Distinct features between MLH1-methylated and unmethylated colorectal carcinomas with the CpG island methylator phenotype: implications in the serrated neoplasia pathway.
ABSTRACT: The presence or absence of MLH1 methylation may critically affect the heterogeneity of colorectal carcinoma (CRC) with the CpG island methylator phenotype (CIMP). Here, we investigated the differential characteristics of CIMP-high (CIMP-H) CRCs according to MLH1 methylation status. To further confirm the MLH1-dependent features in CIMP-H CRC, an independent analysis was performed using data from The Cancer Genome Atlas (TCGA). In our CIMP-H CRC samples, MLH1-methylated tumors were characterized by older patient age, proximal colonic location, mucinous histology, intense lymphoid reactions, RUNX3/SOCS1 promoter methylation, BRAF mutations, and microsatellite instability-high (MSI-H) status. By contrast, MLH1-unmethylated tumors were associated with earlier age of onset, increased distal colorectal localization, adverse pathologic features, and KRAS mutations. In the TCGA dataset, the MLH1-silenced CIMP-H CRC demonstrated proximal location, MSI-H status, hypermutated phenotype, and frequent BRAF mutations, but the MLH1-non-silenced CIMP-H CRC was significantly associated with high frequencies of KRAS and APC mutations. In conclusion, the differential nature of CIMP-H CRCs depends primarily on the MLH1 methylation status. Based on the current knowledge, the sessile serrated adenoma/polyp may be the major precursor of MLH1-methylated CIMP-H CRCs, whereas MLH1-unmethylated CIMP-H CRCs may develop predominantly from KRAS-mutated traditional serrated adenomas and less commonly from BRAF-mutated traditional serrated adenomas and/or sessile serrated adenomas/polyps.
Project description:Colorectal cancer (CRC) mostly develops from a variety of polyps following mainly three different molecular pathways: chromosomal instability (CIN), microsatellite instability (MSI) and CpG island methylation (CIMP). Polyps are classified histologically as conventional adenomas, hyperplastic polyps, sessile serrated adenomas/polyps (SSA/P) and traditional serrated adenomas (TSA). However, the association of these polyps with the different types of CRCs and the underlying genetic and epigenetic aberrations has yet to be resolved. In order to address this question we analyzed 140 tumors and 20 matched mucosae by array comparative genomic hybridization, by sequence analysis of the oncogenes BRAF, KRAS, PI3K3CA and by methylation arrays. MSI was tested indirectly by immunohistochemistry (IHC) and a loss of MLH1, MSH2, MSH6 or PMS2 was assigned as high microsatellite instability (MSI-H), while low microsatellite instability (MSI-L) was defined as MGMT IHC negativity only. CIN was detected in 78% of all MSI-H CRCs, most commonly as a gain of chromosome 8. Methylation data analyses allowed classification of samples into four groups and detected similar methylation profiles in SSA/P and MSI-H CRC. TSA also revealed aberrant methylation pattern, but clustered more heterogeneously and closer to microsatellite stable (MSS) CRCs. SSA/P, TSA and MSI-H CRCs had the highest degree of promotor methylation (CIMP pathway). Chromosomal instability, in contrast to the established doctrine, is a common phenomenon in MSI CRCs, yet to a lower extent and at later stages than in MSS CRCs. Methylation analyses suggest that SSA/P are precursors for MSI-H CRCs and follow the CIMP pathway.
Project description:Colorectal cancers arising via the serrated pathway are often associated with BRAF V600E mutation, CpG island methylator phenotype (CIMP), and microsatellite instability. Previous studies have shown a strong association between BRAF V600E mutation and serrated polyps. This study aims to evaluate CIMP status of all the serrated polyp subtypes and its association with functionally important genes such as MLH1, p16, and IGFBP7. CIMP status and methylation were evaluated using the real-time based MethyLight assay in 154 serrated polyps and 63 conventional adenomas. Results showed that CIMP-high serrated polyps were strongly associated with BRAF mutation and proximal colon. CIMP-high was uncommon in conventional adenomas (1.59%), occurred in 8.25% of hyperplastic polyps (HPs), and became common in sessile serrated adenomas (SSAs) (51.43%). MLH1 methylation was mainly observed in the proximal colon and was significantly associated with BRAF mutation and CIMP-high. The number of samples methylated for p16 and IGFBP7 was the highest in SSAs. The methylation panel we used to detect CIMP is highly specific for CIMP-high cancers. With this panel, we demonstrate that CIMP-high is much more common in SSAs than HPs. This suggests that CIMP-high correlates with increased risk of malignant transformation which was also observed in methylation of functionally important genes.
Project description:Colorectal cancer (CRC) is a heterogeneous disease in terms of molecular carcinogenic pathways. Based on recent findings regarding the multiple serrated neoplasia pathway, we revised an eight-marker panel for a new CIMP classification system.1370 patients who received surgical resection for CRCs were classified into three CIMP subtypes (CIMP-N: 0-4 methylated markers, CIMP-P1: 5-6 methylated markers and CIMP-P2: 7-8 methylated markers). Our findings were validated in a separate set of high-risk stage II or stage III CRCs receiving adjuvant fluoropyrimidine plus oxaliplatin (n=950).A total of 1287/62/21 CRCs cases were classified as CIMP-N/CIMP-P1/CIMP-P2, respectively. CIMP-N showed male predominance, distal location, lower T, N category and devoid of BRAF mutation, microsatellite instability (MSI) and MLH1 methylation. CIMP-P1 showed female predominance, proximal location, advanced TNM stage, mild decrease of CK20 and CDX2 expression, mild increase of CK7 expression, BRAF mutation, MSI and MLH1 methylation. CIMP-P2 showed older age, female predominance, proximal location, advanced T category, markedly reduced CK20 and CDX2 expression, rare KRAS mutation, high frequency of CK7 expression, BRAF mutation, MSI and MLH1 methylation. CIMP-N showed better 5-year cancer-specific survival (CSS; HR=0.47; 95% CI: 0.28-0.78) in discovery set and better 5-year relapse-free survival (RFS; HR=0.50; 95% CI: 0.29-0.88) in validation set compared with CIMP-P1. CIMP-P2 showed marginally better 5-year CSS (HR=0.28, 95% CI: 0.07-1.22) in discovery set and marginally better 5-year RFS (HR=0.21, 95% CI: 0.05-0.92) in validation set compared with CIMP-P1.CIMP subtypes classified using our revised system showed different clinical outcomes, demonstrating the heterogeneity of multiple serrated precursors of CIMP-positive CRCs.
Project description:A subset of aggressive colorectal cancers exhibit BRAF mutation, MLH1 methylation, and a CpG island methylator phenotype (CIMP), but precursors are poorly established. In this study, we determined the status of these markers in colorectal polyps and evaluated associated risk factors. The study included 771 polyp cases and 1,027 controls who were ages 24 to 80 years, part of a group health program, received a colonoscopy from 1998 to 2007, and completed a structured questionnaire assessing risk factors. Following standard pathology review, polyps were assayed for BRAF mutation (V600E) and tested for MLH1 and CIMP methylation, the latter including the genes, CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1. Polytomous logistic regression was used to estimate ORs and 95% confidence intervals for the association between molecularly defined subsets of polyps and potential risk factors. There were 580 conventional adenomas and 419 serrated lesions successfully assayed. For adenomas, the prevalence of each marker was ?1%. In contrast, 55% of serrated lesions harbored mutant BRAF, 26% were CIMP-high, and 5% had methylated MLH1. In these lesions, the highest prevalence of markers was in sessile-serrated polyps (SSP) of ?10 mm that were in the right-side/cecal regions of the colon. Risk factors for CIMP-high-serrated lesions included Caucasian race, current smoking status, and a history of polyps, whereas for serrated lesions with mutant BRAF, the significant risk factors were male sex, current smoking status, obesity, and a history of polyps. Our results suggest that SSPs and other large, right-sided serrated lesions have a unique molecular profile that is similar to CIMP-high, BRAF-mutated colorectal cancers.
Project description:Hyperplastic polyposis syndrome (HPS) is characterized by the presence of multiple colorectal serrated polyps and is associated with an increased colorectal cancer (CRC) risk. The mixture of distinct precursor lesion types and malignancies in HPS provides a unique model to study the canonical pathway and a proposed serrated CRC pathway in humans. To establish which CRC pathways play a role in HPS and to obtain new support for the serrated CRC pathway, we assessed the molecular characteristics of polyps (n = 84) and CRCs (n = 19) in 17 patients with HPS versus control groups of various sporadic polyps (n = 59) and sporadic microsatellite-stable CRCs (n = 16). In HPS and sporadic polyps, APC mutations were exclusively identified in adenomas, whereas BRAF mutations were confined to serrated polyps. Six of 19 HPS CRCs (32%) were identified in a serrated polyp. Mutation analysis performed in the CRC and the serrated component of these lesions showed identical BRAF mutations. One HPS CRC was located in an adenoma, both components harboring an identical APC mutation. Overall, 10 of 19 HPS CRCs (53%) carried a BRAF mutation versus none in control group CRCs (P = 0.001). Six BRAF-mutated HPS CRCs (60%) were microsatellite unstable owing to MLH1 methylation. These findings provide novel supporting evidence for the existence of a predominant serrated CRC pathway in HPS, generating microsatellite-stable and microsatellite-instable CRCs.
Project description:Sessile serrated adenomas with BRAF mutation progress rapidly to cancer following the development of dysplasia (SSAD). Approximately 75% of SSADs methylate the mismatch repair gene MLH1, develop mismatch repair deficiency and the resultant cancers have a good prognosis. The remaining SSADs and BRAF mutant traditional serrated adenomas (TSA) develop into microsatellite stable cancers with a poor prognosis. The reason for this dichotomy is unknown. In this study, we assessed the genotypic frequency of the MLH1-93 polymorphism rs1800734 in SSADs and TSAs to determine if the uncommon variant A allele predisposes to MLH1 promoter hypermethylation.We performed genotyping for the MLH1-93 polymorphism, quantitative methylation specific PCR, and MLH1 immunohistochemistry on 124 SSAD, 128 TSA, 203 BRAF mutant CRCs and 147 control subjects with normal colonoscopy.The minor A allele was significantly associated with a dose dependent increase in methylation at the MLH1 promoter in SSADs (p =?0.022). The AA genotype was only observed in SSADs with MLH1 loss. The A allele was also overrepresented in BRAF mutant cancers with MLH1 loss. Only one of the TSAs showed loss of MLH1 and the overall genotype distribution in TSAs did not differ from controls.The MLH1-93 AA genotype is significantly associated with promoter hypermethylation and MLH1 loss in the context of SSADs. BRAF mutant microsatellite stable colorectal cancers with the AA genotype most likely arise in TSAs since the A allele does not predispose to methylation in this context.
Project description:To clarify molecular alterations in serrated pathway of colorectal cancer (CRC), we performed epigenetic and genetic analyses in sessile serrated adenoma/polyps (SSA/P), traditional serrated adenomas (TSAs) and high-methylation CRC. The methylation levels of six Group-1 and 14 Group-2 markers, established in our previous studies, were analyzed quantitatively using pyrosequencing. Subsequently, we performed targeted exon sequencing analyses of 126 candidate driver genes and examined molecular alterations that are associated with cancer development. SSA/P showed high methylation levels of both Group-1 and Group-2 markers, frequent BRAF mutation and occurrence in proximal colon, which were features of high-methylation CRC. But TSA showed low-methylation levels of Group-1 markers, less frequent BRAF mutation and occurrence at distal colon. SSA/P, but not TSA, is thus considered to be precursor of high-methylation CRC. High-methylation CRC had even higher methylation levels of some genes, e.g., MLH1, than SSA/P, and significant frequency of somatic mutations in nonsynonymous mutations (p?<?0.0001) and insertion/deletions (p?=?0.002). MLH1-methylated SSA/P showed lower methylation level of MLH1 compared with high-methylation CRC, and rarely accompanied silencing of MLH1 expression. The mutation frequencies were not different between MLH1-methylated and MLH1-unmethylated SSA/P, suggesting that MLH1 methylation might be insufficient in SSA/P to acquire a hypermutation phenotype. Mutations of mismatch repair genes, e.g., MSH3 and MSH6, and genes in PI3K, WNT, TGF-? and BMP signaling (but not in TP53 signaling) were significantly involved in high-methylation CRC compared with adenoma, suggesting importance of abrogation of these genes in serrated pathway.
Project description:Colorectal cancers (CRCs) with the CpG island methylator phenotype (CIMP) often associate with epigenetic silencing of hMLH1 and an activating mutation in the BRAF gene. However, the current CIMP criteria are ambiguous and often result in an underestimation of CIMP frequencies in CRCs. Because BRAF and KRAS belong to same signaling pathway, we hypothesized that not only mutations in BRAF but mutant KRAS may also associate with CIMP in CRC.We determined the methylation status in a panel of 14 markers (7 canonical CIMP-related loci and 7 new loci), microsatellite instability status, and BRAF/KRAS mutations in a collection of 487 colorectal tissues that included both sporadic and Lynch syndrome patients.Methylation analysis of 7 CIMP-related markers revealed that the mean number of methylated loci was highest in BRAF-mutated CRCs (3.6) vs KRAS-mutated (1.2, P < .0001) or BRAF/KRAS wild-type tumors (0.7, P < .0001). However, analyses with 7 additional markers showed that the mean number of methylated loci in BRAF mutant tumors (4.4) was the same as in KRAS mutant CRCs (4.3, P = .8610). Although sporadic microsatellite instability high tumors had the highest average number of methylated markers (8.4), surprisingly, Lynch syndrome CRCs also demonstrated frequent methylation (5.1).CIMP in CRC may result from activating mutations in either BRAF or KRAS, and the inclusion of additional methylation markers that correlate with mutant KRAS may help clarify CIMP in future studies. Additionally, aberrant DNA methylation is a common event not only in sporadic CRC but also in Lynch syndrome CRCs.
Project description:Most colorectal cancers (CRCs) containing activated BRAF (BRAF[V600E]) have a CpG island methylator phenotype (CIMP) characterized by aberrant hypermethylation of many genes, including the mismatch repair gene MLH1. MLH1 silencing results in microsatellite instability and a hypermutable phenotype. Through an RNAi screen, here we identify the transcriptional repressor MAFG as the pivotal factor required for MLH1 silencing and CIMP in CRCs containing BRAF(V600E). In BRAF-positive human CRC cell lines and tumors, MAFG is bound at the promoters of MLH1 and other CIMP genes, and recruits a corepressor complex that includes its heterodimeric partner BACH1, the chromatin remodeling factor CHD8, and the DNA methyltransferase DNMT3B, resulting in hypermethylation and transcriptional silencing. BRAF(V600E) increases BRAF/MEK/ERK signaling resulting in phosphorylation and elevated levels of MAFG, which drives DNA binding. Analysis of transcriptionally silenced CIMP genes in KRAS-positive CRCs indicates that different oncoproteins direct the assembly of distinct repressor complexes on common promoters.
Project description:BACKGROUND:Smoking and alcohol increase risk for colorectal malignancies. However, colorectal cancer (CRC) is a heterogenic disease and associations with the molecular pathological pathways are unclear. METHODS:This population-based case-control study includes 2444 cases with first-diagnosis CRC and 2475 controls. Tumour tissue was analysed for MSI (microsatellite instability), CIMP (CpG island methylator phenotype), BRAF (B-Raf proto-oncogene serine/threonine kinase gene) and KRAS (Kirsten rat sarcoma viral oncogene homologue gene) mutations. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were estimated for associations between alcohol and smoking and CRC molecular subtypes and pathways. RESULTS:Current smoking showed higher ORs for MSI-high (OR?=?2.79, 95% CI: 1.86-4.18) compared to MSS (OR?=?1.41, 1.14-1.75, p-heterogeneity (p-het)?=?0.001), BRAF-mutated (mut) (OR?=?2.40, 1.41-4.07) compared to BRAF-wild type (wt) (OR?=?1.52, 1.24-1.88, p-het?=?0.074), KRAS-wt (OR?=?1.70, 1.36-2.13) compared to KRAS-mut (OR?=?1.26, 0.95-1.68, p-het?=?0.039) and CIMP-high (OR?=?2.01, 1.40-2.88) compared to CIMP-low/negative CRC (OR?=?1.50, 1.22-1.85, p-het=0.101). Current smoking seemed more strongly associated with sessile serrated pathway (CIMP-high?+?BRAF-mut; OR?=?2.39, 1.27-4.52) than with traditional pathway CRC (MSS?+?CIMP-low/negative?+?BRAF-wt; OR?=?1.50, 1.16-1.94) and no association was observed with alternate pathway CRC (MSS?+?CIMP-low/negative?+?KRAS-wt; OR?=?1.08, 0.77-1.43). No heterogeneity was observed in alcohol consumption association by molecular subtypes. CONCLUSIONS:In this large case-control study, smoking was more strongly associated with MSI-high and KRAS-wt CRC and with cases showing features of the sessile serrated pathway. Association patterns were less clear for alcohol consumption.