Cytokeratin Profile in Exfoliative Cytology of Smokers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

COORDINATE EXPRESSION OF CYTOKERATIN-7 AND CYTOKERATIN-20 DEFINES UNIQUE SUBSETS OF CARCINOMAS

We tested the hypothesis that the coordinate expression of cytokeratin 7 (CK 7) and cytokeratin 20 (CK 20) could distinguish among carcinomas arising from different primary sites. A total of 384 cases of carcinomas primary to various organs, as well as 16 cases of malignant mesothelioma, were evaluated using commercially available monoclonal antibodies and an avidin-biotin immunoperoxidase technique. The subset of tumors strongly expressing both CK 7 and CK 20 included virtually all bladder transitional cell carcinomas and the majority of pancreatic adenocarcinomas; the tumors negative for both CK 7 and CK 20 were largely restricted to hepatocellular, prostate, and renal cell carcinomas in addition to squamous cell and neuroendocrine carcinomas of lung. The CK 7-/CK 20+ immunophenotype, however, was highly characteristic of adenocarcinomas of colorectal origin, whereas CK 7+/CK 20- immunophenotype was typically seen in the vast majority of carcinomas arising from other sites, including ovary, endometrium, breast, and lung, as well as malignant mesothelioma. Gastric carcinomas were the most heterogeneous subgroup with respect to CK 7/CK 20 immunophenotype. In the subset of mucinous tumors, striking immunophenotypic differences were noted among those primary to the breast (CK 7+/CK 20-), gastrointestinal tract (CK 7-/CK 20+), and ovary (CK 7+/CK 20+). In all cases investigated, this CK immunophenotype was invariant in metastatic vs. primary tumors. It is concluded that, in the appropriate clinical setting, the CK 7/CK 20 immunophenotype of carcinomas is a valuable diagnostic marker in the determination of primary site of origin.

DO GLIOMA-CELLS EXPRESS CYTOKERATIN

There have been several recent reports of cytokeratin immunoreactivity in glial cells and tumors. We wanted to further test these tissues for cytokeratin immunoreactivity, and to determine whether antibody positivity corresponded to true cytokeratin expression. In the first set of experiments, a series of 10 formalin-fixed, frozen sections of glial tissue were employed; positive immunostaining with a cocktail of monoclonal anti-cytokeratin antibodies was seen only when a pepsin predigestion step was included in the immunostaining procedure. In the second set of experiments, 30 cases of malignant glioma fixed in both methacarn and formalin fixation were employed. Using a panel of three different anti-cytokeratin monoclonal antibodies (35 beta H11, 34 beta E12, CAM5.2), no positive immunostaining was observed in any of the methacarn-fixed tissues; positive immunostaining in the corresponding formalin-fixed tissue was frequently found, but only using the antibodies (35 beta H11, 34 beta E12) requiring enzyme predigestion. In the third set of experiments, immunoblots were performed on cytoskeletal extracts of human gliomas; no bands corresponding to known cytokeratins were observed in any cases. It is concluded that glial tissues and tumors do not, in fact, truly express cytokeratins, despite the fact that it is possible to obtain positive immunostaining of glial tumors and tissues using certain anti-cytokeratin antibodies under certain laboratory conditions.

Culture of mature trophoblastic giant cells from bovine placentomes

The mostly binucleate trophoblast giant cells (TGC) found in bovine placentomes, in addition to synthesizing and releasing hormones play an important role in fetal development and maternal adaptation to pregnancy. Placentomes from early gestation were collected, and for isolation of mature TGC, three cellular disaggregation methods, mechanical (MECH), enzymatic by trypsin (TRYP) or collagenase (COLL) were compared to each other. Further on, the cell survival in culture medium (DMEM) supplemented with either 10% fetal calf serum (FCS) or 10% serum replacement (SR) on culture plates free of any substrate was evaluated over a period of 90 days by trypan blue exclusion. The cells were further characterized by HOECHST 33342 nuclear staining, and immunocytochemical staining with monoclonal antibodies against vimentim and cytokeratin. A mean total rate of TGC survival of 82.56% was recorded. Statistical analysis showed significantly higher survival rates after enzymatic disaggregation with COLL (86.23%) than following MECH (80.38%) or TRYP (80.91%) treatment. Supplementation of DMEM with FCS resulted in significantly higher cellular survival rates (87.13%) when compared to the addition of SR (77.73%). Analysis of the influence of both, disaggregation method and medium supplementation on TGC survival revealed statistically significant differences between the following groups: MECH-SR (71.09%) was significantly lower than all other groups; TRYP-SR (78.03%) was significantly different from all other groups; TRYP-FCS (83.43%) and COLL-SR (84.08%) were significantly lower than MECH-FCS (89.98%) which together with COLL-FCS (88.25%) showed the highest cellular survival rate. In summary, our results show that TGC isolated from early gestation placentomes may be viable for more than 90 days of culture. However, whether these TGC produce placental lactogen throughout this period has yet to be determined. (c) 2006 Elsevier B.V. All rights reserved.

Invasion of epithelial mammalian cells by Paracoccidioides brasiliensis leads to cytoskeletal rearrangement and apoptosis of the host cell

Paracoccidioides brasiliensis (Pb) yeast cells can enter mammalian cells and probably manipulate the host cell environment to favor their own growth and survival. We studied the uptake of strain Pb 18 into A549 lung and Vero epithelial cells, with an emphasis on the repercussions in the cytoskeleton and the apoptosis of host cells. Cytoskeleton components of the host cells, such as actin and tubulin, were involved in the P. brasiliensis invasion process. Cytochalasin D and colchicine treatment substantially reduced invasion, indicating the functional participation of microfilaments (MFs) and microtubules (MTs) in this mechanism. Cytokeratin could also play a role in the P. brasiliensis interaction with the host. Gp43 was recognized by anti-actin and anti-cytokeratin antibodies, but not by anti-tubulin. The apoptosis induced by this fungus in infected epithelial cells was demonstrated by various techniques: TUNEL, DNA fragmentation and Bak and Bcl-2 immunocytochemical expression. DNA fragmentation was observed in infected cells but not in uninfected ones, by both TUNEL and gel electrophoresis methods. Moreover, Bcl-2 and Bak did not show any differences until 24 h after infection of cells, suggesting a competitive mechanism that allows persistence of infection. Overexpression of Bak was observed after 48 h, indicating the loss of competition between death and survival signals. In conclusion, the mechanisms of invasion of host cells, persistence within them, and the subsequent induction of apoptosis of such cells may explain the efficient dissemination of P. brasiliensis. (C) 2004 Published by Elsevier SAS.

Timoma em cão. Relato de caso

Uma cadela, Dachshund, quatro anos, foi encaminhada ao H.V. da FMVZ-UNESP-Botucatu-SP com histórico de prostração, emagrecimento progressivo, anorexia, cansaço e tosse seca. Ao exame clínico observou-se apatia, secreção nasal serosa bilateral e dispnéia intensa. Radiografia torácica demonstrou efusão pleural, edema pulmonar acentuado, desvio dorsal da traquéia e possível massa mediastinal. A ultra-sonografia, após drenagem torácica, confirmou a presença de massa mediastinal, sendo classificada como neoplasia maligna de origem epitelial pela citologia aspirativa por agulha fina. O animal veio a óbito logo após o diagnóstico de neoplasia. À necropsia constatou-se massa mediastinal encapsulada, consistência macia e superfície de corte com coloração branco-acinzentada e áreas necrótico-hemorrágicas, localizada na região ântero-ventral do tórax. O exame histopatológico demonstrou células epiteliais neoplásicas, células linfóides (timócitos) com morfologia normal, vasos sangüíneos de pequeno e médio calibre, formações císticas com conteúdo eosinofílico e corpúsculos de Hassall. A imunohistoquímica apresentou positividade para citoqueratina AE1/AE3 e UCHL, confirmando o diagnóstico de timoma.

Cell blocks allow reliable evaluation of expression of basal (CK5/6) and luminal (CK8/18) cytokeratins and smooth muscle actin (SMA) in breast carcinoma

Objective:Gene expression studies have revealed several molecular subtypes of breast carcinoma with distinct clinical and biological behaviours. DNA microarray studies correlated with immunohistochemical profiling of breast carcinomas using cytokeratin (CK) markers, Her2/neu, oestrogen receptor (ER), and basal myoepithelial cell markers have identified five breast tumour subtypes: (i) luminal A (ER+; Her2/neu-), (ii) luminal B (ER+; Her2/neu+), (iii) Her2 overexpression (ER-; Her2/neu+), (iv) basal-like (ER-; Her2/neu-, CK5/6 and 14+), and (v) negative for all markers. Luminal carcinomas express cytokeratins in a luminal pattern (CK8/18), and the basal-like type expresses CK5/6 and CK14 or basal epithelial cell markers. CK5/6, CK8/18, and smooth muscle actin (SMA) expression were assessed in cell blocks and compared with expression in surgical specimens.Methods:Sixty-two cases of breast carcinoma diagnosed by fine needle aspiration cytology with cell blocks and available surgical specimens were included. Cell blocks containing at least 10 high-power fields each with at least 10 tumour cells and surgical specimens were immunostained for CK5/6, CK8/18 and SMA.Results:Percentage sensitivity, specificity, positive predictive value, negative predictive value and accuracy were, respectively, 77, 100, 100, 92 and 94 for CK5/6; 98, 66, 96, 80 and 95 for CK8/18; and 92, 96, 85, 98 and 95 for SMA.Conclusion:The identification of CK5/6, CK8/18 and SMA by immunohistochemistry in cell blocks can be a reliable method that yields results close to those obtained in surgical specimens, and can contribute to the classification of breast carcinomas with luminal and basal expression patterns, providing helpful information in the choice of treatment and in the evaluation of prognostic and predictive factors.

Immunohistochemistry of small blue round cell tumors

Small blue round cell tumors (SBRCTs) are a set of malignancies that have a particular proclivity for the pediatric age group. These tumors are notoriously difficult to distinguish by histologic evaluation alone, and in recent years a number of new immunohistochemical markers have emerged that can aid in the correct categorization of these lesions. Myogenin, a muscle-restricted nuclear transcription factor, has been demonstrated to be a highly sensitive and specific marker of rhabdomyosarcoma, and is superior to previous markers such as myoglobin, muscle actins, and desmin. The FlI-1 gene product is expressed as part of the EWS/FLI-1 novel chimeric protein that results from the t(11;22)(q24;q12) translocation that occurs in approximately two-thirds of cases of PNET/Ewings sarcoma. Immunohistochemical detection of the FLI-1 gene product can thus complement detection of CD99/MIC2 for the positive identification of PNET/Ewings sarcoma. Markers of neuroblastoma include neural markers, such as chromogranin A, neurofilaments, and synaptophysin. Desmoplastic small round cell tumor (DSRCT) is a tumor with an unusual immunophenotype, including co-expression of cytokeratin, vimentin, and desmin; recent studies have also documented the use of antibodies to the WT-1 gene product as a marker of the chimeric EWS/WT-1 protein formed as a result of the t(11;22)(p13;q12) translocation that characterizes this unique tumor. In summary, there now exists a panel of antibodies defining immunohistochemical markers of individual SBRCTs that can identify rhabdomyosarcoma, PNET/Ewings sarcoma, neuroblastoma, and DSRCT with high sensitivity and specificity.

Expressão antigênica em carcinomas do plexo coróide humano: relato de dois casos

Neoplasias provenientes do epitélio de revestimento do plexo coróide são inco-muns, tendo sido descritos 6 padrões morfológicos. O padrão anaplásico, também denominado carcinoma do plexo coróide, é o de menor freqüência e pode dar metastases fora do SNC. A distinção histológica desses tumores, particularmente da variedade anaplásica, com outras neoplasias primárias e metastáticas no SNC pode ser difícil. O uso de técnicas imunocitoquimicas em parafina tem-se mostrado útil no esclarecimento das linhagens tumorais. Os papilomas do plexo coróide têm, no entanto, sido objeto de controvérsia, por sua complexa expressão antigênica. Usando a técnica de imunoperoxidase (sistema avidina-biotina-peroxidase) pesquisaram-se, em dois casos da variedade anaplásica, os seguintes marcadores: proteína glial fibrilar ácida (GFAP) com anticorpo monoclonal e policlonal; ceratinas de 40-50kDa, ceratinas de 60-70kDa (callus ceratina), enolase neuronal específica (NSE) e proteína S-100, com anticorpos monoclonais. Os dois tumores mostraram positividade para NSE, proteína S-100 e ceratina de 40-50kDa; uma das duas neoplasias mostrou diferenciação glial, revelando positividade para GFAP tanto com anticorpo monoclonal quanto policlonal.

Adenocarcinoma of the parotid salivary gland in a cow

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Immunohistochemical and morphological features of a small bowel leiomyoma in a black crested macaque (Macaca nigra)

Background: Spontaneous gastrointestinal neoplasms in non-human primates are commonly seen in aged individuals. Due to genetic similarities between human and non-human primates, scientists have shown increasing interest in terms of comparative oncology studies.Case presentation: The present study is related to a case of an intestinal leiomyoma in a black crested macaque (Macaca nigra), kept on captivity by Mateca a Zoo, Pereira City, Colombia. The animal had abdominal distension, anorexia, vomiting, diarrhea and behavioral changes. Clinical examination showed an increased volume in the upper right abdominal quadrant caused by a neoplastic mass. The patient died during the surgical procedure. Necropsy revealed several small nodules in the peritoneum with adhesion to different portions of the small and large intestines, liver, stomach and diaphragm. Tissue samples were collected, routinely processed and stained by H&E. Microscopic examination revealed a mesenchymal tumor limited to tunica muscularis, resembling normal smooth muscle cells. Neoplastic cells were positive for alpha-smooth muscle actin and vimentin, and negative for cytokeratin AE1/AE3 by immunohistochemistry. Those morphological and immunohistochemical findings allowed to diagnose the intestinal leiomyoma referred above.Conclusion: Neoplastic diseases in primates have multifaceted causes. Their manifestations are understudied, leading to a greater difficulty in detection and measurement of the real impact provides by this disease.

Cutaneous myxosarcoma in a Syrian hamster (Mesocricetus auratus)

The pathological findings in a 2-years-old Syrian hamster (Mesocricetus auratus) with a cutaneous myxosarcoma are described. Grossly, there was a large cutaneous mass in the right cervical region. Microscopical evaluation revealed a myxosarcoma characterized by pleomorphic, fusiform cells loosely arranged, randomly distributed, and presenting a moderate amount of basophilic amorphous stroma. There were hemorrhagic areas within the tumor. The basophilic amorphous stroma was positive to Alcian blue confirming the presence of a mucopolysaccharide matrix. Immunohistochemically, the neoplastic cells expressed vimentin, and were negative for cytokeratin or glial fibrillary acidic protein (GFAP).

Isolamento, caracterização e diferenciação de células-tronco mesenquimais do líquido amniótico equino obtido em diferentes idades gestacionais

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)