Two distinct pathways of p16 gene inactivation in gallbladder cancer

AIM： TO examine the mechanism of inactivation of the p16 gene in gallbladder cancer, and to investigate p16 alterations and their correlation with clinicopathological features.
METHODS： Specimens were collected surgically from 51 patients with gallbladder cancer. We evaluated the status of protein expression, loss of heterozygosity（LOH）, homozygous deletion and promoter hypermethylation using immunohistochemistry, microsatellite analysis, quantitative real-time polymerase chain reaction （PCR） and methylation-specific PCR, respectively. In addition, mutations were examined by direct DNA sequencing.
RESULTS： Homozygous deletions of the p16 gene exon2, LOH at 9p21-22, p16 promoter hypermethylation, and loss of p16 protein expression were detected in 26.0% （13/50）, 56.9% （29/51）, 72.5% （37/51） and 62.7% （32/51）, respectively. No mutations were found. LOH at 9p21 correlated with the loss of p16 protein expression （P 〈 0.05）. Homozygous deletion of the p16 gene, a combination LOH and promoter hypermethylation, and multiple LOH at 9p21 were significantly correlated with the loss of p16 protein expression （P 〈 0.05）. LOH at 9p21 and promoter hypermethylation of the p16 gene were detected in 15.4% （2/13） and 92.3% （12/13） of the tumors with homozygous deletion of the p16 gene, respectively. P16 alterations were not associated with clinicopathological features.
CONCLUSION： Our results suggest that LOH and homozygous deletion may be two distinct pathways in the inactivation of the p16 gene. Homozygous deletion, a combination of LOH and promoter hypermethylation, and multiple LOH are major mechanisms of p16 inactivation in gallbladder cancer.     

Aberrant p16 methylation, a possible epigenetic risk factor in familial esophageal squamous cell carcinoma

AIM: Detection of methylation in the p16 gene, an inhibitor of cyclin D-dependent protein kinase, as a new tumor marker for early detection of esophageal squamous cell carcinoma (ESCC) in DNA derived from blood and serum. METHOD: A large family with clustering of ESCC was assessed in Khorasan province in northeastern Iran. The family had three histologically proven cases of ESCC in two consecutive generations and several other deceased cases with histories of ESCC. DNA from blood of 28 living family members in three consecutive generations, 30 sporadic ESCC cases (from serum, blood, and tumor tissues), and 30 healthy volunteers (from blood) were examined for the methylation status of p16 promoter using methylation-specific PCR (MSP). RESULTS: Aberrant p16 promoter methylation was found in 64.3% (n = 28) of ESCC family members and none (n = 30) of our normal volunteers. Five of the 28 family members with esophageal cancer symptoms had negative endoscopy results for ESCC, while four of these members had p16 hypermethylation in their blood. The family members with negative endoscopy and positive p16 promoter methylation are being monitored closely for signs of ESCC development through regular check-ups and chromoendoscopies. In sporadic ESCC in northeastern Iran, 73.3% (n = 30) of tumor tissue samples had p16 hypermethylation. Serum and blood samples from the same patients showed p16 hypermethylation in 26.6% and 43.3% of the samples, respectively. CONCLUSION: Aberrant p16 methylation may be a valuable diagnostic tool as a tumor marker for the early identification of individuals in high risk ESCC families.     

p16 promoter hypermethylation： A useful serum marker for early detection of gastric cancer

AIM： TO determine p15 promoter hypermethylation in gastric tumoral tissue and serum samples, its impact on p16-protein expression, and correlation with clinical and histological features. METHODS： Samples were obtained from 52 histologically confirmed cases of gastric adenocarcinoma. Gastric tissue and serum of 50 age- and sex-matched individuals with normal gastroscopy and biopsy were obtained as control samples. Methylation-specific polymerase chain reaction （MSP） was used to evaluate methylation status of p16 promoter, p16-protein expression was analyzed by immunohistochemical staining on paraffin-embedded sections. RESULTS： Methylation was detected in 44.2% （23/52） of tumoral tissues. 60.9% of them were also methylated in serum, i.e., 26.9% of all patients （14/52）. Methylation was not detected in tissue and sera of control samples. p16-protein expression was decreased in 61.5% of cases （32/52）, and was significantly associated with promoter hypermethylation （P 〈 0.001）. Methylation was significantly more frequent in higher pathological grades （P 〈 0.05）. Methylation was not associated with other clinicopathological features and environmental factors including Hpylori infection and smoking. CONCLUSION： p16 promoter hypermethylation is an important event in gastric carcinogenesis. It is the principle mechanism of p16 gene silencing. It is related to malignant tumor behavior. Detection of DNA methylation in serum may be a biomarker for early detection of gastric cancer.     

p16基因启动子区甲基化在人嗜铬细胞瘤和副神经节瘤中的变化

目的 检测嗜铬细胞瘤(PHEO)和副神经节瘤(PGL)中p16基因突变和启动子区DNA甲基化改变,分析其与患者临床特征之间的关系.方法收集34例(PHEO 20例、PGL14例)组织标本及患者临床资料,通过甲基化特异性PCR(MSP)测定p16基因启动子区甲基化状态,DNA测序检测基因序列以及RT-PCR方法测定其mRNA表达.结果 (1)34例肿瘤组织中未发现p16基因纯合缺失及点突变;(2)35.3%(12/34)的患者存在p16基因高甲基化,p16基因甲基化阳性标本中,PHEO和PGL分别占25%和75%,两者差异有统计学意义(P=0.005);p16基因甲基化在恶性、单发肿瘤、发病年龄早的亚组中有增高趋势(P＞0.05);(3)甲基化与非甲基化肿瘤组织之间p16 mRNA表达无统计学差异;不同特点的肿瘤中其mRNA表达亦无统计学差异,但恶性肿瘤p16 mRNA表达与良性肿瘤相比有下降的趋势(0.83±0.65对1.12±0.81,P=0.278).结论人PHEO和PGL中,p16基因纯合缺失和突变并不常见,但p16基因启动子区甲基化是其失活的主要形式.     

Aberrant p16 methylation, a possible epigenetic risk factor in familial esophageal squamous cell carcinoma

Aim. Detection of methylation in the p16 gene, an inhibitor of cyclin D-dependent protein kinase, as a new tumor marker for early detection of esophageal squamous cell carcinoma (ESCC) in DNA derived from blood and serum. Method. A large family with clustering of ESCC was assessed in Khorasan province in northeastern Iran. The family had three histologically proven cases of ESCC in two consecutive generations and several other deceased cases with histories of ESCC. DNA from blood of 28 living family members in three consecutive generations, 30 sporadic ESCC cases (from serum, blood, and tumor tissues), and 30 healthy volunteers (from blood) were examined for the methylation status of p16 promoter using methylation-specific PCR (MSP). Results. Aberrant p16 promoter methylation was found in 64.3% (n=28) of ESCC family members and none (n=30) of our normal volunteers. Five of the 28 family members with esophageal cancer symptoms had negative endoscopy results for ESCC, while four of these members had p16 hypermethylation in their blood. The family members with negative endoscopy and positive p16 promoter methylation are being monitored closely for signs of ESCC development through regular check-ups and chromoendoscopies. In sporadic ESCC in northeastern Iran, 73.3% (n=30) of tumor tissue samples had p16 hypermethylation. Serum and blood samples from the same patients showed p16 hypermethylation in 26.6% and 43.3% of the samples, respectively. Conclusion. Aberrant p16 methylation may be a valuable diagnostic tool as a tumor marker for the early identification of individuals in high risk ESCC families.     

Detection of hypermethylation of the p16(INK4A) gene promoter in chronic hepatitis and cirrhosis associated with hepatitis B or C virus

BACKGROUND/AIM: Inactivation of the p16(INK4A) (p16) tumour suppressor gene by promoter region hypermethylation has been demonstrated not only in many types of tumours, including hepatocellular carcinoma (HCC), but also in early preneoplastic lesions in the lung, colon, oesophagus, and pancreas. The aim of this study was to examine the methylation status of the p16 promoter in pre- and/or non-neoplastic liver diseases. PATIENTS/SUBJECTS/METHODS: The methylation status of p16 was evaluated in 22 HCC, 17 cirrhosis, 17 chronic hepatitis, nine primary biliary cirrhosis (PBC), eight autoimmune hepatitis, seven drug induced liver disease, six fatty liver, and three normal liver tissues using methylation specific polymerase chain reaction (MSP). p16 protein expression was also examined by immunohistochemical staining. RESULTS: Methylation of the p16 promoter was detected in HCC (72.7%, 16/22) and also in cirrhosis (29.4%, 5/17) and chronic hepatitis (23.5%, 4/17), all of which were positive for hepatitis B or C virus infections. Methylation was not detected in any of the other samples. All methylation positive HCC, cirrhosis, and chronic hepatitis samples showed loss of p16 expression, and a significant correlation was found between methylation and loss of expression. Analysis of serial samples from individual patients with methylation positive HCC revealed that loss of p16 expression with promoter methylation occurred in 18 of 20 patients at the stage of chronic hepatitis without clinically detectable carcinoma. CONCLUSIONS: Our results suggest that methylation of the p16 promoter and the resulting loss of p16 protein expression are early events in a subset of hepatocarcinogenesis and that their detection is useful in the follow up of patients with a high risk of developing HCC, such as those with hepatitis B or C viral infections.     

食管鳞癌组织p16基因调控区甲基化及其蛋白表达研究

目的探讨p16基因在食管癌变过程中表达缺失与其启动子区甲基化的关系。方法采用MSP免疫组化方法,检测环太行山地区45例食管鳞癌患者癌组织p16基因启动子区甲基化状态及蛋白表达情况。结果p16基因在癌组织中表达异常41例（91．1％）,间变组织中表达异常38例（84．4％）,发生纯合型甲基化的组织分别为33例（73．3％）（癌组织）和32例（71．1％）（间变组织）,而其周围正常组织26例（57．8％）均发生了p16启动子区的杂合型甲基化。p16基因纯合型甲基化与癌组织、间变组织、p16蛋白表达缺失相关（P〈0．05）。结论该地区食管癌组织p16基因在癌前病变中p16启动子区即发生了纯合型甲基化、食管癌变的早期事件。p16基因启动子区甲基化可单独影响p16蛋白的正常表达。     

原发胃癌中p16基因及其甲基化状态、表达异常的研究

目的：检测胃癌组织中p16基因及启动了甲基化状态和p16蛋白表达情况。方法：选择p16基因及启动子区域,用PCR－SSCP、MSP（甲基化特异的PCR）法、测序和免疫组化等方法对100例胃癌患者的癌组织和癌旁组织进行检测。结果：71％的病例p16表达阴性,54％的病例具有p16基因启动子区的高甲基化,50％的病例同时有p16表达阴性和p16基因启动子区的高甲基化,无突变和纯合缺失检出。结论：提示p16基因启动子区域高甲基化是胃癌中p16基因失活的主要原因。     

p16 inactivation by methylation of the CDKN2A promoter occurs early during neoplastic progression in Barrett's esophagus

BACKGROUND & AIMS: The potential role of p16 inactivation by CDKN2A/p16 promoter hypermethylation and/or loss of heterozygosity (LOH) of the CDKN2A gene was investigated in neoplastic progression of Barrett's esophagus. METHODS: CDKN2A promoter hypermethylation was studied by methylation sensitive single-strand conformation analysis and sequencing using bisulfite modified DNA in Barrett's esophageal adenocarcinomas, premalignant lesions, and normal squamous esophageal epithelium. All of the lesions of interest were sampled by microdissection from paraffin-embedded fixed tissue sections. RESULTS: No methylation of the CDKN2A promoter was found in normal esophageal squamous cell epithelia, whereas methylation was detected in 18 of 22 (82%) adenocarcinomas and 10 of 33 (30%) premalignant lesions, including 4 of 12 (33%) samples with intestinal metaplasia only. LOH at the CDKN2A gene locus was found in 68% of adenocarcinomas and in 55% of premalignant lesions. Of 28 samples without p16 immunoreactivity, 25 (89%) showed CDKN2A promoter hypermethylation with or without LOH of CDKN2A. Only 2 (8%) samples expressing p16 protein were found to be methylated; these showed a mixture of completely methylated and unmethylated CDKN2A promoters. In 7 of 19 (37%) informative samples without LOH of CDKN2A, the CDKN2A promoter was found to be methylated at both alleles. Loss of p16 protein expression was strongly associated with CDKN2A promoter hypermethylation (P < 0.00001), but not with LOH (P = 0.33). CONCLUSIONS: Our results indicate that methylation of the CDKN2A promoter is the predominant mechanism for p16 inactivation. This hypermethylation is a very common event in esophageal adenocarcinoma and occurs as early as metaplasia.     

p16 gene methylation in colorectal cancers associated with Duke's staging

AIM To explore the association of methylation of the CpGisland in the promotor of the p16 tumor suppressor genewith the clinicopathological characteristics of thecolorectal cancers.METHODS Methylation-specific PCR(MSP)was used todetect p16 methylation of 62 sporadic colorectal cancerspecimens.RESULTS p16 methylation was detected in 42% of thetumors.Dukes' staging was associated with p16methylation status,p16 methylation occurred morefrequently in Dukes' C and D patients(75.9%)than inDukes' A and B patients(12.1%).CONCLUSION p16 methylation plays a role in thecarcinogenesis of a subset of colorectal cancer,and itmight be linked to poor prognosis.     

Abnormalities of tumor suppressor gene <Emphasis Type="Italic">p16</Emphasis>in pancreatic carcinoma: immunohistochemical and genetic findings compared with clinicopathological parameters

Background. Abnormalities of the tumor suppressor gene p16 have been reported in a variety of human tumors but are rare in pancreatic carcinoma except for cancer cell lines and xenografts. Their clinicopathological significance remains unknown. The purpose of this study was to examine immunohistochemical and genetic alterations of p16 in primary pancreatic carcinoma tissues and to investigate the relation between abnormalities of p16 and clinicopathological parameters to elucidate their clinicopathological significance. Methods. We investigated p16 expression in 60 pancreatic carcinoma cases by immunohistochemistry using a monoclonal antibody clone G175-405. In addition, we analyzed genetic alterations of the p16 gene using DNA extracted from microdissected tissue of pancreatic carcinoma, by polymerase chain reaction, nonradioisotopic single-strand conformation polymorphism (non-RI-SSCP), DNA sequencing, and hypermethylation analyses using restriction enzymes. We compared the abnormalities of p16 alterations with clinicopathological parameters to elucidate their significance. Results. On immunohistochemical study, staining for p16 protein was strongly positive in 22 (37%) of 60 pancreatic carcinoma cases, weakly positive in 24 (40%), and negative in 14 (23%). In contrast, p16 mutations were recognized in 9 (15%) of the 60 pancreatic carcinoma cases. The incidence of p16 mutations was 2 (9%) in 22 cases of pancreatic carcinoma with strongly positive staining, 4 (17%) in 24 with weakly positive staining, and 3 (21%) in 14 with negative staining. Hypermethylation of p16 was detected in the two pancreatic carcinoma cases with weakly positive staining, although homozygous deletions were not found in any case. There was no significant correlation between the expression of p16 protein and any of the clinicopathological parameters. However, there was a tendency for the tumor to be larger in patients with decreased expression of p16 protein than in those with normal expression levels. In contrast, the tumor was significantly larger and the survival period significantly shorter for patients with pancreatic carcinoma with p16 mutation or hypermethylation than for those with pancreatic carcinoma with an intact p16 gene (P 0.05). Conclusions. These findings suggest that p16 alterations may participate in the aggressiveness of pancreatic carcinoma.     

Possible down regulation of the p16 gene promoter in individuals with hepatocellular carcinoma

Background

The p16 tumor suppressor gene is an important negative regulator of the cell cycle. Inactivation of p16, especially via promoter hypermethylation, has been found in numerous human cancers such as breast, lung, colorectal, and liver.

Objectives

To determine the role of epigenetic methylation in p16 regulation in Iranian patients with hepatocellular carcinoma (HCC).

Patients and Methods

The methylation pattern in the p16 gene promoter was analyzed by bisulfite direct sequencing in 43 paraffin-embedded formalin-fixed tissues from patients with HCC. In addition, normal specimens from liver graft donors were used as the control group.

Results

The bisulfite direct sequencing showed heterozygous hypermethylation in 13.9% of individuals with HCC. Homozygous methylation within the GC-box IV was detected in another 58.1% of the patients.

Conclusions

It is proposed that methylation, but not necessarily hypermethylation, may play a role in the down-regulation of the p16 gene promoter at least in some Iranian patients with HCC.     

p16INK4A gene alterations are not a prognostic indicator for survival in patients with hepatocellular carcinoma undergoing curative hepatectomy

BACKGROUND AND AIM: Hepatocellular carcinoma (HCC) is a common malignancy worldwide that is highly associated with chronic hepatitis B or C infection and cirrhosis. The tumor suppressor gene p16INK4A is an important component of the cell cycle and inactivation of the gene has been found in a variety of human cancers. The present study was performed to determine genetic and epigenetic alterations in the p16INK4A tumor suppressor gene and the effect of these on HCC progression. METHODS: The status of p16INK4A was evaluated in 117 HCC tumoral nodules and 110 corresponding peritumoral tissues by loss of heterozigosity (LOH) at the 9p21-22 region, homozygous deletions, single-strand conformation polymorphism-polymerase chain reaction (PCR) mutational analysis and methylation specific PCR. RESULTS: The most frequent inactivation mechanism was hypermethylation of the promoter region, which was found in 63.2% of the tumor samples and in 28.2% of the peritumoral samples. Loss of heterozygosity at the 9p21 region was detected in 27.3% and 10% of tumor and peritumoral tissues, respectively. Homozygous deletions and mutations were less common events in hepatocarcinogenesis. The authors found 5.9% of the tumor cases with exon 2 homozygous deletions and 8.6% with mutations. Two polymorphisms were detected, one at codon 148 (GCG --> ACG, Ala --> Thr) in three cases and the other in exon 3 at 540 bp (34.2% of the samples). No association was found between inactivation of p16INK4A and clinicopathological characteristics or prognosis. CONCLUSION: p16INK4A is altered frequently and early in HCC, being the predominant mechanism of inactivation promoter hypermethylation. The present results suggest that the p16INK4A gene plays an important role in the pathogenesis of HCC.     

老年肺癌病人痰标本中p16基因异常甲基化的研究

目的分析老年肺癌病人痰标本中p16基因启动子区域异常高甲基化的改变情况,评价该指标作为肺癌辅助诊断分子标记物的价值。方法运用半巢式甲基化特异性聚合酶链反应技术,检测94例老年原发性肺癌病人痰标本和部分对应肿瘤组织,以及10例慢性肺炎病例痰标本中p16基因启动子区域的甲基化改变。结果74%的肺癌病人痰标本中检测到了p16基因异常高甲基化,与传统细胞学(46.8%)相比,痰标本中p16基因异常高甲基化对肿瘤的检出率(74.5%)灵敏度更高(P<0.01);痰细胞中p16甲基化检测和细胞学相结合,对肿瘤的检出率可达86.17%(81/94)。如果痰标本中p16基因启动子区域发生高甲基化,其对应的肿瘤组织中p16基因亦为高甲基化。10例慢性肺炎病人痰标本中仅3例检测出p16基因甲基化。结论痰标本中p16基因甲基化是临床肺癌辅助诊断的有效分子生物标记物之一。     

Aberrant promoter CpG island hypermethylation of the adenomatosis polyposis coli gene can serve as a good prognostic factor by affecting lymph node metastasis in squamous cell carcinoma of the esophagus

There has been no clear evidence demonstrating whether DNA hypermethylation can affect the prognosis of esophageal cancer. We collected tissue from 50 cases of squamous cell carcinoma of the esophagus and tested them for DNA hypermethylation using methylation-specific polymerase chain reaction. CpG island hypermethylations were observed in 10% for p16, 34% for RARβP2, 46% for adenomatosis polyposis coli (APC), 14% for RASSF1A, 84% for FHIT, and 8% for hMLH1. APC promoter hypermethylation was frequently found in patients without lymph node metastasis compared with those with lymph node metastasis (62.5% : 30.8%, P  = 0.025). The number of metastatic lymph nodes were lower in patients with APC promoter hypermethylation (0.87 ± 0.30 : 3.07 ± 0.72, P  = 0.008). Excluding operative mortalities and incomplete resections, 42 patients were analyzed for long-term outcome. During the mean follow-up period of 35 months, 17 developed recurrence and 14 died of cancer. Ten patients died of other causes. In univariable analysis, unmethylation of APC ( P  = 0.0015) and FHIT ( P  = 0.0044), as well as presence of lymph node metastasis ( P  = 0.0038), were risk factors for recurrence. In multivariable analysis, lymph nodes metastasis ( P  = 0.050) and unmethylation of APC promoter ( P  = 0.023) remained as significant risk factors. In conclusion, promoter hypermethylation of the APC gene is related to a lower number of metastatic lymph nodes and to superior prognosis in terms of recurrence, which suggests it might be involved in the process of lymph node metastasis in esophageal cancer.     

Hypermethylation of the CDKN2/p16 promoter during neoplastic progression in Barrett's esophagus

Background & Aims: Inactivation of the CDKN2/p16^INK4A tumor-suppressor gene is one of the most frequent genetic alterations in human malignancies. In esophageal adenocarcinomas, mutations of the p16 gene or homozygous deletions of the gene locus 9p21 are rare. This study investigated whether p16 promoter hypermethylation is an alternative mechanism for p16 gene inactivation during neoplastic progression in Barrett's esophagus. Methods: A methylation-specific polymerase chain reaction protocol was applied. A total of 95 specimens from 14 patients with Barrett's esophagus were analyzed longitudinally. The p16 promoter status was compared with histomorphological findings. Results: p16 promoter hypermethylation was detected in 9 of the 10 patients who had displayed dysplasia at some time during surveillance, whereas none of the patients who had not displayed dysplasia during surveillance had p16 promoter hypermethylation. p16 promoter hypermethylation was detected in 3% (2 of 67) of the samples without dysplasia, 60% (3 of 5) of the samples with lesions indefinite for dysplasia, 55.6% (10 of 18) of the specimens with low-grade dysplasia, and 75% (3 of 4) of the specimens with high-grade dysplasia. Conclusions: These data suggest that p16 promoter hypermethylation is a common mechanism of p16 gene inactivation during neoplastic progression in Barrett's esophagus.GASTROENTEROLOGY 1998;115:1381-1386