CpG island methylator phenotype in cancer

DNA hypermethylation in CpG-rich promoters is now recognized as a common feature of human neoplasia. However, the pathophysiology of hyper-methylation (why, when, where) remains obscure. Cancers can be classified according to their degree of methylation, and those cancers with high degrees of methylation (the CpG island methylator phenotype, or CIMP) represent a clinically and aetiologically distinct group that is characterized by 'epigenetic instability'. Furthermore, CIMP-associated cancers seem to have a distinct epidemiology, a distinct histology, distinct precursor lesions and distinct molecular features.     

Aberrant methylation in gastric cancer associated with the CpG island methylator phenotype.

Aberrant methylation of 5' CpG islands is thought to play an important role in the inactivation of tumor suppressor genes in cancer. In colorectal cancer, a group of tumors is characterized by a hypermethylator phenotype termed CpG island methylator phenotype (CIMP), which includes methylation of such genes as p16 and hMLH1. To study whether CIMP is present in gastric cancer, the methylation status of five newly cloned CpG islands was examined in 56 gastric cancers using bisulfite-PCR. Simultaneous methylation of three loci or more was observed in 23 (41%) of 56 cancers, which suggests that these tumors have the hypermethylator phenotype CIMP. There was a significant concordance between CIMP and the methylation of known genes including p16, and hMLH1; methylation of p16 was detected in 16 (70%) of 23 CIMP+ tumors, 1 (8%) of 12 CIMP intermediate tumors, and 1 (5%) of 21 CIMP- tumors (P<0.0001). Methylation of the hMLH1 gene was detected in three of five tumors that showed microsatellite instability, and all three of the cases were CIMP+. The CIMP phenotype is an early event in gastric cancer, being present in the normal tissue adjacent to cancer in 5 of 56 cases. These results suggest that CIMP may be one of the major pathways that contribute to tumorigenesis in gastric cancers.     

CpG island methylator phenotype (CIMP) in cancer: causes and implications

Strong evidence exists for a subgroup of tumours, from a variety of tissue types, exhibiting concordant tumour specific DNA methylation: the "CpG island methylator phenotype" (CIMP). Occurrence of CIMP is associated with a range of genetic and environmental factors, although the molecular causes are not well-understood. Both increased expression and aberrant targeting of DNA methyltransferases (DNMTs) could contribute to the occurrence of CIMP. One under-explored area is the possibility that DNA damage may induce or select for CIMP during carcinogenesis or treatment of tumours with chemotherapy. DNA damaging agents can induce DNA damage at guanine rich regions throughout the genome, including CpG islands. This DNA damage can result in stalled DNA synthesis, which will lead to localised increased DNMT1 concentration and therefore potentially increased DNA methylation at these sites. Chemotherapy can select for cells which have increased tolerance to DNA damage due to increased lesion bypass, in some cases by mechanisms which involve inactivation of genes by CpG island methylation. CIMP has been associated with worse patient prognosis, probably due to increased epigenetic plasticity. Therefore, further clinical testing of the diagnostic and prognostic value of the current CIMP markers, as well as increasing our understanding of the molecular causes underlying CIMP are required.     

CpG island methylator phenotype associates with low-degree chromosomal abnormalities in colorectal cancer

PURPOSE: Aberrant promoter methylation and genomic instability occur frequently during colorectal cancer development. CpG island methylator phenotype (CIMP) has been shown to associate with microsatellite instability, and BRAF mutation and is often found in the right-side colon. Nevertheless, the relative importance of CIMP and chromosomal instability (CIN) for tumorigenesis has yet to be thoroughly investigated in sporadic colorectal cancers. EXPERIMENTAL DESIGN: We determined CIMP in 161 primary colorectal cancers and 66 matched normal mucosae using a quantitative bisulfite/PCR/ligase detection reaction (LDR)/Universal Array assay. The validity of CIMP was confirmed in a subset of 60 primary tumors using MethyLight assay and five independent markers. In parallel, CIN was analyzed in the same study cohort using Affymetrix 50K Human Mapping arrays. RESULTS: The identified CIMP-positive cancers correlate with microsatellite instability (P = 0.075) and the BRAF mutation V600E (P = 0.00005). The array-based high-resolution analysis of chromosomal aberrations indicated that the degree of aneuploidy is spread over a wide spectrum among analyzed colorectal cancers. Whether CIN was defined by copy number variations in selected microsatellite loci (criterion 1) or considered as a continuous variable (criterion 2), CIMP-positive samples showed a strong correlation with low-degree chromosomal aberrations (P = 0.075 and P = 0.012, respectively). Similar correlations were observed when CIMP was determined by MethyLight assay (P = 0.001 and P = 0.013, respectively). CONCLUSION: CIMP-positive tumors generally possess lower chromosomal aberrations, which may only be revealed using a genome-wide approach. The significant difference in the degree of chromosomal aberrations between CIMP-positive and the remainder of samples suggests that epigenetic (CIMP) and genetic (CIN) abnormalities may arise from independent molecular mechanisms of tumor progression.     

Advances of CpG island methylaton phenotypes in gastric cancer

CpG island methylator phenotype (CIMP) refers to a set of multiple gene promoter CpG island methylation phenotype which exists in tumor at the same time. Many studies show that CIMP is ubiquitous in gastric cancer, which is related to the pathogenesis,diagnosis, patient's condition, prognosis and curative effect of gastric cancer.     

MSH6 G39E polymorphism and CpG island methylator phenotype in colon cancer

The MSH6 G39E germline polymorphism is not associated with an increased risk of either microsatellite stable or unstable sporadic colorectal cancer. Other than microsatellite instability, however, most genetic and epigenetic changes of tumors associated with this common variant have not been studied. The objective of our investigation was to evaluate associations between the MSH6 G39E (116G>A) polymorphism and CpG island methylator phenotype (CIMP) and BRAF V600E mutations in tumors from a sample of 1048 individuals with colon cancer and 1964 controls from Utah, Northern California, and Minnesota. The G39E polymorphism (rs1042821) was determined by the five prime nuclease assay. CIMP was determined by methylation‐specific polymerase chain reaction (PCR) of CpG islands in MLH1, methylated in tumors (MINT)1, MINT2, MINT31, and CDKN2A. The BRAF V600E mutation was determined by sequencing exon 15. In microsatellite stable tumors, homozygous carriers of the G39E polymorphism had an increased risk of CIMP+ colon cancer (odds ratio (OR) 2.2, 95% confidence interval (CI) 1.1, 4.2) and BRAF V600E mutation (OR 3.1, 95% CI 1.01, 9.7) in a case–control comparison. This finding was not observed in unstable tumors; however, power may have been low to detect an association. Age at diagnosis, family history, and alcohol use did not interact with MSH6 G39E and CIMP. The MSH6 G39E germline polymorphism may be associated with CIMP+ colon cancer. © 2009 Wiley‐Liss, Inc.     

胃癌CpG岛甲基化表型研究进展

CIMP是指一组在肿瘤中有同时存在的多个基因启动子CpG岛甲基化表型.许多研究证实在胃癌中CIMP是经常存在的现象,与胃癌的发病机制、诊断、病情、预后及治疗疗效等方面均有一定关系.     

Diet and lifestyle factor associations with CpG island methylator phenotype and BRAF mutations in colon cancer

It has been proposed that dietary factors such as folate, alcohol and methionine may be associated with colon cancer because of their involvement in DNA methylation processes. Data from a large population-based case-control study of incident colon cancer were used to evaluate whether intake of dietary, obesity, physical activity and nonsteroidal antiinflammatory drugs are associated with a CpG island methylator phenotype (CIMP). The BRAF V600E mutation and 5 CpG island markers (MINT1, MINT2, MINT31, p16 and hMLH1) were assessed in 1154 cases of colon cancer. We hypothesized that dietary factors involved in DNA methylation, cruciferous vegetables and use of aspirin/NSAIDs would be associated with CIMP-high tumors. Dietary folate, vitamins B(6) and B(12), methionine and alcohol were not associated with increased likelihood of colon tumors with the CIMP-high (2 or more markers methylated) phenotype. Dietary fiber, physical activity and aspirin and other nonsteroidal antiinflammatory drugs were inversely associated with both CIMP-low and CIMP-high tumors. Our results also suggested non-CIMP pathways as well. Obese individuals were at 2-fold increased risk of having a CIMP-low tumor. Alcohol was associated with an increased risk of tumors that were MSI+ and CIMP-low. In the presence of smoking 20 or more cigarettes per day, use of NSAIDs did not protect against a BRAF mutation. Our data suggest multiple pathways to colon cancer. They do not support a unique role for dietary folate, alcohol, vitamins B(6) and B(12) and methionine in a CpG island methylator phenotype.     

High CpG island methylator phenotype is associated with lymph node metastasis and prognosis in gastric cancer

Several studies have found that the promoter CpG island is frequently methylated in gastric cancer. The CpG island methylator phenotype (CIMP) defines concordant methylation of multiple promoter CpG island loci in a subset of gastric cancer. However, the relationship between CIMP and lymph node metastasis in gastric cancer is unknown. Our study aimed to characterize the role of CIMP in lymph node metastasis. Clinical specimens from 120 patients were analyzed and PCR was used to detect the methylation status of five genes (ALX4, TMEFF2, CHCHD10, IGFBP3, and NPR1). We measured the level of mRNA for the five genes by real-time RT-PCR. Microsatellite instability and Helicobacter pylori infection status were assayed by capillary electrophoresis and real-time PCR, respectively. DNA methylation in the five genes was correlated with low expression of the respective mRNA. With CIMP as the dependent variable, CIMP-high gastric cancer tended to show more distant lymph node metastasis, higher pathologic tumor classification, more pathologic metastasis, and higher pathologic TNM status. Microsatellite instability and H. pylori status were not significant predictors of prognosis. CIMP-high gastric cancer showed significantly worse survival compared with that of CIMP-low/CIMP-negative gastric cancer (P < 0.001). Our results show that there is an association between CIMP status and lymph node metastasis in gastric cancer and CIMP-high was an independent prognostic factor.     

CpG island methylator phenotype association with upregulated telomerase activity in hepatocellular carcinoma

CpG island methylator phenotype (CIMP) involves the targeting of multiple genes by promoter hypermethylation. Telomerase plays an important role in the development of cellular immortality and oncogenesis. To gain insight into the role of epigenetic aberration of telomerase-related genes in hepatocarcinogenesis, we determined a hypermethylation profile in HCC. We examined the promoter methylation status of 9 genes associated with telomerase activity in 120 HCC, 120 cirrhosis tissues and 10 normal liver tissues by methylation-specific PCR. Assay of telomerase activity was by TRAP-ELISA. The frequency of promoter methylation of each gene was P21 63.3%, P15 42.5%, P16 62.5%, P53 14.2%, RB 32.5%, P27 48.3%, WTI 54.2%, E2F-1 70.8% and P300 65.8% of 120 HCC. Methylation status of P21, P15, P16, WTI and E2F-1 was significantly associated with HCC and nontumor tissues (p < 0.05). CIMP+ was detected in 61.7% (74/120) HCC and 15% (18/120) cirrhosis tissues, no CIMP+ was present in normal liver tissues (p < 0.001). A significant difference between CIMP status and metastasis was been found in HCC (p < 0.001). Results showed that 94.6% (70/74) HCC and 55.6% (10/18) cirrhosis patients with CIMP+ show expression of high telomerase activity than 45.5% (10/22) HCC and 6.25% (1/16) cirrhosis patients with CIMP- (p < 0.001). CIMP lead to high levels of expression of telomerase activity through the simultaneous inactivation of multiple genes associated with telomerase activity by concordant methylation.     

BRAF mutation is associated with the CpG island methylator phenotype in colorectal cancer from young patients

This study investigated the relationship between BRAF mutation, the CpG island methylator phenotype (CIMP+) and APC methylation in colorectal cancer (CRC) from young patients. The V600E BRAF mutation was found in 7% of cases and was strongly associated with the tumour features of proximal site, advanced stage and poor histological grade. More than half (53%) the tumours with BRAF mutation were also CIMP+ as evaluated by a standard panel of markers, compared to only 4% of tumours with wildtype BRAF (P<0.0001). In contrast to CIMP+, APC methylation was inversely correlated with BRAF mutation (P=0.02). BRAF mutation and CIMP+ are therefore likely to be involved in an alternate, albeit rare, pathway to APC inactivation during the development of CRC in younger patients.     

Association of the CpG island methylator phenotype with family history of cancer in patients with colorectal cancer

Methylation of promoter CpG islands in colorectal cancer (CRC) falls into two categories: age related and cancer specific. Most cancer-specific methylation at CpG islands occurs in a subset of cases that display the CpG island methylator phenotype (CIMP). The underlying cause of CIMP is not known. Using methylation-specific PCR, we studied 47 CRC patients for methylation at five loci to determine whether the methylation status of CpG islands is associated with family history of cancer. Four of the loci were differentially methylated between the CRC cases with a family history and those with no family history. Patients with methylation at all four loci were 14 times more likely to have a family history of cancer than patients with methylation at none of the four loci. These findings suggest that there may be a genetic component to CIMP in CRC.     

Marked and independent prognostic significance of the CpG island methylator phenotype in neuroblastomas

The CpG island methylator phenotype (CIMP) was closely associated with poor overall survival (OS) in Japanese neuroblastoma (NBL) cases in our previous study. Here, in German NBL cases, CIMP(+) cases (n=95) showed markedly poorer OS (hazard ratio (HR)=9.5; P<0.0001) and disease-free survival (DFS) (HR=5.4; P<0.0001) than CIMP(-) cases (n=50). All the 23 cases with N-myc amplification had CIMP. Among the remaining cases without N-myc amplification, CIMP(+) cases (n=27) had a poorer OS (HR=4.5; P=0.02) and DFS (HR=5.2; P<0.0001) than CIMP(-) cases (n=95). In multivariate analysis, CIMP and N-myc amplification had an influence on OS and DFS independent of age and disease stage. CIMP had a stronger influence on DFS than N-myc amplification while N-myc had a stronger influence on OS.     

CpG island methylator phenotype association with elevated serum alpha-fetoprotein level in hepatocellular carcinoma.

PURPOSE: CpG island methylator phenotype (CIMP) involves hypermethylation targeted toward the promoters of multiple genes. To gain insight into the role of epigenetic aberration of tumor-related genes in hepatocarcinogenesis, we determined a hypermethylation profile in hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: We examined the promoter methylation status of nine genes in 50 HCCs, 50 paired nontumor tissues, and 6 normal liver tissues by methylation-specific PCR. CIMP+ was defined as having five genes that are concordantly methylated. RESULTS: The frequency of promoter methylation of nine genes in 50 HCCs varied from 10% in P53 to 94% in c-Myc. The methylation status of P14, P15, P16, ER, RASSF1A, WT1, and c-Myc was significantly correlated with HCC and nontumor tissues (P<0.05). Hypermethylation of one or more genes was found in 96% of HCC. CIMP was more frequent in HCC than in nontumor tissues (70% and 12%, P<0.001). There is a significant association between CIMP and methylation of P14, P15, P16, ER, RSAAF1A, and WT1 (P<0.05) and serum alpha-fetoprotein (AFP) level (P=0.017). CIMP+ was more frequent in HCC with AFP>or=30 microg/L than those with AFP<30 microg/L (P=0.005). In addition, the promoter hypermethylation of P15 and P16 was associated with elevated serum AFP levels in 35 HCC samples with CIMP+ (P<0.05). CONCLUSIONS: Positive correlation of CIMP and AFP levels in HCC suggests that CIMP can serve as a molecular marker of late-stage HCC development.     

Low expression of gamma-glutamyl hydrolase mRNA in primary colorectal cancer with the CpG island methylator phenotype

The CpG island methylator phenotype (CIMP+) in colorectal cancer (CRC) is defined as concomitant and frequent hypermethylation of CpG islands within gene promoter regions. We previously demonstrated that CIMP+ was associated with elevated concentrations of folate intermediates in tumour tissues. In the present study, we investigated whether CIMP+ was associated with a specific mRNA expression pattern for folate- and nucleotide-metabolising enzymes. An exploratory study was conducted on 114 CRC samples from Australia. mRNA levels for 17 genes involved in folate and nucleotide metabolism were measured by real-time RT-PCR. CIMP+ was determined by real-time methylation-specific PCR and compared to mRNA expression. Candidate genes showing association with CIMP+ were further investigated in a replication cohort of 150 CRC samples from Japan. In the exploratory study, low expression of gamma-glutamyl hydrolase (GGH) was strongly associated with CIMP+ and CIMP+-related clinicopathological and molecular features. Trends for inverse association between GGH expression and the concentration of folate intermediates were also observed. Analysis of the replication cohort confirmed that GGH expression was significantly lower in CIMP+ CRC. Promoter hypermethylation of GGH was observed in only 5.6% (1 out of 18) CIMP+ tumours and could not account for the low expression level of this gene. CIMP+ CRC is associated with low expression of GGH, suggesting involvement of the folate pathway in the development and/or progression of this phenotype. Further studies of folate metabolism in CIMP+ CRC may help to elucidate the aetiology of these tumours and to predict their response to anti-folates and 5-fluorouracil/leucovorin.     

LINE-1 hypomethylation is inversely associated with microsatellite instability and CpG island methylator phenotype in colorectal cancer

Two pathways leading to vulvar squamous cell carcinoma (SCC) exist. The expression of proliferation- and cell-cycle-related biomarkers and the presence of high-risk (hr) HPV might be helpful to distinguish the premalignancies in both pathways. Seventy-five differentiated vulvar intra-epithelial neoplasia (VIN)-lesions with adjacent SCC and 45 usual VIN-lesions (32 solitary and 13 with adjacent SCC) were selected, and tested for hr-HPV DNA, using a broad-spectrum HPV detection/genotyping assay (SPF(10)-LiPA), and the immunohistochemical expression of MIB1, p16(INK4A) and p53. All differentiated VIN-lesions were hr-HPV- and p16-negative and in 96% MIB1-expression was confined to the parabasal layers. Eighty-four percent exhibited high p53 labeling indices, sometimes with parabasal extension. Eighty percent of all usual VIN-lesions were hr-HPV-positive, p16-positive, MIB1-positive and p53-negative. Five (of seven) HPV-negative usual VIN lesions, had an expression pattern like the other HPV-positive usual VIN lesions. In conclusion, both pathways leading to vulvar SCC have their own immunohistochemical profile, which can be used to distinguish the 2 types of VIN, but cannot explain differences in malignant potential.     

The CpG island methylator phenotype is not associated with a personal or family history of cancer

Colorectal cancers with widespread CpG island methylation display a number of distinct clinicopathological features, and it has been suggested that the condition has an inheritable genetic component. To address this possibility, histories of cancer were obtained from 562 individuals undergoing curative surgery for unselected colorectal cancer at one institution. Microsatellite status and methylation at p16, MINT1, 2, 12, and 31 loci were determined on fresh tumor tissue using standard methods. Fifty-five of 562 probands in this study provided a personal history of at least one other colorectal cancer, 10 reported at least one extracolonic cancer of hereditary nonpolyposis colorectal cancer type, and 84 individuals had another type of cancer. Age was strongly associated with the risk of multiple cancers, but there was no evidence that microsatellite instability or the CpG island methylator phenotype were independent risk factors for their development, either in the colorectum or elsewhere. Of the 547 individuals with knowledge of their family history, 80 (14.6%) reported a family history of colorectal cancer in a first-degree relative, and 60% of individuals reported a history of any cancer in a first-degree relative. Neither tumor CpG island methylator phenotype status nor microsatellite instability was predictive of a positive history of cancer in first- or second-degree relatives. The probability of a positive family or personal history of cancer did not increase with increasing number of methylated loci. Epigenetic silencing of multiple genes seen in some tumors is at best rarely the result of an inherited defect in the methylation apparatus. There is no justification for altering the personal or family cancer screening recommendations on the basis of tumor CpG island methylator phenotype status.     

APC gene methylation is inversely correlated with features of the CpG island methylator phenotype in colorectal cancer

The notion of a CpG island methylator phenotype (CIMP) was proposed to describe a subset of colorectal cancers (CRC) displaying frequent and concordant methylation of CpG islands located within gene promoter regions. Some workers have failed to observe associations between CIMP and specific clinicopathological features of CRC, possibly because of the choice of genes used to define this phenotype. The aim of the current study was to determine whether the aberrant methylation of 6 genes implicated in CRC development was associated with the same phenotypic features of this tumour type. The MethyLight assay was used to provide quantitative estimates of MLH1, P16, TIMP3, P14, DAPK and APC methylation levels in 199 unselected colorectal tumours. The methylation of MLH1, P16, TIMP3 and P14 was highly concordant (p < 0.0001 for each pair) but that of DAPK and APC was not. An inverse association was observed between the methylation of APC and TIMP3 (p = 0.004). Methylation of the MLH1, P16, TIMP3 and P14 genes was associated with tumour infiltrating lymphocytes (p < 0.05), microsatellite instability (p < 0.001), BRAF mutation (p < 0.0001) and elevated concentrations of the methyl group carriers tetrahydrofolate (THF) and 5,10-methylene THF (p < 0.05). In contrast, APC methylation was associated with wildtype BRAF (p = 0.003) and with lower concentrations of methyl group carriers (p < 0.05). These findings highlight the importance of gene selection in studies that aim to characterize the biological features and clinical behaviour of CIMP+ tumours.