XXVII International Congress of the International ...

Poster Presentations

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XXVII International Congress of the International Academy of Pathology October 12–17, 2008, Athens, Greece POSTER PRESENTATIONS AUTOPSY PATHOLOGY 001 Histochemical and immunohistochemical characteristics of amyloid deposits ´ Apa´thy2 M Be´ly1, A 1 Policlinic of the Order of the Brothers of Saint John of God in Budapest, 2 National Institute of Rheumatology and Physiotherapy, Budapest, Hungary Background: The aim of this study was to define the histological, histochemical and immunohistochemical characteristics of various types of amyloid deposits (systemic AA, systemic AL-l, localized AL-l, and systemic b2-microglobulin amyloidosis (Ab2M). Design: The tissue specimens studied included colon and rectum with systemic AA amyloidosis; heart, bone marrow and periarticular soft tissues with systemic AL-l amyloidosis; epipharynx with localized AL-l amyloidosis; and synovial membrane of both hip joints with hemodialysis associated b2-microglobulin associated amyloidosis (Ab2M). Tissue blocks were fixed in 8% formaldehyde solution and embedded in paraffin. Serial sections were cut and stained with Hematoxylin and Eosin or Congo red according to Romhańyi (1971), without alcoholic differentiation, and sealed with gum Arabic. The amyloid deposits were determined and characterized histochemically by Congo red staining after performate pretreatment according to Romhańyi (1979), or by KMnO4 oxidation according to Wright (1977) and viewed under polarized light. The disintegration of amyloid deposits was analyzed according to Be´ly and Apa´thy (1998), Be´ly (2006). The classical histochemical results were confirmed by immunohistochemical staining. Results: All types of amyloid deposits were eosinophilic, congophilic and birefringent, showing specific apple green color under polarized light. There was no basic histological difference between the systemic AA and systemic AL amyloidosis; both types of amyloid were deposited within the wall of the blood vessels and along the collagen and reticulum fibers of the tissues. The immunohistochemical staining for amyloid A-component was specific. The evaluation of anti-human immunoglobulin light chains was less than satisfactory because of the strong background staining. The localized AL amyloid deposits (with, or without proliferation of undifferentiated immunoblasts and plasmablasts) were never found within the vessel walls; the amyloid deposits were exclusively localized to extravascular tissues. Under polarized light the localized AL deposits exhibited in some areas extremely intensive ‘crystalloid-like’ accumulations of amyloid filaments. The synovial Ab2M deposits were eosinophilic, congophilic and globular in shape. Under polarized light a superficial (peripheral) ring of birefringence was noticed, which showed a ring-like positive staining with a-hu beta-2-microglobulin. Conclusions: The production of amyloid precursors related to the cardiovascular system becomes generalized via the bloodstream, resulting systemic forms of amyloidosis. The local production of immunoglobulin light chains is not directly related to the systemic circulation and remains a localized process with organ- or tissue-

limited isolated amyloidosis. The (2-microglobulin containing amyloid deposits are different from systemic AA, systemic AL, or localized AL deposits histologically. Moreover, their appearance under polarized light is noticeably different.

002 Regional (mediastinal) AA amyloidosis associated with bronchoalveolar carcinoma in rheumatoid arthritis ´ Apa´thy2 M Be´ly1, A 1 Policlinic of the Order of the Brothers of Saint John of God in Budapest, 2 National Institute of Rheumatology and Physiotherapy, Budapest, Hungary Background: Systemic AA amyloidosis should be regarded as one of the most insidious complications of rheumatoid arthritis (RA). This study presents a rheumatoid arthritis patient with regional AA amyloidosis limited to the lower mediastinum of the pleuro-pericardial region. Design: RA was diagnosed clinically according to the criteria of the American College of Rheumatology (ACR). AA amyloidosis was diagnosed histologically. Results: Inspection of the interphalangeal, metacarpo-, metatarsophalangeal joints and knee joints confirmed the clinical diagnosis of RA at autopsy. Different (acute, subacute, and chronic, fibrous) stages of interstitial pneumonitis supported the diagnosis of RA. Microscopic examination of the lung revealed a small nodule of peripheral bronchoalveolar carcinoma, 20–35 mm in diameter, with bronchopneumonia and microabscesses in the adjacent lung parenchyma. There was fibrinous pericarditis without hemorrhage; macroscopically it was considerd to be a part of polyserositis complicating RA. Microscopically the pericarditis proved to be carcinomatous, characterized by sporadic, scattered tumor cells within the inflamed epicardium; a single distinct circumscribed metastasis of bronchoalveolar type was found histologically. No other gross or microscopic evidence of metastases was found. Atherosclerosis was pronounced only in the lower portion of the abdominal aorta, with sclerotic and markedly narrowed iliac arteries, which led to the gangrene of booth feet. The chronic duodenal ulcer and periduodenal adhesions were confirmed at autopsy, without direct causal role in death. The retrospective detailed analysis of tissue samples revealed a regional deposition of amyloid A protein, exclusively localized to the pleuro-pericardial region of the lower mediastinum. No other organs or regions were involved. The amyloid deposits were positive for anti-human (a-hu) amyloid A component, and positive for a-hu amyloid P-component. (Negative for a-hu k-, or j-light chain, and negative for a-hu beta-2-microglobulin). Conclusions: RA was associated with a small peripheral bronchoalveolar carcinoma and carcinomatous pericarditis. The carcinoma was not recognized clinically. The patient died of cardiorespiratory

2008 The Authors. Journal Compilation 2008 Blackwell Publishing Ltd, Histopathology, 53 (Suppl. 1), 1–432

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insufficiency due to pericarditis in combination with bronchopneumonia, which was regarded as the direct cause of death at autopsy. RA was complicated by regional AA amyloidosis localized to the mediastinum. Regional AA amyloid deposit is rare and unusual complication of RA. (Systemic AA amyloidosis was excluded by detailed histological examination of tissue samples of the GI tract, heart, kidney, spleen, liver and the adrenal gland, lung, pancreas, thyroid gland, aorta, skeletal muscle, synovial membrane, lymph nodes, peripheral nerves, bones, skin, brain, etc). The precursor of amyloid A deposits of the tissues is the serum amyloid A (SAA) produced by the liver. SAA proteins spread via the blood stream and deposit throughout the body. Systemic AA amyloidosis starts – according to our previous study – in the vessel walls of the gastrointestinal (GI) tract (1). Other organs like lungs, pancreas, thyroid gland, aorta, skeletal muscle, etc. are involved later and less frequently, in these organs amyloid A deposition starts later. The unusual mediastinal localization of AA amyloid deposits can be explained by local accumulation of amyloid A, due to special circumstances caused by abnormal circulation, pleuro-pericardial adhesions and its limited degradation by macrophages.

003 Lymphoid interstitial pneumonia of the lung – a retrospective clinicopathologic study of 234 autopsy patients M Be´ly1, A Apa´thy2 1 Policlinic of the Order of the Brothers of Saint John of God in Budapest, 2 National Institute of Rheumatology and Physiotherapy, Budapest, Hungary Background: Lymphocytic interstitial pneumonia of the lung is a well known entity. It is characterized by an infiltrate of mononuclear cells, mostly mature lymphocytes, restricted to the interstitium. The mononuclear cells mainly infiltrate the alveolar walls, with or without involvement of the peribronchial areas. Lymphocytic interstitial pneumonia may progress to an end stage lung disease (characterized by non-specific interstitial fibrosis), a usually indolent condition that gradually progresses to respiratory failure. The aim of this study was to determine: (i) the prevalence of lethal lymphocytic interstitial pneumonia (LIP) in rheumatoid arthritis (RA); and (ii) its histological and clinical characteristics. Design: A randomized (non-selected) autopsy population of 234 in-patients with RA was studied. RA was confirmed clinically according to the criteria of the American College of Rheumatology. Causes of death do to associated diseases, or complications of RA were histologically diagnosed post mortem. The retrospective analysis of clinical complaints was based on clinical records. Results: (i) Lethal LIP was observed in three (1.28 %) of 234 RA patients; (ii) Histologically the lungs in two of three patients showed scattered irregular foci of lobular and/or sublobular pneumonia, which was not centered on terminal and respiratory bronchioles. Disseminated, peribronchiolar accentuated lymphoid infiltration with secondary follicles was noted. The bronchioles, and terminal bronchioles were compressed, narrowed, and occluded. In some areas this was accompanied by intraluminal epithelial cell proliferation, or desquamation. Lobular-sublobular atelectasis developed, with or without inflammation, respecting the anatomical bor-

ders. In one of three patients a more diffuse interstitial lymphoid infiltration was present, without peribronchial nodular accentuated lymphocytic infiltration and without secondary follicles. Prominent lobular-sublobular atelectasis and inflammation, respecting the anatomical borders, did not complicate this form of LIP. Conclusions: Lymphocytic interstitial pneumonia (sometimes also called as obliterative bronchiolitis and confused with the organizing pathological processes) is a rare form of bronchial obstruction complicating RA. LIP may be associated with Sjo¨gren’s syndrome, AIDS, or may be accompanying heart-lung transplantation. It can be a complication of rheumatoid arthritis, too. There is a possible relationship between penicillamine, auranofin, bucillamine, sulfasalazine, or cyclooxigenase-2 inhibitor celecoxcib treatment. In some cases the interstitial lymphocytic infiltration is less pronounced (or absent), and the inflammation is predominantly peribronchial, with or without secondary lymphoid follicles. The small bronchioles may be compressed, narrowed and/or obliterated, followed by lobular atelectasis. In the case of secondary infection of the atelectatic area, multifocal, lobular or sublobular inflammation occurs. Antibiotics, bronchodilators, or steroids bring no relief, and there is no response to standard antibiotic treatment, but reportedly patients have recovered with high doses of oral corticosteroids. Conclusions: Lobular-sublobular pneumonia caused by LIP, (i) shows nodular lymphoid hyperplasia; (ii) has no hemorrhagic character; (iii) is not accompanied by thrombembolism or vasculitis. It may be distinguished from bronchopneumonia, because the inflammatory foci are smaller, and the inflammation respects definite anatomic borders. The lobular-sublobular infarction pneumonia is hemorrhagic in character, and thrombembolism can be found. LIP may be distinguished from the vasculitic, lobular-sublobular pneumonia too, because in case of focal pneumonia due to LIP there is no vasculitis.

004 An analysis of virchow’s cellular pathology 150 years on L Bignold, B Coghlan, H Jersmann University of Adelaide, Adelaide, Australia At the beginning of the nineteenth century, ‘theory’ in pathology consisted of various widely differing speculative ‘systems’ based mainly on (i) disturbed local nutrition (Descartes and followers), (ii) altered local ‘irritability’/‘stimulability’ (von Haller, Gaub, Broussais), (iii) altered overall control of the body by the nervous system (Hoffmann, Cullen) and (iv) defective constituents of the blood constituents (or ‘balances’ of them, i.e. ‘dyscrasias’; Rokitansky). With few exceptions, medical opinion discounted ‘contagious’ theories (i.e. ideas of self-replicating living microorganisms causing disease). Rudolf Virchow (1821–1902) trained in the same department as Schwann, who had described the ‘Cell Theory’ in 1839. Virchow wanted to develop a ‘system of pathology’ incorporating Cell Theory which would replace all other theories. By the early 1850s, Virchow had shown that the various connective tissue cell types can metamorphose into each other, and he believed that connective tissue cells can also change into epithelial cells, blood cells and cells of all pathological types. In 1855, Virchow adopted the idea that all cells come from other cells (denied by Schwann, but supported



by Raspail, Remak and others), and suggested that all diseases are reducible to disturbances of cells. However Virchow still could not explain how external agents might cause disease. In 1858, he incorporated ideas of ‘irritation theory’ from Gaub and Broussais into his system to create the ‘Cellular Pathology’. The essential idea was that all disease involves local irritation of pre-existing connective tissue cells so that they produce other cell types by ‘histological substitutions’/metamorphoses – all steps being only alterations of normal cell physiological processes. The key to analysing ‘Cellular Pathology’ (1858) is that it was based on lectures which were designed to convince post-graduate doctors (most of whom may have heard little of ‘Cell Theory’) that what they had previously learned of disease – i.e. ‘nutritional’, ‘irritability’ and ‘blood-related’ theories – should be replaced with a system of pathology based on the behavior of cells at the site of lesions. The long sections in ‘Cellular Pathology’ on basic histology, the blood, ‘nutrition’ and the nervous system were designed to show his audience that the phenomena previously attributed to abnormalities of those systems could be better explained in terms of cells and their behavior. The book was successful because it was correct in this major principle. Nevertheless, within 30 years, the ‘connective tissue’, ‘irritation’ and ‘abnormal physiology only’ concepts were found to be false, and micro-organism-caused diseases - which Virchow had not believed - were found to be true. Thus the fourth edition (1870) of ‘Cellular Pathology’ was the last. One hundred and fifty years on, Virchow’s ‘Cellular Pathology’ should be recognized as the beginning of modern pathology by contrasting it with medical theories of the early nineteenth century, and not judged by any shortcomings which were only discovered in the late nineteenth century, when its main message had been fully absorbed.

005 David Paul Hansemann and the role of chromosomes in cancer L Bignold, B Coghlan, H Jersmann University of Adelaide, Adelaide, Australia Chromosomes were described, named and identified as the basis of the cellular hereditary material in the 1870s–80s. Abnormal including asymmetric mitoses were discovered in tumour cells at almost the same time. In the same period Weismann’s theories of ‘plasmas’ and ‘differentiation by chromosomal loss’ were widely believed. Also, early studies of chromosomes had identified loss of chromosomes (as ‘polar bodies’) in oogenesis. In 1890, David Paul Hansemann (1858–1920), was an Assistent to Rudolf Virchow (1821–1902) in Berlin and noted asymmetric mitoses in cancer cells. Hansemann recognised the paramount features of tumours as loss of tissue differentiationi and increased capacity for independent existence (i.e. ‘‘autonomy’’ ability to grow in remote tissues and form metastases). Probably because Virchow insisted that tumour formation must involve only an abnormality of a physiological tissue process (not a new process), Hansemann looked for a cell process which might be a counterpart of this particular combination of changes i.e. a normal cell process in which changes in chromosomal content, reduction of differentiation and greater autonomy all occurred. Hansemann proposed that oogenesis was the prototype process because (i) the egg comes about by reduction divisions (ii)

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it is less differentiated than ovarian epithelial cells (in fact, it is ‘dedifferentiated’) and (iii) the egg can survive for days free in the endometrial cavity. Hansemann called the normal process anaplasia, and proposed that it could occur to variable degrees in different cases of tumour according to the degrees of chromosome imbalance in the tumour cells. Unfortunately for Hansemann’s theory, within ten years, Weismann’s ‘chromosome loss theory’ was found to be incorrect, and ejection of polar bodies was found to be a very specific phenomenon associated with the formation of (hapoid) ova. However, for practising pathologists, Hansemann’s ideas provided useful terminology. This was because previous classifications had divided tumours into only two types, for example homologous vs heterologous or homeoplastic vs heteroplastic without offering words to describe variations of abnormalities. Hansemann’s terminology provided a way of describing the continuous variation of abnormalities which tumours exhibit. Thus anaplasia and (de) differentiation became popular and have been used to describe tumours ever since, even though the original cell biological ideas on which they were founded are no longer believed. In the early twentieth century, the directions of cancer research moved towards investigating Mendelian genetics in relation to tumours, and the mechanisms of action of viral, physical and chemical carcinogens. Only in the last 30 or so years, have the roles of chromosomal abnormalities in tumour formation which were first considered in detail by Hansemann – again received significant attention.

006 Dengue hemorragic fever in brazil: histopathological findings in two fatal cases R Dos Santos1, V Vendramini1, N Mattos2, C Kanamura3, L Viegas de Carvalho1 1 School of Medicine–Lusiada Foundation, Santos, Brazil, 2Guilherme Alvaro General Hospital, Santos, Brazil, 3Adolfo Lutz Institute, Saõ Paulo, Brazil Background: Dengue fever (DF) is a mosquito-borne infection which in recent years has become a major international public health concern. In the Americas, Aedes aegypti breeds primarily in concrete cisterns used for domestic water storage, as well as discarded plastic food container, used automobile tires and other items that collect rainwater. Dengue hemorrhagic fever (DHF) is a potentially fatal manifestation and mainly occurs in children and young adults. Because of the social and economic costs of DHF, many countries in Asia and South America have initiated public health measures aimed at vector control. Despite these measures, DHF incidence rates do not appear to be declining. A total of 4 243 049 dengue cases have been reported in Brazil between 1981 and 2006, including 5817 cases of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) and a total of 338 fatal cases. In 2007, 438 949 cases of DF and 926 DHF were registered with 98 lethal cases. Although all Brazilian regions have been affected, the Northeast and Southeast regions have registered the highest number of notifications. Design: We studied the pathological features of two fatal cases of DHF occurred in Praia Grande – Baixada Santista in Sao Paulo state of Brazil: Case 1: A 18-year old white girl, presented with an


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acute onset of fever, jaundice, lethargy and respiratory distress. Liver injury was detected by laboratory investigation. Tissue fragments (liver, brain, spleen, lung, heart, kidney and lymph nodes) were collected for light microscopy studies and stained by standard methods. Histopathological study revealed a fulminant hepatitis and severe tissue damage in lung and spleen caused by intense hemorrhage, interstitial edema and inflammation. Case 2: A white boy of 12 years presented an acute onset of fever, abdominal pain, vomiting and diarrhea, dehydration and heart failure. Gross examination has showed subendocardial, gastric and intestinal hemorrhage, liver and spleen enlargement. On microscopy, multiple hemorrhagic foci were observed, congestion and interstitial edema. Surprisingly, sickle red cells were found on histology and the electrophoresis studies of his hemoglobin showed the present of AS hemoglobinopathy (heterozygote HbAS). In both cases the serological tests for hepatitis and yellow fever viruses were negative and the MAC-ELISA for dengue was positive. Some tissue sections were also processed with the immunoperoxidase reaction, which revealed the dengue viral antigen. Conclusions: In Brazil, dengue fever is a nationwide public health problem. We have described the pathological findings in two lethal cases of DHF occurred in Sao Paulo state (Southeast region) that were confirmed on autopsy and we also have discussed the association between dengue fever and hemolytic anemias.

007 Precautions during post-mortem examination of Creutzfeld– Jacobs disease patients N Goutas, E Sakelliadis, D Vlachodimitropoulos, S Papadodima, C Spiliopoulou Department Forensic Medicine and Toxicology, Medical Faculty, University of Athens, Athens, Greece Background: To describe all necessary preventive measures for a ‘lege-artis’ post-mortem examination of Creutzfeld–Jacobs Disease (CJD) patients. Design: Review of the literature and of the experience gathered from the cases referred to the Department of Forensic Medicine & Toxicology of the University of Athens. Results: Post mortem examinations remain an essential element in confirming the clinical diagnosis and the cause of death as TSE. Ideally, three people should be present during the examination: the pathologist assisted by one technician, and one further person to handle and label specimen containers. Except for training purposes, observers should be prohibited or kept to a minimum. All personnel should be made aware of the relevant history of the patient and fully informed of procedures for such post mortem examinations. To the extent possible, disposable protective clothing should be worn including surgical cap and gown, apron, double gloves, and a face visor which completely encloses the operator’s head to protect the eyes, nose and mouth. Consideration should be given to the use of hand protection, such as armored or cut-resistant gloves. Disposable or dedicated re-useable instruments are recommended in order to minimize the risk of environmental contamination. Manual saws are recommended in order to avoid the creation of tissue particulates and aerosols and for ease of decontamination after use.

Conclusions: Restricted post mortem examinations on TSE cases can be undertaken in any mortuary. A full post mortem examination is discouraged except in dedicated facilities, unless special circumstances warrant the added difficulty of infectivity containment.

008 Right ventricular arrhythmogenic dyplasia (ARVD) in a patient with diagnosed myotonic dystrophy N Goutas, S Sakelliadis, D Vlachodimitropoulos, S Papadodima, C Spiliopoulou Department Forensic Medicine and Toxicology, Medical Faculty, University of Athens, Athens, Greece Background: Our case is a 59 years old woman referred to the Department of Forensic Medicine & Toxicology of the Medical Faculty of the University of Athens, for medico-legal investigation. Design: The findings from the autopsy, from the histopathological examination, the review of international literature were employed. Results: Right ventricular arrhythmogenic dysplasia is a rare disease, that is Histologically is characterized by progressive replacement of normal myocardial cells by fibrous and adipose tissue. The anamnesis of the deceased included bradyarrhythmia, syncope, pacemaking and last but not least, myotonic dystrophy. The deceased was consecutively transferred to three hospitals in bad clinical condition, because of thrombosis of the superior vena cava. Macroscopically it was not possible to ascertain the cause of death, so tissue samples were collected for histopathological examination. The histological examination of the heart revealed focally up to intense intermediate edema with fibrous positions and islets of adipose tissue. The myocardial fibers presented variable size enlargement with large hyperchromatic nuclei. In the intermediate tissue, diffuse lymphocytic infiltration was observed. Especially in the right ventricular wall an icreased infiltration by adipose tissue, with focal fibrosis, was detected. This finding is pathognomonic of right ventricular arrhythmogenic dysplasia. Conclusions: The replacement of normal myocardial tissue by fibroadipose tissue is a serious predisposing factor for electrical instability and malignant ventricular arrhythmias. In a patient with an already bad health condition, such as our case, the burden of right ventricular arrhythmogenic dysplasia was too heavy to handle. Indeed every clinician should bear under consideration of the differential diagnosis the possibility of right ventricular arrhythmogenic dysplasia.

009 Trends in infant and child mortality N Goutas, T Konstantinidou, M Konstantinidou, D Vlachodimitropoulos, E Sakelliadis, S Papadodima, T Kontogiannis, C Spiliopoulou Department Forensic Medicine and Toxicology, Medical Faculty, University of Athens, Athens, Greece Background: Worldwide, both infant and child mortality rates tend to decline over the last decades. Specifically, between 1980 and



2003, death rates dropped by 46% for infants, 51% for ages 1–4, 44% for children ages 5–14 and 32% for teens ages 15–19. However, infant mortality rate remained ten times higher in the Less Developed Countries (LDC) compared with that of the More Developed Countries (MDC), as estimated in 2001. Design: Review of the literature and our experience on the field. Results: Infant’s main causes of death include congenital anomalies, SIDS, prematurity and low birth weight. They can also be attributed to respiratory, circulatory, neoplastic and other reasons that are less likely to occur in those ages under normal circumstances. SIDS still accounts for about 25% of all deaths between 1 month and 1 year of age in MDCs, although its rates have dropped to half of the last decade, a fact that is partly attributed to the ‘back to sleep’ campaign. Concerning children’s mortality, while neonatal disorders, diarrhea, pneumonia, and malaria, as well as malnutrition and underweight account for most of the children deaths around the world, children’s health discussions in Europe and the USA focus on issues such as asthma, neurodevelopmental disorders, male genital malformations and childhood cancer. According to autopsies, children’s causes of death, in previously healthy or nearly healthy children, occurring in a children’s hospital are primarily infections (53%), neoplasias (15%) and previously unrecognized congenital disorders (10%). Diseases of the nervous and cardiovascular systems, account for one third of the sudden, unexpected and non violent deaths of children aged 1–19 years. Last but not least, accidental injuries, child abuse, homicides and teenage suicides increase the mortality rates, especially among young children and teenagers, depending on geographical and social parameters. Conclusions: In any case, autopsy provides valuable information on infant and children deaths, leading to a substantially contributing, causal determination.

010 Anthrax epidemic Sverdlovsk 1979: questions and answers L Grinberg Ural State Medical Academy, Yekaterinaburg, Russia Background: At the entry of Sverdlovsk (now Yekaterinburg) tragedy 30th anniversary some finalization of the largest in the modern history human inhalation Anthrax epidemic long-term studies is advisable. Obviously not all questions can be answered, as most of the studies are not based on the real material, authors are operating conjectures and sometimes intentionally misrepresent available information. Only the series of morphologic, epidemiologic and special (material from paraphine blocks PCR) investigations are based on the real samples and data. Design: Try to give accurate answers to some questions on ‘‘Sverdlovsk-1979’’ phenomena. Results: It confidently was the Anthrax epidemic, verified by many investigations including studies of Bacillus anthracis (BA) DNA isolation from paraphine blocks when pathogen several genetic variants antigens were found, confirming artificial infect genesis hypothesis. 1. It’s not known for certain how many people were infected by Anthrax. The number of deaths in some publications is estimated by thousands that is obviously not true. Experts consider the

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number of 64 mortal cases, based on pathology data [42 cases are included in our study (F. Abramova, L. Grinberg, 1992–1993)]. The number can be slightly increased by deaths during the first week of the epidemic when centralized autopsy system wasn’t organized yet. But the difference is unlikely more than 10 cases. We positively can confirm that general number of deaths is not more than 100 people. 2. Considered evident that an accident at the military object, where BA culture was studied, resulted in BA spores aerosol throw out. There are descriptions of the event itself, but there is no official approval of the fact. In publications since 2001 officials mention so called «meat version» and suggest the version of terrorist attack held to defame the military object. 3. Date of aerosol throw out: April 2, 1979 – evident by classical investigation of M. Mezelson group of experts (1993). However in some sources March 30, 1979 is mentioned when the wind rose was similar. 4. Our study used the concept of one-moment infection. In this case incubation can be prolonged to 8 weeks that was confirmed by previous experimental studies. But the concept does not consider the possibility of secondary inhalation of BA spores raised from earth by wind during disinfection. 5. In 41 cases from 42 autopsies (in collaboration in further studies with D. Smith & D. Walker, 2001) signs of inhalation Anthrax were found: hemorrhagic-necrotic intrathorax lymph-nodes lymphadenitis, hemorrhagic mediastenitis, bilateral hemorrhagic pleurisy. Large-seat BA pneumonia found in 11 cases with shown development stages. Extrathoracal changes: the prevalence of GI tract involvement (diffuse, seat and diffuse-seat gastroenterocolitis and mesadenitis) and hemorrhagic meningoencephalitis was found. Shown that after 24 h of intensive antibacterial therapy BA in tissues of died can’t be found by routine methods. Major role in tanathogenesis belongs to septic shock, meningoencephalitis and lung damage with bilateral pleurisy. Conclusions: We have mentioned only some questions of exceptional meaning considering special BA role in contemporary world actual bioterrorism problem.

011 Age estumation of dental attrition in mummies SY Ha1,4, HK Kim2,4, KH Jeong2, CL Yoon3 1 Department of Pathology, Gachon University Gil Medical Center, 2 Korea University, 3Institute of Forencic Odontology, College of Dentistry, Chosun University, 4Korea Lung Tissue Bank, Korea Background: Because of the rarity of the mummy in Korea and the difficulty in obtaining samples from the mummy, studies to determine the mummies’ ages are uncommon especially in Korea. This study was performed to determine ages of the mummies using the information obtained by nondestructive methods to minimize damages on mummies. Design: Three mummies excavation between 2002 and 2004 and were kept at Korea University were used. Three-dimensional reconstructed images of total teeth were obtained from images by CT scanning. Age at death was determined according to ‘Age Estima-


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tion Table of Dental Attrition’ by Takei. For comparison, three teeth extracted from each of three mummies were examined. Results: The ages at death of three mummies were 23.57 year (Yoon mummy), 51.01 year (Bong mummy), and 64.45 year (Black mummy). These results are similar to the ages determined by Gustafson method. Conclusions: Age determination method using CT scan and the three-dimensional reconstruction may be a valuable method because it minimizes the damages on valuable mummies and it gives a reliable data similar to that obtained by other standard method.

012 Nephrogenic systemic fibrosis: an autopsy case series A Koreishi, R Mandal, R Nazarian, A Saenz, V Klepeis, A McDonald Massachusetts General Hospital, Boston, USA Background: Nephrogenic systemic fibrosis (NSF) is a rare but serious disorder initially described as a purely dermatologic process and recently linked to patients with renal dysfunction. While no definite causality has yet been established, there remains a strong epidemiological link to a history of gadolinium-based contrast administration. Clinically, the disease manifests as skin hardening, hyperpigmentation, thickening and tethering to the underlying fascia, with rapid progression to debilitating joint contractures, pain and death. Isolated autopsy reports have described multiorgan involvement by this disease. We describe our autopsy experience of patients with NSF encountered at Massachusetts General Hospital with emphasis on history of renal dysfunction, gadolinium exposure, specific laboratory parameters, and extent of systemic involvement identified by postmortem examination. Design: We searched the autopsy records of the Department of Pathology at the Massachusetts General Hospital from 2004–2008 for the diagnostic terms ‘nephrogenic systemic fibrosis’ and ‘nephrogenic fibrosing dermopathy.’ We found six patients with the appropriate diagnosis and with ample material for analysis. We obtained clinical information from the patient’s electronic medical record, which included: age, gender, significant past medical history including history of renal disease and dialysis, history of exposure to gadolinium contrast (measured in days) and lifetime doses, BUN levels, creatinine levels, calcium levels, phosphate levels, parathyroid hormone (PTH) levels, and thyroid stimulating hormone (TSH) levels on final hospital admission. Results: Gadolinium dose and time of gadolinium exposure did not correlate with clinical findings (indicated by the laboratory parameters BUN, creatinine, calcium, phosphate, PTH, and TSH, and extent of non-cutaneous organ involvement by NSF) and outcome (measured in days from time of exposure to gadolinium to date of death). All patients showed cutaneous manifestations of the disease, with some developing calcification and fibrosis at various sites, including dura mater, thyroid, heart, and lungs. Patients with histologic calcification had concurrent hyperparathyroidism or high serum PTH levels. Conclusions: This case series underscores the systemic manifestations of NSF, which can lead to significant morbidity and mortality. Further work is necessary to understand the underlying pathogenesis, especially with respect to gadolinium exposure, and for the development of effective treatment strategies.

013 Pulmonary embolism among hospitalized patients: our 10-year-autopsy experience K A Kovacs 2nd Department of Pathology, Semmelweis University, Budapest, Hungary Background: Pulmonary embolism is one of the most frequent complications among hospitalized patients. The aim of this study was: (i) to carry out a detailed clinico-pathological analysis of all cases with postmortem diagnosed pulmonary embolism during the period of 1997–2006; (ii) to identify the main risk factors of pulmonary embolism by comparison of two cohorts including the cases with and without pulmonary embolism; and (iii) to examine the correlation between the clinical and autopsy findings regarding the diagnosis of pulmonary embolism. Design: All postmortem reports with diagnosis of pulmonary embolism from our digitalized database were selected. The control group included the cases of the last 3 years without pulmonary embolism. Clinical and pathological data of the two cohorts were transformed to excel file. Chi squared test was used for the statistical calculations. Results: During the 10-year period 4297 autopsies were performed. Pulmonary embolism was found in 522 cases (12.1%) and was recorded as the cause of death in 275 cases (6%). Autopsy identified the origin of pulmonary embolism in 60% of all cases, the most predilected area was the deep veins of the lower extremities. Compared with the control group, patients with pulmonary embolism were older with longer durations of hospitalization. The most significant differences were found in the ratio of the >80-year-old patients and those who were hospitalized for >20-day-long duration. Regarding the underlying diseases, the ratio of malignant tumor and stroke were significantly higher in cases with pulmonary embolism. The proportion of false negative and false positive clinical diagnoses was 81% and 71%, respectively regarding all cases, and 75% and 64% respectively regarding only the fatal pulmonary embolism. Conclusions: Age, duration of hospitalization and type of underlying disease are the most important risk factors of pulmonary embolism. Affected patients typically have an advanced disease. The accuracy of the clinical diagnosis is very low, although pulmonary embolism is one of the most common causes of unexpected events among hospitalized patients.

014 Co-existent lymphoma with tuberculosis and kaposi’ sarcoma with tuberculosis occurring in lymph node in patient with aids: report of two cases D Lanjewar Sir J. J. Hospital Grant Medical College, Mumbai, India Background: Although there have been few reports of simultaneous infections and neoplasm in patients with HIV infection, no reports of co-existent tumor and infection occurring in the same lymph node have been described. In this report we describe two cases of HIV infection, which showed co-existent lymphoma with tuberculosis in one case and Kaposi’s sarcoma with tuberculosis in another case.



Design: Paraffin fixed Formalin fixed and paraffin embedded tissue of lymph node obtained at postmortem examination is studied by H&E and Ziehl Nielsen’s stain. Case Reports: Case 1: A 40 year old HIV infected male developed cutaneous lesions of Kaposi’s sarcoma. In the past patient was diagnosed as tuberculous psoas abscess for which he was treated however, there was no response to treatment. Subsequently his clinical condition deteriorated due to which he died. At post mortem examination lymph node showed Kaposi’s sarcoma and large areas of necrosis which on special stain showed numerous tubercle bacilli. Case 2: A 42 year HIV infected male was diagnosed as anaplastic large B cell lymphoma. In the past, patient was diagnosed as a case of pulmonary tuberculosis. Subsequently his clinical condition deteriorated and he died. Autopsy findings showed enlarged lymph nodes in a cervical, axillary, inguinal and para aortic region. Microscopic examination of the lymph node showed lymphoma and necrotic areas containing numerous tubercle bacilli. Conclusions: Reports of co-existing Lymphoma with tuberculosis and Kaposi’s sarcoma with tuberculosis are rare and to the best of our knowledge are not described in the literature. Co-existent lesions pose problems of diagnostic difficulties particularly in FNAC, when aspirate from different areas of enlarged lymph node is not obtained and if routine staining for microorganisms is not carried out. Co-existing lesions are also likely to be misdiagnosed even in biopsy material if special stains for demonstration of microorganisms are not performed. Hence FNAC/biopsy material obtained from HIV infected patients should be studied by battery of special stains for demonstration of microorganism. Accurate diagnoses of co-existing lesions as described herein have implication on therapeutic management of patients with HIV/AIDS.

015 Leukemic children dying in remission. An autopsy study of 48 cases E Lopez-Corella, C Ridaura-Sanz National Institute of Pediatrics, Mexico City, Mexico Background: Advances in management of leukemia have resulted in a significant proportion of patients attaining remission. These patients are potentially cured of their disease. Nevertheless this is a labile population which for a variable length of time is prone to a diversity of complications aside from the possibility of reappearance of the original disease, a leukemic relapse. We reviewed our experience with leukemic children dying in remission and in whom an autopsy was performed. This was done with two questions in mind: (i) Why do these children, cured of a deadly disease, die?; and (ii) Are these patients really in remission? Design: We reviewed 48 autopsies performed on children dying in leukemic remission from a population of 6460 autopsies performed over 30 years. Results: Our case material of 6460 autopsies included 257 patients with leukemia, 48 of these were in hematological remission. Forty two were acute lymphoid leukemias and six were acute myeloblastic leukemias. The primary disease process was infection in 35 patients and in five additional cases, there was a combination of infection and hemorrhage. In three cases multifo-

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cal hemorrhage was considered the cause of death, effects of chemotherapy was determinant in three, massive leukemic infiltrate in the brain was present in one patient and no identifiable cause of death was found in one. Infection was mainly bacterial in 20 cases, fungi or yeasts were determinant in 11, virus infection in seven and parasites in two. Viral infections included varicella (three), measles (two) and hepatitis (two). Yeasts, fungi and parasites included Candida (six), Pneumocystis (two), Zygomycetes (Mucor) (two), Aspergillus (one), Cysticercus (one), Balantidium (one) and Strongiloides (one). Post mortem examination disclosed leukemic activity in seven patients. Sites of leukemic infiltration were the brain in one case and testis in another. In the remaining five cases, infiltrates were focal, involving the bone marrow in two patients. Conclusions: Autopsy reveals clinically undetected leukemic activity in 7/48 patients considered in remission. This may represent and unidentified relapse or an incomplete remission. Hemorrhage is not an important determinant of death; this contrasts with leukemic patients in initial activity or in induction in whom hemorrhage plays a prominent role. Infection was a major determinant of demise and the etiology was mainly bacterial. Infection with acknowledged opportunists such as Cytomegalovirus, Pneumocystis and Histoplasma, frequently encountered in our immunodeficient or immunosuppressed patients, is absent or infrequent in this population. These patients form a group with different susceptibilities to infectious agents from those associated with other immunocompromised states.

016 Secondary amyloidosis caused by x-linked agammaglobulinemia of bruton K Magdolna1, K Margit1, T Andra´s2 1 2nd department of Pathology, Semmelweis University, Budapest, Hungary, 21st department of Medicine, Semmeiweis University, Budapest, Hungary Background: Bruton disease is one of the most common forms of primary immundeficiency, which is usually recognized early in childhood. In our case X-linked agammaglobulinemia was diagnosed in an adult by nephrotic syndrome indicated kidney biopsy in which secondary amyloidosis was revealed. Design: The case history of the 49-year-old male patient revealed several, recurrent pneumonia in his childhood, hypogammaglobulinemia and gluten sensitive enteropathy. The patient was symptom-free and capable of work under a gluten-free diet. Later he was treated for pleuropneumonia and purulent bronchitis and then a kidney biopsy was performed because of high level of serum kreatinin and nephrotic syndrome. Secondary amyloidosis was diagnosed. Jejunal biopsy was also performed, subtotal villus atrophy and amyloidosis in the small vessels of jejunum were also revealed. Inspite of antibiotic therapy high fever, hypotonia, black-outs developed due to pseudomonas infection. A cardiogenic shock then led to death. Results: Congenital agammaglobulinemia associated with generalized secondary amyloidosis was diagnosed (post mortem) in association with purulent bronchitis and ascites.


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Conclusions: The merit of our case is it’s rarity: X-linked agammaglobulinemia was diagnosed in adulthood due to secondary amyloidosis associated with recurrent inflammation leading to nephrotic syndrome.

017 Sudden unexpected death in south western nigeria; an autopsy audit C Okolo, J Ogunbiyi Department of Pathology, University College Hospital, Ibadan, Nigeria Background: Sudden unexpected deaths are becoming more frequent in occurrence amongst Nigerians. A 10 year autopsy audit of sudden unexpected natural deaths presenting in a Nigerian teaching hospital is presented. The purpose of this study was to determine by autopsy audit, the pattern and commonest causes of sudden unexpected deaths in South Western Nigeria. For this study, sudden unexpected natural death is defined as death in an individual who was apparently well but died within 0–24 h of onset of any symptoms except those related to accidents. Design: One hundred and thirty one (131) cases of persons who died suddenly and unexpectedly and were presented for coroner’s autopsy at the Department of Pathology, University College Hospital Ibadan, Nigeria between January 1996 and December 2005 were included in this study. Their case files and coroners autopsy reports were reviewed and the provisional clinical diagnosis, autopsy diagnosis as well as demographic data were extracted and subjected to simple proportional analysis. Results: A total of 131 cases fulfilled the recruitment criteria (representing 2.24% of all coroner deaths during those 10 years) with age ranging between 2 and 80 years and a male female ratio of 1:8:1. Hypertension and its complications resulted in the highest number of sudden deaths, (68.7%); [cerebrovascular accident (CVA) (82.3%), acute left ventricular failure (14.4%) and myocardial infarction (3.3%)]. This was followed by meningitis, (8.4%) and acute gastroenteritis (3.8%). The remainder consisted of sundry other causes and accounted for 19.1% of the cases. Conclusions: Hypertension related complications are responsible for most cases of sudden unexpected deaths in South Western Nigeria and cerebrovascular accident is the most common of these. Further studies need to be done to delineate why CVA predominates over other hypertension related complications as the cause of sudden unexpected death in hypertensive Nigerians.

018 Extraordinary autopsy case of overwhelming disseminated cryptococcosis in HIV/AIDS F Rana1, A Kalebi2 1 Aga Khan University Hospital, Aga Khan University, 2National Health Laboratory Services University of the Witwatersrand, Johannesburg, South Africa Background: Cryptococcosis is a common and major opportunistic fungal infection in HIV/AIDS which frequently presents as severe

meningo-encephalitis in the immunocompromised. However, pulmonary cryptococcosis and other forms may be clinically missed due to elusive non-specific presentation. It may also be disseminated with multi-organ involvement, which in the absence of antifungal therapy may progress rapidly to death. We present this extraordinary example of overwhelming disseminated cryptococcosis which was clinically unrecognized in a child with AIDS. Design: Autopsy case report. Clinical information was obtained from attending clinicians and relatives of the deceased. A full autopsy examination was performed as routine, with histology and special stains done. Results: Clinical details: The patient is a 10 year-old HIV positive African male with a CD4 of 11 · 109/L and viral load 411 330 RNA copies/mL. He was had been on antiretroviral therapy for about 1 year and had completed empirical treatment for pulmonary tuberculosis 2 months prior to death. He was clinically noted to have meningism and was also suspected to have pneumonia when he presented at his community comprehensive care center. He was referred to the hospital where he collapsed and died on arrival. Gross findings at autopsy: The body showed marked wasting with a total body weight of 20 kg. There was generalized lymphadenopathy and oro-pharygeal candidiasis. The lungs were congested with patchy consolidations. The liver and spleen were both enlarged with multiple nodules on cut surfaces. The brain exhibited features of meningitis. The small and large intestines had areas of mural thickening. The rest of the examination was normal. Histopathology: H&E sections of the spleen, lungs, liver, brain and some of the enlarged lymph nodes revealed extensive cryptococcal infection with a prominent gelatinous pattern and minimal host tissue inflammatory reaction. The cryptococcal yeasts were confirmed on special stains (PAS, Grocott and Mucicarmine stains). Some of the tissues showing greater than 50% replacement of tissue-volume by cryptococcal organisms in aggregates. Conclusions: This is an extraordinary case of overwhelming disseminated cryptococcosis with CNS and pulmonary involvement being the likely primary contributors to causation of death. The extensive gelatinous pattern of cryptococcal tissue infection is a testament to the patient’s much diminished immune status which probable led to a sub-clinical response that contributed to failure of the infection being suspected clinically, facilitation of appropriate laboratory investigation and institution of timely antifungal therapy.

019 Lysosomal disease. Frequency in a pediatric autopsy population C Ridaura-Sanz, E Lopez-Corella National Institute of Pediatrics, Mexico City, Mexico Background: The frequency of lysosomal disease in the general population is difficult to ascertain. Adequately documented cases are usually concentrated in specialized institutions and case reports give no indication of the actual prevalence of these conditions. Registries of inborn errors of metabolism suffer from underdiagnosis inasmuch as these diseases are often misinter-



preted, screening systems have not been developed for many of them and enzymatic or biochemical diagnosis is not widely available. Many lysosomal diseases have distinctive histologic features that lead to diagnosis with a high degree of certainty. Post mortem examination can identify cases unsuspected clinically. Being genetic conditions, lysosomal diseases exhibit variations in different populations and genetic groups. It is therefore important to report the frequency of these diseases in a general pediatric autopsy population. Design: Cases identified as metabolic disease were retrieved from the autopsy files of the Department of Pathology of the National Institute of Pediatrics at Mexico City. Slides from these cases were reviewed and those with morphologic features diagnostic or suggestive of lysosomal disease were selected. Results: Twenty cases of lysosomal disease were detected among a population of 6902 pediatric autopsies performed between 1970 and 2006. Leading in frequency were Mucopolysaccharidoses and Gaucher disease with four cases each. Other conditions included Niemann Pick (two), Pompe (two), Wolman (two), Gm1 gangliosidosis (two), Gm2 gangliosidosis (one), Schindler (one) and two cases storage disease not otherwise specified. A correct clinical diagnosis was established in seven cases (35%); this was in all four cases of mucopolysaccharidosis, in 2/4 cases of Gaucher disease and metabolic disease was the working diagnosis in one of the two cases of unspecified metabolic disease. Conclusions: Postmortem examination led to an accurate diagnosis in most cases of lysosomal disease. These findings support the impression that the frequency of these conditions is underestimated as a result of clinical misdiagnosis.

020 Autopsy pathology in mass disaster incidents E Sakelliadis, S Papadodima, D Vlachodimitropoulos, N Goutas, C Spiliopoulou Department Forensic Medicine and Toxicology, Medical Faculty, University of Athens, Athens, Greece Background: To describe the lege artis forensic approach to the investigation of incidents involving mass casualties and to present our experience in the field. Design: Review of the Greek and of the International literature, as well as our experience. Results: Incidents involving mass casualties may appear unexpectedly, thus imposing the need to local forensic services to prepare a relevant protocol. In Europe such protocols already exist, but unfortunately in Greece no such protocol exists. For example, during last August, Greece experienced an unprecedented ‘wave’ of wildfires involving many different geographical regions and inflicting many human casualties. In such incidents, the role of the forensic pathologist is more crucial for the determination of the identity of the victims, than for ascertaining the cause of death which can be deducted from the incidents (e.g. wildfire, plane crash). In similar incidents, it is essential that the forensic pathologist performs a thorough scene investigation, which may yield important evidence

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that can help during the autopsy. The main forensic issue in hand, besides the positive identification of the victims, is whether the deceased were alive or already dead during the incident. The meticulous search for injuries on the regions of body remaining relatively intact, if any is present, can be of particular interest. Forensic anthropology, DNA sampling or use of forensic odontology may prove necessary to identify the victims. Conclusions: A forensic pathologist must be well aware of the importance of correct and timely corpse identification as well as of the complete investigation and must always keep in mind, the impact that has his practice on the families of the victims. The need to develop a Greek protocol concerning mass fatalities incidents, becomes more evident as the need to provide accurate and fast forensic work, is day by day, increasing.

021 Ethical and deontological consideration in pathology practice in Greece E Sakelliadis, S Papadodima, D Vlachodimitropoulos, N Goutas, C Spiliopoulou Department Forensic Medicine and Toxicology, Medical Faculty, University of Athens, Athens, Greece Background: To describe the ethical and deontological issues arising in the field of practice of pathological anatomy in Greece. Design: Review of the Greek and of the International literature, as well as our experience in the field. Results: The field of pathological anatomy is very important in the diagnosis and subsequent treatment of virtually the majority of diseases, either they concern oncological entities or inflammatory processes. The pathologist is required to validate, or in many cases, ascertain the diagnosis and by means of his work to provide guidelines for the treatment to follow. Because of this coordinating role, a pathologist should be a person of intact moral integrity, that can be in a position to provide consultation both for the clinicians and the patients. In Greece, many patient’s families do not wish the patient himself, to be informed, about the results of histopathological examinations, especially if they concern a neoplasm. This can pose a serious issue for the pathologist, as no rarely they require a ‘false’ diagnosis to be provided for purposes of concealing the truth from the patient. Has the pathologist the right to do so, or is he obliged to inform his patient of the result? The issue of proper archiving is again of grave importance as it is necessary to have every result available in the future for reevaluation. The problem that sometimes arises is the re-evaluation procedure itself. A pathologist often faces a patient with microscope slides for re-evaluation. The way this is performed, the handling of a possible misdiagnosis by the previous pathologist or the reluctance of pathologists to provide slides for re-evaluation, though they are issues of grave ethical and deontological importance, in everyday practice often cause many misunderstandings. Conclusions: A pathologist must be well aware of the importance of his work as well as of the impact his work has on the patient and his colleagues. He should always bide by the rules of deontology both towards his patient and his colleagues.


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022 Identification of victims after mass disaster incidents E Sakelliadis, S Papadodima, K Moraitis, D Vlachodimitropoulos, N Goutas, N Mitsea, C Spiliopoulou Department Forensic Medicine and Toxicology, Medical Faculty, University of Athens, Athens, Greece Background: To describe the lege artis identification procedures of mass casualties’ victims. Design: Review of the Greek and of the international literature as well as use of our own experience in the field, were performed. Results: Incidents involving mass casualties like last Augusts’ wildfires in the Peloponnese peninsula cost the lives of many citizens thus raising the problem of correct corpse identification. As already known, often victims are in such a condition that normal identification procedures employed for routine cases (visual identification, fingerprints, etc) cannot be employed. For this reason, the forensic pathologist who has the legal responsibilty to document and identify the dead can request the assistance of experts in anthropology, dentistry, and DNA. The forensic anthropologist, the first to be called upon, in addition to assisting in the location and recovery of human remains, may provide information concerning the biological characteristics (e.g., sex, ancestry, age, and stature) of the deceased, and facilitate the forensic pathologist in determining the circumstances surrounding the death of the individual. The second specialist to be called upon is the forensic dentist. Forensic odontology can prove to be of great assistance but in order to be so, dental records of the ‘alleged’ victims must be available. Finally the last but not least, resort in the field of identification and perhaps the most definitive is the DNA analyst. However, the positive identification of the victims by means of DNA analysis is feasible only when appropriate sampling is applied and when biological samples are available either previously collected from the victim or to be collected from his/her relatives. Conclusions: Experience from recent tragedies like airplane crashes, transportation accidents, building collapse, industrial accidents, etc have shown the need of a multidisciplinary team of forensic practitioners in order to positively identify the victims as soon as possible. Concerning the identification procedure, we must bear in mind that the more the pressure is that to achieve fast results, the less accurate they may be.

023 Post-mortem histological pulmonary analysis in patients with HIV/AIDS A M Soeiro, A Hovnanian, E Parra, M Canzian, V L Capelozzi Department of Pathology School of Medicine, University of Saõ Paulo, Saõ Paulo, SP, Brazil Background: Different aspects of pulmonary pathology in HIV/AIDS are unknown in autopsies. This study describes the demographic data, etiologic and histological pulmonary findings in different associated pathologies of 250 autopsies of patients with HIV/AIDS and acute respiratory failure (ARF) as the cause of death.

Design: The following data were obtained: age, sex, and major associated diseases (found at the autopsy). Pulmonary histopathology was categorized as: diffuse alveolar damage (DAD); pulmonary edema (PE); alveolar hemorrhage (AH); and acute interstitial pneumonia (AIP). Odds ratio (OR) of the AIDS-associated diseases developing a specific histopathological pattern was determined by logistic regression. Results: A total of 197 men and 53 women were studied. The mean age was 36 years. Bacterial bronchopneumonia was present in 36% (91 cases) and Pneumocystis jiroveci pneumonia (PJP) in 27% (68). Pulmonary histopathology showed AIP in 40% (99), DAD in 36% (89), PE in13% (33) and AH in 12% (29) of the patients. Multivariate analysis showed a significantly positive association between PJP and AIP (OR, 4.51; 95% CI, 2.46–8.24; P < 0.001), severe sepsis and/or shock septic and DAD (OR, 3.60; 95% CI, 1.78–7.27; P < 0.001), cytomegalovirus and AIP (OR, 2.22; 95% CI, 1.01–4.93; P = 0.05). Conclusion: This is the firt report of an autopsy study that includes the demographic data, etiologic diagnosis and respective histopathological findings in patients with HIV/AIDS and ARF. Further studies are necessary to elucidate the complete pulmonary physiopathological mechanism involved with each AIDS-associated disease.

024 An autopsy case of active Epstein–Barr virus infection S Suzuki1, M Toriumi1, T Tsukamoto2, N Ohta3, M Kudo4, S Uchino1,4 1 Clinical Welfare College, 2Ebetsu City Hospital, 3Tokyo Medical and Dental University, 4Tokyo Medical University, Tokyo, Japan A 22-year-old male was admitted under the diagnosis as acute hepatitis with severe bilirubinuria. Two weeks prior to the admission, his chief complaints included generalized fatigue, high fever and throat pain. Treatment for acute tonsillitis was ineffective. Physical findings on admission include jaundice, redness of pharynx and tonsils and the cervical lymph adenopathy. Continuous remittent fever swelling of the cervical lymph nodes and high-titer (2 · 106) of EBV-DNA led to diagnosis as Epstein–Barr virus infection. Steroid-pulse therapy, gamma-globulin therapy and chemotherapy using dexamethasone, ciclosporin etc. were applied immediately after the diagnosis. A patient suffered from adult respiratory distress syndrome (ARDS) and was treated under an intensive care unit (ICU). However, a disease worsened despite treatments including plasma-exchange therapy, a patient died of septic shock. Postmortem examination revealed massive multiple foci of acute hemorrhagic necrosis with bacterial and/or fungal infections in the major organs as bone marrow, lungs, spleen and kidneys, which are interpreted as results of septic shock and following multiple organ failure (MOF). Histopathological findings revealed hemophagocytic syndrome, though, no findings as malignant lymphoma were found. This case fulfils the diagnostic requirements for active EB virus infection, i.e., viral genome was found in the lungs, liver, spleen, lymph nodes and skin using in situ hybridization. An immediate cause of death is severe diffuse alveolar damage (DAD) complicated with fungal and bacterial infections besides EB virus, followed by septic shock and MOF.



025 Biphasic type malignant mesothelioma related to inhalation of asbestos, an autopsy case T Tsukamoto1, S Suzuki2 1 Department of Pathology, Ebetsu City Hospital, Ebetsu, Hokkaido, Japan, 2Preclinical medicine division, Clinical Welfare College, Tokyo, Japan Background: An autopsy case of biphasic type malignant mesothelioma is presented. The close pathogenic relation between asbestos and malignant mesothelioma has been pointed out. As we experienced a case of malignant mesothelioma with an evidence of asbestos inhalation, the case is presented here with results of various methods of analysis. Case: The patient is 76 years old male. He was admitted to the hospital due to dyspnea. Chest roentgenogram proved thickening of the right pleura. Transcutaneous biopsy revealed malignant mesothelioma. Despite chemotherapy including cisplatin and gemcitabine, he expired 15 months after admission. Results: Postmortem examination disclosed malignant mesothelioma, diffuse and biphasic type of the right pleura, infiltrating the right lung, ribs, chest wall, diaphragm and liver. Infiltration along the pathway of transcutaneous biopsy is characteristic to malignant mesothelioma. Distant metastases were found in the left lung and right adrenal gland. No metastasis to the lymph node was found. Other notable findings include pleural plaques on the left parietal pleura and diaphragmatic pleura, which are findings are said to be related with inhalation of asbestos. Quantitative analysis for asbestos in the lung revealed 23 852 rods of asbestos per gram (dry weight) of the left lower lobe (National limit of asbestos is 5000 per gram in Japan). No pulmonary fibrosis (asbestos lung) was found. Conclusions: The pathogenic relation between malignant mesothelioma and asbestos are still indefinite, however, association of massive inhalation of asbestos to the lung was proved in our case of malignant mesothelioma. We will present various analytic data of the patient.

026 Case report: Fatal pneumococcal Waterhouse-Friderichsen syndrome in a vaccinated adult with congenital asplenia C Vincentelli, E Molina, M Robinson Mount Sinai Medical Center, Miami Beach, USA Overwhelming postsplenectomy infection (OPSI) is a low incidence entity with a high mortality rate despite aggressive therapy. While initial symptoms may be mild and non-specific, it can progress rap-

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idly to Waterhouse-Friderichsen syndrome with full blown septic shock and disseminated intravascular coagulation (DIC). OPSI is known to occur at any time after splenectomy, even in patients who have received pneumococcal immunization and/or chemoprophylaxis. Although the term OPSI gives the impression of a ‘postsurgical’ complication, it has been seen in association with conditions predisposing to functional hyposplenism and in children with congenital asplenia. To our knowledge, there has been no previously reported case of OPSI in a pneumococcal vaccinated adult with congenital asplenia. We report a 67 year old woman with congenital asplenia and current pneumococcal immunization who presented to our emergency room with low-grade fever and nonspecific symptoms that evolved to DIC, refractory hypotension and shock in less than 24 h. The diagnosis of Waterhouse-Friderichsen syndrome was made at autopsy. The causative organism was Streptococcus pneumoniae.

027 A very rare case of spinal nerve aplasia in a female newborn D Vlachodimitropoulos, E Sakelliadis, N Goutas, S Papadodima, C Spiliopoulou Department Forensic Medicine and Toxicology, Medical Faculty, University of Athens, Athens, Greece Background: We describe a rare case of spinal cord malformation in a newborn full-gestation infant. Design: The study includes the complete autopsy material from the newborn child. Results: A full-gestation (41 weeks) female infant was born after Cesarian operation. The mother was 29 year old, with another healthy child. The diagnosis of congenital diaphragmatic hernia was established and urgent operative reduction was performed. Severe hypoplasia of the left lung was also observed. The infant died a few hours after the operation and her body was referred to our Department for autopsy. At postmortem examination, the newborn child was of normal weight (3.650 g) and externally normal, but during extraction of the brain from the cranial cavity, the spinal cord spontaneously came out of the spinal channel. The most relevant macroscopically evident finding was the absence of connections between the spinal cord and spinal nerves. Microscopic examination of the spinal cord disclosed a normal forward and posterior median fissure. The number of nerve cells in the gray matter was reduced, and neurons either were short or absent. Especially reduced number of nerve cells is observed in the posterior crura. Conclusions: It is a very rare case of pathology not previously encountered by our Department, that presents particular interest and appears to be also not often observed worldwide.


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BONE PATHOLOGY 028 Can low doses of simvastatin enhance fracture healing? an experimental study in rabbits G Agrogiannis1, D Chissas2, G Stamatopoulos2, A Papalois3, D Verettas4, K Kazakos4, E Kallitsis5, A Asimakopoulos2, E Patsouris6 1 1st Department of Pathology Medical School, University of Athens, 2 General Hospital ‘‘G.Genimmatas’’, Athens, Greece 2nd Department of Orthopaedic and Trauma Surgery, 3Elpen Pharmaceuticals, Athens, Greece Department of Experimental Research, 4Demokritus University of Thrace, School of Medicine, Alexandroupolis, Greece, Department of Orthopaedics and Trauma Surgery, 5National and Kapodistrian University of Athens, School of Medicine, Athens, Greece 1st Department of Pathology, 6Medical School, University of Athens, Greece Background: Several observational and experimental studies have investigated the potential skeletal effects of statins on undisturbed bone but only a few recent studies have examined their effects on fracture healing. The overall goal of the present study was to investigate whether the systemic administration of simvastatin in low doses (based on earlier safety and efficacy studies on undisturbed bone), could accelerate the healing of non-critical-sized ulnar osteotomies in rabbits. Design: Fifty-four skeletally mature male New Zealand White rabbits were used for this study. The rabbits were assigned to one of three experimental groups: a control group, and two groups that were orally administrated a diet with either 10 or 30 mg/kgr/day of simvastatin. A complete biochemical blood count was performed to exclude drug-induced complications. Half of the animals of each group were sacrificed at 15 days and the other half at 30 days after surgery, resulting in six subgroups. Healing quality was assessed at 2 and 4 weeks after fracture. The bones were subjected to biomechanical testing, peripheric quantitative computed tomography and histomorphometric analysis. For the last, and the qualitative as well as the quantitative assessment of the fracture callus, safranine O fast green stain was used. The slides were then digitalized and transferred to a computer equipped with the appropriate image analysis software. Results: Animals received simvastatin of 30 mg/kg/day resulted a significant reduction of BMD, stiffness, and energy absorbed to failure. At 15 days, the amount of cartilaginous callus formation was reduced as it was calculated by image analysis, and the void space was significantly increased, in animals administered with 10 mg/ kg/day (P < 0Æ05) and 30 mg/kg/day (P < 0Æ05) of simvastatin when compared with the control group. Conclusions: The results of this study suggest that simvastatin doses of 30 mg/kg/day may have a negative effect on callus formation in rabbits, whereas doses of 10 mg/kg/day do not produce a significant advantage or disadvantage, especially at the early stages of fracture remodeling.

029 Preliminary study of morphologic changes in acetabular roof due to interaction with hip prosthesis components D Anusca1, I Plesea1, C Georgescu2, M Ghilusi2, O Pop2, V Dascalu2 1 University of Medicine and Pharmacy Craiova, 2Emergency County Hospital Craiova, Craiova, Romania Background: The aim of this study is a preliminary assessment of changes in acetabular roof (AR) structure induced by the interaction with the hip prosthesis. Design: The material consisted of acetabular bone tissue sampled from 14 cases with femoral head prosthesis and revision of the prosthesis. AR stress state and pressure area (PA) were determined with the finite element method (FEM) on the plane models. For FEM, two calculus models were developed considering a frontal section (FS) and a sagital one (SS) through the upper part of a bipolar prosthesis and the surrounding zone of the AR. Numerical simulations were performed for both one and two legs standing positions (OLSP and TLSP) for both models. Main stress and equivalent stress fields distribution in the AR were obtained after processing of calculus results and the corresponding curves were plotted using a special post-processing program. Then, for each case were sampled: one bone fragment from the central zone of the maximal PA of the AR and one bone fragment from each of the medial, anterior and posterior aspects outside the maximal PA, at a distance of 1 cm. Bone tissue samples were fixed and decalcified in Duboscq-Brazil solution, embedded in paraffin wax, then stained with hematoxylineosin, van Gieson thrichrome and Masson thrichrome. Results: In OLSP, PA is ovoid, with the greater diameter in the SS and shifted towards the anterior aspect of the AR. The pressure values have a decreasing trend from lateral to medial. In TLSP, PA is like an antero-posterior narrow strip, near the acetabular rim with decreasing values from posterior to anterior. Maximal PA was characterized by areas of bone necrosis in direct aposition with the prosthetic elements, mineralized lamellae alternating with poorly mineralized ones and focal presence of bone lacunae occupied by loose vascular connective tissue in the cortical layer and areas with thin bone lamellae circumscribing large lacunar spaces occupied by a small amount of bone marrow in the trabecular area. A large cellular population consisting of osteoblasts and osteoclasts, areas with a collagen network in which fibers gradually show a lamellar disposition around wide channels filled with vascular connective tissue and lined by osteoblasts producing this osteoid material, as well as areas with gradual mineralization of collagen lamellae, simultaneous with the narrowing of the vascular connective channels and shaping of new haversian systems were also observed. Outside the maximal PA, in both compact and trabecular components, the bone structure showed characteristics of normal bone. Conclusions: Acetabular bone is hosting degenerative and regenerative changes seen in both compact and trabecular components but only inside the maximal PA of the AR. Our preliminary morphological study revealed the existence of an adaptation effort to the mechanical stress materialized through a dinamic process of bone remodelation in the maximal PA.



030 Collagen I, II, III, V and XI participation in joints remodeling of experimental diabetes S Atayde, W Teodoro, D Nascimento, S Catanozi, E Parra, E Nakandakare, A Velosa, V Capelozzi, N Yoshinari Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo, Brazil Background: Changes in extracellular matrix of tendons and ligaments are frequently observed in diabetic patients. It can result in angiogenesis, healing, inflammatory response and cells proliferation delays, what may be responsible for chronic tendonitis and limited joint locomotion. The aim of this study was to evaluate the joint remodeling after development of diabetes in rats. Design: We used 30 Wistar rats (n = 30), weighting 200–250 g divided in two groups: diabetic (DG = 15) and control (CG = 15) groups. Diabetes was induced by intraperitoneal streptozotocin injection (35 mg/kg) and controls received only physiological solution. The animals were monitored to weight and blood glucose during 10 weeks. After euthanasia, the femorotibial joints, ligaments and tendons were isolated, immersed in formalin 10%, decalcificated, included in paraffin and stained with HE and Picrosirius. The quantitative analysis in ligaments, tendons and cartilage was determined by image analysis system in different randomly selected fields by Picrosirius staining. Collagen I, III and V distribution in ligaments and tendons and II and XI of joint cartilage were evaluated by immunofluorescence. Results: It was found higher levels blood glucose (P < 0.01) in the DG (426.2 ± 65.4 mL/dL) when compared to CG (97.46 ± 6.7 mL/ dL). The final weight of DG was significantly lower (263 ± 19.97 g) than in CG (460.46 ± 65.43 g). The qualitative analysis in DG showed mild inflammation, but higher deposition of collagen, being III and V in tendon/ligaments, and II and XI in joint cartilage. Total collagen in DG was represented by significant higher amount of thin fibers in tendons/ligaments (P = 0.03) and cartilage (P = 0.01), compared to thick fibers. Also a significant increase of chondrocytes (12.78 ± 2.64 vs. 9.43 ± 1.38; P = 0.01) and decrease of cartilage area (93.46 vs. 68.40; P = 0.001) was found in DG compared to CG. Conclusions: We concluded that tendons, ligaments and cartilage remodeling occurs by substitution and increased collagen deposition, probably justifying joint dysfunction in diabetes.

031 Uncommon presentations of chondroblastoma: a destructive aneurysmal bone cyst of the calcaneus and a massive craniofacial mass D Athanazio1, L Barreto2, A Guedes3, B Barreto3, L Siqueira4, N Silva2, M Motta2, P Athanazio1 1 Federal University of Bahia, 2Escola Bahiana de Medicina e Sau´de Pu´blica, 3Hospital Santa Izabel, 4Hospital Espanhol, Bahia, Brazil Background: Chondroblastoma is an uncommon neoplasm accounting for less than 1% of bone tumors. It is usually located in the epiphysis or metaphysis of long bones. Case reports: Case 1: A 26-year old male patient sought medical assistance for a long term swelling in the region of his left ankle.

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Imaging tests revealed an aggressive tumor with extensive osteolytic involvement of the calcaneus. The lesion had a multiloculated cystic appearance and an expansive growth pattern. The patient underwent removal of his entire left calcaneus. The surgical specimen consisted of a 6.0 · 5.5 · 4.5 cm lesion with the macroscopic appearance of a typical aneurysmal bone cyst. The patient has been followed up for 10 months with no symptoms and no sign of recurrence of the disease. Case 2: A 37-year old male patient reported pain in his right hemiface. Initially, he sought dental care, which resulted in the extraction of three teeth. The patient was referred to an otorhinolaryngologist when he first presented epistaxis. Imaging tests revealed an extensive tumor affecting his right maxillary sinus, sphenoidal sinus, the base of his skull, greater wing of the sphenoid bone and temporal bone. Attempts to obtain biopsy specimens by nasal and oral route were unsuccessful. Diagnosis was only possible following transcranial biopsy. The patient refused to undergo surgical resection and currently reports hemifacial pain that hampers sleep and makes deglutition difficult and painful. In the two cases outlined above, microscopy revealed findings typical of chondroblastoma. Conclusions: Chondroblastomas should not be excluded from the differential diagnosis of short/flat bone tumors. Both tumors reported here caused massive and destructive lesions.

032 Primary mixed tumor of bone H Iwasaki, K Nabeshima, M Akamatsu Department of Pathology, Fukuoka University, Faculty of Medicine, Fukuoka, Japan Background: Mixed tumor of bone is very rare neoplasm of unknown histogenesis. We present a case of primary mixed tumor involving the sacrum of a 68-year-old man. Design: Paraffin sections prepared from the bone tumor was utilized for histologic and immunohistochemical studies. Results: A 68-year-old male patient complained of persistent pain and numbness of the right buttock and thigh for 4 months prior to the diagnosis. Imaging examinations revealed a huge pelvic mass with a large cystic change, which grew from the sacrum and invaded the ilium. Microscopically, the tumor showed a lobulated pattern composed of an admixture of atypical epithelial, myoepithelial and mesenchymal elements. The epithelial elements showed irregular ductal formation accompanied by myoepithelial cells. Foci of squamous metaplasia with keratin pearls were observed. The mesenchymal element exhibited fibromyxoid and cartilaginous foci with an abundant chondroid matrix. Areas of spindle cell proliferation, hyalinized stroma, and osseous differentiation were also found. The tumor infiltrated into the adjacent bones, muscles, and adipose tissue. Foci of necrosis and hemorrhage, and some mitotic figures were encountered in the tumor. Immunohistochemically, the tumor cells were positive for S-100 protein and cytokeratins (AE1/AE3, CAM5.2). Conclusion: The mixed tumor reported herein was considered to be malignant because of the presence of moderate to severe nuclear atypia, invasive growth, and necrosis. Although the cellular origin of mixed tumor is unknown, possible candidates include heterotopic


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embryonic remnant of skin appendages migrating to the bone, and pluripotent mesenchymal stem cells in the bone marrow with divergent differentiations into epithelial and mesenchymal elements.

033 Condylar hyperplasia in children: report of two cases I Carrasquero1, A Merino1, A Rodriguez-Morales2, M Del Pilar-Pallares3 1 Dental Unit, Medical Service of SENIAT, Ministry of Finances, Caracas, Venezuela, 2Razetti Medical School, Central University of Venezuela, Caracas, Venezuela, 3Medical Service of SENIAT, Ministry of Finances, Caracas, Venezuela Background: Condylar hyperplasia poses a problem in planning treatment because it is a self-limiting process for some but not all patients. Continued growth creates a progressive deformity that requires condylectomy, whereas an enlarged condyle can be left in place after hyperplastic growth ceases, even if ramus surgery is needed to correct asymmetry. It is more frequent in adults than in children, where is considered extremely rare. For these reasons we report two consecutive cases in children. Case reports: Case 1: a male, 3 year-old, presenting with facial asymmetry, with a history of trauma at 10 months old. A radiology and CT-scan revealed a condylar fracture. The bone scan with 99 mtechnetium phosphate evidenced an asymmetric condylar caption, more prominent in the left side, then diagnosed as active left condylar hyperplasia. Case 2: a female, 8 year-old, presenting with facial asymmetry, with a history of trauma at 5 year-old. A radiology and CT-scan revealed a condylar fracture. The bone scan with 99 mtechnetium phosphate evidenced an asymmetric condylar caption, more prominent in the left side, then diagnosed as active left condylar hyperplasia. Conclusions: Bone scan with 99mtechnetium phosphate can be used to assist in making the differential diagnosis. In the two patients reported here, abnormal metabolic activity revealed by bone scans supported clinical and historical evidence that the condylar hyperplasia was active and required surgical correction, including condylectomy which should wait until they reach 18 year-old to avoid recurrence due to growth, then the orthodontic management and follow-up is done until there.

034 Myopericytoma of bone: a case perort and review of litterature K Diamantopoulou1, S Chranioti1, E Nikolakakis2, E Thoma-Tsangli1 1 Department of Pathology, 2Department of Orthopaedics, General Hospital Asklepieio Voulas, Athens, Greece Background: Primary myopericytomas of bone are extremely rare. Many spindle cell neoplasms of bone have focal areas with perivascular myoid differentiation, simulating a hemangiopericytomatous pattern. The complexity of recent changes in the nomenclature for these tumors, comprising soft tissue counterparts, is related to consideration of adjuvant therapy. This fact explains the necessity of recognition of these rare tumors.

Design: Male, aged 62 years old, presented with a lesion of the 1st phalanx of left foot with cystic radiologic appearance. The lesion measuring 1 cm in greatest diameter was surgically excised by curettage. Paraffin embedded tissue was stained with hematoxylin – eosin, Alcian-Blue, PAS, PAS-Diastase and immunohistochemistry was performed using the antibodies: Vimentin, S-100, EMA, CEA, CD34, CD99, CD31, SMA, CK7, 8/18/19 and Ki-67(MIB-1). Results: Neoplastic tissue was revealed, consisting of small round to spindle cells arranged in an angiocentric perivascular pattern of growth in a myxomatous Alcian Blue (+) background. Glycogen granules (PAS+) were variably present. Rare intranuclear cytoplasmic inclusions and mitoses were observed. Immunohistochemically the tumor cells showed prominent positivity for SMA, Vimentin and CD34, suggesting a perivascular myoid (pericytic) differentiation of the tumor cells. Conclusions: There is close histological relationship between myopericytoma, myofibromatosis, solitary myofibroma and infantile hemangiopericytoma. According to recent literature this group of lesions constitute a morphological spectrum with differentiation towards perivascular myoid cells (pericytes). Currently, myopericytoma is the most accepted term for the spectrum embracing all these lesions. They are considered as benign tumors with 17% percentage of reccurence. Surgical excision with further excisions for local reccurence is the treatment of choice. Malignant myopericytomas are also reported and are associated with aggressive clinical behavior.

035 Histopathological lesions in a case of ochronotic hip arthropaty E Gramada1, C Socoliuc1, D Simeanu1, I Tudose1, A Slavnea1, L Tudorica1, M Cohal1, F Staniceanu1,2, R Marinescu3 1 Department of Pathology, Colentina University Hospital, Bucharest, Romania, 2University of Medicine and Pharmacy ‘‘Carol Davila’’, Bucharest, Romania, 3Department of Orthopedics, Colentina University Hospital, Bucharest, Romania Background: The term ‘ochronosis’ was introduced by Virchow in 1866 to designate the yellow or yellow-brown microscopic discoloration of tissues obtained from pigmented areas of cartilages, ligaments, tendons and the intima of the large blood vessels from patients with alkaptonuria (AKU). AKU is a rare inherited disease with a frequency less than 1:200 000 in population, characterized by deficiency in homogentisic acid oxydase, leading to accumulation of homogentisic acid resulted from degradation of phenylalanine and tyrosine. Most cases of ochronotic arthropathy occur after 40 years of age, with a peak incidence in the fifth decade. Men are affected about two times more often than women. Due to the accumulation of homogentisic acid, the cartilage loses its elasticity, becomes brittle and parts of it may separate forming loose bodies. Cyst formation and small bone infarctions may occur adjacent to the joint. Design: We present the case of a 58 years old male diagnosed with ochronosis who underwent total right hip joint arthroplasty for severe arthritic disease. The patient had arthritis signs and symptoms also in his left hip joint and in both knees. We received in the Pathology Department 14 synovial specimens with brown-black pigmented areas and nodules, the femoral



head, with brown-black pigmented rim at the articular capsule insertion and one small bone fragment excised from a cyst in the femoral dyaphysis. Tissue samples were fixed in 10% formalin and the bone fragments were decalcified. All fragments were routinely processed and paraffin embedded. The slides were stained with Hematoxylin-Eosin. Special stains (Congo Red and Perls) were performed for differential diagnosis of pigmented deposits, and Van Gieson stain to demonstrate the presence of ochronotic pigment within the elastic fibers. Results: Microscopic examination of synovial fragments showed the interstitial presence of numerous yellow-brown pigmented hyaline cartilage fragments, some surrounded by chondroclasts, granulation tissue, fibrin exudation, and marked interstitial fibrosis. Yellow-brown discoloration of adjacent fibrocartilage and tendon was observed. Femoral head examination revealed atrophy and discoloration of the covering hyaline cartilage, small subcondral cysts, small subcondral area of avascular necrosis, areas of immature bone formation and accumulation of pigmented macrophages. No amiloid or iron pigment were identified on special stains. Conclusions: Among AKU patients, almost half will develop ochronotic arthropaty of a variable intensity, some with severe forms that will dramatically impair their lifestyle. Pathologic examination in ochronosis can give additional information which help the clinician to better appreciate the degree of osteoarthrotic remodeling and to exclude other associated diseases (rheumatoid arthritis, gout, chondrocalcinosis) with impact on future treatment. We present this case not only because of the rarity of the disease, but also for its rare association with cyst formation and avascular bone necrosis. These lesions are probably best explained in the general context of arthritic bone remodeling and degeneration but a possible pathogenic correlation with homogentisic acid deposition cannot be excluded.

036 Dedifferented chondrosarcoma. A clinicopathological study of sixteen cases from a single institute N Jambhekar1, S Uppin1, R Kumar1, M Divatia1, M Agarwal2, A Puri2, N Merchant3 1 Department of pathology Tata Memorial Hospital, 2Department of Orthopedic Oncology Tata Memorial Hospital, 3Department of Radiology Tata Memorial Hospital, Mumbai, India Background: Dedifferentiated chondrosarcoma is a distinct variant of chondrosarcoma with a bimorphic histological appearance, comprising a well-differentiated cartilage tumor juxtaposed to a high-grade non-cartilaginous sarcoma. This study documents the experience at a single institute with 16 differentiated chondrosarcomas. Design: A retrospective study of 16 cases of dedifferentiated chondrosarcomas accessioned over a 16-year period (1993–2008) was done. The clinical features, radiologic findings, surgical details were abstracted from the patient’s charts. All histological slides were reviewed to: (i) confirm the diagnosis of dedifferentiated chondrosarcoma; (ii) grade the chondroid component; (iii) classify the histologic subtype of the non-cartilaginous sarcomatous component; and

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(iv) assess the relative percentage of chondroid and sarcomatous components. Follow-up was available in ten patients and ranged from 2 months to 6 years. Results: The patients’ were between 21 to 76 year old (median 46 years); there were ten males and six females. Ten of 16 patients had a denovo-dedifferentiated chondrosarcoma whereas the previous history of the remaining six revealed multiple exostosis in two, well-differentiated chondrosarcomas in three and long standing enchondroma in one. The most frequent site of involvement was pelvis (10), followed by femur (two), tibia (two), scapula (two) and sternum (one). Histologically, the cartilaginous component was Grade I in two and Grade II in fourteen cases. The non-cartilaginous sarcomatous component was malignant fibrous histiocytoma (MFH) in 10, osteosarcoma in three, fibrosarcoma in two and rhabdomyosarcoma in one case. The average proportion of the sarcomatous component was 61.08 % (median: 68.5% range: 16%–95%) of the tumor. Thirteen of 16 patients underwent surgery (wide excision – nine, amputation – four), whereas two had palliative chemotherapy and one underwent pulmonary metastatecomy. The average follow-up was 16 months (range: 0–73 months). At the last follow-up, four patients were alive without evidence of disease at 6, 8, 49 and 73 months. Three patients died of disease at 2, 12 and 30 months after the diagnosis of dedifferentiated chondrosarcoma. Local recurrence was noted in three cases at 11, 24 and 42 months after initial surgery. Metastatic disease was noted in seven patients; this involved the lung in five, deposit in the soft tissue of the leg in one, and the seventh patient developed multiple skeletal and lung metastasis. Two of the five patients with lung involvement had pulmonary deposits at first presentation itself. Conclusions: Dedifferentiated chondrosarcoma is an uncommon and aggressive variant of chondrosarcoma. Histologically the cartilaginous component was grade II in 87.5% of cases and MFH was the most common sarcomatous component comprising 62.5% of cases. The outcome was poor with death due to disease in three, local recurrence in three and lung metastasis in five patients as noted in the short period of observation. No statistically significant correlation was found between the proportions of chondroid component vs. tumor however, a poor outcome was noted when metastatic disease was evident at presentation, and when the dedifferentiated component was MFH.

037 The morphologic and pathologic study of collagen-induced rheumatoid arthritis (CIA) and swimming exercise group with CIA in rats J Kim1, M Lim2, S Shim2, D Sheen2, C Kim3 1 Department of Pathology, Catholic University of Korea College of Medicine Daejeon Saint Mary’s Hospital, Daejeon, South Korea, 2 Department of Internal Medicine, 3Department of Physiology and Biophysics, School of Medicine, Eulji University, Daejeon, South Korea Background: Rheumatoid arthritis (RA), a chronic inflammatiory autoimmune disease, is characterized by progressive synovial inflammation and joint destruction. Gradual destruction of the joints leads to functional impairments and so it is the most critical part of strategy for treatment. It also leads to muscle weakness and


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limitation of range of motion, therefore exercise may be a countermeasure for them. However, it is unclear whether exercise is beneficial in patients with RA, especially from the viewpoint of joint destruction. Collagen-induced arthritis (CIA), a representative animal model for the human rheumatoid arthritis (RA), could be useful to follow up joint destruction. The purpose of this study is to clarify whether swimming exercise can delay inflammatory progress and prevent joint destruction. Design: We induced CIA in Lewis female rats at the age of 8 weeks using chicken CII. Rats were assigned to one of three groups: Normal Control (n = 5); CIA without exercise (n = 6); CIA with exercise (n = 10). Exercise group had carried out a swimming protocol, 30 min/day, 5 days/week, during 3 weeks from 21st days after the primary immunization, around the maximal morphologic score. Rats were observed for swelling and erythema of their four limbs for 67 days after first injection of CII via morphologic score, which is semi-quantitative and ranged from 0 to 4 each limb. The microscopic evaluation was performed without knowledge of specific interventions and consists of synovial hyperplasia (score range: 0– 2), infiltration of inflammatory cells in the subsynovial tissue (0–3), pannus formation (0–1), destruction of cartilage (0–3), and bony erosion (0–3). Results: In final morphologic score, there is no significant difference between groups (Control vs. Exercise: 2.17 ± 0.80 vs. 2.05 ± 0.29, P > 0.05). Swimming exercise group showed significantly fewer incidences of pathologic findings than CIA without exercise: synovial hyperplasia (Control vs. Exercise: 89% vs. 25%), infiltration of inflammatory cells in the subsynovial tissue (78% vs. 25%), pannus formation (67% vs. 0%), destruction of cartilage (67%, severe vs. 50%, moderate), and bony erosion (67%, severe vs. 50%, moderate). In microsocpic score, exercise group showed significantly improvement than control: synovial hyperplasia (Control vs. Exercise: 1.2 ± 0.79 vs. 1.0 ± 0.58), infiltration of the subsynovial tissue with inflammatory cells (1.9 ± 1.37 vs. 0.5 ± 0.42), pannus formation (1.4 ± 1.26 vs. 1.0 ± 0.57), destruction of cartilage (1.4 ± 1.26 vs. 1.0 ± 0.57), and bony erosion (0.6 ± 0.51 vs. 0.0 ± 0.00). Conclusions: 1. Swimming exercise group did not show significant improvement in morpholgic findings. 2. However, we can confirm the improvements of joint destruction in swimming exercise group. 3. So we conclude that swimming exercise in CIA rats could have preventive effect on joint destruction rather than the progress of inflammation.

038 Chondromyxoid fibroma of bone: clinicopathologic appearance in ten cases E Konishi1, Y Nakashima2, A Yanagisawa1 1 Department of Pathology Kyoto Prefectural University of Medicine, 2 Laboratory of Pathology Kyoto University Hospital, Kyoto, Japan Background: Chondromyxoid fibroma (CMF) is a rare bone tumor, and it may not easy for the general pathologists to diagnose it correctly. Herein, we will show ten cases of CMF with a review of their clinicopathological features. Design Six of ten cases were of our

own. Remaining four cases were consultation cases. Pathological slides were available in all cases. Radiographs were available in eight. Each author reviewed all glass slides and radiographs. Results: Six of ten were male and four were female. The patients ranged in age from 14–60 years, with a mean of 25.2 years and a median of 21.5 years. Eight patients were in the second or third decades. Five lesions were located at the proximal tibia, two were at the proximal fibula, and one each was at the distal femur, rib, and iliac wing. All eight cases involving the long bones, were present around knee. Chief complaint was available in nine cases. Pain was most common (six cases). Swelling was observed in three cases. Radiograph showed a lytic lesion with sclerotic margin in seven of eight. Eccentric involvement was observed in five of six cases affecting long bones. Trabeculated lobular pattern was also noted. Cortical thinning or effacement was seen in eight cases. Two cases originated in the cortex of the tibia. Dot-like calcification was noted in only one. The signals of MRI showed iso- or hypointense on T1-weighted images and diffuse or inhomogeneous hyperintense on T2-weighted images. The lesions were enhanced well with Gadolinium. One case showed multilocular cystic change with fluid-fluid levels. Macroscopic photos were available in three cases in which resection of the lesions was done. In two, the lesions showed well-bordered, lobulated gray to yellowish white discoloration. Semitransparent appearance was present. One case had multilocular hemorrhagic cysts. In microscopic, vague lobular arrangement of bluish myxoid matrix was seen. Lobules were surrounded by hypercellular area with occasional osteoclastic giant cells. The tumor cells were mononuclear spindle shape or stellate appearance. Sometimes, epithelial cell-like cells with oval cleaved nuclei were noted. Cellular atypia was not found except one case, in which pseudosarcomatous change of the nuclei was noted. One case showed secondary aneurysmal bone cyst. Three cases were treated with resection of the lesions. Remaining seven were with curettage. Follow up was available in seven cases and all did not have recurrence. Conclusions: Typical clinicopathological features of CMF in our file are as followed: 1) 2nd or 3rd decades of life, 2) Involvement of long bones around the knee, 3) Eccentric metaphseal location, 4) Lobulated lytic lesion with marginal sclerosis in radiographs, 5) Lobules of yellowish white semitransparent matrix in gross, 6) Hypo- and hypercellular areas showing lobular arrangement with myxoid matrix in low power view, 7) Spindled or stellate tumor cells, rarely showing pseudosarcomatous change, 8) Osteoclastic giant cells in cellular area.

039 Rare locations of giant cell tumor of the bone G Liapi-Avgeri1, S Zimaris2, K Kouzelis2 1 Department of Pathology, 2Neurosurgery Clinic, General Hospital of Eleusis ‘‘Thriasio’’, Greece Background: Giant Cell Tumor (GCT) of the bone is a tumor with a rather characteristic location at the epiphyses of long bones, a fact that makes the diagnosis already suspected by imaging. Unusual locations may cause false interpretation with consequence to the appropriate therapeutic management. First case: Female 46 years old with hypothyroidism since 8 years and a hysterectomy for ade-



nocarcinoma of the cervix admitted to be operated for a lytic sellar tumor with destruction of its base, supposed to be pituitary adenoma. The tumor was composed of a plethora of multinucleated CD68 positive giant cells and spindle cells without production of osteoid or any other fundamental material. The diagnosis of GCT of the bone was based on the imaging and histologic findings. Second case: Female 46 years old admitted with lumbar pain I1-I2 without any neurologic deficiencies. CT and MRI demonstrated a lytic lesion in the area of I1-I2 with nearly total destruction of the vertebras and with contrast enhancement. A mass with well circumscribed margins of 10 cm in greater dimension and ‘soap-bubble’ appearance was protruding in the pelvis. The tumor material was a thick fibrous capsule attached with osteoclastic type multinucleated giant cells, CD68 positive, and spindle cells. The diagnosis of GCT of the bone was based on the imaging and histologic findings. Conclusions: GCT of the bone is an aggressive or low-grade malignant bone tumor and in the majority of cases its usual site of development is the epiphysis of long bones. Infrequently it occurs in the spine from the cranial base up to above the sacrum and exceptionally at the sellar region, this site have been reported in isolated cases. In any case imaging examination presents a lytic lesion with expansion and even destruction of the affected bone, trabecular or ‘bubbling’ appearance, because of the intramedullary development, and with variable degree of contrast enhancement. A soft tissue mass is usually detected without periostic reaction and new bone formation at the periphery of the lesion. Histologically it consists of neoplastic mononuclear cells, that are supposed to derive from primitive mesenchymal cells, variably intermingled with non-neoplastic osteoclastic type multinucleated giant cells. Coexistence of secondary lesion may cause dilemma in the differential diagnosis as they can present features of aneurysmal bone cyst, may be rich in foam cells resembling fibrous histiocytoma or they may show osteoid production reminiscent of osteoblastic tumor in the case of pathologic fracture. The well established usual presence of intravascular emboli of the neoplastic cells, sometimes atypical, avoids a false interpretation of a malignant process. Positive reaction of the giant cells against CD68 beside the negative to S-100 protein is useful in the distinction from other bone tumors rich in multinucleated giant cells. Conclusions: Careful examination of the radiographic features combined with the histologic findings identifies the correct diagnosis especially in the cases of unusual locations leading to the appropriate therapeutic management.

040 Unusual cytogenetic findings in a Ewing’s sarcoma der(X)t(X;18) with same breakpoint as synovial sarcoma H Moura, K Abe, R Kalil Department of Surgical Pathology, SARAH Network of Rehabilitation Hospitals, Brasilia, Brazil Background: Small round cell tumors (SRTC) comprise a diverse group of malignant neoplasms affecting children and young adults. Tumors most commonly included in the differential diagnosis of SRCT include Ewing’s sarcoma/primitive neuroectodermal tumor (ES/PNET) and poorly differentiated synovial sarcoma among others. These tumors may be histologically indistinguishable,

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particularly in the setting of a small biopsy. Immunohistochemistry may play a valuable role in the distinction of these entities. However, overlap exists among the immunophenotypes of SRTC group of tumors and many cases may require additional ancillary studies for definitive diagnosis, especially cytogenetic and molecular studies. For most of SRTC group, the presence of chromosome translocation or its fusion product is not only a sensitive marker for the tumor but is specific for the diagnosis. Design: The purpose of the current study is to report the finding of a chromosome der(X)t(X;18)(p11;q11) on cytogenetic analysis and EWS-FLI 1 fusion on molecular study in a case of SRCT that proved difficult to diagnose histologically and immunohistochemically. Case Report: An 8-year-old girl presented with a four-month history of a painless swelling in the region of the right foot. She experienced pain only on exercising. No history of trauma or other systemic illnesses was elicited. Laboratory tests were within normal limits. Imaging studies of the calcaneus showed an osteolytic lesion with cortical permeation and soft tissue extension. A needle biopsy was performed. Microscopy showed an undifferentiated round cell neoplasm, with tightly packed cells with minimal cytoplasm. Immunohistochemical reactions were positive for CD56, Bcl2, EMA, CD99 and p75 and chromosome analysis showed der(X)t(X;18) (p11;q11), findings strongly favoring the diagnosis of synovial sarcoma. The breakpoint detected by routine cytogenetic study performed was the same as in synovial sarcoma, although der(18) was part of rearrangements involving others chromosomes. However, fluorescent in situ hybridization (FISH) using dual color LSI SYT break apart probe did not show the disrupted signal usually found in synovial sarcoma. Molecular analysis by RT-PCR failed to detect SYT-SSX1, SYT-SSX2 and SYT-SSX4 fusion. Furthermore, RT-PCR detected EWS-FLI 1 fusion although cytogenetic abnormalities involving chromosomes 11 and 22 were not found. Conclusions: The literature describes many tumors with the translocation t(X;18) including one case of ES/PNET, but their breakpoints were different from synovial sarcoma. There are also reports of one malignant fibrous histiocytoma and one ependymoma with t(X;18) in the same synovial sarcoma breakpoint. This study reports a case of ES/PNET with a immunophenotype overlapping synovial sarcoma’s and with der(X)t(X;18) on cytogenetic studies. The final diagnosis was established by molecular studies. ES/PNET is a diagnostically challenging malignant round cell tumor and often requires the use of many ancillary diagnostic tools for definitive diagnosis, especially molecular analysis.

041 Expression of ILK and its binding partners in chondrosarcoma: association with prognosis D Papachristou1, V Gkretsi1, U Rao1, O Papaefthymiou2, C Wu1, A Papavassiliou3 1 Department of Pathology University of Pittsburgh, Medical Center (UPMC), Pittsburgh, PA, USA, 2Department of Orthopedics University of Athens Medical School, Athens, Greece, 3Department of Biological Chemistry of Athens Medical School, Athens, Greece Background: Integrin-linked kinase (ILK) is an important component of cell–matrix adhesions implicated in vital processes of many


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cells, including chondroblasts. Furthermore, it has been associated with anchorage-independent growth, cell scatter, and carcinogenesis. A considerable volume of in vivo and in vivo data has shown that ILK plays a significant role in the pathogenesis and progression of several human malignancies (colon, gastric, lung cancer, melanoma, Ewing’s sarcoma). ILK binds the focal adhesion proteins aparvin, b-parvin and PINCH, forming a stable adhesion complex. Mig-2 and migfilin are recently identified adhesion molecules that affect cell-ECM/cell-cell adhesions, cytoskeletal organization and migration. Recent studies have shown that the ILK–PINCH–parvin complex interacts with Mig-2. Although the expression and function of ILK have been studied in several tumors, the expression of its binding companions has not been studied thoroughly. Herein we assessed the expression levels of the ILK–parvin complex, Mig-2 and migfilin in cartilaginous tumors and investigated their role in chondrosarcoma (CHS) pathobiology. Additionally, we evaluated these molecules as possible biomarkers for the prediction of high-grade tumors and correlated their expression levels with patient survival. Design: Sixty formalin-fixed paraffin-embedded, central CHS of all grades and 25 enchondromas (ECH) were examined immunohistochemically. We generated the following mouse monoclonal antibodies against ILK (20 lg/ml, dil: 1/100), a-parvin (7 lg/ml, dil: 1/ 100), b-parvin (12.4 lg/ml, dil: 1/100), Mig-2 (12.46 lg/ml, dil: 1/100), and Migfilin (1.4 lg/ml, dil: 1/70). Stain intensity was scored on a scale of 0–3+, according to the percentage of positive tumor cells. Statistical analyses (Chi-squared, Mann–Whitney Utest, Kendall’s s, binary logistic regression tests, Kaplan–Meier and Cox-multivariate survival analyses) were performed with SPSS 12.0 for Windows. Results: (i) Positive nuclear and cytoplasmic immunostaining for ILK, a-parvin, b-parvin and Mig-2 was observed in 93.3%, 95%, 87.1%, and 85% of CHS and in 36%, 72%, 40% and 16% of ECH, respectively; Migfilin was not detected under the conditions employed, (ii) The expression levels of the examined molecules were significantly higher in CHS compared to ECH (P < 0.001). The cellular levels of ILK and Mig2 were significantly augmented in highgrade (G2/3) compared to low-grade (G1) CHS and in low-grade tumors compared to ECH (P < 0.005). Normal chondrocytes were negative for all proteins tested, (iii) The expression levels of ILK, aparvin and Mig2 were significantly and positively correlated to each other (P < 0.01), (iv) ILK expression levels could predict accurately high-grade CHS (sensitivity 77%, specificity 85%, PPV 91%, NPV 65% and overall accuracy 82%), and (v) On survival analysis tumor grade and Mig-2 expression levels were found to be associated with worse overall survival (P = 0.01 and 0.002, respectively). Conclusions: (i) The ILK–a-parvin–Mig2 complex is expressed and maybe functional in chondrogenic tumors, (ii) ILK and Mig2 are associated with CHS development/progression, (iii) ILK can be used as molecular marker to identify grade-based aggressive CHS, (iv) Mig-2 could be promising prognostic indicator separating patients at higher risk, and (v) Highly-selective small-molecule agents that selectively disrupt the ILK-a-parvin interactions at focal adhesions might have great benefit in CHS patients’ treatment.

042 Mesenchymal stem cells can ameliorate adjuvant arthritis in a dose and time dependent manner A Papadopoulou1, E Yannaki1, E Athanasiou1, I Batsis1, A Athanasiadou1, A Xagorari1, A Anagnostopoulos1, A Fassas1, M Yiangou2 1 Gene and Cell Therapy Center, Hematology-BMT Unit, George PapanicoIaou Hospital, 2Department of Genetics, Development and Molecular Biology, School of Biology, Aristotle University, Thessaloniki, Greece Background: Mesenchymal stem cells (MSCs) obtain the potential to differentiate into a broad spectrum of tissues beyond germ layers, holding promise for repairing damaged tissues. We aimed to explore the effect of rat MSCs on cultured fibrobIast-like synoviocytes (FLS) and T-cells from the spleen after adjuvant arthritis induction (AA) as well as their in vivo immunomodulatory potential in a rat model of AA resembling human rheumatoid arthritis. Design: FLS were isolated from the synovial membrane and T-cells from the spleen. Bone marrow MSCS were cultured and expanded up to 2–4 passages from normal Fisher (used as syngeneic MSCs) or Lewis (used as allogeneic MSCs) rats. AA was induced by intradermal tail injection of Freund’s adjuvant in Fisher rats. Results: When AA-FLS were cultured in the presence and absence of syngeneic (syn) or allogeneic (allo) MSCs supernatant (sup), a dose-dependent inhibitory effect on FLS proliferation was observed by MSCs sup (P < 0.022) as measured by incorporated radioactivity. The addition of either syng or allo MSCs sup also reduced the proliferation of ConA-stimulated AA T-cells in a dose-dependent manner (P < 0.018). Cell to cell contact of syn or allo MSCs with activated AA T-cells resulted in more profound inhibition as compared to the sup effect (P < 0.000004). A strong inhibitory effect was observed at all MSCs concentrations tested even at the minimal ratio (MSCs : T cell = 0.05:1). In in vivo experiments there was no clinical benefit when low doses of MSCs (0.5–5 · 10^5cell/recipient) were administered i.v., intrasplenic or intrabone marrow, at single or multiple infusions. In contrast, repeated, higher dose (6 · 10^6cell/rat), iv infusions of syn or allo Y+MSCs to female recipients, before the onset of AA (day 4 and day 9 post AA), resulted in significantly lower arthritic scores in MSC-treated as compared to control rats. The histopathologic score directly correlated with the clinical score, where MSCs-treated animals preserved a rather normal joint architecture without evident bone destruction or chondroplasia, no pannus formation or synovial membrane thickening, but only focal synovial hyperplasia. Additionally, the number of CD3 cells and the number of small vessels were decreased. At the time of sacrifice (day 30 post AA) no Y+ cells were detected in the spleen, bone marrow or in cultured FLS from synovial membrane, by PCR, immunohistochemistry or FISH, implying that the benefit was not due to MSCs homing to the target tissues but mainly by immunomodulation. Conclusions: Our data suggest that MSCs may serve as a new therapeutic approach for autoimmune arthritis however, dosing and timing need to be determined before these results could be clinically translated.



043 Epidermal growth factor receptor (EGFR) expression in chordoma, fish and IHC studies K Ptaszynski, A Mrozkowiak, K Suszylo, M Pekul, W Olszewski MCS Cancer Centre-Institute, Warsaw, Poland Backround: Chordoma is a rare neoplasm arising from the embryonal remnants of a notochord. The most common location is a sacral bone, cervical spine and skull. Surgery and radiation therapy are the most commonly used treatment modalities. Epidermal Growth Factor Receptor (EGFR) overexpression has been found in various tumors and has a prognostic value. Design: In this study we evaluate an EGFR expression status in 20 cases of chordoma diagnosed at the MCS Oncology Centre-Institute between 2002 and 2008. Fluorescence In Situ Hybridization (FISH) and immunohistochemistry (IHC) with anti EGFR antibody were performed on slides obtained from the paraffin blocks. Results: FISH studies show increased copy number of EGFR gene in our cases of chordoma, immunohistochemical study showed a positive reaction in 10 cases of this neoplasm. Conclusions: In many cases of chordoma surgical and radiotherapy treatment is not effective and new treatment modalities are needed. EGFR may be one of the targets for anticancer therapy in some cases of chordoma (cetuximab, gefitinib).

044 Cytogenetic findings in 13 benign cartilaginous neoplasms N Sakai-JR, C Dantas Nogueira, A Rosembaum Benedetti, D Afonso Cornelio, K Terada Abe, L Lia Menezes Formigli, M Fernandes Pereira, R Karam Kalil Sarah Network of Rehabilitation Hospitals, Brası´lia, Brazil Background: Benign cartilage tumors (enchondromas and periosteal chondromas) represent some 10–25% of all primary bone tumors. The differential diagnosis between chondroma and lowgrade chondrosarcoma is one of the most difficult in bone tumor pathology. Histologic grade has been shown to be of some prognostic importance; however, discrepancies between histologic appearance and biologic behavior are not infrequent. Chromosomal studies have proven to be of value in the diagnostic assessment of many mesenchymal neoplasms. Cytogenetic analysis is a complementary tool to yield more biologic information on cartilaginous tumors. To date, in the Catalog of Chromosome Aberrations in Cancer there are 29 cytogenetically abnormal bone and soft tissue chondromas published. Design: In this study, the karyotypic findings of eight enchondromas and five periosteal chondromas and a review of the literature are presented. Results: Between 2001 and 2007, 33 chondromas were collected in our pathology department, which 27 were enchondromas and six periosteal chondromas. Tumor biopsies of specimens from 13 patients were received directly from surgery for cytogenetic analysis. Five patients were male and eight were female. The age ranged from one to 51 years old. All the specimens studied were primary and untreated lesions. After examination the tumors were classified by histological subtype, there were no histological features suspect

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of malignant transformation and were done correlation between histopathological and radiological findings. Seven tumors were enchondromas, five were periosteal chondromas and one was soft tissue chondroma. None of the patients had Mafucci syndrome or Ollier disease. Cytogenetics analyses of the 13 chondromas specimens were successful. Only normal karyotypes were detected in five patients (four enchondromas and one periosteal chondroma), whereas clonal chromosome aberrations were found in eight patients. Diploid or near-diploid clones were observed in all cases. One soft tissue chondroma had a four cellular clones 44~46,XY,t(6;12)(q12;p11.2)[cp4]/40~46,XY,t(3;7)(q13;p12)[cp3]/ 46,XY,der(2)t(2;18)(p11.2;q11.2)/46,XY; three periosteal chondromas had a cellular clone with random structural aberrations of chromosomes 2, 3, 6, 7, 11, loss of chromosome 13 and normal clone. Three enchondromas had a cellular clone with chromosome losses and normal clone. One enchondroma had a cellular clone with 46,XY, inv(2)(p21;q31),t(12;15;21)(q13;q14;q22). A correlation between histological appearance and presence of complex chromosomal abnormality was not observed. Conclusions: We conclude that considerable cytogenetic heterogeneity exists among benign chondromatous tumors. We think that cytogenetic studies of benign cartilaginous neoplasms may contribute to the solution of clinical problems, such as the differential diagnosis between benign and low-grade cartilaginous lesions.

045 Primary aneurysmal bone cyst-our experience on 253 patients E Santini-Araujo, L Olvi, M Gonzalez, R Cabrini Laboratory of Orthopaedic Pathology, Department of Pathology, School of Dentistry, University of Buenos Aires, Buenos Aires, Argentina Background: The term aneurysmal bone cyst (ABC) was introduced by Jaffe and Lichtenstein in 1942. ABC was defined as a non neoplastic cystic lesion of bone composed of blood filled spaces separated by connective tissue septa containing fibroblasts, osteoclasttype cells and reactive woven bone. The etiology of ABC remaines unknown. In the last years cytogenetic data cast a new light on its etiology. ABC may arise de novo in bone (primary ABC) or be associated with other benign and malignant bone conditions (secondary ABC). Design: In our laboratory, 253 cases of ABC were recorded in 21 years (1986–2007). In all cases were studied the images, and the routine histopathologic slides. In 10 cases were performed genetic studies. Results: The lesion was slightly more common in females 130 than in males 123. The great majority occurred in the first three decades and 58% of patients (138) were 10–20 years old. Almost every bone may be involved, but primary ABC usually affects long bones, specially the distal femur (48), proximal tibia (39) and proximal humerus (28) in our series. It is also common in the spine (22) (with preference in cervical spine) and pelvis (28). In long bones it usually affects the metaphysis. The usual X-ray picture is that of a radiolucent expanding ‘blown-out’ eccentric or less commonly a central lesion which usually arises in the medullary cavity in the metaphysis or diaphysis of a long bone. However the lesion is more frequently intramedullary, it may be cortical or periostical. Histolog-


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ically the lesion shows multiple spaces contain blood or empty, separated by fibrous septae composed of well differentiated benign spindle cells and variable number of multinucleated giant cells. The blood spaces are not lined by endothelium but by flattened fibroblasts. Mitosis is a frequent finding, however, atypical forms are absent. Newly formed osteoid or bone trabeculae resemble that seen in reactive processes. A characteristic feature is the deposition of fine fibrillary osteoid beneath the lining of the septum. Another relatively specific feature of this lesion is the presence of peculiar chondroid-like zones in solid portions of septa. Lace-like or powdery calcification is seen within the septa. The differential diagnosis of ABC includes giant cell tumor, telangiectatic osteosarcoma, and low grade osteosarcoma. Conclusions: ABC was historically considered a nonneoplastic process, but recent cytogenetic data have shown rearrangements of chromosome band 17p13 is a characteristic genetic feature of some ABCs. This translocation may be usefull for differential diagnosis of ABC, however most of them can be diagnoses confidently with HE. It is specially important to determine if the ABC is primary or secondary (which is specially difficult when greater part of the primary lession is cystic) because the treatment is related with the primary lession.

046 Chromosomal instability in giant cell tumor of bone (GCTB) Z Sapi1, M Szendroi2, L Kopper1, T Krenacs1, L Moskovszky1 1 1st Department of Pathology and Experimental Cancer Research Semmelweis University, Budapest, 2Department of Orthopedics Semmelweis University, Budapest, Hungary Background: Though 80% of GCTB is benign, local recurrences and metastases can occur, even in cases with a previous benign histology. To date there are no recognized clinical, radiographic, biological or histological factors that are predictive of the clinical course of GCTB. Design: Analyzing 53 frozen samples from our tumorbank, we performed image-cytofotometry on smears, measuring the ploidy-value only of the mononuclear cell population. After immunocytochemistry on smears using CD68 as a histiocytic marker, we marked and separated the CD68+ and negative cells on the smear using Bioview Duet scanning station. Thereafter we performed FISH on the same – already scanned – slides hibridizing centromeric and telomeric probes for chromosome 11 and on a separate slide four centromeric probes simultaneously (X, 3, 4, 6) after scanning. Results: According to the ploidy value and the clinical outcome we distinguished five different groups. Besides the diploid non recurrent and the tetraploid recurrent cases, we found two marginally groups: the recurrent though diploid and the non recurrent though tetraploid GCTBs. Between CD68+ histiocytes only a very low percentage of number- and telomeric-alterations could be detected for the examined chromosomes. In the tumor cell population, the diploid non recurrent group contained low percentage of aneusomy. In tetraploid recurrent group the aneusomy was more frequent, but we found it a balanced tetrasomic aneusomy. Tetraploid non recurrent group showing slight aneusomy, can featured by so called

euploid poliploidization which is a known benign feature in other type of tumors. Within diploid recurrent group the aneusomy was more frequent, but we found it balanced chromosomal instability. The malignant cases showed aneusomy. TAS or 11p deletion was present in all of our cases. Benign GCTBs contained 11p telomeric alterations regardless of ploidy-value or recurrency-disposition. Nevertheless TAS or 11p deletion appeared more often in malignant GCTBs. Conclusions: We concluded that the histiocytic cells of GCTB are normal disomic cells. Tetraploid non recurrent cases showed eusomic polisomy, which is a benign phenomenon and diploid cases showed balanced aneusomy. The determination of ploidy value combined with FISH analysis can predict wether a GCTB will recurre or not. We could demonstrate possible TAS by interphase-FISH. TAS or 11p deletion was present in all of our cases. Benign GCTBs contained 11p telomeric alterations regardless of ploidy-value or clinical outcome. TAS or 11p deletion appeared more often in malignant cases.

047 Clinical and morphological aspects in osteoarticular tuberculosis. A 12 year study P Sarbu1, D Radulescu1, S Stolnicu2, T Petres1, E Carata3, P Botez1 1 University of Medicine and Pharmacy ‘‘Gr. T.Popa’’ Iasi, Romania, 2 University of Medicine and Pharmacy Targu Mures, Romania, 3 Techical University ‘‘Gh. Asachi’’ Iasi Romania Background: Osteoarticular tuberculosis diagnosis is not so frequent. The authors present 16 cases of osteoarticular tuberculosis which was diagnosed in a 12 year period. The distribution among sexes was almost even while the mean age was of 39 years (range between 18 and 72). The lesions showed various locations: shoulder joint (one case), elbow joint (three cases), hip joint (one case), knee joint (three cases), sacrum-ilium joint (five cases), wrist joint (two cases) and metatarso-phalangeal joint of the hallux (one case). Design: Four patients recorded tuberculosis history, one of them being diagnosed with fistulized elbow osteoarthritis and reactivated pulmonary tuberculosis. The onset of the articular disease varied as follows: 11 patients had an insidious onset, four patients showed traumatic episodes and one presented a fracture (elbow joint) which led to diagnosis. In nine cases, the diagnosis was suggested by clinical and radiological examination, later confirmed by histopathologic examination. In four cases, clinical exam suggested the tumor aspect (of the first metatarseal head – one case, knee joint – one case), of an arthrosynovial cyst (one case) or of a fracture (of the humeral head), diagnosis being sustained exclusively by histopathological examination. A specific treatment was applied for a period of minimal 6 months together with surgical treatment (synovectomy, exeresis of epiphyseal-metaphyseal foci). Results and conclusion: The results consisted in healing with various degrees of articular functional limitation. Arthrodesis was necessary in one case with metatarso-phalangeal arthritis with severe cartilage and bone destructions. One case with severe secondary coxarthrosis received a total hip arthroplasty at few years after onset.



048 Massive bleeding after biopsy of plasmocitoma. A case report P Sarbu1, D Radulescu1, S Stolnicu2, T Petreus1, E Carata3, P Botez1 1 University of Medicine and Pharmacy ‘‘Gr. T.Popa’’ Iasi, Romania, 2 University of Medicine and Pharmacy Targu Mures, Romania, 3 Techical University ‘‘Gh. Asachi’’ Iasi Romania The authors show the case of a 37 old man with a lytic lesion of the right trochanteric region which suggested a bone cyst, after CT examination and full skeletal bone survey. During surgical biopsy by minimal approach, a massive life-threatening bleeding had occurred; it was controlled with difficulty with pressure and packing with fibrin sponge. After pathologic finding of a plasma cell tumor, the studies of serum and urinary proteins and examination of bone marrow showed a multiple myeloma. A pathological fracture occurred and was treated by intramedullary osteosynthesis without opening the fracture site; the patient followed a protocol with radiation and chemotherapy. It is most likely that the excessive bleeding was related to the friable nature of tumor rather than a systemic coagulopathy. In summary, when investigated a patient who possibly has an osseous plasmocytoma, the risk of massive bleeding with biopsy should be considered. Appropriate serum and urinary protein studies and examination of bone marrow may then make the biopsy unnecessary.

049 Membranous lipodystrophy: case report F Souza, H Moura, M Lima, A Monteiro Jr, R Kalil Sarah Network of Rehabilitation Hospitals, Brazilia, Brazil Background: Membranous lipodystrophy (MLD) or Nasu-Hakola disease is a rare autosomal recessive disorder most commonly reported in Japanese and Scandinavian populations. It is characterized by the deposition of lipid eosinophilic membranous material in the central nervous system and bones. The pathogenesis is unknown but it is believed to be an enzyme defect of glicolipid metabolism. Design: We report a case of a 33-year-old man with multiple fractures at the toes, ankle and right hand due to MLD and emphasize the importance of the morphological diagnosis in patients with multiple and symmetric lytic bone lesions associated with progressive neurological disorders. Results: A 33-year-old man presented with multiple fractures at the toes, ankle and right hand. Roentgenograms showed diffuse areas of bone resorption and magnetic resonance imaging (MRI) showed high intensity signal at these sites, with fat tissue characteristics. Bone biopsy from the left foot showed a histological and ultrastructural pattern characteristic of the disease: abnormal material between fat cells of the bone marrow in the shape of amorphous, eosinophilic undulating membrane-like structures and fluid-containing cavities. In neuropsychological examination there was memory loss for recent facts. Computed tomography of the brain showed calcifications in the bilateral lentiform nucleus and thalami, and MRI imaging showed brain-cerebellar volume reduc-

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tion. Parents are first grade cousins and they had two children deceased with neurological regression. Conclusions: The coexistence of neuropsychiatric symptoms associated with multiple symmetric bone lesions suggests the diagnosis of MDL. We present a case, with bone and brain image lesions consistent with this diagnosis, confirmed by histological and ultrastructural analysis.

050 Unicameral bone cysts contain amianthoid-like fibers S Vernon, B Acar Department of Pathology, University of Miami, Miami, FL, USA Background: Unicameral bone cysts (UBC’s) have a distinctive radiographic and histologic appearance, typically presenting as expansile, fluid-containing defects of tubular and flat bones. In most cases the cysts have a lining composed of thin fibrous tissue, with scant inflammatory cells, and up to half of the cases may contain eosinophilic, fibrin-like material which may become ossified, resulting in the incorrect appellation ‘cementoma’ of long bones. In this study we investigated the nature of this fibrin-like eosinophilic material in clinical samples, using conventional light microscopy, immunohistochemistry, and transmission electron microscopy. Design: All cases of unicameral bone cysts from the University of Miami/Jackson Memorial Hospital Department of Pathology from 1995 to 2006 were reviewed; 14 cases were found to be acceptable as UBC’s. Slides and paraffin blocks were reviewed, and sections were stained with hematoxylin and eosin, trichrome, phospho-tungstic acid hematoxylin (PTAH) and reticulin stains. Immunohistochemical staining for fibrin/fibrinogen was performed using standard techniques. Samples were obtained from the blocks, deparaffinized, and re-processed for electron microscopy. Ultra-thin sections were stained with uranyl acetate and lead citrate, and viewed on a Joel Transmission electron microscope. Results: All cases of UBC shared the following features: (i) absence of any epithelial or synovial lining of the cyst cavity; (ii) flattened to cuboidal cells in the lining and adjacent tissues; (iii) histiocytes and scattered inflammatory cells; and (iv) scattered small blood vessels. In seven of the 14 cases there was also amorphous, eosinophilic, usually fibrillar material, often blending imperceptibly with ossified tissue. The pattern was not typical of woven bone, but rather one of a more subtle mineralization of the eosinophilic material. Trichrome and reticulin stains of this material were consistent with the characteristics of collagen, and a PTAH stain did not show the typical staining pattern for fibrin. Immunohistochemical staining for fibrin/fibrinogen was also negative. By electron microscopy, the material was seen to be composed of thick giant collagen fibers, with an unusual branching pattern, similar to those reported in osteoarthritic cartilage or chondrosarcoma, and previously designated as ‘amianthoid fibers’. Conclusions: Our findings substantiate that the eosinophilic material often seen in UBC’s is composed of altered collagen bundles. This conclusion is supported by light microscopic observations, immunohistochemistry, and transmission electron microscopic findings. Contrary to suggestions in many previous publications, this material is not fibrin or old fibrin coagula, and


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does not resemble cementum, but rather more closely resembles amianthoid fibers, with some cases showing gradual ossification. Similar collagen profiles have been described in spindle cell tumors now designated as ‘palisaded myofibroblastoma with amianthoid fibers’. The precise nature of these fibers is uncertain, but some authors believe they are the result of aging or degenerative processes.

051 Expression of c kit and c erbB2 in osteosarcoma Ewing’s sarcoma rhabdomyosarcoma and correlation with clinical parameters G Yu¨zer, B Bilgiç, K Kiroglu Department of Pathology, Istanbul University, Istanbul Medical Faculty, Istanbul, Turkey Background: Osteosarcoma, Ewing’s sarcoma and rhabdomyosarcoma are the three most common bone and soft tissue sarcomas in children and young adults. Almost 20 different prognostic factors have been analyzed in many studies of these group tumors over the last 20 years. The conventional treatment of these tumors includes neoadjuvant chemotherapy, surgery and sometimes, addititional irradiation. Despite of the fact that advances in the therapy of these tumors increase the overall survival, still a substantial number of patients will soon develop a metastatic disease and/or respond weakly to chemotherapy, with the result of poor outcome. Since the recent development of biologic agents targeting oncogenes, such as Trastuzumab, which targets the epidermal growth factor receptor 2 (C-erbB2) and Imatinib mesylate, which is a specific inhibitor of tyrosine kinases including c-kit, abl

and platelet derived growth factor receptor, increasing attention has been focused on determining the role of these receptors in the pathogenesis of tumors. The aim of our study were two-fold: first, to assess the frequency of c-erbB2 and c-kit expression in a series of osteosarcoma, Ewing’s sarcoma and rhabdomyosarcomas and second to correlate the results with clinical parameters and prognosis. Design: Our study included 25 osteosarcoma, 25 Ewing’s sarcoma, 25 rhabdomyosarcoma patients who were diagnosed and treated in Istanbul University Medical Faculty, from 2000 to 2006. Expression was assessed using immunohistochemical stains for c-erbB2 and ckit on formalin-fixed, parafin- embedded tissue sections. Additionally we performed CISH (Chromojenic in situ hybridization) to six rhabdomyosarcoma biopsies which were showed positive immmunostaining to c-erbB2. Results: Positive staining of c-kit was observed in 11 of 25 (44%) osteosarcomas, 11 of 25 (44%) Ewing’s sarcomas and 15 of 25 (60%) rhabdomyosarcomas. Only the overexpression of c-kit in osteosarcomas was significantly associated with poor prognosis (P < 0.05). In all osteosarcomas and Ewing’s sarcomas cases the immunostaining for c-erbB2 were negative. Positive cytoplasmic staining of c-erbB2 was observed in seven of 25 (28%) rhabdomyosarcomas. In one of six rhabdomyosarcomas c-erbB2 gen amplification by CISH was detected. There were no any correlation between c-erbB2 and c-kit expression between clinical parameters in these group tumors. Conclusions: Although expression of c-kit in osteosarcomas was found to be a negative prognostic factor, therapies based on antibodies directed against c-kit and c-erbB2 are unlikely to have much value in the treatment of osteosarcoma, Ewing’s sarcoma and rhabdomyosarcomas.



BREAST PATHOLOGY 052 Study of expression of P-glycoprotein (MDR-1) in breast infiltrating ductal carcinoma (NOS) and its correlation with some clinicopathologic parameters R Al-Areqee Faculty of Medicine, Alexandria University, Alexandria, Egypt Background: In Egypt, breast cancer is the most common cancer among women, representing 18.9% of total cancer patients. Breast cancer is often considered to be one of the most chemoresponsive solid tumors and adjuvant chemotherapy is an integral part of multimodality approach for the local and systemic management of locally advanced and metastatic breast cancer. The failure of the curative treatment of the breast cancer patients often occurs as a result of intrinsic or acquired drug resistance of the tumor to chemotherapeutic agents. One of the most important mechanisms of drug resistance is P-glycoprotein (P-gp) expression in breast cancer cells. To Determine the expression of P-glycoprotein in the primary female breast infiltrating ductal carcinoma (IDC) patients and in the recurrent IDC patients. To correlate the expression of P-glycoprotein with other clinical & pathological Parameters: age, histological grade, nuclear grade, lymph node status, molecular subtypes, ER, PR and Her2. To correlate the expression of P-glycoprotein and other clinical & pathological parameters with the response to endocrine and/or chemotherapies. To ascertain whether pretreatment detection of P-glycoprotein in patients with breast cancer could be utilized as a reliable predictor of response to adjuvant chemotherapy. Design: This is a retrospective study. Sixty-three patients were classified into two main groups. Group-1: thirty nine cases of IDC. Group-2: twenty-four cases of recurrent IDC. After data collection, the Group-1 cases were followed retrospectively for period of two after the mastectomy operation. Immunohistochemical study for [Pgp(MC57, Chemicon), ER, PR and Her2(Dako)] were done. Classification of Group-1 cases into molecular subtypes according to immunohistochemical results into: luminal A, luminal B, Her2/neu and triple-negative. Histological examination before and after Immunohistochemistry were performed. Univariate and multivariate (logistic regression) analysis of predictors of recurrence was performed. Cox regression model for prognostic factors of recurrence was also done. Results: Positive correlation between P-gp and Her2 was found (P = 0.027 odds ratio 8.8). About 66.6% of Group-1 expressing Pgp whereas 62.5% of Group-2 cases expressing P-gp. Significant association was found between high nuclear grade (GradeIII) and ER negativity (P = 0.002), PR negativity (P = 0.017) and Her2/ neu molecular subtype (P < 0.0001) in both Group-1 and Group-2 cases. ER negativity (P = 0.005), high nuclear grade (P = 0.06) and triple negative molecular subtype (P = 0.05) were significant predictors for local recurrence in univariate analysis while triple negative (P = 0.045, Odds ratio = 9.05) was independent predictor for local recurrence in multivariate analysis. Positive lymph nodes was significant predictor for distant metastasis (P = 0.03). High nuclear grade (P = 0.0065), positive lymph node status (P = 0.0137) and Her2/neu and triple-negative molecular subtypes (P = 0.045 and 0.04 respectively) were associated with poor dis-

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ease free survival (DFS). ER negativity (P = 0.009), nuclear grade three (P = 0.06) and triple negative subtype (P = 0.017) were associated with poor local recurrence free survival (LRFS). Her2 overexpression (P = 0.06) was associated with poor distant-metastasis free survival (DMFS). Patient with Luminal B, Her2/neu or triple-negative who expressed P-gp were associated with poor DFS (P = 0.05, 0.005 and 0.0075 respectively). Patients who were P-gp positive and received FAC regimen (fluorouracil, doxorubicin and cyclophosphamide) were associated with poor DFS (P = 0.009). Cox regression hazard model for DFS revealed that patients with Her2/neu IDC had 10.5 times risk having recurrence compared with luminal A patients [P = 0.015 hazard ratio(HR)=10.5 at 95% CI] whereas patients with triple-negative subtype had 6.3 times risk having recurrence (P = 0.007 HR = 6.3) compared with luminal A IDC patients. Conclusions: Significant positive correlation between P-gp expression and Her2 overexpression in patients with IDC. P-gp overexpression is associated with failure of FAC regimen in patients with Her2 positive and triple negative IDCs. Her2/neu and triple negative molecular subtypes were significant independent factors of poor DFS in patients with IDC. Other factors such as high nuclear grade, ER-negativity and positive lymph node status were significant predictors for recurrence. ER negativity, high nuclear grade and triple-negative molecular subtype were associated with poor LRFS. We recommend pretreatment detection of P-gp in patients with Her2-overexpressing and triple negative IDCs.

053 Differential expression of cytokeratins 903 and 5/6 antibodies in breast cancer tissue compared to adjacent benign epithelium C Albarracin, R Ricketts, S Willer, Y Li, L Huo, Y Wu, E Resetkova Department of Pathology, University of Texas, MD Anderson Cancer Center, Houston, Venezuela Background: The development of breast cancer is a multi-step process and samples often contain a spectrum of changes including benign hyperplasia, atypia, in situ carcinoma, and invasive carcinoma. Several reports suggest that antibodies to keratins, cytokeratins 903 (CK903) and cytokeratin 5/6 (CK5/6), are differentially expressed in benign breast tissue vs. cancerous tissues. Our goal is to determine whether CK903 and CK5/6 immunostaining are progressively altered in carcinoma and adjacent benign glands within the same case. Design: Eighty two patients with invasive carcinoma, 53 with invasive lobular (ILC) and 29 with invasive ductal (IDC), were enrolled in our study. Whole tissue sections containing cancerous (invasive and in situ component) and adjacent benign breast tissue within one slide were stained in 22 ILC and 21 IDC patients, respectively. Ductal carcinoma in situ (DCIS) was present in 19 cases. Additional 31 ILC and eight IDC cases were incorporated into tissue microarrays (TMA). Immunohistochemistry was performed using monoclonal antibodies to CK903, which react with cytokeratins 1, 5, 10, and 14 (clone 34bE12, dilution 1:100, Dako, Carpinteria, CA, USA) and CK 5/6 (clone D5/16 B4, dilution 1:50, Dako, Carpinteria, CA, USA), on formalin-fixed paraffin-embedded tissues


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using the Bond Max IHC staining protocol with iView DAB detection and antigen heat retrieval. Positivity for both keratins was defined as >10% of stained cells. The percentage of immunostained cells within benign glands, and in situ and invasive carcinoma were determined. Results: CK903 staining was positive in adjacent benign glandular epithelium and in 17/21 (81%) IDC and 18/19 (95%) of DCIS in whole tissue sections. On TMA, CK903 was positive all eight IDC (100%). CK903 was also positive in 20/22 (91%) of ILC cases in whole sections and in 23/31 (74%) of ILC on TMA. CK5/6 was positive in benign glands and showed heterogeneous staining of epithelial cells and prominent staining of myoepithelial cell layers. Myoepithelial staining for CK5/6 was also positive in DCIS but neoplastic epithelial cells were negative. CK5/6 was negative in all 21 IDC patients in the whole tissue sections and six of eight IDC on TMA. CK5/6 was also negative in all 53 ILC, irrespective of tissue sample. Conclusions: Our study demonstrated that CK903 staining is positive in the benign glandular epithelium as well as adjacent invasive ductal and lobular carcinomas in 37/43 (86%) of cases. In contrast, CK5/6 staining was negative in neoplastic cells of both invasive ductal and lobular carcinomas as compared to adjacent benign glands. These results support the utility of CK5/6, but not CK903, in marking the transition of benign to malignant carcinoma.

054 The expression of CD44 in papillary and ductal invasive carcinomas of the breast D Anestakis1, I Dimitriadis1, C Tsobanidou1, G Sibilidis2, F Patakiouta1 1 Pathology Department ‘‘Theagenio’’ Cancer Hospital, 2Surgery Department ‘‘Theagenio’’ Cancer Hospital, Thessaloniki, Greece Background: Expression of D44 has been shown to correlate with the progression and prognosis of some malignant tumors. The purpose of this study was to evaluate the expression of CD44 in papillary and ductal invasive carcinomas of the breast and to analyze the potential of this expression as a diagnostic and prognostic indicator. Design: In this preliminary report, 48 patients with tumors of the breast treated surgically were examined retrospectively. All patients were female with an age range of 34–80 years (mean age 57 years). The tumors were 19 invasive papillary carcinomas and 29 ductal invasive carcinomas of the breast and were graded according to the modified Bloom and Richardson criteria. The surgical tumor specimens were formalin fixed, paraffin embedded and immunostained for CD44 (clone DF1485 Dako dilution 1:50). The positivity of CD44 was based on percentage of stained epithelial cells and was scored and categorized as £10% (I), 10–70% (II), >70% (III). The normal breast tissues surrounding the lesions served as control. Results: All the cases of the papillary carcinomas showed positivity (100%) for CD44. The majority of them (14 of 19) expressed CD44 in 70%). In contrast the majority of papillary carcinomas cases (14 of 19) expressed this marker in 70%)

14 (73.7%)

5 (26.3%)

0

3 (10.4%)

5 (17.2%)

7 (24.1%)

Conclusions: As shown in this study, the expression of CD44 was more commonly found in invasive papillary carcinomas than in invasive ductal carcinomas of the breast. In contrast, ductal carcinomas have a higher proportion of positive cells than papillary carcinomas. These results suggest that the study of expression of CD44 could probably help in more precise diagnostic approach of carcinomas of the breast. Expression of CD44 could also be a prognostic factor in patients with invasive breast carcinoma and in certain cases could contribute in better planning of the therapeutic confrontation of the patient.

055 Intraductal papilloma with ductal carcinoma in situ: analysis of four cases A Arakawa, T Hayashi, Y Horimoto, T Yao Department of Human Pathology and Breast Center, Juntendo University School of Medicine, Tokyo, Japan Background: Papillary lesion of the breast is still one of the most challenging category in breast pathology. There are many different terminologies to describe in papillary lesions. Particularly, it is very difficult to make diagnosis of the lesions of intraductal papilloma with atypical ductal hyperplasia or with ductal carcinoma in core needle biopsy, even if in surgically resected specimens. We studied that the role of immunohistochemical stains as a tool of diagnosis of these lesions, and studied clinicopathological features of these lesions. Design: We selected four cases of intraductal paplloma with ductal carcinoma in situ, which was surgically resected at the breast center of our university hospital during 2005–2007. Immunohistochemical stains for myoepithelial marker (smooth muscle actin, CD10), proliferative marker (ki-67), p53, D2-40 and neuroendocrine markers (synaptophysin, chromogranin, NSE, CD56). Results: Mean age of our patients with ductal carcinoma in situ in ductal papillama was 41.25 years old. The maximum diameter of ductal carcinoma in situ in papilloma was 5–7 mm. In one case,



neuroendocrine markers were positive in ductal carcinoma in situ area. No patient with these lesion recurred. Conclusions: The patients with intraductal papilloma with ductal carcinoma in tissue were relatively in young age. The foci of ductal carcinoma in situ were very small area in each papillary lesions. There is no patient with invasive ductal carcinoma component in these specimens. Myoepithelial markers and neuroendocrine markers were helpful to evaluate these lesions, particularly in ductal carcinoma in situ component. Although many papillary lesions can be made a diagnosis accurately by using examination of H&E stained sections alone, other markers (particularly staining for myoepithelial marker) are required to reach correct diagnosis of these papillary lesions. And it will be also very useful and helpful to perform immunostains for myoepithelial markers and neuroendocrine cell markers to make diagnosis in core needle biopsy specimens.

056 Candidate stem cell marker ALDH1 expression and relationship with BRCA-mutations and basal-like differentiation in breast carcinoma J Arnes1, W Foulkes2, L Akslen1 1 The Gade Institute, Section for Pathology The University of Bergen, Bergen, Norway, 2Department of Medicine McGill University, Montreal, Canada Background: The cancer stem cell hypothesis indicates that malignant tumors are supported by a small population of tumorigenic cancer stem cells, and characterization of tumor stem cells could identify relevant treatment targets. Aldehyde dehydrogenase 1 (ALDH1), which detoxifies intracellular aldehydes, can identify hematopoietic and neural stem cells, as well as putative cancer stem cells in the breast. Basal-like cancers (ER- Her2- CK5+) share some characteristics with mammary progenitor and stem-cells. BRCA1 has been proposed as a stem cell regulator in breast tissue, and is associated with basal-like cancers. Research by Liu, et al. found that ALDH1 over-expression was related to loss of BRCA1 function, and ALDH1 was also an adverse prognostic factor. Our study explores immunohistochemical expression of ALDH1 in breast carcinoma, using a series of BRCA1 mutated, BRCA2 mutated and non-BRCA mutated breast cancers. Design: In a case-control design, a series of breast carcinomas containing 53 cases with BRCA1 mutations and 45 cases with BRCA2 mutations was compared to a control group of 104 cases with no mutations. Mutation carriers were all Canadian Ashkenazi Jewish women, while the control group was supplemented by Norwegian cases. Using tissue micro-arrays (TMA), slides were stained for ALDH1, and staining results were scored using a staining index (SI) defined as the product of intensity (0–3) and area scores (1 50%). Where appropriate, the series median was used as cut-point. SPSS version 15 was used for statistical analysis. Results: One hundred and ninety-two cases could be evaluated. Stains were clear with a cytoplasmatic staining pattern. Various stromal components were ALDH1+ (microvessels, fibroblasts, lymphocytes and macrophages). 31/192 cases (15.3%) were ALDH1+ in tumor cells. We found significant associations with higher histologic grade (P = 0.015), mitosis counts (P = 0.029), p53

25

(P = 0.027) and the core basal-like profile (ER- Her2- CK5+) (P = 0.040). A highly significant association with VEGF-A expression (P = 0.003, OR 5.22) was also found. There were no significant associations between BRCA-mutation status and ALDH1. Conclusions: New research links ALDH1 to breast somatic and cancer stem cells, as well as BRCA1 dysfunction. In a case-control design, our studies indicate an association with basal-like cancers, but not with BRCA1 mutations as such. Interpretation of ALDH1 immunostaining in tumor cells must be done with care to exclude stromal elements including microvessels. The scarcity of positive tumor cells would be in accordance with an expected low fraction of cancer stem cells, and could support the concept of basal-like cancers resulting from stem-cell dysfunction. The strong association with VEGF-A expression and the staining of ALDH1 in tumor stromal cells could indicate a role of ALDH1 as a marker of an angiogenic phenotype. Further studies are warranted to explore these issues.

057 Analysis of BRCA1 expression patterns in breast carcinomas and correlation with other molecular markers M Aschie, A Papuc, A Craciun, A Dobre, A Iliesiu, I Poinareanu, D Badiu, A Cretu, I Aschie Constanta Clinical County Emergency Hospital, Constanta, Romania Background: Breast carcinomas still represent an important cause of death in women, despite the important discoveries in the genetics and etiology of this malignancy. BRCA1 is an important tumor suppressor gene involved in the etiology of familial and sporadic breast carcinoma and participates together with BRCA2 in DNA doublestrand break repair. Design: This paper investigates the immunohistochemical detection of BRCA1 protein expression in sporadic breast carcinomas and its relations with other molecular markers including ER, PR and HER2 status. For this purpose a total of 58 cases of formalin-fixed paraffin – embedded tissues of breast carcinoma were selected and immunostained using monoclonal antibody to BRCA1, ER, PR, HER2, p53 and Ki67. Results: We found high-level expression of BRCA1 protein in normal mammary epithelium and various degrees of reduced expression in breast carcinoma. 53% of breast carcinoma showed reduced BRCA1 protein expression. This reduced expression correlates with ER and PR negative status, HER2 over expression, high expression of p53 and Ki 67. Also we observed an association between reduction of BRCA1 expression and poor carcinoma differentiation. Conclusions: Our results suggest that BRCA1 may play an important role in sporadic breast carcinogenesis and emphasizes the potential value as prognostic marker.


26


058 Cytoplasmic expression of ALDH1 correlates with breast cancer subtypes S Badve1, M Thorat1, J Reis-Filho2 1 Department of Pathology and Laboratory Medicine IU School of Medicine, Indianapolis, USA, 2Molecular Pathology Laboratory, The Breakthrough Breast Cancer Research Centre Institute of Cancer Research, London, UK Background: Cancer stem cells (CSCs) have been hypothesized to cause initiation, progression and recurrence of cancer. CSCs are identified by expression of CD44 and CD24 surface markers. Alcohol Dehydrogenase (ALDH1), an enzyme linked cellular differentiation through retinoic acid synthesis, has been recently identified as a CSC marker in breast cancer. We analyzed ALDH1 expression in breast cancer by immunohistochemistry (IHC), studied its correlation with various clinico-pathological and molecular variables and survival. Design: IHC using mouse monoclonal ALDH1 antibody (dilution 1:200, BD Biosciences, San Jose, CA, USA) was performed on a tissue microarray (TMA) comprising 245 invasive breast carcinomas obtained from patients treated with curative surgery and adjuvant anthracycline chemotherapy. Intensity (0, 1, 2, and 3) and percentages of cells showing nuclear and cytoplasmic expression were recorded. Intensity scores of 1, 2 or 3 were considered as positive staining. Data were analyzed for correlation with clinico-pathological and molecular variables; survival analysis was performed for both cytoplasmic and nuclear staining. Results: Interpretable ALDH1 staining was obtained in 194 cases; 35 and 16 cases showed cytoplasmic and nuclear expression of ALDH1 respectively. After adjusting for multiple comparisons, cytoplasmic ALDH1 expression correlated positively only with basal and HER2 subtypes (P = 0.012); it did not correlate with other parameters like tumor size, histology, grade, nodal status, LVI, ER, PgR, HER2, MIB-1, basal cytokeratins, p53, EGFR, TOPO2A amplification cyclin-D1, MYC and caveolin-1, 2. No statistically significant correlations were observed between nuclear ALDH1 expression and any variables studied. Cytoplasmic or nuclear expression did not correlate with disease-free, metastasis-free or overall survival. Conclusions: ALDH1, a putative CSC marker, correlates with basal and HER2 subtypes. Lack of correlation with tumor size, nodal status, disease-free and metastasis-free survival questions the role of ALDH1 expressing cells in breast cancer progression and recurrence. It is possible that ALDH1 is a marker of basal cells with high proliferation rather than true CSCs.

059 The morphological and immunophenotypic analysis of estrogen negative invasive breast carcinomas F Bahadır, Z Gu¨cin, N Dursun, M Ozcan, E Bozkurt Ministery of Health Istanbul Training and Research Hospital Pathology Department, Istanbul, Turkey

special type (IDC-NST). The majority of those have a luminal phenotype, that is they express similar proteins to those found in the normal proteins to those found in the normal inner luminal cells of breast, ducts and acini and such as express ER, CK8,18,19. But 2– 18% also coexpress proteins normally found in the outer contractile myoepithelial or basal layer of normal breast duct and acini. These proteins include high molecular weight cytokeratins: CK14, CK5/6, CK17, Smooth muscle markers; such as SMA, Calponin, p63. These tumors are usually ER(-), PR(-), HER-2(-) (Triple negative tumors). There are not present identified in routine practice and there is no consensus terminology about these tumors. In recent years, gene expression studies using DNA microarrays have identified several distinct breast cancer subtypes that are associated with different clinical outcomes. Design: The purpose of this study was to determine the characteristics of 48 ER-negative breast cancers through analysis of several morphological, prognostic variables and immunophenotypical profile and research the HER-2 positive and triple negative/basal like subsets and their frequency, common and diverse features by routin morphologic and immunohistochemical examination. 48 ER (-), 15 ER(+) invasive breast carcinoma cases with retrievable histological material (blocks and slides) were included in this study. An H&E stained section from a representative tumor block was examined for the various morphological parameters such as appearance of tumor margin, the presence of lymphoid stromal infiltrate, clear cell changes, basaloid change, adenoid cystic pattern, undifferentiated/small cell pattern, comedo-type necrosis, squamoid or spindle cell changes, presence of tumor giant cells and prominent central fibrosis/necrosis. A representative tissue sections sample was stained to study the expression of various antigens by immunohistochemistry. The antibodies utilized included ER, PR CK5/6, CK18, CK14,CK17, SMA, BRCA1, CerbB2, p63 and Beta cathenin. Calponin, SMA, p63 were utilized to identify myoepithelial differentiation, CK5/6,CK14,CK17 which identify basal, CK18 luminal epithelial differentiation respectively were also included. Results: High grade, lymphoid stroma, comedo-type necrosis and central fibrosis/necrosis are the dominant morphological findings. In addition, squamoid, clear cell and spindle cell differentiation may be identified. The majority of these lesions are grade 3 carcinomas, a finding that correlates with the presence of lymphoid stroma and comedo-type necrosis. Alterations in protein expression of basal cytokeratins, SMA, calponin and BRCA-1, Beta Cathenin are also more commonly seen than ER(+) invasive breast cancers. Conclusions: These results strongly support the notion that different tumor subtypes originate from different cell types, ie, luminal epithelial cells and basal/myoepithelial cells. These subtypes are: luminal A, luminal B, Basal-like, HER2 overexpressing and Normal breast like. The worst prognosed group is Basal like and HER-2 (+) group tumor. Basal like berast carcinomas can escape observation in routine practice or not realized because of wrong identification however these tumor groups are candidates for future treatment models.

Background: Breast carcinoma are categorized with in a single, histological diagnostic category, of invasive ductal carcinoma of no 2008 The Authors. Journal Compilation 2008 Blackwell Publishing Ltd, Histopathology, 53 (Suppl. 1), 1–432


060 EXpression of deleted in breast cancer 1 and SIRT1 are associated with poor prognosis of breast carcinoma H Baik, K Kim, S Noh, H Kim, H Park, M Chung, M Kang, D Lee, W Moon, K Jang Department of Pathology, Chonbuk National University, Medical School and Center for Healthcare Technology Development, Jeonju, Chonbuk, Korea Background: Sirtuin1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent deacetylase. In recent reports, cancer tissues have shown higher endogenous levels of SIRT1 expression compared with normal tissues. Deleted in breast cancer 1 (DBC1), initially cloned from a region 8p21 homozygously deleted in breast cancer, promotes p53-mediated apoptosis through specific inhibition of SIRT1. However, recent DNA microarray datasets revealed that DBC1 was upregulated in breast cancers compared with normal breast tissues. The expression of DBC1 and SIRT1 and their roles in breast carcinoma have not yet been examined, thereby forming the basis for this study. Design: To assess the clinical significance of the expression of DBC1, SIRT1 and p53 in breast cancer patients, immunohistochemical staining for DBC1, SIRT1, p53, and estrogen receptor (ER) were performed by using a 3 mm core from 122 breast cancer patients for tissue microarray. Immunohistochemistry for ER were used for the classification of breast carcinoma into ER-negative and ER-positive types. Results: Positive expression of DBC1 and SIRT1 were seen in 73% (89/122) and 72% (88/122) of patients. Expression of DBC1 was significantly correlated with expression of SIRT1, p53, and ER (P < 0.001, P = 0.043 and P = 0.042 respectively). In total breast cancer patients, high tumor stage (P = 0.003) and expression of ER (P = 0.04), DBC1 (P = 0.014), SIRT1 (P = 0.013) and p53 (P = 0.001) were significantly associated with shorter overall survival (OS) by univariate analysis. Tumor stage, DBC1 and p53 were also an independent prognostic factor by multivariate analysis (P < 0.001, P = 0.012 and P = 0.003 respectively). In the ER-negative type, only tumor stage was associated with a significantly shorter OS by univariate analysis (P = 0.007). In the ER-positive type, p53 expression was associated with a significantly shorter OS by univariate analysis (P < 0.001) and expression of DBC1 and SIRT1 were marginally significant shorter OS by univariate analysis (P = 0.062 and P = 0.069 respectively). Conclusions: A high percentage of breast carcinomas showed expression of DBC1 and SIRT1; this expression was correlated with the OS of breast cancer patients, especially for the ER-positive type.

061 Can KI-67 and p53 expression predict the molecular subtypes of primary invasive mammary carcinoma? R Bhat, S Soundararajan, F Garcia Department of Pathology, Drexel College of Medicine, Philadelphia, USA Background: To study the of value Ki67 and p53 expression in different subtypes of invasive mammary carcinoma as defined by immunohistochemistry (IHC).

27

Design: 1641 consecutive primary invasive breast carcinoma cases which cancer prognostic panel performed at DUCOM Pathology from 1997–2008 were divided into Luminal A (ER+, and/or PR+, HER2), Luminal B (ER+ and/or PR+, HER2), Basal like (ER-, PR-, HER2), and HER2 (ER-, Her2) as defined by IHC, FISH (HER2) and image analysis. The image analysis values for each specific subtype were recorded. Statistical analysis was done with one-way ANOVA (P < 0.05). Results: The cases were classified into four subtypes: Luminal A: 909 (55.42%); Luminal B: 146 (8.9%); Basal like: 414 (25.2%) and HER2: 172 (10.5%). Luminal A and B show significantly lower expression of Ki67 and p53 compared to Basal and HER2 subtypes. Basal like tumors express the highest level of Ki67, with Luminal A expressing the least. There is no significant difference in p53 expression between Basal and HER2 subtypes. Luminal A Number of

Luminal B

909 (55.42%) 146 (8.9%)

Basal like

Her2/neu

414 (25.2%)

172 (10.5%)

cases Ki-67 in %,

18.4 ± 18.41

24.25 ± 16.6*$ 44.51 ± 28.73*$ 39.25 ± 23.15$

6.16 ± 17.17

12.87 ± 21.25* 32.34 ± 37.82$

33.43 ± 33.84$

6.16 ± 17.17

12.87 ± 21.25* 32.34 ± 37.82$

33.43 ± 33.84$

Mean ± SD p53 in %, Mean ± SD p53 in %, Mean ± SD One way ANOVA, between groups P < 0.001 *Luminal A vs. Luminal B; Basal vs. HER2 P < 0.05 $Luminal A vs. Basal, Luminal A vs. HER2, Luminal B vs. Basal, Luminal B vs. HER2 P < 0.01

Conclusions: (1) Luminal A appears to be the most frequent subtype followed by Basal like. (2) The finding of high Ki-67 and p53 in a mammary carcinoma predicts either Basal like or HER2 subtypes.

062 Prediction of nodal status of pt1b and pt1c primary breast carcinoma using KI-67 and P53 immunohistochemistry R Bhat, S Soundararajan, F Garcia Department of Pathology, Drexel College of Medicine, Philadelphia, USA Background: To examine the role of Ki67 and P53 expression in predicting nodal status in (pT1) breast carcinomas. Design: All PT1b and PT1c primary invasive breast carcinoma cases with known nodal status (171 out of 1641) at DUCOM, Pathology from 1997–2008 were used for this study. These were classified into PN0 and PN1or higher. Mean Ki67 and P53 expression levels were recorded for each group. Statistical analysis was done with one-way ANOVA (P < 0.05). Results: A significant increase in Ki67 expression was seen in PT1b-N1 tumors as compared to PT1b-No (27.4 ± 21.94 vs. 13.31 ± 13.25; P = 0.02). Increased P53 expression was seen with N1 status as compared to N0 status in both PT1b and PT1c tumors 24.86 ± 27.62 vs. 2.26 ± 4.15; P = 0.0002 for PT1b and 21.32 ± 30.96 vs. 16.03 ± 24.22; P = 0.043 for PT1c.


28


PT1b-N1

No. of

PT1b-No

or higher

26

11

13.31 ± 13.25

27.4 ± 21.94

PT1c-N1 P value

PT1c-No

or higher

83

51

20.56 ± 20.12

24.14 ± 18.34

P value

cases Ki-67 %

p53 %

(0–51.52)

(3.3–56.18)

2.26 ± 4.15

24.86 ± 27.62

(0–19)

(0–68.5)

0.02*

(0–83) 0.0002**

16.03 ± 24.22 (0–96)

0.3

(0–92) 21.32 ± 30.96

Conclusions: Membranous expression of E-cad in Paget cells associated with an underlying ductal malignancy, while less than that of surrounding keratinocytes, was nonetheless positive in all cases examined. These findings indicate that loss of E-cadherin expression is not the mechanism responsible for epidermal migration in mammary Paget disease with underlying ductal carcinoma. It is unclear what role it plays in rare cases with underlying lobular carcinoma.

0.043*

(0–99)

Conclusions: Ki-67 and p53 predict lymph node metastasis better in small (pT1b) tumors than large tumors (pT1c).

063 Mammary paget disease: E-cadherin and the Paget phenotype E Brooks, C Aubertine, H Blaszyk, D Weaver Department of Pathology Fletcher Allen Health Care / University of Vermont, Burlington, USA Background: The majority of mammary Paget disease has been mechanistically attributed to epidermotropic migration of neoplastic cells from an underlying malignancy, usually ductal in nature. E-cadherin (E-cad) is a calcium-regulated molecule important in epithelial cell-cell adhesion. Loss or reduced expression of E-cad has been found in several aggressive tumors. Little to no data has been published examining whether loss of E-cad expression in Paget cells may contribute to epidermotropic spread. In the present study, we evaluate the immunohistochemical expression pattern of E-cad in mammary Paget disease to assess whether E-cad loss plays a pathogenetic role in the disease. Design: Computer diagnosis search yielded 21 cases of mammary Paget disease accessioned from 1996–2006. Hematoxylin & Eosin stained slides were reviewed independently by two pathologists. E-cad immunohistochemistry (IHC) was performed on one block selected from each case. Results: All patients were female with a mean age of 57 years (range 35–84). The majority of cases (86%) had an underlying ductal carcinoma in addition to Paget disease, including ductal carcinoma in situ (DCIS) (6/21, 28.6%), invasive ductal carcinoma (IDC) (1/21, 4.8%), or both DCIS and IDC (11/21, 52.4%). Of the remaining cases, one was associated with an underlying DCIS and lobular carcinoma in situ (LCIS), one with invasive lobular, and one had no underlying malignancy. Of the cases with underlying DCIS, 16/18 (88.9%) had predominantly solid and/or comedo patterns; the next most common pattern was cribriform (7/18, 38.9%). All cases of both DCIS and IDC had moderate or high nuclear grade. IHC for E-cad revealed membranous expression in Paget cells in all cases associated with underlying ductal carcinoma (95% CI: 0.854–1.0) and in the single case with no underlying malignancy. In the many cases in which ductal carcinoma was present in the same section as Paget disease, the ductal carcinoma cells were also noted to express E-cad. In the single case of invasive lobular carcinoma, both lobular carcinoma cells as well as Paget cells showed no reactivity for E-cad (95% CI: 0.05–1.0).

064 Genotypic heterogeneity in breast carcinoma with Her-2/neu amplification sec. ASCO/CAP criteria M Brunelli, E Manfrin, K Miller, S Gobbo, A Eccher, G Martignoni, A Remo, D Reghellin, F Bonetti Department of Pathology, University of Verona, Italy, UK National External Quality Assessment Scheme, University College London, UK Background: To evaluate the genotypic intratumoral heterogeneity of Her-2/neu gene status in breast cancer with amplification using the recent ASCO/CAP recommended criteria. Design: We selected thirty consecutive invasive ductal breast carcinomas with Her-2/neu gene amplification on whole tissue sections by FISH and built two microarray blocks which targeted three different neoplastic areas. Specimens were determined to be amplified if the ratio of Her-2/neu signals to chromosome 17 centromere signals was higher than 2.2 according to ASCO/CAP recommended criteria. Polysomy of chromosome 17 (without Her-2/neu gene amplification) was defined as centromeric chromosome 17 spot count 3.0 or more in at least 80% of tumor cells. Moreover, among Her-2/neu amplified cases we distincted those with high grade (ratio ‡4.0) vs. those with a low grade amplification (ratio >2.2 · 80% of cells). Overall 2/46 (5%) triple negative breast carcinomas showed EGFR gene amplification. No one case negative for EGFR immunoreaction showed gene amplification. Conclusions: We report here that triple negative breast carcinoma usually do not show EGFR molecular overexpression; however EGFR molecular status has to be evaluated in order to select a small subset suitable for the tyrosine kinase inhibitor gefitinib.

066 Phyllodes tumor of the breast: a clinicopathologic and immunohistochemical study of 70 cases S Bulimbasic1, F Knezevic2 1 Department of Pathology Dubrava University Hospital, 2Pathologist Clinic for tumors Zagreb, Zagreb, Croatia Background: Phyllodes tumors (PTs) are rare fibroepithelial tumors of the breast with wide biological potential. Based on evaluation of stromal component, PTs are currently classified as benign, borderline and malignant. However, even with the use of the most recent classification recommended by World Health Organization, uniformity and reproducibility have not been entirely achieved. Design: Seventy cases of breast PT diagnosed at the University Hospital for Tumors, Zagreb in the period 1995–2006 have been analyzed. Immunohistochemical evaluation of Ki-67 proliferation index (PI) and p21 labeling index (LI) in both stromal and epithelial component, as well as assessment of mean vascular density (MVD) using CD31 have been performed, in order to determine whether these markers correlate with histological grades and if they can be

29

used for improvement of classification of PT. Differences in clinicopathologic features (age, localization, size of the tumor) and additional histological criteria (hyperpastic and metaplastic epithelial changes, pseudoangiomatous stromal hyperplasia, multinuclear stromal cells) have also been analyzed. Results: The study included 40 (57%) benign, 21 (30%) borderline and nine (13%) malignant PTs. Patients with borderline and malignant PT were older than those with benign PT (means 47.5 vs. 38.5 years; ANOVA P = 0.0074). Although borderline and malignant PT were larger than benign PT (means 5.0 vs. 4.1 cm), this difference was not statistically significant (ANOVA P = 0.0865). Differences in localization of the tumor and in additional histological parameters have not been found. There were significant differences in stromal Ki-67 PI and p21 LI among the three groups (ANOVA Ki-67 P < 0.0001; p21 P = 0.002), and significant correlation with malignant features of PT (Spearman Ki-67 P < 0.0001; p21 P = 0.0001), while epithelial Ki-67 PI and p21 LI showed no significant differences. Interestingly, differences in intensity of p21 staining were also noted. Overall, higher intensity of the nuclear staining has been observed in epithelial component, while stromal component showed more variable, less intensive pattern of nuclear staining. MVD showed significant difference among the three groups (ANOVA P < 0.0001) as well as significant correlation with tumor malignancy (Spearman P < 0.0001). Logistic regression model showed that stromal Ki-67 PI and MVD are the best predictors and in present sample with 97% confidence define tumor malignancy (Sensitivity 0.9667; Specificity 0.9750). Conclusions: Our results support previous studies and suggest that stromal Ki-67 PI and MVD are valuable additional parameters for histological grading of PT. They are applicable not only to surgically excised specimens, but also might be a useful tool for improvement of preoperative diagnosis of PT on core biopsies. Data concerning p21 expression in different types of PTs are limited. Although positive correlation of p21 PI and malignant type of PT was observed in our series and has been indicated by few previous studies, the small number of analyzed cases does not allowed us to make definitive conclusion. Further studies are needed to confirm those findings.

067 Invasive lobular carcinoma arising in malignant phyllodes tumor of the breast: a case report S Bulimbasic1, S Sitic2, I Penavic2, R Loncaric2, I Bobus-Kelcec2 1 Department of Pathology Dubrava University Hospital, 2Pathologist Clinic for tumors Zagreb, Zagreb, Croatia Background: Phyllodes tumor (PT) of the breast is a rare neoplasm that accounts for 1% of all breast tumors. According to World Health Organisation’s recommendation, PTs are classified into three groups: benign, borderline and malignant. Many PTs exhibit epithelial hyperplasia, but the epithelial abnormality rarely reaches a level of in situ or invasive ductal or lobular carcinoma. Design: A 63-year-old woman presented with a palpable, painless, well-circumscribed mass in the left breast. An ultrasound study and mammography results were suggestive of a fibroepithelial neoplasm. Palpation of the supraclavicular, internal mammary and


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axillary lymph nodes revealed no adenopathy. The remaining examination was unremarkable. Results: A segmental mastectomy revealed a 3 · 2 · 1.5 cm wellcircumscribed, firm, grey to tan tumor, with a few grossly identifiable large clefts containing leaf-like projections. Microscopic examination showed biphasic neoplasm consistent with malignant PT. The stroma comprising leaf-like projections was cellular, with large pleomorphic nuclei, numerous multinuclear cells, focal fibrosarcomatoid growth pattern and 10 mitotic figures per 10 hpf. The epithelial cells lining the cleft-like spaces exhibited all degrees of epithelial hyperplasia. Moreover, within the epithelial component, there was a proliferation of monomorphic cells that filled acinar structures, focally infiltrated stroma and grow in dishesive manner or forming indian-file structures. The individual cells had round, regular nuclei with inconspicuous nucleoli, homogenous pale cytoplasm and showed strong positive reaction to cytokeratin, ER and PR. Reaction to vimentin, E-cadherin and Her-2 was negative. Although the pushing growth pattern was predominant, both stromal and malignant epithelial component focally spread beyond the grossly appreciable tumor margins and infiltrated the surrounding breast tissue. Overall narrow, focally positive surgical margins and presence of malignant epithelial component required additional surgical treatment. Microscopic examination of additional segment showed granulation tissue and areas of lobular neoplasia, while axillary dissection performed in the same time revealed 19 lymph nodes with no signs of metastases. Conclusions: Malignant alteration of epithelial component is a rare complication, seen in less than 1% of all PTs. The most common type is lobular carcinoma in situ followed by intraductal carcinoma, presented in up to 50% and 20% of cases, respectively. If present, invasive epithelial component mostly shows features of ductal carcinoma NOS, while invasive lobular carcinoma arising in PT is extremely rare. According to some authors, carcinoma arising in malignant PT represents a distinct subgroup of carcinosarcoma of the breast. The clinical behavior of carcinoma arising in PT is difficult to predict. Few small series reported that if in situ or invasive carcinoma is confined to PT, it has less likelihood of recurring. However, prognosis of carcinoma spreading beyond PT remains uncertain. Six months after surgery, our patient is disease free, on adjuvant chemotherapy according to AC protocol, and being followed up closely.

068 Expression of estrogen receptor B1 and B2 in hyperplastic and neoplastic breast disease N Chantzi1,2, A Stylianidou3, H Karaiossifidi3, C Kittas1, M Alexis2, D Tiniakos1 1 Laboratory of Histology and Embryology, Medical School, National and Kapodistrian University of Athens, 2Molecular Endocrinology Programme, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation (NHRF), 3Histopathology Department, ‘‘Helena Venizelou" General and Maternity District Hospital, Athens, Greece Background: The action of estrogens is mediated by the two subtypes of estrogen receptor (ER), ERa and ERb. While ERa is a well documented prognostic marker for breast cancer, the clinical

significance of ERb is not clear and little is known on its role in benign hyperplastic breast lesions. Among the different isoforms of ERb, ERb1 (the estradiol-binding isoform) and ERb2 (does not bind estadiol) are those mostly expressed in mammary epithelial cells. In this study, we have investigated for the first time the immunohistochemical expression of ERb1 and ERb2 in hyperplastic and neoplastic breast lesions and correlated it with clinico-pathological features and cell proliferation. Design: Paraffin sections from breast tissue specimens from 37 patients (age range 37–75 years, median age 49 years) were used. These included breast tissue with fibrocystic changes and apocrine metaplasia (AM) (n = 13), epithelial hyperplasia [hyperplasia of usual type (HUT) n = 13, atypical ductal hyperplasia (ADH) n = 5, atypical lobular hyperplasia (ALH) n = 1] and neoplasms [papilloma n = 4, ductal carcinoma in situ (DCIS) n = 8, lobular carcinoma in situ (LCIS) n = 1] and five normal breast biopsies. IHC was performed using, monoclonal antibodies and rabbit antisera specific for ERb1 and ERb2 that were produced and characterized in NHRF, and commercial antibodies for ERa and the proliferative marker Ki67. We used Histoscore to analyze semiquantitatively the nuclear immunostaining intensity and the percentage of ER+ epithelial cells. Fisher’s exact test was used for statistical analysis of the data. Results: All AM cases were ERa-, while 92% were ERb1+ and 42% were ERb2+. ERb1 positivity was observed in 92%, 80% and 100% of HUT, ADH and DCIS, respectively, while the respective ERb2 positivies were 39%, 20% and 25%. The HUT cases were more frequently ERb1+ than ERa+ (P = 0.001) or ERb2+ (P < 0.001). The majority of ADH and DCIS were ERb1+ and ERb2- (P = 0.047 and P = 0.041, respectively). All papillomas were ERa+ and ERb1+, while only 50% were ERb2+. There was no correlation between ERb1 or ERb2 expression and the Ki67 labeling index. Conclusion:In AM, ERa is not expressed, while ERb1 is more often expressed than ERb2. The most frequently expressed ER in hyperplastic and non-invasive neoplastic lesions of the breast is ERb1, while ERb2 expression is less frequent. The ratio ERa/ERb2-expressing breast epithelial cells increases progressively in HUT, ADH and DCIS. This finding supports the view that in breast tissue deviation from normal increases, the more ERb2 is unable to repress the action of ERa. The differential expression and potentially distinct significance of ERb1 and ERb2 in breast lesions indicates that the use of isoform-specific antibodies is essential for studying the role of ERb isoforms in proliferative disorders of the breast.

069 Estrogen receptor beta expression in triple negative breast cancer Y Choi Department of Pathology, Yale School of Medicine, Bridgeport Background: Currently, ER-alpha is the main diagnostic and therapeutic receptor in breast cancer. Recently, the ER-b isoform has been identified and shown to have separate genetic regulation along with tissue distribution, ligand binding characteristics, and responsiveness to antiestrogens distinct from ER-a. Also, in contrast to ER-alpha, ER-beta is abundant not only in epithelial cells but also in myoepithelial and stromal cells in normal breast tissue. Although there is not yet a consensus regarding the



usefulness of ER-beta as a diagnostic or therapeutic marker for breast cancer, most studies have suggested that ER-beta is associated with prognosis and response to endocrine therapy. In this regards, Tamoxifen was shown to be more effective than raloxifene at activating protein site-1with ER-alpha, whereas Raloxifene was superior at activating protein site-1 with ER-beta. Our preliminary studies of ER-alpha positive breast cancers showed an association between the expression of ER-beta and Her-2 and p53 expression and also the presence of ER-beta expression in a significant number of ER-alpha negative tumors, suggesting a potentially larger role for ER-beta. This study aims to study ERbeta expression in ER, Progesteron Receptor (PR) and Her-2 negative (triple negative) tumors. Design: A total of 233 breast cancer tissues were obtained from a period of 10 years and retrospectively evaluated for ER, PR and Her-2 expression. Then all triple negative breast cancers were tested for the expression of ER-beta (ERb88, 1:40 dil, Biogenex, CA) using standard immunohistochemistry (IHC). These cases were further analyzed for histopathology type and associations between ER-b and p53, Ki-67, IgF-1, Androgen receptor, PS-2, Bcl-2 and Cathepsin D expression. Immnunoreactive cells with >10% expression were considered positive. Results: Of 233 breast cancer cases, 57 cases (24.5%) were classified as exhibiting triple negative tumors. Of these 57 cases, ER-beta was expressed in the epithelial cells in 70.2% (40/57) of the cases. When tumor contains stromal cells, ER-beta was expressed in these cells. The majority of the 40 cases were of ductal type cancer: 70.0% (28/40) with NOS, 17.5% (7/40) medullary or basaloid type, 10.0% (4/40) with apocrine features and 2.5 % (1/40) other types. They were poorly differentiated carcinomas with Nottingham grade three in 87.5% (35/40). Prognostic biomarkers were expressed as follows: Ki-67 in 45% (18/40), p53 in 50% (20/40), Cathepsin D in 50% (20/40), PS2 in 25% (10/40), IGF-1 in 67.5% (27/40), Bcl-2 in 10% (4/40) and androgen receptor in 7.5% (3/ 40). There was significant associations between ER-beta expression and p53 (P < 0.0375) and Bcl-2 (P < 0.0090) expression. Conclusions: ER-beta is expressed in a significant number of triple negative breast cancers. The marked presence of ER-beta expression suggests that this sub-population of breast cancer cases have a distinct endocrine expression pattern, although androgen receptors were present in a small number of cases. In addition, the carcinogenesis of breast cancer in these patients may not necessarily be dependent on ER-alpha, but may function through ER-beta or nongenomic pathways. Further studies on ER-beta in this group of patients may benefit the future of endocrine therapy, specifically ER-beta ligand treatment and management.

070 Expression of estrogen receptor-beta in various malignant lesions Y Choi Department of Pathology, Yale School of Medicine, Bridgeport Background: Estrogen receptor (ER) beta is expressed in epithelial and stromal cells in many different organs. ER-beta has been studied mostly in breast cancer but only in a few other malignancies. The results of ER-beta research in breast cancer are variable and

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inconsistent; some studies reported ER-beta to be a favorable biomarker but others concluded that it is a poor prognostic marker. However, the majority of studies in breast cancer suggested that ER-beta plays an important role in tumorigenesis and/or in developing targeted therapy. To further explore the role of ER-beta, the expression of ER-beta was evaluated in normal and malignant tissues in many different organs. Design: A tissue microarray (TMA) block was prepared from 270 normal tissues and 270 malignant lesions from many different organs. TMA slides were tested for ER-beta (1:40 dilution, Biogenex, CA, USA), ER-alpha, Ki-67 and p53 expression using standard IHC (immunohistochemistry). The percentage of nuclear staining was scored in each core. Less than 5% expression was considered negative. Results: ER-beta expression in normal tissues from different organs was variable: high expression in breast, kidney, brain, thyroid, lung, endometrial, bladder, pancreas and prostate tissues and low in ovary, muscle, and lung. There was no expression of ER-beta in normal colon and liver tissues. ER-beta was decreased in malignant tumors of the kidney, thyroid, prostate, endometrium, and stomach in comparison with increased expression in corresponding normal tissue, demonstrating an inverse correlation in these organs. ERbeta was highly expressed in both normal and malignant lesions of the ovary, lung, bladder, skin (squamous cancer), and brain (glioblastoma and astrocytoma). ER-beta was increased in malignant colon tissue in comparison with that of normal tissue. ER-beta expression was not demonstrated in either the normal or malignant liver tissues. When ER-alpha was present in any tissues, ER-beta was co-expressed in 30% of the cases. In addition, approximately 20% of ER-alpha negative breast cancers showed ER-beta expression. There was no association between ER-beta expression and the grade of the malignant lesions, Ki-67 or p53 expression. Conclusions: ER-beta expression is highly variable depending on the types of tissues and organs: there was an inverse relationship between its expression in normal tissues vs. malignant tissues in some organs where ER-beta was highly expressed in normal tissues. On the other hand, some organs showed high expression in both normal and malignant tissues. There appears to be no one mechanism of ER-beta expression in various lesions although some reported that ER-beta plays a protective role. In addition, ER-beta expression in a significant number of ER-alpha negative breast cancers may have an important clinical implications in relation to a potential therapeutic target for this group of patients. Further studies are needed to explore such potential in other malignancies with ER-beta expression.

071 An interesting case of colonization of a nerve twig by sclerosing adenosis in a breast J Choudhury, A Al-Dawoud, W Salman, Z Twaij Department of Pathology, Burnley General Hospital, Burnley, UK Background: Sclerosing adenosis is an organoid lobular enlargement within the breast. The acinar structures may infiltrate adjacent connective tissue and occasionally nerves and may lead to an erroneous diagnosis of malignancy. This is a report of such a unique case.


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Design: A 65 year old female had a wide local excision of the left breast following a diagnosis of ductal carcinoma in situ (DCIS) on a needle core. Histological examination confirmed DCIS but also revealed a nerve fibre interrupted by breast glands. This case was sent for an expert opinion. Results: The admixed tubular structures seen in association with the nerve twig were formed from cells with an apocrine morphology and didn’t show as marked atypia as in the adjacent DCIS. Elsewhere, foci of sclerosing adenosis colonized by apocrine change was present. This feature was of assistance in concluding that, in addition to the DCIS, this case also represented sclerosing adenosis with apocrine change infiltrating around a nerve twig. Conclusions: Utmost diagnostic diligence should be given when examining nerve fibres interrupted by tubular structures, it may represent a benign entity mimicking malignancy.

072 Immunohistochemical coexpression of BRCA1 and TOP2A in triple negative breast cancer M Comanescu1, C Ardeleanu1,2, F Vasilescu1, D Terzea1,2, M Mihai1, C Iosif1, A Georgescu1, F Andrei1, C Dobrea1,2, D Ene1, M Neagu1, M Ceausu1,2, F Cionca1, A Balan1, F Staniceanu2 1 ‘‘Victor Babes’’ National Institute of Pathology, Bucharest, Romania, 2 ‘‘Carol Davila’’ University of Medicine and Pharmacy, Bucharest, Romania Background: Triple negative (TN) breast cancer is immunophenotypically characterized by absence of ER, PGR and Her2/neu protein expression and has an aggressive biologic behavior. Topoisomerase II alpha (TOP2A) is involved in the anthracyclins responsiveness and can be expressed in TN breast carcinoma. We studied the BRCA1 immunohistochemical expression in TOP2A positive TN mammary carcinomas, in association with prognostic markers. Design: We analyzed retrospectively 102 consecutive mammary carcinomas positive for TOP2A (archives of ‘Victor Babes’ Institute), tested also for ER, PGR (Novocastra), cerbB2, Ki67 and p53 (Dako). The TN tumors were selected and tested also for BRCA1 (Biogenex). The IHC bistadial indirect technique was performed using producer’s development kit. In selected cases of TN tumors FISH (PATH-Vision) was performed for TOP2A gene amplification. Results: From 102 breast carcinomas (G2 and G3) (age range:28– 83 years), 18 TN cases were founded (median age, 47 years). BRCA1 was positive in all TN cases tested. The BRCA1 expression in tumoral cells was associated in all cases of TN tumors with a high index of proliferation (>20%) of Ki67 and a strong positivity for p53. Three cases out of 10 TN cases tested showed TOP2A gene amplification. Conclusions: In our series of TOP2A positive TN selected mammary carcinomas, the patients median age was higher than usually for TN tumors. The expression of BRCA1 in TN cases was associated with poor prognostic markers (high Ki67 index and p53 protein) and in some cases with TOP2A gene amplification; the BRCA1 determination in mammary tumors could have a prognostic significance.

073 Sentinel node ITC/micrometastases and non-sentinel node involvement with different interpretations of the TNM definitions G Cserni1, S Bianchi2, V Vezzosi2, P van Diest3, C van Deurzen4, I Sejben1, P Regitnig5, M Asslaber5, M Foschini6, A Sapino7, I Castellano7, G Callagy8, E Arkoumani9, J Kulka10, C Wells9 1 Department of Pathology, Bacs-Kiskun County Teaching Hospital, Kecskemet, Hungary, 2Department of Human Pathology and Oncology, University of Florence, Firenze, Italy, 3Department of Pathology University of Utrecht, Utrecht, The Netherlands, 4Department of Pathology, St. Antonius Hospital, Nieuwegein, The Netherlands, 5Institute of Pathology, Medical University Graz, Graz, Austria, 6Department of Pathology, Bellaria Hospital, University of Bologna, Bologna, Italy, 7Department of Biological Science and Human Oncology, University of Turin, Turin, Italy, 8Department of Pathology, National University of Ireland, Galway, Ireland, 9Department of Cellular Pathology, The Royal London Hospital, London, UK, 102nd Department of Pathology, Semmelweis University, Budapest, Hungary Background: To compare the rates of isolated tumor cells (ITC) and micrometastasis in the sentinel nodes (SLNs) of breast cancer patients according to two different interpretations of the definitions of the Tumor Node Metastasis staging categories and to evaluate the potential differences in non-SLN involvement. Design: Cases of low-volume SLN involvement and a minimum of six non-SLNs removed from nine centers were categorized as having either micrometastasis or ITC in the SLNs according to two classification systems. The first classification used the European Working Group interpretation of these staging categories published in Cancer, 2005; 103: 358–367, and the second used the definitions of a more recent study published by Turner et al. in J Clin Oncol, 2008; 26: 258–263. Results: Of the 517 cases reviewed, 82 had ITC and 435 micrometastasis on the basis of the first classification, and the number of ITC increased to 207 with 310 micrometastases on the basis of the second. Approximately 24% of the cases were discordantly categorized. The rates of non-SLN metastases associated with SLN ITCs were 8.5% and 13.5%, respectively (Pearson chi-square not significant, P = 0.24). Conclusions: Although the second interpretation of the low-volume nodal stage categories has better reproducibility, which is a basic requirement in tumor staging, it may to some extent underestimate the rate of non-SLN involvement when compared to the first interpretation. The TNM definitions of low-volume nodal metastases need to be better formulated and supplemented with visual information in the form of multiple sample images.



074 Molecular characterization of breast cancer in young Brazilian women L De Carvalho1, V Tarricone1, E Pereira2, L Frappart3, M Boniol4, S V Tavtigian4, M C Southey4,5 1 School of Medicine-Lusıáda Foundation/Hospital Guilherme A´lvaro, Santos-Brazil, 2Salomaõ and Zoppi Laboratories, Saõ Paulo-Brazil, 3´ LHoˆpital Edouard Herriot, Lyon, France, 4International Agency for Research on Cancer, Lyon, France, 5Department of Pathology, The Melbourne University, Victoria, Australia Background: Breast cancer represents a heterogeneous group of lesions in terms of clinical presentation, pathological features and outcome reflecting their molecular complexity and biological diversity. Gene expression profiling has defined five molecular subgroups of breast cancer with different prognosis that include Luminal A and Luminal B types, Basal-like type, ERBB2-overexpressing type and normal breast tissue-like. Basal-like tumor corresponds to the group of estrogen receptor-negative, progesterone receptor-negative and HER2-negative tumors that express at least one basal marker. Design: We evaluated the distribution of molecular subtypes of breast tumors diagnosed in Brazilian women under the age of 40 years using an immunohistochemical panel of biomarkers and loss of heterozygosity (LOH) analysis within BRCA1 in order to evaluate the frequency of loss of heterozygosity in BRCA1 among different molecular subtypes of early-onset breast cancer. Results: We found 15 cases (21%) that had an immunohistochemical profile consistent with being basal-like. Forty cases (55%) were of Luminal A type; 11% (8 cases) were of Luminal B type and 13% (9 cases) were HER2-overexpressing tumors. Four of the 16 informative cases at D17S1322, one of the four informative cases at D17S855, and none of the five informative cases at D17S1323 displayed LOH (four basal-like and one Luminal A). Microsatellite instability (MSI) at D17S855 and D17S1322 was found in two cases (one a basal-like and one luminal A). Conclusions: LOH in BRCA1 and MSI in these breast cancers is not frequent but may indicate a small group of breast cancers with a specific molecular makeup.

075 Topoisomerase II Alpha and HER-2/NEU expression in node positive breast ductal invasive carcinomas I Dimitriadis1, A Nikolaidou1, D Anestakis1, I Moisidis1, A Bousoulegas2, F Patakiouta1 1 Pathology Department, 2Surgery Department ‘‘Theagenio’’ Cancer Hospital, Thessaloniki, Greece Background: Topoisomerase (Topo II alpha) plays a key role in DNA replication and is a target for multiple chemotherapeutic agents. In breast cancer, Topo II alpha expression has been linked to cell proliferation and HER2/neu protein overexpression. The HER-2 oncogene is the most frequently amplified oncogene in breast cancer, and its overexpression is associated with poor clinical outcome. Design: The purpose of this study was to evaluate the relationship between Topo II alpha and Her-2/neu expression in patients with

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node positive invasive breast cancer. Formalin-fixed, paraffinembedded specimens from 60 women with an age range of 30– 83 years (mean age 57 years) with node positive invasive breast cancer were stained for Topo II alpha (clone Ki-S1, Dako dilution 1 : 50) and Her-2/neu (polyclonal rabbit, Dako dilution 1 : 100). Our aim was to assess the proliferative activity, expressed as Topo II alpha immunoreactivity and Her-2/neu expression in relation to morphologic features of ductal invasive carcinomas (DIC). The study included 60 DICs, which were reclassified according to the recommendations of Consensus Conference. Results: We have studied the immunohistochemical expression of Topo II alpha in breast cancer and its correlation with grade and Her-2/neu expression. The percent of positive cells at the area of highest staining was recorded as Topo index (T.I.) For each case, a Topo II alpha index was determined as the number of positivestaining tumor cells divided by the total number of tumor cells counted, times 100. Tumors with a T.I. of more than 1 were considered positive, and those with a T.I. of 1 or less were considered negative. Topo II alpha was consistently negative in normal ductal epithelium. The Topo II alpha expression in breast cancer ranges from low (Topo II alpha index 50%. 12 cases (54.5%) were positive for cyclin D1. 10 cases (45.4%) were positive for CK 5/6. 7 cases (31.7%) were positive for S-100. 17 cases (77.27%) were positive for P-Cadherin. 11 cases (59.1%) were positive for p53. 9 cases (40.9%) were positive for EGFR. 14 cases (63.6%) were positive for E-Cadherin. 10 cases (45.4%) proved to display a basal-like phenotype according to the criteria of Nielsen et al.

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Conclusions: Our results are in many respects similar to those of other investigators suggesting that a high percentage of AMC has a basal-like phenotype.

080 A novel quantitative real-time PCR method to detect HER-2/NEU gene amplification in formalin fixed, paraffin embedded breast cancer K Egervari, Z Szollosi, Z Nemes Department of Pathology, University of Debrecen Medical and Health Science Center, Debrecen, Hungary Background: Over-expression or gene amplification of the HER-2/ neu (HER-2) is an independent prognostic marker of clinical outcome of breast cancer, however the real significance lies in the potential to predict response to the anti-HER-2 monoclonal antibody, trastuzumab. Therefore determination of the HER-2 status has become an integral part of the pathological workup of breast cancer. Measurement of the HER-2 gene copy number has proved to be the most reliable predictor of response to targeted therapy. The ways of measuring the copy number of the HER-2 gene in tumor cells comprise in situ hybridization techniques and quantitative real-time PCR (qPCR), the latter being a relatively new technique for assessing HER-2 gene amplification with high sensitivity. Since the HER-2/neu DNA Quantification Kit developed by Roche designed for a LightCycler 1.5 platform had been withdrawn from the commercial market, we have developed an alternative LightCycler based custom method aimed to offer the desired level of reliability. Design: Fifty-five breast cancer cases with known HER-2/neu status have been examined with the original Roche HER-2/neu DNA Quantification kit (Roche PCR) and our custom qPCR assay (Custom PCR). Histological, immunohistochemical (IHC) and fluorescence in situ hybridization (FISH) data were retrieved from the routine files of the Department of Pathology, University of Debrecen Medical and Health Science Center. Concordances between the immunohistochemical and genetic data were calculated. Taking FISH as the endpoint, both qPCR methods were characterized by their sensitivity, specificity, positive and negative predictive values (PPV, NPV) and overall accuracy. Results: The newly developed, Custom PCR showed sensitivity of 90%, specificity of 85.71%, PPV and NPV of 78.26% and 93.75%, respectively, and accuracy of 87.27%, whereas the Roche PCR assay showed values of 84.21%, 80.56%, 69.56%, 90.63% and 81.81%, respectively. Concordances with the IHC data of the FISH and the Roche PCR results were both 69.1%, while the Custom PCR presented 72.72% concordance to IHC. Conclusions: We have described a novel real-time PCR technique for the relative quantification of the HER-2/neu gene on a LightCycler 1.5 platform. We have determined, that our method is eligible and ideal for the supplement of regular FISH reactions, concerning its high sensitivity and reliability. Further studies with larger number of cases are needed in order to determine whether real-time qPCR may take the place of in situ hybridization in the routine clinical setting regarding HER-2 status determination.


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081 Mammoglobin: a novel tumor marker for breast cancer M Elshaer1, N Fathy1, S El-Sharkawy1, W Abd El-Aal1, M Youssef2 1 Pathology Department, National Research Centre, Cairo, Egypt, 2Clinical Pathology Department, Ain Shams University, Cairo, Egypt Background: Breast cancer is a major problem among females all over the world. Despite apparent curative resection, subsequent development of metastatic spread presents a major clinical problem in about 30% of all breast cancer patients. Aim: To investigate the clinical reliability of mammaglobin m-RNA (MAG m-RNA) as a marker of circulating cancer cells in breast cancer patients and to study the relevance of its expression in blood and expression of its protein in breast tissues, with the pathological parameters and its value in evaluating efficiency of treatment. Also, the usefulness of image processing techniques in immunohistochemistry is evaluated. Method: This study was conducted on 48 breast cancer patients and 28 controls (10 healthy controls and 18 patients controls: 6 with fibroadenoma, 4 with uterine carcinoma, 4 with ovarian carcinoma and 4 with cancer colon). For histopathological study, the healthy control group included the normal breast tissue adjacent to fibroadenoma. All breast cancer patients were of the infiltrating duct carcinoma type and 10 of them had associated areas of intraductal carcinoma. The patient group was classified into 26 patients with localized breast cancer and 22 patients with metastases (9 patients had axillary lymph node metastases and 13 patients had distant metastases). Breast cancer patients were reclassified according to the histologic grade into grade I (8 patients), grade II (26 patients) and grade III (14 patients). All individuals included in this study were subjected to detection of MAG m-RNA in circulating tumor cells in peripheral blood using nested PCR technique. Breast tissue expression of MAG was investigated using immunohistochemistry. Blood and tissue MAG expression were correlated with estrogen receptor and Ki-67 proliferation index. Ki-67 immunostaining was evaluated using Leica Image Processing and Analysis System. In each case, the analysis was done on areas expressing quantitatively the highest number of immunoreactive nuclei (10– 20 microscopic fields at ·400 magnification were measured for each case). The results were expressed as Ki-67 proliferation index which is defined as the percentage of positively stained nuclei divided by the total number of the counted nuclei. Ki-67 proliferation index was either £20 or >20. Results: Circulating MAG m-RNA is a highly specific (100%) tumor marker. The detection rate was significantly associated with the histologic grades, ER positivity and low proliferative rate of tumors. The detection rate decline after receiving chemotherapy. Immunohistochemically, the pattern of expression of MAG in breast cancer tissues was characteristically different than that in non-cancer tissues (being diffuse cytoplasmic in the former and scattered in the latter). MAG overexpression in breast tissue was significantly higher in low grade tumors (I and II) than in high grade ones (III). The strong staining intensity was more frequently detected in low grade tumors. Also MAG expression in breast tissue was significantly correlated with ER positivity and low Ki-67 proliferation index of the tumors.

Conclusions: MAG is a promising specific tumor marker of breast cancer that could predict the prognosis of breast cancer and its response to hormonal treatment. Ki67 (proliferative activity) is an important criterion for assessment of prognosis and therapy of malignant tumors and its evaluation is reliable with image processing techniques.

082 Correlation between histologic grading of invasive ductal carcinoma of breast and infiltration of mast cell A Fakhrjou, V Montazery, N Farmani Anosheh Faculty of Medicine, Tabriz University of Medical Science, Tabriz, Iran Background: Breast cancer is the most frequent malignant tumor of women. Neoplastic transformation evokes an immune response, which morphologically manifests as peritumoral and intratumoral inflammatory cell infiltrations. Inflammation releases host cells, including fibroblast, macrophages and mast cells, which are recruited and activated by tumoral cells. Exprimentally induced tumors display mast cell accumulation close to the tumor cells. Mast cells are round to oval cells of connective tissue, measuring 20–30 micrometer in diameter with cytoplasmic granules containing glycosaminoglycans resulting in metachromes and histamine, neutral proteases, platelet activating factor and many others. The purpose of this study was the correlation between histologic grade of breast ductal carcinoma and mast cells infiltration. Many studies on the association between mast cells counts, angiogenesis and prognosis of the tumor have been performed. Design: The study population composed of 75 female breast invasive ductal carcinoma which had been surgically diagnosed at Emam Khomaini hospital between March 1, 2007 and March 1, 2008. According to the Bloom and Richardson system, the tubular differentiation and nuclear pleomorfism and size, and mitoses were graded as grade 1, 2, 3. In each grade 25 ones were selected. Mast cells were highlighted by toluidine blue. Stained cells were counted at 10 areas at 400· magnification. Statistical analysis were performed using SPSS package version 11.5 and were composed one-away ANOVA. Results: Mast cells were mainly distributed in between tumoral nests and non tumoral interstitial tissue. Among 75 invasive breast ductal carcinoma s, the mast cells ranged from 6 to 96. The mast cell counts had a tendency to increase with the higher histological grades, with statistical significance (P value < 0.005). Conclusions: With the introduction and advancement of different techniques for the management of breast cancer, knowledge of the course have been increasing important in assigning patients identified. In this study mast cell counts revealed statistical significance with the histological grade. This study suggests that increasing numbers of mast cells be activated by malignant tumor cells. Further studied of the correlation between other motility factor may be warranted.



083 Malignant papillary lesions diagnosed by core needle biopsy of the breast M Fischer, T Sheridan, J Warner, T Ali, O Ioffe Department of Pathology, University of Maryland, Baltimore, USA Background: Ductal carcinoma in situ of papillary type can present challenges on limited core needle biopsy (CNB) material. We have retrospectively reviewed CNBs and excisions of all malignant papillary lesions diagnosed at our institution by CNB. Design: From 1995 to 2008, 238 lesions in 217 women (age 30– 90, median 54.5) were diagnosed as papillary. Twenty-seven (11%) were called malignant. All malignant lesions were excised. Five presented with microcalcifications, and the remaining as mass lesions. Results: Malignancy was confirmed on excision in all but one case, which on review was a papilloma with florid ductal hyperplasia. Nineteen cases were diagnosed as intracystic papillary carcinomas on CNB, and eight as intraductal carcinoma (DCIS) involving a papilloma. Eleven cases (41%) were upgraded to invasive carcinoma (ICA), including two invasive solid papillary CA, and two medullary CA with cystic change. All but one of the ICAs were seen at the site of biopsy. No lymph node metastases were found on excision, although in one case transported DCIS cells were seen in the sentinel lymph node. Five of 15 (33%) low-grade lesions had ICA on excision, as did two of seven (29%) intermediate/high grade lesions (NS). Five of 15 (33%) intracystic papillary carcinomas and two of seven (29%) papillomas with DCIS had ICA on excision (NS). Five of 17 (29%) solitary and two of five (40%) multiple papillary lesions showed ICA. Conclusions: We describe potential pitfalls in the diagnosis of malignant papillary lesions on CNB. The rate of upgrade to invasive carcinoma is higher in papillary DCIS than the reported overall rates for DCIS on CNB. Nuclear grade and type are not predictive of invasion in papillary DCIS diagnosed on CNB, unlike in usual intraductal carcinoma; multiple papillary lesions are more likely to harbor invasion.

084 Mammary tubular adenomyoepitheliomas (TAME) with cellular stroma – a spectrum of tumors with propensity for phyllodes tumor like recurrences T Shet Department of Pathology, Tata Memorial Hospital, Parel, Mumbai, Maharashtra, India Background: Recurrences in a fibroadenoma of breast are rare and a less commonly discussed issue in breast pathology. Design: Reported herein is a short series of adenomas of the breast selected from archives of fibroadenomas with distinct clinicopathologic features and tendency for recurrences. The term TAME was used given their histological similarity to tubular variants of adenomyoepitheliomas and propensity for recurrences. The epithelial element though dominant always showed myoepithelial cell layer and was benign though nuclei of cells often showed prominent nucleoli. The distinct feature was the cellular fibroblastic myofibroblastic stroma which surrounded these epithelial units with sprinkling of masts cells and eosinophils.

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Results: Fourteen such tumors were retrieved from our archives over last 15 years. This spectrum of tumors had three subtypes each with propensity to be associated with another or recur as another. These lesions were histologically divided into three subtypes 1) Tubular adenoma and sclerosing adenosis type or group I (n = 3) – Composed of tumors resembling tubular adenoma and sclerosing adenosis but with cellular stroma. Mitotic count within the stroma was 1– 2/10 hpf. 2) Tubular adenomyoepithelioma rich in myoepithelial cells- TAME group II (n = 6). These tumors had patulous ductolobular units with striking myoepithelial cell proliferation. Three of these had a leiomyomatous stroma. 3) Tubular adenoma with mitotically active stroma- TAME group III (n = 4), which showed stroma with mild overgrowth but with atypical and brisk mitosis ranging from 7–20/10 hpf. 4) Mixed type: With morphological combination of group I and II (n = 1) Age of patients ranged from 16–49 years, with group III tumors occurring in older women. While patients in group I presented with multiple tumors, group III patients had very large solitary tumors. One patient with group II was an HIV positive adolescent girl. Most tumors were enucleated except for group III patients who required mastectomy based on their large size. Recurrences occurred in seven patients which including 2 of group I, 2 of group II and 3 group III patients. Recurrences were similar in 4 patients, while one group II recurred as malignant phyllodes tumor, 1 group I recurred as group III with phyllodes like areas and one group II recurred as group III. None of patients had metastasis or died from disease. All patients with recurrences had to undergo mastectomy eventually. Conclusion: To conclude the TAME spectrum includes a group of tumors with Tubulolobular units with cellular stroma with distinct propensity for stromal transformation towards a phyllodes tumor. Awareness of these tumors will lead to excision with wider margins and may curb their recurrences.

085 Breast cancer patients in Argentina: HER2 over-expression and histological features correlation I Frahm1, G Acosta2, S Sarancone3, A Arra4, J Mosto5, I Bravo6, A Lopez Presas7, G Marraco8, D Martins9, N Lago10, M Paradelo11, P Calafat11, T Glastein12, M Urbano Martıńez13, J Zoppi14, S Colombo15, V Caceres16 1 Pathology Sanatorio Mater Dei, 2Pathology Hospital Marıá Curie, 3 Pathology Laboratorio Quantum, 4Pathology Hospital Santojanni, 5 Pathology Laboratorio Privado de Patologıá, 6Pathology Hospital Eva Peroń, 7Pathology Hospital Especializado, 8Pathology Hospital Durand, 9 Pathology Hospital Paroissien, 10Pathology Hospital Aeronaútico Central, 11Pathology Hospital San Roque, 12Pathology Hospital Ne´stor Lencinas, 13Pathology Centro de Citologıá y Patologıá, 14Pathology Hospital Interzonal, 15Pathology Hospital Zenoń Santillań, 16Pathology Productos Roche Background: HER2 over-expression was found to be an independent overall survival and time to relapse predictor; furthermore,


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correlates with poor prognostic features variables. HER2 National Program enabled access to HER2 testing country-wide with standardized and replicate technique. Internal as well as independent and external quality control has assured accurate results. Background: To correlate HER2 over-expression in our program population with age and classic histological features, such as: histological type and grade, tumor size and hormone receptors status. Design: 1316 H&E-stained, formalin fixed, paraffin embedded invasive breast carcinoma tissue samples with required criteria were collected. Routine histological parameters were assessed according to WHO Tumor classification. HER2 analysis was performed using polyclonal antibody anti-Her 2 (DAKO), microwave antigen retrieval, detection system EnVision (Dako) and developed with diaminobenzidine. Results were interpreted as hercepTest guideline’s. ER and PR were screened by IHC analyses and interpreted as positive when more than 10% of tumor cells showed positive nuclear staining. Results: HER2 over-expression in all samples (1316): Score 0: 735 (55.9%), 1+: 399 (30.3%), 2+: 56 (4.3%), 3+: 126 (9.6%). We compared age and histological features in HER2 - (0–1) vs. HER2 + (3) patients: HER2-

HER2+

P value

(n1134)

(n126)

univariate analysis

Age (median)

55.9 years

55.4 years

0.999

T size cm.(median)

2.17 cm

2.23 cm

0.721

ER+

787

93

0.306

ER-

347

33

0.306

PR+

728

88

0.208

PR-

406

38

0.208

GH3

546

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10% of luminal cells was taken as a cut-off for positivity. Attenuated continuous or discontinuous basal staining was considered negative. Preserved foci in group 3, 4 & 5 also served as internal controls. Results: Results of the present study are summarized in Table-1. As shown in the table, staining pattern in control groups 1 & 2 corroborated the results of previous studies showing either negative or only basal attenuated, linear staining for these markers in DCIS and diffuse cytoplasmic labeling of luminal and basal cells in UDH. Staining pattern of the crushed margins in all the 11 cases of UDH corresponded with the staining pattern noted in the preserved foci of UDH. Though the percentage and intensity of labeled cells varied from 20% to 100% and 2+ to 3+ respectively, a specific pattern of circumferential, continuous, non linear, cytoplasmic staining of basal cells with streaming of stain in the center of the crushed ducts was maintained in all the cases. Ten of 11 cases of group 4 DCIS showed absence of staining for CK5/6 and HMWK in the luminal cells of crushed ducts confirming the involvement of margins by DCIS. A single case deviating from the expected DCIS pattern in this group was falsely positive due to the presence of central necrotic cells. In the study group 5, 9/11 cases exhibited a staining pattern similar to that seen in UDH establishing a negative margin, whereas the remaining two cases showed a negative stain (DCIS pattern), indicating the positive surgical margins in the latter two cases. As compared to CK5/6, staining for HMWK was less intense, overall. Conclusions: Our results strongly suggest that presence or absence of luminal staining for CK5/6 and HMWK is a useful tool in determining the status of surgical margins by DCIS in the event of severe crush artifacts.

122 Significance of overexpression and amplication of Nek2 and centrosome tubulin in breast carcinogenesis Y Niu1, X Yang1, H Wang2, T Liu1, R Niu1, Y Yang1, X Ding1 1 Breast Pathology Department and Research Laboratory, Breast Cancer Research Key Laboratory of National Education Ministry, Cancer Institute and Hospital, Tianjin Medical University, 2Medical College, NanKai University, Tianjin, China Background: Nek2 is a cell cycle-regulated serine/threonine protein kinase with maximal activity at the onset of mitosis that localizes to the centrosome in which a-tubulin is the structural components. Functionally, it is implicated in control of centrosome separation and bipolar spindle formation in mitotic cells. It is up-regulated in human cancers. In immortalized breast epithelial cell lines, increased Nek2 protein leads to accumulation of multinucleated cells with supernumerary centrosomes. However, the contribution of Nek2 abnormalities to breast tumorogenesis has not been previously evaluated. Design: We presented the first study of the protein expression levels and DNA copy numbers of Nek2 in atypical ductal hyperplasia (ADH), ductal carcinomas in situ (DCIS), and invasive ductal carcinomas (IDC) of breast. Immunohistochemistry (IHC) was performed on 40 cases of normal breast tissue (NBT), 40 cases of ADH, 40 cases of DCIS and 40 cases of IDC to analyze Nek2 protein expression. Real time quantitative PCR was performed on 20 cases of NBT, 20 cases of ADH, 20 cases of DCIS and 20 cases of IDC selected randomly from each groups by IHC to determine the DNA copies numbers of Nek2. Based on our previous studies of centrosome aberration in ADH and carcinoma of the breast, the relationship between Nek2 and centrosome protein a-tubulin in breast tumorogenesis was analyzed. Results: The positive signal of Nek2 protein was located in the nucleus and cytoplasm, which was observed as yellow-brown staining. Nek2 protein profile or DNA copy numbers was increasingly expressed from NBT to ADH, DCIS, and IDC, respectively, with the highest expressions being found in IDC. There were significant difference in the Nek2 protein expression and DNA copy numbers among four groups (v2 = 25.030, P = 0.000 and F = 36.600, P = 0.000, respectively). However, there were no significant differences between DCIS and IDC. The protein expression and DNA copy numbers of Nek2 showed positive correlation (r = 0.887, P < 0.01). The level of a-tubulin DNA copy numbers varied with different groups (F = 25.543, P = 0.000) and showed a rising tendency from NBT to ADH, DCIS, and IDC. The median values of DNA copy numbers of Nek2 and a-tubulin were showed in TABLE-1, and they presented positive correlation (r = 0.796, P < 0.01) among four groups. Conclusions: The aberration of centrosome related factor Nek2 may play a key role in the early stage of breast tumorogenesis and could be contributory factor in breast cancer progression. The overexpression of Nek2 might play a causal role in the centrosome abnormality in the development of breast cancer. With the ADH–carcinoma sequence of breast cancer, the gene amplification of Nek2 might lead to protein over expression. Nek2 might be recognized as the early marker in tumorigenesis of the breast.



123 SISH (silver in situ hybridization) a new method to detect HER2/ NEU gene amplification in breast cancer. comparison with fish S Papadopoulos1, M Perdiki1, D Papaioannou1, I Karyda2, I Evangelou1, E Razis3, E Skarpidi1, E Tsoli1, P Kosmidis3 1 Department of Pathology, Hygeia Hospital-Harvard Medical International, Athens, Greece, 2Breast Unit, Hygeia Hospital-Harvard Medical International, Athens, Greece, 3Oncology Department, Hygeia HospitalHarvard Medical International, Athens, Greece Background: HER2 gene amplification is associated with aggressive breast cancer and poor prognosis. Accurate HER2 testing of patients with breast cancer before treatment is important to ensure that as many patients with HER2-positive breast cancer as possible will receive the most appropriate treatment and that women with HER2-negative disease will avoid a potentially toxic therapy. The purpose of this study is the comparison of two HER2 detection methods: the fluorescence in situ hybridization (FISH) and the silver in situ hybridization (SISH). Design: The HER2 status of 40 breast cancer samples was determined by FISH and SISH analysis, scored as number of signals or a ratio. For the FISH technique the PathVysion HER2 DNA Probe Kit was used and for the SISH the Ventana Probe Kit and the XT Automated System, according to the manufacturers’ protocols. The scoring criteria for FISH were based on the guideline for HER2 testing in breast cancer (Arch Pathol Lab Med- Vol 131, Jan 2007). The scoring criteria for SISH were the same as the ones described by Patrick C. Roche on 2007. Results: Twelve samples were positive with both FISH and SISH techniques, two samples were positive with the SISH technique and negative with the FISH technique, two samples were both FISH and SISH equivocal whereas 24 samples were negative with both techniques. These data presented a good correlation between FISH and SISH technique, with a concordance of 95%. The advantages of SISH technique, in comparison to FISH, is that SISH is automated, viewable by light microscopy with no need of fluorescence and completed within shorter period of time. Conclusions: According to our data the SISH technique showed to be a method with a good correlation to the widely used and accepted FISH for the detection of HER2 status in patients with breast cancer and maybe a reasonable potential alternative.

124 Evaluation of routine pathologic examination of sentinel lymph nodes in breast cancer surgery with emphasis on intraoperative cytological smear assessment D Papaioannou1, I Evangelou1, I Karydas2, E Skarpidi1, M Perdiki1, S Papadopoulos1 1 Histopathology Department, 2Breast Center, ‘Hygeia’ Hospital Harvard Medical International, Athens, Greece Background: Excisional biopsy and pathologic assessment of sentinel lymph node has been widely incorporated in routine breast cancer surgery in recent years. The objective of this study is to evaluate the pathologic assessment of sentinel lymph nodes in early stage breast cancer surgery, as it is routinely performed in a busy,

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general private hospital and to pin-point areas for potential further improvement. Design: We reviewed all 103 specimens of breast cancer surgery, from 102 patients, where pathologic examination of sentinel lymph nodes was performed in ‘Hygeia’ Hospital, from January 2006 to May 2008. The number of sentinel lymph nodes per specimen ranged from 1 to 6 (median 2) and the maximum diameter from 0.4 to 3.5 cm (mean 1.7 cm). Pathologic assessment consisted of (1) intraoperative identification and measuring of all lymph nodes included in each specimen, (2) intraoperative cutting of all lymph nodes in multiple sections vertical to their long axis and macroscopic observation for areas suspicious for metastasis, (3) intraoperative assessment of cytological smears from all sections, prepared by scraping and stained with a quick H&E stain, (4) preparation of at least 4 levels of H&E stained permanent sections and (5) immunohistochemical staining (IHC) with Cytokeratins (one or more of AE1/AE3, 7, 8 and 19) in all tumour-free lymph nodes on routine H&E microscopy. Retrospective Cytokeratin IHC was performed on multiple paraffin sections of (1) negative sentinel lymph nodes and (2) negative axillary dissection lymph nodes from positive sentinel lymph nodes to confirm absence of metastasis in deeper levels. Results: On H&E microscopy 27/103 (26%) specimens were found positive for metastatic carcinoma. After IHC, micrometastases or tumour clusters were identified in another five specimens, increasing the total number of positive cases in 32/103 (31%). Intraoperative cytological smear assessment was reported positive or suspicious in 21 of these specimens (sensitivity 78% compared to conventional H&E histology and 66% compared to histology after IHC). In 71/103 specimens no evidence of metastatic disease was identified histologically and immunohistochemically. The 69/71 of these specimens had been reported negative during intraoperative cytological smear assessment while the remaining two of 71 specimens had been reported suspicious for malignancy (specificity 97%). IHC performed retrospectively on the morphologically negative axillary dissection lymph nodes from positive sentinel lymph nodes confirmed their negativity while retrospective IHC on deeper levels of immunohistochemically negative sentinel lymph nodes revealed no micrometastases. Conclusions: Intraoperative assessment of cytological smears in breast cancer surgery sentinel lymph node excisional biopsy is a sensitive method of detecting metastatic disease in multiple lymph nodes, allowing for minimal tissue destruction. IHC increases the sensitivity of pathologic examination, particularly in cases of micrometastases. IHC at multiple levels of paraffin sections may be expected to further increase the sensitivity, however this hypothesis was not supported in our material.

125 Mutational analysis of beta-catenin in invasive breast carcinomas and its correlation with invasion and angiogenesis E Papanikolaou, I Theohari, I Giannopoulou, E Mylona, L Nakopoulou 1st Department of Pathology, Medical School, University of Athens, Greece Background: b-catenin plays a central role in cellular adhesion as well as in cellular communication, being a member of Wnt signaling pathway. While mutations in b-catenin gene have been


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detected in numerous carcinomas, in breast cancer they have not been reported. Most studies, however, had focused in exon 3 of the gene. The detection of a smaller molecule of b-catenin in breast cancer cells lines derived from proteolytical cleavage of the protein N-terminus, led us to study the existence of possible mutations at the 5¢ end of the gene, especially in exons 1 and 2. Also, the finding that human breast cancer cell lines show reduced or lack of bcatenin protein expression prompted us to study b-catenin gene copies. The results were then examined for the existence of possible correlations with the clinicopathologic parameters of the disease, patients’ survival and markers of biological behavior. Design: A total of 80 specimens embedded in paraffin were used, obtained from invasive breast cancer patients. DNA extraction from the specimens was performed using the Qiamp DNA Mini Kit (Qiagen), while polymerase chain reaction (PCR) was done using Platinum Pfx read-proof DNA polymerase (Invitrogen). Real-time PCR was performed for the detection of b-catenin gene copies. An immunohistochemical method was previously performed for the detection of p53, VEGF-A, VEGF-B, FLT-1, metalloproteinases MMP-7 and MMP-11, their inhibitor TIMP-3 and uPAR. Results: Mutations in exon 1 were detected in 28% and in exon 2 in 40% of patients and included missing bases, point mutations and aberrant gene copies of b-catenin. Specifically for exon 2, a certain substitution at position 405 was detected in 70% of patients and was positively correlated with cytoplasmic expression of VEGF-A in cancer cells (P = 0.013). Mutations in exon 2 were inversely associated with p53 (P = 0.031) and uPAR (P = 0.048). Mutations in exon 1 were positively associated with VEGF-B (P = 0.027), FLT-1 (P = 0.043), MMP-7 (P = 0.032) and MMP-11 (P = 0.016) expression and inversely with TIMP-3 (P = 0.023). b-catenin gene copy number was lower in 64.1% of cases. No associations were observed between mutations in exons 1 and 2, clinicopathologic parameters and patients’ survival. Conclusions: This is the first report of mutations in exons 1 and 2 of b-catenin gene. Our results show that the existence of mutations in exon 1 of b-catenin gene is indirectly correlated with invasion, metastasis and angiogenesis, through the positive correlation with MMPs, the inverse correlation with TIMP-3 and positive associations with VEGFs (-A, -B) and their receptor (FLT-1) and as a consequence, possibly with an aggressive phenotype. On the other hand, correlations of mutations in exon 2 with low expression of p53 and uPAR indicate that these mutations are probably associated with a less aggressive phenotype. Further study of these mutations in a larger line of patients needs to be performed for possible development of new targeted therapies.

126 Stromal derived factor 1 (SDF1) and CXC chemokine receptor 4 (CXCR4) expression in invasive breast cancer H Papatheodorou1, H Kalofonos2, N Alexandrou2, C Scopa3, J Varakis1, E Papadaki1 1 Department of Anatomy, 2Department of Medicine-Division of Oncology, 3Department of Pathology, School of Medicine, University of Patras, Patras, Greece

growth, angiogenesis and metastasis. Therefore, it is crucial to understand their contribution in breast cancer biologic behaviour and progression of disease. The goal of present study is to demonstrate immunohistochemical distribution of SDF-1 and CXCR4 in invasive breast carcinomas and possible correlation of their expression patterns with pathological parameters. Design: We investigated paraffin embedded tissue samples from 96 invasive breast tumors using immunohistochemistry for SDF-1 and CXCR4 expression. Differences regarding the expression of the aforementioned molecules in normal and cancerous tissue were compared using the Wilcoxon signed ranks test. The relationships between different expression patterns of SDF-1 and CXCR4, with estrogen receptors (ER), progesterone receptors (PR), Ki67, p53, HER2/c-erB-2 and tumor grade were evaluated by the Kendall’s tau correlation test. The correlation of SDF-1 and CXCR4 expression with tumor size were assessed by logistic regression models. P < 0.05 was considered statistically significant. Results: Strong membranous and cytoplasmic SDF-1 expression in cancer cells was identified in 92% and 98% of the cases respectively. Normal mammary epithelial cells showed high membranous immunopositivity in 44% and cytoplasmic in 55% of the cases. SDF-1 expression was statistically significant higher in breast carcinomas compared to non neoplastic breast tissue (P < 0.001). There was a significant correlation between membranous and cytoplasmic SDF1 expression in normal mammary epithelial cells (r = 0.56, P < 0.01). Tumor size was greater in patients with low membranous expression of SDF1 in normal epithelial cells (P < 0.05). A significant statistical correlation between SDF1 expression in fibroblasts of normal breast tissue and tumor grade was noticed (r = 0.20, P < 0.01). SDF-1 expression in cancer fibroblasts tended to be correlated with PR expression status (r = 0.22, P = 0.06) while HER2/c-erB-2 expression was reversibly correlated with expression of SDF-1 in normal vessels (r = -0.21, P = 0.04). Correlation of membranous SDF-1 immunoreactivity in normal epithelial mammary cells with Ki67 expression was also of borderline significance (r = 0.20, P = 0.07). Strong nuclear and cytoplasmic expression of CXCR4 was found in cancer cells in 69% and 45% of the cases respectively. The majority of normal mammary epithelial cells showed no expression of CXCR4. CXCR4 expression was statistically significant higher in breast carcinomas compared to non neoplastic breast tissue (P < 0.001). Tumor size was greater in cases with CXCR4 expression in fibroblasts of tumor stroma (P < 0.05). Nuclear expression of CXCR4 in cancerous cells tended to be correlated with expression of PR (r = 0.20, P = 0.07) and ER (r = 0.21, P = 0.07). Conclusions: Our data demonstrate that SDF-1/CXCR4 ligand/ receptor pair had statistically significant higher expression in cancerous compared to normal breast tissue and specific expression patterns of these molecules are correlated with certain prognostic markers and tumor size. Further studies may provide important information regarding the role of SDF-1/CXCR4 interaction on breast cancer biological behaviour and will be crucial to further understanding of the cancer chemokine network.

Backround: G protein-coupled receptor CXCR4 and its ligand SDF-1 are involved in various steps of tumorogenesis such as tumor 2008 The Authors. Journal Compilation 2008 Blackwell Publishing Ltd, Histopathology, 53 (Suppl. 1), 1–432


127 Extrapleural solitary fibrous tumour with haemangiopericytic pattern of the breast. An uncommon neoplasm D Parada, K Penã Servicio de Patologıá, Hospital Vargas. Servicio de Patologıá, Instituto Oncolo´gico ‘Dr Luis Razetti’, Caracas, Venezuela Background: An extrapleural solitary fibrous tumor is a mesenchymal tumor of probably fibroblastic type. The breast is an uncommon site for an extrapleural solitary fibrous tumor. This report documents the clinical, histological and immunohistochemical characteristics in a case of breast solitary fibrous tumor; additionally we discuss about the probable origin of this neoplasm. Design: A 43-year-old woman presented with a painless growing mass at the left breast mammary gland with a ten-month evolution. Left breast mammoghraphy showed a dense, nodular, well delimited lesion 10 cm in diameter. A left mastectomy without axillary dissection was performed. After surgical treatment the whole tumor was submitted to histopathological study. Results: The tumor was formed by a proliferation of fusocelular cells haphazardly distributed and varying degrees of stromal collagenization. Medium-sized thin-walled blood vessels in a haemangiopericytic growth pattern were observed. An immunohistochemical study showed strong CD34 and CD99 positivity. Conclusions: Our case represent a typical extrapleural solitary fibrous tumor with haemangiopericytoma pattern. Our findings support the perilobular or interlobular stroma as probable origin.

128 Molecular classification of 123 breast invasive carcinomas: a tissue microarray study A Pires1,3, N Canedo1,2, M Andrea1 1 Fonte Medicina Diagno´stica Ltda., 2Universidade Federal do Rio de Janeiro, Department of Pathology, 3Universidade Federal Fluminense, Department of Pathology, Niteroí, Rio de Janeiro, Brazil Background: Recently, molecular classifications for breast carcinoma have been presented, based on the immunohistochemical pattern of reactivity with hormonal receptors, Her2-neu oncoprotein and basal cytokeratins, distinguishing between basal and non-basal carcinomas, but with different definitions. These classifications aim to reflect the subtypes identified by gene expression profiling and also to provide a better correlation with clinical outcome. Design: A tissue microarray (TMA) was constructed using 123 cases of invasive breast carcinomas (one tissue core with 0.8 mm diameter for each case) and submitted to immunohistochemistry with estrogen and progesterone receptors (ER and PR), Her2-neu and cytokeratins 5/6 (CK5/6) antibodies. Cut-off values were: any nuclear positivity for ER and PR analysis and most of the neoplastic cells positive for CK5/6. All cases were previously submitted to whole section analysis of ER, PR and Her2-neu and these results compared with TMA. Results: The rates for basal subtype were: 26.3% (using ‘triple negative classification’, basal subtype = negative for ER, PR and Her2-neu), 5.1% (using ‘cytokeratin classification’, basal subtype = positive for CK5/6) and 4.4% (using ‘triple

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negative + cytokeratin classification’). There were little discordance between TMA and whole tissue analysis for ER, PR and Her2-neu (6.8%, 8.6% and 1.7%) and minimal core loss (8.1%), mostly due to absence of neoplastic tissue on cores. Conclusions: The usage of different classifications/criteria to define basal carcinoma subtype led to variable rates of diagnosis. More studies using these molecular classifications and comparing with clinical data are needed. The use of TMA to study the molecular classification in large population is validated.

129 Centralized HER2 breast carcinoma testing in Slovakia: 5-years experiences with examinations of 8740 cases L Plank, K Kajo, M Barthova´, L Janakova, Z Kviatkovska Department of Pathology, Jessenius Medical Faculty, Comenius University and Faculty Hospital, Martin, Slovakia Background: A standardized testing of HER2 status represents an essential component of the invasive breast carcinoma diagnosis. The aim was to develope a national system of a standardized HER2 protein and gene testing for all diagnosed breast carcinomas and its quality control based on the principles of a cooperation of pathology departments in the Slovak Republic. Design: A national network system of the breast carcinoma biopsy diagnosis provided by regional centralization of biopsies in originally 11 departments of pathology in Slovakia using a standardized biopsy diagnostic approach, included immunohistochemical HER2 protein testing was introduced in January 2003. For cases showing 2+ protein positivity, HER2 gene amplification testing is provided centrally, since January 2006, by the coordination center of the project first by CISH technique and for cases showing low amplification also by FISH. The quality of HER2 protein testing was controlled in 2003 and 2006 by using blind testing cases being examined in the participating departments and subsequently evaluated centrally by a panel of ‘experts’. Results: On the basis of quality controls and numbers of the examined cases (973 in 2003, 1287 in 2004, 1655 in 2005, 1932 in 2006 and 2893 cases in 2007), the program was further centralized to nine regional centers. Technical and interpretational problems related to HER2 protein expression examination and all the experiences are regularly evaluated and discussed by educational conferences and joint meetings. From all 8740 cases tested in January 2003–December 2007, the HER2 protein positivity was proved in 2107 cases (24.1%): 1112 cases (12.7%) showed 3+ and 995 (11.4%) 2+ positivity. From 531 cases tested in 2006– 2007, centrally, by in situ hybridization techniques, 78 were found to show HER2 gene amplification: 66 of 427 cases with HER2 protein 2+ positivity and 12 of 13 cases with HER2 protein posivity 3+. The cases with HER2 protein 0 and 1+ expression, tested for different reasons by in situ hybridization techniques did not show HER2 gene amplification. Correlation of HER2 status with other parameters (e.g. grading, staging, HR expression) seems to be significant. Conclusions: The described program offers standardized results representing a good basis for the therapeutical considerations and for further optimization of HER2 testing and studies of all the


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parameters of the disease. The program is supported by web-based register of the cases.

130 Pancreatic metastasis from malignant phyllodes tumor of breast. A case report and review of the literature R Rabah, S Sheikh Dhahran Health Center, Dhahran, Saudi Arabia Background: Malignant phyllodes tumor is a rare neoplasm comprising 10% of all phyllodes tumors. Approximately 22% of these malignant neoplasms metastasize, usually within the first few months, and often containing only mesenchymal elements. Metastasis of malignant breast tumors to the pancreas is infrequent, with only two cases reported to date in English literature of malignant phyllodes tumor metastasizing to the pancreas. Case Report: A 40-year-old woman presented with a 5 year history of a breast mass. Multiple fine needle aspirations were performed, all interpreted as fibroadenoma. As the mass progressively enlarged, a lumpectomy was performed that showed a 17 · 16 · 13 cm malignant phyllodes tumor with stromal fibrosarcomatous overgrowth. Since the status of the margins was not clear, she underwent a completion mastectomy revealing multiple residual tumor masses of fibrosarcoma. She was treated with chest wall radiotherapy. Eighteen months later she presented with acute pancreatitis and radiological investigation revealed an 8 cm pancreatic head mass. A core biopsy was done that confirmed the presence of metastatic fibrosarcoma. The patient received palliative chemotherapy and ten months later died of hemorrhagic shock and multiorgan failure. Conclusions: Metastasis to the pancreas is uncommon with frequency of secondary pancreatic tumors varying between 3% and 12% of all pancreatic malignancies. Clinical symptoms of pancreatic metastasis are similar to primary pancreatic tumors and commonly patients present as primary pancreatic tumors. The most common primary tumors to give rise to metastasis to pancreas are carcinoma of lung, breast, gastrointestinal tract, prostate, and kidney. Less commonly metastasis from soft tissue sarcomas to the pancreas have been reported. Phyllodes tumor is the most commonly occurring non-epithelial tumor of the breast however representing only about 1% of all tumors in the breast. As per WHO classification phyllodes tumors are classified into benign, borderline, and malignant tumors. Recurrent malignant tumors behave more aggressively than the original ones. Metastasis from malignant phyllodes tumor has been reported in nearly all internal organs with most common sites being lungs and bones, followed by heart, liver, and CNS. Axillary lymph nodes metastasis is extremely rare occurring in 25% of the cells and 3+ = membranous staining >50% of the cells).

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Cytoplasmic staining without distinct membranous staining was considered negative. Immunopositivity in histological specimens was assessed according to the HerceptTest guidelines. Results: Immunohistochemically, three cases (12%) were positive, four cases (16%) were equivocal and 18 cases (72%) were negative. Of the positive cases, two were ICC 3+ and one 1+, and only one proved to be amplified in CISH (both in cyto- and histology). Of the four IHC equivocal cases, one was ICC 3+, one 2+ and two negative, while three were highly amplified and one low grade amplified in CISH (cytology). Of the negative cases, three were low grade amplified in cytology, but not amplified in histology. One negative case was ICC 3+. Conclusions: Our preliminary data suggest that: CISH is a useful technique to determine HER-2/neu oncogene status in cytologic specimens. There is a good concordance of lack of gene amplification by CISH in both cytologic and histologic specimens. Negative ICC is highly predictive of lack of HER-2 amplification, while positive ICC needs further evaluation by molecular methods. The FDAapproved histological scoring system cannot be reliably applied to cytopathology, making interpretation by the arbitrarily-selected scoring system in cytologic material, highly subjective. Nevertheless, cases with cytologic scores 3+ and 2+ are more likely to be amplified. Of note, is the higher detection rate of HER-2/neu amplification in cytologic material. A higher detection rate of HER-2/neu protein expression in cytologic specimens in comparison with surgical specimens, has been reported by some authors, possibly due to better quality of antigen preservation and wider representativity of the tumor areas in cytology. In our cases, it could be due to probable exfoliation of more aggressive tumor cell clones in the imprints. The results are preliminary and need to be confirmed by a larger study.

153 Evaluation of intralaboratory reproducibility of the immunohistochemical assessment of HER2 expression in breast carcinoma H Trihia, O Tzaida, T Gavressea, C Valavanis, G Stanc-Giannakopoulou, P Arapantoni-Dadioti Department of Pathology, ‘Metaxas’ Cancer Hospital of Piraeus, Greece Background: Immunohistochemistry is commonly used for evaluating HER-2/neu protein expression in breast cancer in daily routine practice. Accurate assessment of HER-2 status is essential to ensure that all patients who may benefit from Herceptin are correctly identified. The use of laboratory assays as the sole determinant for therapy selection poses a significant challenge for pathologists performing and interpreting the results. Despite attempts within the international Pathology community to improve the status of HER-2 testing, inaccuracy remains a major issue with both IHC and molecular methods, with as high as 20% of HER-2 testing being inaccurately assessed. Various factors can affect the results achieved, including the assay antibody/probe, the methodology and the experience of personnel. The purpose of this study was to establish the intralaboratory variability of immunohistochemical assessment of HER-2, in a major anti-Cancer hospital. Design: A set of 600 hundred slides stained for HER-2 protein expression with the HerceptTest (DAKO) were randomly retrieved from the archives of the Pathology Department and submitted for


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evaluation to two experienced Pathologists (observer A & B) of the Department with special interest in Breast Pathology. The cases were independently reviewed, blindly to the initial report. The cases of disagreement were further evaluated by a third Pathologist (observer C), the opinion of whom was considered as the diagnostic ‘gold’ standard. The percentage of the positive cases was also calculated. Results: The observer A assessed positive expression (3+) in 12.8%, equivocal (2+) in 13.3% and negative (0.1+) in 73.8%. Accordingly, the observer B assessed 3+ in 17.6%, 2+ in 14.5% and negative in 67.8%. There was agreement in 89.5% (537 cases) and disagreement in 10.5% (63 cases). The observer C assessed 3+ in 17%, 2+ in 14.5% and negative in 68.5%. Agreement between A & C was established in 91.6% (P < 0.001) and disagreement in 8.3%. Accordingly, agreement between B & C was established in 97.8% (P < 0.05) and disagreement in 2.2%. In the original reports, 3+ was assessed in 18.2%, 2+ in 18.6% and negative in 63.1%. Of the 63 cases of disagreement, 30 were initially assessed as 2+. Twenty one of these were further tested for gene amplification by chromogenic in situ hybridization, with the following results: three cases with low grade amplification (LGA) and 18 cases with no amplification. Conclusions: No assay currently available is perfectly accurate to identify all patients expected to benefit, or not, from anti-HER-2 therapy. The study confirms the subjectivity of the proposed immunohistochemical criteria to assess the HER-2 status. Furthermore, evaluating the accuracy of a test requires comparison to a gold standard, although internationally there is no gold standard at present. The frequency of HER-2 positive breast cancer appears to be lower in a general unselected population, since the original data came largely from high-risk early-stage breast cancer cohorts and from patients with metastatic disease.

154 FISH analysis of 1P36/1Q25 gain, EGFR amplification and P-53 deletion in phyllodes tumors and comparison with immunohistochemical profile G Trucco1, N Nicisia Esposito1, U Surti1, L Hoffner2, E Kouri2, S Kounelis1, D Dabbs1, M Jones1 1 Magee-Womens Hospital, University of Pittsburgh, 2Helena Venizelou Hospital, Pittsburgh, USA Background: Phyllodes tumors (PTs) are rare biphasic neoplasms classified as benign, borderline and malignant (WHO Classification). Since morphologic criteria do not always identify tumors that recur or metastasize, immunohistochemical (IHC) and molecular studies may help to determine aggressive behavior. Our aim in this study was therefore to identify molecular alterations by fluorescence in-situ hybridization (FISH) analysis involving: gain of 1p and/or 1q, p53 gene deletion and amplification of the epidermal growth factor receptor (EGFR) gene, and to compare these results with immunohistochemical tumor profiles using p53 and EGFR antibodies. Design: Cases of phyllodes tumors were retrieved from the pathology archives at Magee-Womens Hospital of UPMC. After IRB approval and H&E review of the cases, a single paraffin-embedded tissue block from each case was selected. Four-micron thick deparaffinized

sections were used from representative tissue blocks for FISH analysis using standard methods and DNA-labeled probes for p53, EGFR and 1p36/1q25. Hybridization was visualized using Zeiss Axiophot Fluorescence Microscope with Cytovision Image Analysis. The cells analyzed ranged from 50 to 138. Immunohistochemical staining for p53 (Ventana, clone DO7) and EGFR (Ventana, clone 3C6) was performed using standard avidin-biotin immunoperoxidase method. Nuclear positivity for p53 and membrane positivity for EGFR were evaluated in the stromal cells and scored by two independent observers as follows: 0 (no staining), 1+ (70% positivity). Results: A total of 35 cases were collected from the archived pathology files at Magee-Womens Hospital of UPMC, and included 6 benign, 15 borderline and 14 malignant PTs. 1p and/or 1q abnormalities were detected in 1/6 (16.7%), 0/4 (0%), and 6/14 (42.9%) benign, borderline, and malignant tumors, respectively. The most common abnormality detected was 1q gains (2 malignant PTs) and 1p losses (3 malignant PTs). Chromosome 1 polyploidy was seen in the benign PT and in 1 malignant PT. None of the cases displayed definite EGFR amplification, however chromosome 7 polysomy was seen in three cases: 1 borderline and 2 malignant PTs. Three cases with p53 gene deletion were detected: one borderline tumor in a 16 y.o. girl, and two in malignant PTs in a 49 and 76 y.o. women. Both tumors were also p53 positive by IHC. EGFR was positive by IHC in the majority of tumors, though the predominant pattern of staining was weak cytoplasmic with the exception of the two malignant tumors that displayed chromosome 7 polysomy – both displayed 2 to 3+ membrane-predominant or mixed cytoplasmic/membranous staining. Conclusion: Our study corroborates previous data in that 1q gains and p53 mutation are more common in malignant phyllodes tumors. In contrast to recently published data, EGFR amplification does not appear to be a common phenomenon in phyllodes tumors. However, chromosome 7 polysomy is present in a subset of tumors, and correlates with moderate to strong membrane staining by IHC. These results suggest that cytogenetic analysis, in addition to morphologic criteria, may be useful in identifying tumors with potentially aggressive behavior, for which EGFR may be a potential candidate for targeted therapy.

155 Expression of HER-2/NEU in Paget’s disease of the vulva and female breast C Tsobanidou1, I Dimitriadis1, E Stergiou2, G Sibilidis3 1 Department of Pathology, 2Clinical Department, 3Surgery Department, Theagenio Anticancer Hospital, Thessaloniki, Greece Background: Cytogenetics and molecular studies have provided evidence that multiple genetic lesions occur during the pathogenesis of Paget’s disease of the vulva and breast. Her-2/neu is an oncogene that encodes a transmembrane glycoproten receptor. Its clinical significance is under evaluation. The aim of this study was to examine the expression of Her-2/neu by immunohistochemistry on resected tumors of Paget’s disease of the vulva and breast. Design: Nine cases of Paget’s disease of the vulva including two cases with an associated invasive adenocarcinoma and 29 cases of



Paget’s disease of the breast including 28 with available associated ductal carcinoma in situ and/or invasive carcinoma of the breast were evaluated immunohistochemically for the expression of Her-2/ neu. Section of the lesions from each case were fixed in 10% formalin embedded-block and immunostained for Her-2/neu using Hercep Test (clone CB11, dilution 1 : 40 Novocastra). Following the FDAapproved scoring guide lines, only membrane staining intensity and pattern were evaluated using the 0 to 3+ scale as illustrated in the Hercep Test kit guitlines: 0 for no staining, 1+ for only partial, weak staining of the cell membrane of more than 10% of the tumor cells, 2+ for moderate staining of the complete cell membrane in more than 10% of tumor cells, 3+ for intense staining of the complete membrane of more than 10% of the tumor cells. In accordance with Hercep Test kit guit, the Her-2/neu overexpression assessment was negative for score 0 to 1+ and positive for score 2+ and 3+. Results: Her-2/neu expressed in five out of nine (55.5%) cases of both noninvasive and invasive Paget’s disease of the vulva and only one from these patients who had underlying deeply invasive adenocarcinoma showed positivity 3+. Overexpression of Her-2/neu can be demonstrated in 25 of 29 (86.2%) invasive Paget’s disease of the breast. From these patients only one without underlying adenocarcinoma showed positivity 2+. We found that in vulva Paget’s disease, Her-2/neu is less frequently detected and staining is weaker than in mammary Paget’s disease. Conclusions: The positivity of Her-2/neu may provide reliable information for the patients with Paget’s disease of the vulva or of the breast and could help to elaborate a therapeutic strategy in cases with underlying carcinoma.

156 A rare case of mucinous breast carcinoma of columnar cells N Tsoukalas1, N Apostolikas2, V Barbounis1, G Koumakis1, G Lypas1, C Papadimitriou1, S Droufakou1, C Panopoulos1, A Efremidis1 1 2nd Department Clinical Oncology, 2Department Pathology, ‘‘Saint Savvas’’ Anticancer Hospital, Athens, Greece Background: To report a rare case of mucinous breast carcinoma of columnar cells. Design: A 74-year-old woman presented with a left breast tumor, measuring 7 · 7 cm, in the upper-lateral quadrant, with inflammatory components (peau d’orange). She reported a personal history of cholecystectomy, diabetes mellitus under medication and allergic predisposition and no family history. Four months later, a core biopsy showed infiltrating ductal carcinoma, of no specific type, grade II, ER = (-), PR = (-), and c-erbB-2 = (-) (triple negative). The TNM clinical staging was T4cN3M0, (Stage IIIc). She was treated with 3 cycles of Cyclophosphamide and Adriamycin, followed by 2 cycles of Cisplatin – 5 FU, due to progressive disease, with axillary lymph node block. The re-evaluation showed minimal response and the tumor was considered marginally operable. A left mastectomy with an attempt of radical axillary resection was performed.

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Results: The histology showed two tumors in the tail of Spence which consisted of lymph tissue, infiltrated by adenocarcinoma with high columnar cells. There were foci of mucous production and extended necrosis. In addition there was infiltration of the axillary lymph nodes. The immunohistochemistry was positive for Keratin-7 and CEA, focally positive for Keratin-20 and negative for Vimentin, CA 19-9, CA125, ER, PR, c-erbB-2, GCDEP-15 and TTF-1. The immunohistochemistry, in conjunction with the tumor morphology, is compatible either with metastatic ovarian cancer, or primary mucinous breast carcinoma of columnar cells. The work up failed to reveal any suspicious lesion (neither ovarian, nor pulmonary). During local therapy with radiotherapy there was a tumor progression (cutaneous nodules). The patient died 16 months after diagnosis with pulmonary metastases and anterior chest wall infiltration. Conclusions: The primary mucinous breast carcinoma of columnar cells is a rare entity. It is a subtype of mucinous producing carcinomas of the breast. The clinical features are similar with the common infiltrating ductal breast adenocarcinomas.

157 Is malignancy of fibroepithelial breast tumors really corresponding to the negative skewness of their normalized volume distribution? an answer by monte carlo method Mitrovic´ D1, Govedarovic´ V1, Markovic´-Lipkovski J1, Jovanovic´ D2 1 Institute of Pathology, Faculty of Medicine, University of Belgrade,2National Cancer Research Institute, Belgrade, Serbia Background: Human breast fibroadenomas (FA) and phyllodes tumors (PT) have similar morphology, but different growth kinetic potential and biological behavior. Subsequently, malignant and border line PT have negative asymmetry of lognormal volume frequency distribution, but benign PT and FA have positive ones. Purpose: Calculate probability that empirical data sets of tumors (natural logarithm of their volumes) still retains negative or positive skewness using computer intensive resampling method. Design: Empirical skew ± SE of first extirpated tumors, measured on the National Cancer Research Institute of Serbia, were 0.516 ± 0.149 in 264 cellular FA, 0.288 ± in 264 benign PT, 0.176 ± 0.477 in 21 border line PT and -0.224 ± 0.297 in 63 malignant PT. Using Statistics101 software with 10 000 artificial samplings of the same size for such original data sets we were calculating virtual skewness and probability that skew is less than zero. Results: Probability that the skew is negative, with 99% confidence interval, was between 0.873 and 0.852 for malignant PT, 0.684 and 0.656 for border line PT, 0.0044 and 0.0012 for benign PT, 0.0033 and 0.0007 for cellular FA. Conclusion: Malignancy of fibroepithelial breast tumors is well represented with negative skewness of their volume frequency distribution. Probability that benign fibroepithelial tumors have negative skewness is less than 0.005.


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158 Columnar cell lesions of the breast are associated with mamographic density G Turashvili1, S McKinney1, L Martin2, K Gelmon1, P Watson1, N Boyd2, S Aparicio1 1 Molecular Oncology, BC Cancer Research Centre, Vancouver, 2Campbell Family Institute of Breast Cancer Research, Ontario Cancer Institute, 610 University Avenue, Toronto, Canada Background: Mammographic density is the third largest risk factor for ductal carcinoma in-situ (DCIS) and invasive breast cancer. However, the question of whether risk-mediating precursor histological changes, such as columnar cell lesion (CCL), can be found in dense but non-malignant breast tissues has not been systematically addressed. We hypothesized that CCL may be related to breast composition, in particular breast density, in non-tumor containing breast tissue. Design: We examined randomly selected tissue samples obtained by bilateral subcutaneous mastectomy from a forensic autopsy series, where tissue composition was assessed, and in which there had been no selection of subjects or histological specimens for breast disease. We reviewed H&E slides for the presence of CCLs and correlated with histological features measured using quantitative microscopy. Depending on the number of cell layers and presence or absence of cytological pleomorphism, the CCLs were placed into four groups: non-atypical and atypical columnar cell change (CCC) and non-atypical and atypical columnar cell hyperplasia (CCH). Results: CCLs were seen in 40 out of 236 cases (17%). The presence of CCLs was found to be associated with several measures of breast tissue composition, including radiographic density: high Faxitron Wolfe Density (P = 0.037), high density estimated by percentage non-adipose tissue area (P = 0.037), high percentage collagen (P = 9.2e-05) and high percentage glandular area (P = 2e-05). DCIS was identified in two atypical CCL cases. The extent of CCL was not associated with any of the examined variables. Presence of CCL was also associated with early menarche, a well-established risk factor for breast cancer. Conclusions: Our study is the first to report a possible association between CCLs and breast tissue composition, including mammographic density. Our data suggest that prospective elucidation of the strength and nature of the clinicopathological correlation may lead to an enhanced understanding of mammographic density and evidence based management strategies.

159 Male breast cancer: a clinicopathological study of forty cases O Tzaida1, T Gavressea1, H Trihia1, M Terzi1, D Kondylis2, G StancGiannakopoulou2, P Arapantoni-Dadioti1 1 Pathology Department, 2Surgical Clinic, METAXA’S Anticancer Hospital, Piraeus, Greece Background: Male breast cancer is an uncommon neoplasm among men accounting for not more than 1% of all breast cancers and less than 1% of all cancer deaths in men. Although it has identical histological features to that seen in the female breast, some differences in the epidemiological and clinical data are reported. The prognosis

of the disease is mainly depended on the clinical stage. The recently proposed classification based on gene expression patterns, identifying biologically and clinically distinct groups of breast cancers, prompted us to carry out this retrospective clinicopathological study. Design: A total of 43 cases of male breast cancer was diagnosed in our Department during a 20-year period. We studied 40 cases and in the 37 invasive ones we performed an immunohistochemical investigation with the following panel of antibodies: AR, ER, PR, cerbB-2, EGFR, p53, TopoIIa, MIB1, CK5/6, CK14. Results: A. Clinical: The average patient age at presentation was 58 years (range 31–85). A modified radical mastectomy was performed in 25 (62.5%) patients while the rest of them (37.5%) had a partial mastectomy. Fourteen patients received adjuvant radiation therapy or/and chemotherapy. After a mean follow-up of 46 months (range 12–228) six patients experienced tumor local recurrence or distal metastases and eight died (6 of them of their disease). B. Histological: Our cases included 37 invasive and three in situ carcinomas (one of which with microinvasive component). The histological types of the infiltrative cases were as follows: 33 (89.2%) ductal not otherwise specified, 2 (5.4%) papillary, 1 (2.7%) lobular and 1 (2.7%) mixed carcinoma. In 10.8% of the cases we identified the presence of a metaplastic component. Among them 54% were grade III, 43.2% grade II and 2.7% grade I. Lymph node metastases were present in 56% of the 25 modified radical mastectomy specimens with one to sixteen positive lymph nodes included. 36% of the cases were of clinical stage III. C. Immunohistochemical: Positive expression was found in: ER:91.9%, PR:89.2%, AR:89.2%, c-erbB-2:13.5%, EGFR:16.2%, p53:43.2%, TopoIIa:56.7%. MIB1 was positively expressed in 58,3% of the cases. Basal type cytokeratins (CK5/6 jai CK14) had a partial expression in 29,7% and 10,8% of the cases, respectively (co-expression in 8.1%). We identified 3 (8.1%) triple negative (ER, PR, c-erbB-2) tumors with positive either CK5/6 or EGFR (one case with coexpression of the two markers). Two of the three triple negative tumors were also AR negative and accompanied by a high (>10%) MIB1 and positive p53 expression. Conclusions: Male breast cancer is a rare malignancy that presents in an advanced clinical stage (stage III: 36%), characterized by a high histological grade (grade III>50%) and exhibits a common expression of negative prognostic factors (MIB1 and p53) and basal type CKs. These findings are probably related to an aggressive biological course while the frequent hormone receptors expression indicates possible modes of therapeutic approach of the disease.

160 Mixed epithelial/mesenchymal metaplastic carcinoma/ carcinosarcoma of the breast with secretory component O Tzaida, T Gavressea, H Trihia, M Britsou, G Veccini, P Arapantoni-Dadioti Pathology Department, METAXA’S Anticancer Hospital, Piraeus, Greece Background: Metaplastic carcinomas of the breast are heterogeneous group of biphasic tumors, with epithelial and mesenchymal components. Undifferentiated spindle cells and squamous elements may form part of the tumor. When the mesenchymal component



is malignant the designation carcinosarcoma is used. Secretory type carcinoma of the breast is a very rare form of breast cancer, mostly seen in children and young adults, with characteristic histological features. A case of a metaplastic Ca of the breast with a malignant epithelial secretory component, mixed with frankly sarcomatous elements, is presented. As far as we know, this combination has not been previously reported in the literature. Design: A 66-year-old woman presented to our hospital with weakness for over 2 years and a right ulcerated breast mass rapidly enlarged during the last 3 months. At the time of diagnosis the patient had already had pulmonary metastases (chest CT). A right modified radical mastectomy was performed. The patient received systemic chemotherapy and radiotherapy. Two years after the initial diagnosis the tumor recurred and the patient was re-treated with chemotherapy. Results: Gross examination revealed a tumor of 9.5 · 9 · 8 cm. Histologically, the tumor was consisted of a mixture of both epithelial and mesenchymal components. The two elements were distinct from each other, or intimately admixed in areas. The carcinomatous component showed macro-/microcystic, papillary and cribriform patterns with an extensive intraepihelial (in situ) ductal element. An intraluminal secretory material was evident in all the tubular formations. The mesenchymal component was hypercellular, composed of undifferentiated atypical spindle cells intermingled with multinucleated giant cells of osteoclast-type. Areas of ossification were also present. The features were strongly in favor of osteosarcoma. Three lymph nodes had metastatic deposits, consisted exclusively of the epithelial component of the tumor. Immunohistochemical findings: CKAE1/AE3, CK7, EMA, e-cadherin were exclusively expressed by the epithelial neoplastic cells, while spindle cell elements showed a focal weak reactivity in vimentin. S100 was expressed in both tumor cell components. MIB1 was expressed up to 30% in mesenchymal cells and 5% in epithelial ones. Conclusions: Metaplastic changes in breast cancer is a rare feature, with an incidence of 2 and >5% of the neoplastic cells respectively. Mitoses in all cases were rare. In the largest neoplasm necrosis was also identified. Axillary lymph node dissection was performed on two patients (with neoplasm diameter 3 cm and 1.1 cm). In only one case (with 3 cm maximum diameter, and >20% Ki-67+ cells) one lymph node with metastatic neoplasm was identified. Conclusions: Pathologists occasionally face neuroendocrine breast tumors in routine clinical practice. Familiarity with their morphological features is essential in order to suspect their neuroendocrine nature and perform the appropriate immunohistochemical stains. Increased Ki-67 positivity possibly correlates with more aggressive clinical behavior. Based on the immunophenotype, it is hypothesized that neuroendocrine tumors of the breast originate from ductal cells.


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CARDIOVASCULAR PATHOLOGY 166 Intravascular fasciitis of the cephalic vein. Report of a case G Agrogiannis, E Agapitos, S Kalitsis, V Stoukas, E Patsouris 1stDepartment of Pathology, School of Medicine, National and Kapodistrian University of Athens, Greece Background: Intravascular fasciitis (IVF) is a rare and unusual variant of nodular fasciitis which is characterized by reactive myofibroblastic proliferation arising from the superficial or deep fascia and involving arteries and/or veins. Although its distinctive histological features, diagnostic difficulties arise in cases with increased cellularity and mitotic activity. Case Report: A 38-year old male presented at the Department of Internal Medicine, Heraklion University Hospital, with fever (37.5–38C) for about a week and general discomfort. There was no history of weight loss, trauma or cutaneous lesions. The past history included only surgery for appendicitis. Laboratory data were in normal limits. Physical examination revealed no significant findings except from a painless firm mass at the anterior side of the forearm. The mass was located along the cephalic vein, was not fixed to the overlying skin and was rather mobile. A simple segmental excision took place and the intraoperative observation suggested venous thrombosis. A part of the cephalic vein which was involved with the mass was excised. The postoperative course was uneventful. The excised vein was 4.5 cm in length and the lumen was almost fully obstructed by an intravascular tumor like lesion which was partially attached to the wall. Following formalin fixation, the vein was transversely cut. Histopathological examination revealed increased cellularity with uniform fibroblast like spindle cells within a myxoid stroma. Mononuclear cell infiltrates were also present. The lesion was encapsulated by a surrounding thin fibrous cap and no extravascular involvement was present. Studies with Masson trichrome and immunohistochemistry for S100 protein, ki67 and CD34 were carried out. Masson confirmed the presence of fibrotic tissue within the lesion while staining for S100 protein was negative. The mitotic activity as it was indicated by ki67 was 1–2% in 10 hpf. CD34 staining demonstrated some capillary network. Conclusions: The spindle cell intravascular lesion had features of intravascular fasciitis as first described in 1981 by Patchevsky and Enzinger in order to distinct this entity as a variant of nodular fasciitis. The most common site is the upper extremity, followed by the head – neck, the lower extremity and finally the trunk. Both arteries and veins were involved in different cases and in a few of them both types of vessel were affected at the same time. All the patients in the series of Patchevsky and Enzinger had a simple local excision and few of them had recurrence. The etiology of IVF is unknown and although trauma maybe implicated, very few patients (as well as our one) reported such history. Viral etiology has been hypothesized but no viral cytopathic effects have been recorded. Follow up at 10 months in our case revealed no evidence of recurrent disease and the patient was free of any symptoms. Conclusions: The recognition of intravascular lesions as IVF and not as a malignant vascular neoplasm is necessary to avoid overtreatment of these rare cases of reactive fibroblastic proliferation.

167 Emilin is less expressed in aneurysms, but not dissections, of the ascending aorta in non-marfan patients L Borges, J Blini, A Pereira, R Dias, N Stolf, P Gutierrez Heart Institute (InCor), Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo, Brazil Background: Ascending aorta aneurysms (AscAA) and dissections (AD) have similar histopathological features, namely fragmentation of elastic tissue and increase in mucoid content, creating the aspect known as mediocystic necrosis. Although Marfan syndrome (MFS) may be linked to both conditions, most affected patients do not have this syndrome, but rather systemic arterial hypertension (SAH). In MFS there is a genetic defect in fibrillin, a component of elastic system microfibrils; the causes underlying AscAA and AD in patients without MFS have not been clarified so far. Emilin is another microfibril associated to the elastic system; it shares with fibrillin the property of regulating TGF-beta signalling, that could be involved in the pathogenesis of AscAA and AD. Moreover, mice knocked out to emilin have been described to present SAH. Thus we analyzed the tissue expression of emilin in human AscAA and AD. Design: Five micrometer-thick sections of ascending aorta samples obtained at surgery were submitted to immunoperoxidase reactions to detect emilin. Positive areas of the medial layer positive were quantified by color detection using a computerized image analysis system and the results displayed as the area fraction relative to the total medial layer. Aortas from 10 patients with AscAA and 10 with AD (all without MFS) were included as cases, and from patients submitted to coronary artery bypass surgery, either with or without SAH (10 cases in each group, all without AD, AscAA, or MFS) were analyzed as controls. ANOVA was used to verify possible differences between groups, with Tukey test for pairwise comparisons; significance was established in P < or = 0.05. Results: Mean proportion of the aortic medial layer positive to emilin was 0.25 ± 0.06 in normotensive patients, 0.22 ± 0.05 in hypertensive patients, 0.21 ± 0.06 in AD patients and 0.15 ± 0.08 in AscAA patients. There was a significant difference between groups (P = 0.01); patients with AscAA had less emilin expression than the normotensive controls. Conclusions: A defect in emilin may be involved in AscAA in patients without MFS. Interestingly, emilin is not diminished in AD; in spite of histological similarities between AscAA and AD, different physiopathological mechanisms should be operating in these conditions.

168 Three-dimensional structure of collagen and elastic tissue in ascending aorta aneurysms and dissections L Borges, J Blini, R Dias, N Stolf, P Gutierrez Heart Institute (InCor), Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo, Brazil Background: Ascending aorta aneurysms and dissections (AscAAD) have many point in common, leading to the phenotypes of chronic wall dilation (aneurysms) or acute intramural cleavage at the



external half of the medial layer (dissections). Besides the association with other conditions like systemic arterial hypertension, Marfan’s syndrome and other genetic disturbances, they share the histopathological characteristics, namely fine elastic fragmentation and the presence of mucoid (proteoglycan) accumulations in the medial layer, in the absence of significant inflammatory infiltrate. We previously showed a decrease in the collagen, in the whole media in aneurysms and at the external half in dissections. Design: In this study, we searched if alterations in the three-dimensional histoarchitecture of collagen and elastin could be involved in AscAAD. Aortic specimens obtained at surgery of these diseases (n = 4) and on coronary artery bypass surgery (controls, n = 4) were formalin-fixed and submitted to either maceration with NaOH, that preserves collagen, or enzymatic treatment with KOH plus collagenase, which maintains mostly the elastic structure. These samples were examined by scanning electron microscopy after metallization. Results: In all groups most of collagen fibers are packed in laminar structures very similar to the elastic sheets. In most cases (5/8, either AscAAD or normal groups) the space between two of such structures is more loose at the external half of the media than in the region close to the intima. Elastic tissue, as already known, is distributed in large sheets (that defines the aortic lamellae) with fenestrations. Between them, there are irregularly distributed, smaller branches of the elastic system. Differently from collagen, no difference could be perceived between the internal and external region of the aortic media concerning the amount of elastic tissue. In three out of the four AscAAD cases, there were transversal clefts along the media, a feature not found in any of the control aortas. Conclusions: Collagen fibrils are arranged in structures similar to the elastic lamina, and less packed in the outer than in the inner medial layer. This difference can explain at least partially why aortic dissection occur always at the external portion of the artery. Regarding the elastic system, our most striking finding is that the elastic fragmentation, which at light microscopy seems to be located at random, constitutes clefts, irregular but approximately transversal to the main axis of the wall. The recognition of this pattern facilitates to understand why the wall is weak as to produce the aneurysms and mostly the dissections.

169 Familial arrithmogenic right ventricular cardiomyopathy Z Dolenc Strazar1, P Rakovec2 1 Institute of Pathology, Medical Faculty, University of Ljubljana, Slovenia, 2Clinical Department of Cardiology, University Medical Centre Ljubljana, Slovenia Background: Arrithmogenic right ventricular cardiomyopathy (ARVC) is an important cause of sudden death in young, mostly men, usually 30-fold above normal lever. 1131-metaiodobenzylguanidine (MIBG) scintigraphy showed there was a high uptake mass in right atrium. CT scan and MRI confirmed the tumor located in right atrium. The patient underwent a very successful operation and all symptoms disappeared soon after that. Pathologically, an encapsulated tumor measured in 6.0 · 4.9 · 4.0 cm in diameter and 41.7 g in weight. Microscopically, the tumor consisted of small polygonal cells with abundant eosinophilic cytoplasm, which formed typical zellballen pattern. The nuclei were blandness and no mitotic figure found. IHC stain demonstrated that tumor cells were strongly reacted with chromogranin A. The MIB-1 labeling index was 2 million expressed Tags for each staged embryonic heart, allowing us to identify, quantify, and annotate expressed genes on the level of the whole genome in the dissected hearts. Conclusions: This study allowed us to analyze the spatial and temporal contribution of endocardial cells to the mesenchymal progenitors of the AVC during development. Furthermore, our immunohistochemical analysis is enhanced by our ability to measure changes in gene expression in both a qualitative and quantitative manner. This initial set of experiment provides a detailed description of changes at the morphological and molecular levels during normal valve formation. By employing this same analysis to mutant embryos defective in AVC development, we can advance our understanding of the signaling networks required for valve formation that may underlie many forms of CHD.

176 Cellular proliferation index in carotid atherosclerotic plaques P Manolakou1, C Bakoyannis2, R Angelopoulou1, E Bastounis2 1 Department of Histology and Embryology, Medical School National and Kapodistrian University of Athens, 2First Department of Surgery, Medical School, Laiko Hospital National and Kapodistrian University of Athens, Athens, Greece Background: Proliferation in atheromatic plaque cellular populations is considered a fundamental constituent for plaque progression and stability, since it can affect the extracellular matrix constitution of the fibrous cap and the local thrombogenicity of atherosclerotic lesions. As such, the purpose of this study was to analyze cellular proliferation in relation with other histological characteristics of advanced atherosclerosis in carotid artery samples. Design: The study included 10 formalin-fixed and paraffin-embedded carotid samples from patients that underwent carotid endarterectomy. The samples were stained with hematoxylin-eosin and Van Gieson’s stain for connective tissue elements. Cellular proliferation was analyzed using an antibody for proliferating cell nuclear antigen (PCNA) Clone PC10 (Dako). Estimation of reactive/ total cell ratio [Labeling Index (LI)], per cell type (foam or endothelial cells) and visual field was achieved using the Image Pro Plus Software, following detection of reactive cells with Zeiss Axiolab microscope. Results: In all samples a variety of advanced atherosclerotic lesions was observed. These included congregation of macrophages loaded with fat (foam cells), thickening of the intimal layer with newly formed vessels along with a varying necrotic core (in size, location and constitution) and in some cases the presence of intraplaque hemorrhage. Immunohistochemical analysis revealed actively proliferating cellular populations with an abundance of foam and endothelial cells with stained nuclei, including those lining the newly formed vessels. Conclusions: Advanced atherosclerotic plaques display a wide range of proliferating cell populations, with implications for their role in plaque stability and the patients’ clinical outcome.

177 Prenatally diagnosed sporadic cardiac rhabdomyoma: a case report and review of literature O Mete, S Ozturk, T Tansel, N Buyukbabani, D Yilmazbayhan Department of Pathology, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey Background: Rhabdomyomas are the most common cardiac tumor in infancy and childhood followed by fibromas. It usually occurs in patients with tuberous sclerosis, but may be sporadic or arise in patients with structural congenital heart disease. Although it may regress spontaneously, surgical excision is usually curative, especially in sporadic and single tumors. We report herein a case of sporadic cardiac rhabdomyoma diagnosed prenatally by fetal echochardiography and excised surgically, immediately after birth. Authors regard their case worthy of presenting for discussing the entities that should be remembered in the differential diagnosis of congenital cardiac tumors and also to review the literature. Design and Results: Our case was a newborn male and his prenatal echochardiography revealed circular and hipoechogenic lesion in the left ventricle, with a 5 cm in its largest dimension. A biopsy from the white, solid and well-circumscribed nodule was performed for an intra-operative examination. The remaining tumor was resected and the histopathological examination revealed several vacuolated eosinophilic cells with radial cytoplasmic extensions, known also as ‘spider cells’. Anti-desmin immunopositivity lead us to diagnose the case as a cardiac rhabdomyoma with the presence of typical histopathological features. Clinical examinations results and the family records were free of tuberous sclerosis manifestations. For this reason, the patient is accepted to be a sporadic form of cardiac rhabdomyoma. Following the first day of surgery the patient died. Conclusions: Besides rhabdomyoma and fibroma, benign and malignant caridac tumors are very rare in infancy and childhood. As a histological point of view, glycogen storage disease (especially Pompe’s disease), histiocytoid cardiomyopathy and granular cell tumor are the main differential diagnosis of cardiac rhabdomyoma. The formation of distinct tumor nodules, the absence of epicardial surface involvement alone and the presence of radial cytoplasmic extensions in vacuolated cells (known also as spider cells) are the main morphological clues for diagnosing cardiac rhabdomyoma. Furthermore, the strong relationship with tuberous sclerosis manifesting with intracranial hamartomas, facial angiofibromas, subungual fibromas, linear epidermal nevi, renal angiomyolipomas and other hamartomas should be keep in mind in patients diagnosed as cardiac rhabdomyoma.

178 )765 G > C polymorphism in the cyclooxygenase-2 gene is not associated with rupture of the free ventricular wall after myocardial infarction K Odar1, N Zidar1, D Glavacˇ1, D Sˇtajer2 1 Institute of Pathology, Medical Faculty, University of Ljubljana, Ljubljana, Slovenia, 2Centre for Intensive Internal Medicine, University Medical Centre, Ljubljana, Slovenia Background: Rupture of the free ventricular wall (RFW) is an important cause of death in patients with myocardial infarction (MI), but



its pathogenesis is poorly understood. In patients with RFW after MI, we found a more intensive local inflammatory response than in those without RFW1. We hypothesized that genetic predisposition might be one of the factors responsible for this intensive local inflammation in patients with RFW. One of the key enzymes in the inflammatory response is cyclooxygenase-2 (COX-2). A -765 G > C polymorphism in the COX-2 gene was reported to be a protective factor against MI2. Furthermore, carriers of the C allele were shown to have a less intensive inflammatory response. In this study, we investigated the association between the -765 G > C polymorphism in the COX-2 gene and the RFW after MI. Design: We investigated DNA, isolated from formalin-fixed paraffin embedded autopsy livers of 54 MI patients divided into 2 groups: group A (26 patients without RFW) and group B (28 patients with RFW). Blood samples of 72 healthy blood donors were included as controls. Genotyping was performed by singlestrand conformational polymorphism and direct sequencing of samples with different mobility patterns. For statistical analysis, Fisher’s exact test was performed and P < 0.05 was considered statistically significant. Results: Genotype prevalences were as follows. All MI patients: GG = 88.89%, GC = 9.26%, CC = 1.85%. Group A: GG = 88.23%, GC = 7.69%, CC = 3.85%. Group B: GG = 89.28%, GC = 10.71%, CC = 0%. Controls: GG = 72.22%, GC = 27.78% and CC = 0%. Allele frequencies were as follows. All MI patients: 0.935 for the G and 0.065 for the C allele. Group A: 0.923 for the G and 0.077 for the C allele. Group B: 0.946 for the G and 0.054 for the C allele. Controls: 0.861 for the G and 0.138 for the C allele. We found no statistically significant differences in genotype distribution and allele frequencies between groups A and B. The GG/GC ratio was significantly higher in MI patients than in controls (P = 0.0129). Allele frequencies did not differ significantly (P = 0.066). We found four additional heterozygotic changes in the sequence of the amplified region in three MI patients: -907 G > C, -867 C > T, -705 G > A and -674 G > A alterations. Conclusions: We found no association between the -765 G > C polymorphism in the COX-2 gene and the RFW after MI. It is possible that other variants of the COX-2 gene and/or other inflammatory genes are associated with a more intensive local inflammation observed in patients with RWF. The prevalence of the -765 G > C polymorphism in patients with MI in this study is comparable to the one described in a large series of patients with MI2. However, the difference in allele frequencies between MI patients and controls was not significant, which could be attributed to a small sample size. The potential functional importance of the described additional alterations in the nucleotide sequence remains to be determined. References: 1. Zidar et al. Cardiovasc. Pathol. 2005; 14: 247–50. 2. Cipollone et al. JAMA 2004; 291: 2221–8.

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179 Idiopathic arterial calcification of infancy: difficulty with diagnosis, variation in presentation and the importance of autopsy M Sheppard, N Sebire, S Ho, K Hault CRY Centre for Cardiac Pathology, Royal Brompton Hospital, London, UK Background: Arterial calcification is common in patients with atheroma but there is a disease called idiopathic calcification of coronary arteries which is a rare hereditary condition occurring usually in infants. Calcification occurs due to mutations in enzymes essential in the inhibition of calcification. The calcification leads to occlusion of the vessels especially coronary and renal vessels resulting in cardiac ischemia and hypertension. Design: Cases are difficult to diagnose during life which is emphasised by three cases in our files which masqueraded as other cardiac diseases. Results: Five month old female presented with respiratory failure and hypertension and died within 24 h of admission. All the coronary arteries were thick walled with narrow lumen. The aorta, great vessels and renal arteries also showed thickening of the wall. Histology confirmed calcium in the internal elastic lamina of all vessels. Case two was a 2 month old girl diagnosed with a large VSD. She died suddenly prior to surgery and at autopsy the right coronary artery orifice was reduced to a pinhole. The coronary arteries had white patches which were calcified with associated ventricular infarction. Case three was an 11 year old female who presented with cardiac failure and was diagnosed as dilated cardiomyopathy. Two weeks later, she died suddenly. The coronary arteries were patent, but firm with calcification and narrowing associated with ventricular infarction. Conclusions: Idiopathic arterial calcification of infancy can occur in a wider age group than originally suspected and should always be considered in infants and children presenting with hypertension, cardiac failure or sudden death. Both pediatric and forensic pathologists need to be aware of its existence, and once diagnosis is made, screening of other family members is essential.

180 Clinicopathologic study of primary cardiac tumors J Silva, B Chiu Lab-Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada Background: Surgically resected primary cardiac tumors were searched in our database and clinicopathologic features in these patients were reviewed. Design: The pathology of surgically resected cardiac tumors from a tertiary-care center was reviewed for a 12-year period. Clinical and pathologic data were collected from the Capital Health’s pathology database and electronic health records. Results: Between 1996 and early 2008, 82 tumors from 77 patients (mean age = 55, range=1–82, F:M ratio = 2:1) were reviewed. The tumors were of mean size 4.3 cm (range 0.5– 8.0 cm). Left-sided tumors were more common than right-sided (left


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n = 65, 79%, right = 17, 21%), with some tumors involved multiple cardiac sites. Myxomas were the most common (n = 56) from 53 patients (mean age = 56, F:M=2:3:1) found mainly in the atria (LA n = 50, RA n = 8 with biatrial tumors n = 2). This was followed by papillary fibroelastoma (n = 17); 15 of 17 were found involving the cardiac valves (AV = 7, MV = 5, TV = 3, PV = 1, one case involved both AV and MV). There were four sarcomas in three patients, and one each of epithelioid hemangioendothelioma, fibroma, inflammatory pseudotumor and two lipomas. In cardiac myxomas, the myxoma cells were immuno-positive for CD34, S100 and vimentin and one case contained benign glandular component in the atrial myxoma. Four patients had recurrent/multifocal myxomas and three patients from the same family presented with Carney’s Complex. Cardiac symptomatologies from obstruction and systemic embolization were seen in 12 patients and from larger tumors (mean size = 5.2 cm). The sarcomas were widely invasive with systemic embolization. Conclusions: Primary cardiac tumors were rare. Our study showed that cardiac myxomas were the most common, involving predominantly the left atrium. This was followed by cardiac papillary fibroelastoma, involving mainly the cardiac valves. The patients frequently presented with non-specific symptomatology. The family with Carney’s Complex presented with recurrent multifocal myxomas and one member with systemic embolism. Furthermore, as reported in other studies, large tumor size and multifocality in primary cardiac tumors might contribute to adverse clinical events.

181 Analysis of causes of death in patients after aortocoronary bypass operation R Stevanovic, S Glumac, J Sopta, M Stefanovic, J Vasiljevic Insitute of Pathology, Medial School, Belgrade, Serbia Backround: Evaluation of causes of death after aorto-coronary bypass grafting (CABG) might help to elucidate disease mechanisms, early and late complications, and can be useful for developing treatment modalities. Design: A retrospective study was performed in order to determine causes of death within 1 year survival after CABG. In 31 patients, who died within the first year after CABG, autopsy was done at the Institute of Pathology in Belgrade, during 4 years period. All cases was divided in two groups: patients with early postoperative complications (dead within a first month after CABG: 23 patients) and patients with late postoperative complications (other eight patients). Results: Both groups had similar results in respect to age, with the mean age of 64.16 ± 8.18. Out of 31 cases, there where 64.5% men, and 35.5% women. One vascular graft was implanted in 13% of cases with early postoperative complications, two in 36%, three in 48%, and four in 3% of cases. In group with early post-operative complications 48% had myocardial infarction as cause of death; 22% died of congestive heart failure, and 30% died of non-cardiac causes (sepsis, thromboembolism of pulmonary artery, adult respiratory distress syndrome, acute renal failure and bleeding peptic ulcer). Late postoperative complications had more often non-cardiac origin (87%), leaving only one case dying from heart failure. The frequency of cardiac death was significantly different between early

and late CABG postoperative complications (P < 0.05). 70% of patients with early postoperative complications died of cardiac causes. During first postoperative month, the most frequent cardiac death was in following groups: with 2 grafts (55%) and 3 grafts (82%). Conclusions: Our results show that most of patients died within first month after CABG surgery (70%) and nearly 50% due to acute myocardial infarction. In contrast, late postoperative CABG complications are mainly related with non-cardiac causes of death.

182 Molecular histopathological study of HSP25 and HSP72 on atherosclerotic lesions in apolipoprotein E deficient mice S Taniguchi, N Hashikawa, H Murakami, S Doi, Y Kihara, H Yamaguchi, K Imamura, C Yutani Department of Life Science Okayama University of Science, Okayama, Japan Background: In contrast to Hsp65 and HSP72, Hsp25 has not been much studied for its relationship with atherosclerosis, and expression and plasma level of both HSP25 and HSP72, presumably through its multiple intracellular protective effects on a cell’s response to stress. Because there has not been a thorough examination of Hsp25 and HSP72 in atherosclerotic lesions on Apoliproprotein E Deficient mice, we immunohistchemically identified its localization in the atherosclerotic plaques and assessed the expression of Hsp25 and HSP72, and PTX-3 (a new mediator in vascular inflammatory responses) signaling by western blot analysis. Design: To determine whether positive association exists between HSP 25 and HSP72 expression and its localization in the atherosclerotic lesions, we investigated that its localization by using anti-HSP 25/72 antibodies produced in our laboratory were immunohistochemically examined on endothelium, macrophage, and smooth muscle cells of aortic sinus atherosclerotic lesions and performed western blot analysis. Apo E-deficiency mice fed a standard chow diet were assigned into age-matched groups that divided into three groups according to developmental week, namely, 6-week-old group as Type I and/or II atherosclerosis classification of AHA, 15-week-old group as Type III, 33-week-old group as Type IV and/or V, and control group. Results: At 6-week-old group, immunohistochemical analysis revealed that the localization of HSP 25, HSP72 and PTX-3 were stained mainly on the smooth muscle cells and partially stained on the endothelium and macrophage of the aortic sinus atherosclerotic lesions. At 15-week-old group, macrophages were strongly stained, partially stained on smooth muscle cells. No stainability was seen on necrobiosis areas of macrophage. At 33-week-old group, lipid core areas were not stained, but fibrolipid caps were positively stained by Hsp25/72 and PTX-3. Western blot analysis showed that HSP25/72 and PTX-3 expression were significantly increased in the atherosclerotic lesions of 6-week-old group compared with the plaques of the 15-week-old group, and 33-week-old group disclosed moderate increase of their identification. These results mean that because PTX-3 expression was correlate with that of HSP25/ 72, initial lesions of atherosclerosis (Type I atherosclerosis) was most affected by inflammatory reaction of atherogenesis and its protecting effects.



Conclusions: Expression of HSP25/72 in atherosclerotic lesions might be dependent upon their histopathological localization, therefore the clinical evaluation of the plasma level of HSP25/72 will be needed further investigation.

183 Expression of cyclo-oxygenase (COX-2) and Bax protein in acute myocardial infraction A Tsipis, A Athanassiadou, P Athanassiadou, N Kavantzas, G Agrogiannis, E Patsouris 1st Department of Pathology, Medical School, University of Athens, Athens, Greece Backround: Cyclo-oxygenase-2 (COX-2) is one of the two isoforms of cyclo-oxygenase, the rate limiting enzyme in prostaglandin synthesis, catalysing the conversion of arachidonic acid to prostaglandin H2. Myocardial expression of COX-2 has been reported in response to stress, such as ischemia and in the end stage of heart failure. Recently the involvement of apoptosis in myocardial infarctions has been suggested. Bax, a member of the bcl-2 family is an inducer of apoptosis and is activated by ischemia. COX-2 expression has been variably

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associated with apoptosis in neoplastic disorders but in myocardium there are no available data. The aim of the present study was to investigate the expression of COX-2 in acute myocardial infarction and the relation with proapoptotic protein Bax. Design: We studied myocardial samples of hearts with histologic findings of acute myocardial infarction (group A, n = 20), and myocardial samples of normal heart (control group, n = 10). An immunohistochemical method was performed with the use of polyclonal antibodies against COX-2 and Bax protein in order to investigate their expression in ischemic cardiac disorders. Results: Bax overexpression in myocytes surrounding the infarcted areas in the regions at risk was observed in 25% (group A, 5/20 positive samples). COX-2 overexpression in cardiomyocytes at the same area was found in 35% (group A, 7/20 positive samples). High concordance of COX-2 and Bax expression was detected (71.5% of cases, P = 0.020). Absence of COX-2 positive cells was confirmed in normal control myocardium. Conclusions: The expression of COX-2 co-localises with proapoptotic protein Bax in the risk area of the hearts with acute infarction. These findings suggest the association between COX-2 expression and apoptosis in cases of acute myocardial infarction.


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Continued

CYTOPATHOLOGY 184 The large spectrum of pleural primary effusion lymphoma: from positive to negative human herpes-8 associated infection. Report of two cases L Antonangelo, A Cora, E Genofre, F Vargas, M Acencio, V Capelozzi Pathology and Cardiopneumology Departments, Heart Institute, University of Saõ Paulo, Sao Paulo, Brazil Background: Primary effusion lymphoma (PEL) is a rare HIV-associated non-Hodgkin’s lymphoma characterized by the predilection to arise as lymphomatous growth in a liquid phase of body cavities. PEL cells are morphologically variable with predominance of null lymphocyte immunophenotype and evidence of human herpes virus (HHV)8 infection. The report of PEL cases in HIV-negative patients and in HHV-8 non-infected patients is more uncommon. We relate two atypical cases of PEL arising from pleural cavity. Design: Case 1: A 65-years-old man was admitted to our hospital for dyspnea. A computed tomography of the chest and abdomen showed a left pleural effusion, with no evidence of tumor masses, lymph node enlargement, or hepatosplenomegaly. The patient was HIV-negative and had a previous history of congestive heart failure. Cytological analysis of the pleural effusion revealed a high grade lymphoma with round nuclei, prominent nucleoli and basophilic cytoplasm. Polymerase chain reaction (PCR) performed on the pleural effusion was positive for HHV-8 and negative for EBV. The immunophenotypic profile is shown on the table below. The patient died after 3 months from the diagnosis. Case 2: A 39-years-old HIV-positive man also admitted to our hospital for dyspnea and diffuse thoracic pain. In this case, the computed tomography of the chest and abdomen showed a right pleural effusion with no solid masses. Cytological analysis of the pleural effusion showed a lot of large-sized lymphoid cells with abnormal nuclei and basophilic cytoplasm. PCR and serology were negative for HHV-8 and EBV. The immunophenotypic profile is shown on the table below. The patient died 20 days after the diagnosis. Results: Both patients with PEL were male with similar clinical picture and pleural involvement. However, the age, the immunological status and the viral profile were different. The first was an immunocompetent man with HHV-8 co-infection. Immunophenotypically PEL cells display a ‘null’ lymphocyte phenothype and negative CD45 antibody. The second was an immunossupressed patient, with no-related HHV-8 infection. The immunophenotype revealed a ‘null’ lymphocyte phenotype, but positive CD45. Patient no. 1

Patient no. 2

Age (year)

65

39

Involved body cavity

Pleural

Pleural

Clinical characteristics

Pleural findings Radiological

LPE

RPE

Oncotic cytology (Pleural fluid)

88% of lymphoid

62% of lymphoid

Biopsy

Non-specific

malignant cells chronic pleuritis

malignant cells Atypical lymphoid cells

Patient no. 1

Patient no. 2

HHV-8

Positive

Negative

EBV

Negative

Negative

HIV

Negative

Positive

CD45

Negative

Positive

CD19, CD20, CD 22 and CD79b

Negative

Negative

Light chains immunoglobulins

cykappa

cylambda

Virus-associated

Immunophenotyping

CD3, CD4, CD5, and CD8

Negative

Negative

CD16, and CD 56

Negative

Negative

CD138

Positive

Positive

Conclusions: PEL and PEL variants have been more and more described in the last years. A firm diagnosis of PEL can be established by the examination of cells from the lymphomatous effusion by a combination of cytology, molecular genetics and phenotypic features.

185 A retrospective analysis of discrepant results between liquid based Pap tests (LBPT) and high risk HPV testing (HR-HPV) combined with adjuvant dna ploidy measurement S Bellefqih, S Caudroy, G Evrard, M Lorenzato, V Dalstein, P Birembaut Labortatoire Pol Bouin CHU REIMS, Hoˆpital Maison Blanche, Reims, France Background: To determine the reasons for discrepancies between LBPT and HR-HPV. At our Institution this conflicting results (normal smears with HR-HPV and abnormal smears without HR-HPV) are systematically controlled by DNA Image Cytometry. This approach may be useful for quality improvement. Design: In a period of 6 months (September 2005 to March 2006), of 3396 Pap Tests associated to HR HPV testing with Hybrid Capture 2 assay (HC2), 331 discordant LBPTs were reviewed blindly by two cytopathologists and one pathologist, according to 2001 TBS. The gold standard was the consensus review. Ploidy evaluation was performed using DNA image cytometry performed on an other slide obtained from the same sample of the LBPTs. The ploidy profiles were suspect in cases of aneuploidy, multiploidy or presence of cells with a DNA content >5 C and or >9 C. Results: The mean age of patients was 37 years (range 17–74). Of the 331 women, 35% (116) had previous intraepithelial cervical lesions, 35.6% (118) underwent a second smear and 5.7% (19) had undergone colposcopic evaluation before biopsy, at the follow up (4–12 months). Of the 331 cases, we found 252 NILM (76.1%), 60 ASC-US (18,10%), two ASC-H (0.60%), 13 LSIL (3.90%), four HSIL (1.2%). The diagnoses were changed in 63 cases (19%): 25 were upgraded and 38 undergraded: 22 NILM (12 DNA normal and 10 DNA suspects) were modified (20 to ASC-US and two to LSIL). 31 ASC-US (26 DNA normal and five DNA suspect) were modified, (28 to NILM and three to LSIL). One ASC-H (DNA nor-



mal) became NILM, six LSIL (six DNA normal) were changed into two ASC-US and four NILM. Three HSIL (DNA suspect) were modified (one to ASC-H, one LSIL and one NILM). HR-HPV status and DNA ploidy profile were highly linked to the diagnosis changes (P < 0.0000 and P < 0.0012 respectively). For HR-HPV, the sensitivity (Se) was 88%, specificity (Sp) and positive predictive value (PPV) were 100%, and negative predictive value (NPV) was 92.7%. For DNA status, Se was 48%, Sp 84.2%, PPV 67% and NPV 71.1%. Among the 268 smears for which the diagnosis was not modified, 48 (17.9%) were DNA suspect and 220 (82.1%) had a normal ploidy profile (P = 0.056). Conclusions: This study demonstrate the potential use of the combination of HPV testing and DNA ploidy measurement for the detection of cervical squamous atypical lesions that have to be reviewed for more efficient follow-up.

186 Diagnostic values of endobronchial ultrasound-guided fine needle aspiration biopsy (EBUS-FNAB) in assessment of mediastinal lymphadenopathy G Cai, W Khalbuss, L Teot Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA Background: Evaluation of mediastinal lymph nodes status is important for the tumor staging in patients with lung cancer as well as for etiologic assessment of primary mediastinal lymphadenopathy. Excisional lymph node biopsy under mediastinoscopy is considered as the gold standard approach. The EBUS-FNAB is an emerging minimally invasive means to sample to mediastinal lymph node. This study is to review the diagnostic values of EBUS-FNAB in evaluating mediastinal lymph nodes in patients with or without diagnosis of lung carcinomas. Design: Sixty-three patients underwent EBUS-FNAB and a total of 120 mediastinal lymph nodes were biopsies. The biopsied lymph nodes included subcarinal, paratracheal, hilar and others. Of the 120 lymph nodes biopsied, 55 nodes from 26 patients were subsequently subject to mediastinoscopic excisional biopsy. Results: The EBUS-FNAB was considered as adequate in 84 lymph node biopsies (70%), which showed metastatic carcinoma, granuloma, or significant numbers of lymphoid cells with or without pigmented histiocytes. Metastatic carcinomas and granulomas were identified in 28 (23%) and 10 (8%) lymph nodes, respectively. In 55 lymph nodes with excisional biopsies, 12 nodes showed either metastatic carcinoma (two cases) or granuloma (10 cases). Eleven of these 12 nodes were not adequately sampled by EBUS-FNAB. Conclusions: Mediastinal lymph node status can be accurately assessed with EBUS-FNAB. Practice to increase sampling adequacy is the key to increase diagnostic value of EBUS-FNAB in the assessment of mediastinal lymphadenopathy.

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187 Assessment of estrogen receptor (ER), progesterone receptor (PR) and Her2/neu status in fine needle aspirates of metastatic breast carcinomas G Cai, S Monaco, L Teot Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA Background: The ER, PR and Her2/neu status are important prognostic factors and may guide clinical management of breast cancers. The ER, PR and Her2/neu status in breast cancers may be altered with tumor progression, evolution and metastasis. On the other hand, accurate assessment of hormone receptor and Her2/ neu status may be challenging in small samples such as fine needle aspirates. This study is to assess ER, PR and Her2/neu status in the fine needle aspirates of metastatic breast carcinomas as compared to that of primary breast cancers. Design: Fifty-seven cases of metastatic breast carcinomas diagnosed by fine needle aspiration biopsy were retrieved from the cytopathology archives at the University of Pittsburgh Medical Center. The metastatic sites include soft tissue, lymph nodes, lung, liver and bone. The ER, PR and Her2/neu status was assessed with immunostains in all cases. The Her2/neu immunostain was scored as 0– 3+. The Her2/neu gene amplification was further assessed in 32 (56%) cases. The ER, PR and Her2/neu status in primary breast cancers were available to compare in 29 cases (51%). Results: The immuno-positivities for ER, PR and Her2/neu were seen in 31 (54%) and nine (16%) cases of metastatic breast carcinomas, respectively. The Her2/neu immunoreactivity was scored as 0–1+ in 35 (61%), 2+ in eight (14%) and 3+ in 14 (25%) cases. All cases with scores 0–2+ were negative for Her2/neu gene amplification and four of 14 (29%) cases with score 3+ also showed negative FISH results. The concordance rates between metastatic and primary breast carcinomas in ER, PR and Her2/neu immunostains were 76%, 48% and 55%, respectively. Conclusions: Assessment of ER, PR and Her2/neu status in fine needle aspirates shows higher concordance rates for ER than PR and Her2/neu. Addition of the FISH study for Her2/neu gene amplification in fine needle aspirates is beneficial in avoiding false positive for assessment of Her2/neu status in metastatic breast carcinomas.

188 Cytopathologic differential diagnosis of small cell carcinoma and non-small cell carcinoma in bronchial lavage specimens by using a regression analysis E Cakir, F Demirag, M Aydın Department of Pathology, Ataturk Chest Diseases and Chest Surgery Education and Research Hospital, Ankara, Turkey Background: A distinction between small cell carcinoma and nonsmall cell lung carcinoma is often enough for the clinician to decide upon further treatment since surgery is not performed in cases of small cell carcinoma. Such a distinction is often possible by cytology alone because these tumors have a rather characteristic cytologic features. The aim of this study is to determine the significance of cytologic features in distinguishing small cell carcinoma from non-small cell carcinoma in bronchial lavage specimens.


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Design: The bronchial lavage cytologies from 35 cases of small cell carcinoma and 63 cases of non-small cell carcinoma diagnosed at Ataturk Chest Diseases and Chest Surgery Education and Research Hospital were reviewed retrospectively. Three cytologic features which are regarded as useful in the differential diagnosis of small cell carcinoma and non-small cell carcinoma were assessed. Results: Although no single criterion displayed 100% sensitivity and specificity for small cell carcinoma, univariate statistical analysis indicated that three criteria; nuclear molding, small cell size, salt and pepper chromatin were more than 90% sensitive and more than 80% specific in cases of small cell carcinoma. Logistic regression analysis demonstrated that the most effective model for seperating small cell carcinoma from non-small cell carcinoma is using the criteria: small cell size, salt and pepper chromatin and papilla formation. When these selected variables were used sensitivity for small cell carcinoma is 94.3% and specificity 96.8%, sensitivity for non-small cell carcinoma is 96.8 % and specificity 94.3%. Conclusions: We conclude that bronchial lavage cytology is of considerable diagnostic value in lung cancer and there are several cytologic features which are highly sensitive and specific for distinguishing small cell carcinoma from non-small cell carcinoma.

189 Fine needle cytology of salivary gland tumors: a retrospective analysis of 1232 cases D Daskalopoulou1, E Klapsinou1, K Kyriakou1, P Helen2, N Vasileios2 1 Cytology Department, 2Pathology Department, Saint Savvas Oncologic Anticancer Institute, Athens, Greece Background: To evaluate the diagnostic accuracy of Fine Needle Cytology (FNC) in salivary gland tumors over a period of 17 years. Design: Between the years 1991 and 2007, 1232 patients with salivary gland tumors were referred to our Cytology Department by the Maxillofacial Surgery Department of our hospital and other Head and Neck Surgery Departments of the country. Of these, 129 were evaluated between the years 1991 and 1996, 255 between the years 1997 and 1999, and 848 between the years 2000 and 2007. In all patients, a FNC was performed by cytologists on an outpatient basis. The cytologic results from these patients were retrospectively collected and analyzed. The patients had a median age of 56 years, ranging from 12 to 92 years. The cytologic material was collected with a simplified method of fine-needle sampling without aspiration. Needles of 21 gauges were used, and smears were stained with Hemacolor and Papanicolaou stains. Results: Cytologically, 86% of the tumors were classified as benign, and 14% as malignant. Of the 1232 patients, 780 were subsequently operated, and the histopathologic examination of the tumors could be compared with preoperative cytologic diagnosis. The comparison between histology and cytology revealed five falsenegative and two false-positive results. In 16 other cases, a discrepancy in the exact type of lesion was found. The overall diagnostic accuracy of the method was found to be 97%, whereas the sensitivity was 96.7% and the specificity 99.6%. Conclusions: The improvement of the sampling technique and the increase of experience, due to the increase of patients diagnosed with FNC over the years, accounts for the high diagnostic accuracy

of FNC in the material (97%) and the significant increase in sensitivity, compared to previous studies. Our accumulated experience has enabled us to accurately diagnose even uncommon salivary gland tumors, such as sebaceous carcinoma, carcinoma ex-pleomorphic adenoma, small cell carcinoma, basal cell carcinoma, melanoma, non-Hodgkin lymphoma, chloroma, neurilemmoma, neurosarcoma, Kaposi’s sarcoma, acinic cell carcinoma, carcinoma ex Warthin’s tumor etc.

190 Characterization of lower uterine segment cells on thin preparation and cell block E Elishaev, A Kanbour, A Kanbour-Shakir Department of Pathology, Magee Women’s Hospital, University of Pittsburgh School of Medicine, Pittsburgh, USA Background: The cytomorphologic criteria of lower uterine segment (LUS) cells in Conventional Pap smear (CP) have been described. Whereas, the presence of LUS cells in thin prep (TP) poses a dilemma in differentiating these cells from atypical glandular of endocervical or endometrial origin and/or HSIL. This problem is increasing due to the use of endocervical cytobrush in acquiring liquid based Pap smears. This in turn may subject the patient to unnecessary surgical procedures. Our objectives are to identify and characterize the cytomorphologic features of LUS cells in TP, to compare them to their counterpart in the CP smear and to differentiate them from atypical glandular cells. Design: Liquid based Pap test of 20 patients; ages 23–44 years, containing endometrial cells suggestive of LUS with and without atypical glandular cells (AGUS) were re-reviewed and compared to clusters of LUS on 30 conventional Pap smears of similar age group. In addition, cell blocks were prepared from remaining available thin prep fluid in 15 cases. Results: Of the 20 TP examined, five were diagnosed as benign and 15 (75%) were interpreted as atypical glandular cells (endocervical or endometrial). The prepared H&E sections from the 15 cell blocks confirmed the presence of atypical glandular cells in three (20%). The cells were pleomorphic with abnormal nuclei of endometrial or endocervical origin. The pathologic findings were subsequently confirmed by biopsy. In nine (60%) cases the cell block sections revealed endometrial tissue with physiologic changes and three (20%) cases demonstrated benign reactive endocervical epithelium without endometrial or LUS cells. High grade SIL, when present, is easily recognizable on cell block sections and can be easily differentiated from LUS cells. In comparisons to the CP, TP- LUS cells and tissue fragments depicted (with/without stroma), are smaller in size and lesser in number. Their cells are cytologically benign small uniform with crowded palisaded nuclei, compared to the large number of branching tissue fragments in CP. Gland opening is rarely seen in TP cell clusters, whereas single cells are often present. Conclusions: The cytomorphologic criteria of LUS cells in TP are similar to those described for their counterpart in CP. In difficult TP cases, when the origins of the glandular cells in thin prep are unknown and questionable, a cell block preparation is a helpful next step and will usually help in providing a more definitive inter-



pretation. In addition sections prepared from the cell block can be used for ancillary studies.

191 Primary non-hodgkin’s lymphoma of the parotid gland. A case report M Fotou, E Koniaris, V Oikonomou, T Drouveli, E Kotzia Department of Clinical Cytology, ‘‘Hippokratio’’ General Hospital of Athens, Athens, Greece Background: Primary non-Hodgkin’s Lymphoma (PnHL) of the parotid gland is a rare entity with only a few cases described so far. According to statistic reports the male and female ratio is 1:1:7 and the median patient age is 57. Parotid gland is the most frequently involved among all salivary glands. Design: Our material was received from a 77-year old patient that admitted to the O.R.L. department of our hospital with a painless mass in the parotid region. The physical examination showed a firm mass at the right parotid gland, measuring 3 · 2 cm. The US and CT scans revealed a mass with peripheral vasculature. The lesion was aspirated and the material was examined by a specialized cytopathologist. Results: The result of the needle biopsy of the lesion was a nonHodgkin’s lymphoma of the parotid gland. The material was composed of many single, neoplastic cells with irregular nuclei of varied size and shape and scant to indiscernible cytoplasm. The patient underwent surgical resection of the parotid gland. After histological examination the final diagnosis confirmed the initial cytologic one. Conclusions: PnHL of the parotid gland presents in the majority of the patients with an early stage disease and wide excision followed by local radiotherapy usually suffices. In our case the patient after local excision received also chemotherapy. One year after the patient is well with no signs of recurrence. In conclusion Fine Needle Aspiration Cytology of the parotid gland is a diagnostic method that can be used to identify the vast majority of parotid lesions including such rare neoplasms like primary non-Hodgkin’s lymphoma.

192 The decalogue of babes-papanicolaou test E Gramada1, F Staniceanu1, G Micu, S Zurac1, R Andrei, A Georgescu1, A Ene1, M Gafton1, L Cosmin1, A Popa2, A Florescu 1 Department of Pathology, 2Department of Gynecology, Colentina University Hospital, Bucharest, Romania Background: Today the importance of cytological Babes-Papanicolaou (B-P) test is well known for detecting the preneoplasic and neoplasic lesions especially for low genital tract, test that may indicate by cells description, the presence or absence of pathological cells. Method: The authors performed a retrospective study in the Department of Pathology Colentina University Hospital 2002– 2004, studying a group of 2186 women, 20–70 years old. The

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study aimed to display the cellular changes in patients with gynecological investigations for low genital tract disease, or after multiple treatments like: laser/cryotherapy, tamoxifen, colonic and bladder neoplasia. The smears were stained with Papanicolaou stain and the 2001-revised Bethesda System was used to interpret the results. Results: After cytological evaluation our group segregated as follows: 7.43% normal Pap smears, 49.58% B-Pap II, of them 4.71% had changes post-irradiation, 21.09% cytological changes of mild dysplasia, 17.63% changes of severe dysplasia and 4.37% presented ASC-US /AGUS changes. The results were correlated in a perfect addition to the clinical and paraclinical data and evolutive status of the patients after treatment. Conclusions: Based on our analysis, due to the variety and complexity of treatment after the B-Pap test results we formulated the main reasons advocating the necessity and the importance of BPap test: B-Pap test is responsible for diagnosis and further recommendations (according to Bethesda System 2001). Cytological evaluation is necessary to detect the preneoplasic and neoplasic lesions of the uterine cervix. Surgery for any genital unknown bleeding must be preceded by B-Pap test. Presence of altered cells may highlight the presence of HPV-related cytological cervical changes, situations that require supplementary tests for detection of oncogenic HPV and further decision for HPV vaccination. Cervico-vaginal cytological investigation must be included in the follow-up protocol of the LSIL cases after cryo/laser therapy. The follow-up protocol for cervical uterine neoplasia after surgery and radiotherapy must include B-Pap test. Women treated with tamoxifen for breast cancer should be investigated by cervical cytological investigation test in order to prevent the secondary effects of the treatment. B-Pap test must be included in the follow-up protocol of the patients operated for colonic and bladder cancer. Pregnant women should be screened for cellular alterations of the cervical cytology for early detection of preneoplastic/neoplastic lesions. A good psychosocial health will always be the result of a perfect body health that may be assured by continuous education of young people about genital diseases.

193 Argyrophilic nucleolar organizer region (AgNOR) count in exfoliative cytology of buccal mucosa in opium addicts, smokers and nonsmokers M Kadivar, M Attar Iran University of Medical Sciences, Tehran, Iran Background: Squamous cell carcinoma, accounts for over 90 percent of the oral and oropharyngeal malignancies. A strong relationship exists between smoking and developing oral squamous cell carcinoma. There is little data about cytologic evaluation of the effects of smoking and opium consumption on proliferative activities of normal buccal mucosa. The aim of this study is to compare the argyrophilic Nucleolar Organizer Region (AgNOR) count of cells collected from the normal buccal mucosa of cigarette smokers, opium addicts, and controls (nonsmokers). Design: Exfoliative cytologic smears of buccal mucosa from 25 smokers, 25 addicts and 25 nonsmokers were stained for AgNORs according to the Ploton‘s method. The AgNOR count was per-


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formed on 100 cells. These AgNOR counts were compared and analyzed using the SPSS 13 program and one-way ANOVA test. Results: Statistically, the highest mean of AgNOR count (mAgNOR) was in cells from opium addicts (9.21 ± 2.95) and the lowest in cells from nonsmokers (4.35 ± 1.62) (P < 0.0001). For smokers, this value was in midrange (5.68 ± 2.17). The percentage of cells with six or more AgNORs (pAgNOR ‡6) was the best discriminator among the different groups (P < 0.0001). Conclusions: Our study reveals a higher frequency of mean AgNOR counts in smokers than in nonsmokers. Similar findings were demonstrated by sampaio et al. In our study, however, the mean AgNOR count in both groups is higher than that reported by Sampaio et al. Our explanation for this difference is that living in a polluted city (Tehran) might contribute to higher AgNOR counts. The results obtained in the present study suggest that the oral mucosa is susceptible to cigarette smoking. This is reflected by an increase in cell proliferation activity as demonstrated by an increase in AgNORs per nucleus. This study reveals the additive effect of opium consumption with smoking on the proliferative activity of normal buccal mucosa. The connection between the AgNOR evaluation and malignant transformation remains unknown. Further studies are necessary to evaluate the significance of this increase in the development of dysplasia and malignant neoplasia.

194 Intraocular lymphoma: a series of six cases with emphasis on diagnostic challenges in vitrectomy specimens interpretation S Kondratiev1, F Khokhar1, E Yakirevich2, M Pilichowska1, N Laver1 1 Department of Pathology, Tufts Medical Center, Boston, 2Rhode Island Hospital, Providence, USA Background: Intraocular lymphomas (IOL), including primary intraocular lymphoma (PIOL) and ocular involvement by systemic lymphoma, are uncommon neoplasms usually of B-cell origin. Clinically, the diagnosis is suspected when bilateral, treatment-resistant uveitis presents. Our study demonstrates the clinicopathological features of six IOL and 12 uveitis cases with emphasis on the problems in differentiating between malignant and reactive lymphoid proliferations on vitreous biopsies. Appropriate handling of the vitreous specimens is also described. Design: Eighteen patients with a diagnosis of vitritis were evaluated. Six patients were diagnosed as suspicious or positive for IOL on vitrectomy biopsies and 12 patients were diagnosed with uveitis. Clinical follow-up was available in all cases. Cytology slides were obtained by direct smear and SurePath, processed according to routine protocols, and stained with H&E, PAS, DiffQuik, and PAP stains. Immunoglobulin gene rearrangement PCR analysis was performed in four IOL cases. Results: All IOL cases, presented with vitritis, were diagnosed as Bcell lymphoma, large cell type. Five IOL patients had PIOL and one had secondary involvement by diffuse large B-cell lymphoma. The mean age of presentation was 69 years (range 57–88 years) with an equal number of men and women. Three IOL samples showed a mixture of reactive lymphocytes and lymphoma cells; two of them showed necrosis and cellular debris obscuring the diagnostic cells;

one case had a mixed lymphoid population along with macrophages, neutrophils, giant cells, apoptotic lymphoma cells, and necrosis. One case was non-diagnostic initially due to low cellularity with predominately small lymphocytes; it was diagnosed as IOL on a latter biopsy. Two cases showed atypical lymphoid cells suspicious for lymphoma. Cytologic evaluation was diagnostic or suspicious for IOL when large lymphoid cells with a size at least twice of small lymphocyte, high N/ C ratio, nuclear membrane irregularity, centrally located or membrane-bound nucleoli, and occasional mitotic figures were found. Necrosis was often observed. PCR clonal immunoglobulin gene rearrangement was detected in three IOL cases and was non-diagnostic in one case. DiffQuik and PAP stained slides were most helpful for diagnosis along with evidence of clonal immunoglobulin heavy chain rearrangement by PCR. The reactive vitritis was diagnosed more frequently in females than in males, with a female-to-male ratio of 2:1. The patients’ age was slightly younger, albeit not significantly, than in IOL group (mean 66 years; range 46–86 years). These cases contained reactive small lymphocytes, histiocytes, vitreous fibers, and pigment-laden macrophages. Conclusions: IOL can be diagnosed on vitrectomy cytology. Challenges in diagnosis include a small sample, few viable lymphoma cells, reactive changes and necrosis. The presence of large atypical lymphoid cells with or without a reactive background is a key finding for diagnosis. Optimal handling of the vitreous specimens and examination of the slides by an experienced cytopathologist are critical in the diagnostic workup of IOL.

195 Comparison of urine cytology and DNA ploidy in the prediction of persistence or recurrence of urothelial carcinoma (UC) S Lu, B Varadarajalu, M Punar, C Sheehan, T Nazeer, J Ross, K Linos Department of Pathology and Laboratory Medicine, Albany Medical College, Albany, USA Background: Although urinary cytology is the most common method for detection and monitoring of bladder cancer it is widely regarded as being insensitive and not definitive. This study compared the accuracy of cytology and DNA ploidy status to predict biopsy and resection results in patients with UC. Design: Two hundred forty urinary bladder washings and biopsies from 119 patients with UC were evaluated by conventional cytology using the ThinPrep processing technique (Cytyc, Inc., Marlboro, MA, USA), for DNA content on Feulgen stained aliquots using the CAS 200 Image Analyzer, and by H&E stained histology slides. Patients were observed from 3 to 250 months. Cytology was classified either as positive or non-positive (includes negative, atypical, and markedly atypical). A non-diploid (aneuploid) histogram was defined as either a DNA index of greater than 1.23, a tetraploid peak greater 25% of total cells, or the resence of multiple polyploid peaks (excluding viral effects). Corresponding tumor biopsies or resections were classified as negative (including non-diagnostic) or positive: either low grade or high grade (ISUP system), and either non-invasive or invasive (into lamina propria or muscularis propria). Result: Cytology and DNA ploidy were concordant in 174 of 240 (72.5%) cases. Of the 134 UC persistence or recurrences, 34 were



detected as cytology positive (sensitivity = 25.3%), 62 were nondiploid (sensitivity = 46.2%), and 65 were either cytology positive or non-diploid (sensitivity = 48.5%). Although the positive predictive value (PPV) for cytology was higher than DNA ploidy (91.9% vs. 83.8%), DNA ploidy status was significantly more sensitive than cytology for UC detection (P = 0.0004), especially for high grade (P = 0.0008) vs. low grade (P = 0.04), and for non-invasive (P = 0.005) vs. invasive (P = 0.03) lesions. Of 59 cytology atypical or markedly atypical results, 27 (45.8%) were non-diploid (positive) on DNA ploidy histograms. Conclusions: In this study, DNA ploidy increased the overall sensitivity of urine cytology by ~20% for detection of persistence or recurrence of UC. DNA ploidy analysis significantly out-performed conventional cytology, particularly for non-invasive or high-grade UC. When the cytology is reported as atypical but not definitive, DNA ploidy status provides important additional information that can be used to guide follow-up studies and overall clinical management for patients with UC.

196 Usefulness of various diagnostic techniques during fiberoptic bronchoscopy for endoscopically non visible lung lesions N Maounis, D Melemeni, N Trakas, A Lekkakou, E Ellina, M Aggelidou, M Chorti, A Blana, A Emmanouilidou Department of Clinical Cytology, Sismanoglio General Hospital, Athens, Greece Background: The contribution of various cytological techniques towards the diagnosis of lung cancer, in cases with no visible endobronchial alterations at endoscopy, was evaluated. Design: In a 12 month period flexible fiberoptic bronchoscopy (FB) was performed in 387 patients with various clinical and radiological findings having no visible endoscopic lesions. Histologic examination of forceps biopsy (FB) specimens was performed in 43 (11.11%) patients. Cytological evaluation on bronchial brushing (BB), bronchial washings (BW) and post-bronchoscopic sputum smears were performed on 158 (40.8%), 387(100%) and 367 (94.8%) cases respectively. Results: Eighty five patients had at least one of the techniques positive for lung malignancy (21.96%). Mean age ± SD: 64.59 ± 14.00; range 16–90 years. Two hundred thirteen (57.3%) of our patients were smokers and 59 (15.9%) ex smokers. Of the above 85 tumors, 28 were diagnosed as squamous cell carcinoma (32.94%), 10 as adenocarcinoma (11.76%), one as bronchoalveolar carcinoma (1.176%), eight as small cell lung carcinoma (9.41%), 35 as nonsmall cell lung carcinoma (41.17%) one as mixed (1.176%) and two as metastatic (2.35%). Collection of washing specimens in addition to post-bronchoscopic sputum increased the diagnostic yield of bronchoscopy from 4.9% to 11.7% (P < 0.0001). Furthermore, collection of BB to the above techniques increased the diagnostic yield to 21.7% (P < 0.0001). Finally, the addition of forceps biopsy increased the diagnostic yield of the combination of the three cytological techniques from 21.7% to 34.9% (P < 0.0001). Conclusions: For the diagnosis of lung cancer in cases with no visible endobronchial lesions, a complementary role of cytology and histology can be postulated by our data.

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197 Cytological features of melanoma in fluid specimens R Murali1,2,3, N Loughman1, P McKenzie1, G Watson1, J Thompson2,4, R Scolyer1,2,3 1 Department of Anatomical Pathology, Royal Prince Alfred Hospital, Sydney, Australia, 2Sydney Melanoma Unit, Sydney Cancer Centre, Royal Prince Alfred Hospital, Sydney, Australia, 3Discipline of Pathology, Faculty of Medicine, The University of Sydney, Sydney, Australia, 4 Discipline of Surgery, Faculty of Medicine, The University of Sydney, Sydney, Australia Background: Melanoma shows a wide range of morphological appearances. The features of melanoma in fine needle biopsy specimens have been well described in the literature, along with the differential diagnoses and difficulties in accurate diagnosis in this setting. However, the cytological appearances of melanoma in fluid specimens and potential diagnostic pitfalls in such specimens have not been previously described. Design: Cases of melanoma diagnosed between January 1993 and April 2008 in cytology specimens of fluids (pleural fluid, ascitic fluid, cerebrospinal fluid and other fluids), but excluding fine needle biopsy specimens, were extracted from the files of the Department of Anatomical Pathology, Royal Prince Alfred Hospital. Results: A total of 31 fluid specimens (from 26 patients) were identified. The cellularity ranged from low to high, but was moderate in the majority of cases. The cells were singly dispersed in all cases, and tumor cell aggregates were identified in four (12.9%) cases. The cell shape was epithelioid in all but one case composed of spindle cells. Binucleation and multinucleation were identified in 18 (58.1%) and five (16.1%) cases, respectively. Nuclear pleomorphism was moderate to severe, and nucleoli were moderately to markedly prominent in most cases. A single case showed scattered bizarre giant tumor cells. Mitotic figures and intranuclear cytoplasmic invaginations (pseudoinclusions) were identified in 11 (35.5%) and six (19.4%) cases, respectively. Necrotic material was present in two (6.5%) cases, both of which were tumor fluid samples from metastatic melanoma in brain. Melanin pigment was seen in nine (29.0%) cases and was located in the cytoplasm of tumor cells in all nine cases, as well as in macrophages (five cases, 16.1%) and extracellularly (one case, 3.2%). The presence of melanoma was confirmed by immunochemistry for S-100, HMB-45 and/or MelanA in cell block preparations in 24 (77.4%) cases, and by histological examination of biopsied tissue in three additional cases (9.7%). In some body cavity fluid specimens, mesothelial cells and histiocytes, particularly when exhibiting reactive changes, showed some resemblance to epithelioid melanoma cells, as they were rounded or polygonal in shape, contained rounded or oval nuclei centrally or eccentrically located within the cells, and exhibited varying degrees of cytological atypia and moderate to abundant amounts of cytoplasm. However, careful examination revealed greater cytological atypia in the melanoma cells and immunochemistry was helpful in arriving at the correct diagnosis. Conclusions: Melanoma is rarely diagnosed in fluid cytology specimens. Reactive mesothelial cells and histiocytes, particularly in ascitic and pleural fluid specimens, may mimic epithelioid melanoma cells. Careful assessment of the cytological features with confirmatory immunochemistry usually enables accurate diagnosis.


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Awareness of the morphological features and diagnostic pitfalls of melanoma in fluids is important in avoiding the potentially deleterious consequences of misdiagnosis.

198 Presence of cell cannibalism itself does not rule out renal glomerular disease: cytologic-histologic correlation study H Ohsaki1, R Haba2, T Matsunaga2, M Nakamura3, H Kiyomoto4, E Hirakawa1 1 Department of Medical Technology Kagawa Prefectural College of Health Sciences, 2Department of Diagnostic Pathology University Hospital, Faculty of Medicine, Kagawa University, 3Department of Pathology National Hospital Organization Zentsuji Hospital, 4Department of Nephrology and Dialysis Faculty of Medicine, Kagawa University, Kiga, Kagawa, Japan Background: The presence of cannibalism in voided urine cytology has been considered an important cytologic parameter for differentiation of urothelial carcinoma from benign lesions. Most studies reported a lack of cannibalism in benign lesions of voided urine cytology. Recently, however, we observed cannibalism in voided urine obtained from patients with renal glomerular disease. It mimicked the cannibalism of urothelial carcinoma and posed a potential diagnostic pitfall. The purpose of this study was to determine the frequency, cytomorphologic characteristics and immunocytochemical reaction of vimentin for cannibalism in voided urine cytology. Design: Fifty-four cytologic specimens of voided urine were examined and compared with the cytologic and histologic findings. Forty-four of these voided urine samples from patients histologically diagnosed as having renal glomerular disease were reviewed. In addition, we compared the cytomorphologic and immunocytochemical differences of the cannibalism between renal glomerular disease and urothelial carcinoma. Also, 10 voided urine samples from patients histologically diagnosed with urothelial carcinoma of the bladder were evaluated. Cannibalism was assessed with the following four parameters: (i) diameter of cannibalizing cell; (ii) chromatin pattern; (iii) vimentin reactivity; and (iv) background (necrosis, isomorphic erythrocytes and dysmorphic erythrocytes). Results: A total of 54 voided urine samples were cytologically analyzed. Cannibalism was found in three cases of renal glomerular disease (three of 44, 6.8%) and in all cases of urothelial carcinoma (10 of 10, 100%). Maximum diameter of cannibalizing cells in renal glomerular disease was 24.3–33.0 lm (mean 29.8 lm) vs. 18.0–30.4 lm (mean 23.3 lm) in urothelial carcinoma. Heterochromatin pattern was seen in one case of renal glomerular disease (one of three, 33.3%) and seven cases of urothelial carcinoma (70.0%). Euchromatin pattern was identified in two cases of renal glomerular disease (two of three, 66.6%), and three cases of urothelial carcinoma (three of 10, 30.0%). Necrosis and isomorphic erythrocytes were absent in renal glomerular disease, whereas in urothelial carcinoma, necrosis and isomorphic erythrocytes were found in two cases (two of 10, 20.0%) and 10 cases (10 of 10, 100%), respectively. Dysmorphic erythrocytes were identified in three cases (three of three, 100%) of renal glomerular disease. Vimentin reactivity was found in three cases (three of three, 100%)

of renal glomerular disease, but never in urothelial carcinoma cases. In pathologic evaluation of histologic specimens of renal biopsy, the reactive renal tubular cells showed vimentin reactivity and a striking morphologic similarity to the cannibalism found in the voided urine. Conclusions: Although cannibalism in voided urine cytology has been considered an important cytological finding in urothelial carcinoma, our results show that it is present not only in urothelial carcinoma but also in renal glomerular disease. We conclude that the cannibalism in voided urine does not itself rule out renal glomerular disease in cases of urothelial carcinoma. Our results also show that vimentin reactivity of cannibalism and background (necrosis, isomorphic erythrocytes and dysmorphic erythrocytes) were accurate, but that cytomorphologic parameters (diameter, chromatin pattern) of cannibalism were not very accurate for specific parameters of differential diagnosis between renal glomerular disease and urothelial carcinoma.

199 Is really useful the rapid evaluation (RE) of the FNAC biopsies in thyroidal lesions? V Oikonomou, T Drouveli, M Fotou, E Koniaris, E Kotzia Clinical Cytology department, ‘‘Hippokratio’’ General Hospital, Athens, Greece Background: The purpose of this study was to compare the Rapid Evaluation (RE) of the FNAC biopsies from the thyroid gland to the Non-Rapid Interpretation (NRI). The NRIs were performed by the clinician or radiologist. While all REs, including the preparation of the smears were performed by a cytopathologist. Design: This study describes our 3-year experience from 1.01.05 to 31.12.07 of 1063 FNAs of thyroid gland biopsies. From those, 817 were RE-FNA biopsies (76.86%) that were performed by a specialized cytopathologist and 246 (23.14%) were NRI-Fnas received from the outpatient Pathology Department. All the obtained slides were stained with May-Grunwald-Giemsa and Papanikolaou stains. While in all the FNAs performed by cytopathologist, at least two air-dried, Diff-Quick stained smears were prepared for immediate microscopic evaluation. Both RE and NRI-FNAs were reviewed at final interpretation by three cytopathologists. Results: Of the 817 RE-FNAs samples, the 740 (90.57%) were adequate for supporting cytological diagnoses, while only 77 cases (9.43%) were non diagnostic. Furthermore from the total 246 of NRI cases only 137 (55.69%) were diagnostic and 109 cases (44.31%) were inadequate for cytological report. Conclusions: A successful FNA requires a specimen with adequate cellularity, high quality preparation and an experienced cytopathologist. According to our findings we believe that RE-FNA thyroid biopsy offer these three conditions, guiding further clinical investigations and decisions as well as determining further ancillary studies (flow cytometry, molecular studies and immunocytochemistry.



200 Role of post-chemoradiation endoscopic brush cytology and biopsy in predicting residual esophageal adenocarcinoma H Peng1, B Greenwald2, V Manucha1, S Nugent1, W Rodgers1, C Sun1 1 Department of Pathology, 2Division of Gastroenterology and Hepatology, Department of Medicine, University of Maryland, School of Medicine, Baltimore, USA Background: Adenocarcinoma of the esophagus and esophagogastric junction (EGJ) is aggressive and has a poor prognosis. The majority of these patients present with locally advanced tumors or regional lymph node metastasis. Combination chemoradiation followed by esophagectomy has been used to treat locoregionally advanced carcinomas. Pathologic complete response in the resected esophagus is achieved in about 20–30% of cases, and these patients tend to have a longer survival than those with less than pathologic complete response. Identifying this subset of patients has potential implications for management. Post-chemoradiation upper GI endoscopic brush cytology and biopsy have been used to diagnose the presence of residual tumor before a definitive resection is performed. However, the clinical value of these two diagnostic methods in predicting the presence of residual carcinoma is not clear, and data on the accuracy of these techniques is lacking. Design: Forty-six patients with locally advanced esophageal or GEJ adenocarcinoma, who underwent preoperative chemoradiation therapy and had post-chemoradiation endoscopic brush cytology and biopsy followed by esophagectomy were identified from the database at the University of Maryland Medical Center from 1995 to 2006. Using histological diagnosis of the esophagectomy specimen as the gold standard, we evaluated the performance of cytology and biopsy in diagnosing residual carcinoma. We compared the cytomorphology of adenocarcinoma before and after chemoradiation in 12 cases with residual carcinoma who have available cytology and/or histology slides before and after treatment. Two pathologists independently reviewed all false negative and false positive cases. Disagreements between the two reviewers were resolved by consensus review. Results: The sensitivity, specificity, positive and negative predictive values and accuracy for cytology to diagnose residual carcinoma were 27%, 92%, 90%, 33% and 46% respectively. These rates for biopsy were 9%, 85%, 60%, 27% and 30% correspondently. Sampling error accounted for false negative diagnosis in two third of cytology cases and 97% of biopsy cases. One third of false negative cytology cases and one false negative biopsy case were due to under recognition of tumor cells. The contributing factors included obscuring acute inflammation, necrosis, tumor cells mimicking benign cells with radiation atypia and under recognizing mucincontaining adenocarcinoma cells. Conclusions: Both brush cytology and biopsy are specific but not sensitive diagnostic methods to predict residual cancer after chemoradiation for esophageal adenocarcinoma, however cytology is superior. Biopsy can be replaced by brush cytology alone in postchemoradiation patients to diagnose residual carcinoma. The utility of brush cytology along with molecular markers in predicting response to preoperative treatment should be explored.

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201 Preliminary study regarding the endoscopic-ultrasound guided fine needle aspiration role in the assessment of the mediastinal tumors F Popescu1, A Saftoiu2, V Comanescu1, E Plesea3, M Ghilusi1, A Ioncica4 1 Morphopathology and Cytopatology Department, Emergency County Hospital, Craiova, Romania, 2Department of Gastroenterology, University of Medicine and Pharmacy, Craiova, Romania, 3Department of Morphopathology, University of Medicine and Pharmacy, Craiova, Romania, 4 Research Center of Gastroenterology and Hepatology, University of Medicine and Pharmacy Craiova, Romania Background: Endoscopic ultrasound (EUS) – guided fine-needle aspiration (FNA) is a minimally invasive technique, increasingly used for the assessment of digestive organs. However, this technique is important to determine the diagnosis and staging of pulmonary cancer by assessing the posterior mediastinal masses. Design: Our preliminary study included 26 patients visualized by EUS at the Research Center of Gastroenterology and Hepatology, University of Medicine and Pharmacy Craiova. The patients were initially examined by chest x-ray, computer tomography scans and bronchial endoscopy, without a tissue confirmation of malignancy. All the patients were assessed by EUS-FNA followed by cytology examination and imunocytochemistry. From the material obtained by fine needle aspiration we prepared smears which have been stained by Giemsa stain (at least four smears per case) and Papanicolaou stain (at least four smears per case). It has been assessed the following cytopathological parameters: smears cellularity; presence, disposition, shape and number of the cells with malignant features; presence of other benign cells (inflammatory cells, red cells). The smears were categorized as: benign, suspicious probably malignant and malignant. Imunocytochemistry was performed on cell blocks obtained from 15 patients and we used the following antibodies: (i) for detecting the primary pulmonary tumor: anti-CK 7, anti-TTF1, anti-CEA, anti-chromogranin, anti-synaptophysine; (ii) for detecting the local metastases: anti-ER, anti-PR, anti-CK 20, anti-CA 19-9, anti-CA 125; and (iii) for the evaluation of the proliferation rate of the tumors: anti-Ki67, anti-PCNA. Results: Of the 26 patients, 21 had a positive FNA, confirming the diagnosis of malignancy, the samples being obtained from the lymph nodes at level 5 (the aorto-pulmonary window) or level 7 (the subtraheal space), as well as from the primary mediastinal tumors. One of the patients could not be assessed by EUS-FNA because of the difficulty to find a suitable puncture route, without interfering with the main vessels (the aorta, the pulmonary artery). In this patient, the diagnosis was confirmed by EUS-guided FNA of the left adrenal gland. Four patients had negative EUS-FNA and the diagnosis was confirmed by EUS-guided large needle (Trucut). On imunocytochemistry, tumor cells from all cases expressed CK 7 and CEA, and were negative for ER, PR, CK 20, CA 19-9 and CA 125. In addition, in six cases, there was a slight and focal expression of TTF1 and synaptophysin within tumor cells. The Ki67-LI was higher in tumor cells with severe nuclear atypia and was not correlated with PCNA-proliferative index. Conclusions: In conclusion, EUS is highly sensitive for diagnosing lung cancer, while FNA improves not only the specificity, but also


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the accuracy. In addition, EUS-FNA is an excellent minimal invasive technique, especially when the standard techniques of histological diagnosis are negative, influencing the therapeutic actions and preventing unnecessary invasive techniques such as: thoracotomy, thoracoscopy and mediastinoscopy.

202 Accuracy of transthoracic FNAB in parenchymal lung lesions: retrospective analysis of 2 years (242 cases) S Ramazanoglu1, C Cakalir1, B O¨z1, C Akman2, A Demirkaya3, K Kaynak3, R Dashti4 1 Department of Pathology, 2Department of Radiology, 3Md Istanbul University Cerrahpasa Medical Faculty, Department of Thoracic Surgery, 4 Department of Neurosurgery, Istanbul University Cerrahpasa Medical Faculty, Istanbul, Turkey Background: Fine Needle Aspiration Biopsy (FNAB) is the first method of choice in patients with a lung mass and it is a reliable method. We wanted to find out the sensitivity/specificity, positive/ negative predictive values and the accuracy of FNAB of parenchymal lung lesions diagnosed in Istanbul University, Cerrahpasa Medical Faculty, Department of Pathology. Design: In our study, we retrospectively evaluated 242 FNAB of parenchymal lung lesions (from 238 patients) performed and diagnosed (both cytologically and histologically) in Istanbul University Cerrahpasa Medical Faculty, in the years 2006 and 2007 (FNAB of mediastinal masses and consultation cases were omitted). All of the FNAB were done at Radiology Department under the guidance of computerized tomography, where a pathologist evaluated the adequacy of the aspirated materials on site. The slides were smeared and fixed in alcohol for Papanicolau stain and some air dried for MayGru¨nwald-Giemsa stain. Immunocytochemical stains were applied to paraffin embedded cell blocks which were fixed in formalin. The FNAB results were analyzed from archival cytopathology reports and all of the patients were searched for subsequent resection/core needle biopsy either of the lesion or excision of the lymph node/distant organ metastasis (brain, esophagus, pleura). All of the FNAB diagnoses (242) were then compared with histology (n = 64). For the calculation of accuracy and other parameters, consequent and although concurrent cytology specimens were omitted. In addition, six cases which were cytologicaly grouped as suspicious for a tumor and/or malignancy and two cases insufficient for diagnosis with subsequent histologic diagnoses, were not included in the analysis. Results: We grouped our cases as malignant (67.35%), benign (14.88%), suspicious for a tumor and/or malignancy (9.09%), necrotic material (with no further comment) (5.37%) and insufficient for diagnosis (3.31%). FNAB diagnoses of the malignant group (n = 163), showed that adenocarcinomas (61.35%, n = 100) comprised the majority. The second most common histologic type was squamous cell carcinoma (14.11%, n = 23). Benign group included, parenchymal changes, nonspecific inflammations, tuberculosis (acid fast bacilli demonstrated by Ehrlich-Ziehl-Nielsen stain), hydatid cysts and a hamartoma. In our study the sensitivity of FNAB in lung lesions was 92% and the specificity 83%; positive predictive value was 97.9% and negative predictive value 55.6% and the accuracy was found to be 91.1%.

Conclusions: FNAB in lung lesions is a reliable method with high sensitivity and accuracy rate.

203 Cytology – the bridge between the clinician and the pathologist E Szekely, R Istok, T Szekely, B Jaray 2nd Department of Pathology, Semmelweis University, Budapest, Hungary Background: Although cytology is a cheap, minimally invasive method among the numerous available diagnostic possibilities, it is not well appreciated by many clinicians. The possible cause of this attitude is multiple: (i) clinicians are unaware of the fact that sample taking is not as simple as it seems to be, and in many cases cytologists are given inappropriate samples for examination, which are unsuitable for diagnosis; (ii) clinicians are unaware of the fact that without appropriate clinical data correct diagnosis cannot be given in most cases, or conversely, with appropriate clinical data, associated with correctly put questions by the clinicians, a more appropriate diagnosis and answer can be given by the cytopathologist; and (iii) unfortunately cytology is practiced by many unexperienced pathologists, who give false positive/false negative diagnoses, further destroying the reputation of this diagnostic modality. Design: In our department we have about 2500 ultrasound guided fine needle aspiration cases (liver 330, pancreas 50, kidney 30, retroperitoneum 20, breast 420, thyroid 550, lymph nodes 450, salivary glands 100, other sites 500) yearly. The aspirates are used not only for conventional cytological examinations, but 10 percent of the cases are examined by immunocytochemistry, and on certain occasions, fluorescent in situ hybridization is also performed on specifically prepared smears. Two-thirds of the aspirations are performed by the cytologist, who examines the aspirate. Results: The results are strikingly better when the sample is taken by the cytologist, since personal experience is important to recognize which smear seems to be inappropriate macroscopically, needing further sampling (3.5/20 % inadequate smears in case of lymph node aspirates, taken by clinicians or cytologists, respectively). Conclusions: We wish to reiterate and underline the most important steps during sampling and diagnosing smears to reposition cytology to it’s worthy place in the diagnostic cascade. We would like to illustrate our statements with some of our favourite cases.

204 VEGF expression in cytologic material from squamous cell carcinomas of the oral cavity Z Tatsiou1, K Boglou1, R Valeri1, A Patrikidou2, K Vahtsevanos2, N Kehagias2, C Destouni1 1 Department of Cytopathology, 2Department of Maxillofacial Surgery, Theagenion Cancer Hospital, Thessaloniki, Greece Background: Angiogenesis is a crucial step in tumor growth, progression and metastasis. Vascular Endothelial Growth Factor (VEGF) is a multifunctional cytokine that plays a pivotal role in angiogenesis.



Also, it has been shown that VEGF is overexpressed in many forms of carcinomas, including oral cancer, with a positive rate of 60–80%, on immunohistochemical studies. Oral squamous cell carcinomas (SCC) represent a group of diseases with high mortality and increasing rates among young individuals. The aim of this study was to evaluate the expression of VEGF in cytologic material from SCC of the oral cavity. According to recent bibliographic data, this is the first immunocytochemical analysis of VEGF expression in cytological material of oral SCC. Design: We examined 30 cases of SCC of the oral cavity (tongue: six cases, buccal: four cases, upper lip: 12 cases and lower lip: eight cases), which were obtained by brush and FNA biopsy. The material was processed using both conventional and Liquid Based Cytology (ThinPrep) technique. Immunocytochemical investigation by using monoclonal antibody of VEGF (clone VG1, Diagnostic Biosystems) was followed. Results: VEGF cytoplasmic immunoexpression was evident in almost 60% of oral SCC. No difference was detected in immunoexpression as far as the location of the tumors is concerned. Conclusions: 1. ThinPrep technique seems to provide a better cytomorphology with a cleaner background as well as easier application of immunocytochemistry, comparing to conventional smears. 2. VEGF expression was evident in a high proportion of SCC of the oral cavity, approximately 60%. 3. Therefore, it could be used as a potential biomarker for oral neoplasia in order to plan targeted therapy.

205 Cytologic detection of adenocarcinoma of the female genital tract: a retrospective cohort study comparing ThinPrep, SurePath, and conventional papanicoulau preparations C Teman, J Bentz Department of Pathology, University of Utah, Salt Lake City, Utah, USA Background: The incidence of adenocarcinoma of the uterine cervix, a tumor with an aggressive clinical course, is rising. While these lesions are now well-recognized as glandular entities in the 2001 Bethesda System for Reporting Cervical Cytology (AGC, AIS, Adenocarcinoma), they have been difficult to detect with conventional cervicovaginal cytology. The current study was performed to assess the accuracy of liquid-based cytology (LBC) in detecting cervical and endometrial adenocarcinomas. Design: The subject group consisted of all LBC specimens (SurePath or ThinPrep) identified as AGC, AIS or adenocarcinoma diagnosed between July 1997 and April 2008, for which histologic follow-up was available. Conventional smears (CS) collected in the same time period comprised the control group. A case was called positive if the histologic follow-up showed either dysplasia or invasive carcinoma. Results: Of a total of 455 cases identified as AGC, AIS or adenocarcinoma, 163 (35.8%) were conventional Pap smears, 233 (51.2%) were SurePath preparations, and 59 (13.0%) were ThinPreps. On histologic follow-up, the SurePath method identified the highest number of malignancies (18% vs. 16.6% CP vs. 8.5% TP) as well as overall cases with significant pathology (31% vs. 24%). The

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ThinPrep method identified the highest number of dysplasias (CIN13 and glandular dysplasias) (15.2% vs. 13% SP vs. 7.4% CP) and there was no difference between CP and TP in overall incidence of significant pathology. Specimen

Mean Total

type

age

SurePath

50.4

Thinprep

49.4

Conventional Pap 52.7 Total

51.2

Positive for Positive for Total

Total

negatives dysplasia

malignancy positives cases

160

31

42

73

(68.7%)

(13.3%)

(18.0%)

(31.3%) (51.2%)

233

45

9

5

14

(76.3%)

(15.2%)

(8.5%)

(23.7%) (13.0%)

59

122

12

27

39

(76.0%)

(7.4%)

(16.6%)

(23.9%) (35.8%)

163

329

52

74

126

(72.3%)

(11.4%)

(16.3%)

(27.7%)

455

Conclusions: SurePath liquid-based cytology specimens categorized as AGC, AIS or adenocarcinoma appear to have an improved accuracy for histologically-proven significant pathology than either ThinPrep or conventional cervicovaginal preparations.

206 PCR detection of human papillomavirus infection in tunisian women with normal cytology H Tounsi-Guettiti1, L Attia2, D Chelly3, A Chachia2, T Laassili1, E Enaifer-Jerbi1, S Boubaker4 1 Research Unit ‘‘Human Papillomavirus’’, Department of Pathology Institut Pasteur de Tunis, 2Department of Gynaecology Charles Nicolle Hospital –Tunis, 3Department of Gynaecology La Rabta Hospital –Tunis, 4 Pathology department Institut Pasteur de Tunis, Tunisia Background: Detection of human papillomavirus infection (HPV) becomes increasingly attractive as a primary screening tool, because of sensitivity and cost-effectiveness. As a matter of fact, different proportions of HPV infected women show normal cytology in many studies all over the world. This data are not yet available in Tunisia and their knowledge is a necessary step before considering a new screening approach. Aim: To estimate the frequency of HPV infected women with normal cytology in Tunisia. Design: Study group: 480 Tunisian women between 18 and 72 years old are collected between June 2005 and July 2007. They were attending by two hospital centers of Tunis for a wide spectrum of gynaecological complaints, with a normal. physical examination. Conventional Pap smear does not show any abnormality. Design: For each woman DNA sampling was performed by dry cotton swabs. For HPV detection a PCR (MY09/MY11) and a linear array were used. Cytological examination was investigated on the bases of the Bethesda system 2001. Results: Nineteen women (3.95%) were HPV infected with 13 single infections and six mixed infections. Twelve different HPV types were detected. Most frequent types were HPV 16 and HPV 11 equally (36.8%). 52.6% of all infections were related to high risk types.


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Conclusions: HPV infection with normal cytology shows road variations between countries: 1.4% in Spain; 25.6% in Nigeria (one). Our data are nearly the same as northern Mediterranean region (1.4–9.2%) but lower than Asian, South American and sub-Saharian African countries. More than half of normal Pap smears bear high risk HPV types and most frequently HPV 16 (36.8%). This confirms that a primary HPV testing approach screening is more sensitive than cytology alone. However, the absolute risk for cervical cancer for high risk HPV types infected women with normal cytology needs to be well defined. Reference: 1. Clifford G M, et al. Worldwide distribution of human papillomavirus types in cytologically normal women in the International Agency for Research on Cancer HPV prevalence surveys: a pooled analysis. Lancet 2005; 366: 991–998.

207 Determination of human Papilloma Virus L1 capsid protein expression levels in Pap smears based on computerized image analysis E Tsiambas1, L Kampas1, A Karameris2, R Valeri1, C Destouni1 1 417 VA Hospital (NIMTS), Athens, Greece, 2‘‘Theagenion ’’ Anti-Cancer Hospital, Thessaloniki, Greece

grade (LGSILs, HGSILs) dysplastic lesions seem to be a prognostic factor for the progression of HPV infection in squamous epithelia of uterine cervix. Our aim was to evaluate its expression based on a quantitative digital image analysis system. To our knowledge this is the first experimental study regarding the marker. Design: Using thin prep fixed Pap smears derived from invasive squamous cell carcinoma (n = 5), HGSILs (n = 15), LGSILs (n = 15) and normal cytological samples (n = 10) we performed immunohistochemical analysis based on a panreactive HPV L1 antibody. Digital image analysis was applied for the evaluation of L1 protein expression results (staining intensity levels and Nuclear Labeling Index-NLI). Results: HPV staining intensity levels were not associated significantly with the types of examined squamous cell lesions (P = 0.67). In contrast to this observation, invasive carcinomas and also HGSILs demonstrated low NLIs compared to LGSILs (P = 0.03). Conclusions: The total amount of HPV L1 immunostained cells and not the corresponding staining intensity levels may be is an objective criterion for a rational evaluation of squamous lesions in uterine cervix associated also to their progression.

Background: Detection of Human Papilloma Virus L1 (HPV L1) protein expression in Pap test smears demonstrating low or high



ELECTRON MICROSCOPIC DIAGNOSTIC PATHOLOGY 208 Ultrastructural characteristics of amyloid deposits M Be´ly1, A Apa´thy2, P Kapp1 1 Policlinic of the Order of the Brothers of Saint John of God in Budapest, 2 National Institute of Rheumatology and Physiotherapy, Budapest, Hungary Objective: The aim of this study was to define the ultrastructural characteristics of various types of amyloid deposits (systemic AA, systemic AL-k, localized AL-k, and systemic b2-microglobulin amyloidosis (Ab2M). Design: The tissue specimens studied included colon and rectum with systemic AA amyloidosis; heart, bone marrow and periarticular soft tissues with systemic AL-k? amyloidosis; epipharynx with localized AL-k amyloidosis; and synovial membrane of both hip joints with hemodialysis associated b2-microglobulin associated amyloidosis (Ab2M). The tissues studied with the light microscope were also examined electron microscopically. The ultrastructure of amyloid deposits of different types was examined with a JEM 100C· electron microscope. Results: Electron microscopically there was no difference between various amyloid deposits in morphology of filaments and fibrils. The amyloid deposits were present as extracellular ‘lakes’ showing a loose filamentous deposition, alternating with electron dense areas of condensed or fragmented filaments. There were differences in involvement of vessel walls, arrangement (condensation) of filaments, and cellular response of phagocytes. In case of systemic AA, or systemic AL amyloidosis the arterioles and small arteries were always involved. The amyloid filaments within the vessel walls or around the capillaries were more compact than the more distant interstitial amyloid deposits. In case of localized AL amyloidosis the vessel walls were free of deposits, and the condensation of filaments was more pronounced than in systemic ALk or systemic AA amyloidosis. Areas of loose filamentous amyloid deposits alternated with electron dense territories of clusters composed of regularly (parallel) arranged, closely packed filaments and fibrils. In some cases the ‘lakes’ were bordered by circularly condensed fibrils at the periphery, followed by a mid-zone in which, perpendicular to the peripheral ring, clusters of closely packed fibrils were radially arranged, while in the center only loosely deposited filaments were present. The circular zone at the periphery and the radial middle zone of closely packed fibrils correspond to the ‘crystalloid like’ birefringence of the localized AL amyloid deposits. This type of deposition is characteristic of localized AL amyloid electron-microscopically. Electron microscopically the extracellular amyloid deposits of Ab2M deposits showed a loose filamentous-fibrillar arrangement. The deposits were inhomogeneous and one or several focal dense, core-like accumulations of filaments were seen, surrounded by a loose interdigitating, or peripheral marginal zone. The dense cores were composed of compactly arranged or fragmented filaments of different sizes and variable thickness. The loose filamentous zone at the periphery of amyloid deposits corresponds to the superficial (peripheral) ring of birefringence observed under polarized light. Conclusions: The production of amyloid precursors related to the cardiovascular system becomes generalized via the bloodstream,

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resulting systemic forms of amyloidosis. The local production of immunoglobulin light chains is not directly related to the systemic circulation and remains a localized process with organ- or tissuelimited isolated amyloidosis. Electron microscopically the closely packed or fragmented filaments may appear as single or multiple, core like, electron dense structures within the loose filamentous amyloid deposits. According to our interpretation these represent the stage dependent maturation (or fragmentation) of deposited amyloid protein filaments, indicative of the advanced stage of deposition.

209 Ultrastructural localization of adhesive protein molecules of Pneumocystis carinii to type i alveolar epithelial cells K Kwon1, Y Kim2, J Park1, H Jung1, S Kwon1, M Choi1, Y Kang1, S Kim1, S Lee1 1 Department of Pathology Keimyung University School of Medicine, 2 Central Electron Microscopy Laboratory Keimyung University School of Medicine, Deagu, South Korea Background: Both fibronectin and vitronectin bind to Pneumocystis carinii (P. carinii) and mediate the attachment of the organisms to host respiratory epithelial cells. Surfactant A and D play a role in the interaction between P. carinii and alveolar epithelial cells. In this study we examined by immunohistochemistry and immunoelectron microscopy, utilizing the pre-embedding method, the expression of fibronectin, vitronectin, surfactant-A and D, and their role in the interaction between P. carinii and alveolar epithelial cells. Design: The experimental rat model of P. carinii pneumonia was induced by administration of low protein diet (8%) and drinking water containing dexamethasone (2 mg/L) for 6–8 weeks. The primary antibodies for light and electron microscopic immunohistochemistry were monoclonal antibodies against fibronectin (1:100) and vitronectin (1:100), and polyclonal antibodies against surfactant A (1:50) and D (1:50). Results: Light microscopic immunohistochemistry for the fibronectin, vitronectin, surfactant-A and D showed strong expression of these molecules on the P. carinii and on the type I alveolar epithelial cells. However the exact localization for these molecules could not be done at light microscopic level. The electron-microscopic examination showed localization of the fibronectin and vitronectin along the surface pellicles and the tubular extensions of P. carinii trophozoites, as well as the surface membranes of the type I alveolar epithelial cells. Additionally, the surfactant-A and D proteins were strongly expressed on the pellicles of P. carinii and the surface membranes of the type I alveolar epithelial cells, but they were weakly expressed on the free-floating surfactant residues. Conclusions: Our results indicate that the trophozoites and the cysts of P. carinii are mostly attached to type I alveolar epithelial cells. On the basis of the light and electron microscopic immunohistochemical findings, it is presumed that fibronectin, vitronectin, surfactant-A and D molecules play enhancing roles for binding between the P. carinii organisms and the type I alveolar epithelial cells.


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210 Male breast nipple adenoma with DCIS followed 9 years later by invasive carcinoma P Rao, S Shousha Department of Histopathology, Charing Cross Hospital and Imperial College, London, UK Background: Nipple adenoma is a distinct rare benign lesion which is very uncommon in males. The first reported case in a male dates back to 1965. Malignant change in these lesions has been rarely reported. Interestingly AFIP fascicle of tumors states that nipple adenoma co exists with carcinoma in 50% of male patients, indicating its precancerous potential. Design: A study of a case of nipple adenoma in a male patient with foci of DCIS that reached the excision margins but was not reexcised; and was followed by an invasive carcinoma in the same area 9 years later. Results: A 76 year old male patient presented with a 4 month history of itching and blood stained discharge from the left nipple. The nipple appeared sore and crusted. No axillary lymph nodes were palpable. An excisional biopsy was carried out. Histopathological examination revealed a nipple adenoma with marked sclerosis of the stroma and foci of intermediate grade micropapillary DCIS. No invasive carcinoma was seen. No reexcision was done. Nine (9) years later the patient presented with a lump in the left breast at the previous biopsy site. A core biopsy of the lump revealed a poorly differentiated invasive ductal carcinoma. A wide local excision of the lump was done. On microscopic examination a grade three invasive ductal carcinoma was seen, with satellite tumor deposits in the overlying skin, but no Paget’s disease was seen. Areas of low grade DCIS and lymphovascular invasion were also noted. Conclusions: This uncommon case supports a possible link between male nipple adenoma and carcinoma. It also highlights the importance of complete excision of such lesions. The time scale of nine (9) years between the diagnosis of DCIS and the detection of invasive carcinoma is consistent with that provided in the literature concerning female patients.

211 A new variant of ciliopathy syndrome: oralfacialdigital-like with respiratory tract symptoms from birth and ultrastructurally ciliary dysplasia U Stenram1, C Cramnert1, H Axfors-Olsson2 1 Department of Pathology, University, Lund, Sweden, 2Central Hospital, Va¨xjo¨, Sweden Background: Primary ciliary dyskinesia is a ciliary disease giving rise to respiratory tract infections and often situs inversus. It is less known that most cells of the body have a single, as a rule immobile cilium, monocilium or primary cilium. Mutations in their genes give strange malformation syndromes. Respiratory tract symptoms are seldom reported, except in Alstro¨m syndrome (sinusitis, chronic bronchitis) and Bardet-Biedl syndrome (asthma), but ultrastructural studies of respiratory tract cilia have not been reported in these entities.

Case Report: A girl presented with polydactyly, high arched palate, tongue lipoma and dysmorphic face. She had respiratory tract problems from birth, atelectasis, pneumonia and later bronchiectasis, and viscous secretion in the middle ears. The fourth ventricle is slightly enlarged. At 4 years of age she takes only a few steps without a walker and speaks very few words. Electron microscopy of nasal brush biopsies at four occasions revealed almost total ciliary aplasia, but long, sometimes branched microvilli. A few cilia were seen in the cytoplasm and also lumens. Most basal bodies/centrioles were found deep in the cytoplasm, often surrounded by vesicles. Gene analysis is in progress. Results: The case has resemblances to but does not quite tally with the Bardet-Biedl and the oralfacialdigital syndromes (OFDS). These and three other syndromes are due to mutations affecting the basal bodies. The present case has especially similarities to the OFDS but in addition respiratory tract problems from birth. There seems to be a decreased ability to form cilia. Conclusions: Such a case has not previously been described. It has been approved by POSSUM as well as London dysmorphology databases as OFDS, Stenram type.

212 Ultra structural lesions study of testicles of alloxan induced diabetes mellitus in dog M Valilou1, I Sohrabi2, J Soleimani3, D Mohammadnejhad4 1 Department of pathology, faculty of Veterinary medicine Islamic Azad University, Shabestar branch, 2Department of Pathology, Faculty of Specialized Veterinary medicine Islamic Azad University, Science and Research Campus, Tehran, 3Faculty of Medicine and Drug Applied Research Centre Tabriz University of Medical Sciences, 4Department of Histology Tabriz University of Medical Sciences, Shabestar, Iran Background: Diabetes mellitus is one of the most common diseases of endocrine glands in body that is diagnosed by malfunction in natural metabolism of carbohydrate, fat and protein. This disease involves most tissues of the body and the consequent deficiencies reduce their efficiency, cause infections and disease in body. Alloxan is a chemical substance which is used in creating experimental diabetes in animals. In this research, nine apparently healthy German shepherd dogs were provided, five of which was considered as our experimental group and the remaining four was considered as our control group. The necessary examinations were conducted to guarantee their health and we approved the absence of diabetes with intravenous Glucose Tolerance Test (IVGTT). We injected intravenously Alloxan monohydrate with 100 mg/kg in experimental group. Presence of diabetes was approved by IVGTT. In experimental group, If the symptoms were indicating the death and in control group (after end of experimented group) we took sample tissue of testicles. We scrutinized microscopic and ultra structural deficiencies. We could see vacuolization of cytoplasm, cell degeneration and big interval space in spermatogonium cells. Spermatogenesis process was faced derangement in testicles and was stopped in one of them. Leydig cells was decreased.



ENDOCRINE PATHOLOGY 213 Primary thyroid non-hodgkin lymphomas: histopathological and immunohistochemical analysis of seven patients H Aki1, R Ramazanoglu1, E Akyildiz1, H Demir1, F Aksoy1, N Tuzuner1, Y Bukey2 1 Istanbul University, Cerrahpasa Medical Faculty, Department of Pathology, 2Istanbul University, Cerrahpasa Medical Faculty, Department of General Surgery, Istanbul, Turkey Background: Primary thyroid lymphoma is a rare tumor representing approximately 1–5% of all thyroid malignancies and comprises less than 2% of all extranodal lymphomas. Thyroid lymphoma almost exclusively occurs in patients with Hashimoto’s thyroiditis. It usually presents as a rapidly progressive mass in the the thyroid gland. Therefore, progressively enlarging goitre and symptoms of compression, are the most common clinical manifestations. Design: We retrospectively analyzed pathological and immunohistochemical features of seven cases of primary thyroid lymphomas diagnosed between the years 2000–2007, in Istanbul University Cerrahpasa Medical Faculty, Department of Pathology. Patients’ records and archival pathology specimens were reviewed and CD20, CD10, bcl-2, bcl-6, MUM-1 and Ki67 antibodies were applied immunohistochemically to histologic sections. Results: Median age of the seven cases was 58 (range 34–82). Male to female ratio was 2:5:1. Common symptom in all cases was a neck mass and one of them had signs of compression. Total thyroidectomy was performed in five cases whereas in two, subtotal thyroidectomy was done. Six of the cases were diagnosed as large B cell lymphoma and one as follicular center cell lymphoma. Immunophenotypically, CD20 was positive in all cases, CD10 in three, bcl-2 in one, bcl-6 in three, MUM-1 in two cases. Ki-67 score was high in all of the cases (80–100%). Four of the cases showed germinal center B cell phenotype (GCB) whereas two were non-germinal center B cell (non-GCB). The remaining case was only positive for CD20 and did not show any staining for GCB and non-GCB phenotype antibodies. Conclusions: Primary thyroid lymphomas are rare neoplasms and their prognosis depends upon the histologic subtype, immunophenotype and stage. Up to now in literature, thyroid lymphomas with germinal center B cell phenotype shows better prognosis than noncenter B cell phenotype. Therefore, in a case of lymphoma CD20, CD10, bcl-2, bcl-6, MUM-1 should be performed immunohistochemically for immunophenotypic expression and Ki-67 score should be evaluated for prognostication of patients.

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logical categories: oncocytoma, oncocytic neoplasm of uncertain malignant potential and oncocytic carcinoma. Oncocytic carcinoma of the adrenal cortex (OAC) is an exceptional pathological entity, which often appears as locally advanced disease or with distant metastases. Design: The aim of this study was to investigate the clinical, histolopathological and immunohistochemical features of an OAC in a 54-year-old man. The patient was referred to our hospital with metastatic lung disease. He had a history of right adrenalectomy with partial hepatectomy due to a malignant adrenal tumor. Initially, he underwent a wedge resection and later was submitted to three more consecutive resections due to recurrent metastases in both lungs and the adjacent mediastinal vessels and lymph nodes. We studied the pathological material of all metastatic foci in paraffin-embedded tissues stained with hematoxylin-eosin and special immunohistochemical stains. Results: Histological examination revealed a neoplasm with an oxyphillic cell population and a solid (mainly), rosette-like and papillary growth pattern. Focal necrosis was present. Moderate nuclear atypia and a moderate number of mitotic figures were found. The proliferation index (MIB-1/Ki-67) was positive in 10–20% of tumor cells and p53 oncoprotein was weakly expressed only in a few cells. Immunohistochemically, there was positivity for CK8-18 and AE3 (dot-like paranuclear expression), Vimentin, Melan-A, Calretinin and Synaptophysin. Inhibin and CD56 were expressed focally. CK7, CK20, EMA, CEAm, CEAp, TTF-1, Chromogranin, S-100, CD31, CD34 and FVIII were negative. The patient history combined with the neoplasm morphological and immunohistochemical features supported a metastatic oncocytic adrenocortical carcinoma diagnosis. At the follow-up, 3.5 years after the first operation, a right hip joint metastasis was revealed and concurrent chemoradiation treatment was applied. He stayed in a good performance status for 14 months but he finally passed away 5 years after the first diagnosis of his disease. Conclusions: The OAC of our case showed aggressive behavior with constant metastases and this fact may be assumed to originate in carcinomatous emboli that entered the inferior vena cava. There is no absolute agreement among different authors on the rules applying to the histological categorization of oncocytic adrenocortical tumors depending on their biological behavior. It is expected that acquisition of experience from a larger series of patients will contribute to the solution of this problem in the future.

214 Oncocytic adrenocortical carcinoma with recurrent metastases P Argyriou, C Zisis, M Kopaka, P Kara, F Malta, C Petraki Department of Thoracic and Vascular Surgery, Department of Nephropathology, Evangelismos Hospital, Athens, Greece

215 Universal markers of thyroid malignancies: galectin-3, HBME-1 and cytokeratin-19 F Barut1, N Onak Kandemir1, S Bektas1, B Bahadir1, S Keser2, H Hancer3, S Ozdamar1 1 Zonguldak Karaelmas University Faculty of Medicine, Department of Pathology, Zonguldak, Turkey, 2Lutfi Kırdar Kartal Training and Research Hospital, Department of Pathology, Istanbul, Turkey, 3Zonguldak Karaelmas University Faculty of Medicine, Zonguldak, Turkey

Background: Oncocytic adrenal neoplasms represent unusual lesions composed entirely or almost entirely of cells with abundant, granular, eosinophilic cytoplasm and are divided into three histo-

Background: Difficulties in the diagnosis of thyroid lesions, even with histologic analysis, are well known. This study has been carried on to evaluate the role of immunohistochemical markers;


96


galectin-3, a beta-galactoside-binding protein; HBME-1, monoclonal antibody against mesothelial cell surface protein and cytokeratin-19 in the diagnosis and differential diagnosis of benign and malignant thyroid lesions. Design: The expression of galectin-3, HBME-1 and cytokeratin-19 have been tested in formalin-fixed, paraffin-embedded tissues from 460 surgically resected thyroid lesions including non-neoplastic lesions (n: 363) (283 hyperplastic nodules; three granulomatous, 62 lymphocytic and 15 Hashimoto’s thyroiditis), benign neoplastic lesions (n: 30) (22 follicular adenomas, eight Hu¨rthle cell adenomas), and malignant neoplastic lesions (n: 67) (55 papillary carcinomas, 10 follicular carcinoma, one insular carcinoma, one medullary carcinoma). Immunostaining, with standard avidin-biotin complex technique has been performed by using monoclonal antibodies, galectin-3, HBME-1 and cytokeratin-19. For each antibody, immunoreactivity has been scored as negative (less than 10% cells positive), 1+ (poor-focal positive), 2+ (strong-focal positive), 3+ (poor-diffuse positive), 4+ (strong-diffuse positive), based on the extent of the reaction regardless of previous diagnosis. Diffuse staining patterns were accepted as significantly positive, and sensitivity and specificity were assessed for each marker in the diagnosis of malignant neoplastic thyroid lesions. Results: In malignant neoplastic thyroid lesions, galectin-3, HBME1 and cytokeratin-19 were mostly diffusely expressed. Diffuse expression rates of these three markers were 74.6% (50/67), 71.6% (48/67), 85.0% (57/67), respectively. The sensitivity and specificity for galectin-3, HBME-1, and cytokeratin-19 expression were 77.0%, 77.0%, 79.7% and 99.2%, 99.7%, 98.2%, respectively, in malignant lesions. In papillary and follicular carcinomas, the sensitivity for galectin-3, HBME-1, and cytokeratin-19 expression were 77.5%, 76.4%, 88.7%, and 83.3%, 90.1%, 55.5%, respectively. The specificity for galectin-3, HBME-1, and cytokeratin-19 expression were 99.2%, 99.8%, 98.2%, and 99.2%, 99.8%, 98.2% respectively, in papillary and follicular carcinomas. These three markers were mostly negative or focally expressed in almost all benign and nonneoplastic thyroid lesions. Conclusions: The use of monoclonal antibodies, galectin-3, HBME1 and cytokeratin-19 provide significant contributions in the differential diagnosis of malignant thyroid tumors. Although focal galectin-3, HBME-1, and cytokeratin-19 expression may be encountered in benign lesions, diffuse positive reactions for these three markers are characteristic for malignant lesions. Cytokeratin-19 was the most sensitive, whereas HBME-1 was the most specific antibody in papillary carcinomas. Meanwhile, HBME-1 was the most sensitive and the most specific antibody in follicular carcinomas.

216 Immunohistochemical approach to thyroid nodules with diagnostic challenge P Bo¨rcek, A Poyraz Gazi University Faculty of Medicine Department of Pathology, Ankara, Turkey Background: Differential diagnosis of thyroid nodules constitutes an important problem in surgical pathology routine especially for follicular lesions, where follicular adenoma, follicular carcinoma,

adenomatous nodules and follicular variant of papillary carcinoma compose the diagnostic spectrum. Three immunohistochemical markers, namely CK19, HBME-1 and galectin-3, are being intensely investigated in recent years for their utility to differentiate between malignant and benign thyroid masses. The present study is performed to determine the diagnostic value of these three markers in thyroid nodules with diagnostic difficulty. Design: All thyroidectomy specimens examined between the years 2004 and 2007 in our department were re-evaluated without the information on the final diagnosis. 88 cases which present diagnostic difficulty including suspicious nuclear features for papillary thyroid carcinoma, suspicious invasion in a follicular lesion, lesions combining both features, and papillary architecture with insufficient nuclear features for the diagnosis of papillary thyroid carcinoma were selected. 20 cases of clear malignancy diagnosed as papillary and follicular thyroid carcinoma were also included to serve as controls. All of the 108 cases included in the study were immunohistochemically stained with CK19, HBME-1 and galectin-3 antibodies. The results were statistically analyzed by SPSS 12.0 package program for statistics. Results: All three markers were more commonly expressed in malignant lesions than in the benign ones and the positivity ratios for tumors with uncertain malignant potential were between benign and malignant nodules. The highest ratios of positivity in all three markers were observed in papillary thyroid carcinoma. There was no difference between the staining patterns of the nodules with nuclear suspicion which were finally diagnosed as papillary carcinoma and the nodules with nuclear suspicion which were finally diagnosed as benign nodules for all three antibodies. It was detected that positivity with all three antibodies is more commonly observed in malignant nodules and negativity with all three antibodies is more common in benign nodules. However, false positive and false negative cases made up a considerable proportion. Conclusions: CK19, HBME-1 and galectine-3 have only a contributory role in the diagnosis of diagnostically challenging thyroid nodules. The most accurate results may be obtained by careful examination of the histologic features of a given nodule on hematoxylin and eosin stained sections.

217 Are giant cells always predictive for a bad evolution in papillary thyroid carcinoma? A Borda1, A Loghin2, J Hommell3, M Decaussin-Petrucci4, D Sava1, S Stolnicu2, N Berger3 1 Department of Histology University of Medicine and Pharmacy, Tıˆrgu Mures¸ , Romania, 2Department of Patology: Medical University Targu Mures, Romaˆnia, 3Department of Pathology Hopital Lyon Sud, Lyon, France, 4Department of Pathology Hospices Civils de Lyon, Universite´ Lyon, France Background: Giant cells associated with papillary thyroid carcinoma (PTC) are usually recognized as a feature of dedifferentiation in conventional papillary thyroid carcinoma (CPTC) and evolution towards anaplastic carcinoma.



Design: We present the case of a young man of 36 years old, with a history of a growing mass in the right lobe of the thyroid. The fine needle aspiration specimen was reported as PTC and consequently a total thyroidectomy was performed. Macroscopically the 4 cm diameter nodule was whitish and well defined from the normal tissue but not totally encapsulated. In microscopy the architecture was follicular and trabecular, without solid component and the nuclear features were typical for a CPTC. The particularity of the case was that in some areas we noticed islets of very pleomorphic giant cells, admixed with the CPTC. The cytoplasm was abundant and more eosinophilic than in neighboring cells, but not oncocytic-like. The nuclei of these cells were enlarged, irregular in shape and size, some of them truly monstrous, with coarse chromatin and prominent nucleoli. Some of these giant cells were multinucleated. Neither mitosis nor necrosis was found. Immunohistochemistry with anti-thyroglobulin and antiTTF1 antibodies was performed. Results: The CPTC cells and also all the giant cells strongly expressed both thyroglobulin and TTF1. After 6 years of follow up the patient is alive, with no recurrence or metastases. Conclusions: The WHO classification and the majority of the authors recommend to consider the pleomorphic giant cells areas as a feature of dedifferentiation in thyroid carcinoma, with a bad evolution and a high rate of mortality. In our case the positivity for thyroglobulin, the absence of necrosis, the preservation of the trabecular architecture and especially the 6 years free disease survival of the patient were markers of a good prognosis tumor. These data are consistent with our opinion that not all giant cell components are predictive for dedifferentiation in PTC. When all morphologic features mentioned above are gathered, these giant cells may represent only dystrophic changes.

218 Expression of angiogenic factors in solid cell nests (ultimobranchial remnants) of the thyroid gland J Cameselle-Teijeiro1, I Abdulkader1, M Alfonsıń-Barreiro2, R ReyesSantıás1, P Soares3, J Forteza1, M Sobrinho-Simoes3 1 Hospital Clıńico Universitario University of Santiago de Compostela, Spain, 2Hospital do Meixoeiro de Vigo, 3IPATIMUP University of Porto, Portugal Background: Solid cell nests (SCNs) of the human thyroid, usually considered as the remnants of the ultimobranchial body, may play a critical stem cell-like role in the genesis of important clinical disorders of the thyroid gland. To further our insights concerning SCNs we have investigated the expression of five angiogenic factors in the SCNs of eight thyroid glands. Design: The immunohistochemical studies were performed on paraffin sections using a peroxidase-conjugated labeled-dextran polymer (EnVision Peroxidase/DAB, DAKO, Glostrup, Denmark), to avoid misinterpreting endogenous biotin or biotin-like activity as positive staining. Commercially available monoclonal and polyclonal antibodies for VEGF (clone VG1, dilution 1:10, water bath, Dako), VEGFR-1 (Fit-1, 1:1000, water bath, Santa Cruz, California, USA), VEGFR-2 (polyclonal rabbit, 1:2000, water bath, Santa Cruz), VEGFR-3 (KLT9, 1:10, water bath, Novocastra, Newcastle upon Tyne, UK) and HIF-2a (MAB3472, 1:100, water bath,

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Chemicon, Hampshire, UK) were used. As additional markers of SCNs monoclonal antibodies for p63 (4A4, 1:50, water bath, Dako) and galectin-3 (9C4, 1:200, Novocastra) were also used. Results: Strong positivity for p63 and galectin-3 was found in main cells of all SCNs. No immunoreactivity was observed in SCNs nor in follicular cells for VEGF, VEGFR-1, nor VEGFR-3. Both the main cells of SCNs and follicular cells were positive for VEGFR-2, but immunoexpression of HIF-2a was only found in SCNs. Conclusions: The exclusive expression of HIF-2a in the main cells of SCNs is an important finding which merits special consideration.

219 The expression of KI 67, p53 and cyclin d1 in thyroid carcinomas S Coban, N Yalcin Department of Pathology, Pamukkale University School of Medicine, Denizlil, Turkey Background: Thyroid carcinomas, comprising 1 % of all the cancer cases all over the world, are the most frequently experienced malignity of endocrine system and are those that cause mortality in the same manner. Most neoplasias seen in thyroid are primary and epithelial tumors. In this study we compared the clinical and histopathological data with the expressions of Ki-67, a proliferative marker of thyroid carcinomas, P 53, the suppressor gene of tumor and Cyclin D1, the regulator of cell cycle; and tried to find out the answer whether these three immunohistopathological markers are important in the estimation of the prognosis. Design: The cycle of Ki-67, P 53 and Cyclin D1 have been evaluated together with the clinical and histopathological data in 52 cases of thyroid carcinoma (38 of which were Papillary Carcinomas, nine Follicular Carcinomas, three Medullary Carcinomas and two Anaplastic Carcinomas). The statistical analysis of the obtained data has been carried out at SPSS 10.0 statistical package. Results: In comparison to the benign differential thyroid carcinomas, proliferative rate of Ki-67 has been found increased in the cases of anaplastic thyroid carcinomas. The proliferative rate of Ki-67 has not shown any meaningful differentiation between the cases of Papillary carcinoma, Follicular carcinoma, Medullary carcinoma and the variants of Papillary carcinoma. However, it has been noticed that the proliferative rate of Ki-67 has increased in thyroid carcinomas with vascular invasion. It has also been determined that the sighting risk of vascular invasion has increased in the cases where the proliferative rate of Ki-67 is higher than 5%. The difference between the groups is not meaningful when the rate of tumor cells in which the expression of nuclear P 53 and Cyclin D1 were determined are analyzed in comparison to the tumor groups. What is more, no meaningful relationship has been statistically determined between prognostic parameters and the expression of P 53 and Cyclin D1. Conclusions: As a result, it has been found that when the differentiation in thyroid carcinoma decreases, the proliferative rate of Ki67 increases. However, it has also been noticed in the cases of differentiated tumors that Ki-67 does not have any contribution to distinctive diagnosis. When the rate of Ki-67 is higher than 5%, it has been concluded that this rate might be stimulating in terms of vascular invasion. It has been determined that P 53 and Cyclin D1


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do not have any effect on the distinctive diagnosis of thyroid carcinoma and clinicopathological prognostic parameters. It has also been considered that the clinicopathological parameters (age, gender, the type and diameter of the tumor, the vascular invasion, the spread of tumor out of capsula, lymph node – distant metastasis and the stage of tumor) are more important in thyroid carcinomas to estimate prognosis.

220 Papillary thyroid carcinoma with nodular fasciitis-like stroma: a case report with BRAFV600E mutation C Colato1, M Gobbato1, C Cannizzaro1, P Brazzarola2, G El-dalati3, M Davı`4, G Francia4, M Ambrosetti3, F Basolo5, M Ferdeghini3, F Menestrina1 1 Dipartimenti di Patologia, 2Scienze Chirurgiche e Gastroenterologiche, 3 Scienze Morfologico-Biomediche, 4Scienze Biomediche e Chirurgiche, Universita` di Verona e, 5Dipartimento di Chirurgia, Universita` di Pisa, Italy Background: Papillary carcinoma (PTC), the most common malignant tumor of the thyroid, has several morphological variants, some with clinical and prognostic implications. We described one case of an unusual histologic subtype of PTC characterised by prominent stromal cell proliferation, resembling fibromatosis of soft tissues or fibroadenoma/phyllodes tumor of the breast. This variant has been named as PTC with fibromatosis-like stroma, PTC with nodular fasciitis-like stroma or PTC forming myofibroblastic nodular tumor. The histogenesis of stromal proliferation and clinicopathological significance of this PTC variant have not been fully clarified because of its rarity. Clinical Data: A 52 years old man presented to our institution for evaluation of a large fore-neck mass. Ultrasound studies of the neck showed a well-defined solid mass with a cystic peripheral space. Total thyroidectomy was performed after fine-needle aspiration yielded a diagnosis of PTC. On gross examination, the resected tumor was well demarcated, measuring 11 cm in diameter with a pseudocystic peripheral space. On the cut surface, the tumor was greyish-white in color with fibromatous appearance. Results: Microscopically the tumor was composed of epithelial and stromal components simulating fibroadenoma or adenosis of the breast. The abundant stromal component (60% of the neoplasia) had irregular and interlacing fascicles of spindle cells lying in a fibrous/fibromyxoid matrix. Stromal cells had neither atypia nor mitotic figures. Spindle cells were positive for a-smooth muscle actin, vimentin, WT1 but negative for S100, CD34, Thyroglobulin, TTF-1, Cytokeratin and p53, suggesting a myofibroblastic nature. Epithelial component was arranged in anastomosing tubules, lobular glandular structures, papillae and follicles. A leaflet-like growth pattern was detected at the tumor periphery and isolated round cells were also scattered in the stroma. Epithelial cells showed typical nuclear features of PTC. This component was positive for Galectin-3, HMBE, CK19, Claudin-1, GLUT-1 and WT1. At molecular level this tumor carried BRAFV600E mutation detected by PCR-SSCP and direct DNA sequencing. Metastases to two periglandular lymph nodes showed epithelial component devoid of stroma.

Conclusions: This case represents a rare morphological variant of PTC characterized by peculiar spatial arrangement of the epithelial and stromal components simulating tumors as fibroadenoma/phillodes tumor, (cysto)adenofibroma, adenosarcoma or carcinosarcoma of the female genital tract and breast. The pathogenesis of stromal cell proliferation is still an open question regarded as reactive in origin or autonomous proliferation stimulated by cytokines. To date, 18 cases have been reported. Recognition of this variant is important both to avoid diagnostic misinterpretations of cytologic and histologic specimens and of intraoperative consultation and to enable appropriate management. It must be distinguished from PTC with anaplastic transformation and carcinosarcoma and from benign conditions as fibrous variant of Hashimoto’s thyroiditis, Riedel’s thyroiditis and de Quervain’s thyroiditis. This variant usually implies a good prognosis, as classic PTC. Only one case showed an unusual aggressive behavior, linked to a progressive transformation of conventional PTC into poorly differentiated component. To the best of our knowledge, this is the first case with BRAFV600E mutation that is reported. Patient’s follow-up is needed to evaluate influence of oncogenic BRAF on clinical course.

221 The misleading thyroid solid cell nest (SCN) J Cote1, C Marin1, R Karkouche1, Z Merabet2, B Franc1 1 Service d’Anatomie et de Cytologie Pathologiques, CHU Ambroise Pare´, AP-HP, Universite´ Versailles St-Quentin en Yvelines, Boulogne-Billancourt, France, 2Hoˆpital Henri-Mondor, AP-HP, Cre´teil, France Background: Micropapillary thyroid carcinomas (PTC) are generally indolent but show attributes which are candidate markers for malignancy such as HBME-1, CK19, Galectin-3, and loss of TPO. This group of carcinomas represents no less than 45% of new thyroid carcinoma cases per year. They are difficult to distinguish from benign conditions such as SCN and rare squamous thyroid metaplasia (STM) due to confusing histological features and similar immunohistochemical staining. Design: We present the immunophenotypic profile of a series of five SCN and two STM taking into consideration several features of SCN solid and follicular components, as well as the surrounding normal follicular thyroid cells. The expression of the following antibodies was tested: HBME-1, Galectin-3, CK19, TPO, TTF-1, p63 and thyroglobulin (TG). The immunostaining was evaluated as follows: negative: less than 50% stained cells and positive: greater than 50% stained cells. Results: The solid components of the SCN (5/5) and STM (2/2) are almost 100% positive for CK19, Galectin-3 and p63 and negative with TPO and TG. HBME-1 staining is positive in the two STMs and in the solid component of 1/5 SCNs, weak/rare in 1/5 solid SCN, and absent in 3/5 solid SCN. Weak nuclear expression of TTF-1 is seen in 2/5 solid SCN. Two of the three SCNs negative for nuclear TTF-1 show positive cytoplasmic TTF-1 staining. In contrast, the follicular components of the five SCN are negative for Galectin-3 in 4/5 specimens, negative for CK19 in 2/5 and positive for p63 in 3/4 available cases. TG and TTF-1 are positive in the SCN follicular component. TPO is partly or totally absent in 3/5 follicular SCN components. The STMs are positive for TTF-1 in less



than 50% of the nucleus, weaker than in the adjacent thyroid follicles. As expected, normal thyroid follicles are only positive with TTF-1, TPO, and TG. Conclusions: The antibodies in this series have not been previously tested together on SCN or STM. Separate studies have shown similar results except for TTF-1, while HBME-1 and TPO were not tested. SCN are a source of confusion in conventional thyroid pathology. Complementary results add to the confusion. Strict morphological criteria should be applied before concluding a diagnosis of malignancy.

222 Gene expression profiles in archival thyroid carcinoma using preamplification Q-RT-PCR and immunohistochemistry K Denning, P Smyth, R Flavin, S Finn, S Russell, E Conlon, J Li, S Aherne, J OLeary, S Orla Department of Histopathology, University of Dublin, Trinity College, Dublin, Ireland Background: Thyroid carcinoma is the most frequently occurring endocrine malignancy. Previous work carried out by our group using whole genome expression microarrays indicated a cohort of gene markers capable of discriminating between variants of thyroid carcinoma. This study investigated the utility of these markers in an expanded cohort of archival specimens at both gene and protein levels of expression using RT-PCR and immunohistochemistry. Design: Two hundred five cases of archival thyroid specimens (51 PTC, nine Hu¨rthle cell, 10 FTC, one MTC, one ATC, nine thyroiditis, 51 hyperplastic, 26 benign, 47 normal) were analyzed for gene expression of TOP2A, APOE, BAX, MMP11, and LYN using TaqMan Real Time PCR. Laser Capture Microdissection was used to procure pure cell populations for extraction. In addition, a novel multiplex pre-amplification technique was used to facilitate analysis of multiple targets. Utilizing immunohistochemistry, protein expression analysis of TOP2A, APOE, BAX, MMP11, and LYN was performed on a tissue microarray (TMA) encompassing the same 205 cases.

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Results: Specimens considered unsuitable for analysis of protein (due to loss of/inadequate TMA cores) or gene expression (inadequate Ct values) were removed from the final study cohort resulting in parallel gene and protein data in 179 cases for TOP2A, 180 cases for LYN, 161 cases for MMP11, 178 cases for BAX and 177 cases for APOE. Statistical analysis (non-parametric Mann–Whitney U-test) showed significance at both gene and protein levels of expression for all five targets across a range of diagnostic comparisons, which are shown in Table 1. Conclusions: TOP2A, APOE, BAX and MMP11 have discriminating power as biomarkers of thyroid neoplasia, based on significant up-regulation at gene and protein expression levels in the study cohorts analysed. LYN also shows potential as a biomarker for differentiating between follicular lesions in thyroid carcinoma.

223 Expression of somatostatin receptors in gastroenteropancreatic neuroendocrine tumors E Diakatou1, G Kaltsas2, G Kontogeorgos1 1 Department of Pathology, ‘‘G. Gennimatas’’ Athens General Hospital, Athens, Greece, 2Department of Pathophysiology, University of Athens, Athens, Greece Background: Gastroenteropancreatic neuroendocrine tumors (GEP NETs) originate from cells of the diffuse endocrine system in the gastrointestinal mucosa or the pancreatic islets. They form a heterogenic group of neoplasms with different biology and functional activity, differentiation and clinical course. The recent WHO classification has developed a system to access the prognosis of these tumors according to tumor size, Ki-67 labeling index (LI), presence of necrosis, vascular invasion and presence of metastases grading GEP-NETs as: (i) well-differentiated neuroendocrine tumors (WD-NETs); (ii) well-differentiated neuroendocrine carcinomas (WD-NECs); and (iii) poorly differentiated neuroendocrine carcinomas (PD-NECs). A distinctive feature of the well-differentiated neoplasms is the presence of somatostatin receptors (sst). There are five receptor types, which are encoded by five different genes; the sst2

Table for 222 TOP2A

APOE

BAX

MMP11

LYN

protein/gene P-values





Normal (no hyperplasia) vs. Malignant

0.002/0.004

0.0024//=++). A statistically significant (ss) positive association was observed between KLK10 IE and tumor stage (P = 0.015) and liver metastases (P = 0.035). Paradoxically, a ss negative association was observed between KLK10 IE and tumor grade (P = 0.009). Kaplan–Meier and univariate analysis showed that KLK10 IE and stage had ss relation with DFS (P = 0.030 and P < 0.001, respectively) and OS (P = 0.010 and P = 0.001, respectively). Cox multivariate analysis showed that KLK10 IE and stage were independent unfavorable predictors for DFS (P = 0.057 and P = 0.001, respectively) and OS (P = 0.009 and P = 0.001, respectively). Conclusions: KLK10 IE may contribute to the prediction of disease relapse and patients survival in CRC.

359 The frequency of variability of duodenal histologic lesion in celiac disease K K Prasad, B R Thapa, C K Nain, K Singh Divisions of GE Histopathology and Pediatric Gastroenterology, Department of Superspeciality of Gastroenterology Postgraduate Institute of Medical Education and Research, Chandigarh, India Background: It is generally believed that in CD, mucosal lesions may have a patchy distribution. The aim of this study was to evaluate the frequency of histologic lesion variability of the duodenal mucosa in Indian children with CD.

Design: Sixty seven children with suspicion of CD and positive tTGab test were enrolled in a prospective fashion. During esophagogastroduodenoscopy (EGD) at least two biopsies each were taken from duodenal bulb (B) and descending duodenum distal to the papilla of Vater (D2). The histologic lesions were classified according to the Oberhuber classification after modification as proposed by our group. Results: Sixty-seven children (42 boys and 25 girls; mean age 6.3 ± 0.4 years) were recruited with a mean duration of symptoms 2.9 ± 0.2 years. Recurrent diarrhea, 83.6%; anemia, 76.1%; failure to thrive (FTT), 62.7%; abdominal distension, 40.3%; and abdominal pain, 22.4% was the main clinical manifestations in CD. All celiacs had evidence of villous atrophy (VA)–type3a, 3b, or 3c–in at least one biopsy site. Similar degree of VA in both biopsy sites was seen in 23.9% celiacs, whereas 64.2% had a ‘mixed’ type3 lesion characterized by a different degree of VA at different biopsy sites. The identical type of lesion (1, 2 or 3) in both biopsy sites was seen in 88.1% celiacs. No CD patients show exclusively type or type1 lesion in both biopsy sites. Two different types of histologic lesion (type1 + type2 or type2 + type3) were present in 11.9% celiacs. Overall variability of histologic lesion (variability in the grade of VA, and coexistence of different types of lesion) was seen in 76.1% of celiacs. The variability of histologic lesion in celiacs has no relationship either with age; sex; duration of symptoms; clinical manifestations like recurrent diarrhea, anemia, FTT, abdominal distension and abdominal pain; or duodenal IEL count (P > 0.05). Conclusions: There is high frequency of histologic lesion variability of the duodenal mucosa in Indian children with CD. Therefore, during EGD at least four duodenal biopsies (2 from distal duodenum and 2 from duodenal bulb) should be obtained in order to avoid the risk of underdiagnosis or misdiagnosis due to the fact that CD has a variable distribution of histologic lesion.

360 Study of duodenal mucosal enzyme activities and morphological changes in irritable bowel syndrome K K Prasad, S K Sinha, S Bhonchal, C K Nain, A K Sharma, K Singh Department Gastroenterology and Histopathology, Postgaraduate Institute of Medical Education and Research, Chandigarh, India Background: Irritable bowel syndrome (IBS) is a disease entity characterized by recurrent abdominal pain or discomfort associated with altered bowel movement, in which no obvious pathogenetic lesion is identified to explain the symptoms. The involvement of the duodenal mucosa rarely been systematically studied in IBS. Therefore, the purpose of this study was to evaluate the enzyme activities and morphological changes in duodenal mucosa of patients with IBS. Design: In this prospective study duodenal biopsy from diagnosed cases of IBS (n = 21) were studied for brush border enzymes (disaccharidases, ALP, LAP, LDH, GGT), intracellular enzymes (G-6-PDH, ICDH, G-6-P), membranous enzyme (Na+-K+ ATPase) activities and morphological (light microscopic and ultrastructural) changes. The duodenal biopsy from patients with GERD (n = 29) were taken as control. The results were expressed as number and percentage or mean ± SEM. Comparisons of quantitative measurements between groups were performed with Student’s t test. Informed written con-



sent was obtained prior to participation in study. The study was approved by the ethical committee of our Institute. Results: The mean age (66.7% males, 33.3% females) of IBS patients was 33.1 ± 10.6 years. In duodenal mucosa of IBS and GERD patients the mean enzymatic activities (Mean ± SEM; IU/g protein) of lactase was 22.3 ± 1.4 vs. 24.4 ± 1.1 (P > 0.05), sucrase was 46.3 ± 2.2 vs. 42.5 ± 1.9 (P > 0.05), maltase was 77.1 ± 3.9 vs. 76.1 ± 2.6 (P > 0.05), LAP was 128.5 ± 5.5 vs. 131.9 ± 4.6 (P > 0.05), LDH was 91.6 ± 7.5 vs. 104.9 ± 4.1 (P > 0.05), GGT was 104.2 ± 7.7 vs. 99.4 ± 4.3 (P > 0.05), ALP was 901.6 ± 48.8 vs. 830.7 ± 30.3 (P > 0.05), G-6-PDH was 11.4 ± 0.5 vs. 11.0 ± 0.2 (P > 0.05), ICDH was 24.7 ± 0.7 vs. 23.2 ± 1.6 (P > 0.05), G-6-P was 1.6 ± 0.05 vs. 1.7 ± 0.05 (P > 0.05) and Na+-K+ ATPase was 2.2 ± 0.1 vs. 2.5 ± 0.1 (P > 0.05). IBS patients showed normal villous structure (95.2%), intraepithelial lymphocyte count of 20.4 ± 1.1 with intraepithelial lymphocytosis in 19.1% and an excess of lamina propria infiltrate (42.9%) in the duodenal mucosa. Scanning electron microscopy showed normal villous structure and well oriented microvilli in IBS patients. All duodenal mucosal histological parameters of IBS patients were not significantly different from GERD patients. Conclusions: Irritable bowel syndrome patients were not showed any significant alteration in various enzyme activities indicating normal function of enterocytes. This was substantiated by light microscopic and ultrastructural changes.

361 Synchronous adenocarcinomas of colorectal and extra colorectal origin mimicking advanced colorectal carcinoma: report of two unusual cases T P Quek1, T P Thamboo2, A Wee2, G C Raju2, D Lim1 1 Department of Pathology, National University Hospital, Singapore, 2 Department of Pathology, Yong Loo Lin School of Medicine, Singapore Background: Colorectal adenocarcinoma is one of the most common malignancies worldwide. Rarely, it may co-exist with malignancies from non-colorectal sites. Distinguishing local or metastatic spread of colorectal adenocarcinoma from an extra-colorectal synchronous malignancy can be difficult, especially if the co-existing tumor is also an adenocarcinoma. This may be a potential pitfall in the accurate staging and prognostication of these tumors, which can affect the treatment of these patients. We report two cases of colorectal adenocarcinoma with synchronous adenocarcinomas from unusual primary sites, both of which have never been described in the English literature. Case 1: A 67-year-old man presented with a change in bowel habits. Colonoscopy revealed a rectal tumor, confirmed on biopsy to be a moderately differentiated adenocarcinoma. CT and MRI scans of the abdomen showed a lesion in segments 5 and 8 of the liver, suggestive of either a metastatic deposit or a haemangioma. The patient underwent an anterior resection with central hepatectomy. Intraoperative frozen sections performed on cores of the liver lesion showed an adenocarcinoma. Histological examination of the resected specimens showed features of a moderately differentiated adenocarcinoma in both sites, although the two tumors were histomorphologically distinct. The rectal tumor showed diffuse strong staining for CK20 and villin, focal weak staining for CK7 and

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CK19, and was negative for 34bE12, Hep Par and CD56; consistent with a primary rectal adenocarcinoma. The liver tumor however, showed strong staining for CK7, CK19 and CD56, patchy staining for villin and 34bE12 and no staining for CK20 or Hep Par, consistent with a primary cholangiocarcinoma. Case 2: A 68-year-old woman presenting with constipation underwent colonoscopy, which showed an extrinsic stricture in the sigmoid colon with normal-looking mucosa. Serum CA-125 was raised. A CT scan of the abdomen and pelvis showed narrowing of the sigmoid colon lumen from a large adjacent tumor mass with omental caking and numerous peritoneal deposits. Biopsy of the peritoneal nodules showed a serous adenocarcinoma, which was CK7 positive, CK20 and CDX2 negative. The patient was treated with chemotherapy, which led to significant size reduction of the peritoneal lesions and serum CA-125 levels. The patient then underwent a low anterior resection, total hysterectomy with bilateral salpingo-oophorectomy, omentectomy and appendectomy. Histological examination showed multiple tumor deposits within the colonic wall, omentum, lymph nodes, appendix as well as a solitary 5 mm deposit on the capsular surface of the right ovary. These had similar morphologic and immunophenotypic characteristics as the initial peritoneal tumor. Incidentally, a 2.5 cm tumor mass with a villoglandular morphology, confined to the mucosa and submucosa of the bowel, was also identified at the area of colonic constriction. This tumor was CK20 and villin positive and CK7 negative, which was consistent with a primary colonic adenocarcinoma. Conclusions: A high index of suspicion, together with attention to morphologic details and judicious use of immunohistochemical techniques, may aid in the distinction of advanced colorectal carcinoma from synchronous tumors of colorectal and extra-colorectal origin. This is of importance because of the prognostic and therapeutic implications.

362 The diagnostic value of immunohistochemistry in gastrointestinal stromal tumors (GIST) D Radulescu1, S Stolnicu2, L Ungureanu1, L Jalba3, S Dumitriu1 1 Department of Pathology, UMF ‘Gr. T. Popa’, Iasi, Romania, 2Department of Pathology, UMF Targu Murres, Targu Murres, Romania, 3 Department of Pathology, Spitalul Judetean Botosani, Botosani, Romania Background: To determine the immune profiles of 9 cases of GISTs (8 in the stomach and 1 in the large bowel). Design: Cases diagnosed as GIST were examined by both light microscopy and IHC. Tumors were diagnosed as benign and malignant by considering the tumor size and number of mitoses. We also used these antibodies: Desmin, SM Actin, S-100 protein and CD 34. Results: Most mesenchymal neoplasms were gastrointestinal stromal tumors (GIST) or smooth muscle types. Six cases resembled smooth muscle tumors histologically as well as grossly, and in three cases the tumors had moderate cellularity and focal nuclear palisading that can resemble nerve sheath tumors. When the results of both light microscopical and immunohistochemical


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analysis were considered toghether, five tumors with SM differentiation showed CD34 expresion as well as SM markers. Also two tumors, IHC showed SM profile in one and neural in the other. Conclusions: This study showed that IHC is very helpful to determine the differentiation pattern and also that CD34 positivity and neural differentiaton seem to correlate with features of malignancy observed in light microscopy.

363 Gallbladder cancer in a high risk country. Results of a prospective study of 1366 patients I Roa1, X De Aretxabala2, O Tapia3 1 Department Of Pathology, Clinica Alemana, Faculty of Medicine, Universidad del Desarrollo, Santiago, Chile, 2Department of Surgery, Clinica Alemana, 3Department of Pathology, Faculty of Medicine, Universidad de la Frontera, Temuco, Chile Background:Gallbladder cancer (GBC) is one of the most epidemiologically significant neoplasias in Chile, and for the last 20 years it has been the leading cause of death by malignant tumor in women. We aimed to show the results obtained from 17 years of standardized study on GBC patients in the Hospital in Temuco, Chile. Design: A study of 25 971 cholecystectomies: 79% women with an average age of 45.4 years (SD 15.9 years), and 21% men with an average age of 53.6 years (SD 16.4 years) (P < 0.0001). An analysis was conducted of macroscopic and microscopic variables as well as digitized images, follow-up and survival in 1366 patients with GBC. Results: 1138 were women and 228 were men, with an average age of 61.5 years (60.7 for women and 65.5 for men). 29% were of Mapuche origin. 346 cases were early carcinomas (25.3%) and 1020 were advanced. Mucosal carcinomas: 213 cases (15.6%), muscular: 132 cases (9.7%), subserosal: 316 cases (23.5%) and serosal: 382 cases (28%). The remaining cases saw compromise in the adjacent organs. 95.6% were adenocarcinomas and 1.5% adenosquamous. Only 21.5% were well differentiated tumors. The overall survival rate was 38% at 5 years. Unapparent carcinomas had the best prognosis at 92% vs. 25% at 5 years (P < 0.001). Papillary and nodular mucosa was present in 80% of the cancers and cholesterolosis in 1% of the cancers (P < 0.0001). The tumors associated with acute inflammation and a greater deformation of the gallbladder had a worse prognosis (P = 0.03 and P = 0.001). The best differentiated tumors had a significantly higher survival rate at 5 years than those will less differentiation (P < 0.0001). A gallbladder wall with a thickening > de 10 mm was associated with poorer survival, as was a gallbladder less than 9 cm long (P = 0.001). Patients with carcinomas with venous vascular compromise did not survive beyond 36 months. Conclusions: There are many factors present in gallbladder specimens with cancer associated with the prognosis and these must be taken into consideration before complementary therapies are established.

364 Excision repair cross-complementing 1(ERCC1) expression in gallbladder cancer is a prognostic factor? I Roa1, X De Aretxabala2, S Lantadilla1 1 Department of Pathology, Clinica Alemana, Faculty of Medicine, Universidad del Desarrollo, Santiago, Chile, 2Department of Surgery, Clinica Alemana, Santiago, Chile Background: Gallbladder cancer (GBC) has been one of the main causes of death by malignant tumor in women in Chile for the last 20 years. ERCC1 is a highly preserved protein within the DNA repair nucleotides, and its importance has been demonstrated in other tumors such as lung cancer. There is no information with respect to the significance of ERCC1 expression in GBC. We aimed to determine the expression and the significance of ERCC1 expression as a prognostic factor in GBC. Design: Tissue microarrays (TMA) were made using 200 gallbladder cancers and 76 non-tumor gallbladder samples as controls. In 190 cases, ERCC1 could be determined. Immunohistochemistry techniques were used on the ERCC1 protein [monoclonal antibody ERCC1 Ab-2 (clone 8F1) Labvision]. Nuclear staining of at least 50% of the tumor cells or an intense positivity in more than 10% of the tumor cells was considered positive staining (according to other authors). Results: The total study group consisted of 166 women and 24 men with an average age of 62.7 years (61.7 years in women and 69.8 years in men). In 120 cases (63%), positive staining was proven in the tumor cell nuclei. The best differentiated tumors showed positive staining of greater intensity (P < 0.05) with respect to those that were poor differentiated. Significant differences between tumor infiltration level in the gallbladder wall and the positivity in ERCC1 expression were not observed. Nor were any relations observed between the positivity of the staining and other microscopic or macroscopic variables. The actuarial survival at 5 years was over 90% in the early cancers, and in the advanced cancers it was 27%. No relation was observed between the positivity of ERCC1 and survival in early carcinomas, but in patients with advanced carcinomas, the positive expression in the tumor cells was associated with a significantly better survival (P = 0.01). Conclusions: A high percentage of GBC show positive expression for ERCC1. Our preliminary results demonstrate that patients with advanced gallbladder cancers who have not undergone complementary treatments present better survival than those with negative expression of this protein. The importance of this finding has still not been validated and gene transcription studies will be required to confirm these findings.

365 Immunoexpression of COX-1, COX-2 AND P53 in Crohns disease M Romero, R Artigiani-Neto, H Costa, C Oshima, S Miszputen, M Franco Department of Pathology and Department of Gastroenterology, Federal University of Saõ Paulo, Brazil Background: Crohn’s disease (CD) accompanied by nonspecific or idiopathic ulcerative proctocolitis corresponds to a condition



called intestinal inflammatory disease. The immunoexpression of COX-2 in CD becomes more marked with progression of the disease and the presence of wild-type p53 suppresses the transcription of COX-2. Design: To investigate the immunoexpression of COX-1, COX-2 and p53 in Crohn’s ileocolitis and to correlated this expression with clinical and histopathological parameters. Forty-five cases of CD, 16 cases of actinic colitis (diseased-control group) and 11 cases without a history of intestinal disease (normal control group) were studied. Hematoxylin/eosin-stained sections were submitted to histopathological analysis and the immunohistochemical expression of COX-1, COX-2 and p53 was evaluated by the streptavidin-biotin-peroxidase method. Results: Sixty percent of the CD patients were women and 40% were men, with 75.5% whites and 25.5% non-whites. The disease involved the terminal ileum in 44.5% of cases, ileum in 33.3%, colon in 20% and duodenum-ileum in 2.2%. A significant association was observed between COX-2 immunoreactivity and age £40 years (P = 0.049). Histopathological analysis of CD samples showed mild or moderate crypt distortion (57.8% and 35.6% of cases), atrophy (6.6%), mild, moderate and marked chronic inflammation (46.7%, 26.7% and 20%), acute inflammatory activity (93.3%), ulceration (24.4%), mucin depletion (37.8%), Paneth’s cells (24.4%), intraepithelial lymphocytes (93.3%), and subepithelial collagen (6.7%). In the CD group, COX-1 immunoreactivity in epithelial and inflammatory cells was observed in 26.7% and 22.2% of cases, respectively. COX-2 immunoreactivity was detected in epithelial cells in 68.9% of cases and in inflammatory cells in 46.7%. A significant difference in COX-2 reactivity was observed between epithelial (P = 0.0877) and inflammatory cells (P < 0.001) in association with acute inflammatory activity and increase in intraepithelial lymphocytes. Comparison of the date among the three groups (CD, actinic colitis and normal controls) showed a higher proportion of cases presenting COX-2 immunoreactivity in inflammatory cells in the CD group (P < 0.001). No p53 reactivity was observed in all cases. Conclusions: (1) COX-2 immunoexpression is high in CD, which suggest a possible role of the protein in the pathogenesis of the inflammation; (2) The absence of epithelial dysplasia in all CD samples was correlated with the lack of expression of p53.

366 Pleomorphic phenotypes and genotypes of gastrointestinal stromal tumors (GISTS) at metastatic sites; 26 autopsy cases without imatinib treatment K Sakamoto, S Sakurai, T Sano Department of Diagnostic Pathology, Graduate School of Medicine, Gunma University, Gunma, Japan Background: GISTs are the most common mesenchymal tumors found in the GI tract. A molecular targeting drug, imatinib mesylate, has recently been reported as effective against advanced and/ or metastatic GISTs. However, the efficacy of imatinib depends on the mutational status of c-kit or PDGFRA genes in GISTs. Moreover,

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secondary resistance to the drug has been reported in most GISTs after several years of administration. Secondary mutations in the ckit gene in GISTs have been detected in some of the tumors showing acquired resistance to imatinib and are thought to be a cause of resistance. However, these have not been found in over half of the GISTs with secondary imatinib resistance; thus the resistance mechanism remains unknown in those cases. Understanding the characteristics of advanced and/or metastatic GISTs is necessary for deciding a suitable treatment strategy for these GISTs. In this study we analyzed the histological and immunohistochemical phenotypes and genotypes of GISTs at different metastatic sites without imatinib treatment and clarified the pleomorphism of metastatic GISTs. Design: We examined 26 autopsy cases where the patient died of multiple metastases without imatinib treatment. Five metastatic sites were selected from an autopsy case and then examined by histological morphology, the expression of c-kit gene product (KIT), CD34 and loss of heterozygosity (LOH) of the c-kit gene. Results: From immunohistochemical findings, in 9 and 11 of 26 cases, tumors at some metastatic sites lacked KIT and CD34 expressions, respectively. Histology of these metastatic sites differed from KIT positive tumors in the same cases. LOH of the c-kit gene was observed in 2 of the 10 informative cases. Conclusions: The histological morphology and immunohistochemical phenotypes of metastatic sites vary among lesion without imatinib treatment. This knowledge could lead to specially tailored therapies, that would consider the specific histological features, for treatment of metastatic GISTs. We are now carrying out gene analyses of secondary c-kit mutations.

367 Apoptosis detected by cleaved cytokeratin 18 (M30) immunohistochemistry in colorectal cancer is related to acidic tumoral microenviroment C Scapulatempo1, A Longatto Filho2,3, K Simoes4, C Pinheiro2, F Schmitt5, F Baltazar2, V Alves4 1 Fundaçao Pio XII – Hospital do Cancer de Barretos, 2Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal, 3Instituto Adolfo Lutz, Saõ Paulo, Brazil, 4 LIM14, Department of Anatomic Pathology, Saõ Paulo University School of Medicine, Saõ Paulo, Brazil, 5IPATIMUP, Porto, Portugal Background: Gradual progression from normal mucosa through adenoma to colorectal cancer is a paramount model to understand tumor progression. Most of the genetic alterations contributing to colorectal carcinogenesis affect normal tissue homeostasis and involve alterations in apoptosis and proliferation signaling pathways. In the other hand, acid efflux through MonoCarboxylate Transporters (MCTs) constitute one of the most important mechanisms involved in maintenance of intracellular pH and also directly influence tumoral microenvironment. The goal of this study was to correlate M30, a well-accepted apoptotic marker with MCTs isoforms in colorectal cancer. Design: Paraffin blocks from 139 cases of colorectal carcinoma were retrieved from the files of Department of Pathology, Hospital das Clinicas, University of Saõ Paulo School of Medicine. From these cases, 43 had hepatic metastasis. Immunohistochemical reactions were performed for the following markers: M30 (a neo-epitope formed after


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cytokeratin 18 cleavage by caspases), p53, Ki-67, EGFR, MCTs 1, 2 and 4, D2-40 (for lymphatic vessel highligthing) and these were correlated with clinico-pathological parameters. Result: Pathological parameters showing significant correlation with tumors giving rise to hepatic metastasis were: serous infiltration (P = 0.047), deepness of tumor invasion (P = 0.019), infiltrative tumor borders (P = 0.0004) and lymph node metastasis (P = 0.003). M30 immunoexpression was similar between normal mucosa and adenoma, but was significantly different between them and primary tumor (P = 0.016 and P = 0.001, respectively). M30 expression was also significantly different between primary tumors and metastasis (P = 0.044). Interestingly, M30 significantly correlated with p53 expression (P = 0.01) but not with Ki-67 (P = 0.071). Newly, M30 showed a positive correlation with expression of MCT1 that importantly participate of tumoral microenvironment acidification and correlates with colorectal carcinoma aggressiveness. This is important because we found that tumoral size is related to M30 expression (P = 0.005), p53 expression (P = 0.03), and MCT4 expression (another key player in tumoral microenvironment acidification). Conclusions: Our results showed for the first time that apoptosis, as here detected by M30 marker, can be influenced by microenvironment acidification and be related to the more aggressive phenotype of colorectal carcinoma. This study is supported by grants from FAPESP and Fundaçaõ Faculdade de Medicina, Brazil.

368 Tumor stage dependent expression of leptin receptor in human colonic adenocarcinoma D Schaeffer1, I Tai2, D Owen1 1 Department of Pathology, University of British Columbia, Vancouver, BC, Canada, 2Department of Medicine University of British Columbia, Vancouver, BC, Canada Background: Obesity is associated with an increased risk of developing colorectal cancer. Circulating levels of leptin, produced by adipocytes, are elevated in obesity and are strongly correlated to levels of insulin. The leptin receptor is expressed in normal, premalignant and malignant colonic epithelia and leptin itself is considered to be a growth factor for colonic epithelial cells. Leptin may also play a role in regulation of the intestinal barrier function by direct stimulation of leptin receptors in the colonic epithelium. In addition, leptin stimulates proliferation and inhibits apoptosis in human colon cancer via JAK2, PI3 kinase and JNK. Leptin has also shown to induce downregulation of STAT3 and PAP-1. Given the overwhelming evidence for leptin as an important biochemical mediator in colon cancer, this study aimed to determine the expression of leptin receptors in human colonic tumors and study its relationship to tumor stage. Design: A tissue microarray (TMA) composed of 143 primary colonic adenocarcinomas, 45 adenomas, 62 metastasis of colonic cancer and 63 normal colonic control tissues were immuno stained for leptin receptor. Slides were reviewed by two pathologists and

graded for extent of leptin receptor staining. Tumor stage was obtained from the original pathology report. Positive immunostaining was scored, as either negative, focal (50%). For statistical analysis the ‘positivity cut-off’ included only moderate and high positivity cases. The Spearman rank-order correlation coefficient (r) was used to assess bivariate association. Results: Of the 143 colonic Adenocarcinomas 130 cases expressed leptin receptors, 63% (n = 82) demonstrated high positivity, while 29% (n = 39) showed low positivity for the leptin receptor. Furthermore, the high level expression of leptin receptor was noted to be stage dependent with AJCC Stage III tumors exhibiting an overall positivity of 94% (n = 44) compared to 89% (n = 34; r = 0.06) in Stage II and 88% (n = 42; r = 0.08) in Stage I respectively. Only low level positivity was recorded in adenomas 9/45 (20%) and colonic metastasis 18/62 (29%). Normal colonic control tissue exhibited only minimal amounts 4/63 (6.35%) of leptin receptor positivity. Conclusions: This study demonstrates a tumor stage dependent expression of leptin receptors within colonic adenocarcinoma. These data are consistent with a biologically plausible mechanistic link between elevated levels of leptin and an increased risk for the development of colon cancer. These findings add to the growing body of evidence that leptin and leptin antagonism may have potential efficacy in cancer therapy.

369 E-cadherin expression in intraductal papillary mucinous neoplasms of the pancreas S Serra, R Chetty Department of Pathology, University Health Network University of Toronto, Toronto, Canada Background: E-cadherin is important in cell adhesion and its normal immunohistochemical localization is at the cell membrane. Both cytoplasmic and nuclear immunostaining and loss of E-cadherin has been reported in several tumors. Loss of membrane staining and/or nuclear staining for E-cadherin has been seen in 100% of cases of solid pseudopapillary tumors (SPTs) of the pancreas. Design: The aim of this study was to evaluate the expression of Ecadherin protein in intraductal papillary mucinous neoplasms of the pancreas and correlate with clinicopathological parameters. A tissue microarray of 23 cases of IPMN was stained with two different antibodies for E-cadherin protein that recognize the extracellular and cytoplasmic domains of the protein. Results: The IPMNs were classified as 10 adenomas, 3 borderline and 10 cases with carcinoma in situ and/or invasive carcinoma, occurring in 15 females and 8 males, with an overall age range of 45–74 years. E-cadherin expression was strong and membrane in location in 8 of 10 adenomas. There was progressive decrease in membrane staining of E-cadherin in borderline lesions, carcinomas in situ, and invasive carcinomas. No difference in staining pattern was seen when the two antibodies were compared.



Conclusions: We show that E-cadherin decreases with progression from adenoma to carcinoma; however no differences in pattern of E-cadherin expression were noted with the two different antibodies.

370 Microsatellite instability analysis of adenocarcinoid tumors of the appendix: an immunohistochemical and molecular genetic study P Sharma, Z Gatalica, J Knezetic Department of Pathology, Creighton University School of Medicine, Omaha, NE Background: Adenocarcinoids of appendix are rare mixed endocrine-exocrine neoplasms containing both argentaffin cells and mucus secreting cells. Some of these tumors show significant mucin production leading to mucinous peritoneal carcinomatosis. The genetic mechanisms involved in the tumorigenesis of these neuroendocrine tumors are poorly understood. Their proximal location, mucin production, histologic resemblance to true signet ring cell carcinoma and younger age of onset prompts us to investigate whether adenocarcinoids of appendix display the characteristic MSI phenotype of Lynch syndrome (Hereditary Non-Polyposis Colorectal Cancer). Design: Genomic DNA was isolated from formalin-fixed, paraffinembedded tumor and matched normal tissues after manual microdissection to obtain enriched cancer sample. Microsatellite instability (MSI) testing was performed using multiplex PCR amplification (Promega) with a panel of five markers (BAT25, BAT26, NR21, NR24 and MONO27). Immunohistochemical (IHC) detection of DNA mismatch repair proteins MLH1, MSH2, MSH6 (Zymed) and PMS2 (Becton-Dickinson) was also performed. Additional IHC tests included p53 (Cell Marque), EGFR (Zymed) and COX-2 (Cayman Chemical) expression. In addition, immunohistochemical stains were performed on formalin fixed paraffin embedded sections using monoclonal antibodies raised against MLH1, MSH2, MSH6 and PMS2. A panel of prognostic and therapeutic immunohistochemical markers including Ki67, p53, EGFR and COX-2 was also performed. Results: Thirteen appendiceal mixed adenocarcinoma-carcinoid (adenocarcinoid) tumors (M : F = 9 : 4) were identified from 155 patients evaluated for peritoneal carcinomatosis (8%). Mucinous peritoneal carcinomatosis was present in seven patients, nonmucinous carcinomatosis in five and one case was localized to periappendiceal tissue. Average patients’ age was 44 years. No tumor showed instability in any of the five DNA markers (microsatellite stable tumors) and no loss of mismatch repair proteins were seen. Overexpression (>20%) of p53 was seen in 10, Epidermal Growth Factor Receptor (‡2+) in 8 and COX-2 (‡1+) in 10 cases, respectively. All cases showed high Ki67 proliferation index (>20%). Conclusions: Adenocarcinoids of appendix consistently express DNA mismatch repair proteins and do not exhibit DNA microsatellite instability. Despite young age at presentation, right-sided location and frequent mucinous histology, the likelihood that this tumor type is a part of the Lynch syndrome is low. Our findings

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support the concept that adenocarcinoids of appendix evolve through molecular different pathway. High expression of p53, COX2 and EGFR offer potential for targeted therapeutic interventions.

371 EGFR and KRAS mutations in pseudomyxoma peritonei P Sharma1, B Loggie2, Z Gatalica1 1 Department of Pathology, Creighton University School of Medicine, Omaha, NE, 2Department of Surgery, Creighton University School of Medicine, Omaha, NE Background: Pseudomyxoma Peritonei (PMP) is a clinical syndrome characterized by intraperitoneal accumulation of mucus produced by mucin producing neoplasm. Most commonly, the origin is an adenoma or well-differentiated adenocarcinoma of the appendix. Mucinous tumors presenting as PMP are histologically classified as disseminated peritoneal adenomucinosis (DPAM), peritoneal mucinous carcinomatosis (PMCA), or PMCA with intermediate/discordant features (PMCA-I/D). The epidermal growth factor receptor (EGFR), and its family members play an important role in the development and progression of various cancers such as lung, colon and ovarian cancers. It has been reported that somatic mutations in the tyrosine kinase domain of the EGFR genes occur in these cancers. However, the role of such mutations in the pathogenesis of PMP is unclear. We searched for mutations of the EGFR and KRAS genes to determine the prevalence of these mutations in PMP cases. Design: Forty-five cases of PMP were retrieved from surgical pathology files. The cases were classified into DPAM (16 cases), PMCA-I (3 cases), and PMCA (26 cases), according to the criteria of Ronnett et al. Cases classified as PMCA-I/D were grouped with PMCA cases. Mutations of the EGFR and KRAS genes were determined by polymerase chain reaction-based direct sequencing in all cases. We sequenced exons 18, 19 and 21 of EGFR gene and exons 2 and 3 of KRAS gene in our panel of 45 cases of PMP. Result: EGFR mutations were present in 17/45 cases of PMP (38 %). Fifty percent low grade neoplasms (DPAM) while 33% of high grade tumors (PMCA) showed EGFR mutations. The difference was not significant. KRAS mutations were seen in 18/45 cases of PMP (40%). Thirteen out of 29 PMCA cases (44%) and 5 out of 16 DPAM cases (31%) had KRAS mutations. Two thirds of KRAS mutations were exclusive from EGFR mutations. Conclusions: These findings suggest that the EGFR and KRAS mutations are frequent in Pseudomyxoma peritonei. The findings presented herein support the hypothesis that at least two distinct molecular pathways are involved in the pathogenesis of PMP, one involving EGFR tyrosine kinase domain mutations and the other involving KRAS gene mutations. EGFR tyrosine kinase targeted therapy may have a role in management of PMP patients.


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372 Mismatch repair enzymes (MMR) immunohistochemistry in tissue microarrays (TMA) for screening of gastric cancer samples can replace large sections K Simoes1, I Cauduro Soares1, A Wakamatsu1, R Albergaria Ressio1, S Nonogaki2, A Longatto Filho2,3, V Alves1 1 LIM14, Department Anatomic Pathology, Saõ Paulo University School of Medicine, Saõ Paulo, Brazil, 2Instituto Adolfo Lutz, Saõ Paulo, Brazil, 3 Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Portugal Background: Gastric cancer remains a clinical concern in Brazil due to its high prevalence and aggressiveness. Nowadays, progressive molecular understanding of gastrointestinal tract neoplasias is helping researchers to stratify tumors according to the type of genomic instability present, e.g. chromosomal vs. microsatellite instability. Chromosomal instability results from significant genetic aberrations, such as loss of chromosomal arms, large deletions, insertions and rearrangements. Conversely, microsatelite instability (MSI) is a phenotype generated by mutations occuring at specific repetitive sequences due to DNA mismatch repair (MMR) activity loss. For tumors of different organs, immunohistochemical assessment of MMR enzymes expression in tumoral samples has been recommended as a tool in predicting microsatellite instability (MSI) phenotype prior to additional and expensive molecular studies. The aim of the present study was to compare MMR enzymes immunoexpression results in samples contained in Tissue MicroArrays (TMA) with those obtained when using conventional large sections, as well as explore correlations between the three enzymes immunohistochemically analyzed (hMLH1, hMSH2, hMSH6). Design: Paraffin blocks from 142 gastrectomy specimens were retrieved from the files of Department of Pathology, Hospital das Clinicas, Sao Paulo University, School of Medicine, compared to respective hematoxylin-eosin slides, thus selecting specific areas for spotting at TMA (Beecher Instruments). Immuno-detection of MLH1, MSH2 and MSH6 was based on primary monoclonal antibodies (BD Biosciences Pharmingen) and polymer-based amplification (Novolink, Novocastra, UK). Spots from non-neoplastic and neoplastic samples were assigned as negative or positive, and negative samples additionally confirmed on large sections. Correlations between pairs of markers, as well as for TMA vs. large section results, were determined. Result: Positive immunoreactivity to MLH1, MSH2 and MSH6 in non-neoplastic mucosa was seen in 142, 139 and 140 samples, respectively. Immunonegativity for MLH1, MSH2 and MSH6 in neoplastic samples was found in 29/140 (20.7%), 24/142 (19.01%) and 27/139 (19.42%). Significant correlations occurred between MLH1/MSH2, MLH1/MSH6 and MLH2/MSH6 in TMA obtained results (Spearman´s rho values = 0.424, 0.422 and 0.691 respectively, all P < 0.0001). Concordant negativities between TMA and large sections were obtained for MLH1, MSH2 and MSH6 (rho 0.834, 0.426 and 0.542, P < 0.0001, P = 0.03 and P = 0.004, respectively). Conclusions: Our results showed significant correlation between TMA and conventional large sections results regarding MMR enzymes immunoexpression. Furthermore, significant correlations

occurred between different MMR enzymes when analysis was performed considering pairs of them. Taken together, these results support the use of immunohistochemistry in TMA samples in substitution to conventional large sections for MMR enzymes screening as a valid, low-cost and high-throughput research tool to be employed prior to additional molecular studies.

373 Increased apoptosis in gastric carcinogenesis progression can be influenced by mechanisms involved in tumoral microenviroment pH control K Simoes1, A Longatto Filho2,3, C Pinheiro2, F Schmitt4, R Albergaria Ressio1, F Baltazar2, V Alves1 1 LIM14, Department of Anatomic Pathology, Sao Paulo University School of Medicine, Saõ Paulo, Brazil, 2Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Portugal, 3Instituto Adolfo Lutz, Saõ Paulo, Brazil, 4IPATIMUP, Porto, Portugal Background: Progression from normal mucosa to gastritis, atrophy, intestinal metaplasia, dysplasia and invasive carcinoma is a well established pathway in gastric carcinogenesis, and genetic alterations and relationship between biochemical pathways are far from be completely understood. In evolution of this process, apoptosis evasion is one of key mechanisms confering growth advantage for neoplastic cells. Furthermore, monocarboxylate transporters (MCTs) involved in regulation of intracelular and tumoral microenviroment pH have recently been described by our group as an important player in colon cancer progression. The aims of the present study was to compare different steps of gastric carcinogenesis regarding expression of APAF-1 (involved in apoptotic intrinsic pathway), cleaved caspase 3 (from common final effector phase) and cleaved cytokeratin 18 (M30), as well as to explore associations between these molecules with clinicopathological parameters and different monocarboxylate transporters isoforms (MCTs). Design: Paraffin blocks from 142 gastrectomy specimens were retrieved from Department of Pathology, Hospital das Clinicas, Saõ Paulo University School of Medicine, and used for TMA construction. Immunohistochemistry employed specific monoclonal antibodies and polymer or avidin-biotin based detection. Normal and metaplastic mucosa, as well as primary tumor and metastatic samples, were semi-quantitated for APAF-1, caspase 3 and M30 expression from (0) negative, (1+) 50% of cells stained. MCT1 and MCT4 were evaluated as positive or negative. Expression of these markers was compared between different tissues as well with clinico-pathological parameters through Pearson´s chi-square analysis and significance considered at P < 0.05. Association between different MCTs isoforms and apoptosis related markers was also explored. Results: Normal and metaplastic mucosa showed lower caspase 3 and M30 than primary tumor (P < 0.0001 and 0.001). Interestingly, metaplastic mucosa showed higher APAF1 expression than primary tumor (P = 0.010). Comparison between tumor



and metastasis for caspase 3, APAF1 and M30 showed no significant results (P = 0.95; 0.86 and 0.17). Tumor location in stomach and pT were significantly associated to APAF1 expression (P = 0.001 and 0.004). Comparison between other clinico-pathological variables and APAF1, caspase 3 or M30 gave no significant results. Importantly, tumors showing MCT4 membranous expression had increased expression of caspase 3 and M30 (P = 0.019 and 0.040). Conclusions: Results from this study support increased expression of pro-apoptotic mediators (caspase 3) and its final identifiable products (M30) along gastric carcinogenesis steps. Notably, although yet paradoxically, APAF1 reduced expression in tumor when compared to metaplastic mucosa can represent an apoptosis evading mechanism needed to neoplastic progression. Increased apoptosis in membrane MCT4 expressing tumors suggest a yet unidentified influence of intracelular/microenviroment pH balance in metabolic machinery leading to programmed cell death.

374 A case of gastric small cell carcinoma associated with adenocarcinoma-loss of heterosigosity (LOH) analysis confirmed monoclonal nature of the tumors Y Sugiura, H Fujii, M Takase, T Matsumoto, O Hino Department of Pathology, Juntendo University School of Medicine, Tokyo, Japan Background: Gastric small cell carcinoma is rare, accounting for only 0.1% of gastric cancer specimen. The present case had multiple cancers in the esophagus and the stomach. Three esophageal cancers were all squamous cell carcinomas. Two gastric carcinomas were adenocarcinomas. In one of two gastric adenocarcinomas, there was a focus of small cell carcinoma in the mucosa and submucosa which was adjacent to the mucosal adenocarcinoma. The transition from an adenocarcinoma component to a small cell carcinoma component was also seen. Morphologically, we speculated that gastric adenocarcinoma progressed to small cell carcinoma. We performed molecular genetic analysis to determine the clonality of the tumors. LOH analysis demonstrated a monoclonal nature of the two tumor components. Design: We performed immunohistochemical stainings and PCRbased LOH analysis using radioisotope-labeled microsatellite markers located one 13 chromosomal arms. Results: Immunohistochemically, gastric adenocarcinoma component was negative for neuroendocrine markers, while the transitional part from adenocarcinoma to small cell carcinoma and the small cell carcinoma component were positive for NCAM (CD56), synaptophisin and NSE. On LOH analysis, both tumor components showed identical LOH of 9p, 13q, 17p and 22q. Conclusions: In the present case, we verified a monoclonal nature of the gastric adenocarcinoma and a closely associated small cell carcinoma component. Although we failed to detect progression of LOH, we speculated that gastric small cell carcinoma component could have arisen from adenocarcinoma by genetic progression.

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375 Comparison of Giemsa staining, immunohistochemistry and fluorescent in situ hybridization in detecting Helicobacter pylori infection in human gastric biopsy specimens I Szirtes, E Sze´kaćs, Z Kramer, J Hala´sz, A Kiss, Z Schaff, G Lotz Second Department of Pathology, Semmelweis University, Budapest, Hungary Background: Histopathological examination of gastric mucosal biopsy specimens is the gold standard in diagnosing Helicobacter pylori (H. pylori) infection, which is one of the main causes of dyspeptic symptoms. Among routine staining methods such as Giemsa and methylene-blue, nowadays immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) techniques are also available to detect the bacteria. The advantage of the FISH method is that it shows not only the presence of the bacteria, but also the possible genetic mutations which cause their resistance to clarithromycin antibiotics, the main drug of first line treatment. The aim of this study was to compare the sensitivity and specificity of the H. pylori detection methods Giemsa, IHC, and FISH. Design: Gastric mucosal biopsy specimens examined in our Department in the year 2006 were stained with haematoxylin-eosin (HE), Giemsa staining and IHC (Dako B0471). On those which were positive with these methods, we also applied the FISH test (SeaPro SeaFast H. pylori Combi Kit / Izinta Kft.). Results: Analysis of the 1026 specimens showed 280 cases positive for H.pylori with Giemsa staining, and 273 positive also with IHC. Out of the 273 Giemsa and IHC positive cases 271 proved to be positive with FISH, in one FISH negative case the bacteria were carved out of the block, and in another case Helicobacter heilmanii was detected (these bacteria can not be distinguished from H. pylori with IHC). Examining the seven Giemsa positive and IHC negative cases, one specimen proved to be positive with FISH, and six were negative also with this technique. From the 746 Giemsa negative cases 43 specimens showed positivity with IHC, and 41 cases were confirmed also with FISH. Our preliminary results showed that the sensitivity of the FISH method for detecting H. pylori is in accordance with IHC, therefore no FISH analysis was performed on the 703 Giemsa and IHC negative specimens. Considering IHC as reference, the sensitivity of the diagnosis of H.pylori with Giemsa staining was 86%, while the specificity was 99%. Conclusions: The detection of H. pylori with the routinely used Giemsa staining method has enough specificity, but its sensitivity is not sufficient, therefore we recommend applying IHC in the everyday diagnostics. Performing FISH on the IHC positive cases would be useful for showing the clarithromycin resistance before administering first line H. pylori eradication treatment.

376 Foregut duplication cyst of the stomach ZT Sever1, L S¸ en2, Ç Ataizi Çelikel, C Yeg˘en2 1 Department of Pathology, Marmara School of Medicine, Istanbul, Turkey, 2Department of Surgery, Marmara School of Medicine, Istanbul, Turkey Background: Foregut duplication cysts are rare congenital anomalies, and more exceptional are those occurring in the stomach.


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A gastrointestinal duplication is characterized by a smooth muscle coat lined by alimentary epithelium. In adults, the symptoms are usually nonspecific like nausea and vomiting and may cause diagnostic confusion and delay. Herein we report a duplication cyst of stomach incidentally found during a Whipple operation in a patient with an ampulla vateri tumor. Design: A 57-year-old man was admitted to our institute with weight loss, fatigue and jaundice. The abdominal MR scan demonstrated approximately 16 and 10 mm dilatation in the suprapancreatic portion of bile duct and the head of pancreas region, respectively. During the operation, beside a firm mass in the distal part of the choleduct, a 10 cm cystic lesion leading to external pressure on the greater curvatura of the stomach was coincidentally seen. The cyst had no communication with the lumen. Gross examination of the resection specimen of the Whipple procedure revealed a 2 · 1 cm beige colored, irregular contoured, granular lesion located in the papilla and a 1.1 · 1 cm nodular area in the duodenal mucosa. The cystic lesion of the stomach was 13 · 12 · 5 cm with a 0.1 cm thick capsule and filled with viscous yellow-tan fluid. Histopathological examination showed the cyst consisted of smooth muscle wall and lined by a pseudostratified, ciliated and columnar epithelium and diagnosed as foregut duplication cyst. Seroumucous glands were apparent in the subepithelium. In addition the nodular lesion in duodenum was microscopically identified as heterotopic pancreatic tissue. Results: The case was reported as a grade 2, stage T3N1 (AJCC 2002) adenocarcinoma of ampulla vateri accompanied by foregut duplication cyst in the greater curvatura of stomach and heterotopic pancreatic tissue in duodenum. Conclusions: Gastric duplications can be tubular and cystic and the cystic type does not communicate with the lumen. The therapeutic approach in these cysts is complete resection. There are few case reports about the coexistence of pancreatic heterotopic tissue and foregut duplication cysts. However these cases may indicate that these two malformations in the intrauterine period of life have a connection. Besides other anomalies can be seen in cases with duplication cysts.

377 Prognostic value of HGF expression in colorectal adenocarcinomas A Talvane1, R Artigiani-Neto2, S Saad2, D Matos2, A Logullo2 1 Hospital Saõ Judas Tadeu Fundaçaõ Pio XII, 2Unifesp-EPM Background: To evaluate the prognostic value of the HGF expression in colorectal adenocarcinomas through immunohistochemical essay. Design: A retrospective descriptive study of 286 patients with colorectal adenocarcinoma, who have been diagnosed ant surgically treated at Barretos Cancer Hospital, from 1993 to 2002. The tissular expression of the tumoral marker was evaluated using the cmet/HGF anti-protein monoclonal antibody through the estreptavidin-biotinperoxidase technique. The protein expression was semiquantitatively and carried out by three pathologists with no previous knowledge on clinical and histopathological data.

Results: Out of a total of 286 patients, HGF protein expression was positive in 236 (78.8%) and negative in 50 (21.2%). It was different between stages l and lV (P = 0.004), and correlated to global life span (P = 0.009), and to cancer mortality rate (P = 0.022); however, there was no association between the HGF expression and recurrence (P = 0.89) or disease-free period (P = 0.91). Conclusions: c-met/HGF has shown significant prognostic value in colorectal adenocarcinomas and further analysis with larger series should be performed to confirm the promising results.

378 Signaling profiles in pancreatic cancer progression C Tanase1, M Nicolescu1, L Albulescu1, M Cruceru2, A Popa2, S Mihai1, M Neagu1, M Poroschianu1, S Dima3, R Albulescu1, M Hinescu1,2 1 Victor Babes National Institute of Pathology, Bucharest, 2Carol Davila University of Medicine and Pharmacy, Bucharest, 3Fundeni Clinical Institute, Bucharest, Romania Background: Multi-stage development of molecular aberrations affecting signaling pathways regulating cancer growth and progression is a key process in the pathogenesis of pancreatic ductal adenocarcinoma (PDAC). The purpose of this study was to gain a better understanding of the abnormal signaling that occurs in PDAC compared with normal duct epithelia. Design: Signaling profile was analyzed using xMAP array technology (Luminex 200) on samples derived from: Capan and MIA PaCa cell lines, cell cultures established from patients’ tissue, tumoral and peritumoral tissue from PDAC patients and normal pancreas. Results: Expression levels of signaling molecules were significantly increased in tumoral tissue from PDAC patients compared to normal tissue: (1) cell receptors: c-KIT and EGFR were 1,5-2,0 fold increased; (2) kinases: p70, p38, ERK/MAPK, JNK/SAPK were 1,7-4,0 fold increased; (3) non-enzymatic signaling molecules: IRS1 was 2x increased while HSP27 over 100x; (4) transcription modulators: STAT3 and CREB were around two times increased. Similar variations were recorded in some of the peritumoral samples. Cell cultures showed enhanced levels of expression, with a more pronounced increase in cell lines compared to primary cell cultures established from patients’ tissue. Conclusions: Our results indicate that multiple signaling proteins are over expressed and provide a higher amplification level of growth signals in PDAC. Increased levels of signaling molecules expression in peritumoral samples could be correlated with the invasivity of PDAC. Our study suggests that proteins form major cellular signaling pathways can be targets for pharmacological modulators developing thus new strategies in cancer therapy and improve the prognosis of pancreatic cancer.



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379 Thrombomodulin expression in gastrointestinal stomal tumors (GISTS): a novel finding with diagnostic implications T P Thamboo1, M Nga1, R Soong1,2, G S D Lim3, M Salto-Tellez1,2 1 Department of Pathology, Yong Loo Lin School of Medicine, Singapore, 2 Oncology Research Institute, Yong Loo Lin School of Medicine, Singapore, 3Department of Pathology, National University Hospital, Singapore

380 A new approach of P73 implication in colorectal carcinogenesis A Toumi Arfaoui1, L Kria Ben Mahmoud1, A Lahmer2, S Djerbi2, T Khalfallah3, S Regaya Mzabi2, S Bouraoui1,2 1 Unite´ de recherche (M.E.S.R.S), 2Laboratoire d’Anatomie et de Cytologie Pathologique, CHU Mongi Slim La Marsa, Tunisie, 3Service de Chirurgie geńe´rale, CHU Mongi Slim La Marsa, Tunisie

Background: Gastrointestinal stromal tumors (GISTs) are one of the commonest mesenchymal tumors arising from the gastrointestinal tract. Due to their varying clinical behavior and responsiveness to the tyrosine kinase receptor inhibitor Imatinib, there has been an increasing need amongst histopathologists to diagnose them accurately. Immunohistochemical (IHC) markers such as CD117 (c-kit) and CD34 have traditionally been used to characterize these tumors, however no marker is completely specific for this entity. It has recently been shown that vascular endothelial growth factor (VEGF) overexpression may be predictive of malignant behavior in GISTs. The use of anti-VEGF therapy, alone or in combination with Imatinib, may be beneficial in patients with expression of VEGF protein. Adding to this concept of GIST vascularity as a possible therapeutic target, the expression of thrombomodulin (TML) by the tumor cells raises the novel possibility of another potential therapeutic target. The expression of TML in GISTs was studied using a tissue microarray (TMA). The results were correlated with VEGF gene mutation analysis as well as with the expression of endothelium-related markers such as vascular endothelial growth factor (VEGF), flt-1 and flk-1, and also with a panel of non-endothelium related markers, previously analyzed in another study on GISTs. Clinical follow-up were also obtained for a majority of cases studied. Design: Immunohistochemistry was performed on sections from a TMA constructed from formalin-fixed and paraffin-embedded tissue including 37 cases of GISTs and seven smooth muscle tumors. Scoring of TML staining was done by three pathologists in an independent and blinded fashion. The extent and intensity of staining were scored separately. VEGF gene mutation analysis was performed using both PCR-denaturing high performance liquid chromatography analysis and conventional sequencing. Results: Eight of the thirty-seven cases of GIST (22%) showed moderate to strong cytoplasmic staining for TML. No statistically significant difference in malignant behavior, disease-free survival, overall survival, expression of endothelium related markers or frequency of VEGF gene mutations was seen between the TML-positive and TML-negative cases. Interestingly, prominent nuclear palisading was seen in 63% of the TML-positive cases, compared to only 3% in the TML-negative cases (statistically significant, P = 0.0007). None of the cases showed co-expression of calretinin or HBME-1. All seven smooth muscle tumors were negative for TML. Conclusions: A significant subset of GISTs shows strong expression of TML, which appears to be independent of VEGF expression. The role of TML in the development and progression of GISTs, with potential therapeutic implications, is as yet unknown. In addition, awareness that a significant proportion of GISTs shows strong immunohistochemical expression of TML is important to avoid diagnostic errors in histopathological practice.

Background: Colorectal cancers (CRC) still represent a paradigm for the elucidation of the cellular, genetic and molecular mechanisms that underlay solid tumor initiation, progression and metastasis. Along this tumorigenic pathway, mutational activation of oncogenes and inactivation of tumor-suppressor genes define stages of carcinogenesis. In this frame, Tp73 recently individualizes gene, seems to present many uncertainties concerning its exact role in colorectal carcinogenesis. Indeed, several authors grant to him, like Tp53, a tumor-suppressor function though their structure and sequence homology, but others investigators say the opposite and suggest to him an oncogenic function like P21-ras. Aim: In this study, we are the first to examine the expression profile of P73 in different subtypes and different stages of CRC comparatively to P53 and P21-ras for better elucidating its implication in this neoplasm. Design: We immunohistochemically analyzed the expression of P73, P53 and P21-ras in 200 cases of CRC subdivided into 53 mucinous adenocarcinomas (MA) and 147 common adenocarcinomas (CA). We compared the intensity and the distribution of the immunoreactivity in the tumor according to the type, differentiation, and staging, with both the adjacent and distanced healthy mucosa. Results: The immunohistochemical analysis showed that within the (CA), expression profile of P73 is completely different from its homologous (P53), but it approaches more to P21-ras. In fact, we note a moderate, nuclear positivity in depth of crypts in the adjacent and distant normal mucosa. This positivity is accentuated in the well-differentiated ADK and increased progressively according to the decreasing degree of differentiation. As to (MA), we found that the expression profile of the three proteins decreases meaningfully in the mucinous component of the tumor when compared to the normal mucosa and to mucinous free territories. Conclusions: According to our results, P73 could be an indicator for poorer prognosis and also an etiopathogenic factor by supporting the tumorigenesis through its up-expression during the neoplasic process. So, it would be more judicious to allot to P73 an oncogenic function instead tumor-suppressor function. Many indirect mechanisms which could start this oncogenic action, knowing that TP73 gene is rarely genetically mutated in the human cancers. Thus, probably there would be upstream or downstream mutations of this gene, or a mutation at the level of the splicing system. Consequently, it lead to more expression of the short isoforms (DN-P73) than the longs ones (TA-P73). In fact, (DN-P73) inhibits apoptosis induction though a negative dominating mechanism. However, within the (MA), expression profile of the three protein does not show any significant difference, where we note the total absence of proteins. This results suggests a distinct molecular feature from common ADK to mucinous type implying the mucin which would interfere with these proteins. In conclusion this study showed a


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new etiopathogenic approach for CRC including a P73 as a new oncoprotein implied in its genesis.

381 An interesting case of a pseudomyxoma peritonei in a woman N Tsoukalas1, N Apostolikas2, G Koumakis1, C Panopoulos1, G Lypas1, C Papadimitriou1, N Pistamalntzian1, S Droufakou1, V Barbounis1, A Efremidis1 1 nd 2 Department Clinical Oncology, ‘Saint Savvas’ Anticancer Hospital, Athens, Greece, 2Department of Pathology, ‘Saint Savvas’ Anticancer Hospital, Athens, Greece Background: We present a rare and interesting case of peritoneal pseudomyxoma in a woman. Design: A 51-year-old woman presented with unclear ascites with gradually deterioration. An upper and lower abdomen CT, 5 months after the first symptoms, revealed an ovary enlargement. At that period of time, the levels of serum tumor markers were: CEA = 312.9 ng/mL, CA125 = 72.9 IU/mL, CA19-9 = 520.6 ng/ mL. During the investigational laparotomy, a tumor was found in the right iliac fossa, which infiltrated the right ovary, the right fallopian tube, the fallopian interspace, and the omentum. In addition to that, a tumor was found in the left ovary. Consequently, an extrafascial total abdominal hysterectomy, bilateral salpingo-oophorectomy and resection of the ifracolic omentum was carried out. Results: Macroscopically, the right iliac tumor was described as a mucinous, cystic form (5 · 5 · 3 cm) with irregular inner surface and multilobar, mucinous, grey-brown outer surface. The second tumor sallied out from the left ovary, laterally, towards the fallopian interspace, infiltrating, almost, the entire left fallopian tube. It was a multilobar, mucinous, grey-brown tumor (3 · 3 · 2.5 cm). The pathology report of these tumors was mucinous cystadenocarcinoma, in the context of pseudomyxoma peritonei. The immunohistochemistry report was CK20 positive, and CK7 negative. Regardless the multiple surgical sampling, no histological evidence of appendix residue was found. It was considered as colorectal cancer, stage IV, and the patient was treated with Oxaliplatin and 5 FU (FOLFOX4 regiment). The response was poor and after three cycles of chemotherapy, Bevacizumab was administered in addition to FOLFOX4. After six cycles of chemotherapy, the re-evaluation showed, virtually, stable disease. The patient was, then, treated with Irinotican and 5 FU (FOLFIRI regiment) plus Bevacizumab. Three months later, the patient underwent plastic surgery for omphalocele and white line hernia. The pathology report showed infiltration with mucinous cystadenocarcinoma, with extracellular and intracellular mucin production (signet-ring cells). The patient died 30 months after the primary diagnosis. Conclusions: The discovery of the primary origin of a peritoneal pseudomyxoma is very important because this origin indicates the appropriate therapy regiment. The revelation of this primary origin is not quite easy. If no histological evidence of appendix residue is found then we must consider the primary origin as colorectal cancer. In this case the immunohistochemistry findings (CK20 positive and CK7 negative) as well as the tumor markers (elevated CA19-9 and CEA) were strongly evidence of the colorectal origin of this pseudomyxoma peritonei.

382 Expression of estrogen receptors a and b and steroid receptor coregulators (AIB-1, TIF-2) at the invasion front of colorectal carcinomas V Tzelepi1,2, P Grivas3, Z Kefalopoulou1, H Kalofonos3, M Melachrinou1, G Sotiropoulou-Bonikou2 1 Department of Pathology, 2Department of Anatomy and Histology – Embryology, 3Department of Internal Medicine, Division of Oncology, Medical School, University of Patras, Patras, Greece Background: The invasion front (periphery) of carcinomas is an area of active cross-talk between malignant epithelial cells and benign cancer-associated myofibroblasts that promotes local tumor invasion. Epidemiological and molecular data suggest that estrogens may be involved in colorectal carcinogenesis, mainly through estrogen receptor beta (ERb)-dependent signaling. AIB1 (Amplified in Breast Cancer1) and TIF2 (Transcription Intermediary Factor-2) enhance the transcriptional activity of nuclear receptors, including estrogen receptors. The expression of ERb in colorectal carcinomas has been extensively investigated, albeit with controversial results. However, its role and correlation with ER-associated co-regulators at the invasion front of colorectal carcinomas has not been previously studied. Design: Expression of ERa, ERb, AIB1 and TIF2 in epithelial cells and myofibroblasts of 90 colorectal carcinomas was examined, using immunohistochemistry on formalin-fixed paraffin-embedded tissue sections. The central parts of the tumors (inner tumor mass) and the invasion front were separately assessed. For specific detection of myofibroblasts in cancer-associated stroma, a-SMA immunostaining was performed in serial tissue sections. The percentage of positive cells was evaluated in each case. Results: ERa was expressed in 50% of tumor cells showed positive immunostain,


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respectively. Since the median point of claudins 1, 4, 7 expression was 2+ and of claudin-5 expression was 1+, these points were used as cut-off values to determine low and high expression during analysis of the data. Mean follow-up time was 45.3 months (range 3.5– 140 months). The patients had received no chemotherapy or radiotherapy prior to or after surgery. Results: Results are shown in the table. All claudins studied, were more frequently expressed in intestinal type GC compared to diffuse type. Low claudin 1 expression and increased claudin-4 expression were correlated with worse disease-free survival on univariate analysis (P = 0.012 and P = 0.018 respectively). Spearman rank analysis revealed strong correlation between claudin 1 and claudin 5 expression (P = 0.002) and between claudin 4 and claudin 5 expression (P < 0.001). Cox regression analysis revealed that tumor type, stage and claudin 4 expression were independent prognostic factors (P = 0.03, P = 0.019 and 0.02 respectively).

Claudin-1 Claudin-4 Claudin-5 Claudin-7 Total

Stain intensity

Intestinal type (# cases)

Diffuse type (# cases)

0* 1 0* 1 0 1 0* 1

29 63 23 69 39 53 38 54 92

15 9 13 11 20 4 17 7 24

p 0.034 0.017 0.004 0.034

*0 (low expression): £30% of the cells exhibit positive immunostain, 1 (high expression): >50% of the cells exhibit positive immunostain. 0 (negative expression): £1% positive cells, 1 (positive expression): >1% positive cells. Conclusions: This study demonstrates that dedifferentiation of gastric carcinomas is accompanied by reduction in claudins 1, 4, 5 and 7 expression. This possibly implies that deregulation of tight junction proteins play a role in determining the diffuse phenotype and loose cohesion of cells in diffuse type gastric carcinoma. Additionally, claudins 1 and 4 expression may be important prognostic indicators of recurrence in gastric carcinomas.

385 Microvascular density in T1–T4, N0, M0 colorectal carcinomas and prognosis A R Uribarrena, J Ortego, J Fuentes, N Ravento´s, P Parra, E R Uribarrena Departments of Gastroenterology, Hospital Universitario Miguel Servet, and of Pathology, Hospital Clinico, Universitario Lozano Blesa, Zaragoza, Spain Background: Aproximatelly 30% of patients operated on for colorrectal cancer (CRC), with an expectedly favourable prognosis (Dukes A-B/T1–T4, N0, M0 cases) suffered recurrence and/or died.

Design: In order to determine if tumor microvascular density (MVD) is a contribuiting factor of recurrence/death because of CRC, samples from tumors of 85 of patients patients apparently cured after undergoing surgery for CRC (Dukes A-B) and without undergoing pre nor postoperatory radio/chemotherapy, immunohistochemistry was performed for anti-CD34 antibody to visualize tumor vascularization. MVD was expressed as the total number of vessels in eight selected fields within the microvascular hot spots by every case and as the percentage of vascularization area in relation to the total tumor area of the each field. We calculated MVD semiautomatically, with a morphometry program (ContImUZ, Zaragoza University) and performed descriptive, bivariate inferential, and survival statistics on MVD relative to other examined variables. Results: The mean total number of vessels was 37.37/200· field, and the vascular area was the 3.972 %. With the survival and bivariate analyses in relation to the total number of vessels, we observed that 30% of the patients with 37 vessels/field, experienced recurrence/death. The 35% of patients with