Expressão imuno-histoquímica dos biomarcadores p16 e Ki-67 na lesão intraepitelial cervical de alto grau: revisão de estudos / Immunohistochemical expression of biomarkers p16 and Ki-67 in high-grade cervical intraepithelial lesion: review of studies

O câncer do colo do útero é o quarto tumor mais comum entre mulheres no mundo e o terceiro no Brasil. A detecção precoce e a identificação das lesões cervicais são essenciais no rastreamento do câncer cervical. Nos últimos anos, vários marcadores têm sido apresentados como possíveis candidatos para a triagem eficiente de exames citológicos com anormalidades das células epiteliais. O objetivo deste trabalho é analisar a correlação com a expressão imuno-histoquimica dos biomarcadores p16 e Ki-67 com lesão intraepitelial cervical de alto grau na detecção molecular DNA/HPV de alto risco. A metodologia de pesquisa empregada é uma revisão sistemática, realizada por meio de buscas nas bases de dados eletrônica Literatura Latino-Americana em Ciência e Saúde (LILACS), Scientific Electronic Library Online (SCIELO), National Library of Medicine (MEDLINE) de artigos publicados no período de 2005 a 2019 nos idiomas português, inglês e espanhol. Concluiu-se que o uso das proteínas p16 e Ki67 auxilia na identificação das mudanças que acontecem durante a progressão da lesão cervical, aprimorando os métodos de rastreio atuais. O gene p53, a pRb e ciclinas também têm um papel crítico na carcinogênese e, desta maneira, também têm sido indicados para entrar nos painéis de estudo.

Cervical cancer is the fourth most common tumor among women in the world and the third in Brazil. Early detection and identification of cervical lesions are essential in screening for cervical cancer. In recent years, several markers have been presented as possible candidates for efficient screening of cytological exams with abnormalities of epithelial cells. The objective of this work is to analyze the correlation with the immunohistochemical expression of the biomarkers p16 and ki-67 with high-grade cervical intraepithelial lesion in high-risk DNA / HPV molecular detection. The research methodology employed is a systematic review, carried out by searching the electronic databases Latin American Literature in Science and Health (LILACS), Scientific Electronic Library Online (SCIELO), National Library of Medicine (MEDLINE) of published articles from 2005 to 2019 in Portuguese, English and Spanish. It was concluded that the use of proteins p16 and Ki67, help to identify the changes that happen during the progression of the cervical lesion, improving the current screening methods. The p53 gene, the retinoblastoma protein ­ pRb and cyclins also plays a critical role in carcinogenesis and thus, they have also been indicated to enter the study panels.

Metilación de genes supresores tumorales en pacientes venezolanos con cáncer colorrectal: relación con estadio clínico de la enfermedad / Relationship of Methylation of Tumor Suppressor Genes with Clinical Stage of Colorectal Cancer in Venezuelan Patients

Resumen El cáncer colorrectal es una enfermedad heterogénea, en cuya aparición se involucran factores hereditarios y ambientales. En las formas heredadas existen genes responsables de incrementar el desarrollo tumoral en los portadores, y se consideran a los factores medioambientales como responsables de gran parte de las formas esporádicas. El objetivo de este estudio fue analizar el estado de metilación de 5 genes implicados en la carcinogénesis colorrectal y su relación con los distintos estadios clínicos de estos tumores. Por una parte, nuestro análisis reveló que el estado de metilación de los promotores de los genes HMLH1 (human mut homologue 1), APC (adenomatous poliposis coli), P15, P16 y CDH1, considerados como unas de las alteraciones más tempranas en este proceso; fluctuaron entre 13,3 % para hMLH1 y 56,6 % para APC. También reveló que la inactivación epigenética de los genes APC y P16 podrían ser responsables de la aparición y de la progresión de los tumores ya que se encontraron en pacientes con estadio II. Por otra parte, los genes APC y p15 resultaron estar mutados en todas las etapas de la carcinogénesis, por lo que se involucrarían en todos los procesos tanto de inicio como de invasión y metástasis. Por último, nuestros resultados apoyan la utilización de la identificación de la metilación de los genes supresores ya que se están identificando dianas epigenéticas para el desarrollo de nuevos tratamientos de quimioterapia y está emergiendo como una estrategia con gran potencial dado que, en principio, las alteraciones epigenéticas son potencialmente reversibles.

Abstract Colorectal cancer is a heterogeneous disease which involves hereditary and environmental factors. The inherited forms have genes which are responsible for increasing the tumor development in carriers. Environmental factors are considered responsible for many sporadic forms. The objective of this study was to analyze the methylation status of five genes involved in colorectal carcinogenesis and their relationships with the various clinical stages of these tumors. Our analysis revealed that the methylation status of the promoters of genes HMLH1, APC, P15, P16 and CDH1, considered to be among the earliest alterations in this process, ranged from 13.3% for HMLH1 to 56.6% for APC. In addition, epigenetic inactivation of APC and P16 genes could be responsible for the appearance and progression of tumors since inactivation was found in stage II patients. On the other hand, the APC and p15 gene were mutated in all stages of carcinogenesis, so they could be involved throughout the processes of initiation, invasion and metastasis. Finally, our results support using identification of methylation of suppressor genes since they identify epigenetic targets for development of new chemotherapy treatments. This is emerging as a strategy with great potential since epigenetic alterations are, in principle, potentially reversible.

Evaluation of p16ink4a expression and presence of HPV-16 DNA by real time PCR in patients with oral leukoplakia / Avaliação da expressão da p16Ink4a e a presença do DNA do HPV-16 pela Real time PCR em pacientes com leucoplasia bucal

Objetivo: Avaliar a expressão do p16Ink4a por imunohistoquímica e a presença do HPV16 pela Real time PCR em tecido fresco, plasma e saliva de pacientes com leucoplasia bucal (LB) e hiperplasia fibrosa inflamatória (HFI). Material e métodos: Foram incluídos 67 pacientes com o diagnóstico de LB e 44 pacientes com diagnóstico de HFI no estudo. Foram coletados dados sociodemográficos, clinicopatológicos, amostras de tecido fresco, sangue e saliva, que foram armazenados em um freezer a -80o C para realização de análise molecular. Também foi utilizado o tecido parafinizado para realização da imunohistoquímica para a p16Ink4a. As amostras de materiais biológicos obtidos dos pacientes foram submetidas à detecção do DNA do HPV-16 pela Real time PCR. O tecido parafinizado dos mesmos pacientes foram utilizados para avaliar a expressão da p16Ink4a pela imunohistoquímica. Resultados: Dos 67 pacientes incluídos de LB no estudo, 55,2% eram do sexo masculino com uma média de idade de 57,1 anos. Os pacientes idosos (>45 anos) compuseram 86,6% da amostra. As localizações anatômicas mais acometidas por LB foram a mucosa jugal (35,8%) e língua (20,9%). Sobre o tabagismo, 71,8% dos pacientes eram fumantes, onde a maioria (47,8%) foi classificada como tabagista leve. Em relação ao consumo de álcool, 47,4% eram alcoolistas, sendo a sua maioria classificada como alcoolista leve (59,3%). O grupo HFI foi composto por 54,5% pacientes do sexo masculino com uma média de idade de 57,3 anos. 90,9% dos pacientes eram idosos (>45 anos). A ocorrência das lesões foram em mucosa jugal (31,8%) e rebordo alveolar (25%). A expressão para p16Ink4a na LB foi fraca (41,8%) e para o grupo HFI, majoritariamente a expressão foi forte (68,2%). Real time PCR não detectou HPV-16 em nenhumas das amostras analisadas. Conclusão: Os achados em nosso estudo mostraram que a detecção de HPV de alto risco em tecido fresco, plasma e saliva para LB e HFI não se correlacionaram com a superexpressão da p16Ink4a(AU)

Objective: To evaluate the expression of p16Ink4a by immunohistochemistry and the presence of HPV-16 by Real time PCR in fresh tissue, blood plasma, and saliva of patients with oral leukoplakia (OL) and inflammatory fibrous hyperplasia (IFH). Material and methods: Sixty-seven patients with the diagnosis of OL and 44 patients with the diagnosis of IFH were included in the study. Sociodemographic, clinicopathological, fresh tissue, blood, and saliva samples were collected and stored at - 80o C for molecular analysis. Paraffinized-embedded tissue was also used to perform the immunohistochemistry for p16Ink4a. Samples of biological material obtained from the patients were submitted to HPV-16 DNA detection by real time PCR, both with specific probes. Paraffinized-embedded tissue from the same patients were used to evaluate the expression of p16ink4a by immunohistochemistry. Results: Out of the 67 included OL patients, 55.2% were male with a mean age of 57.1 years. Elderly patients (> 45 years) comprised 86.6% of the sample. The prevalence of lesions was highest in the buccal mucosa (35.8%) followed by the mobile tongue (20.9%). 71.8% were smokers and the majority (47.8%) was classified as light smoker. Regarding alcohol consumption, 47.4% were alcoholics, most of whom were classified as light alcoholics (59.3%). The IFH group was composed of 54.5% of male patients with a mean age of 57.3 years. 90.9% of the sample were elderly patients (> 45 years). The highest occurrence of the lesions was in buccal mucosa (31.8%) and alveolar ridge (25%). The expression for p16Ink4a in LB was mostly weak (41.8%) and for the IFH group, the expression was mostly strong (68.2%). There was no positivity in any of the samples for HPV-16 by real time PCR. Conclusion: Our findings showed the HR-HPV detection in fresh tissue, plasma blood and saliva for OL and IFH does not correlate with p16Ink4a immunoexpression in paraffinized-embedded tissue(AU)

Knockout of p16INK4a promotes aggregative growth of dermal papilla cells

Summary Objective: Dermal papilla cells (DPCs) are located in the hair follicles and play an important role in hair growth. These cells have the ability to induce hair follicle formation when they display aggregative behavior. DPCs derived from the androgenetic alopecia (AGA) area undergo premature senescence in vitro, associated with p16INK4a expression. The aim of the current study was to investigate the expression of p16INK4a in aggregative and non-aggregative DPCs and the effect of p16INK4a down-regulation in these cells by adenovirus-mediated RNA interference (RNAi). Method: DPCs were isolated and cultured from healthy human scalp. p16INK4a gene and protein were detected in aggregative and non-aggregative cells. Expression of p16INK4a in DPCs was silenced by infection with rAd5-CDKN1A-1p2shRNA. Cell fate was monitored after infection. The growth of cells was measured by MTT assay. Cell cycle was evaluated by flow cytometry (FCM). Results: DPCs were isolated by digestion and showed aggregative behavior for six passages. The expression of p16INK4a showed a clear upward trend in non-aggregative cells when compared with aggregative group. p16INK4a expression was silenced by rAd5-CDKN1A-1p2shRNA (p<0.05). The p16INK4a-silenced cells grew more rapidly and exhibited a trend towards aggregative growth. There was an increase in the proportion of cells in G1 phase, while those in S phase were reduced after p16INK4a gene silencing (p<0.05). Conclusion: Our results suggest that p16INK4a plays an important role in the premature senescence and aggregative behavior of DPCs. These observations can lead to novel therapeutic strategies for treatment of AGA.

Genetic Basis of Gastric Cancer / 中国医学科学杂志(英文版)

Gastric cancer is the result of multiple risk factors, including environmental factors, genetic factors and the interaction between them. The environmental factors mainly include dietary, Helicobacter pylori infection and family history of gastric cancer. Genetic factors mainly refer to the susceptible genes that cause epigenetic alterations in oncogenes, tumor suppress genes, cell cycle regulators, DNA repair genes and signaling molecules. This paper summarizes the susceptible genes of gastric cancer and explores the genetic basis of it.

p16 Hypermethylation and KRAS Mutation Are Independent Predictors of Cetuximab Plus FOLFIRI Chemotherapy in Patients with Metastatic Colorectal Cancer / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Hypermethylation of the CpG island of p16(INK4a) occurs in a significant proportion of colorectal cancer (CRC). We aimed to investigate its predictive role in CRC patients treated with 5-fluorouracil, leucovorin, irinotecan (FOLFIRI), and cetuximab. MATERIALS AND METHODS: Pyrosequencing was used to identify KRAS mutation and hypermethylation of 6 CpG island loci (p16, p14, MINT1, MINT2, MINT31, and hMLH1) in DNA extracted from formalin-fixed paraffin-embedded specimens. Logistic regression and Cox regression were performed for analysis of the relation between methylation status of CpG island methylator phenotype (CIMP) markers including p16 and clinical outcome. RESULTS: Hypermethylation of the p16 gene was detected in 14 of 49 patients (28.6%) and showed significant association with KRAS mutation (Fisher exact, p=0.01) and CIMP positivity (Fisher exact, p=0.002). Patients with p16-unmethylated tumors had significantly longer time to progression (TTP; median, 9.0 months vs. 3.5 months; log-rank, p=0.001) and overall survival (median, 44.9 months vs. 16.4 months; log-rank, p=0.008) than those with p16-methylated tumors. Patients with both KRAS and p16 aberrancy (n=6) had markedly shortened TTP (median, 2.8 months) compared to those with either KRAS or p16 aberrancy (n=11; median, 8.6 months; p=0.021) or those with neither (n=32; median, 9.0 months; p < 0.0001). In multivariate analysis, KRAS mutation and p16 methylation showed independent association with shorter TTP (KRAS mutation: hazard ratio [HR], 3.21; p=0.017; p16 methylation: HR, 2.97; p=0.027). CONCLUSION: Hypermethylation of p16 was predictive of clinical outcome in metastatic CRC patients treated with cetuximab and FOLFIRI, irrespective of KRAS mutation.

Correlation between methylation and expression level of P15 and P16 genes during differentiation of cord blood stem cells into erythroid lineage mediated by erythropoietin

Several influential factors such as transcription factors and intracellular signaling components are involved in differentiation of stem cells into a specific lineage. P15 and p16 proteins are among these factors. Accumulating evidences has introduced the epigenetic as a master regulator of these factors during lineage specification. The main objective of this study is to determine the correlation between the expression level and methylation pattern of P15 and P16 genes in erythroid lineage after in vitro differentiation by erythropoietin [EPO]. The purified and expanded CD34+ cord blood stem cells were differentiated into erythroid lineage in the presence of EPO. DNA was isolated from both cord blood stem cells and differentiated cells. The Real-Time PCR performed using cDNA and the isolated DNA was used in methylation Specific PCR [MSP] reaction for methylation pattern analysis in both pre and post differentiation stages. The study demonstrated that P15 and P16 genes have partial methylation after erythroid differentiation by EPO. The Expression of P15 gene was higher after differentiation and the expression of P16 gene had a slightly decreased level in post differentiation stage. Significant increase in P15 gene expression after differentiation to erythroid lineage, suggests the remarkable efficacy of this gene in erythroid function. According to upregulation of P15 gene after differentiation despite unchanged methylation status and slight down regulation of P16 gene with slight hyper-methylation of the gene it can be suggested that although the methylation can affects the expression level of P16 gene, the P15 gene is not affected by this mechanism during erythroid differentiation mediated by EPO

Detection of p16 by fluorescence in-situ hybridization and immunohistochemistry in malignant mesothelioma / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the role of p16 gene mutation status as detected by fluorescence in-situ hybridization (FISH) and p16 protein expression as detected by immunohistochemistry in differential diagnosis of malignant mesothelioma and benign mesothelial hyperplasia.</p><p><b>METHODS</b>p16 gene mutation status and protein expression were detected by FISH and immunohistochemistry respectively in 55 cases of pleural malignant mesothelioma and 30 cases of benign mesothelial hyperplasia.</p><p><b>RESULTS</b>FISH study showed that the rate of p16 deletion in malignant mesothelioma (81.8%,45/55) was higher than that in benign mesothelial hyperplasia (3.3%,1/30). The difference was statistically significant (P<0.05). Immunohistochemical study showed that the rate of p16 protein expression in malignant mesothelioma (23.6%) was lower than that in benign mesothelial hyperplasia (76.7%). The difference was also statistically significant. The sensitivity and specificity of FISH in distinguishing between mesothelioma and reactive mesothelial hyperplasia were higher than those of immunohistochemistry.</p><p><b>CONCLUSIONS</b>In contrast to reactive mesothelial hyperplasia, p16 gene is deleted and p16 protein is not expressed in malignant mesothelioma. The sensitivity and specificity of FISH are higher than those of immunohistochemistry in the distinction.</p>

Clinical features and expressions of p16, p53 protein of human papillomavirus-related tonsillar carcinoma / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To analyze the clinical characteristics, prognosis and molecular biological changes of tonsillar squamous cell carcinoma (TSCC).</p><p><b>METHODS</b>Retrospective analysis of 61 TSCC cases treated from January 1999 to December 2012. Demographic data and clinical charts, including histologic grade of tumor, treatment and outcome of the patients, were reviewed.Human papillomavirus (HPV)-DNA were detected using SPF10-DNA enzyme immunoassay and LiPA genotyping method. Expressions of p16 and p53 proteins were examinated by immunohistochemistry. Survival rate was calculated with SPSS 19.0 software using the Kaplan-Meier method.</p><p><b>RESULTS</b>There were 55 males and 6 females, with a median age of 57 years. Of the 61 TSCC, 21 were with well differentiation, 19 with moderate differentiation and 21 with poor differentiation, including 7 patients at stage II, 10 at stage III and 44 at stage IV. HPV-positive rate of TSCC was 29.5% (18/61) and high-risk HPV-16 subtype accounted for 72.2% (13/18). The percentage of famel patients in HPV-positive TSCC was higher than HPV-negative TSCC (22.2% vs 4.7%).HPV-positive TSCC was more common in non-smoking patients (50.0% vs 79.1%, χ(2) = 5, 155, P = 0.023) and non-drinking patients (27.8% vs 51.2%, χ(2) = 4.346, P = 0.037). HPV-positive TSCC mostly presented with high expression of p16 protein (88.9% vs 16.3%, χ(2) = 28.481, P = 0.000), and low expression of p53 protein (72.7% vs 46.5%, χ(2) = 5.028, P = 0.025). The prognosis of patients with HPV-associated TSCC was significantly better than non-HPV-associated TSCC, and The 3-year and 5-year overall survival rates of patients with HPV-positive TSCC were higher than those of patients with HPV-negative TSCC (87.7% vs 49.5% and 78.9% vs 33.0%, respectively).</p><p><b>CONCLUSION</b>HPV-associated TSCC had unique clinicopathological and molecular biological features, showing better prognosis compared to HPV-negative TSCC.</p>

Promoter hypermethylation patterns of P16, DAPK and MGMT in Oral Squamous Cell Carcinoma: A systematic review and meta-analysis.

Background: Oral squamous cell carcinoma (OSCC) is a common cancer world‑wide that is highly lethal due to its recurrence and metastasis. Methylation is a common epigenetic mechanism that leads to gene silencing in tumors and could be a useful biomarker in OSCC. The prevalence of P16, death‑associated protein kinase (DAPK) and O6‑methylguanine‑DNA‑methyltransferase (MGMT) promoter hypermethylation in OSCC has been evaluated for several years while the results remain controversial. Objective: The aim of this systematic review is to critically analyze and perform a meta‑analysis on the various studies in the literature that have reported the promoter hypermethylation of P16, DAPK and MGMT genes in OSCC. Search Strategy: Articles were searched and selected through PubMed. Hand search from the relevant journals was also performed. Articles were reviewed and analyzed. Results: The estimated prevalence of P16 methylation was 43%, DAPK methylation was 39.7% and MGMT methylation was 39.8%. Heterogeneity in methylation prevalences and correlations with the clinical outcomes of the disease prevailed in various studies. Conclusion: We can conclude from our systematic review that a higher prevalence of methylation of P16, DAPK and MGMT occur in OSCC. Further studies are required to substantiate the role of methylation of P16, DAPK and MGMT as a marker in OSCC.

Diagnostic value of p16 methylation for malignant pleural effusion a meta-analysis / Valor diagnóstico de la metilación p16 en el derrame pleural maligno un meta-análisis

OBJECTIVE: To evaluate the overall diagnostic performance of the p16 methylation for diagnosing malignant pleural effusion (MPE). METHODS: All published literature in English and Chinese were reviewed. Sensitivity, specificity, likelihood ratio and diagnostic odds ratio (DOR) were pooled by using random-effects model or fixed-effects model. Summary receiver operating characteristic (SROC) curve was used to evaluate the overall diagnostic value. RESULTS: Six studies were included with a total of 378 cases. The sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR) and DOR of p16 methylation in the diagnosis of MPE were 0.41 [95% confidence interval (CI) 0.35, 0.48], 0.97 [95% CI 0.93, 0.99], 9.57 [95% CI 4.53, 20.20], 0.61 [95% CI 0.45, 0.82] and 19.82 [95% CI 8.35, 47.04], respectively. The area under the curve (AUC) was 0.864. CONCLUSION: Pleural p16 methylation test plays a useful role in the diagnosis of MPE.

OBJETIVO: Evaluar el rendimiento diagnóstico general de la metilación p16 para el diagnóstico del derrame pleural maligno (DPM). MÉTODOS: Se revisó toda la literatura publicada en inglés y chino. La sensibilidad, especificidad, razón de verosimilitud, y el odds-ratio diagnóstico (DOR) fueron agrupados mediante el modelo de efectos aleatorios o el modelo de efectos. La curva de las características operativas de resumen del receptor (SROC) fue usada para evaluar el valor diagnóstico general. RESULTADOS: Se incluyeron seis estudios con un total de 378 casos. La sensibilidad, especificidad, razón de verosimilitud positiva (PLR), razón de verosimilitud negativa (NLR) y el DOR de la metilación p16 en el diagnóstico de DPM, fueron 0.41 [95% intervalo de confianza (IC) 0.35 0.48], 0.97 [95% IC 0.93, 0.99], 9.57 [95% IC 4.53, 20.20], [95% IC 0.45, 0.82] 0.61 y 19.82 [95% IC 8.35, 47.04], respectivamente. El área bajo la curva (AUC) fue 0.864. CONCLUSIÓN: La prueba de metilación p16 pleural desempeña un papel útil en el diagnóstico del DPM.

p16 gene CpG methylation in cervical squamous cell carcinoma and HPV16 infection in Uyghur women / 中南大学学报(医学版)

OBJECTIVE@#To determine the correlation between p16 gene CpG methylation sites in the promoter region and HPV16 infection in cervical squamous cell carcinoma in Xinjiang Uyghur women.@*METHODS@#MALDI-TOF MS was used quantitatively to analyze p16 gene promotor methylation status of CpG islands in 20 cervix squamous cell carcinomas and 20 corresponding non-cancerous tissues in Uyghur women. HPV16 infection was detected by polymerase chain reaction (PCR) in both groups.@*RESULTS@#Among the 16 CpG sites in the p16 gene promoter region, CpG1-2 and CpG 6 sites were different between the 2 groups, and the levels of CpG1-2 and CpG6 methylation sites in the cervical squamous cell carcinoma were higher than those in the control group. The presence of HPV16 infection was significantly different between the cervix squamous cell carcinoma tissue and non-cancerous tissues (P<0.05). There was no significant correlation between p16 gene CpG methylation sites and HPV16 infection of cervical squamous cell carcinoma in Uyghur women.@*CONCLUSION@#P16 gene CpG 1-2, CpG 6 hypermethylation and HPV16, which are independent of one another, play an important role in cervical squamous cell carcinogenesis in Uyghur women.

Disappearance of Serum Methylated p16 Indicates Longer Survival in Patients with Gastric Cancer

PURPOSE: The aim of this study was to assess clinical correlations with postoperative alteration of p16 DNA methylation, and to clarify whether postoperative changes in the serum DNA methylation status of p16 could be used as a reliable prognostic factor for gastric cancer. MATERIALS AND METHODS: Fifty-three consecutive gastric adenocarcinoma patients who underwent gastric resection (Chung-Ang University Hospital, Seoul, Korea) were included. DNA methylation of p16 was evaluated by methylation-specific polymerase chain reaction using serum DNA preoperatively and at the 10th postoperative day. The correlation between changes in methylation status and patients' prognosis was analyzed. RESULTS: p16 was methylated in 79.2% of preoperative serum DNA and in 54.7% of postoperative serum DNA, respectively. Methylation in p16 disappeared more frequently in patients who underwent standard D2 lymphadenectomy compared to those who underwent modified D1+ lymphadenectomy (P=0.016). Whereas methylation of preoperative serum DNA was not correlated with survival, patients with postoperative disappearance of p16 methylation showed longer survival than those without postoperative disappearance of p16 methylation in the patients who had gastric cancer with lymph node metastasis (P=0.042). CONCLUSIONS: Postoperative disappearance of p16 methylation could be an available prognostic factor for node-positive gastric cancer.

Primary Squamous Cell Carcinoma of the Upper Genital Tract: Utility of p16INK4a Expression and HPV DNA Status in its Differential Diagnosis from Extended Cervical Squamous Cell Carcinoma

BACKGROUND: Primary squamous cell carcinoma (SCC) of the upper genital tract, including the endometrium, fallopian tubes, and ovaries, is extremely rare. It must be distinguished from the mucosal extension of primary cervical SCC because determination of the primary tumor site is important for tumor staging. However, patients with SCC of the fallopian tubes or ovarian surface have often undergone prior hysterectomy with inadequate examination of the cervix, making it difficult to determine the primary site. METHODS: We compared histologic findings, p16INK4a expression, and human papillomavirus (HPV) DNA status in four patients with primary SCC of the upper genital tract and five patients with primary cervical SCC extending to the mucosa of the upper genital tract. RESULTS: All five SCCs of cervical origin showed strong expression of p16INK4a, whereas all four SCCs of the upper genital tract were negative, although one showed weak focal staining. Three of the five cervical SCCs were positive for HPV16 DNA, whereas all four primary SCCs of the upper genital tract were negative for HPV DNA. CONCLUSIONS: Although a thorough histological examination is important, immunonegativity for p16INK4a and negative for HPV DNA may be useful adjuncts in determining primary SCCs of the upper genital tract.

Comparison of clinical implications of p16 deletion in childhood and adult B-lineage acute lymphoblastic leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate and compare the clinical implications of p16 deletion in childhood and adult B-lineage acute lymphoblastic leukemia (B-ALL).</p><p><b>METHODS</b>A total of 129 cases of de novo childhood (73 cases) and adult (56 cases) B-ALL were examined genetically and immunologically using G-banding techniqhe, interphase fluorescence in situ hybridization (I-FISH) and immunophenotyping by flow cytometry, and their clinical data were retrospectively analyzed.</p><p><b>RESULTS</b>Of 73 childhood cases, the prevalences of homozygous deletion, hemizygous deletion and no deletion of p16 were 24.7% (18 cases), 6.8% (5 cases) and 68.5% (50 cases) respectively, and of 56 adult cases, the incidences as of 14.3% (8 cases), 8.9% (5 cases) and 76.8% (43 cases) respectively. The incidence of p16 deletion between the two groups had no significant difference (P = 0.338). In both groups, patients with or without p16 deletion had no significant difference in terms of white blood cells (WBC) count at diagnosis, BM blast percentage, chromosome karyotype, extra-infiltration and CR1 rate. Of note, there were 2 cases, each in childhood and adult, showed no deletion at the time of diagnosis, their p16 deletions occurred at relapse. The deletion of p16 was associated with poor overall survival and event-free survival (EFS) in both childhood and adults. According to the standard of NCI risk stratification, we divided patients of two groups into standard and high risk category respectively, and performed further analysis. The significance of different risk category in children and adults was disparity. The overall survival (OS) rates of deletion and no deletion of p16 were 45.3% and 79.8% (P = 0.006) in children, and 7.7% and 22.6% (P = 0.002) in adults, respectively. EFS rates of deletion and no deletion of p16 were 33.5% and 58.1% (P = 0.008) in children, and 0 and 10.9% (P < 0.01) in adults, respectively. Of the standard risk category in children, OS rates of deletion and no deletion of p16 were 46.8% and 89.3% (P = 0.015) respectively, and EFS rates of deletion and no deletion of p16 as of 40.9% and 82.1% (P = 0.007) respectively. Of the high risk category in children, OS rates of deletion and no deletion of p16 were 41.7% and 67.4% (P = 0.193) respectively, and EFS rates of deletion and no deletion of p16 were 25.0% and 25.6% (P = 0.305) respectively. Of the standard risk category in adults, OS rates of deletion and no deletion of p16 were 20.0% and 46.9% (P = 0.092) respectively, and EFS rates of deletion and no deletion of p16 were 0 and 25.0% (P = 0.062) respectively. Of the high risk category in adults, OS rates of deletion and no deletion of p16 were 0 and 12.4% (P < 0.001) respectively, and EFS rate of deletion and no deletion of p16 was 0 and 4.8%(P < 0.001), respectively.</p><p><b>CONCLUSION</b>This study indicated that deletion of p16 was associated with poor prognosis in both childhood and adult B-ALL, which highlighted an important significance to define the status of p16 in both childhood and adult B-ALL for predicting prognosis and guiding clinical intervention.</p>

Hepatitis B virus X promotes HepG2 cell cycle progression and growth via downregulation expression of p16 protein / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effects and related mechanisms of hepatitis B virus X (HBx) protein on cell cycle and growth in hepatocellular carcinoma.</p><p><b>METHODS</b>A human hepatocyte HepG2 cell line stably expressing a green fluorescent protein (GFP)-tagged HBx (HepG2/GFP-HBx cells) was used for the experiment, and HepG2 parental and HepG2/GFP cells was used as the controls. Effect of HBx on cell growth was evaluated by the MTT cell proliferation assay and on cell cycle progression by flow cytometry analysis of cells with or without treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR; 5 pmol/L). Effect of HBx expression on promoter methylation status of the p16INK4A tumor-suppressor gene was detected by methylation-specific polymerase chain reaction and on p16 protein level was analyzed with western blotting.</p><p><b>RESULTS</b>The HepG2/GFP-HBx cells showed significantly higher cell proliferation at 72 hrs of culture (3.225+/-0.038 A490) than either control (HepG2: 2.012+/-0.022 A490, t = -46.86, P less than 0.001; HepG2/GFP: 2.038+/-0.029 A490, t = 42.51, P less than 0.001). The HepG2/GFP-HBx cells also showed significantly lower proportion of cells in the G0/G1 phase (16.45%+/-0.45%) than either control (HepG2: 44.81%+/-1.36%, t = -34.202, P less than 0.001; HepG2/GFP: 42.76%+/-1.58%, t = -28.88, P less than 0.001). However, 5-Aza-CdR treatment did lead to a significant amount of HepG2/GFP-HBx cells being arrested in the G0/G1 phase (33.25%+/-0.79%, t = 31.85, P less than 0.001). The p16INK4A promoter was methylated in the HepG2/GFP-HBx cells, and became demethylation after treatment with 5-Aza-CdR. However, no methylation of p16INK4A promoter was observed in both HepG2 and HepG2/GFP cells. The p16 protein level was significantly lower in the HepG2/GFP-HBx (vs. HepG2 and HepG2/GFP cells) and this level increased after treatment with 5-Aza-CdR.</p><p><b>CONCLUSION</b>HBx protein promotes hepatocellular carcinoma cell cycle progression and growth by shortening the G0/G1 phase, and the underlying mechanism may involve inducing p16INK4A promoter methylation and downregulating p16 protein expression.</p>

Expression of multiple tumor suppressor gene p16 and its relationship with prognosis of gastric cancer patients / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the relationship between p16 expression and cell proliferation and prognosis in gastric cancer patients.</p><p><b>METHODS</b>Gastric cancer cell lines SGC-7901, MKN45, MKN28, human embryonic kidney cell line HEK293, human fibroblast cell line MRC-5, and surgical specimens of gastric carcinoma and adjacent normal gastric mucosa from 65 patients were included in this study. RT-PCR, MTT and FCM assays were used to detect p16 expression in gastric cancer cell lines and surgical specimens of gastric cancer. MTT assay was used to determine cancer cell viability and FCM to detect cell cycle. Kaplan-Meier survival curve and Log-Rank statistics were used to analyze the relationship between p16 expression and survival of petients with gastric cancer.</p><p><b>RESULTS</b>Gastric cancer cell lines were mostly negative for p16 expression, and p16 was re-expressed after the cells transfected with p16 gene by adenovirus AdCMV-p16. p16 re-expression resulted in the decrease of cancer cell viability and cancer cell cycle arrest with increased G(1) phase and decreased S phase. p16 expression in cancer specimens was 32.3% (21/65), significantly lower than the 81.5% (53/65) in normal mucosa (χ(2) = 32.124, P < 0.001). The disease-free survival was significantly shorter in p16-negative patients than that in p16-positive patients (P < 0.01), but not the overall survival (P > 0.05). p16 expression was significantly correlated with differentiation and lymph node metastasis, but not significantly correlated with sex, age, tumor size or invasion depth of the gastric cancer.</p><p><b>CONCLUSIONS</b>p16 gene is important for cancer cell proliferation. The inactivation gives cancer cells a high activity for proliferation and metastasis, and then influences the disease-free survival of gastric cancer patients.</p>

Primary Endometrial Squamous Cell Carcinoma: A Case Report and Review of Relevant Literature on Korean Women

Primary endometrial squamous cell carcinoma (PESCC) is an extremely rare tumor with unclear pathogenesis. A 54-year-old postmenopausal woman presented with a 6-month history of vaginal bleeding. The patient was provisionally diagnosed with uterine submucosal leiomyoma. This was followed by total hysterectomy with a bilateral salpingo-oophorectomy under the laparoscopic guidance. Histopathologically, the tumor was PESCC which was accompanied by a lack of the tumor in the uterine cervix. The tumor showed positive immunoreactivity for p16INK4a. But there was no evidence of human papillomavirus (HPV) on in situ hybridization and HPV DNA chip analysis. We also present a review of the relevant literature on Korean women.

DNA methylation status of RARβ2 and p16(INK4α) in peripheral blood and tumor tissue in patients with esophageal squamous cell carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the methylation status of retinoic acid receptor β2 (RARβ2) and p16(INK4α) genes in peripheral blood and tumor tissues and the perioperative dynamic changes of free RARβ2 and p16(INK4α) in the peripheral blood, and to investigate the relationship between RARβ2 and p16(INK4α) methylation in peripheral blood and clinicopathological characteristics of esophageal squamous cell carcinoma (ESCC) and their value in evaluating the completeness of surgical resection.</p><p><b>METHODS</b>Real-time methylation specific polymerase chain reaction (real-time MSP) technique was used to detect the methylation status of RARβ2 and p16(INK4α) in tumor tissue, adjacent normal tissue and peripheral blood perioperatively in 76 cases of ESCC. Sixty age-matched healthy volunteers were randomly selected as a control.</p><p><b>RESULTS</b>RARβ2 and p16(INK4α) hypermethylation presented in both tumor tissue [72.4% (55/76) and 86.8% (66/76)] and peripheral blood [63.2% (48/76) and 71.1% (54/76)] in the ESCC patients, showing a good agreement between them. RARβ2 and p16(INK4α) hypermethylation was significantly related with pathological stage, lymph node metastasis, and invasion of nerves and vessels (P < 0.05). The DNA methylation rate in peripheral blood was increasing first and then decreasing in the preoperative, intraoperative and postoperative periods. Moreover, the RARβ2 methylation in peripheral blood was shown to be significantly associated with family history of cancer (P = 0.023).</p><p><b>CONCLUSION</b>RARβ2 and p16(INK4α) methylation in the peripheral blood in ESCC patients may reflect the tumor-bearing status in the body, and may serve as a valuable marker in assessment of the degree of completeness of surgical resection in ESCC patients.</p>