Evaluation of Telomerase (hTert), Ki67 and p16ink4a expressions in low and high-grade cervical intraepithelial lesions / Avaliação da expressão de Telomerase (hTert), Ki67 e p16 ink4a em lesões intraepiteliais cervicais de baixo e alto graus

ABSTRACT Objective : to study the association between the histological grading of cervical intraepithelial neoplasia (CIN I, CIN II and CIN III) and the immunohistochemical expression for p16ink4a, hTert and Ki67, as well as to evaluate the relationship of these markers with the risk of recurrence after surgical treatment. Methods : we studied a historical cohort of 94 women with intraepithelial lesions CIN I (low grade), CIN II and CIN III (high grades) submitted to conization or electrosurgical excision of the transformation zone. We evaluated all surgical specimens for immunohistochemical expression of p16ink4a, hTert and Ki67. Results : the mean age was 38.2 years; p16ink4a was absent in most CIN I cases. In patients with CIN II or I/II (association of low and high-grade lesions), we observed p16ink4a ≤10%. In patients with CIN III, we found a higher expression frequency of p16ink4a >50%. In CIN I, the majority had Ki67≤10% and low frequency of Ki67>50%. In the CIN III category, there were fewer patients with Ki67≤10%, and Ki67 was absent in most patients of CIN II and III groups. There was no association between hTert expression and histologic grade. There were no statistically significant differences between the expression of the markers in patients with and without recurrence. Conclusion : there was a statistically significant association of p16ink4a and Ki67 with histological grade. The markers' expression, as for disease recurrence, was not statistically significant in the period evaluated.

RESUMO Objetivo: estudar a associação entre a graduação histológica das neoplasias intraepiteliais cervicais (NIC I, NIC II e NIC III) e a expressão imuno-histoquímica para p16ink4a, hTert e Ki67, assim como, avaliar a relação destes marcadores com o risco de recorrência após tratamento cirúrgico. Métodos: estudo de coorte histórica de 94 mulheres portadoras de lesões intraepiteliais NIC I (baixo grau), NIC II e NIC III (altos graus), submetidas à conização ou à excisão eletrocirúrgica da zona de transformação. Todas as peças cirúrgicas foram avaliadas quanto à expressão imuno-histoquímica para p16ink4a, hTert e Ki67. Resultados: a média de idade das pacientes foi 38,2 anos. Nas pacientes NIC I, a p16ink4a estava ausente na maioria dos casos; nas pacientes NIC II ou I/II (associação de lesões de baixo e alto graus), observou-se frequência de p16ink4a≤10%. Nas pacientes NIC III, observou-se maior frequência de expressão de p16ink4a>50%. Na categoria NIC I, a maioria apresentava Ki67≤10% e baixa frequência de Ki67>50%. Na categoria NIC III houve menor número de pacientes com Ki67≤10%, sendo que a maior parte das pacientes tinha Ki67 ausente nos grupos NIC II e III. Não houve associação entre a expressão do marcador imuno-histoquímico hTert e a graduação histológica. Não houve diferenças estatisticamente significativas entre as expressões dos marcadores em pacientes com e sem recorrência. Conclusão: houve associação estatisticamente significativa apenas de p16ink4a e Ki67 com a graduação histológica. A expressão dos marcadores em relação à recorrência da doença não foi estatisticamente significativa no período avaliado.

Immunohistochemical Expression of the Tumor Suppressor Protein p16 INK4a in Cervical Adenocarcinoma / Expressão imunhistoquímica da proteína de supressão tumoral p16 INK4a em adenocarcinoma cervical

ABSTRACT Objective: To evaluate the diagnostic utility of the p16ink4a protein expression as a marker for adenocarcinoma of the cervix. Methods: In a cross-sectional study, p16ink4a expression was evaluated in 30 cervical biopsies from patients diagnosed with invasive adenocarcinoma from 2 reference clinics in Brazil, and compared with 18 biopsies of endocervical polyps (control cases). The performance of the tests for p16ink4a was evaluated using a conventional contingency table, and the Kappa (k) index was used to evaluate the agreement of the marker with the tissue diagnosis. Results: In total, 66% of the invasive adenocarcinoma cases were positive for p16ink4a. All of the adenomatous polyps cases used as negative controls were shown to be negative for p16ink4a. The marker showed a high sensitivity and a high negative predictive value. The Kappa index was good for p16ink4a (k 1/4 0.6). Conclusion: Considering the strong association between the p16ink4a marker and the cervical adenocarcinoma, its use represents an important tool for reducing incorrect diagnoses of adenocarcinoma and thereby avoiding overtreatment.

RESUMO Objetivo: Avaliar a utilidade diagnóstica da expressão da proteína p16ink4a como marcador de adenocarcinoma do colo. Métodos: Em estudo transversal, a expressão de p16ink4a foi avaliada em 30 biópsias cervicais de pacientes diagnosticadas com adenocarcinoma invasivo de colo uterino provenientes de dois serviços de referência no Brasil, comparando com achados em 18 biópsias de pólipos endocervicais (grupo de controle). Para avaliar a performance do teste, foi utilizada tabela de contingência convencional, e para avaliar a concordância com o diagnóstico, foi aplicado o índice de Kappa (k). Resultados: No total, 66% dos casos de adenocarcinoma invasivo foram positivos para p16ink4a. Todos os pólipos adenomatosos foram negativos para p16ink4a. O marcador mostrou uma alta sensibilidade e alto valor preditivo negativo. O índice de Kappa foi bom para p16ink4a (k 1/4 0.6). Conclusion: Considerando a forte associação entre o marcador p16ink4a e o adenocarcinoma cervical, seu uso representa uma ferramenta importante para reduzir o risco de diagnóstico incorreto de adenocarcinoma e, por conseguinte, evitar o excesso de tratamentos.

Expression of Ki-67 and P16 INK4a in chemically-induced perioral squamous cell carcinomas in mice / Expressão KI-67 e P16 INK4a em carcinomas espinocelulares periorais quimicamente induzidos em camundongos

ABSTRACT Objective: to evaluate the influence of Ki-67 and P16INK4a proteins immunohistochemical expressions on the clinical and morphological parameters of perioral squamous cell carcinoma induced with 9,10-dimethyl-1,2-benzanthracene (DMBA) in mice. Methods: we topically induced the lesions in the oral commissure of ten Swiss mice for 20 weeks, determining the time to tumors onset and the average tumor volume up to 26 weeks. In histopathological analysis, the variables studied were histological malignancy grade and the immunohistochemical expression of Ki-67 and P16INK4a proteins. The correlation between variables was determined by application of the Spearman correlation test. Results: the mean time to onset of perioral lesions was 21.1 ± 2.13 weeks; mean tumor volume was 555.91 ± 205.52 mm3. Of the induced tumors, 80% were classified as low score and 20% high score. There was diffuse positivity for Ki-67 in 100% of lesions - Proliferation Index (PI) of 50.1 ± 18.0. There was a strong direct correlation between Ki-67 immunoreactivity and tumor volume (R = 0.702) and a low correlation with the malignancy score (R = 0.486). The P16INK4a protein expression was heterogeneous, showing a weak correlation with tumor volume (R = 0.334). There was no correlation between the immunohistochemical expression of the two proteins studied. Conclusion: in an experimental model of DMBA-induced perioral carcinogenesis, tumor progression was associated with the tumor proliferative fraction (Ki-67 positive cells) and with tumor histological grading, but not with P16INK4a expression.

RESUMO Objetivo: avaliar a influência da expressão imuno-histoquímica das proteínas Ki-67 e p16INK4a sobre parâmetros clínico-morfológicos em carcinomas espinocelulares periorais quimicamente induzidos com 9,10-dimetil-1,2-benzantraceno (DMBA) em modelo murino. Métodos: as lesões foram induzidas topicamente na comissura labial de dez camundongos Swiss durante 20 semanas, sendo determinado o momento de surgimento dos tumores e volume tumoral médio até 26 semanas. Na análise histopatológica, as variáveis estudadas foram gradação histológica de malignidade tumoral e expressão imuno-histoquímica das proteínas Ki-67 e p16INK4a. A correlação entre as variáveis estudadas foi determinada pela aplicação do teste de correlação de Spearman. Resultados: o tempo médio de surgimento das lesões periorais foi 21,1±2,13 semanas. Volume tumoral médio foi de 555,91±205,52mm3. Dos tumores produzidos, 80% foram classificados como de baixo escore e 20%, alto escore. Evidenciou-se positividade difusa para Ki-67 em 100% das lesões - índice de marcação (PI) de 50,1±18,0. Verificou-se correlação direta forte entre a imunoexpressão do Ki-67 e o volume tumoral (R=0,702) e fraca correlação com o escore de malignidade (R=0,486). A expressão da proteína p16INK4a foi heterogênea, mostrando fraca correlação com o volume tumoral (R=0,334). Não houve correlação entre a expressão imuno-histoquímica das duas proteínas estudadas. Conclusão: Em modelo experimental de carcinogênese perioral DMBA-induzida, a progressão tumoral está associada à fração proliferativa do tumor (células ki-67 positivas) e com a gradação histológica tumoral, porém não com a expressão da p16INK4a.

p16INK 4a and Ki-67 expression in human papilloma virus-related head and neck mucosal lesions / Expresión de p16INK 4a y Ki-67 en las lesiones de cabeza y cuello relacionados con el virus del papilloma humano

Human papilloma virus (HPV) is postulated as a risk factor in the etiology of some specific mucosal pathologies in the head and neck regions. Despite the frequent use of p16INK4a as a surrogate marker for HPV-infection, there is still controversy with respect to its reliability. This study has been undertaken to assess the potential role of p16INK 4a and Ki-67 expression in HPV-related lesions. The study was conducted on 71 specimens of oral, tonsillar and laryngeal lesions which comprised 25 dysplasia and 46 papilloma specimens. Specimens were immunohistochemically stained for p16INK4A and Ki-67 proteins. HPV DNA was determined by one step multiplex polymerase chain reaction. HPV DNA was detected in 33.8% of all lesions. Tonsil and larynx lesions showed significant differences with oral lesions for HPV positivity (p<0.001). p16INK 4a over-expression was seen in 56.5% of papilloma and 60% of dysplasia specimens. HPV status showed a positive correlation with p16INK 4a expression in tonsillar dysplasias (p<0.001). p16INK 4a expression may have a value as a marker in high risk HPV induced dysplasias, but not in low risk infected lesions. The proliferation index is not related to HPV-induced lesions and may be evaluated as an independent marker in head and neck premalignant lesions.

El virus del papiloma humano (VPH) se postula como un factor de riesgo en la etiología de algunas patologías de la mucosa, específicas en las regiones de cabeza y cuello. A pesar de usar con frecuencia el p16INK4A como un marcador sustituto para la infección por VPH, todavía existe controversia con respecto a su fiabilidad. Este estudio se ha llevado a cabo para evaluar el papel potencial de la expresión de p16INK 4a y de Ki-67 en las lesiones relacionadas con el VPH. El estudio se realizó en 71 muestras de lesiones orales, tonsilares y laríngeas que comprendían 25 displasias y 46 especímenes de papiloma. Los especímenes fueron teñidos inmunohistoquímicamente para p16INK4a y Ki-67. El ADN del VPH se determinó mediante una PCR multiplex de un paso. ADN del VPH se detectó en el 33,8% de todas las lesiones. Las lesiones de la amígdala y laringe mostraron diferencias significativas con lesiones orales para la positividad de VPH (p <0,001). Sobre-expresión de p16INK 4a se observó en 56,5% de las muestras de papiloma y 60% de las muestras de displasia. El estatus del VPH mostró una correlación positiva con la expresión de p16INK4a en displasias tonsilares (p <0,001). La expresión de p16INK4a puede tener valor como marcador en las displasias inducidas por VPH de alto riesgo, pero no en las lesiones infectadas de bajo riesgo. El índice de proliferación no está relacionado con las lesiones inducidas por VPH y puede ser evaluado como un marcador independiente en las lesiones premalignas de la cabeza y del cuello.

P16(INK4A) expression in Bowen's disease and Bowenoid papulosis.

BACKGROUND: Bowen's disease (BD) is a skin carcinoma in situ occurring over the entire body surface. It shares similar histopathological features with Bowenoid papulosis (BP) of the genitalia, but differs in etiology and clinical course. Increased p16(INK4A) (p16) tumor suppressor protein expression has been demonstrated in relation to the progression of cutaneous squamous neoplasms. OBJECTIVE: To evaluate the difference in p16 expression between Bowen's disease and Bowenoid papulosis. MATERIAL AND METHOD: Biopsies of 46 cases of BD in the period 1994 - 2003 and 14 cases of BP during 1987 - 2003 in the Anatomical Pathology Unit, Department of Pathology, Faculty of Medicine, Prince of Songkla University, Thailand were studied by immunohistochemical methods using the P16 kit (CINTec Histology Kit, clone E6H4, Code-Nr. K5334, DakoCytomation, Denmark). Nuclear/cytoplasmic immunoreactivity in more than 10% of neoplastic cells was considered positive. RESULTS: P16 expression was positive in 37 of 46 BD cases (80.4%) which was higher than that of BP (6 of 14 cases or 42.9%) (p value < 0.05, Chi-square test). The expression among the three groups of BD: extragenital (28 of 35), chronic arsenical-related (7 of 8) and genital lesions (2 of 3) was not significantly different (p value = 0.734, Chi-square test). CONCLUSION: P16 expression was more frequent in BD than BP. This suggests a possible association between p16 expression and tumorigenesis of these lesions.