Avaliação da via do c-kit no adenoma pleomórfico e carcinoma adenoide cístico das glândulas salivares: estudo da expressão das proteínas da via, das mutações e da regulação pós-transcricional do gene kit / c-Kit signaling cascade in pleomorphic adenoma and adenoid cystic carcinoma of salivary glands: evaluation of cascade proteins, mutations and post-transcriptional regulation of gene Kit

Introdução: As neoplasias das glândulas salivares têm amplo espectro histológico resultante da múltipla diferenciação celular tumoral. O adenoma pleomórfico (AP) e o carcinoma adenoide cístico (CAC) são as mais comuns neoplasias benignas e malignas provenientes do ducto intercalado, respectivamente, além de serem compostas por estruturas luminais e células mioepiteliais. Em estudo realizado previamente pelo nosso grupo, detectamos que a proteína c-kit está envolvida nos processos da morfogênese das glândulas salivares e no adenoma pleomórfico. A proteína c-Kit tem papel importante no desenvolvimento de muitos processos embrionários, incluindo a gametogênese, melanogênese e hematopoiese, e também na biologia de tumores. Sua ativação induz diversas respostas intracelulares através de cascatas de sinalização de vias como PI3K/AKT e MAPK. Em tumores da glândula salivar ainda há poucos estudos sobre as alterações do gene KIT e das proteínas relacionadas a sua via de sinalização, assim como sua regulação pós-transcricional, realizada principalmente por meio dos microRNAs. O presente estudo avaliou, em APs e CACs (a) a localização das proteínas das vias PI3K/AKT/mTOR e MAPK por meio da técnica de imunoistoquímica; (b) a expressão dos microRNAs 221 e 222, relacionados ao gene KIT (c) a associação dos achados laboratoriais com variáveis clínicas, patológicas e sobrevida. Resultados: Nos casos de AP a proteína c-Kit foi identificada em formações luminais e em raras células isoladas no parênquima tumoral. Já nos CAC, observou-se positividade na membrana das células ductais. Para a via de PI3K/AKT/mTOR, no AP, a proteína PI3K beta mostrou-se parcialmente positiva no citoplasma das células próximas à capsula tumoral, e as proteínas AKT e mTOR fosforiladas, foram expressas especialmente nas células epiteliais e em poucas células mioepiteliais. Já no CAC, a proteína PI3K beta e AKT fosforilada mostraram-se negativas na maioria dos casos, e a proteína mTOR fosforilada foi expressa no citoplasma das células epiteliais e em algumas células mioepiteliais. Para a via MAPK, as proteínas RAS, MEK-1 fosforilada e ERK 1/2 foram negativas na maioria dos AP e CAC; B-Raf e MEK-2 fosforilada foram observadas nas células luminais dos AP. Nos CAC, estruturas luminais neoplásicas foram positivas para a proteína MEK-2 fosforilada; B-Raf foi positivo nas células luminais e mioepiteliais. Além disso, os pacientes que expressaram as proteínas mTOR e MEK-2 fosforilada apresentaram sobrevida câncer-específica significativamente aumentada (p=0,040 e p=0,005, respectivamente). Na análise do microRNAs, a expressão do miR-221 foi variável nas 13 amostras analisadas, tendo baixa expressão em 30,77% dos casos, expressão normal em 38,46 e expressão aumentada em 30,77% dos casos. Já nos APs o miR-221 foi detectado em 19 amostras, sendo 36,84% com baixa expressão, 52,63% com expressão normal e expressão aumentada foi vista em 10,53% dos casos. A expressão do miR-222 foi detectada em 14 CACs, sendo que a maioria dos casos (8 casos ­ 57,1%) a expressão do miR-222 foi semelhante ao observado nas amostras não neoplásicas. Nos APs, o miR-222 foi detectado em 22 amostras, sendo 31,8% com baixa expressão, 31,8% com expressão normal e 36,4% com expressão aumentada. Conclusão: Apesar de a proteína c-Kit ser expressa em ambas as neoplasias ­ AP e CAC, sua influência sobre as vias de sinalização MAPK e PI3K/AKT/mTOR ainda permanece por ser estabelecida. Ainda, os microRNAs 221 e 222 não mostram correlação consistente com a expressão de c-Kit nos tipos tumorais estudados.

Introduction: Salivary gland tumors present broad histological spectrum resulting from multiple tumor cell differentiation. Pleomorphic adenoma (PA) and adenoid cystic carcinoma (ACC) are the commonest benign and malignant salivary gland neoplasms originated from the intercalated duct region, respectively, and are composed by luminal structures and myoepithelial cells. In a previous study we detected that protein c-kit is involved in the process of salivary gland morphogenesis and PA. c-Kit protein is important during embryogenesis, including gametogenesis, melanogeneis and hematopoiesis as well as in tumorigenesis. Its activation induces various intracellular responses through pathways such as MAPK and PI3K/AKT/mTOR signaling cascades. In salivary gland neoplasms, only a few reports have shown that alterations in KIT gene are present and proteins related to its signaling pathway as well as its post-transcriptional regulation. This study has aimed at evaluating in PA and ACC: (a) the proteins location of PI3K/AKT/mTOR and MAPK pathways using immunohistochemistry (IHC); (b) expression of miR-221 and miR-222, related to KIT gene; and (c) the association of these findings with clinical, pathological and survival data of patients. Results: In PA c-kit was positive in isolated luminal cells; in ACC, neoplastic luminal structures were positive for c-Kit. In PA, PI3K beta protein was shown to be partially positive in the cytoplasm of cells near the tumor capsule and phosphor AKT and phospho mTOR, are specifically expressed in epithelial cells and in a few myoepithelial. In ACC, PI3K and phosphor AKT protein showed to be negative in most of cases. Phospho mTOR protein was expressed in the cytoplasm of epithelial cells and some myoepithelial cells. In MAPK pathway, Ras, ERK1/2 and phosphor MEK-1 proteins were negative in most PAs and CACs; B-Raf and phospho MEK-2 were detected in luminal cells of PA. In ACC neoplastic luminal structures were positive for phospho MEK-2; B-Raf was also positive in myoepithelial and epithelial cells. In addition, cases with expressed phospho-mTOR and phosphor MEK-2 proteins were significantly associated with higher cancer-specific survival (p = 0.040 and p = 0.005, respectively). Moreover, expression of miR-221 was detected in 13 CAC samples and 19 PA samples. In CAC, expression of miR-221 was downregulated in 30,77% of the samples, upregulated in 30,77% samples, and normal in 38,46% samples. In PA, miR-221 expression was downregulated in 36,84% samples, upregulated in 10,53% samples, and normal in 52,63% samples. Expression of miR-222 was detected in 14 CAC samples and 22 PA samples. In the majority of CAC samples, the expression of miR-222 was similar to that observed in non-neoplastic samples. In PA samples, expression of miR-222 was downregulated in 31,8% samples, upregulated in 36,4% samples, and normal in 31,8% samples. Conclusion: Although c-Kit expression is detected in PA and ACC, its influence on the MAPK e PI3K/AKT/mTOR signaling cascades remains to be established. miR-221 e -222 did not show a robust correlation with c-Kit expression in the tumors studied.

Expression of Autophagy and Reactive Oxygen Species-Related Proteins in Lacrimal Gland Adenoid Cystic Carcinoma

PURPOSE: To investigate the difference of expression of autophagy and reactive oxygen species (ROS) related proteins in adenoid cystic carcinoma (ACC) of lacrimal gland in comparison with ACC of salivary gland. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded tissue samples from patients pathologically diagnosed as lacrimal gland ACC (n=11) and salivary gland ACC (n=64) were used. Immunochemistry was used to measure expression of autophagy related proteins [beclin-1, light chain (LC) 3A, LC3B, p62, and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3)] and ROS related proteins [catalase, thioredoxinreductase, glutathione S-transferasepi (GSTpi), thioredoxin interacting protein, and manganese superoxide dismutase (MnSOD)]. The prognostic factors related to disease-free and overall survival (OS) in lacrimal gland ACC by log-rank tests, were determined. RESULTS: GSTpi in stromal cells was more highly expressed in lacrimal gland ACC (p=0.006), however, MnSOD in epithelial cells was expressed more in salivary gland ACC (p=0.046). LC3B positivity and BNIP3 positivity in epithelial component were associated with shorter disease-free survival (both p=0.002), and LC3A positivity in stromal component was the factor related to shorter OS (p=0.005). CONCLUSION: This is the first study to demonstrate the expression of autophagy and ROS related proteins in lacrimal gland ACC in comparison with the salivary gland ACC, which would provide a basis for further study of autophagy and ROS mechanism as novel therapeutic targets in lacrimal gland ACC.

Expression of beclin 1 in primary salivary adenoid cystic carcinoma and its relation to Bcl-2 and p53 and prognosis

Beclin 1 plays a critical role in autophagy and functions as a haploinsufficient tumor suppressor. The expression and prognostic significance of beclin 1 in head and neck adenoid cystic carcinoma (ACC) are largely unexplored. Therefore, we investigated the expression of beclin 1, Bcl-2, and p53 in head and neck ACC tissue. Tissue samples from 35 cases (15 females, 20 males) of head and neck ACC were utilized for immunohistochemistry. Beclin 1 expression was observed in 32 cases (91.4%) and considered to be high in 15 cases (42.9%) and low in 20 cases (57.1%). Beclin 1 expression was significantly correlated with a histological growth pattern (P=0.046) and histological grade (P=0.037). Beclin 1 expression was inversely correlated with Bcl-2 expression (P=0.013) and significantly associated with overall survival (P=0.006). Bcl-2 and p53 expression were observed in 21 cases (60.0%) and 16 cases (45.7%). Bcl-2 expression was significantly correlated with perineural invasion (P=0.041) and not associated with overall survival (P=0.053). p53 expression was directly correlated with beclin 1 expression (P=0.044). Our results indicated that beclin 1 may be a novel, promising prognostic factor for clinical outcome in head and neck ACC patients and may play a part in the development of head and neck ACC by interacting with Bcl-2 and p53.

Intraoral mucoepidermoid carcinoma of salivary glands: lack of association among clinicopathological features and immunoexpression of c-erbB-2 in 29 cases

The association among clinicopathological features and c-erbB-2 oncoprotein expression was evaluated in twenty-nine cases of intra-oral mucoepidermoid carcinoma (MEC). MEC was prevalent in the female gender (79.3 percent), tumors were more frequent in ages between 21 and 40 years (48.3 percent), and the palate was the most commonly affected site (72.4 percent). Microscopically, 27 cases (93.1 percent) were classified as low grade of malignancy. The c-erbB-2 expression was considered positive in 9 (31 percent) cases and no significant association (p>0.05) was found among protein expression and gender nor between patient age and site or histological grade of the lesion. c-erbB-2 expression in MEC may reflect intrisinc biologic properties of salivary gland neoplasms and may be linked to histogenesis and cellular differentiaton.

Fueron evaluados 29 casos de carcinoma mucoepidermoide intraoral en sus aspectos clínico-patológicos, además de la expresión de la oncoproteina c-erbB-2. El carcinoma mucoepidermoide fue predominante en las mujeres (79,3 por ciento), siendo más frecuente en individuos entre 21 y 40 años de edad (48,3 por ciento). El paladar fue el sitio más comunmente afectado (72,4 por ciento). Microscópicamente, 27 casos (93,1 por ciento) fueron clasificados como de baja malignidad. La expresión del c-erbB-2 se consideró positiva en 9 (31 por ciento) casos y no fue observada ninguna asociación significativa (p>0,05) entre la expresión de la proteína y género, ni entre la edad de los pacientes y el sitio o el grado histológico de la lesión. La expresión de la c-erbB-2 en el carcinoma mucoepidermoide puede mostrar las propiedades biológicas intrísecas de las neoplasias de las glándulas salivales.

Immunohistochemical expression of vimentin, calponin and HHF-35 in salivary gland tumors

Myoepithelial cells present a complex immunophenotype, with the expression of proteins varying according to the stage of normal or neoplastic differentiation of the cell. In order to evaluate the immunohistochemical markers expressed by these cells, a panel of antibodies composed of vimentin, calponin and HHF-35 was applied to 28 salivary gland tumors. The results demonstrated a higher percent sensitivity of vimentin and calponin compared to HHF-35. However, calponin and HHF-35 presented a focal labeling pattern in contrast with the diffuse distribution of vimentin. The cells predominantly stained by all tested antibodies included nonluminal cells in duct-like and tubular structures, such as those seen in pleomorphic adenomas and adenoid cystic carcinomas, as well as cells in the cords and nests of polymorphous low-grade adenocarcinomas and peripheral cells of sheets and nests of myoepitheliomas. In conclusion, the combination of calponin and vimentin is suggested for the identification of myoepithelial cells in salivary gland tumors.

As células mioepiteliais apresentam um imunofenótipo complexo, variando a expressão de suas proteínas na dependência do seu estágio de diferenciação normal ou neoplásico. Com o objetivo de avaliar comparativamente marcadores imuno-histoquímicos para estas células, um painel de anticorpos composto pela vimentina, calponina e HHF-35 foi aplicado em 28 tumores de glândulas salivares. Os resultados demonstraram que a vimentina e a calponina foram percentualmente mais sensíveis que o HHF-35; entretanto, a calponina e o HHF-35 apresentaram padrão de distribuição focal diferentemente da distribuição difusa da vimentina. As células predominantemente marcadas, por todos os anticorpos utilizados, foram as não luminais presentes nas estruturas ductiformes e tubulares, vistas no adenoma pleomórfico e no carcinoma adenóide cístico, bem como as células dos cordões e ninhos dos adenocarcinomas polimorfo de baixo grau e periferia de lençóis e ninhos dos mioepiteliomas. Em conclusão, sugere-se que se faça associação da calponina com vimentina para identificação de células mioepiteliais em neoplasias de glândula salivar.

Mioepitelioma de glândula salivar menor: uma análise imunohistoquímica de quatro casos / Myoepithelioma of minor salivary gland: an immunohistochemical analysis of four cases

Introdução e Metodologia: Realizou-se um estudo imunohistoquímico em 4 casos de mioepiteliomas, visando traçar seu perfil quanto ao grau de diferenciação das células através dos anticorpos alfa-SMA, CK 14 e vimentina, bem como investigar o índice de proliferação celular pelo anti-PCNA, além de comparamos a imunorreatividade com o tecido glandular normal adjacente ao tumor. RESULTADOS: No tecido glandular normal as células mioepiteliais exibiram marcação para alfa-SMA e CK 14, enquanto que nas células ductais somente a presença da CK 14 foi verificada. Em todos os casos foi verificada positividade para CK 14 e vimentina, porém a CK 14 esteve presente somente em células epitelióides e fusiformes, enquanto que a vimentina mostrou-se positiva também nas células plasmocitóides. A alfa-SMA não foi detectada nas células neoplásicas. Imunopositividade para o PCNA foi observada em mais de 75 por cento do componente celular dos tumores analisados, independente do tipo. CONCLUSÕES: Concluiu-se que não houve diferença na atividade proliferativa entre os tipos celulares presentes nos mioepiteliomas e, ainda, que os resultados deste estudo sugerem que as células constituintes desta neoplasia realmente representam células da linhagem mioepitelial, mas em diferentes estágios de diferenciação.

Introduction and Methods: We performed an immunohistochemical study in four cases of myopitheliomas with objective to realize a profile in respect of differentiation grade by the monoclonal antibodies CK14, vimentin and alph-SMA, besides to investigate the cell proliferation by anti-PCNA, besides, we compare the immunoreactive with glandular normal tissue. RESULTS: In the glandular normal tissue the myoepithelials cells had shown expression for alpha-SMA and CK 14, while that in the ductals cells, only the presence of CK 14 was verified. All the cases was verified positivy for CK 14 and vimentin, however, CK 14 had been present only in epithelioid and fusiform cells, while that the vimentin revealed positive also in the cytoplasm of the plasmocytoid cells. alpha-SMA was not detected in the neoplasic cells. Immunopositivity for the PCNA was observed in more than 75 percent of the cellular component of the analyzed tumors, independent of the cellular type. CONCLUSIONS: We concluded that it did not have difference in the proliferative activity among the cellular types presents in the myoepitheliomas and, still, the results of this study suggest that the constituent cells of this neoplasia one really represent cells of the mioepitelial ancestry, but in different stages of differentiation.

Immunohistochemical evaluation of p27 (kip1) in pleomorphic adenomas and adenoid cystic carcinomas of the minor salivary glands.

BACKGROUND: p27(kip1), a universal cyclin-dependent kinase inhibitor, is a useful marker for predicting clinical aggressiveness with various human tumors. In this study, p27 expression was investigated in pleomorphic adenomas (PAs) and adenoid cystic carcinomas (ACCs) of minor salivary glands to evaluate its utility for differentiation purposes. At the same time, the correlation between p27 and ACC grading was evaluated. MATERIALS AND METHODS: Clinicopathological features of 22 patients (11 ACCs, 11 PAs), including age, sex and size of tumor were obtained from medical records. Immunohistochemical staining with p27(kip1) was performed for each specimen and p27 labelling indices were determined with a computer-assisted image-analyzing system (CAS 200). Pearson's correlation coefficient, Spearman's correlation coefficient, Students t-test, Kruskal-Wallis test and ANOVA were applied for statistical analyses using SPSS 11.5. RESULTS: p27 LIs for all PAs were above 25% whereas for ACCs they were under 25% (except one case). p27 expression (LI and intensity) was significantly lower in ACCs than PAs. The correlation between p27 expression and ACC grading was not significant. CONCLUSION: Overall, these findings suggest that reduced expression of p27 might be correlated with the development of ACC and could be an indicator of malignant behavior.

Expressão imuno - histoquímica de p53 na discriminação do comportamento biológico dos tumores de glândulas salivares / Immunohistochemical expression of p53 on the biological behavior of salivary gland tumors

INTRODUÇAO: Mutações no gene TP53 têm sido identificadas em várias neoplasias, incluindo as de glândulas salivares (NGS). Entretanto não existe consenso sobre o significado biológico da expressão de p53 nessas lesões. OBJETIVOS: Avaliar a marcação imuno-histoquímica do antígeno p53 em NGS, investigando possíveis diferenças entre lesões benignas e malignas, entre diferentes tipos histológicos e entre tumores malignos que: 1) não desenvolveram metástases; 2) emitiram metástases; e 3) metástases de NGS. MATERIAL E MÉTODO: Foram avaliados 16 casos de adenomas pleomórficos (AP), 17 lesões malignas que não apresentaram metástase durante a proservação (sete carcinomas adenóides císticos [CAC] e dez mucoepidermóides [CM]), 13 tumores malignos que emitiram metástases (nove CAC e quatro CM) e, ainda, 12 metástases regionais ou distantes (cinco CAC e sete CM). Por marcação imuno-histoquímica (estreptavidina-biotina-peroxidase), índices médios de localização do antígeno p53 foram estabelecidos para cada tumor. Para análise, utilizaram-se testes de Mann-Whitney e Kruskal-Wallis, com nível de significância de 5 por cento. RESULTADOS: Foram observadas diferenças estatisticamente significantes nos índices médios de imunomarcação de p53 entre lesões benignas e malignas (p = 0,03) e entre os diferentes tipos histológicos, notadamente entre o AP e o CM (p = 0,02). Todavia houve marcada sobreposição dos intervalos de índices de expressão entre os grupos testados. Nenhuma diferença de expressão foi observada entre as neoplasias malignas quanto ao seu comportamento metastático. CONCLUSAO: Embora a expressão de p53 tenha sido detectada em todos os subtipos de NGS, a marcação imuno-histoquímica deste antígeno não permitiu discriminar os tumores malignos quanto ao seu comportamento biológico.

Proliferating cell nuclear antigen expression in mucoepidermoid carcinoma of salivary glands

CONTEXT: Among the cytological and morphological properties of mucoepidermoid carcinoma, one of the most important criteria for measuring its biological behavior and aggressiveness is cell proliferation. In this way, immunohistochemical markers of cell proliferation have been found to be useful in tumor classification and have formed part of the prognostic and therapeutic studies of these pathologies. OBJECTIVE: To analyze 11 cases of mucoepidermoid carcinoma (MEC) using the proliferation activity marker (PCNA) and to determine its relationship to the grade of malignancy of these tumors. DESIGN: Correlation study. SETTING: Head and Neck Surgery Service of Heliópolis Hospital, São Paulo, Brazil. SAMPLE: Slides of 11 cases of primary mucoepidermoid carcinomas of salivary glands were prepared according to routine techniques employed in the Oral Pathology Department of the Dentistry Faculty of São Paulo University, Brazil. They were fixed in a 10 percent formaldehyde solution and stained with hematoxylin and eosin. After this preparation the tumors were classified as low, intermediate and high grade of malignancy, according to the criteria established by Seifert & Sobin and Auclair, Goode & Ellis. The slides were sent for immunohistochemical processing to evaluate the positivity of proliferating cell nuclear antigen using the streptavidin biotin technique. MAIN MEASUREMENT: The correlation between proliferating cell nuclear antigen expression and the histological malignancy grade in mucoepidermoid carcinoma of salivary glands. RESULTS: there were 4 cases (36 percent) of low grade, 4 cases (36 percent) of intermediate grade and 3 cases (27 percet) of high grade of malignancy. After a comparative study between histological features and immunohistochemical analysis, significant differences were observed (P < 0.01) for low, intermediate and high grades: 16.04 percent, 26.98 percet and 56.98 percent of proliferating cell nuclear antigen expression in mucoepidermoid carcinoma , respectively. CONCLUSION: The proliferating cell nuclear antigen expression increases with the grade of malignancy in mucoepidermoid carcinoma of salivary glands.

Estudo imuno-histoquímico do carcinoma adenóide cístico de glândula salivar menor / Immunohistochemical study of minor salivary gland's adenoid cystic carcinoma

A expressäo de um painel de anticorpos, constituídos pelas citoqueratinas 7, 8, 10, 13, 14, 18 e 19, vimentina e HHF-35, foi estudada em 21 casos de carcinomas adenóide císticos de glândula salivar menor, de diferentes sub-tipos histológicos, utilizando-se a técnica da imunohistoquímica, pelo método da estreptpavidina-biotina. Os resultados mostraram que nos tumores cribriformes, as células luminas das estruturas ductais foram positivas para as citoqueratinas 7, 8, 14, 18 e 19 e negativas para a vimentina e HHF-35, enquanto as células externas dos ductos, as células que revestiam os espaços pseudo-císticos e as células periféricas dos cilindros foram negativas para as citoqueratinas testadas e positivas para a vimentina e HHF-35. Nos tumores tubulares, enquanto as células internas dos ductos mostraram-se positivas para as citoqueratinas 7, 8, 14, 18 e 19 e negativas para vimentina e HHF-35, as células periféricas das estruturas ductais, apresentaram-se sempre positivas para a vimentina e HHF-35 e ocasionalmente positivas para a citoqueratina 14. Nas neoplasias do subtipo sólido, observou-se positividade esporádica das células, especialmente para as citoqueratinas 7 e 14, bem como para a vimentina. Os nossos achados mostraram que o carcinoma adenóide cístico, de um modo geral, mimetiza, em seu perfil imuno-histoquímico, o segmento de ducto intercalado da glândula salivar normal, apresentando variaçöes que sugerem diferentes graus de diferenciaçäo para os seus três subtipos histológicos. Os tumores tubulares representam a variante mais diferenciada, os tumores sólidos, o subtipo menos diferenciado, e os tumores cribiformes, neoplasias de um grau intermediário de diferenciaçäo entre aquele visto nas outras duas variantes histológicas