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     

胃癌形成过程中p16IN4a、Runx3和O-6-甲基鸟嘌呤-DNA甲基转移酶基因甲基化及其表达研究

目的 研究胃癌形成过程中p16INK4a、Runx3和O-6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)基因启动子区的高甲基化状态,同时检测MGMT的蛋白表达情况.探讨抑癌基因启动子区高甲基化与胃癌发生的关系.方法 选择经透明帽法进行首次黏膜病变切除者43例,其中异型增生27例,早期胃癌16例.选择胃镜活检证实为慢性萎缩性胃炎伴肠上皮化生者14例.另取20例正常胃黏膜活检组织作为对照.采用甲基化特异聚合酶链反应(MSP)检测每例组织中p16INK4a、Runx3和MGMT基因启动子区的甲基化状态,对所有甲基化p16INK4a产物进行测序,免疫组化检测MGMT蛋白表达情况.结果 肠上皮化生、异型增生和早期胃癌中p16INK4a基因甲基化率依次为14.3%(2/14)、22.2%(6/27)和37.5%(6/16);Runx3基因甲基化率依次为14.3%(2/14)、48.1%(13/27)和50.0%(8/16);MGMT基因甲基化率依次为7.1%(1/14)、48.1%(13/27)和50.0%(8/16).20名正常对照均未检出基因甲基化,与异型增生和早期胃癌相比差异有统计学意义(P＜0.05).Runx3和MGMT两种基因在异型增生和早期胃癌中的甲基化率显著高于肠上皮化生组(P＜0.05).各组病变中三种基因甲基化联合分析发现,异型增生和早期胃癌中甲基化的基因种类高于肠上皮化生组.差异有统计学意义(P＜0.01).基因甲基化与息者年龄、性别、幽门螺杆菌感染以及病变部位无相关性,但p16INK4a和MGMT基因甲基化与血清癌胚抗原水平升高显著相关(P值分别为0.003和0.039).MGMT基因启动子区高甲基化与其蛋白失表达密切相关(χ2=12.821,P=0.001).结论 抑癌基因启动子区高甲基化是基因失活的主要机制,可能是胃癌发生的早期分子事件.p16INK4a、Runx3和MGMT基因启动子区高甲基化在胃癌形成过程中起着重要的作用.     

Differential alterations of the genes in the CDKN2A-CCND1-CDK4-RB1 pathway are associated with the development of head and neck squamous cell carcinoma in Indian patients

Purpose The aim of this study was to analyse the alterations of the genes in the CDKN2A/CCND1/CDK4/RB1 pathway in the G1-S phase of the cell cycle during development of head and neck squamous cell carcinoma (HNSCC).Methods The alterations of these genes were analysed in 22 dysplastic lesions, 26 stage-I/II and 33 stage-III/IV HNSCC tumours of Indian patients.Results The alterations [mutation, hypermethylation, homozygous deletion and loss of heterozygosity/microsatellite size alteration (LOH/MA)] in the CDKN2A were found to be highest in 57% of the samples, followed by CCND1 amplification and LOH/MA at the RB1 locus in 14% and 8.5% of the samples, respectively. No dominant CDK4 Arg24Cys mutation was seen in our samples. Comparatively high frequency of CDKN2A alterations (except homozygous deletion) was found in dysplastic head and neck lesions and remained almost constant or increased during progression of the tumour, whereas the homozygous deletion of CDKN2A and the alterations in CCND1 and RB1 genes were seen mainly in the later stages of the tumour.Conclusions Our study suggested that mutation/hypermethylation/allelic alterations (LOH/MA) of CDKN2A were associated with the development of dysplastic head and neck lesions. All the other alterations might provide some cumulative effect during progression of later stages of the tumour to have selective growth advantages.     

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.     

Small adenocarcinomas of the esophagogastric junction: association with intestinal metaplasia and dysplasia

OBJECTIVES: Intestinal metaplasia in Barrett's esophagus predisposes to esophageal adenocarcinoma. Intestinal metaplasia of the cardia is a common finding in persons without cancer. Many adenocarcinomas of the esophagogastric junction are large enough to obliterate any underlying intestinal metaplasia. To estimate how often adenocarcinoma of the esophagogastric junction arises in intestinal metaplasia, we studied small adenocarcinomas of the esophagogastric junction. METHODS: Resection patients had adenocarcinomas 2 cm or smaller, within 2 cm of the esophagogastric junction. Age- and sex-matched controls had resection for squamous carcinoma. Saved and new histological slides from the esophagogastric junction were examined, with additional stains. RESULTS: Intestinal metaplasia was found in 86% (19/22) of adenocarcinoma cases, versus 32% (7/22) of controls (p < 0.001). Intestinal metaplasia with high or low grade dysplasia was associated with 64% (14/22) of adenocarcinomas and with 5% (1/22) of controls (p < 0.001). Excluding four cases with long and three with short Barrett's esophagus, 80% (12/15) of adenocarcinomas had associated intestinal metaplasia, 53% (8/15) with dysplasia. Most adenocarcinoma cases had the incomplete type of intestinal metaplasia with a Barrett type cytokeratin 7/20 staining pattern. Helicobacter pylori were seen in one adenocarcinoma and five control cases. CONCLUSIONS: Most adenocarcinomas of the esophagogastic junction arise in the background of intestinal metaplasia, sometimes in an endoscopically visible Barrett's esophagus, more often in small areas of intestinal metaplasia of the cardia. In cases of adenocarcinoma, the intestinal metaplasia resembled that found in Barrett's esophagus, and was not associated with H. pylori.     

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.     

Associations of risk factors obesity and occupational airborne exposures with CDKN2A/p16 aberrant DNA methylation in esophageal cancer patients

It is known that obesity and occupational airborne exposure such as dust are among risk factors of esophageal cancer development, in particular squamous cell carcinoma (SCC) of esophagus. Here, we tested whether these factors could also affect aberrant DNA methylation. DNAs from 44 fresh tumor tissues and 19 non‐tumor adjacent normal tissues, obtained from 44 patients affected by SCC of esophagus (SCCE), were studied for methylation at the CDKN2A/p16 gene promoter by methylation‐specific polymerase chain reaction assay. Statistical methods were used to assess association of promoter methylation with biopathological, clinical, and personal information data, including obesity and airborne exposures. Methylation at the CDKN2A/p16 gene promoter was detected in 12 out of 44 tumor samples. None of the non‐tumor tissues exhibited the aberrant methylation. Our results confirmed previously described significant association with low tumor stage (P= 0.002); in addition, we found that obesity (P= 0.001) and occupational exposure (P= 0.008) were both significantly associated with CDKN2A/p16 promoter methylation. This study provides evidence that obesity and occupational exposure increase the risk of developing esophageal cancer through an enhancement of CDKN2A/p16 promoter methylation.     

Hypermethylation of p16 gene promoter correlates with loss of p16 expression that results in poorer prognosis in esophageal squamous cell carcinomas

SUMMARY.  The purpose of this study was to analyze loss of p16 expression and its relationship to hypermethylation, clinicopathological parameters and prognosis in patients with esophageal squamous cell carcinoma (ESCC). Tissue samples from 60 ESCC were subjected to histological analysis. Immunohistochemical staining for p16 expression was performed. DNA was extracted from these primary esophageal tumors and from sera from another 38 ESCC patients. The DNA was modified with bisulfite and analyzed for p16 promoter methylation by methylation-specific polymerase chain reaction. Twelve out of the 60 tumors (20%) were methylated at the p16 promoter and 48 tumors (80%) were unmethylated. There were no significant correlations between the methylation of the p16 promoter and clinicopathological parameters. Immunohistochemical staining revealed that 41 of the 60 tumors (68.3%) were p16-negative and 19 tumors (31.7%) were p16-positive. The correlation between negative p16 immunohistochemical staining and methylation was statistically significant ( P =  0.0084). No instances of p16 methylation and p16 positive immunostaining were found. There was a close correlation between loss of p16 expression and poorer prognosis in ESCC ( P =  0.0517 in overall survival, P  = 0.0478 in disease-free survival). The p16 gene promoter hypermethylation was detected in the serum of two of 38 (5.2%) patients with ESCC. This indicates that p16 promoter methylation suppresses p16 expression and that the loss of expression has a close relationship with poor prognosis in patients with ESCC. The present results may lead to the development of new therapeutic strategies, such as p16 ^INK4A gene therapy, to treat patients with ESCC.     

Genetic instability and aberrant DNA methylation in chronic hepatitis and cirrhosis--A comprehensive study of loss of heterozygosity and microsatellite instability at 39 loci and DNA hypermethylation on 8 CpG islands in microdissected specimens from patients with hepatocellular carcinoma

A study was conducted to examine the significance of genetic instability and aberrant DNA methylation during hepatocarcinogenesis. Genomic DNA was extracted from 196 microdissected specimens of noncancerous liver tissue that showed no marked histologic findings or findings compatible with chronic hepatitis or cirrhosis, and 80 corresponding microdissected specimens of hepatocellular carcinoma (HCC) from 40 patients. Loss of heterozygosity (LOH) and microsatellite instability (MSI) were examined by polymerase chain reaction (PCR) using 39 microsatellite markers, and DNA methylation status on 8 CpG islands was examined by bisulfite-PCR. In noncancerous liver tissues, LOH, MSI, and DNA hypermethylation were found in 15 (38%), 6 (15%), and 33 (83%) of 40 cases, respectively. The incidence of DNA hypermethylation in histologically normal liver was similar to that in chronic hepatitis and cirrhosis, although neither LOH nor MSI was found in histologically normal liver. In cancerous tissues, LOH, MSI, and DNA hypermethylation were found in 39 (98%), 8 (20%), and 40 (100%) of 40 cases, respectively. CpG islands of the p16 gene and methylated in tumor 1, 2, 12, and 31 clones were frequently methylated in cancerous tissues, although neither the thrombospondin-1 nor the human Mut L homologue (hMLH1) gene was methylated. Absence of silencing of the hMLH1 gene by DNA hypermethylation is consistent with the low incidence of MSI in HCCs. The results of this study indicate that LOH and aberrant DNA methylation contribute to hepatocarcinogenesis; DNA hypermethylation in particular, which precedes or may even cause LOH, is as an early event during hepatocarcinogenesis.     

p16基因高甲基化在胃癌发展中的作用

目的:通过检测胃癌、癌前病变和正常对照组中p16基因启动子区CpG岛甲基化水平及其表达,并结合临床病理资料,分析他们在胃癌发生、发展中的作用.方法:用甲基化特异性聚合酶链反应(methylation-specific PCR,MSP)检测41例胃癌组织、40例癌前病变组织和38例正常对照组织中p16基因启动子5′CpG岛甲基化;应用免疫组化检测基因的蛋白表达.结果:胃癌组织中p16基因甲基化阳性率为56.1%(23/41),癌前病变组织中为17.5%(7/40),而正常对照组织中为2.6%(1/38),前组与后两组之间的差异有显著性(P<0.05).胃癌组织中p16基因表达阳性率为51.2%,癌前病变组织中为90.0%,正常对照组织中为100.0%,前组与后两组之间的差异有显著性(P<0.05).低分化型胃癌组织中的p16基因甲基化阳性率明显高于高分化型(81.3%vs 40.0%,P<0.05).有淋巴结转移的胃癌组织中,p16基因甲基化阳性率与无转移组的差异有显著性(81.0%vs 30.0%,P<0.05).浸润深达浆膜层的胃癌组织中,甲基化阳性率与未达浆膜层组无统计学差异(60.0%vs 52.4%,P>0.05).胃癌组织中p16基因甲基化阳性组的蛋白表达阳性率显著低于甲基化阴性组(26.1%vs 83.3%,P<0.01).结论:胃癌组织中存在有p16基因启动子5′CpG岛高甲基化,并导致其基因表达率显著低于正常对照及癌前病变组织.p16基因的高甲基化与胃癌分化程度、淋巴结转移相关.p16基因甲基化的发生,从正常、癌前病变到胃癌有逐渐增加的趋势,提示其基因CpG岛高甲基化有可能作为诊断早期胃癌的一项较为敏感的指标.     

Epigenetic inactivation of the CIP/KIP cell-cycle control pathway in acute leukemias

Dysregulation of the cell cycle is important in oncogenesis. We analyzed the potential inactivation of the CIP/KIP family of the cyclin E/CDK/RB pathway by gene promoter hypermethylation in leukemias. The methylation-specific polymerase chain reaction (MSP) with primers for methylated (M-MSP) and unmethylated (U-MSP) alleles of the p21, p27, and p57 genes was used to study five leukemic cell lines, 50 acute myeloid leukemia (AML) samples, and 25 acute lymphoblastic leukemia (ALL) samples. p21 was hemizygously methylated in Raji and Jurkat but remained unmethylated in U937, HL60, and NB4. p27 was hemizygously methylated in Raji but unmethylated in the other cell lines. p57 was completely methylated in Raji and NB4, hemizygously methylated in U937, and unmethylated in HL60 and Jurkat. At diagnosis, p21 methylation was not detected in any case of AML or ALL. p27 methylation occurred in 2 (4%) AML patients and in 1 (4%) ALL patient. p57 methylation occurred in 1 (2%) AML patient and in 1 (4%) ALL patient. Therefore, methylation inactivation of the INK4/CDK/RB pathway in leukemia is infrequent. A review of the literature showed a marked variation in the frequencies of methylation of these genes, which might be attributable to difference in methodologies used to detect gene methylation.     

CDX1 is an important molecular mediator of Barrett's metaplasia

The molecular pathogenesis of Barrett's metaplasia (BM) of the esophagus is poorly understood. The change to an intestinal phenotype occurs on a background of esophagitis due to refluxing acid and bile. CDX1, an important regulator of normal intestinal development, was studied as a potential key molecule in the pathogenesis of BM. CDX1 mRNA and protein were universally expressed in all samples of BM tested but not in normal esophageal squamous or gastric body epithelia. This tissue-specific expression was attributable to the methylation status of the CDX1 promoter. Conjugated bile salts and the inflammatory cytokines TNF-alpha and IL-1beta were all found to increase CDX1 mRNA expression in vitro. These effects were primarily mediated by NF-kappaB signaling but only occurred when the CDX1 promoter was unmethylated or partially methylated. The data suggest that CDX1 is a key molecule linking etiological agents of BM to the development of an intestinal phenotype. Although the initial trigger for CDX1 promoter demethylation is not yet identified, it seems likely that demethylation of its promoter may be the key to the induction and maintenance of CDX1 expression and so of the BM phenotype.     

Helicobacter pylori infection in relation to E-cadherin gene promoter polymorphism and hypermethylation in sporadic gastric carcinomas

AIM: To study Helicobacterpylori (H pylori) infection in relation to E-cadherin (E-cad) promoter polymorphism and hypermethylation in GCs. METHODS: Specimens were taken from representative cancerous lesions and adjacent non-cancerous epithelia of 67 resected GCs. H pylori was detected by real-time PCR of the cagA gene from non-neoplastic epithelium. E-cad promoter polymorphism and hypermethylation were determined by restriction fragment length polymorphism analysis and methylation-specific PCR, respectively. Expression of E-cad protein was determined by immunohistochemistry. RESULTS: H pylori was found in 57% of patients with GC. H pylori infection was more frequently found in tumors with the -160C/C genotype than those with the -160C/A and -160A/A genotypes (74% vs 47%, P= 0.02). H pylori infection was associated with E-cad methylation in non neoplastic epithelium; however, no significant difference in H pylori was observed between methylated and unmethylated cancerous lesions. CONCLUSION: Patients with the -160C/C genotype might require H pylori infection to promote the inactivation of CDH1, suggesting that H pylori infection might affect GC in an initial stage because polymorphism is germ line. Mechanism of hypermethylation of CDH1 promoter in GC is complex, and H pylori infection might affect it in an initial stage.     

5'-CpG island methylation of the LKB1/STK11 promoter and allelic loss at chromosome 19p13.3 in sporadic colorectal cancer

BACKGROUND: In patients with Peutz-Jeghers syndrome (PJS), causative germline mutations in the LKB1/STK11 gene on chromosome 19p13.3 have been identified. Because of the loss of heterozygosity (LOH) at 19p13.3 in hamartomas and the cancer susceptibility of patients with PJS, LKB1/STK11 is suggested to act as a tumour suppressor. However, the frequency of genetic and epigenetic inactivation of LKB1/STK11 in sporadic tumours is unclear. AIMS: To investigate the LKB1/STK11 gene for promoter hypermethylation and allelic loss in tumour specimens of patients with sporadic colorectal cancer. METHODS: DNA from 50 consecutive paraffin embedded sporadic colorectal adenocarcinomas and corresponding normal epithelium was extracted. After bisulphite treatment, specimens were analysed for methylation of the LKB1/STK11 promoter 5'-CpG island by methylation specific polymerase chain reaction (MSP). In addition, tumours were analysed for LOH of chromosome 19p13.3. In tumours exhibiting LOH, LKB1/STK11 was sequenced. RESULTS: MSP was successful in 48 of 50 tumour specimens. Of those, four (8%) demonstrated hypermethylation of the LKB1/STK11 promoter 5'-CpG island. Moreover, LOH at either D19S886 or D19S878 was observed in five of 38 (13%) informative tumours. All five tumours showing LOH at 19p13.3 were advanced and four of five were located in the left sided colon. There was no correlation between LOH and LKB1/STK11 promoter hypermethylation or somatic mutation. CONCLUSIONS: In sporadic colorectal cancer, hypermethylation of the LKB1/STK11 promoter and allelic loss at the STK 11 gene locus are rare events. LOH at 19p13.3 was associated with advanced tumour stage and left sided location but not with LKB1/STK11 promoter hypermethylation or somatic mutation.     

3p21, 5q21, 9p21 and 17p13.1 allelic deletions are potential markers of individuals with a high risk of developing adenocarcinoma in Barrett's epithelium without dysplasia

BACKGROUND/AIMS: A common genetic abnormality detected in Barrett's adenocarcinoma is LOH (loss of heterozygosity) at the sites of known or putative tumor suppressor genes. Thus, some deletions have also been determined in peritumoral Barrett's epithelium. These findings suggest that a tissue field of somatic genetic alterations precede the histopathological phenotypic changes of carcinoma. We investigated 32 cases of Barrett's esophagus with no evidence of dysplasia for LOH at 5q21 (APC), 3p21, 9p21 (p16) and 17p13.1 (p53) chromosomal regions. METHODOLOGY: Two groups were randomly selected and compared: 16 cases of Barrett's epithelium adjacent to adenocarcinoma and 16 cases of Barrett's epithelium with no evidence of malignant transformation in a 5-10 years follow-up period. In three adenocarcinomas cases several previous endoscopic biopsies of Barrett's esophagus were available. RESULTS: We determined frequent allelic losses in adenocarcinomas at p53 (54%), p16 (50%), 3p21 (40%) and 5q21 (33%). Identical LOH was present in most cases in the Barrett's epithelium adjacent to adenocarcinoma. LOH at these loci was unusual in Barrett's epithelium with no evidence of malignant transformation. However, in cases where sequential endoscopic biopsies were performed in advance to the adenocarcinoma diagnosis LOH was already present in the Barrett's epithelium. CONCLUSIONS: We suggest that LOH at these loci may be present before the onset of the malignant growth and LOH studies may supplement the histopathological evaluation of Barrett's epithelium. LOH at 3p21, 5q21, 9p21 and 17p13 chromosomal regions in cells of Barrett's epithelium without dysplasia may have a role as a potential marker for individuals with a high risk of developing adenocarcinoma.     

Frequency of p16(INK4A) alterations and K-ras mutations in intrahepatic cholangiocarcinoma of the liver

BACKGROUND: Inactivation of the tumour suppressor gene p16 (CDKN2/MTS-1/INK4A) and K-ras mutations are among the most frequent genetic alterations in human malignancies. AIMS: To investigate the tumour suppressor gene p16 and its possible association with K-ras mutations in intrahepatic cholangiocarcinomas of the liver. METHODS: The status of p16 was evaluated in 41 cholangiocarcinomas by methylation specific polymerase chain reaction, microsatellite analysis, DNA sequencing, and immunohistochemical staining. K-ras mutations were determined by direct DNA sequencing analyses after microdissection. The results obtained were correlated with histopathological variables and patient survival. RESULTS: Hypermethylation of the 5' CpG island of the p16 gene was found in 34 of 41 (83%) carcinomas. Homozygous deletion at the p16 region was present in two (5%), and loss of heterozygosity (LOH) in eight cases (20%). We failed to detect p16 gene missense mutations. K-ras mutations were found in 22 of 41 (54%) cholangiocarcinomas and in two cases of tumour surrounding non-neoplastic liver tissue. All 22 cancers with K-ras mutations also exhibited methylated p16. We failed to observe a correlation between K-ras or p16 status and histopathological factors or prognosis of patients. CONCLUSION: These data suggest that inactivation of the p16 gene is a frequent event in cholangiocarcinoma. The most common somatic alteration is promotor methylation of the p16 gene which is closely associated with K-ras mutations. We failed to establish p16 or K-ras status as independent prognostic factors in these tumours.     

Methylation of the p16INK4A promoter is associated with malignant behavior in abdominal extra-adrenal paragangliomas but not pheochromocytomas

Pheochromocytomas and abdominal extra-adrenal paragangliomas are related to endocrine tumors of the sympathetic nervous system. Studies in animal models have shown that inactivation of the products of the cyclin dependent kinase inhibitor 2A (CDKN2A) gene locus, p16INK4A and p14ARF, promotes the development of pheochromocytoma, especially in malignant form. The present study evaluated the involvement of CDKN2A in human pheochromocytomas and abdominal extra-adrenal paragangliomas from 55 patients. Promoter methylation was assessed using quantitative Pyrosequencing and methylation-specific PCR, and mRNA expression was measured by quantitative real-time PCR. For p16, western blot analysis and sequencing were also performed. succinate dehydrogenase complex subunit B (SDHB) sequencing analysis included extra-adrenal paragangliomas, all tumors classified as malignant, and cases diagnosed at 30 years or younger. The p16INK4A promoter was heavily methylated in a subset of paragangliomas, and this was significantly associated with malignancy (P<0.0043) and SDHB mutation (P<0.002). p16INK4A mRNA expression showed moderate suppression in malignant cases (P<0.05). In contrast, very little p14ARF promoter methylation was seen and there was no significant difference in p14ARF expression between tumors and normal samples. The p16 protein expression was reduced in 16 tumors, and sequence variations were observed in four tumors including the missense mutation A57V and the single nucleotide polymorphism (SNP) A148T. The results suggest that p16INK4A, and not p14ARF, is a subject of frequent involvement in these tumors. Importantly, hypermethylation of the p16INK4A promoter was significantly associated with malignancy and metastasis, and SDHB gene mutations. This finding suggests an etiological link and could provide a clinical utility for diagnostic purposes.     

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.