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.     

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.     

Identification of genes uniquely involved in frequent microsatellite instability colon carcinogenesis by expression profiling combined with epigenetic scanning

Gene silencing through CpG island hypermethylation has been associated with genesis or progression of frequent microsatellite instability (MSI-H) cancers. To identify novel methylation sites unique to MSI-H colon cancers in an unbiased fashion, we conducted a global expression profiling-based methylation target search. We identified 81 genes selectively down-regulated in MSI-H cancers using cDNA microarray analysis of 41 primary colon cancers. Forty six of these 81 genes contained CpG islands overlapping their 5'untranslated regions. Initial screening of six genes in 57 primary colon cancers detected the following gene with MSI-H cancer-specific hypermethylation: RAB32, a ras family member and A-kinase-anchoring protein, was methylated in 14 of 25 (56%) MSI-H cancers but in none of 32 non-MSI-H cancers or 23 normal colonic specimens. RAB32 hypermethylation correlated with RAB32 mRNA down-regulation and with hMLH1 hypermethylation. In addition, the protein-tyrosine phosphatase receptor type O gene, PTPRO, was frequently methylated in right-sided tumors. This methylation screening strategy should identify additional genes inactivated by epigenetic silencing in colorectal and other cancers.     

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.     

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 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.     

A LIN28B polymorphism predicts for colon cancer survival

The pathogenesis of sporadic colorectal cancer involves distinct pathways, with characteristic genomic alterations. The first pathway, chromosome instability (CIN), is driven by APC mutations and is typified by Kras mutations, p53 mutation/loss of heterozygosity, and deletions at chromosome 18q. The second pathway is referred to as microsatellite instability (MSI), a genetic hallmark of the accumulated mutations that occur as a consequence of derangements in the mismatch repair genes. Finally, proximal colon cancers may involve methylation of a number of genes, which is frequently referred to as the CpG island methylator phenotype (CIMP), and are associated with B-raf mutations. The ability to stratify colorectal cancers by risk would be facilitated by the identification of polymorphisms that might be utilized as biomarkers. LIN28B is an RNA binding protein that is overexpressed in colon cancers. We find that LIN28B rs314277 is associated with significant recurrence of colorectal cancer in Stage II disease, which may have translational therapeutic implications.     

Targeting aberrant chromatin structure in colorectal carcinomas

Epigenetic processes such as DNA methylation and histone modifications are now recognized as critical events for regulation of gene expression in mammalian cells and affect gene function without a change in coding sequence. Neoplastic cells often show profound epigenetic alterations that contribute to tumorigenesis by altering expression of critical genes. In colorectal tumorigenesis, detailed analysis led to a hypothesis on a critical role for epigenetic changes in age-related cancer susceptibility and separately identified a distinct phenotype termed the CpG island methylator phenotype. CpG island methylator phenotype-positive colorectal cancers have significant associations with female sex, older age, proximal location, mucinous histology, KRAS and BRAF mutations, wild-type p53, and microsatellite instability. Histone modifications that affect chromatin structures are also closely implicated in tumor suppressor gene inactivation and DNA methylation and histone modifications seem to form reinforcing networks for stable gene silencing. Much of the excitement in this field relates to the possibility of therapeutic reversal of epigenetic changes by chromatin-modifying drugs. In CpG island methylator phenotype-positive colorectal cancers, DNA methylation inhibitors restore key silenced pathways in vivo (eg, mismatch repair defects), and hypomethylation can largely abolish tumorigenesis in a mouse model. Drugs that inhibit DNA methylation and histone deacetylation are in use in the clinic and should be tested in colorectal malignancy.     

18q loss of heterozygosity in microsatellite stable colorectal cancer is correlated with CpG island methylator phenotype-negative (CIMP-0) and inversely with CIMP-low and CIMP-high

Background:  

The CpG island methylator phenotype (CIMP) with widespread promoter methylation is a distinct epigenetic phenotype in colorectal cancer, associated with microsatellite instability-high (MSI-high) and BRAF mutations. 18q loss of heterozygosity (LOH) commonly present in colorectal cancer with chromosomal instability (CIN) is associated with global hypomethylation in tumor cell. A recent study has shown an inverse correlation between CIN and CIMP (determined by MINTs, p16, p14 and MLH1 methylation) in colorectal cancer. However, no study has examined 18q LOH in relation to CIMP-high, CIMP-low (less extensive promoter methylation) and CIMP-0 (CIMP-negative), determined by quantitative DNA methylation analysis.     

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.     

Alterations of DNA methylation associated with abnormalities of DNA methyltransferases in human cancers during transition from a precancerous to a malignant state

Alterations of DNA methylation are one of the most consistent epigenetic changes in human cancers. Human cancers generally show global DNA hypomethylation accompanied by region-specific hypermethylation. Alterations of DNA methylation may result in chromosomal instability as a result of changes in chromatin structure. DNA hypermethylation of CpG islands silences various tumor-related genes. Alterations of DNA methylation are frequently observed in cancers associated with chronic inflammation and/or persistent infection with viruses or other pathogenic microorganisms, such as hepatitis B or C viruses, Epstein-Barr virus, human papillomavirus and Helicobacter pylori, or with cigarette smoking. Accumulating evidence suggests that alterations of DNA methylation are involved even in the early and precancerous stages. On the other hand, in patients with cancers, aberrant DNA methylation is significantly associated with poorer tumor differentiation, tumor aggressiveness and poor prognosis. Precancerous conditions showing alterations of DNA methylation may progress rapidly and generate more malignant cancers. DNA methyltransferase (DNMT) 1 over-expression is not a secondary result of increased cell proliferative activity but is significantly correlated with the CpG island methylator phenotype, which is defined as frequent DNA hypermethylation of C-type CpG islands that are usually methylated in a cancer-specific (not age-dependent) manner. Splicing alteration of DNMT3b may result in chromosomal instability through DNA hypomethylation of pericentromeric satellite regions. Alteration of DNA methylation may become an indicator for carcinogenetic risk estimation and early diagnosis of cancers and a biological predictor of poor prognosis in patients with cancers. Correction of DNA methylation status may offer a new strategy for prevention and therapy of cancers.     

DNA methylation of multiple genes in gastric carcinoma: association with histological type and CpG island methylator phenotype

Hypermethylation of CpG islands is associated with silencing of various tumor suppressor genes. Recent studies on colorectal and gastric cancer have identified a CpG island methylator phenotype (CIMP), which involves the targeting of multiple genes by promoter hypermethylation. For determination of association between DNA methylation pattern or histological type and CIMP status in gastric carcinoma, CpG islands in the promoters of hMLH1 and CDH1 genes, CpG islands overlapping exon 1 of MGMT and p16^INK4a genes, and a non-CpG island in exon 1 of the RAR-β gene were studied. The presence of the CIMP was determined by monitoring five methylated in tumor (MINT) loci in 103 gastric carcinomas. Among the 103 gastric carcinomas, DNA hypermethylation was detected in the following frequencies: 14 (14%) for hMLH1 , 26 (25%) for MGMT , 26 (25%) for p16^INK4a , 54 (52%) for CDH1 , and 53 (52%) for RAR-β. Forty-two (41%) of 103 gastric carcinomas were positive for the CIMP. CIMP and hypermethylation of p16^INK4a gene were found more frequently in intestinal and diffuse-adherent types than in diffuse-scattered type ( P =0.013 and 0.017, respectively). In contrast, hypermethylation of the CDH1 and RAR-β genes was more common in the diffuse-scattered type than in the other types ( P =0.008 and 0.007, respectively). In intestinal- and diffuse-adherent-type gastric carcinomas, we found significant associations between the presence of the CIMP and hypermethylation of several genes: hMLH1 ( P =0.006), p16^INK4a ( P =0.018), CDH1 ( P =0.024), and RAR-β ( P =0.044). Our overall results suggest that in some intestinal- and diffuse-adherent-type gastric carcinomas, DNA hypermethylation affects non-specific gene promoters concordantly, at least in part, whereas in diffuse-scattered-type gastric carcinoma, DNA hyper-methylation affects specific genes such as CDH1 and RAR-β.     

DNA hypomethylation arises later in prostate cancer progression than CpG island hypermethylation and contributes to metastatic tumor heterogeneity

Hypomethylation of CpG dinucleotides in genomic DNA was one of the first somatic epigenetic alterations discovered in human cancers. DNA hypomethylation is postulated to occur very early in almost all human cancers, perhaps facilitating genetic instability and cancer initiation and progression. We therefore examined the nature, extent, and timing of DNA hypomethylation changes in human prostate cancer. Contrary to the prevailing view that global DNA hypomethylation changes occur extremely early in all human cancers, we show that reductions in (5me)C content in the genome occur very late in prostate cancer progression, appearing at a significant extent only at the stage of metastatic disease. Furthermore, we found that, whereas some LINE1 promoter hypomethylation does occur in primary prostate cancers compared with normal tissues, this LINE1 hypomethylation is significantly more pronounced in metastatic prostate cancer. Next, we carried out a tiered gene expression microarray and bisulfite genomic sequencing-based approach to identify genes that are silenced by CpG island methylation in normal prostate cells but become overexpressed in prostate cancer cells as a result of CpG island hypomethylation. Through this analysis, we show that a class of cancer testis antigen genes undergoes CpG island hypomethylation and overexpression in primary prostate cancers, but more so in metastatic prostate cancers. Finally, we show that DNA hypomethylation patterns are quite heterogeneous across different metastatic sites within the same patients. These findings provide evidence that DNA hypomethylation changes occur later in prostate carcinogenesis than the CpG island hypermethylation changes and occur heterogeneously during prostate cancer progression and metastatic dissemination.     

Characteristic methylation profile in CpG island methylator phenotype‐negative distal colorectal cancers

Aberrant DNA methylation is involved in colon carcinogenesis. Although the CpG island methylator phenotype (CIMP) is defined as a subset of colorectal cancers (CRCs) with remarkably high levels of DNA methylation, it is not known whether epigenetic processes are also involved in CIMP‐negative tumors. We analyzed the DNA methylation profiles of 94 CRCs and their corresponding normal‐appearing colonic mucosa with 11 different markers, including the five classical CIMP markers. The CIMP markers were frequently methylated in proximal CRCs (p < 0.01); however, RASSF1A methylation levels were significantly higher in distal CRCs, the majority of which are CIMP‐negative (p < 0.05). Similarly, methylation levels of RASSF1A and SFRP1 in the normal‐appearing mucosae of distal CRC cases were significantly higher than those in the proximal CRC cases (p < 0.05). They were also positively correlated with age (RASSF1A, p < 0.01; SFRP1, p < 0.01). Microarray‐based genome‐wide DNA methylation analysis of 18 CRCs revealed that 168 genes and 720 genes were preferentially methylated in CIMP‐negative distal CRCs and CIMP‐positive CRCs, respectively. Interestingly, more than half of the hypermethylated genes in CIMP‐negative distal CRCs were also methylated in the normal‐appearing mucosae, indicating that hypermethylation in CIMP‐negative distal CRCs is more closely associated with age‐related methylation. By contrast, more than 60% of the hypermethylated genes in CIMP‐positive proximal CRCs were cancer specific (p < 0.01). These data altogether suggest that CpG island promoters appear to be methylated in different ways depending on location, a finding which may imply the presence of different mechanisms for the acquisition of epigenetic changes during colon tumorigenesis.     

Screening for genomic fragments that are methylated specifically in colorectal carcinoma with a methylated MLH1 promoter

A subset of colorectal carcinomas (CRCs) is associated with microsatellite instability (MSI) of the genome. Although extensive methylation of CpG islands within the promoter regions of DNA mismatch repair genes such as MLH1 is thought to play a central role in tumorigenesis for MSI-positive sporadic CRCs, it has been obscure whether such aberrant epigenetic regulation occurs more widely and affects other cancer-related genes in vivo. Here, by using methylated CpG island amplification coupled with representational difference analysis (MCA-RDA), we screened genomic fragments that are selectively methylated in CRCs positive for MLH1 methylation, resulting in the identification of hundreds of CpG islands containing genomic fragments. Methylation status of such CpG islands was verified for 28 genomic clones in 8 CRC specimens positive for MLH1 methylation and the corresponding paired normal colon tissue as well as in 8 CRC specimens negative for methylation. Many of the CpG islands were preferentially methylated in the MLH1 methylation-positive CRC specimens, although methylation of some of them was more widespread. These data provide insights into the complex regulation of the methylation status of CpG islands in CRCs positive for MSI and MLH1 methylation.     

A distinct DNA methylation profile associated with microsatellite and chromosomal stable sporadic colorectal cancers

Aberrant DNA methylation, microsatellite instability (MSI) and chromosomal instability (CIN) are well-characterised molecular features of sporadic colorectal cancers (CRCs). In addition to CpG island methylator phenotype (CIMP) associated with MSI, an intermediate methylation subgroup is also a feature of non-MSI cancers. A large proportion of CRCs have no evidence of either MSI or CIN, here called Microsatellite and Chromosomal Stable (MACS), and require their methylation profile to be established. The clinical and molecular features of 170 sporadic CRC patients were investigated and stratified into MSI, CIN and MACS groups. MACS were most often found in the left colon and had a significantly lower BRAF mutation frequency (p < 0.001) compared with MSI. MACS had better survival [hazard ratio (HR) = 0.244, p = 0.017] compared with CIN, but were similar to MSI. The methylation status of 1,505 CpG loci from cancer-related genes was analysed in a subset of CRCs (n = 44 normal-tumour pairs) and compared with CIN, MSI and MACS status. Using two-way hierarchical clustering, three subgroups were identified, which associated with CIN, MSI and MACS status. Using significance analysis of microarray, 16 CpG loci demonstrating methylation changes associated with MACS were identified. A combination of six loci identified MACS with 81% sensitivity and 93% specificity. This result now requires independent validation. Hypomethylation of a CpG locus within the sonic hedgehog (SHH) promoter correlated with increased gene expression and was associated significantly with MACS cancers. In conclusion, we propose that MACS have distinct clinicopathological features and can be distinguished from other CRCs by a specific set of methylation loci.