Differences in DNA methylation signatures reveal multiple pathways of progression from adenoma to colorectal cancer.

BACKGROUND & AIMS: Genetic and epigenetic alterations contribute to the pathogenesis of colorectal cancer (CRC). There is considerable molecular heterogeneity among colorectal tumors, which appears to arise as polyps progress to cancer. This heterogeneity results in different pathways to tumorigenesis. Although epigenetic and genetic alterations have been detected in conventional tubular adenomas, little is known about how these affect progression to CRC. We compared methylomes of normal colon mucosa, tubular adenomas, and colorectal cancers to determine how epigenetic alterations might contribute to cancer formation. METHODS: We conducted genome-wide array-based studies and comprehensive data analyses of aberrantly methylated loci in 41 normal colon tissue, 42 colon adenomas, and 64 cancers using HumanMethylation450 arrays. RESULTS: We found genome-wide alterations in DNA methylation in the nontumor colon mucosa and cancers. Three classes of cancers and 2 classes of adenomas were identified based on their DNA methylation patterns. The adenomas separated into classes of high-frequency methylation and low-frequency methylation. Within the high-frequency methylation adenoma class a subset of adenomas had mutant KRAS. Additionally, the high-frequency methylation adenoma class had DNA methylation signatures similar to those of cancers with low or intermediate levels of methylation, and the low-frequency methylation adenoma class had methylation signatures similar to that of nontumor colon tissue. The CpG sites that were differentially methylated in these signatures are located in intragenic and intergenic regions. CONCLUSIONS: Genome-wide alterations in DNA methylation occur during early stages of progression of tubular adenomas to cancer. These findings reveal heterogeneity in the pathogenesis of colorectal cancer, even at the adenoma step of the process.

CpG island methylator phenotype is associated with response to adjuvant irinotecan-based therapy for stage III colon cancer.

BACKGROUND & AIMS: The CpG island methylator phenotype (CIMP), defined by a high frequency of aberrantly methylated genes, is a characteristic of a subclass of colon tumors with distinct clinical and molecular features. Cohort studies have produced conflicting results on responses of CIMP-positive tumors to chemotherapy. We assessed the association between tumor CIMP status and survival of patients receiving adjuvant fluorouracil and leucovorin alone or with irinotecan (IFL). METHODS: We analyzed data from patients with stage III colon adenocarcinoma randomly assigned to groups given fluorouracil and leucovorin or IFL after surgery, from April 1999 through April 2001. The primary end point of the trial was overall survival and the secondary end point was disease-free survival. DNA isolated from available tumor samples (n = 615) was used to determine CIMP status based on methylation patterns at the CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1 loci. The effects of CIMP on survival were modeled using Kaplan-Meier and Cox proportional hazards; interactions with treatment and BRAF, KRAS, and mismatch repair (MMR) status were also investigated. RESULTS: Of the tumor samples characterized for CIMP status, 145 were CIMP positive (23%). Patients with CIMP-positive tumors had shorter overall survival times than patients with CIMP-negative tumors (hazard ratio = 1.36; 95% confidence interval: 1.01-1.84). Treatment with IFL showed a trend toward increased overall survival for patients with CIMP-positive tumors, compared with treatment with fluorouracil and leucovorin (hazard ratio = 0.62; 95% CI: 0.37-1.05; P = .07), but not for patients with CIMP-negative tumors (hazard ratio = 1.38; 95% CI: 1.00-1.89; P = .049). In a 3-way interaction analysis, patients with CIMP-positive, MMR-intact tumors benefited most from the addition of irinotecan to fluorouracil and leucovorin therapy (for the interaction, P = .01). CIMP was more strongly associated with response to IFL than MMR status. Results for disease-free survival times were comparable among all analyses. CONCLUSIONS: Patients with stage III, CIMP-positive, MMR-intact colon tumors have longer survival times when irinotecan is added to combination therapy with fluorouracil and leucovorin.

Aberrantly methylated PKP1 in the progression of Barrett's esophagus to esophageal adenocarcinoma.

The aberrant DNA methylation of tumor suppressor genes occurs frequently in Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) and likely affects the initiation and progression of BE to EAC. In the present study, we discovered PKP1 as a novel methylated gene in EAC and then investigated the role of loss of PKP1, a constituent of the desmosome complex found in stratified epithelial layers, on the behavior of Barrett's esophagus and esophageal adenocarcinoma cells. By using primary esophageal tissue samples we determined that PKP1 was rarely methylated in normal squamous esophagus (5/55; 9.1%) and BE (5/39; 12.8%) and more frequently methylated in Barrett's esophagus with high-grade dysplasia (HGD) or EAC (20/60; 33.3%; P < 0.05). Furthermore, PKP1 levels were decreased in BE and HGD/EAC cases compared to normal squamous esophagus cases. Knockdown of PKP1 in the BE cell lines CP-A and CP-D (both normally express PKP1) resulted in increased cell motility. Thus, PKP1 loss secondary to promoter methylation, as well as other mechanisms, may promote the progression of BE to EAC in a subset of patients via decreased desmosome assembly and increased cell motility.

Aberrant DNA methylation occurs in colon neoplasms arising in the azoxymethane colon cancer model.

Mouse models of intestinal tumors have advanced our understanding of the role of gene mutations in colorectal malignancy. However, the utility of these systems for studying the role of epigenetic alterations in intestinal neoplasms remains to be defined. Consequently, we assessed the role of aberrant DNA methylation in the azoxymethane (AOM) rodent model of colon cancer. AOM induced tumors display global DNA hypomethylation, which is similar to human colorectal cancer. We next assessed the methylation status of a panel of candidate genes previously shown to be aberrantly methylated in human cancer or in mouse models of malignant neoplasms. This analysis revealed different patterns of DNA methylation that were gene specific. Zik1 and Gja9 demonstrated cancer-specific aberrant DNA methylation, whereas, Cdkn2a/p16, Igfbp3, Mgmt, Id4, and Cxcr4 were methylated in both the AOM tumors and normal colon mucosa. No aberrant methylation of Dapk1 or Mlt1 was detected in the neoplasms, but normal colon mucosa samples displayed methylation of these genes. Finally, p19(Arf), Tslc1, Hltf, and Mlh1 were unmethylated in both the AOM tumors and normal colon mucosa. Thus, aberrant DNA methylation does occur in AOM tumors, although the frequency of aberrantly methylated genes appears to be less common than in human colorectal cancer. Additional studies are necessary to further characterize the patterns of aberrantly methylated genes in AOM tumors.

Comparative analysis of PCR-based biomarker assay methods for colorectal polyp detection from fecal DNA.

BACKGROUND: Aberrantly methylated genes are promising biomarkers for the detection of colon adenomas and colorectal cancers (CRCs). The optimal assay type and specific methylated genes for these assays remain to be determined. METHODS: We used genomewide microarray-based assays to identify methylated genes as candidate biomarkers for colon neoplasms. The frequency of aberrant methylation of these genes in primary tumors was assessed with methylation-specific PCR (MSP). The limits of detection and specificities for different types of PCR-based assays were then assessed with the most promising genes identified in this screen. Finally, we assessed the best-performing MSP assay as an early-detection marker using fecal DNA samples. RESULTS: ITGA4 [integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor)] was identified as a novel gene frequently methylated in CRC. Methylated ITGA4 is present in 75% of colon adenomas (n = 36) and 92% of colon adenocarcinomas (n = 75). Comparison of end point MSP, end point MSP with clamped primers, and quantitative fluorescent MSP (qMSP) approaches revealed that both types of end point MSP assays could routinely detect as little as 70 pg DNA, whereas the qMSP assay could routinely detect as little as 7 pg. A fecal DNA qMSP assay for methylated ITGA4 can detect 69% of individuals with colon adenomas (n = 13) with a diagnostic specificity of 79% (n = 28). CONCLUSIONS: Methylated ITGA4 is a promising marker gene for the early detection of colonic neoplasms. qMSP has the lowest limit of detection of the MSP assay types tested, and a qMSP assay that detects methylated ITGA4 has potential as an early-detection assay for colon neoplasms.

Patterns of DNA methylation in the normal colon vary by anatomical location, gender, and age.

Alterations in DNA methylation have been proposed to create a field cancerization state in the colon, where molecular alterations that predispose cells to transformation occur in histologically normal tissue. However, our understanding of the role of DNA methylation in field cancerization is limited by an incomplete characterization of the methylation state of the normal colon. In order to determine the colon's normal methylation state, we extracted DNA from normal colon biopsies from the rectum, sigmoid, transverse, and ascending colon and assessed the methylation status of the DNA by pyrosequencing candidate loci as well as with HumanMethylation450 arrays. We found that methylation levels of repetitive elements LINE-1 and SAT-α showed minimal variability throughout the colon in contrast to other loci. Promoter methylation of EVL was highest in the rectum and progressively lower in the proximal segments, whereas ESR1 methylation was higher in older individuals. Genome-wide methylation analysis of normal DNA revealed 8388, 82, and 93 differentially methylated loci that distinguished right from left colon, males from females, and older vs. younger individuals, respectively. Although variability in methylation between biopsies and among different colon segments was minimal for repetitive elements, analyses of specific cancer-related genes as well as a genome-wide methylation analysis demonstrated differential methylation based on colon location, individual age, and gender. These studies advance our knowledge regarding the variation of DNA methylation in the normal colon, a prerequisite for future studies aimed at understanding methylation differences indicative of a colon field effect.

Altered RECQ Helicase Expression in Sporadic Primary Colorectal Cancers.

Deregulation of DNA repair enzymes occurs in cancers and may create a susceptibility to chemotherapy. Expression levels of DNA repair enzymes have been shown to predict the responsiveness of cancers to certain chemotherapeutic agents. The RECQ helicases repair damaged DNA including damage caused by topoisomerase I inhibitors, such as irinotecan. Altered expression levels of these enzymes in colorectal cancer (CRC) may influence the response of the cancers to irinotecan. Thus, we assessed RECQ helicase (WRN, BLM, RECQL, RECQL4, and RECQL5) expression in primary CRCs, matched normal colon, and CRC cell lines. We found that BLM and RECQL4 mRNA levels are significantly increased in CRC (P = .0011 and P < .0001, respectively), whereas RECQL and RECQL5 are significantly decreased (P = .0103 and P = .0029, respectively). RECQ helicase expression patterns varied between specific molecular subtypes of CRCs. The mRNA and protein expression of the majority of the RECQ helicases was closely correlated, suggesting that altered mRNA expression is the predominant mechanism for deregulated RECQ helicase expression. Immunohistochemistry localized the RECQ helicases to the nucleus. RECQ helicase expression is altered in CRC, suggesting that RECQ helicase expression has potential to identify CRCs that are susceptible to specific chemotherapeutic agents.

Evidence for the role of aberrant DNA methylation in the pathogenesis of Lynch syndrome adenomas.

Colorectal cancer (CRC) forms through a series of histologic steps that are accompanied by mutations and epigenetic alterations, which is called the polyp-cancer sequence. The role of epigenetic alterations, such as aberrant DNA methylation, in the polyp-cancer sequence in sporadic CRC and particularly in hereditary colon cancer is not well understood. Consequently, we assessed the methylation status of CDKN2A/p16, MGMT, MLH1 and p14(ARF) in adenomas arising in the Lynch syndrome, a familial colon cancer syndrome caused by MLH1 and MSH2 mutations, to determine if DNA methylation is a "second hit" mechanism in CRC and to characterize the role of DNA methylation in the polyp phase of the Lynch syndrome. We found MLH1 and p14(ARF) are methylated in 53 and 60% of the Lynch syndrome adenomas and in 4 and 20% of sporadic adenomas, whereas CDKN2A/p16 and MGMT are methylated in 6 and 14% of the Lynch syndrome adenomas versus 50 and 64% of sporadic adenomas. Therefore, the frequency and pattern of gene methylation varies between the Lynch syndrome and sporadic colon adenomas, implying differences in the molecular pathogenesis of the tumors. MLH1 methylation in the Lynch syndrome adenomas suggests gene methylation might have a role in the initiation of these neoplasms.

CpG island methylation of genes accumulates during the adenoma progression step of the multistep pathogenesis of colorectal cancer.

Genetic alterations occur during the adenoma-carcinoma sequence of colon cancer formation and drive the initiation and progression of colon cancer formation. The aberrant methylation of genes is an alternate, epigenetic mechanism for silencing tumor suppressor genes in colon cancer. The aim of this study was to determine on a global and gene-specific level the role of CpG island methylation in the initiation and progression of colon cancer. Consequently, we assessed the frequency of gene methylation in tumors representative of the commonly recognized histological steps of the adenoma-carcinoma progression sequence through the analysis of eight genes previously identified to be methylated in colon cancer, MGMT, HLTF, MLH1, p14(ARF), CDKN2A, TIMP3, THBS1, and CDH1. We observed that the proportion of tumors carrying methylated alleles increased from adenomas to adenocarcinomas but that the proportion of tumors with methylated alleles was not different between adenocarcinomas and metastases (69% versus 90%, P = 0.01 and 90% versus 81%, P > 0.05). The most substantial difference occurred between early and advanced adenomas (47% versus 84%, P = 0.018). Furthermore, we observed that the frequency of gene methylation at the different steps of the progression sequence varied between genes. Thus, the aberrant methylation of genes appears to increase most significantly during the progression of early adenomas to advanced adenomas, and the frequency of specific gene methylation at the different steps of the adenoma-carcinoma progression sequence varies in a gene-specific fashion.