The 9p21 region in bladder cancer cell lines: large homozygous deletions inactivate the CDKN2, CDKN2B and MTAP genes

Homozygous and hemizygous deletions of 9p21 are the earliest and most common genetic alteration in bladder cancer. The identification of two cell cycle regulators, CDKN2 and CDKN2B, that map to the common region of deletion has prompted the hypothesis that they are critical tumor suppressor genes in this malignancy. However, controversy as to whether these genes are the only or even the most important target in bladder cancer oncogenesis remains. To more clearly determine the effect of these 9p21 alterations, we mapped the homozygous deletions and performed a detailed mutational and expression analysis for CDKN2, CDKN2B and a closely linked gene, methylthioadenoside phosphorylase (MTAP), in 16 established bladder cancer cell lines. Nine of the 16 lines exhibit large (30 to >2000 kb) homozygous deletions on 9p21. All deletions include at least one exon of CDKN2, eight of nine include CDKN2B, and six of nine include MTAP. MTAP function correlates with the genomic deletions. SSCP and sequence analysis does not reveal any inactivating point mutations of CDKN2 or of CDKN2B in any of the cell lines without homozygous deletions, and all express the CDKN2 and the CDKN2B mRNA as well as the encoded p16 protein. The p16 protein levels vary widely and are correlated with absent pRb expression. We conclude that the 9p21 deletions in bladder cancer usually inactivate the CDKN2, CDKN2B, and MTAP genes but that CDKN2 is the most common target. Other mechanisms for inactivating this gene in bladder cancer appear to be uncommon.     

SH3GL1与p-ERK在原发性肝癌中的表达与其对索拉非尼治疗效果的影响

目的 研究SH3GL1和p-ERK在原发性肝癌组织中的表达以及二者对服用索拉非尼患者预后的影响。方法 收集我院自2008年1月至2016年12月期间因原发性肝癌进行手术治疗,并且术后坚持口服索拉非尼的患者组织标本共73例,免疫组化分析患者组织中SH3GL1和p-ERK的表达情况,统计所有患者性别、年龄、术前HBs Ag表达情况、术前AFP值、术前肝功能分级、体力状况评分、肿瘤分化程度、是否有脉管侵犯、是否有肝外转移病灶、肿瘤数量、肿瘤分期在内的多项临床指标,分析SH3GL1和p-ERK在HCC中的表达情况与这些指标的联系及其对患者预后的影响。结果 SH3GL1和p-ERK在原发性肝癌患者组织中阳性表达率高,并且二者的表达水平呈正相关关系(P0.001);SH3GL1的高表达与肿瘤有肝外转移、肿瘤多发和肿瘤分期相关,p-ERK的高表达则与患者肿瘤分期相关;影响肝癌术后服用索拉非尼患者总生存期的危险因素包括年龄低于50岁、SH3GL1高表达,p-ERK高表达、有肝外转移灶、肿瘤多发,其中SH3GL1高表达、有肝外转移和肿瘤多发为影响总预后的独立危险因素。结论 SH3GL1和p-ERK在原发性肝癌中的表达对于肝癌多项临床指标相关,并且二者的高表达是影响服用索拉非尼患者总生存期的危险因素。     

Immunohistochemical Expression of p16INK4A, Ki-67, and Mcm2 Proteins in Gastrointestinal Stromal Tumors: Prognostic Implications and Correlations with Risk Stratification of NIH Consensus Criteria

Background Inactivation of p16^INK4A promotes G1/S progression of cell cycle. Minichromosome maintenance protein-2 (Mcm2), a novel cell proliferation marker, is known to better correlate with clinical outcomes than Ki-67 in many carcinomas. Since gastrointestinal stromal tumors (GISTs) sometimes remains challenging in prognostication, we analyzed the utility of these three markers in GISTs.Methods Immunohistochemistry was performed in tissue microarrays of 277 primary GISTs and correlated with NIH consensus criteria and clinical outcomes.Results The increment of NIH risk levels significantly correlated with increasing labeling indices (LI) of both Ki-67 (P <.001) and Mcm2 (P <.001) and loss of p16^INK4A expression (P <.035). However, the latter aberration did occur in 23% of very low/low-risk GISTs. The relationship between Mcm2 and Ki-67 LIs could be modeled as linear (P <.001, r = 0.697), while Mcm2 LI was considerably higher (P <.001) with a stepwise escalation related to risk levels. Ki-67 LI >5% (P <.0001) and Mcm2 LI >10% (P <.0001) were strongly predictive of inferior disease-specific survival (DSS), while aberrant loss of p16^INK4A only reached a trend (P = .0954). In multivariate analyses, independent adverse factors of DSS were high-risk category (RR = 16.93, P <.0001), metastatic disease (RR = 4.12, P = .0015), Ki-67 LI >5% (RR = 3.55, P = .001), and presence of epithelioid histology (RR = 2.17, P = .0308).Conclusions Prognostic efficacy of NIH consensus criteria is substantiated. P16^INK4A deregulation can occur early in GIST tumorigenesis and marginally correlates with patient survival. Despite Ki-67 LI being an independent prognosticator, simultaneous detection of Mcm2 is recommended as a prognostic adjunct of GISTs, given its better sensitivity and stepwise escalation with increasing risk levels.     

Molecular and Immunohistochemical Cognizance of HPV16 in Oral Leukoplakia,Oral Squamous Cell Carcinoma and Oropharyngeal Squamous Cell Carcinoma

Prior studies have established the carcinogenic role of HPV16 and also demonstrated its unique biological behavior in cervical and oropharyngeal squamous cell carcinoma (OPSCC) but its role in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) is not well explored. Therefore, in the present study, we assessed HPV16 prevalence using PCR and Anti-HPV16 antibodies for the first time and correlated its biological behavior using p16^INK4a and Ki67 proliferation index (PI) in OL, OSCC, and OPSCC. This study included 63 subjects comprising of 25 OL, 26 OSCC, and 12 OPSCC cases. Exfoliated cells were collected and processed for PCR followed by immunohistochemistry with primary antibodies p16^INK4a, Anti-HPV16, and Ki67. The expressions were evaluated and statistical analysis included Chi-square and Spearman’s test. Cumulatively 37% (OL—7%, OSCC—14% & OPSCC—16%) of cases showed positive PCR expression. PCR positivity was observed to be significantly higher (p 0.00) in OPSCC (9/12) than OSCC (9/26) and OL (5/25) cases. Overall immunohistochemical expression of p16^INK4a, Anti-HPV16, and Ki67 were significantly (p 0.02) higher in HPV16 (PCR) positive cases. HPV16 + OSCC cases showed higher grades of p16^INK4a and Ki67 expression. We have demonstrated a prevalence of HPV16 in OL, OSCC, and OPSCC through PCR, which may be concluded as a gold standard for the detection of HPV16 DNA.     

Differential Expression of p16INK4A and Cyclin D1 in Benign and Malignant Salivary Gland Tumors: A Study of 44 Cases

Salivary gland tumors (SGT) are a heterogeneous group of lesions. There is conflicting data concerning the molecular events involving the tumour suppressor retinoblastoma protein (pRb) pathway in these tumors. Few studies examined the alterations in components of the Rb pathway by immunohistochemical (IHC) methods in benign and malignant SGTs. Furthermore, recent evidence implicates human papillomavirus (HPV) in mucoepidermoid carcinoma (MEC) carcinogenesis. The purpose of our study is to examine p16^INK4A and cyclin D1 expression in a variety of benign and malignant salivary gland tumors, and to investigate p16^INK4A expression as a surrogate marker for HPV infection in MEC. Our series includes 30 malignant tumors [14 MEC, 6 acinic cell carcinomas (ACC), 5 polymorphous low grade adenocarcinomas (PLGA), 5 (AdCC)] and 14 benign tumors (4 benign cysts, 5 Warthin tumors and 5 pleomorphic adenomas (PA). All cases were tested by IHC for p16^INK4A and cyclin D1. Testing for HPV wide spectrum (HPV-WS) was performed by in situ hybridization in all MEC cases. Staining intensity was recorded semi quantitatively (on a scale from 0 to 4+). Fisher’s exact test and Pearson X² test with a p < 0.05 were used. Cyclin D1 and p16^INK4A are expressed similarly in malignant and benign tumors (p = 0.146 and p = 0.543, respectively). None of the MEC cases showed nuclear reactivity for HPV-WS. Statistical analysis showed positive correlation between cyclin D1 and p16^INK4A expression. Our findings suggest that p16^INK4A overexpression is likely secondary to cyclin D1 gene upregulation or amplification. Further molecular studies are warranted.     

Absence of p16 gene (CDKN2) deletions in microdissected primary breast carcinoma specimens

Background: The p16 gene (CDKN2), a tumor suppressor gene located on chromosome 9p21, has been demonstrated to be mutated or deleted with high frequency in a variety of tumor cell lines, including breast. While previous studies have not demonstratedCDKN2 mutations in primary breast carcinomas, it is possible that gene deletion in neoplastic DNA was masked by the presence of contaminating normal stromal DNA in breast carcinoma specimens. Methods: We investigated the incidence of homozygous deletion ofCDKN2 by analyzing 20 microdissected pure populations of primary breast carcinoma cells. Using polymerase chain reaction (PCR) techniques, the entire coding region and intervening introns ofCDKN2 were amplified. The PCR products were resolved by agarose gel electrophoresis and single-strand conformation polymorphism (SSCP) analysis. Results: We detected no deletions or mutations of the p16 gene. Conclusions: CDKN2 is not deleted with high frequency in primary breast carcinomas, and the p16 gene does not play a role in breast carcinogenesis via this mechanism. Presented at the 49th Annual Cancer Symposium of The Society of Surgical Oncology, Atlanta, Georgia, March 21–24, 1996.     

Genetic and Epigenetic Changes in Mammary Epithelial Cells May Mimic Early Events in Carcinogenesis

Studies of human mammary epithelial cells from healthy individuals are providing novel insights into how early epigenetic and genetic events affect genomic integrity and fuel carcinogenesis. Key epigenetic changes, such as the hypermethylation of the p16 (INK4a) promoter sequences, create a previously unappreciated preclonal phase of tumorigenesis in which a subpopulation of mammary epithelial cells are positioned for progression to malignancy (Romanov et al. , 2001, Nature , 409:633-637; Tlsty et al. , 2001, J. Mammary Gland Biol. Neoplasia , 6:235-243). These key changes precede the clonal outgrowth of premalignant lesions and occur frequently in healthy, disease-free women. Understanding more about these early events should provide novel molecular candidates for prevention and therapy of breast cancer that target the process instead of the consequences of genomic instability. This review will highlight some of the key alterations that have been studied in human mammary epithelial cells in culture and relate them to events observed in vivo and discussed in accompanying reviews in this volume.