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.

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.

Immunohistochemical expression of MIB-1 and PCNA in precancerous and cancerous lesions of uterine cervix.

BACKGROUND AND OBJECTIVE: The present study was done to analyze the immunoexpression of diagnostic markers (MIB-1: molecular immunology borstel and PCNA: proliferating cell nuclear antigen) in grading cervical intraepithelial lesion (CIN) and squamous cell carcinoma (SCC) in cervix. SETTING AND DESIGN: Total 150 cervical biopsies were divided into four groups respectively; Group I-Normal (n = 32), Group II- CIN (n = 60), Group III- SCC (n = 44), Group IV- CA cervix (n = 14) respectively. MATERIALS AND METHODS: These biopsies were stained with monoclonal antibodies by streptavidin-- biotin method. Mean labeling index was calculated and grading was performed using the I--III scoring system. STATISTICAL ANALYSIS: Findings were correlated with age and menopausal status. Statistical analysis was done by using student sample‘t’ test and analysis of variance (ANOVA) by SPSS 10 package. RESULTS: MIB-1 immunostaining was positive in 112/150 (74.6%) cases and PCNA in 118 /150 (78.6%) cases. Labeling indices showed linear progression from normal to CIN to SCC to cancer lesion. Few cases of low-grade CIN lesion had high proliferative index. A significant positive correlation was found between age and PCNA and MIB-1 values (P < 0.05) when comparison was made for all the cases. CONCLUSION: These markers may be useful in identifying low-grade CIN lesion with high proliferative index. These cases should be kept for follow up studies so that proper intervention can be taken at an early stage. This method is simple and cost effective and can easily be done in formaline-fixed paraffin embedded tissues in a clinical laboratory for grading CIN and SCC lesions in cervix.

Expression of vimentin in breast carcinoma, its correlation with Ki67 and other histopathological parameters.

CONTEXT: Vimentin is a mesenchymal marker, known to express in some epithelial carcinomas. AIMS: 1. To find out the expression of vimentin in infiltrating ductal carcinoma of breast (not otherwise specified), 2. To find out the correlation between expression of vimentin and prognostic markers such as tumor size, tumor grade, lymph node status, proliferation index (measured by Ki 67), and Nottingham prognostic index (NPI). MATERIALS AND METHODS: Study was done at Department of Pathology; 50 cases of infiltrating ductal carcinoma (NOS) were studied for tumor grade; immunohistochemistry was done using antibodies against vimentin and Ki 67. Percentages of positive cells were documented. An immunoscore was also calculated for vimentin. Vimentin expression was correlated with tumor size, lymph node status, Nottingham prognostic index, and Ki 67. Statistical analysis used: statistical correlation was done using Pearson’s chi-square test. A P value less than 0.01 was considered significant. RESULTS: Vimentin expression was seen in 18% of cases. Its expression correlated with high tumor grade and high growth fraction (P value < 0.01). It did not correlate with lymph node status, tumor size, and NPI. CONCLUSIONS: Increased vimentin expression is associated with bad prognostic factors. Immunohistochemistry with vimentin may be helpful in knowing the prognosis in cases of infiltrating ductal carcinoma of breast (NOS).

Prognostic significance of ER, PR, Ki67, c-erbB-2, and p53 in endometrial carcinoma.

OBJECTIVE: To better discern the prognostic significance of estrogen-progesterone (ER-PR) receptor proliferative index, tumor suppressor gene, and over expression of oncogene c-erbB-2 in correlation with survival time and recurrence of tumor. MATERIAL AND METHOD: Paraffin blocks from 65 cases of endometrial carcinoma diagnosed and treatment at Rajavithi Hospital, Bangkok, Thailand with a follow-up time of at least 60 months were immunohistochemical studiedfor ER and PR status, tumor proliferative index (Ki-67), tumor suppressor gene (p53), and overexpression of oncogene c-erbB-2. Survival analysis was performed with the Cox proportional hazards. RESULTS: The mean age of the patients was 54.94 years with a range of 24 to 80 years. The mean follow-up time was 50.35 months. Nine patients (13.8%) had recurrent tumors, 5 years after treatment. Ten patients (15.4%) died of the primary disease during the follow-up period. ER was positive in 50 cases (76.9%) and negative in 15 cases (23.1%). PR was positive in 47 cases (72.3%) and negative in 18 cases (27.7%). Both ER and PR showed significant correlation (p<0.01). Ki-67 showed 27 cases (41.5%) having >35% positive nuclear staining and 38 cases (58.5%) had < or =35% positive nuclear staining. p53 was positive in 31 cases (47.7%) and negative in 34 cases (52.3%). c-erbB-2 was positive in one case (1.5%), equivocal in six cases (9.2%), and negative in 58 cases (89.3%). CONCLUSION: Survival analysis showed that cases with low-stage, low-grade, no recurrent tumor, ER and PR positive, and Ki-67 < or =35% had good survival compared to cases with high-stage, high-grade, presence of recurrent tumor, ER-PR-negative, and Ki-67 > 35% (p<0.05). Cox regression analysis showed ER-PR status and Ki-67 were significant independent prognostic indicators for survival time. Ki-67 expression was also a significant independent prognostic indicator for recurrent tumor p53 and c-erbB-2 displayed no statistical significance related to survival time.

Expression of Pin1 and Ki67 in cervical cancer and their significance / 华中科技大学学报（医学）（英德文版）

In order to investigate the expression levels of Pin1 mRNA and protein in cervical cancer and its association with Ki67 and their clinical significance, amplification of Pin1 gene was examined by RT-PCR, and the expression of both Pin1 and Ki67 protein was detected by immunohistochemistry in cervical cancer tissues. It was shown that the expression levels of Pin1 were higher in cervical cancer than in normal cervical tissues (P 0.05). The expression of Pin1 was significantly higher in adenocarcinoma than in squamous carcinoma of the uterine cervix (P < 0.05). In cervical cancer, the overexpression of Pin1 was positively correlated with that of Ki67 (P < 0.05). These results suggested that the overexpression of Pin1 was closely related with cancer cell proliferation or progression of cervical cancer and contributed to oncogenesis. Pin1 may serve as a potential marker for cervical cancer diagnosis.

Histological spectrum of ependymomas and correlation of p53 and Ki-67 expression with ependymoma grade and subtype.

BACKGROUND: Clinical and histological criteria for ependymoma prognosis are well recognized. Recently few studies have been done based on Immunohistochemistry for prognostication of these tumours. In this study we have correlated the histological spectrum with immmunoexpression of p53 and Ki67 in these tumors. AIMS: To know the incidence of ependymomas; study their morphological spectrum and to evaluate expression of P53 and Ki 67 in different morphological subtypes. MATERIAL AND METHOD: A retrospective study was preformed on 70 ependymomas received in a period between 1994 and 2001. Entire tissue received was processed for routine paraffin embedded H&E stained sections. Immunocytochemistry was performed using antibodies to GFAP, EMA, Pancytokeratin and synaptophysin, to differentiate papillary ependymoma from choroid plexus papilloma; clear cell ependymoma from oligodendroglioma and central neurocytoma; ependymoblastoma from other embryonal tumours. p53 and Ki-67 immunohistochemistry was performed to correlate their expression with various tumour grades and subtypes. RESULTS: There were 3 cases (4.2%) of Grade I ependymoma (2 cases of myxopapillary ependymoma and 1 case of subependymoma); 57 cases (81.5%) of ependymoma grade II (43 of these were of classical variety, 11 of clear cell ependymoma, 2 of papillary and 1 case of cellular ependymoma). There were 9 cases (12.8%) of anaplastic ependymoma (one of these was a clear cell ependymoma and 1 case (1.5%) of ependymoblastoma CONCLUSION: p53 and Ki67 indices can be used in routine diagnostic laboratories to supplement the tumor grade on histology and more studies with follow up should be performed to analyse the prognosis of different subtypes. The expression of Ki 67 and p53 was significantly higher in anaplastic ependymomas. 4 out of 11 cases of clear cell ependymomas showed higher Ki 67 indices as compared to classical grade II ependymomas, thus further highlighting the importance of differentiating the various subtypes.

Thyroid Transcription Factor-1 (TTF-1) Expression in Human Lung Carcinomas: Its Prognostic Implication and Relationship with Expressions of p53 and Ki-67 Proteins

This study was aimed to evaluate the prevalence and prognostic implication of thyroid transcription factor-1 (TTF-1) immunoreactivity in 81 human lung carcinomas, including 65 cases of non-small cell lung carcinoma (NSCLC) and 16 cases of small cell lung carcinoma (SCLC); and also to investigate its relationship with the cell proliferation and regulation by immunostaining of Ki-67 and p53 proteins, respectively. The immunohistochemical staining for TTF-1(clone 8G7G3/1) was performed and several clinicopathologic variables and the follow-up data were obtained. The immuno-staining results for TTF-1 were semiquantitatively interpreted as negative and positive. Of NSCLCs, TTF-1 is highly expressed in adenocarcinomas (76%), whereas squamous cell carcinomas revealed no immunoreactivity (0%). SCLCs showed strong TTF-1 expression (88%). In NSCLC, TTF-1 expression was inversely correlated with Ki-67 proliferative activity and independent of p53 overexpression. TTF-1(+) group tended to show better survival than TTF-1(-) group in NSCLC. Conclusively, these observations suggest that TTF-1 is a sensitive and specific diagnostic marker for pulmonary adenocarcinomas and SCLCs; that TTF-1 might have a good prognostic implication based on its inverse correlation with Ki-67 proliferative activity and tendency for better survival in NSCLC; that this cell lineage marker may play a role in the molecular pathogenesis of lung cancers at the level of transcription.

Expression of Cyclin B1 and cdc2 in Nodal Non-Hodgkin's Lymphoma and its Prognostic Implications

To investigate the role of cyclin B1 and cdc2 in the pathogenesis and progression of malignant lymphoma, 68 cases of nodal non-Hodgkin's lymphoma were examined about the expression of cyclin B1 and cdc2 along with p53 and Ki-67 by immunohistochemical method. The correlation of their expression with various clinicopathologic findings was also analyzed. Cyclin B1 and cdc2 were diffusely expressed in 39 cases (57.4%) and 54 cases (79.4%) out of 68 cases studied, respectively. The mean labeling indices of cyclin B1 and cdc2 in malignant lymphoma were 31.9% and 68.0%, respectively. In normal lymphoid tissues, cyclin B1 and cdc2 were expressed predominantly in the germinal center with mean labeling indices of 13.9% and 28.3%, respectively. The correlation between the expression of cyclin B1 and cdc2 was noted (p=0.013). The expression of Ki-67 was correlated with that of cyclin B1 (p=0.023) and marginally correlated with that of cdc2 (p=0.056). The expression of cdc2 and p53 in complete remission group to chemotherapy was lower than that of progressive disease group (p=0.047, p=0.049). In multivariate analysis, the clinical stage alone showed significance on overall survival (p=0.049). In conclusion, cyclin B1 and cdc2 appeared to be involved in the genesis or progression of malignant lymphoma and cdc2 can be a useful marker for response to chemotherapy.