjeg PM, a 2 pacijenta rupturu prednjeg PM. Kod 8 pacijenata postojale su anatomske varijacije PM. U 8 slučajeva desna koronarna arterija je bila dominanatna, a u ostala 4 slučaja se radilo o balansiranoj cirkulaciji. Zaključak: Morfološke varijante papilarnih mišića(postojanje više odvojenih mišićnih masa) su česte i mogu imati značajnu ulogu u nastanku rupture PM. Ključne reči: papilarni misic, ruptura, infarkt miokarda 1Institute of Pathology, Medical School Belgrade of Pathology, Clinical Centare of Serbia 2Department Aim: The aim of work is to determine whether occlusion of the dominant coronary artery, the anatomic variations and existence of anomalies of PMs may favor the development of postinfarction PMs rupture. Introduction: Rupture of a papillary muscle (PM) is rare but often fatal complication of acute myocardial infarction (AIM). The rupture of the posterior PM is more common than the anterior one, since the posterior PM has a single blood supply in cases of dominant right coronary artery and is associated with posterior wall infarctions.The anterior PM has dual blood supplies. Several factors play an important role in the development of PM rupture: occlusion of the dominant coronary artery, the anatomic variations and existence of anomalies of PMs. Material an methods: Twelve autopsy findings with the diagnosis of acute papillary muscle rupture in myocardial infarction were reviewed to determine pathologic features of this entity. Results: 10 patients had posterior PM rupture and 2 patients the anterior one. The site of AMI was posterior wall of the left ventricle in 11 patients and anterior in 1 out of 12. In 8 patients there was anatomic variations of PMs. In 8 cases the right coronary artery was dominant and in the remain 4 cases a balans circulation was present. Conclusion: Morphological variants of PMs are common and may play an important role in the PMs ruptures Key words: papillary muscle, rupture, myocardial 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 641 POSTER PRESENTATIONS 2/poster prezentacije 2 P49 Prekid aortonog luka –autopsijska studija 27 slučajeva Interupted aortic arch –an autopsy study of 27 cases Radmila Janković1, Sofija Glumac1, Branislav Lekić1, Jelena Marković2, Isidora Ranisavljević-Katuca1, Ivana Savić1, Jovan D. Vasiljević1 Radmila Janković1, Sofija Glumac1, Branislav Lekić1, Jelena Marković2, Isidora Ranisavljević-Katuca1, Ivana Savić1, Jovan D. Vasiljević1 Institut za patologiju, Medicinski fakultet, Univerzitet u Beogradu, Beograd, Srbija 2 Služba za patologiju, Klinički centar Srbije, Beograd, Srbija 1 Cilj: Cilj ovog istraživanja je da se utvrde učestalost i tip prekida aortnog luka (PAL), udružene anomalije i uzroci smrti u slučajevima sa PAL. Uvod: Prekid aortnog luka je realtivno retka urođena srčana anomalija. Od 3 tipa PAL (A, B i C) najčešći je tip B (oko 70%); tip C je najređi (oko 1%). Materijal i metode: Analizirani su autopsijski zapisnici slučajeva sa urođenom srčanom manom (USM) obdukovanih modifikovanom Rokitansky tehnikom na Institutu za patologiju u Beogradu. Analizirani period obuhvata 45 godina (1965 – 2010). Rezultati: USM je nađena u 4,9% autopsijskih slučajeva. PAL je dijafnostikovan u 27 (1,3) slučajeva USM. 8 slučajeva sa PAL je bilo tipa A, dok je tip B dijagnostikovan u 18 slučajeva. Tip C je nađen samo u jednom slučaju.U najvećem broju slučajeva su postojale udružene srčane anomalije (96%), često vise od jedne: ductus arteriosus persistens (96%), defect međukomornog septuma (78%), defect međupretkomornog septuma (30%), bikuspidna aortna valvula (11%), korigovana transpozicija velikih arterija (7%), atrioventrikularni septani defekt (4%). Udružene ekstrasrčane anomalije su bile: anomalije urinarnog (26%) i digestivnog trakta (11%), anomalna lobulacija pluća (18%), polisplenija (15%) i meningocela (4%). Kako ni jedan slučaj sa IAA nije prethodno operisan, uzroci smrti su bili povezani sa srčanom i kadrio-respiratornom insuficijensijom. Zaključak: Naši rezultati su slični rezultatima drugih autora. Pacijeti sa PAL imaju lošu prognozu ukoliko nisu operisani kako zbog prirode PAL, tako i zbog udruženosti sa drugim srčanim i ekstrasrčanim anomalijama. Ključne reči: urođena srčana mana, prekid aortnog luka, udružene anomalije, obdukcija 642 1 Institute of pathology, Faculty of medicine, University of Belgrade, Belgrade, Serbia 2 Department of pathology, Clinical center of Serbia, Belgrade, Serbia Aim: The aim of this study was to investigate frequency and types of interrupted aortic arch (IAA), associated anomalies and causes of death in cases with IAA. Introduction: Interruption of aortic arch is a relatively rare congenital heart anomaly. Out of 3 types of IAA (A, B and C), type is B is the most common (70%); type C is rare (1%). Material and methods: We analyzed autopsy records of cases with congenital heart diseases (CHD) at Institute of pathology in Belgrade. Autopies were performed during 45 years period (1965 – 2010). Modified Rokitansky technique was used for CHD. Results: CHD were found in 4,9% autopsied cases. IAA was present in 27 cases (1,3%) of CHD. We found 8 IAA type A and 18 IAA type B. Only one IAA case was type C. Associated cardiac anomalies were present in majority cases of IAA (96%), often more than one: patent ductus arteriosus (96%), ventricular septal defect (78%), atrial septal defect (30%), bicuspid aortic valve (11%), corrected transposition of great arteries (7%), atrio-ventricular septal defect (4%). Associated extracardiac anomalies were: anomalies of the urinary (26%) and digetive tract (11%), anomalous pulmonary lobulation (18%), polysplenia (15%) and meningocoele (4%). As none of autopsied IAA cases was operated, the causes of death were relates to heart failure or cardio-respiratory insufficiency. Conclusion: Our findings are consistent with majority of large series of IAA. Patients with IAA have poor prognosis without surgical procedure due nature of IAA, but also due associated cardiac and extracardiac anomaies. Key words: congenital heart disease, interrupted aortic arch, associated anomalies, autopsy 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 2/poster prezentacije 2 KOŠTANA I MEKOTKIVNA PATOLOGIJA P50 Parametri u diferencijalnoj dijagnozi bolesti sinovije - prikaz slučaja Jordan Radojičić, Petar Novak, Živojinović Dragan, Miloš Zaric, Jovanka Trifunović, Saša Ristic, Jelena Vrtikapa, Biserka Vukomanović-Đurdević Vojnomedicinska Akademija Beograd, Srbija Cilj: Prikazati bitnosti korelacije rezultata kliničkih pregleda i patohistološke analize. Uvod: Giht predstavlja metaboličko oboljenje. Taloženje kristala urata u tkivu produkuje specifične morfološke promene na zglobovima, bubrezima i drugim organima. Međutim, laboratorijske analize krvi jesu potreban, ali ne i dovoljan uslov za postavljanje dijagnoze bolesti. Materijali i metode: Pacijent u starosti od 52 godine javio se u Kliniku za ortopediju i traumatologiju VMA zbog otežanih pokreta kolena usled bolnog stanja. Rezultati: Na RTG snimcima uočene su subkortikalne ciste i erozije u predelu I metatarzalne kosti obostrano i stiloidnog nastavka ulne jednostrano. Ustanovljeno je povišenje vrednosti reumatoidnog faktora (38,8 IU/ml), anti CCP (30 IU/ml) i mokraćne kiseline (467 ľmol/l). Učinjena je sinoviektomija. Metodom polarizacije preparata tkiva nije utvrđeno prisustvo kristala urata. Standardnim HE bojenjem dokazano je postojanje sinovitisa sa fibrinskim naslagama na površini edematozne, hiperemične sinovije sa resičastim marginama, fokalno sa poljima fibrinoidne nekroze i palisadnim aranžmanom ćelija inflamacije sa predominacijom CD4 i CD8 limfocita uz fokalno prisustvo CD20 limfocita, te je upućivalo na postojanje reumatoidnog artritisa što je koreliralo sa nalazima ostalih kliničkih ispitivanja. Zaključak: Postavljanje dijagnoze bolesti sa morfološkim manifestacijama na sinoviji uključuje korelaciju kako kliničkih ispitivanja tako i detaljnu patohistološku analizu u donošenju definitivne dijagnoze. Ključne reči: giht, limfociti, erozija, kost BONE AND SOFT TISSUE PATHOLOGY Parameters in the differential diagnosis of diseases of the synovia case report Radojicic Jordan, Dragan Zivojinovic, Petar Novak, Milos Zaric, Jovanka Trifunovic, Saša Ristic, Jelena Vrtikapa, Biserka Vukomanovic-Đurdevic Military Medical Academy Belgrade, Serbia Aim: To show the importance of the correlation of results of clinical examinations and the pathohistological analysis. Introduction: Gout represents a metabolic disease. The deposition of uric crystals causes specific morphological alterations in the joints, kidneys and other organs. However, laboratory blood analyses are a necessary, yet not a sufficient condition for the correct diagnosis. Material and methods: A patient aged 52 years was submitted to the Clinic for orthopedics and traumatology of the Military medical academy because of limited mobility of the knee joint caused by pains. Results: The X-ray pictures showed subcortical cysts and erosions in the region of I metatarsal bone bilaterally and, unilaterally, of the styloid processus of the ulna. The levels of the reumatoid factor (38,8 IU/ml), anti-CCP (30 IU/ml) and uric acid (467 ľmol/l) were increased. A synievectomy was performed. Whereas the polarization method did not reveal uric crystals, the standard HE staining confirmed the existence of a synovitis with fibrinous deposits on the edematous, hyperemic synovia with cribriform margins, focal regions of fibrinoid necrosis and a palisad-like arrangement of inflammatory cells, predominantly of CD4 and CD8 lymphocites with a focal presence of CD20 lymphocites, which implied the existence of rheumatoid arthritis, which corresponded with the other clinical examinations. Conclusion: The procedure to a correct diagnosis of diseases with morphological manifestations in the synovia includes the correlation of clinical examinations and a detailed pathohistological analysis. Key words: gout, lymphocites, erosion, bone 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 643 POSTER PRESENTATIONS 2/poster prezentacije 2 P51 Intranodalni palisadirajući miofibroblastom - slučajan nalaz retkog tumora Intranodal palisaded myofibroblastoma as an incidental findings Jelena Urošević1, Anđela Tatarinov1, Dragoslav Nenezić2, Jelena Sopta1 Jelena Urosevic1, Andjela Tatarinov1, Dragoslav Nenezic2, Jelena Sopta1 Cilj: Tačna dijagnostika intranodalnog palisadirajućeg miofibroblastoma je važna iz terapijskih i prognostičkih razloga. Prikazujemo ovaj redak tumor koji je otkriven kao slučajan nalaz tokom operacije aorto-femoralnog by-pass-a. Uvod: Intranodalni palisadirajući miofibroblastom je redak benigni mezenhimalni tumor, koji gotovo uvek vodi poreklo iz ingvinalnih limfnih nodusa. Materijali i metode: Hirurški uklonjen bezbolni ingvinalni tumefakt kod žene stare 66 godina. Rezultati: Makroskopskim pregledom verifikovana je inkapsulirana čvrsta tumorska masa bez hemoragičnih i nekrotičnih područja. Mikroskopskom analizom reperzentatvnih uzoraka iz operativnog materijala utvrđeno je prisustvo fibroblastne proliferacije koja potpuno narušava gradju limfnog nodusa. Tumor čine vretenaste ćelije u palisadnom aranžmanu sa amiantoidnim vlaknima. Imunohistohemijski je dokazana pozitivnost na vimenntin, alfa-SMA I HHF 35, što ukazuje na miofibroblastnu prirodu tumorskih ćelija. Na osnovu morfoloških i imunohistohjemijskih karakteristika tumora postavljena je dijagnoza intranodalnog palisadirajućeg miofibroblastoma. Zaključak: Intranodalni mezenhimalni tumori su izuzetno retki. Diferencijalno dijgnostički intranodalni palisadirajući miofibroblastom treba razlikovati od Kapošijevog sarkoma, neurilemoma ili metastatskih sarkoma. Za razliku od malignih mezenhimalnih neoplazija,ovaj tumor se ponaša kao benigna promena i ne zahteva bilo kakvu dalju terapiju, osim totalne hirurške resekcije. Prikazani pacijent je 2 godine nakon radikalne hirurške ekscizije bez lokalnih znakova bolesti. Ključne reči: benigni tumor, limfni nodus Aim: Accurate diagnosis of intranodal palisaded myofibroblastoma is important for therapeutic and prognostic reasons. We report a case of this rare lesion that was discovered accidentally, as the inguinal tumor during operation of aorto-femoral by-pass. Introduction: Intranodal palisaded myofibroblastoma is a rare benign soft tissue tumor, almost always arising from inguinal lymph nodes. Material and methods: Surgicaly removed a painless inguinal mass in 66 years old female. Results: Macroscopic examination demonstrated incapsulated solid appearance without hemorrhagic and necrotic areas. Microscopic examination revealed intranodal spindle cell proliferation, with amianthoid fibers. It has a deeply eosinophilic core and lighter periphery. Nuclei of the spindle cells displayed a palisaded appearance. Immunohistochemistry was done and the myofibroblastic nature of the tumour cells was proved by the presence of vimentin, SMA and HHF-35. S-100 and CD 34 were negative. In light of these results, the case was diagnosed as “intranodal palisaded myofibroblastoma.” Conclusion: This entity is generally misdiagnosed as intranodal Kaposi’s sarcoma, schwannoma or metastatic lesions. In contrast to Kaposi’s sarcoma and metastatic lesions, it behaves in a benign fashion and does not need any further therapy except total surgical resection of the mass. Presented patient is 2 years after surgical therapy out of disease. Key words: benign tumor, lymph node 1Institut za patologiju, Medicinski fakultet, Univerzitet u Beogradu 2Institut za Kardiovaskularne Bolesti Dedinje, Beograd, Srbija 644 1Institute of Pathology, Faculty of Medicine, University of Belgrade 2Institute for Cardiovascular Diseases Dedinje 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 2/poster prezentacije 2 P52 Kavernozni angiomiom kosti - redak tumor vilice Cavernosus angiomyoma of bone - a rare jaw tumor Andjela Tatarinov1, Jelena Urošević1, Radojica Dražić2, Zvezdana Tepavčević3, Jelena Sopta1 Andjela Tatarinov1, Jelena Urosevic1, Radojica Drazic2, Zvezdana Tepavcevic3, Jelena Sopta1 Cilj: Predstavljanje slučaja retkog intraosealnog tumora koji je pimarno bio dijagnostikovan kao hemangiom kosti Uvod: Angiomiom je benigni mezenhimalni tumor mekog tkiva koji se sastoji od glatkih mišićnih fascikula i zadebljanih zidova krvnih sudova. Njegova intraosealna lokalizacija je izuzetno retka Materijal i metode: Osteolitična lezija u levoj mandibuli koja je dijagnostikovana digitalnim ortopanom kao incidentalni nalaz kod 61 godišnje pacijentkinje. Rezultati: Nakon kiretaže kosti biopsijski material je poslat na patohistološku analizu. Primarna dijagnoza je bila hemangiom kosti. Histološki, tumor se sastoji od dilatiranih krvnih sudova sa zadebljanjem mi šićnog sloja zida vaskulrnih prostora i fokusima zrelih masnih ćelija.Trihromnim bojenjem po Masonu se u zidovima krvnih sudova uočavaju zone mišićnog tkiva. Ćelije zida krvog suda su bile pozitivne na bojenje glatko mišicnim aktinom, dezminom, mišićno-spečifičnim aktinom i vimentinom. Oko tumora koštane gredice su atrofične, istanjene i redukovane. Navedene mikroskopske karakteristike ukazivale su na kavernozni tip angioleiomioma, mada je njegova intraosealna lokalizacija izuzetno retka. Zaključak: Prikazom ovog sličaja želeli smo da naglasimo da skoro svaki tumor mekih tkiva može biti prisutan i intraosealno,o čemu treba misliti pri dijagnostici Ključne reči: angiomiom, benigni, tumor, kost, vilica Aim: To present the case of a very rare intraosseal tumor that is primarily misdiagnosed as bone hemangioma Introduction: Angiomyoma is well known soft tissue benign mesenchymal tumor composed of smooth muscule fascicles and thick-walled vessels.Intraosseal localization of this tumor is very rare and unusual Material and methods: Osteolytic lesion in left mandibula diagnosed as an incidental finding in 61 years old female on digital ortopan Results: After bone curettage bioptical material was sand to pathological analysis. The first diagnosis was bone hemangioma. Histologicaly tumor was composed of dilated vascular channels with little muscular thickening of the walls and foci of mature fat cells. Masson s trichrom stain showed large zone of muscle tissue in the vascular wall. Walls cells were positive for smooth muscle actin, desmin, muscle specific actin and vimentin. Bone tissue was analyzed by tumor, only few bone trabeculs were presented into the biopsy. According to microscopic examination diagnosis were changed into cavernous type angioleiomyoma of bone. Conclusion: Showing this case we wanted to emphasize that almost all soft tissue tumors may be present in the bone Key words: angiomyoma, benign, tumor, bone 1Institut za patologiju, Medicinski fakultet, Univerzitet u Beogradu 2Klinika za oralnu hirurgiju, Stomatološki fakultet, Univerzitet u Beogradu 3Institut za patologiju,Stomatološki fakultet, Univerzitet u Beogradu Institute of Pathology, Faculty of Medicine, University of Belgrade,Belgrade 2Clinic for Oral Surgery,Faculty of Dentistry, University of Belgrade,Belgrade 3Institute of Pathology,Faculty of Dentistry, University of Belgrade, Belgrade 1 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 645 POSTER PRESENTATIONS 2/poster prezentacije 2 NEUROPATOLOGIJA P53 Kraniofaringiomi: 10 godina iskustva Emilija Manojlović – Gačić1, Milica Skender – Gazibara1, Tatjana Gazibara2, Savo Raičević3, Danica Grujičić3 1Institut za patologiju, Medicinski fakultet, Univerzitet u Begradu, Beograd, Srbija 2Institut za Epidemiologiju, Medicinski fakultet, Univerzitet u Begradu, Beograd, Srbija, 3Klinika za neurohirurgiju, Klinicki centar Srbije, Beograd, Srbija Cilj: Analiza histopatoloških tipova kraniofaringioma, njihove distribucije po uzrastu i polu, intrakranijalnoj lokalizaciji i recidivima u desetogodišnjem periodu. Uvod: Kraniofaringiomi su benigni, sporo rastući, lokalno invazivni intrakranijalni tumori porekla epitela Ratkeovog špaga. Pored dva osnovna tipa (adamantinomatozni i papilarni) opisane su i mešovite forme. Prognostički značaj različitih tipova kraniofaringioma je kontroverzan. Materijal i metode: U bazi podataka neurohirurških biopsija u periodu od 2002-2011 registrovano je 89 novootkrivenih slučajeva kraniofaringioma. Tumorsko tkivo je obradeno rutinskim hematoksilin-eozin bojenjem. Korišćcene su deskriptivne statističke metode. Rezultati: Od 89 slučajeva kraniofaringioma (46 ž ena i 43 muškarca), uzrasta izmedu 4 i 76 godina, bilo je 74 adamantinomatoznih (prosečna starost 35.68 godina), 13 papilarnih (prosečna starost 48 godina) i 2 mešovita tipa (prosečna starost 46 godina). Bimodalna uzrasna distribucija je uočena kod dece uzrasta 10-19 godina i odraslih uzrasta 50-59 godina. Svi kraniofaringiomi u uzrastu unutar prve dve decenije ž ivota bili su adamantinomatoznog tipa. Najčešća lokalizacija je bila supraselarna (43.82%), zatim intra/suparaselarna (15.73%), treća komora (14.06%), supraselarna/treća komora (13.48%), intra/supra/paraselarna (6.74%) i intraselarna (5.52%). Recidivi su se javili kod 16 pacijenata (15 adamantinomatozni, 1 mešoviti tip). Tri pacijenta su imala dva recidiva (dva su bili decaci starosti 8 i 11 godina). Jedna pacijentkinja je dva meseca nakon subtotalne resekcije adamantinomatoznog kraniofaringioma preminula usled tromboembolije pluća dokazane na autopsiji. Zaključak: Naši rezultati ukazuju na izvesne razlike u biološkom ponašanju histopatoloških tipova kraniofaringioma. Izgleda da papilarni tip ima bolju prognozu nego adamantinomatozni. Recidivi su se javili skoro isključivo kod adamantinomatoznog tipa, koji dominira u uzorku. Ključne reči: kraniofaringiom, histopatološki podtipovi, recidiv 646 NEUROPATHOLOGY Craniopharyngiomas: 10-year experience Emilija Manojlovic-Gacic1, Milica Skender- Gazibara1, Tatjana Gazibara2, Savo Raicevic3, Danica Grujicic3 1Institute of Pathology, School of Medicine, University of Belgrade, Belgrade, Serbia 2Institute of Epidemiology, School of Medicine, University of Belgrade, Belgrade, Serbia, 3Neurosurgery Clinic, Clinical Centre of Serbia, Belgrade, Serbia Aim: To investigate histopathological subtypes of craniopharyngiomas, their distribution according to the age and gender of patients, intracranial localization and recurrences in 10-year period. Introduction: Craniopharyngiomas are histologically benign, slow-growing, locally invasive intracranial tumors, presumably derived from Rathke pouch epithelium. Beside two primary subtypes (adamantinomatous and papillary) mixed forms have been described. Prognostic significance of these subtypes remains controversial. Material and methods: In the database of neurosurgical biopsies from 2002-2011 we registered 89 newly diagnosed cases of craniopharyngiomas. Tumor tissue was treated by routine hematoxylin-eosin method. Descriptive statistical analysis was performed. Results: Among 89 cases of craniopharyngioma (46 females and 43 males), ranging from 4 to 76 years, we found 74 adamantinomatous (mean age 35.68), 13 papillary (mean age 48) and 2 mixed (mean age 46) subtypes. A bimodal age distribution was present in children aged 10-19 and adults aged 50-59. All craniopharyngiomas in the first two decades were of adamantinomatous type. The most common localization was suprasellar (43.82%), followed by intra/suprasellar (15.73%), third ventricle (14.06%), suprasellar/third ventricle (13.48%), inra/supra/parasellar (6.74%) and intrasellar (5.52%). Sixteen patients developed recurrences (15 of adamantinomatous, 1 of mixed subtype). Of these, three cases had two recurrences (two were boys aged 8 and 11). Woman with partially resected adamantinomatous craniopharyngioma died two months after surgery due to autopsy-proven pulmonary thromboembolism. Conclusion: Our data showed certain differences in biological behavior of histopathological subtypes. It seems that papillary subtype has better prospects than adamantinomatous. Recurrences were noted almost exclusively in adamantinomatous subtype which was predominant in material. Key words: craniopharyngioma, histopathological subtypes, recurrence 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 2/poster prezentacije 2 P54 Deponovanje lipofuscina u neuronima pasa tokom života Lipofuscin deposition in dogs neurons durin the life Sladjan Nešic1, Milijan Jovanović1, Bojan Ristić2 Sladjan Nesic1, Milijan Jovanovic1, Bojan Ristic2 1Fakultet veterinarske medicine, Univerzitet u Beogradu, Beograd 2Veterinarski specijalistički institut, Zaječar, Zaječar, Serbia Cilj: Mogućnost detetekcije lipofuscina u mozgu različitim metodama kod različitih starosnih kategorija pasa. Uvod: Lipofuscin je pigment koji nastaje u organizmu živih bića u procesu starenja. On se nakuplja uglavnom u dugoživećim postmitotičkim ćelijama (neuroni). Pigment sadrži i različite unutarćelijske strukture, a primarno se nakuplja u lizozomima. Materijal i metode: Pregledani su mozgovi 59 obdukovanih pasa. Ispitivani psi su podeljeni na osnovu starosti u 4 grupe: I do 5 godina, II 5-10 godina, III 10-15 godina, IV preko 15 godina. Delovi mozgova (frontalna kora, parijetalna kora, hipokampus, mali mozak i produžena moždina) su fiksirani u 10% neutralnom formalinu a posle toga su standarno procesuirani do parafinskih blokova. Parafinski isečci debljine 5μm bojeni su metodama: hematoksilin-eozin, periodic acid Schiff (PAS) i produženom Ziehl-Neelsen. Rezultati: Granule lipofuscina su uočene u preparatima bojenim sa sve tri metode. U starosnoj kategoriji preko 15 godina pigment je dokazan u 80% slučajeva, dok taj procenat iznosi 30% u prvoj grupi. Kod pasa iz I i II grupe lipofuscin se nakupio u neuronima samo u produženoj moždini. U III i IV grupi lipofuscin je detektovan u različitim procentima u svim ispitivanim regionima mozgova pasa. Zaključak: Prisustvo lipofuscina u neuronima je dokazno kod pasa iz sve četiri kategorije. Broj pozitivnih ž ivotinja se povećavao proporcionalno sa starošću. Lipofuscin se najčešće nagomilava u velikim neuronima pojedinih jedara produžene moždine. Količina nagomilanog pigmenta se povećava sa starošću pasa kao i njegovo pojavljivanje u neuronima različitih regija mozga. Ključne reči: lipofuscin, metode, neuroni, psi 1Faculty of veterinary medicine,University of Belgrade,Belgrade, Serbia 2Veterinary specialized institute Zajecar,Zajecar, Serbia Aim: Possibility of lipofuscin detection using different methods in various age groups of dogs. Introduction: Lipofuscin is a pigment that occurs in the organism in the process of aging. It accumulates mainly in postmitotic long living cells (neurons). The pigment consists of various intracellular substances and accumulates primarily in lysosomes. Material and methods: Brain samples were examined from 59 autopsied dogs. Examined dogs were divided into 4 groups according to the age: I up to 5, II 5-10, III 10-15 and IV over 15 years. Parts of brains (frontal cortex, parietal cortex, hippocampus, cerebellum and medulla oblongata) was fixed in 10% neutral formalin and processed by routine method to paraffin blocks. For staining of 5ľm thick paraffin slices were used: hematoxylin-eosin, periodic acid Schiff (PAS) and long Ziehl-Neelsen techniques. Results: Lipofuscin granules were observed using all three staining techniques. In the age group over 15 years, pigment was detected in 80% of the examined dogs, while this percentage was 30% in the first group. In first and second group, lipofuscin accumulated only in neurons of the medulla oblongata. In the third and fourth group lipofuscin was detected in various percentages in neurons of all examined brain sections. Conclusion: Presence of lipofuscin in neurons was proven in dogs from all age categories. Number of positive animals increases proportionally with age. Lipofuscin most often accumulates in large neurons of medulla oblongata nuclei. The accumulation of lipofuscin pigment in neurons increases with the dog s age as well as widespread in neurons of different brain regions. Key words: lipofuscin, techniques, neurons, dogs 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 647 POSTER PRESENTATIONS 2/poster prezentacije 2 P55 Histoplazmoza centralnog nervnog sistema imitira tumor mozga: prikaz slučaja Central nervous system histoplasmosis mimics a brain tumor: a case report Nanad Miladinović, Milica Lavrnić, Marija Nikolić, Zorana Bokun, Lidija Prijić-Plećević, Sanja M. Milenković Nanad Miladinovic, Milica Lavrnic, Marija Nikolic, Zorana Bokun, Lidija Prijic - Plećevic, Sanja M. Milenkovic Cilj: Prikazujemo slučaj izolovane histoplazmoze centralnog nervnog sistema kod imunokompetentne pacijentkinje Uvod: Mnogi ne-neoplastični procesi u mozgu se manifestuju kao “mass” lezije i ponekad ih je veoma teško razlikovati od tumora mozga. Učestalost CNS histplasmoze u ne-endemskim oblastima raste, posebno kod imunokompromitovanih osoba. Materijal i metode: Opisujemo slučaj 48-godišnje imunokompetentne žene koja je imala glavobolju, meningealne znakove i ataksiju, ali bez dokazane sistemske infekcije. Kompjuterizovana tomografija je pokazala ekspanzivnu masu masu u malom mozgu. Posle biopsije tkiva uzorci su analizirani rutinski uz primenu histohemijskih i imunohistohemijskih bojenja. Rezultati: Histološkom analizom se uočavalo prisustvo malomoždanog parenhima, koji je delom bio očuvan, a delom pokazivao veoma izražene ishemijske promene sa eozinofilnim ishemijskim neuronima i izraženom Bergmanovom gliozom. Dominantan nalaz je intenzivna limfocitna (CD45+) infiltracija uz dominaciju T (CD3+) limfocita. Limfocitni infiltrate je veoma intenzivan, perivaskularan, pokazuje ekstenziju kroz Virchow-Robin-ove prostore penetrirajući u moždani parehnim. Nekroza moždanog parehnima, i kore i bele mase, je praćena histiocitnom reakcijom i mikroglijalnom proliferacijom bez formiranja granuloma Nisu detektovane virusne partikule Human Herpes virusa (tip 8), ni Epstein-Barr-ovog virusa. Bojenjem na PAS se uočavaju lezije granulirnog izgleda, i perivaskularno, i u arahnoideji, ali i u ishemijskom parenhimu. Granule su bile manje od aksonalnih sferoida i bojile su se na Grocott methenamine silverčime smo potvrdili dijagnozu histoplazmoze. Zaključak: Dijagnostika histoplasmoze CNS može biti teška i ponekad zahteva biopsiju moždanog parenhima, kao i primenu histohemijskih i imunohistohemijskih metoda bojenja radi potvrđivanja. Ključne reči: Histoplazmoza, centralni nervni sistem, tumor mozga, imunokompetentnost Aim: We report a case of isolated central nervous system histoplasmosis in immunocompetent patients Introduction: Many non-neoplastic diseases manifest as mass lesions and may be indistinguishable from brain tumors. The frequency of CNS histplasmose in non-endemic areas is growing, especially in immunocompromised persons. Material and methods: We describe a 48-year-old immunocompetent woman who had headache, meningeal irritation signs and cerebellar ataxia but no evidence of systemic infection. Computed tomography imaging showed an enhancing mass in the cerebellum. After biopsy tissue samples were analyzed pathohistological with histochemistry and immunohistochemistry. Results: Histological analysis showed cerebellar parenchyma, which was partly preserved, partly showing pronounced ischemic changes with eosinophil ischemic neurons and expressed Bergman gliosis. The dominant finding is intense lymphocyte (CD45 +) infiltration of the dominance of T (CD3 +) lymphocytes. Lymphocytic infiltrate werw very intense, with extension through the Virchow-Robin spaces and penetrated the brain parehnim. Parehnim necrosis of the brain, and cortex and white matter, was accompanied by histiocytic reaction and microglial proliferation without the formation of granuloma.Viral particles Human herpes virus (type 8) or Epstein-Barr’s virus were not detected. PAS staining showed granular lesions in perivascular and arachnoidal localisation, as well as, in the ischemic parenchyma. Granules were smaller than aksonalnih spheroids and stained with Grocott methenamine silver, which we confirmed diagnosis of histoplasmosis. Conclusion: The diagnosis of CNS histoplasmosis can be difficult, and sometimes performing a parenchymal biopsy and histochemistry and immunohistochemistry staining is necessary to confirm the diagnosis Key words: Histoplasmosis, central nervous system, brain tumor, immunocompetence. Služba kliničke patologije, Kliničko-bolnički centar Zemun, Beograd, Srbija 648 Department of Pathology, Clinical Hospital Center Zemun, Belgrade, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 2/poster prezentacije 2 PERINATALNA I PEDIJATRIJSKA PATOLOGIJA PERINATAL AND PEDIATRIC PATHOLOGY P56 Sijamski blizanci – prikaz slučaja Siamese twins –case report Lidija Vučković, Marija Milić Lidija Vuckovic, Marija Milic, Cilj: Prikaz sijamskih blizanaca kao redak medicinski fenomen Uvod: Sijamski blizanci su identični blizanci kod kojih nije došlo do potpune podele tokom ranog fetalnog razvoja, tako da mogu deliti delove tela i organe. Materijal i metode: Prikaz slučaja Rezultati: Pacijentkinja stara 30 godina, druga potpuno nekotrolisana trudnoća se javlja ginekologu u Opštoj bolnici Subotica zbog bolova u stomaku. Uradi se ginekološki pregled i Usg i ustanovi da je reč o 40/41 nedelji trudnoće. Učini se operacija Sectio Caesarea. Estrahuju se ženski sijamski blizanci (Thoracopagus ), težine 6460 gr, dužine 50 cm, Apgar score 1/0, koji nisu zaplakali. Započete su mere reanimacije i nakon 30 min proglašava se exitus letalis. Sijamski blizanci su poslati na obdukciju gde je nađeno da su spojeni u predelu grudnog koša i abdomena, sa jednim pupčanikom. Pri spoljšnjem pregledu uočava se kratak vrat, niže postavljene ušne školjke i blago uvučene donje usne, normalni ekstremiteti, bez deformiteta prstiju i patoloških linija na šakama. Po razdvajanju grudnog koša i abdomena nađeno je da se pupčanik račva, postoji jedno srce sa dve pretkomore i jednom hipertrofičnom i dilatiranom komorom. Prisutan je jedan timus i jedna uvećana jetra. Ostali organi su parni i pravilne grade. Zaključak: Sijamski blizanci nastaju pri nepotpunom razdvajanju embrionalnog diska kod monozigotnih blizanaca. Ova anomalija je veoma retka u razvijenim zemljama gde se zahvaljujući blagovremenoj ultrazvučnoj dijagnostici takve trudnoće obicno prekidaju. Poslednji slučaj sijamskih blizanaca u Opštoj bolnici Subotice se desio 1987 godine. Ključne reči: sijamski blizanci, urođene anomalije Aim: Presentation of siamese twins as a rare medical phenomenon Introduction: Siamese twins are identical twins, but they had no complete separation during early fetal development so they can share some body parts and organs. Material and ethods: Case review Results: 30 year old patient report to the gynecologist in Subotica general hospital because of the abdominal pain. Pelvic exemination and Usg was made. It resulted that she was 40/41 weeks pregnant. Operation Sectio Caesarea was made. The female Siamese twins were extrachted (Thoracopagus ), weighing 6460 g, length 50 cm, Apgar score 1/0, they didn`t cried. Resusciation measures were initiated and after 30 minutes exitus letalis was declared. Siamese twins were sent for autopsy, where it was found that they were connected to the chest and abdomen, with one umbilical cord. The external review showed short neck, low set ears, slightly recessed lower lip, normal extrimitates without deformity of the fingers and pathological lines on the hands. By separating the chest and abdomen, umbilical cord that branched was found. There was one heart with two atria, one hypertrophic and delated ventricul. There was one thymus and one enlarged liver. Other organs were in pair and properly developed. Conclusion: Siamese twins become as a result of incomplete separation of embryonic disc in monozygotic twins. This anomaly is very rare in developed countries and due to ultrasound diagnosis such a pregnancy is usually interrupted. The last case of Siamese twins in Subotica general hospital occured in 1987. Key words: siamese twins, congenital anomalies Opšta bolnica Subotica, Subotica, Srbija General hospital Subotica, Subotica, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 649 POSTER PRESENTATIONS 2/poster prezentacije 2 P57 Limfomi u dečijem uzrastu - analiza desetogodišnjeg rada jedne ustanove Lymphoma in children - ten-year analysis in a single institution Gordana Samardžija, Slaviša Đuričić, Milena Đukić, Milo Kuzmanović, Dragan Micić, Dragomir Đokić, Gordana Samardzija, Slavisa Djuricic, Milena Djukic, Milos Kuzmanovic, Dragan Micic, Dragomir Djokic Cilj: Procena distribucije limfoma dečijeg doba u odnosu na uzrast, pol, histološki tip i anatomsku lokalizaciju u nacionalnoj pedijatrijskoj ustanovi. Uvod: Postoje razlike u incidenci i međusobnom odnosu histoloških podtipova Hodgkin i non-Hodgkin limfoma (HL, NHL) dečijeg doba izmedu različitih delova sveta. Materijali i metode: Desetogodiš nja retrospektivna anliza (2002-2011) tipova HL i NHL na osnovu medicinske dokumentacije i tkivnih uzoraka. Poređenje rezultata sa podacima iz literature. Rezultati: Lečeno je 117 bolesnika sa limfomom: 58 HL (49.6%), 59 NHL (50.4%). HL: odnos dečaci: devojčice 33:25 (1.32:1), uzrast se kretao od 4 meseca do 18 godina sa medijanom od 14 godina, nodularna skleroza je bio najčešći podtip (86.2%). NHL: odnos dečaci: devojčice 47:12 (3.9:1), uzrast se kretao od 12 meseci do 18 godina sa medijanom od 11 godina. Od svih NHL 62.7% su bili porekla B-ćelija, 28.8% T-ćelija i 8.5% neklasifikovani. Histološka distribucija NHL je bila: Burkitt-ov limfom 49.2%, difuzni B krupnoćelijski 11.9%, anaplastični krupnoćelijski 11.9%, limfoblastni 20.3% i neklasifikovani NHL 6.7%. Zastupljenost lokalizacija glava/vrat i abdomen je bila po 35,5%, medijastinum (19.4%), gonade (4.8%) i druge lokalizacije (4.8%). Nismo mogli da procenimo tačnu zastupljenost ekstranodalne lokalizacije kao primarne po to su neki limfomi dijagnostikovani u višim stadijumima sa ispoljenom i nodalnom i ekstranodalnom zahvaćenošću. Zaključak: Distribucija limfoma u dečjem uzrastu u odnosu na pol, uzrast, histološke tipove i lokalizaciju u našoj seriji malo se razlikuje od objavljenih velikih svetskih serija i nalazi se najčešće izmedu podataka iz ekonomski razvijenih zemalja i zemalja u razvoju. Ključne reči: Hodgkin limfom, Non-Hodgkin limfom, Deca Aim: The assessment of distribution of childhood lymphomas according to age, sex, histological types and anatomic location in a single pediatric institution. Introduction: There are differences in both incidence and proportion of histological subtypes of Hodgkin lymphoma and non-Hodgkin lymphoma (HL, NHL) of childhood in different parts of the world. Material and methods: Ten-year retrospective analysis (2002-2011) of HL and NHL types distribution by reviewing medical documentations and tumor histology. Comparison of results with relevant data from the literature. Results: 117 patients with lymphoma were treated: 58 HL (49.6%), 59 NHL (50.4%). HL: male: female ratio 33:25 (1.32:1), median age 14 years, range 4 months-18 years, nodular sclerosis as predominant type (86.2%). NHL: male: female ratio 47:12 (3.9:1), median age 11 years, range 12 months-18 years. Of all NHL 62.7% were B-cell origin, 28.8% T-cell and unclassified (8.5%). NHL histologic distribution was: Burkitt’s lymphoma 49.2%, diffuse large B-cell 11.9%, anaplastic large cell 11.9%, lymphoblastic 20.3% and unclassified NHL 6.7%. The frequency of localization was head/neck and abdomen 35.5% each, mediastinum (19.4%), gonads (4.8%) and other (4.8%). However, we were not be able to assess the exact percent of primary extranodal localization because some lymphoma were diagnosed in high stage with both nodal and extranodal involvement. Conclusion: Distribution characteristics of childhood lymphomas according to sex, age, histological types and anatomic location in our series are somewhat different from the published series. They are in the middle range between the relevant data from economically developed and developing countries. Key words: Hodgkin lymphoma, Non-Hodgkin lymphoma, Children Institut za zdravstvenu zaštitu majke i deteta Srbije “Dr Vukan Čupić”, Beograd, Srbija 650 Mother and Child Health Care Institute of Serbia “Dr Vukan Cupic”, Belgrade, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 POSTER PRESENTATIONS 2/poster prezentacije 2 P58 Wilms-ov tumor: mutacija β katenin gena Wilms tumor: Mutation in the β katenin gene Gordana Basta-Jovanović1, Brašanac Dimitrije1, Ljiljana Bogdanović1, Sofija Glumac1, Zoran Krstić2, Sanja Radojević-Škodrić1 Gordana Basta-Jovanovic1, Dimitrije Brasanac1, Ljiljana Bogdanovic1, Sofija Glumac1, Zoran Krstic2, Sanja Radojevic-Skodric1 Cilj: Da se analizira ekspresija ß-katenin proteina u primarnim Wims-ovim tumorima i korelirati sa prisustvom somatskih mutacija ß-katenin gena. Uvod: Wnt-signalni put ima važnu ulogu u normalnom razvoju bubrega, ali i u tumorogenezi Wilmsovog tumora (WT). Nuklearna translokacija ß-katenin proteina može biti posledica specifičnih mutacija ß-katenin gena. Materijal i metode: Analizirana je nuklearna ekspresija ß-katenin proteina u 30 slučajeva primarnih WT-a i ovi rezultati su korelirani sa prisustvom mutacija ß-katenin gena u ispitivanim slučajevima. Za imunohistohemijsko bojenje korišćena je streptavidin-biotin tehnika, a za detekciju somatskih mutacija PCR i metoda sekvenciranja. Rezultati: Nuklearna ekspresija ß-katenin proteina je detektovana u 4 od 30 slučajeva WT-a (13,3%). Nuklearno bojenje u ovim slučajevima je bilo jako, ali prisutno u manjem broju ćelija (5 % - 20 % celija). Jedra blastemskih ćelija su u većini slučajeva pozitivna, dok je nuklearna ekspresija ß-katenina u stromalnoj komponenti uocena u 2 slučaja, a u epitelnoj u samo jednom slučaju WT. Osim toga, pozitivne ćelije su nađene u WT-ima svih stadijuma i u ne-anaplastičnim WT-ima. Mutacije ß-katenin gena nisu uočene u analiziranim uzorcima. Zaključak: Činjenica da je u nekim WT-ima uocena nuklearna ekspresija ß-katenina i u odsustvu mutacija ß-katenin gena, ukazuje da mutacije drugih članova Wnt-signalnog puta može doprineti razvoju WT-a. Ključne reči: WT, ß- katenin, mutacije, nuklearna ekspresija Aim: To analyze the expression of ß-catenin protein in primary Wilms’ tumor (WT) specimens and to correlate these results with the mutational status of the ß-catenin gene. Introduction: The Wnt-signaling pathway plays an important role during both normal kidney development and Wilms’ tumorigenesis. Nuclear translocation of the ß-catenin protein may be caused by specific mutations in the ß-catenin gene itself. Material and methods: We have analyzed nuclear expression of ß-catenin protein in 30 primary WTs and correlated these results with the mutational status of the ß-catenin gene in these samples. Immunohistochemistry was performed using the streptavidin-biotin technique. PCR and sequencing analysis were carried out to examine the presence of somatic mutations of ß-catenin gene. Results: Nuclear immunoreactivity for ß-catenin was detected in 4 out of 30 WT (13.3%). Nuclear positivity, in each case, was found to be very strong, but usually present only in a fraction of cells ranging from 5% to 20%. Blastemal cell nuclei preferentially stained positive, whereas cells of stromal component showed positivity in 2 cases and epithelial cells displayed nuclear localization of ß-catenin protein only in single case. Furthermore, nuclear positive cells were found in WTs of all stages and in non-anaplastic WTs. ß-catenin gene mutations were not detected in analyzed WTs specimens. Conclusion: The fact that nuclear expression of ß-catenin was detected in the absence of ß-catenin gene mutations in some analyzed WTs, suggests that genetic defects affecting other members of the Wnt-signalling pathway may contribute to the development of WT. Key words: WT, ß catenin, mutation, nuclear expression 1Institut za patologiju,Medicinski fakultet, Univerzitet u Beogradu, Beograd, Srbija 2Univerzitetska dečija klinika, Beograd, Srbija 1Institute of pathology, Medical School, University of Belgrade, Belgrade, Serbia 2University Children’s Hospital, Belgrade, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 651 POSTER PRESENTATIONS 2/poster prezentacije 2 PULMONALNA PATOLOGIJA PULMONARY PATHOLOGY P59 Uloga imunohistohemijske analize u u dijagnozi malignog mezotelioma pleure The role of immunohistochemical evaluation in the diagnosis of malignant pleural mesothelioma Aleksandra Lovrenski1, Milana Panjković1, Dragana Tegeltija1, Živka Eri, Slavica Knežević Ušaj2, Zoran Nikin2 Aleksandra Lovrenski1, Milana Panjkovic1, Dragana Tegeltija1, Zivka Eri1, Slavica Knezevic Usaj2, Zoran Nikin2 Cilj: Utvrditi značaj imunohistohemijske analize i primene pojedinih antitela u dijagnozi malignog mezotelioma pleure. Uvod: Maligni mezoteliom je agresivan tumor sa visokom stopom mortaliteta. Konačna dijagnoza malignog mezotelioma pleure postavlja se isključivo patohistološkim pregledom bioptiranog materijala koji se rutinski nadopunjuje i upotrebom imunohistohemijske analize. Materijal i metode: Retrospektivno je izvršena analiza klinickih podataka 32 pacijenta kod kojih je u periodu od 2004. do 2009. godine na Institutu za plućne bolesti Vojvodine u Sremskoj Kamenici patohistološki postavljena dijagnoza malignog mezotelioma pleure. Materijal za patohistološku i imunohistohemijsku analizu dobijen je videoasistiranom torakoskopijom (30 uzoraka), transbronhijalnom biopsijom (1 uzorak) i otvorenom biopsijom pluća (1 uzorak). Mikroskopska analiza imunohistohemijski obradenih uzoraka bazirala se na njihovoj pozitivnosti i negativnosti na određeno antitelo, na koji način je postavljena konačna dijagnoza malignog mezotelioma određenog tipa. Rezultati: Citokeratin 5/6 (CK5/6) bio je pozitivan u 63% slučajeva, Calretinin u 94%, a Anti-Human Mesothelial Cell, Clone HBME-1 (HBME-1) u 80% slučejeva. Citokeratin 7 (CK7) se pokazao pozitivnim u 78%, a Epithelial membrane antigen (EMA) u 83% slučejeva. Svi slučajevi (100%) bili su negativni na Thyroid transcription factor 1 (TTF-1), Carcinoembryonic antigen (CEA), Cytokeratin 20 (CK 20), Epithelial Specific Antigen Ab-7, Clone MOC-31 (MOC 31) i Desmin. Zaključak: Imunohistohemija je postala esencijalni dijagnostički postupak u postavljanju dijagnoze i određivanju tipa malignog mezotelioma pleure. Zbog nedostatka samostalnog antitela danas se koristi kombinacija antitela različite senzitivnosti i specificnosti. Ključne reči: Maligni mezoteliom, pleura, imunohistohemijska analiza Aim: Determination of the role of immunohistochemical analysis and some antibodies in the diagnosis of malignant pleural mesothelioma. Introduction: Malignant mesothelioma of the pleura is aggressive neoplasm with a high mortality. The final diagnosis of malignant pleural mesothelioma can be made only by pathohistological examination of tissue biopsy that routinely is followed by use of immunohistochemical analysis. Material and methods: Retrospective analysis of clinical data from 32 patients diagnosed pathohistologically with malignant pleural mesothelioma in period from 2004. to 2009. year at Institute for Pulmonary Diseases of Vojvodina was performed. Material for pathohistological and immunohistochemical analysis was obtained by video assisted thoracoscopic surgery (30 samples), transbronchial biopsy (1 sample) and open lung biopsy (1 sample). The microscopic examination of immunohistochemically processed samples was based on their positivity or negativity to specific antibody, and final diagnosis of malignant pleural mesothelioma of certain type was concluded. Results: Cytokeratin 5/6 (CK5/6) was positive in 63% cases, Calretinin in 94% and the Anti-Human Mesothelial Cell, Clone HBME-1 (HBME-1) in 80% cases. Cytokeratin 7 (CK7) showed positivity in 78% and Epithelial membrane antigen (EMA) in 83% cases. All cases (100%) stainded negative for Thyroid transcription factor 1 (TTF-1), Carcinoembryonic antigen (CEA), Cytokeratin 20 (CK 20), Epithelial Specific Antigen Ab-7, Clone MOC-31 (MOC 31), and Desmin. Conclusion:Immunohistochemistry has become an essential diagnostic tool for the diagnosis and determination of the type of MPM. Since there is no individual antibody, today is in use a combination of antibodies with different sensitivity and specificity. Key words: Malignant mesothelioma, pleura, immunohistochemistry 1Institut za plucne bolesti Vojvodine, Sremska Kamenica, Srbija 2Institut za onkologiju Vojvodine, Sremska Kamenica, Srbija 652 1Institute for pulmonary diseases of Vojvodina, Sremska Kamenica, Serbia 2Institute of Oncology of Vojvodina, Sremska Kamenica, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 P60 Imunohistohemijski pristup u diferencijaciji nemikrocelularnih karcinoma pluća na malim biopsijskim uzorcima: prvi korak u personalizovanoj terapiji Immunohistochemical approach in differentiation of non-small cell lung cancer on small-sized biopsy samples: the first step in personalized therapy Jelena Stojšić1, Jelena Marković1, Irena Jovanić2, Milan Gajić3 Jelena Stojsic, Jelena Markovic1, Irena Jovanic2, Milan Gajic3 Cilj: Precizirati optimalni panel monoklonskih antitela u diferencijalnoj dijagnozi NSCLC na malim biopsijskim uzorcima. Uvod: Ciljna terapija povećava dužinu preživljavanja i kvalitet života kod pacijenata sa karcinomom pluća ali zahteva preciznu tipizaciju nemikrocelularnih karcinoma pluća (NSCLC). Materijal i metode: 50 biopsijskih uzoraka dobijenih tokom bronhoskopije ili FNA biopsijom tokom poslednje dve godine. Prema morfološkom nalazu dijagnostikovana su 2 skvamocelularna karcinoma(SCC), 6 adenokarcinoma(AC), 9NSCLC verovatno SCC, 11NSCLC - verovatno AC i 22NSCLC(NOS) su bili dijagnostikovani. TTF1, Cytokeratin5, Napsin-A, p63 i CD56 i Synaptophisin su korišćeni u diferencijaciji NSCLC. Rezultati: Posle imunohistohemijskog bojenja 13(26.0%)SCC, 27(54.0%) AC, 3(6.0%) NSCLC sa neuroendokrinom diferencijacijom (NSCLC-NE) i 7(14.0%) NSCLC(NOS) su bili dijagnostikovani. 22NSCLC- prethodno neklasifikovanih su bili dijagnostikovani kao 7SCC i 7AC, 2NSCLC sa neuroendokrinom diferencijacijom i 6 NSCLC(NOS) su dijagnostikovani posle imunohistohemije. 8(20.5%)NSCLC versus 15(38.5%)AC, p=0.008. TTF1 je bio eksprimiran 23(85.2%) od 27AC, 1(14.3%) od 7NSCLC(NOS) i 2(66.7%) od 22NSCLC-NE. Napsin-A je bio eksprimiran u 2(15.4%) od 13SCC, 23(85.2%) od AC, 4(57.1%) od 7NSCLC(NOS) i 1(33.3%) od 3NSCLCNE. Cytokeratin 5 je bio eksprimiranu svih 13(100%)SCC, 2(7.4%) od 27AC i 2(28.6%) od7NSCLC(NOS). p63 je bio eksprimiran u svih 13(100%)SCC i u 1(3.7%) od 27AC. TTF-1 i Napsin-A su bili eksprimirani u 85.2%(23/27, oba), a Cytokeratin5 i p63 u 100%(13/13)SCC. Pozitivnost CD56 i synaptophisina u 3NSCLC determinisanih kao NSCLC-NE. Zaključak: Ni jedno monoklonsko antitelo nije u potpunosti specifično za jedan histološki tip tumora i njegovo poreklo. Optimalni panel u diferencijaciji NSCLC su TTF-1, p63, Cytokeratina5, CD56, Synaptophisin ali i Napsin-A ChromograninA. Ključne reči: karcinom pluća, imunohistohemija. Aim: To evaluate 6 monoclonal antibodies in differential diagnosis of NSCLC on small-sized tissue samples. Introduction: Target therapy increases survival rate and quality of life of lung cancer patients but requests precise subtyping of non-small cell lung carcinoma(NSCLC). Material and methods: 50 small-sized tissue samples obtained on bronchoscopy or FNAB. According to morphology before immunohistochemistry 2 squamous cell carcinomas (SCC), 6 adenocarcinomas (AC), 9NSCLC-probably SCC, 11NSCLC-probably AC and 22 unclassified NSCLC were diagnosed. TTF-1, cytokeratin5/6, cytokeratin7, p63 and neuroendocrine markers CD56 and synaptophisin were used in differentiation NSCLC. Results: After immunohistochemistry 13(26.0%) SCC, 27(54.0%)AC, 3(6.0%)NSCLC with neuroendocrine differentiation(NSCLC-NE) and 7(14.0%) NSCLC- unclassified(NOS) were diagnosed. 22NSCLCunclassified were diagnosed as 7SCC and 7AC, respectively, 2NSCLC with neuroendocrine differentiation and 6NSCLC- unclassified(NOS). Significant difference was found between finally diagnosed 8NSCLC and 15AC (20.5% versus 38.5%, p: 0.008). TTF-1 and Cytokeratin7 were expressed in 85.2%(23/27) AC, respectively and Cytokeratin5/6 and p63 in 100%(13/13) SCC, respectively. Positivity of CD56 and synaptophisin in 3NSCLC determinate as NSCLC-NE. Conclusion:No one monoclonal antibody is totally specified for one histological type of tumor and its origin. Optimal panel in differentiation of NSCLC is TTF-1, cytokeratin7, p63, cytokeratin5/6, CD56, Synaptophisin and Napsin-A and ChromograninA in addition. Key words: lung cancer, immunohistochemistry 1Odeljenje torakopulmonalne patologije, Služba za patohistologiju, Klinički Centar Srbije, Beograd 2Služba patohistologije, Institut za onkologiju i radiologiju Republike Srbije, Beograd, Srbija 3Institut za medicinsku statistiku i informatiku, Medicinski fakultet, Univerzitet u Beogradu 1 Department of thoracopulmonary patology, Service of pathology, Clinical centre of Serbia, Belgrade, Serbia 2Service of pathohistology, Institute for Oncology and Radiology of Republic of Serbia, Belgrade, Serbia 3Institute for Medical Statistic and Informatic, Medical Faculty, University of Belgrade, Belgrade, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 653 P61 Imunohistohemijska analiza “non-small” cell carcinoma pluća Immunohistochemical analysis of non-small cell lung carcinoma Žaklina Mijović1, Dragan Mihailović1, Miloš Kostov2, Nikola Živković1, Miljan Krstić1, Zaklina Mijovic1, Dragan Mihailovic1, Milos Kostov2, Nikola Zivkovic1, Miljan Krstic1, Milorad Pavlovic3 Cilj: Cilj ovog rada je imunohistohemijska analiza “non-small” cell karcinoma pluća na bronhoskopskim biopsijama. Uvod: Za razliku od prethodne WHO klasifikacije (2004) u kojoj su primarni dijagnostički kriterijumi bazirani na hematoksilin eozin metodi, nova internacionalna multidisciplinarna klasifikacija adenokarcinoma pluća potencira upotrebu imunohistohemijskih bojenja (adenokarcinomatozni marker -tiroidni transkripcioni faktor, TTF-1 i skvamoidni marker -p63 i/iliCK5/6). Materijal i metode: Bronhoskopske biopsije, fiksirane u formalinu i kalupljene u parafinu, 20 pacijenata sa planocelularnim karcinomom i 20 pacijenata sa adenokarcinomom pluća su izdvojene iz plućnog arhiva Instituta za patologiju Medicinskog fakulteta Univerziteta u Nišu. Serijski histološki isečci debljine 4 μm su bojeni hematoksilin eozinom i imunohistohemijskom metodom DAKO LSAB/HRP na TTF-1 i CK 5/6 antitelo. Za stataističku analizu korišćen je Xi 2 test. Rezultati: Pozitivna imunoreaktivnost na CK5/6 nađena je kod 18 od 20 (90%) slučajeva planocelularnih karcinoma i u jednom od 20 (5%) slučajeva adenokarcinoma (p<0,01). Kod 16 od 20 (80%) slučajeva adenokarcinoma nađen je pozitivan TTF-1 imunofenotip, dok su svi planocelularni karcinomi bili negativni na ovaj marker (0/20)(p<0,01). Zaključak: Preporučeni panel imunohistohemijskih markera CK5/6 i TTF-1 je pouzdan u diferenciranju planocelularnog od adenokarcinoma pluća na bronhoskopskim biopsijskim uzorcima. Ključne reči: Imunohistohemija, „non-small cell karcinom pluća, planocelularni karcinom, adenokarcinom Aim: The aim of this study was immunohistochemical analysis of non-small cell lung carcinoma in bronchoscopic biopsy specimens. Introduction: Unlike previous WHO (2004) classifications where the primary diagnostic criteria were based on hematoxylin and eosin method, the new international multidisciplinary classification of lung adenocarcinoma (2011) emphasizes the use of immunohistochemical stain (adenocarcinoma marker -thyroid transcription factor, TTF-1 and squamous marker -p 63 and/or CK5/6). Material and methods: Formalin-fixed, paraffinembedded bronchoscopic mucosal samples from 20 patients with squamous cell lung carcinoma and 20 patients with adenocarcinoma of the lung were retrieved from pulmonary pathology archives at Institute of Pathology, Medical Faculty University of Ni . Serial histologic sections of 4 ľm thickness were stained with hematoxilin and eosin and immunohistochemical method DAKO LSAB/HRP for TTF-1 and CK5/6 antibodies. The Xi 2 test was used for statistical analysis. Results: Positive immunoreactivity for CK5/6 was found in 18 of 20 (90%) cases of squamous cell carcinomas and in the one case of 20 (5%) adenocarcinomas (p<0,01). In 16 of 20 (80%) cases of adenocarcinomas were found a positive TTF-1 immunophenotype, while all squamous cell carcinomas were negative for this marker (0/20) (p<0,01). Conclusion: The proposed panel of immunohistochemical markers CK5/6 and TTF-1is reliable in distinguishing squamous cell lung carcinoma from adenocarcinoma in bronchoscopic biopsy specimens. Key words: Immunohistochemistry, non smallcell lung carcinoma, squamous cell lung carcinoma, adenocarcinoma 1Institut za patologiju, Medicinski fakultet, Univerzitet u Nišu,Srbija 2Služba za patologiju, Vojna bolnica, Niš, Srbija 3Milorad Pavlović, Služba za patologiju, Opšta bolnica Leskovac,Srbija 654 1Institute of Pathology, Medical Faculty, University of Nis, Serbia 2Department of pathology, Military Hospital, Nis, Serbia, 3Department of pathology, General hospital, Leskovac, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 P62 Pleuralna efuzija na autopsiji Pleural effusion on autopsy Petar Salević, Dragan Mitrović, Radmila Janković Petar Salevic, Dragan Mitrovic, Radmila Jankovic Cilj: Ispitati opštu učestalost, uzročnost, lokalizaciju, zastupljenost tipova i vrednosti količina pleuralnog izliva na obdukciji. Uvod: I pored mnogobrojnih studija i radova u dostupnoj literaturi koji su ispitivali mnogobrojne aspekte pleuralne efuzije do sada nije bilo istraživanja koja su ispitivala karakteristike pleuralne efuzije na obdukciji. Materijali i metode: Retrospektivnom studijom su obuhvaćene 302 od 502 obdukovane osobe, podeljene u dve grupe: grupa obdukovanih pacijenata sa pleuralnom efuzijom (n=229) i kontrolna grupa (n=73) osobe bez pleuralne efuzije kad kojih su bila prisutna različita patološka stanja koja mogu biti uzrok pleuralne efuzije. Rezultati: Pleuralna efuzije bila je ucestalija kod osoba muškog pola (X2=14,19 p: 0,0002). Najveći broj obdukovanih sa pleuralnom efuzijom bio je u dve starosne grupe: 60,0 - 69,9 (23,14%) i 70,0 - 79,9 godina (31,44%). Slabost srca bila je najučestalija medu pojedinačnim uzrocima, dok je u okviru udruženih uzroka naučestalija bila kombinacija srčane slabosti i brohopneumonije. Najzastupljeniji tipovi pleuralnog izliva bili su: transudat (73,80%), eksudat (19,65%) i krv (2,62%). Količine tecnosti u desnom pleuralnom prostoru (MED=120 ml) bile su veće od kolicina tečnosti u levom pleuralnom prostoru (MED=100 ml). Zaključak: Pleuralna efuzija je učestalija kod muškaraca i osoba starosne dobi između 60 i 80 godina. Pleuralna efuzija predstavlja važan klinički nalaz koji najčešće upućuje na srčanu slabost, pneumoniju, maligne neoplazme i plućnu emboliju, ali može biti izazvan i ređim uzrocima koji neposredeno ugrožavaju život bolesnika kao što je krvarenje u pleuralnom prostoru, stoga je u kliničkom radu potrebno posebno obratiti pažnju na mogućnost nastanka pleuralne efuzije kod bolenika sa navedenim bolestima. Ključne reči: Pleuralna efuzija, transudat, eksudat, srcana slabost, pneumonija Aim: To show general frequency, causes, localization, frequencies of different tipes and value of amount of pleural effusion on autopsy. Introduction: Beside noumerous studies in available literature that examined various aspects of pleural effusion there was no studies that examined characteristics of pleural effusion on autopsy. Material and methods: The postmortem retrospective study consisted of 302 persons divided into two groups, group of patients with pleural effusion and control group, patients without pleural effusion who had different pathological condition that might be causes of pleural effusion.Results: Pleural effusion was more frequent in males (X2=14.19 0.0002). Most of the patients with pleural effusion was in two age groups: 60,0 - 69,9 (23.14%) and 70,0 - 79,9 years (31.44%). Heart failure was the most common among single causes, while combination of heart failure and pneumonia was most common among associated causes. The most frequent tipes of pleural effusion were: transudate (73.80%), exudate(19.65%) and blood (2.62%). Amounts of liquid in right pleural space (MED: 120 ml) were above amounts of liquid in left pleural space (MED=100 ml). Conclusion: Pleural effusion is more frequent within males and persons aged 60 80 years. Pleural effusion is important clinical finding that in the most cases implicates on heart failure, pneumonia, malignancy and pulmonary embolism, but also can be caused by some less frequent conditions which immediately endangers patient s life such as bleeding in pleural space. Therefore it is very important in clinical practise to pay attention on patients whith these conditions. Key words: Pleural effusion, transudate, exudates, heart failure, pneumonia. Institut za patologiju, Medicinski fakultet, Univerzitet u Beogradu, Beograd, Srbija Institute of pathology, Faculty of Medicine, University of Belgrade, Belgrade, Serbia 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 655 uputstvo za autore Časopis „Materia Medica” izlazi četiri puta godišnje i objavljuje radove iz različitih oblasti biomedicine. Za publikovanje se primaju sledeće vrste radova: uvodnici (do 5 strana), originalni radovi (do 10 strana), revijalni radovi (do 12 strana), seminarski radovi (do 10 strana) prikazi slučaja (do 5 strana), pisma uredniku (do 2 strane), prikazi knjiga (do 2 strane), dopisi za rubriku u spomen - „In memoriam” (do 5 strana), istorija medicine (do 5 strana) i konferencijska saopštenja (do 5 strana). Uređivački odbor se striktno pridržava principa Dobre naučne prakse. Kada pripremaju rad za publikovanje autori moraju da se pridržavaju uputstva koje je predložio Internacionalni komitet za urednike medicinskih časopisa, a koje je publikovano na web sajtu Internacionalnog komiteta urednika medicinskih časopisa http://www.icmje.org/ UPUTSTVO ZA PRIPREMU RUKOPISA Koristite Time New Roman, font 12, justify orjentaciju (Ctrl + J) i prored 1,5 1. strana Naslov rada (do 12 reči ili 100 slovnih mesta sa proredima, pisati malim slovima poštujući pravopis o velikim slovima, ne stavljati tačku na kraju) Prvi A. Autor 1, Drugi B. Autor2, Treći C. Autor3… (puno ime i prezime sa srednjim slovom) 1 Ustanova iz koje su autori (pun naziv) 2 Ustanova iz koje su autori (pun naziv) 3 Ustanova iz koje su autori (pun naziv) Autor za korespondenciju Ime Prezime, institucija, adresa, telefon, e-mail 2. strana Apstrakt (do 250 reči, strukturiran) Pišite ga u: originalnom naučnom članku, preglednom članku, prikazu slučajačeva, rubrici aktuelno i u rubrici seminarski radovi, a ne pišite ga u uvodnicima i pismima uredništvu Apstrakt treba da sadrži sledeće delove Cilj (Objective, Aims), Metod (Methods), Rezultate (Results) Zaključak (Conclusion). Ključne reči: ili kratke fraze do 10 (obavezno sa MeSH liste koja se može naći na web sajtu www.nlm. nih.gov/tsd/serials/lji.html I SADA PONOVITE SVE NA ENGLESKOM 3. strana Uvod (idealan uvod je uvod do 25 rečenica na jednoj strani A4 formata) 1. Paragraf - 1-2 uvodne rečenice za centralnu rečenicu Centralna rečenica, ključna rečenica prvog paragrafa je odgovor na pitanje „Šta mi znamo” (polje istraživanja). Posle centralne rečenice slede 1-2 završne rečenice za 1. paragraf ili 1-2 prelazne na sledeći paragraf. Poželjno je ovaj deo potkrepiti sa 1-2 reference, ne više od 5, a najbolje je da to budu poglavlja iz udžbenika ili revijalni radovi. 2. Paragraf – 1-2 uvodne rečenice ka centralnoj rečenici drugog paragrafa. Centralna rečenica, ključna rečenica prvog paragrafa je odgovor na pitanje „Šta mi ne znamo” (problem istraživanja). Čitaoca upoznajete sa postojećim podacima (tuđim i sopstvenim) o problemu koji istražujete, o ograničenjima da se taj problem reši i o pitanjima na koja odgovori još nisu dati. Citirati samo one reference koje se neposredno odnose na istraživanje istog predmeta i koja su prethodila vašem istraživanju. 3. Paragraf - Cilj vašeg istraživanja. 656 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 uputstvo za autore Sugestije: Ako preterate sa referencama u Uvodu izgubićete „blago” za diskusiju i opteretićete spisak literature (većina časopisa dozvoljava, pa i mi najviše 25-30 referenci. Prilikom prikupljanja reference neophodno je citirato reference novijeg datuma, naravno da neka stara (“kapitalna”) može naći svoje mesto. Redosled referenci koje citirate treba da sledi logičan raspored paragrafa uvoda. Prve reference su one koje se odnose na uopšteno znanje o problem i reference o istraživačkom problem. Zatim slede reference vezane za nova istraživanja - prethodna, aktuelna istraživanja i njihove limitacije Nikada u Uvodu ne iznositi svoje rezultate Konkretan cilj se obično navodi u jednoj rečenici (poslednjoj rečenici Uvoda) koja postavlja očekivanja zbog kojih je istraživanje započeto i zbog kojeg se rad piše. Vodite računa cilj je prva rečenica strukturiranog apstrakta i poslednja rečenica Uvoda . 4. strana Materijal i metode Opišite kako ste došli do rezultata (precizan dizajn studije, metoda koju ste koristili i kako ste analizirali podatke). Tačni podaci gde je studija sprovedena. Budite koncizni ( ne pišete turistički vodič). Ukoliko koristite standatdni metod citirajte referentnu literaturu. Sve mere koje saopštavate u poglavlju rezultati, u poglavlju metode moraju imati opisan način kao se do njih došlo. Prilikom čitanja ovog metoda, treba omogućiti čitaocima da imaju kritički uvid u vaš radi i da ponove vašu studiju baš na onaj način kako ste je vi uradili. Podnaslovi koji se koriste u poglavlju metoda kao što su: učesnici, dizajn studije, specifične metode, analiza podataka... klasično određuju njen sadržaj. Neophodno je da date detalje o odobrenju vaše studije, koje je dao etički komitet vaše institucije u kojoj je istraživanje sprovedeno. Zbog toga što su etnički principi fundamentalni za dobru istraživačku praksu, mnogi časopisi ne žele da publikuju članke koji ne uključuju detalje o etničkim odobrenjima (Materia Medica je prihvatila Principe dobre naučne prakse). Čitaoci žele da znaju na koji ste način uključili ljude u vašu studiju. Stoga, izbor učesnika mora biti jasno opisan i uključujući i isključujući detalji moraju biti opisani u sitnice. Prilikom opisivanja učesnika studije, njihova privatnost mora biti poštovana. Ne smete uključiti bilo kakve indentifikacione infomacije o njima, u tekstu, tabelama ili fotografijama. Ako se koristi fotografija, pismeni pristanak mora biti uzet od pacijenta ili ako su deca, od njihovih roditelja. Veličinu i karakteristike uzorka, ne stavljajte u poglavlje materijal i metode nego stavite na početak poglavlje rezultati. Mnoge istraživačke studije koriste upitnike pa u poglavlju metode morate dati precizne detalje o upitiniku, koje ste koristili, kako ste ga razvili, i testirali za ponovljivost. U eksperimentalnim studijama, detalji intervencija i kako su primenjeni moraju biti u potpunosti opisane. 5. Strana Rezultati Posle metoda, predstavlja najlakše poglavlje za pisanje. Možete koristiti interesantne kombinacije teksta, tabli i figura da odgovorite na pitanje studije u vidu jasne priče. Ovo poglavlje iz praktičnih razloga je poželjno pisati posle poglavlja metode, a pre pisanja uvoda i diskusije. Osnovno je da sopstvene rezultate učinite jasnim za čitaoca kako bi razumeli šta ste radili i dokle ste stigli. Ovo poglavlje mora voditi čitaova kroz proces istraživanja. Dužina ovog poglavlja je određena isključivo brojem rezultata koje želite da prikažete, a ne onim što vi želite da kažete o tome. Rezultate treba prikazivati postepeno. Prvo se prikazuju elementi deskriptivne statistike koja opisuje karakteristike uzorka studije. To je prvi paragraf poglavlja rezultati i njegov cilj je da precizno i jasno prikaže detalje vašeg uzorka. To je veoma važno, jer epidemolozi žele da znaju kako ste definisali karakteristike vašeg uzorka, a kliničari žele da znaju koliko su učesnici u vašoj studiji slični sa njihovim pacijentima. Po završetku statističke analize podaci i rezultati se mogu prikazati na tri načina: tekstualno, tabelama i figurama. Tekst – pojedine rezultate je bolje prikazati jednostavnim rečenicama sa podacima stavljenim u zagradu. Primer: srednja vrednost proliferativnog potencijala za PCNA (2.20%) je veća nego srednja vrednost za Ki67 P (1.64%) i Cyclin D1 (1.36%). 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 657 uputstvo za autore Tabele – predstavljaju popis brojeva ili teksta u rubrikama pri čemu je svaka rubrika obeležena. Tabele pored prikazivanja podataka na pregledan način omogućavaju i ekonomično raspologanje prostorom u članku. Ne treba ih koristiti da bi se pokazao način kretanja nekih rezultata (trend) ili veza između pojedinih rezultata i to je bolje prikazati figurama (dijagramima). Na primer ukoliko želite da prikažete veličinu uzorka i odnos polova vaših ispitanika bolje je da koristite tabelu. Međutim, ukoliko želite da prikažete način na koji je pol povezan sa uzorkom populacije onda je bolje koristiti dijagrame. Legenda tabele se stavlja ispod tabele, levo orjentisana. U mnogim eksperimentalnim i opservacionim studijama je neophodno da prikažete osnovo upoređivanje studijskih grupa koje takođe definišu sposobnost generalizacije vaših rezultata. Nikada ne nazovite osnovnu karakteristiku vašeg uzorka ,,demografskim“ jer shodno Oksfordskom rečniku, demografija je grana antropolologiju u kojoj se proučava statistika, rođenja, smrti i bolesti i stoga, to nije prikladno za ovaj kontekst. U bilo kojoj studiji, procenat, srednja vrednost i njena standardna devijacija ili medijana i njen rang su najprikladnije metode deskriptivne karakteristike i zavise od informacija koje opisuju. Figure – prikazivanje rezultata figurama podrazumeva korišćenje dijagrama, fotografija, šema, mapa i crtreža kako bi se na jasan i pregledan način prikazali rezultati dobijeni u istraživanju. Postoji više vrsta dijagrama (štapišasti dijagram (engl. bar chart), histogrami učestalosti (engl. histogram) , pogačasti dijagrami (engl. pie chart) , linijski dijagrami (engl. line graph), i grafikoni sa slikama (engl. pictograph) prilagođenih za opisivanje i prikazivanje različitih vrsta obeležja i rezultata. Sledeći paragraf poglavlja rezultati se odnosi na opisivanje bivarijantnih analiza. U trećem paragrafu se opisuju multivarijantne analize i to je mesto gde se završava cilj ili testiranje hipoteze, navedeno na kraju poglavlje uvod. Prilikom pisanja ovog paragrafa jedino je bitno da kažete čitaocu ono što on želi da zna. Nemojte dodavati ili uključivati bilo kakave podatke koji se udaljavalju od glavnog cilja. Podsećamo vas da rezultati i podaci nisu ista stvar, nije potrebno da ponavljate brojeve u tekstu koje ste prikazali u tabelama ili figurama. Čitaoci žele da prime poruku iz tabela ili figura i ne treba im dozvoliti da sami interpretiraju. 6. Strana Diskusija (1/3 vašeg teksta) Diskusija je vrlo često najslabiji deo članka. Pojedine stvari u poglavlju diskusija praktično NE SMETE uraditi: 1. ne ponavljajte činjenice iz uvoda 2. izbegavajte ponavljanje rezultata 3. ne prikazujte rezultate koje niste prikazali u poglavlju rezultati 4. ne postoji ni jedan razlog da podvlačite koliko je „sjajan“ vaš rezultat, dozvolite da čitaoci sami o tome prosude Diskusija ne predstavlja jednostavno ponavljanje rezultata ili potvrde njihove tačnosti. Svaka diskusija iznosi ono izvan očiglednosti (engl. beyond the evidence). Svaki članak sadrži zaključak koji se ne nalazi u poglavlju rezultati. Takođe svaki statistički značajan nalaz nema klinički značaj. Diskusiju bi trebalo započeti, po mogućstvu jednom rečenicom - ponavljanjem glavnog nalaza. 1. paragraf poglavlja diskusija se jednostavno može početi: „Naša studija pokazuje...” i izneti sažeto nalaz naše studije, po mogućstvu u jednoj rečenici. 2. paragraf - treba izneti jasno i precizno (praktično opširno) prednosti i nedostatke studije sa podjednakim naglaskom na oba elementa. Posebno treba imati na umu da će i urednici i čitaoci biti najzainteresovaniji baš za taj paragraf diskusije. Ukoliko urednik ili čitalac otkriju nedostatke u vašoj studiji, a vi ih niste opisali izgubiće poverenje u vašu studiju, jer praktično se postavlja pitanje: „Kolika je snaga vaše studije ako vi niste uočili nedostatak”. 3. paragraf se odnosi na studiju koja je izvedena. Neophodno je izneti doprinos studije. Ne treba iznositi da li je i u kojoj meri bolja od prethodnih studija na osnovu kvaliteta ili nedostataka koje ste izneli u prethodnom paragrafu, nego treba prednosti i nedostatke sopstvene studije uporediti sa prednostima i nedostacimma drugih studija. Vrlo je važno da naglasite zašto ste vi dobili drugačije rezultate od ostalih ukoliko ste ih dobili. Pažnja! U ovom trenutku postoji opasnost da uđete u sferu špekulacija. Ukoliko ne znate zašto se vaši rezultati razlikuju od drugih iznesite to i ne pretendujte da su vaši ispravni, a tuđi pogrešni. 658 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 uputstvo za autore U trećem paragrafu možete diskutovati saglasnost ili kontradiktornost vaših rezultata sa rezultatima drugih studija ili opšteprihvaćenih dogmi. 4. Paragraf Vi sada iznosite šta vaša studija “stvarno” znači. Objasnite jasno doprinos vaših rezultata kliničarima ili drugim čitaocima. Ali, vi ste i ovde na opasnoj zemlji. Mnogi urednici i čitaoci prihvataju vaše rezultate sa opravdanom opreznošću. To je zato što vaši rezultati tek publikovanjem ulaze u fazu kritičkog mišljenja naučne javnosti i budućeg prihvatanja ili odbacivanja. Dozvolite čitaocu da izgradi sopstveni stav o vašem istraživanju. Opišite novinu koju je doneo vaš nalaz u odnosu na rezultate drugih studija. Samo se početnici u pisanju trude da referišu sve članke koje su publikovali identičan aktuelni problem (engl. topic). Sugestije Ni jedan časopis nema dovoljno sredstava da štampa svaki detalj i duge članke. Neophodno je izabrati prikladan broj referenci koje će prikazati željeni uvid (najviše 30). Izbegavajte da citirate apstrakte, a nikada ne citirajte apstrakte starije od dve godine ukoliko nije publikovan originalan članak koji je predstavljen apstraktom na nekom kongresu ili sastanku. Možete sebi dozvoliti još jedan paragraf ukoliko želite da diskutujete o pojedinim pitanjima koja ostaju nejasna i da predložite npr. pravce dajih istraživanja. Na tome će neupadljivo uživati i urednik i čitaoci. Zašto? Urednik vidi potencijalnu mogućnost za nove članke a čitalac ideju za sopstvena istraživanja. Vi naravno ne morate iznositi stavove po pitanju novih istraživanja i vaša diskusija može biti bez tog dela. Praktično ne posedujete argumente za sigurnost onoga što predlažete i može biti domen špekulacija. Sada možete izneti jasan zaključak. Zaključak Kratko i sažeto iznesite potencijalni značaj vašeg nalaza i koliki je njegov doprinos dotadašnjem znanju. Pojedine stvari u zaključku praktično NE SMETE uraditi: 1. Ne iznosite zaključke koji nisu potkrepljeni rezultatima 2. Ne iznosite vaše mišljenje kako da se reši neki problem, a da ga pritom niste analizirali u svojoj studiji 3. Ne pišite pregled svih mogućih mehanizama ako ih niste uključuli u svoju studiju POSLEDNJA STRANA VAŠEG RADA Literatura Izvore za istraživačke ideje i sopstveni rad naučnici nalaze u originalnim naučnim člancima. Pravilno citiranje članaka je važno jer predstavlja sa jedne strane izvor informacija, a sa druge strane način izražavanja poštovanja prema naučniku/cima koji su autori originalnog dela i koji su ga prvi objavilu. Korišćenje ideja drugih istraživača ili bilo kog dela njihovog pisanja kao sopstvenog, čini se težak etički prekršaj poznat kao plagijarizam. Reference numerički navodite kroz članaki u vidu liste na kraju članka, koristeći Vancouver stil Pravila za citiranje originalnih naučnih članaka prema Vancouver stilu podrzumevaju da se autori navode prezimenima posle kojih slede inicijali imena, bez tačke i pojedini se autori odvajaju samo zarezima. Navodi se prvih 6 autora(ako ih ima), a posle toga sledi sentenca ,,at al“. Spisak autora se završava tačkom, pa sledi jedan prazan prostor i tada se piše naslov, koji se završava tačkom pa sledi jedan prazan prostor, pa potom ime časopisa prema previlima index medicus-a. Ne stavlja se tačka, već postoji samo jedan prazan prostor, pa se posle piše godina publikovanja članka, sledi tačka zarez, pa volumen, dvetačke, a potom broj strane i tačka. Broj sveske ili datum volumena nisu uključeni u citiranje. Sve detalje o pravilnom citiranju možete naći na web adresi: http://ncbi.nlm.nih.gov/books/bv.fcgi?rid=citmed.TOC&depth=2. I naravno posetite web adresu International Committee of Medical Journal Editors. Uniform requirements for manuscripts submitted to Biomedical Journals (http://www.icmje.org), za sva dodatna pitanja ILI SE OBRATITE REDAKCIJI. I na kraju to pošaljite na redakciju časopisa kbczpatologija@open.telekom.rs Obavestićemo vas o prispelom radu i proslediti ga u postupak recenziranja, a vama poslati da potpišete izjavu o sukobu interesa. Naši recenzenti će zajedno sa uredništvom pomoći da dođemo do publikovanog članka. 14. Kongres udruženja patologa i citologa Srbije sa međunarodnim učešćem, Beograd 14-16 juna, 2012 14 th Congress of Serbian Association of Pathologists and Cytologists with international participation, Belgrade 14-16 June, 2012 659
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