CpG Island Methylator Phenotype Status Research Articles

The CpG Island Methylator Phenotype Status in Synchronous and Solitary Primary Colorectal Cancers: Prognosis and Effective Therapeutic Drug Prediction.

Synchronous colorectal cancer (sCRC) is characterized by the occurrence of more than one tumor within six months of detecting the first tumor. Evidence suggests that sCRC might be more common in the serrated neoplasia pathway, marked by the CpG island methylator phenotype (CIMP), than in the chromosomal instability pathway (CIN). An increasing number of studies propose that CIMP could serve as a potential epigenetic predictor or prognostic biomarker of sCRC. Therapeutic drugs already used for treating CIMP-positive colorectal cancers (CRCs) are reviewed and drug selections for sCRC patients are discussed.

Novel insights into tumorigenesis revealed by molecular analysis of Lynch syndrome cases with multiple colorectal tumors.

Lynch syndrome (LS) is an autosomal dominant multi-organ cancer syndrome with a high lifetime risk of cancer. The number of cumulative colorectal adenomas in LS does not generally exceed ten, and removal of adenomas via routine screening minimizes the cancer burden. However, abnormal phenotypes may mislead initial diagnosis and subsequently cause suboptimal treatment. Currently, there is no standard guide for the care of multiple colorectal adenomas in LS individuals. We aimed to shed insight into the molecular features and reasons for multiplicity of adenomas in LS patients. We applied whole exome sequencing on nine adenomas (ten samples) and three assumed primary carcinomas (five samples) of an LS patient developing the tumors during a 21-year follow-up period. We compared the findings to the tumor profiles of two additional LS cases ascertained through colorectal tumor multiplicity, as well as to ten adenomas and 15 carcinomas from 23 unrelated LS patients with no elevated adenoma burden from the same population. As LS associated cancers can arise via several molecular pathways, we also profiled the tumors for CpG Island Methylator Phenotype (CIMP), and LINE-1 methylation. All tumors were microsatellite unstable (MSI), and MSI was present in several samples derived from normal mucosa as well. Interestingly, frequent frameshift variants in RNF43 were shared among substantial number of the tumors of our primary case and the tumors of LS cases with multiple tumors but almost absent in our control LS cases. The RNF43 variants were completely absent in the normal tissue, indicating tumor-associated mutational hotspots. The RNF43 status correlated with the mutational signature SBS96. Contrary to LS tumors from the reference set with no elevated colorectal tumor burden, the somatic variants occurred significantly more frequently at C>T in the CpG context, irrespective of CIMP or LINE-1 status, potentially indicating other, yet unknown methylation-related mechanisms. There were no signs of somatic mosaicism affecting the MMR genes. Somatic variants in APC and CTNNB1 were unique to each tumor. Frequent somatic RNF43 hot spot variants combined with SBS96 signature and increased tendency to DNA methylation may contribute to tumor multiplicity in LS.

A Novel and Efficient Digital Pathology Classifier for Predicting Cancer Biomarkers Using Sequencer Architecture

In digital pathology tasks, transformers have achieved state-of-the-art results, surpassing convolutional neural networks (CNNs). However, transformers are usually complex and resource intensive. This study developed a novel and efficient digital pathology classifier called DPSeq to predict cancer biomarkers through fine-tuning a sequencer architecture integrating horizontal and vertical bidirectional long short-term memory networks. Using hematoxylin and eosin-stained histopathologic images of colorectal cancer from two international data sets (The Cancer Genome Atlas and Molecular and Cellular Oncology), the predictive performance of DPSeq was evaluated in a series of experiments. DPSeq demonstrated exceptional performance for predicting key biomarkers in colorectal cancer (microsatellite instability status, hypermutation, CpG island methylator phenotype status, BRAF mutation, TP53 mutation, and chromosomal instability), outperforming most published state-of-the-art classifiers in a within-cohort internal validation and a cross-cohort external validation. In addition, under the same experimental conditions using the same set of training and testing data sets, DPSeq surpassed four CNNs (ResNet18, ResNet50, MobileNetV2, and EfficientNet) and two transformer (Vision Transformer and Swin Transformer) models, achieving the highest area under the receiver operating characteristic curve and area under the precision-recall curve values in predicting microsatellite instability status, BRAF mutation, and CpG island methylator phenotype status. Furthermore, DPSeq required less time for both training and prediction because of its simple architecture. Therefore, DPSeq appears to be the preferred choice over transformer and CNN models for predicting cancer biomarkers.

Abstract 3019: Density of T cell subsets in colorectal cancer in relation to disease-specific survival

Abstract Background. Prior studies have demonstrated that the overall density of T cells in colorectal tumors is favorably associated with colorectal cancer (CRC) survival; however, few studies have considered the potentially distinct roles of heterogeneous T cell subsets in different tissue regions in relation to CRC outcomes. Methods. We used data and colorectal tumor specimens from a subset of participants in three studies (total N=878). We profiled the in situ T cell landscape of CRC using digital imaging, machine learning, and a customized 9-plex multiplexed immunofluoresence panel with antibodies directed against CD3, CD4, CD8, CD45RA, CD45RO, FOXP3, and MKI67. This allowed us to determine the density of T cell subsets defined according to marker co-localization and according to tissue region (i.e., epithelial, stromal). We used Cox proportional hazards models to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for the associations of T cell subset densities in CRC with disease-specific survival, adjusting for age, sex, tumor site, microsatellite instability status, CpG island methylator phenotype status, status of somatic mutations in BRAF and KRAS, stage, and study. Results. Overall, the densities of CD3+CD4+ helper T cells and CD3+CD8+ cytotoxic T cells in CRC were favorably associated with CRC-specific survival. However, in analyses stratified by tissue region, the association of CD3+CD4+ helper T cell density with survival was limited to density in stromal regions (HR for highest vs. lowest density quartile=0.40, 95% CI: 0.26-0.63, ptrend<0.001), whereas the association with CD3+CD8+ cytotoxic T cells was limited to density in epithelial regions (HR=0.48, 95% CI: 0.31-0.76, ptrend=0.001). These significant associations persisted after mutually adjusting region-specific CD3+CD4+ and CD3+CD8+ T cell densities for each other. Associations of memory T cell subsets (i.e., CD3+CD4+CD45RO+ and CD3+CD8+ CD45RO+ T cells) with survival were more modest than associations with overall CD3+CD4+ and CD3+CD8+ T cell densities, and were similar in magnitude across stromal and epithelial tissue regions. Conclusions. The density of T cells in CRC tissue is significantly associated with CRC survival, independent of stage at diagnosis and other evaluated tumor markers; however, the T cell subsets that are associated with CRC outcomes differ according to tissue region. These findings highlight the differing roles of distinct T cell subsets in CRC with regard to cancer outcomes, and support the need for additional work identifying factors that contribute to T cell response profiles in CRC. Citation Format: Amanda I. Phipps, Jonathan Nowak, Yasutoshi Takashima, Daniel Buchanan, Andressa Dias Costa, Steven Gallinger, Jeroen Huyghe, Li Hsu, Conghui Qu, Claire Thomas, Sushma Thomas, Tomotaka Ugai, Ulrike Peters, Shuji Ogino. Density of T cell subsets in colorectal cancer in relation to disease-specific survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3019.

Prognostic role of detailed colorectal location and tumor molecular features: analyses of 13,101 colorectal cancer patients including 2994 early-onset cases.

The pathogenic effect of colorectal tumor molecular features may be influenced by several factors, including those related to microbiota, inflammation, metabolism, and epigenetics, which may change along colorectal segments. We hypothesized that the prognostic association of colon cancer location might differ by tumor molecular characteristics. Utilizing a consortium dataset of 13,101 colorectal cancer cases, including 2994 early-onset cases, we conducted survival analyses of detailed tumor location stratified by statuses of microsatellite instability (MSI), CpG island methylator phenotype (CIMP), and KRAS and BRAF oncogenic mutation. There was a statistically significant trend for better colon cancer-specific survival in relation to tumor location from the cecum to sigmoid colon (Ptrend = 0.002), excluding the rectum. The prognostic association of colon location differed by MSI status (Pinteraction = 0.001). Non-MSI-high tumors exhibited the cecum-to-sigmoid trend for better colon cancer-specific survival [Ptrend < 0.001; multivariable hazard ratio (HR) for the sigmoid colon (vs. cecum), 0.80; 95% confidence interval (CI) 0.70-0.92], whereas MSI-high tumors demonstrated a suggestive cecum-to-sigmoid trend for worse survival (Ptrend = 0.020; the corresponding HR, 2.13; 95% CI 1.15-3.92). The prognostic association of colon tumor location also differed by CIMP status (Pinteraction = 0.003) but not significantly by age, stage, or other features. Furthermore, MSI-high status was a favorable prognostic indicator in all stages. Both detailed colonic location and tumor molecular features need to be accounted for colon cancer prognostication to advance precision medicine. Our study indicates the important role of large-scale studies to robustly examine detailed colonic subsites in molecular oncology research.

Body mass index and molecular subtypes of colorectal cancer.

Obesity is an established risk factor for colorectal cancer (CRC), but the evidence for the association is inconsistent across molecular subtypes of the disease. We pooled data on body mass index (BMI), tumor microsatellite instability status, CpG island methylator phenotype status, BRAF and KRAS mutations, and Jass classification types for 11 872 CRC cases and 11 013 controls from 11 observational studies. We used multinomial logistic regression to estimate odds ratios (OR) and 95% confidence intervals (CI) adjusted for covariables. Higher BMI was associated with increased CRC risk (OR per 5 kg/m2 = 1.18, 95% CI = 1.15 to 1.22). The positive association was stronger for men than women but similar across tumor subtypes defined by individual molecular markers. In analyses by Jass type, higher BMI was associated with elevated CRC risk for types 1-4 cases but not for type 5 CRC cases (considered familial-like/Lynch syndrome microsatellite instability-H, CpG island methylator phenotype-low or negative, BRAF-wild type, KRAS-wild type, OR = 1.04, 95% CI = 0.90 to 1.20). This pattern of associations for BMI and Jass types was consistent by sex and design of contributing studies (cohort or case-control). In contrast to previous reports with fewer study participants, we found limited evidence of heterogeneity for the association between BMI and CRC risk according to molecular subtype, suggesting that obesity influences nearly all major pathways involved in colorectal carcinogenesis. The null association observed for the Jass type 5 suggests that BMI is not a risk factor for the development of CRC for individuals with Lynch syndrome.

CIMP-positive glioma is associated with better prognosis: A systematic analysis.

Background:CpG island methylator phenotype (CIMP) was closely related to the degree of pathological differentiation of tumors, and it’s an important determinant of glioma pathogenicity. However, the molecular and pathological features of CIMP-positive glioma have not been fully elucidated. In addition, CIMP have been reported to be a useful prognostic marker in several human cancers, yet its prognostic value in gliomas is still controversial. Therefore, we aimed to evaluate gene mutations and pathological features of CIMP-positive glioma and explore the prognostic value of CIMP in gliomas.Methods:We comprehensively searched PubMed, Embase, and MEDLINE for studies describing gene mutations, pathological features and overall survival of gliomas stratified by CIMP status. Odds ratios (OR), hazard ratios (HR), and their 95% confidence intervals (CI) were used to estimate the correlation between CIMP and the outcome parameters.Results:Twelve studies with 2386 gliomas (1051 CIMP-positive and 1335 CIMP-negative) were included. Our results showed that CIMP was more frequent in isocitrate dehydrogenase 1 (IDH1)-mutated gliomas (OR 229.07; 95% CI 138.72–378.26) and 1p19q loss of heterozygosis (LOH) gliomas (OR 5.65; 95% CI 2.66–12.01). Pathological analysis showed that CIMP was common in low-malignant oligodendroglioma (OR 5.51; 95% CI 3.95–7.70) with molecular features including IDH1 mutations and 1p19q LOH, but rare in glioblastoma (OR 0.14; 95% CI 0.10–0.19). However, CIMP showed no obvious correlation with anaplastic oligoastrocytomas (OR 1.57; 95% CI 1.24–2.00) or oligoastrocytomas (OR 0.79; 95% CI 0.35–1.76). Concerning the prognosis, we found that CIMP-positive gliomas had longer overall survival (HR 0.57; 95% CI 0.97–0.16) than CIMP-negative gliomas.Conclusions:CIMP could be used as a potential independent prognostic indicator for glioma.

Genetic Profiling of Colorectal Carcinomas of Patients with Primary Sclerosing Cholangitis and Inflammatory Bowel Disease.

BackgroundPatients with primary sclerosing cholangitis (PSC) and inflammatory bowel disease (IBD) run a 10-fold increased risk of developing colorectal cancer (CRC) compared to patients with IBD only. The aim of this study was to perform an extensive screen of known carcinogenic genomic alterations in patients with PSC-IBD, and to investigate whether such changes occur already in nondysplastic mucosa.MethodsArchival cancer tissue and nondysplastic mucosa from resection specimens of 19 patients with PSC-IBD-CRC were characterized, determining DNA copy-number variations, microsatellite instability (MSI), mutations on 48 cancer genes, and CpG island methylator phenotype (CIMP). Genetic profiles were compared with 2 published cohorts of IBD-associated CRC (IBD-CRC; n = 11) and sporadic CRC (s-CRC; n = 100).ResultsPatterns of chromosomal aberrations in PSC-IBD-CRC were similar to those observed in IBD-CRC and s-CRC, MSI occurred only once. Mutation frequencies were comparable between the groups, except for mutations in KRAS, which were less frequent in PSC-IBD-CRC (5%) versus IBD-CRC (38%) and s-CRC (31%; P = .034), and in APC, which were less frequent in PSC-IBD-CRC (5%) and IBD-CRC (0%) versus s-CRC (50%; P < .001). Cases of PSC-IBD-CRC were frequently CIMP positive (44%), at similar levels to cases of s-CRC (34%; P = .574) but less frequent than in cases with IBD-CRC (90%; P = .037). Similar copy number aberrations and mutations were present in matched cancers and adjacent mucosa in 5/15 and 7/11 patients, respectively.ConclusionsThe excess risk of CRC in patients with PSC-IBD was not explained by copy number aberrations, mutations, MSI, nor CIMP status, in cancer tissue, nor in adjacent mucosa. These findings set the stage for further exome-wide and epigenetic studies.

Evaluation of methylated DCR1 as a biomarker for response to adjuvant irinotecan-based therapy in stage III colon cancer: cancer and leukaemia Group B 89803 (Alliance)

ABSTRACT Aberrantly methylated genes contribute to the landscape of epigenetic alterations in colorectal adenocarcinoma. The global CpG Island methylator phenotype (CIMP) and individually methylated genes are potential prognostic/predictive biomarkers. Research suggests an association between methylated DCR1 (mDCR1) and lack of benefit with irinotecan (IFL) treatment. We assessed the association between DCR1 methylation status and survival in patients receiving adjuvant fluorouracil/ leucovorin (5-FU/LV) or IFL. We analysed data from patients with stage III colon adenocarcinoma randomly assigned to adjuvant 5-FU/LV or IFL in CALGB 89803 (Alliance). The primary endpoint was overall survival (OS), and the secondary endpoint was disease-free survival (DFS). Using tumour sample DNA, we evaluated the association between survival, DCR1 methylation status, and molecular subgroups (BRAF, KRAS, mismatch repair status, CIMP status) using Kaplan–Meier estimator and Cox proportional hazard model. mDCR1 was observed in 221/400 (55%) colon cancers. Histopathologic features were similar between mDCR1 and unmethylated DCR1 (unDCR1) colon cancers. There was no difference in OS (p = 0.83) or DFS (p = 0.85) based on DCR1 methylation status. There was no association between methylation status and response to IFL . In patients with unDCR1 and KRAS-wildtype tumours, those who received IFL had a nearly two-fold worse DFS compared to patients who received 5-FU/LV (HR = 1.85, 95% CI (0.97–3.53, p = 0.06). This relationship was not notable among other subgroups. In stage III colon cancer patients, mDCR1 status did not associate with response to irinotecan. Larger studies may suggest an association between the iridocene response and molecular subgroups.

CpG Island Methylator Phenotype Modulates the Immune Response of the Tumor Microenvironment and Influences the Prognosis of Pancreatic Cancer Patients.

Background CpG island methylator phenotype (CIMP), featured with concurrent and widespread hypermethylation of a cluster of CpGs, has been reported to play an important role in carcinogenesis. Limited studies have investigated the role of CIMP in pancreatic cancer (PC). The aim of this study was to explore the CIMP in PC patients and its impact on the immune response of the tumor microenvironment and prognosis. Methods DNA methylation, somatic mutation, mRNA, and corresponding clinical data of PC patients were downloaded from TCGA (184 patients) and the ICGC (264 patients). Univariate and multivariate regression analyses were used to identify prognosis-related CpGs. Consensus clustering analysis was used for identification of the CIMP in PC patients. ESTIMATE and CIBORORT were used for estimation of the tumor microenvironment (TME) in PC patients. Results In the TCGA PC cohort, 22,450 differential CpGs, including 12,937 hypermethylated CpGs and 9,513 hypomethylated CpGs, were identified between 184 PC patients and 10 normal controls. Univariate and multivariate Cox analysis further screened out 72 OS-related CpGs, and three distinct CIMP groups with distinctly different prognosis and molecular features, including the CIMP-L subgroup, CIMP-M subgroup, and CIMP-H subgroup, were identified based on unsupervised consensus clustering analysis of these CpGs. Patients of the CIMP-H subgroup had poorer OS and RFS, while patients of the CIMP-L subgroup had better OS and RFS. The CIMP status was also an independent prognostic factor for OS and PFS. In molecular features, significantly higher somatic mutation burden and tumor mutational burden were found in patients of the CIMP-H subgroup compared to those of the CIMP-L subgroup. Besides, lower stromal score, immune score, and higher cancer stemness indices and tumor purity were also found in patients of the CIMP-H subgroup compared to those of the CIMP-L subgroup. Correspondingly, significant total T cells, total B cells, CD8 T cells, memory CD4 T cells, and higher regulatory T cells were found in patients of the CIMP-H subgroup. Moreover, significantly lower expression of immune checkpoint genes, such as PD-1, CTLA4, CD86, VTCN1, and LAG-3, was also found in patients of the CIMP-H subgroup compared to those of the CIMP-L subgroup. In the end, we validated the CIMP status in PC patients of the ICGC dataset. Conclusion The CIMP may modulate the immune response of the tumor microenvironment and influence the prognosis of pancreatic cancer patients, which may help to make an assertion to provide specific and efficient treatment options for patients of different subtypes.

Multiregional assessment of CIMP in primary colorectal cancers: Phenotype concordance but marker variability.

Intratumor heterogeneity of colorectal cancers (CRCs) is manifested both at the genomic and epigenomic levels. Early genetic aberrations in carcinogenesis are clonal and present throughout the tumors, but less is known about the heterogeneity of the epigenetic CpG island methylator phenotype (CIMP). CIMP characterizes a subgroup of CRCs thought to originate from specific precursor lesions, and it is defined by widespread DNA methylation within promoter regions. In this work, we investigated CIMP in two to four multiregional samples from 30 primary tumors (n = 86 samples) using the consensus Weisenberger gene panel (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1). Twenty‐nine of 30 tumors (97%) showed concordant CIMP status in all samples, and percent methylated reference (PMR) values of all five markers had higher intertumor than intratumor variation (P value = 1.5e−09). However, a third of the CIMP+ tumors exhibited discrepancies in methylation status in at least one of the five gene markers. To conclude, CIMP status was consistent within primary CRCs, and it is likely a clonal phenotype. However, spatial discordances of the individual genes suggest that large‐scale analysis of multiregional samples could be of interest for identifying CIMP markers that are robust to intratumor heterogeneity.

Clinicopathological and Molecular Profiles of Sporadic Microsatellite Unstable Colorectal Cancer with or without the CpG Island Methylator Phenotype (CIMP).

Simple SummaryThe 5’-C-phosphate-G-3’ island methylator phenotype (CIMP) is a phenotype of colorectal cancer associated with microsatellite instability (MSI). The aim of the present study was to analyze the CIMP phenotype and genetic mutations in MSI-high colorectal cancer. Our results demonstrated that among MSI-high colorectal cancer patients, CIMP-high tumors were associated with specific mutation profiles and clinicopahtological features compared with CIMP-low or CIMP-0 tumors, however, the CIMP status was not an independent prognostic factor.Background: The 5’-C-phosphate-G-3’ island methylator phenotype (CIMP) is a specific phenotype of colorectal cancer (CRC) associated with microsatellite instability-high (MSI-high) tumors. Methods: In this study, we determined the CIMP status using eight methylation markers in 92 MSI-high CRC patients after excluding five germline mismatch repair (MMR) gene mutations analyzed by next-generation sequencing (NGS) and confirmed by Sanger sequencing. The mutation spectra of 22 common CRC-associated genes were analyzed by NGS. Results: Of the 92 sporadic MSI-high tumors, 23 (25%) were considered CIMP-high (expressed more than 5 of 8 markers). CIMP-high tumors showed proximal colon preponderance and female predominance. The mutation profiles of CIMP-high tumors were significantly different from those of CIMP-low or CIMP-0 tumors (i.e., higher frequencies of BRAF, POLD1, MSH3, and SMAD4 mutations but lower frequencies of APC, TP53, and KRAS mutations). Multivariate analysis demonstrated that tumor, node, metastasis (TNM) stage was the independent prognostic factor affecting overall survival (OS). Among the MSI-high cases, the CIMP status did not impact the outcome of patients with MSI-high tumors. Conclusions: Only TNM stage was a statistically significant predictor of outcomes independent of CIMP profiles in MSI-high CRC patients. Sporadic MSI-high CRCs with different mechanisms of carcinogenesis have specific mutation profiles and clinicopathological features.

A Qualitative Transcriptional Signature for Predicting CpG Island Methylator Phenotype Status of the Right-Sided Colon Cancer.

A part of colorectal cancer which is characterized by simultaneous numerous hypermethylation CpG islands sites is defined as CpG island methylator phenotype (CIMP) status. Stage II and III CIMP−positive (CIMP+) right-sided colon cancer (RCC) patients have a better prognosis than CIMP−negative (CIMP−) RCC treated with surgery alone. However, there is no gold standard available in defining CIMP status. In this work, we selected the gene pairs whose relative expression orderings (REOs) were associated with the CIMP status, to develop a qualitative transcriptional signature to individually predict CIMP status for stage II and III RCC. Based on the REOs of gene pairs, a signature composed of 19 gene pairs was developed to predict the CIMP status of RCC through a feature selection process. A sample is predicted as CIMP+ when the gene expression orderings of at least 12 gene pairs vote for CIMP+; otherwise the CIMP−. The difference of prognosis between the predicted CIMP+ and CIMP− groups was more significantly different than the original CIMP status groups. There were more differential methylation and expression characteristics between the two predicted groups. The hierarchical clustering analysis showed that the signature could perform better for predicting CIMP status of RCC than current methods. In conclusion, the qualitative transcriptional signature for classifying CIMP status at the individualized level can predict outcome and guide therapy for RCC patients.

Genome-wide identification of CpG island methylator phenotype related gene signature as a novel prognostic biomarker of gastric cancer.

BackgroundGastric cancer (GC) is one of the most fatal cancers in the world. Results of previous studies on the association of the CpG island methylator phenotype (CIMP) with GC prognosis are conflicting and mainly based on selected CIMP markers. The current study attempted to comprehensively assess the association between CIMP status and GC survival and to develop a CIMP-related prognostic gene signature of GC.MethodsWe used a hierarchical clustering method based on 2,082 GC-related methylation sites to stratify GC patients from the cancer genome atlas into three different CIMP subgroups according to the CIMP status. Gene set enrichment analysis, tumor-infiltrating immune cells, and DNA somatic mutations analysis were conducted to reveal the genomic characteristics in different CIMP-related patients. Cox regression analysis and the least absolute shrinkage and selection operator were performed to develop a CIMP-related prognostic signature. Analyses involving a time-dependent receiver operating characteristic (ROC) curve and calibration plot were adopted to assess the performance of the prognostic signature.ResultsWe found a positive relationship between CIMP and prognosis in GC. Gene set enrichment analysis indicated that cancer-progression-related pathways were enriched in the CIMP-L group. High abundances of CD8+ T cells and M1 macrophages were found in the CIMP-H group, meanwhile more plasma cells, regulatory T cells and CD4+ memory resting T cells were detected in the CIMP-L group. The CIMP-H group showed higher tumor mutation burden, more microsatellite instability-H, less lymph node metastasis, and more somatic mutations favoring survival. We then established a CIMP-related prognostic gene signature comprising six genes (CST6, SLC7A2, RAB3B, IGFBP1, VSTM2L and EVX2). The signature was capable of classifying patients into high‐and low‐risk groups with significant difference in overall survival (OS; p < 0.0001). To assess performance of the prognostic signature, the area under the ROC curve (AUC) for OS was calculated as 0.664 at 1 year, 0.704 at 3 years and 0.667 at 5 years. When compared with previously published gene-based signatures, our CIMP-related signature was comparable or better at predicting prognosis. A multivariate Cox regression analysis indicated the CIMP-related prognostic gene signature was an independent prognostic indicator of GC. In addition, Gene ontology analysis indicated that keratinocyte differentiation and epidermis development were enriched in the high-risk group.ConclusionCollectively, we described a positive association between CIMP status and prognosis in GC and proposed a CIMP-related gene signature as a promising prognostic biomarker for GC.

Characterization of Chilean patients with sporadic colorectal cancer according to the three main carcinogenic pathways: Microsatellite instability, CpG island methylator phenotype and Chromosomal instability.

Molecular classification of colorectal cancer is difficult to implement in clinical settings where hundreds of genes are involved, and resources are limited. This study aims to characterize the molecular subtypes of patients with sporadic colorectal cancer based on the three main carcinogenic pathways microsatellite instability (MSI), CpG island methylator phenotype (CIMP), and chromosomal instability (CIN) in a Chilean population. Although several reports have characterized colorectal cancer, most do not represent Latin-American populations. Our study includes 103 colorectal cancer patients who underwent surgery, without neoadjuvant treatment, in a private hospital between 2008 and 2017. MSI, CIN, and CIMP status were assessed. Frequent mutations in KRAS, BRAF, and PIK3CA genes were analyzed by Sanger sequencing, and statistical analysis was performed by Fisher's exact and/or chi-square test. Survival curves were estimated with Kaplan-Meier and log-rank test. Based on our observations, we can classify the tumors in four subgroups, Group 1: MSI-high tumors (15%) are located in the right colon, occur at older age, and 60% show a BRAF mutation; Group 2: CIN-high tumors (38%) are in the left colon, and 26% have KRAS mutations. Group 3: [MSI/CIN/CIMP]-low/negative tumors (30%) are left-sided, and 39% have KRAS mutations; Group 4: CIMP-high tumors (15%) were more frequent in men and left side colon, with 27% KRAS and 7% presented BRAF mutations. Three percent of patients could not be classified. We found that CIMP-high was associated with a worse prognosis, both in MSI-high and MSI stable patients (p = 0.0452). Group 3 (Low/negative tumors) tend to have better overall survival compared with MSI-high, CIMP-high, and CIN-high tumors. This study contributes to understanding the heterogeneity of tumors in the Chilean population being one of the few characterizations performed in Latin-America. Given the limited resources of these countries, these results allow to improve molecular characterization in Latin-American colorectal cancer populations and confirm the possibility of using the three main carcinogenic pathways to define therapeutic strategies.

Postmenopausal Hormone Therapy and Colorectal Cancer Risk by Molecularly Defined Subtypes and Tumor Location.

BackgroundPostmenopausal hormone therapy (HT) is associated with a decreased colorectal cancer (CRC) risk. As CRC is a heterogeneous disease, we evaluated whether the association of HT and CRC differs across etiologically relevant, molecularly defined tumor subtypes and tumor location.MethodsWe pooled data on tumor subtypes (microsatellite instability status, CpG island methylator phenotype status, BRAF and KRAS mutations, pathway: adenoma-carcinoma, alternate, serrated), tumor location (proximal colon, distal colon, rectum), and HT use among 8220 postmenopausal women (3898 CRC cases and 4322 controls) from 8 observational studies. We used multinomial logistic regression to estimate odds ratios (OR) and 95% confidence intervals (CIs) for the association of ever vs never HT use with each tumor subtype compared with controls. Models were adjusted for study, age, body mass index, smoking status, and CRC family history. All statistical tests were 2-sided.ResultsAmong postmenopausal women, ever HT use was associated with a 38% reduction in overall CRC risk (OR =0.62, 95% CI = 0.56 to 0.69). This association was similar according to microsatellite instability, CpG island methylator phenotype and BRAF or KRAS status. However, the association was attenuated for tumors arising through the serrated pathway (OR = 0.81, 95% CI = 0.66 to 1.01) compared with the adenoma-carcinoma pathway (OR = 0.63, 95% CI = 0.55 to 0.73; Phet =.04) and alternate pathway (OR = 0.61, 95% CI = 0.51 to 0.72). Additionally, proximal colon tumors had a weaker association (OR = 0.71, 95% CI = 0.62 to 0.80) compared with rectal (OR = 0.54, 95% CI = 0.46 to 0.63) and distal colon (OR = 0.57, 95% CI = 0.49 to 0.66; Phet =.01) tumors.ConclusionsWe observed a strong inverse association between HT use and overall CRC risk, which may predominantly reflect a benefit of HT use for tumors arising through the adenoma-carcinoma and alternate pathways as well as distal colon and rectal tumors.

Unique characteristics of CpG island methylator phenotype (CIMP) in a Chinese population with colorectal cancer

BackgroundMolecular characteristics of CpG island methylator phenotype (CIMP) in colorectal cancer (CRC) have been well documented in Western, but not in Chinese, populations.MethodsWe investigated the incidence of CIMP, BRAF/KRAS mutation, and microsatellite instability (MSI) in a Chinese population with CRC (n = 401) and analysed associations between CIMP status and clinicopathological and molecular features.ResultsA total of 41 cases, 310 cases, and 40 cases were classified as CIMP-high, CIMP-low, and CIMP-negative, respectively. We detected a significantly low incidence of BRAF mutation in adenomas (2%) and CRC (0.7%), and a relatively low incidence of KRAS mutation (24.9%) compared with that in other populations. We also detected a relatively low incidence of CIMP-high (10.2%), which was significantly associated with younger age (≤49 years of age), female sex, and proximal tumour location.ConclusionsThis study revealed unique characteristics of CIMP in a Chinese population with colorectal cancer. Developing specific CIMP markers based on unique populations or ethnic groups will further help to fully elucidate CIMP pathogenesis.

A prognostic CpG score derived from epigenome-wide profiling of tumor tissue was independently associated with colorectal cancer survival

BackgroundResults of previous studies on the association of the CpG island methylator phenotype (CIMP) with colorectal cancer (CRC) prognosis were inconsistent and mostly based on different CIMP definitions. The current study aimed to comprehensively investigate the associations between DNA methylation on genes previously used to define CIMP status with CRC survival.ResultsPatients with CRC followed up for a median of 5.2 years were divided into a study cohort (n = 568) and a validation cohort (n = 308). DNA methylation was measured in tumor tissue using the Illumina Infinium HumanMethylation450 BeadChip and restricted to 43 genes used to define CIMP status in previous studies. Cox proportional hazard regression models were used to estimate adjusted hazard ratios (HR) and 95% confidence intervals (CI) of survival after CRC, including adjustment for tumor stage, microsatellite instability, and BRAF mutation status. In the study cohort, ten CpG sites were identified to be associated with CRC survival. Seven of these ten CpG sites were also associated with CRC survival in the validation cohort and were used to construct a prognostic score. CRC patients with a prognostic score of the lowest methylation level showed poorer disease-specific survival compared with patients with the highest methylation level in both the study cohort and the validation cohort (HR = 3.11 and 95% CI = 1.97–4.91, and HR = 3.06 and 95% CI = 1.71–5.45, respectively).ConclusionsA CpG panel consisting of seven CpG sites was found to be strongly associated with CRC survival, independent from important clinical factors and mutations associated with CIMP. Further studies are required to confirm these findings.

Cimp-Positive Status is More Representative in Multiple Colorectal Cancers than in Unique Primary Colorectal Cancers

Colorectal cancer (CRC) with CpG island methylator phenotype (CIMP) is recognized as a subgroup of CRC that shows association with particular genetic defects and patient outcomes. We analyzed CIMP status of 229 individuals with CRC using an eight-marker panel (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1); CIMP-(+) tumors were defined as having ≥ 5 methylated markers. Patients were divided into individuals who developed a “unique” CRC, which were subclassified into early-onset CRC (EOCRC) and late-onset CRC (LOCRC), and patients with multiple primary CRCs subclassified into synchronous CRC (SCRC) and metachronous CRC (MCRC). We found 9 (15.2%) CIMP-(+) EOCRC patients related with the proximal colon (p = 0.008), and 19 (26.8%) CIMP-(+) LOCRC patients associated with tumor differentiation (p = 0.045), MSI status (p = 0.021) and BRAF mutation (p = 0.001). Thirty-five (64.8%) SCRC patients had at least one CIMP-(+) tumor and 20 (44.4%) MCRC patients presented their first tumor as CIMP-(+). Thirty-nine (72.2%) SCRC patients showed concordant CIMP status in their simultaneous tumors. The differences in CIMP-(+) frequency between groups may reflect the importance of taking into account several criteria for the development of multiple primary neoplasms. Additionally, the concordance between synchronous tumors suggests CIMP status is generally maintained in SCRC patients.

Abstract LB-141: CIMP is associated with altered microbiota composition in colorectal cancer patients

Abstract DNA methylation changes are important in cancer development and the etiology of the CpG Island Methylator Phenotype (CIMP), the most extreme form of aberrant promoter DNA methylation in cancer, can only partially be explained by genetic changes. An association between CIMP and the gut microbiota has been implicated in colorectal cancer (CRC) pathogenesis but the exact mechanisms underlying the observations are poorly understood. In order to clarify the link between CIMP and the microbiome in CRC, we studied human primary tumor and adjacent mucosal tissues (normal) from CRC patients (n=46). The patient samples were categorized by tumor sites (23 proximal and 23 distal), sex (28 males and 18 females), and age (69 yrs ±11). Bisulfite pyrosequencing and Digital Restriction Enzyme Analysis of Methylation (DREAM) were performed to determine the methylation of CpG sites in the CRC tumors. The tumor samples were selected based on their CIMP status (10 high, 10 low, and 26 negative) and the tumor sites. 16S rRNA gene sequencing using Illumina-Hi-Seq was performed on those samples. Principle Coordinate analysis of the 16S rRNA sequences showed that the microbiota of CIMP-positive (CIMP-high and -low combined) tumors was significantly different from the microbiota of the CIMP-negative tumors (PERMANOVA, P = 0.013). Linear discriminant analysis effect size (LEfSe) showed an enrichment of specific bacterial taxa in the CIMP-positive microbiota. Fusobacterium (LDA score = 4.71, p=0.033) and Erysipelotrichaceae (LDA score = 3.77, p=0.004) were most enriched in the CIMP-positive tumors along with Bacteroides. The microbiota of different tumor sites also showed significant difference with Fusobacterium (LDA score = 4.32, p=0.047) and Erysipelotrichaceae (LDA score = 3.45, p=0.039) being enriched in the proximal tumors compared to the distal tumors. All these bacterial taxa have previous been found to be associated with CRC and/or metabolic disorders. We validated the 16S rRNA data by qPCR analysis using genus-specific probes. In the same samples, Pan-Fusobacterium and Bacteroides fragilis were enriched in the CIMP-positive tumors. The median number of Pan-Fusobacterium per 100 human cells was 12-fold higher in the CIMP-positive tumors (p=0.0015) and 6-fold higher in the case of B. fragilis (p=0.0228). Thus, our data show broad difference in the microbiota of CIMP-positive CRCs compared to CIMP-negative CRCs. The significantly enriched bacterial taxa in CIMP-positive CRC suggest that these specific taxa could play an important role in aberrant DNA methylation modulation in colorectal cancer, and further studies should elucidate the mechanism underlying the CIMP/microbiota link in the context of CRC. Citation Format: Pyoung Hwa Park, Raad Z. Gharaibeh, Lauren Cole, Ang Sun, Woonbok Chung, Jaroslav Jelinek, Jillian L. Pope, Christian Jobin, Jean-Pierre J. Issa. CIMP is associated with altered microbiota composition in colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-141.