CpG Island Methylator Phenotype Research Articles

Clinicopathological, molecular, and prognostic features of colorectal carcinomas with KRAS c.34G>T (p.G12C) mutation.

Evidence indicates that combinations of anti-EGFR antibodies and KRAS p.G12C (c.34G>T) inhibitors can be an effective treatment strategy for advanced colorectal cancer. We hypothesized that KRAS c.34G>T (p.G12C)-mutated colorectal carcinoma might be a distinct tumor subtype. We utilized a prospective cohort incident tumor biobank (including 1347 colorectal carcinomas) and detected KRAS c.34G>T (p.G12C) mutation in 43 cases (3.2%) and other KRAS mutations (in codon 12, 13, 61, or 146) in 467 cases (35%). The CpG island methylator phenotype (CIMP)-low prevalence was similarly higher in KRAS c.34G>T mutants (52%) and other KRAS mutants (49%) than in KRAS-wild-type tumors (31%). KRAS c.34G>T mutants showed higher CIMP-high prevalence (14%) and lower CIMP-negative prevalence (33%) compared with other KRAS mutants (6% and 45%, respectively; p = 0.0036). Similar to other KRAS mutants, KRAS c.34G>T-mutated tumors were associated with cecal location, non-microsatellite instability (MSI)-high status, BRAF wild type, and PIK3CA mutation when compared with KRAS-wild-type tumors. Compared with BRAF-mutated tumors, KRAS c.34G>T mutants showed more frequent LINE-1 hypomethylation, a biomarker for early-onset colorectal carcinoma. KRAS c.34G>T mutants were not associated with other features, including the tumor tissue abundance of Fusobacterium nucleatum (F. animalis), pks+ Escherichia coli, Bifidobacterium, or (enterotoxigenic) Bacteroides fragilis. Among 1122 BRAF-wild-type colorectal carcinomas, compared with KRAS-wild-type tumors, multivariable-adjusted colorectal cancer-specific mortality hazard ratios (95% confidence interval) were 1.82 (1.05-3.17) in KRAS c.34G>T (p.G12C)-mutated tumors (p = 0.035) and 1.57 (1.22-2.02) in other KRAS-mutated tumors (p = 0.0004). Our study provides novel evidence for clinical and tumor characteristics of KRAS c.34G>T (p.G12C)-mutated colorectal carcinoma.

Exploring the interplay between colorectal cancer subtypes genomic variants and cellular morphology: A deep-learning approach.

Molecular subtypes of colorectal cancer (CRC) significantly influence treatment decisions. While convolutional neural networks (CNNs) have recently been introduced for automated CRC subtype identification using H&E stained histopathological images, the correlation between CRC subtype genomic variants and their corresponding cellular morphology expressed by their imaging phenotypes is yet to be fully explored. The goal of this study was to determine such correlations by incorporating genomic variants in CNN models for CRC subtype classification from H&E images. We utilized the publicly available TCGA-CRC-DX dataset, which comprises whole slide images from 360 CRC-diagnosed patients (260 for training and 100 for testing). This dataset also provides information on CRC subtype classifications and genomic variations. We trained CNN models for CRC subtype classification that account for potential correlation between genomic variations within CRC subtypes and their corresponding cellular morphology patterns. We assessed the interplay between CRC subtypes' genomic variations and cellular morphology patterns by evaluating the CRC subtype classification accuracy of the different models in a stratified 5-fold cross-validation experimental setup using the area under the ROC curve (AUROC) and average precision (AP) as the performance metrics. The CNN models that account for potential correlation between genomic variations within CRC subtypes and their cellular morphology pattern achieved superior accuracy compared to the baseline CNN classification model that does not account for genomic variations when using either single-nucleotide-polymorphism (SNP) molecular features (AUROC: 0.824±0.02 vs. 0.761±0.04, p<0.05, AP: 0.652±0.06 vs. 0.58±0.08) or CpG-Island methylation phenotype (CIMP) molecular features (AUROC: 0.834±0.01 vs. 0.787±0.03, p<0.05, AP: 0.687±0.02 vs. 0.64±0.05). Combining the CNN models account for variations in CIMP and SNP further improved classification accuracy (AUROC: 0.847±0.01 vs. 0.787±0.03, p = 0.01, AP: 0.68±0.02 vs. 0.64±0.05). The improved accuracy of CNN models for CRC subtype classification that account for potential correlation between genomic variations within CRC subtypes and their corresponding cellular morphology as expressed by H&E imaging phenotypes may elucidate the biological cues impacting cancer histopathological imaging phenotypes. Moreover, considering CRC subtypes genomic variations has the potential to improve the accuracy of deep-learning models in discerning cancer subtype from histopathological imaging data.

Immune microenvironmental heterogeneity according to tumor DNA methylation phenotypes in microsatellite instability-high colorectal cancers

The detailed association between tumor DNA methylation, including CpG island methylation, and tumor immunity is poorly understood. CpG island methylator phenotype (CIMP) is observed typically in sporadic colorectal cancers (CRCs) with microsatellite instability-high (MSI-H). Here, we investigated the differential features of the tumor immune microenvironment according to CIMP status in MSI-H CRCs. CIMP-high (CIMP-H) or CIMP-low/negative (CIMP-L/0) status was determined using MethyLight assay in 133 MSI-H CRCs. All MSI-H CRCs were subjected to digital pathology-based quantification of CD3 + /CD8 + /CD4 + /FoxP3 + /CD68 + /CD204 + /CD177 + tumor-infiltrating immune cells using whole-slide immunohistochemistry. Programmed death-ligand 1 (PD-L1) immunohistochemistry was evaluated using the tumor proportion score (TPS) and combined positive score (CPS). Representative cases were analyzed using whole-exome and RNA-sequencing. In 133 MSI-H CRCs, significantly higher densities of CD8 + tumor-infiltrating lymphocytes (TILs) were observed in CIMP-H tumors compared with CIMP-L/0 tumors. PD-L1 TPS and CPS in CIMP-H tumors were higher than in CIMP-L/0 tumors. Next-generation sequencing revealed that, compared with CIMP-L/0 tumors, CIMP-H tumors had higher fractions of CD8 + T cells/cytotoxic lymphocytes, higher cytolytic activity scores, and activated immune-mediated cell killing pathways. In contrast to CIMP-L/0 tumors, most CIMP-H tumors were identified as consensus molecular subtype 1, an immunogenic transcriptomic subtype of CRC. However, there were no differences in tumor mutational burden (TMB) between CIMP-H and CIMP-L/0 tumors in MSI-H CRCs. In conclusion, CIMP-H is associated with abundant cytotoxic CD8 + TILs and PD-L1 overexpression independent of TMB in MSI-H CRCs, suggesting that CIMP-H tumors represent a typical immune-hot subtype and are optimal candidates for immunotherapy in MSI-H tumors.

High-level tumour methylation of BRCA1 and RAD51C is required for homologous recombination deficiency in solid cancers.

In ovarian and breast cancer, promoter methylation of BRCA1 or RAD51C is a promising biomarker for PARP inhibitor response, as high levels lead to homologous recombination deficiency (HRD). Yet the extent and role of such methylation in other cancers is not clear. This study comprehensively investigated promoter methylation of eight homologous recombination repair genes across 23 solid cancer types. Here, we showed that BRCA1 methylated cancers were associated with reduced gene expression, loss of heterozygosity (LOH), TP53 mutations and genomic features of HRD. We identified BRCA1 methylation in 3% of the copy-number high subtype of endometrial cancer, and as a rare event in six other cancer types, including lung squamous cell, pancreatic, bladder and stomach cancer. RAD51C promoter methylation was widespread across multiple cancer types, but HRD features were only observed for cases which contained high-level tumour methylation and LOH of RAD51C. While RAD51C methylation was frequent in stomach adenocarcinoma (6%) and low-grade glioma (2.5%), it was mostly detected at a low tumour level, suggestive of heterozygous methylation, and was associated with CpG island methylator phenotype. Our findings indicate that high-level tumour methylation of BRCA1 and RAD51C should be explored as a PARP inhibitor biomarker across multiple cancers.

Epigenetic alterations affecting hematopoietic regulatory networks as drivers of mixed myeloid/lymphoid leukemia

Leukemias with ambiguous lineage comprise several loosely defined entities, often without a clear mechanistic basis. Here, we extensively profile the epigenome and transcriptome of a subgroup of such leukemias with CpG Island Methylator Phenotype. These leukemias exhibit comparable hybrid myeloid/lymphoid epigenetic landscapes, yet heterogeneous genetic alterations, suggesting they are defined by their shared epigenetic profile rather than common genetic lesions. Gene expression enrichment reveals similarity with early T-cell precursor acute lymphoblastic leukemia and a lymphoid progenitor cell of origin. In line with this, integration of differential DNA methylation and gene expression shows widespread silencing of myeloid transcription factors. Moreover, binding sites for hematopoietic transcription factors, including CEBPA, SPI1 and LEF1, are uniquely inaccessible in these leukemias. Hypermethylation also results in loss of CTCF binding, accompanied by changes in chromatin interactions involving key transcription factors. In conclusion, epigenetic dysregulation, and not genetic lesions, explains the mixed phenotype of this group of leukemias with ambiguous lineage. The data collected here constitute a useful and comprehensive epigenomic reference for subsequent studies of acute myeloid leukemias, T-cell acute lymphoblastic leukemias and mixed-phenotype leukemias.

HGG-04. MALIGNANT GLIOMA IN L-2-HYDROXY GLUTARIC ACIDURIA; THOROUGH MOLECULAR CHARACTERIZATION OF A CASE AND LITERATURE REVIEW

Abstract L-2-hydroxyglutaric aciduria (L-2-HGA) is a rare neurometabolic disorder characterized by accumulation of L2-hydroxyglutarate (L-2-HG) due to mutations in the L2HGDH gene. L-2-HGA patients have a significantly increased lifetime risk of central nervous system (CNS) tumors. Here, we present a 16-year-old girl with L-2-HGA who developed a tumor in the right cerebral hemisphere, which was discovered after left-sided neurological deficits of the patient. Histologically, the tumor had a high-grade diffuse glioma phenotype. DNA sequencing revealed the germline L2HGDH mutation as well as inactivating mutations in TP53, BCOR and NF1. Genome-wide DNA-methylation analysis was unable to classify the tumor with high confidence. More detailed analysis revealed that this tumor clustered amongst IDH-wildtype gliomas by methylation profiling and did not show the glioma CpG island methylator phenotype (G-CIMP) (in contrast to IDH-mutant diffuse gliomas with accumulated levels of D-2-HG). These findings were against all our expectations given the inhibitory potential of 2-HG on DNA-demethylation enzymes. Our final integrated histomolecular diagnosis was diffuse pediatric-type high-grade glioma, H3-wildtype and IDH-wildtype. Due to rapid tumor progression the patient died nine months after initial diagnosis. In this manuscript we provide extensive molecular characterization of the tumor as well as a literature review focusing on oncogenetic considerations of L-2-HGA-associated CNS tumors.

Unmasking early colorectal cancer clues: in silico and in vitro investigation of downregulated IGF2, SOCS1, MLH1, and CACNA1G in SSA polyps

Background and aimColorectal cancer (CRC) originates from pre-existing polyps in the colon. The development of different subtypes of CRC is influenced by various genetic and epigenetic characteristics. CpG island methylator phenotype (CIMP) is found in about 15–20% of sporadic CRCs and is associated with hypermethylation of certain gene promoters. This study aims to find prognostic genes and compare their expression and methylation status as potential biomarkers in patients with serrated sessile adenomas/polyps (SSAP) and CRC, in order to evaluate which, one is a better predictor of disease.MethodThis study employed a multi-phase approach to investigate genes associated with CRC and SSAP. Initially, two gene expression datasets were analyzed using R and Limma package to identify differentially expressed genes (DEGs). Venn diagram analysis further refined the selection, revealing four genes from the Weissenberg panel with significant changes. These genes, underwent thorough in silico evaluations. Once confirmed, they proceeded to wet lab experimentation, focusing on expression and methylation status. This comprehensive methodology ensured a robust examination of the genes involved in CRC and SSAP.ResultThis study identified cancer-specific genes, with 8,351 and 1,769 genes specifically down-regulated in SSAP and CRC tissues, respectively. The down-regulated genes were associated with cell adhesion, negative regulation of cell proliferation, and drug response. Four highly downregulated genes in the Weissenberg panel, including CACNA1G, IGF2, MLH1, and SOCS1. In vitro analysis showed that they are hypermethylated in both SSAP and CRC samples while their expressions decreased only in CRC samples.ConclusionThis suggests that the decrease in gene expression could help determine whether a polyp will become cancerous. Using both methylation status and gene expression status of genes in the Weissenberg panel in prognostic tests may lead to better prognoses for patients.

Association between gut microbiota and CpG island methylator phenotype in colorectal cancer

ABSTRACT The intestinal microbiota is an important environmental factor implicated in CRC development. Intriguingly, modulation of DNA methylation by gut microbiota has been reported in preclinical models, although the relationship between tumor-infiltrating bacteria and CIMP status is currently unexplored. In this study, we investigated tumor-associated bacteria in 203 CRC tumor cases and validated the findings using The Cancer Genome Atlas datasets. We assessed the abundance of Bacteroides fragilis, Escherichia coli, Fusobacterium nucleatum, and Klebsiella pneumoniae through qPCR analysis and observed enrichment of all four bacterial species in CRC samples. Notably, except for E. coli, all exhibited significant enrichment in cases of CIMP. This enrichment was primarily driven by a subset of cases distinguished by high levels of these bacteria, which we labeled as “Superhigh”. The bacterial Superhigh status showed a significant association with CIMP (odds ratio 3.1, p-value = 0.013) and with MLH1 methylation (odds ratio 4.2, p-value = 0.0025). In TCGA CRC cases (393 tumor and 45 adj. normal), bacterial taxa information was extracted from non-human whole exome sequencing reads, and the bacterial Superhigh status was similarly associated with CIMP (odds ratio 2.9, p < 0.001) and MLH1 methylation (odds ratio 3.5, p < 0.001). Finally, 16S ribosomal RNA gene sequencing revealed high enrichment of Bergeyella spp. C. concisus, and F. canifelinum in CIMP-Positive tumor cases. Our findings highlight that specific bacterial taxa may influence DNA methylation, particularly in CpG islands, and contribute to the development and progression of CIMP in colorectal cancer.

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.

Unraveling the Progression of Colon Cancer Pathogenesis Through Epigenetic Alterations and Genetic Pathways.

In the modern age, colon cancer has attained a widespread status, affecting a considerable number of people.It develops due to the progressive accumulation of genetic and epigenetic alterations. While genetic mutations have been extensively studied in the context of colon cancer, emerging evidence highlights the pivotal role of epigenetic alterations in its pathogenesis. These alterations ultimately result in the transformation of normal colonic epithelium into colon adenocarcinoma.Key mechanisms of epigenetic modifications include DNA methylation, histone modification, and nucleosome positioning. Research findings have linked these modifications to the development, progression, or metastasis of tumors. Through the assessment of the colon cancer epigenome,it has been discovered that practically all colorectal cancers (CRCs) display gene methylation abnormalities and changes in miRNA expression. Advancements in this area indicate that epigenetic modifications will likely be commonly used in the near future to direct the prevention and treatment of CRC. The maintenance of genome stability is essential for preserving cellular integrity. The development of CRC is primarily influenced by the loss of genomic stability, which allows for the emergence of new mutations contributing to tumor characteristics. Although genetic mutations have been extensively researched in the realm of colon cancer, recent evidence underscores the pivotal role of epigenetic changes in its pathogenesis. The following types of genomic instability will be discussed: chromosomal instability, microsatellite instability, CpG island methylation phenotype, and aberrant DNA methylation.

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.

Weighting estimation in the cause-specific Cox regression with partially missing causes of failure.

Complex diseases are often analyzed using disease subtypes classified by multiple biomarkers to study pathogenic heterogeneity. In such molecular pathological epidemiology research, we consider a weighted Cox proportional hazard model to evaluate the effect of exposures on various disease subtypes under competing-risk settings in the presence of partially or completely missing biomarkers. The asymptotic properties of the inverse and augmented inverse probability-weighted estimating equation methods are studied with a general pattern of missing data. Simulation studies have been conducted to demonstrate the double robustness of the estimators. For illustration, we applied this method to examine the association between pack-years of smoking before the age of 30 and the incidence of colorectal cancer subtypes defined by a combination of four tumor molecular biomarkers (statuses of microsatellite instability, CpG island methylator phenotype, BRAF mutation, and KRAS mutation) in the Nurses' Health Study cohort.

Abstract P43: Optimization of DNA Methyltransferase Inhibitors-based Combination to Target the Epigenome of Gastric Cancer

Abstract Aberrant DNA methylation is one of the critical epigenetic modifications that has become a widespread phenotype in solid tumours. Similar to haematological malignancies, many studies have shown that the DNA methylation landscape of solid tumours exhibits a pattern of either hypermethylation of the promoter regions of tumour-suppressive genes in advanced cancer or global hypomethylation in the early stages of carcinogenesis. Additionally, dysregulated DNA methylation also seems to remodel the tumour microenvironment and affect immune cells' response towards immune checkpoint inhibitors. In gastric cancer, comprehensive DNA methylation analyses have demonstrated that gastric tumours carry the CpG island methylation phenotype (CIMP), resulting in extended molecular-based subgroups such as the extremely high-methylation epigenotype (E-HME), HME, and low-methylation epigenotype (LME). Therefore, conventional histology grading and molecular subtyping are insufficient to address the multifaceted epigenome of gastric cancer. As such, current standard-of-care (SOC) therapy is not able to overcome cancer relapse and tumour metastasis. Clearly, this shows the therapeutic potential of using DNA hypomethylating drugs like DNA methyltransferase inhibitors (DNMTi) for solid tumours such as gastric cancer. In our study, we exploited the engineering-based Quadratic Phenotypic Optimization Platform (QPOP) to identify top-ranking DNMTi-based drug combinations across gastric cancer cell lines (GC-CL) and patient-derived organoids (GC-PDO). Based on QPOP analysis, we showed that a non-SOC drug combination, Docetaxel/5-Azacytidine is a frequently top-ranking. Besides, our present findings suggest that CIMP-high gastric cancer cells are more susceptible towards DNMT inhibitors through the specific reduction of DNMT1 levels. As we surveyed the effects of Docetaxel/5-Azacytidine in a CIMP-high GC-CL via Infinium methylation array, we observed robust gene methylation changes that are involved in non-canonical Wnt Signaling and EMT. On the other hand, proteomic analysis revealed a small subset of our panel of GC lines that are positive for the cancer-testis antigen, MageA4. Interestingly, the protein expression of MageA4 in some CIMP-high lines is restored upon treatment with DNMT inhibitors. This evinces that DNMTi can subject epigenetically silenced MageA4 gastric tumours to autologous T-cell receptor therapy. Taken altogether, we plan to further interrogate the mechanisms of DNMT inhibitors in sensitizing CIMP-high gastric tumours towards Taxanes by acting on the non-canonical Wnt pathway and EMT activators. Citation Format: Lissa Hooi, Vivien Koh, Dexter Kai Hao Thng, Jasmine Goh, Lim Jhin Jieh, Lee Rui Xue, Masturah Bte Mohd Abdul Rashid, Toh Tan Boon, Wei Peng Yong, Edward Kai-Hua Chow. Optimization of DNA Methyltransferase Inhibitors-based Combination to Target the Epigenome of Gastric Cancer [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P43.

Abstract 4410: The CpG island methylator phenotype is closely associated with prognosis in patients with oral squamous cell carcinomas

Abstract Few abnormalities are known to be closely associated with prognosis in OSCC cases. In this study, genomic abnormalities of cancer-related genes and epigenomic abnormalities of genes methylation-silenced in OSCCs were analyzed to identify abnormalities associated with prognosis. Thirty-nine OSCC cases and their surgical materials were analyzed for the presence of mutations using next-generation sequencing. Methylation status of promoter CpG islands of five genes was analyzed by methylation-specific PCR. Mutations in CRG, TP53 and PIK3CA were observed in 64.1%, 38.5%, and 33.3%, respectively, of the cases. However, no significant association between mutation status and overall survival (OS) was observed. Aberrant methylation of CMTM3, NKX2-3, RBP4, EGFLAM, and MAP6 were observed in 51.3%, 41.0%, 66.7%, 38.5%, and 41.0%, respectively. Methylation of NKX2-3, EGFLAM, and MAP6 were significantly associated with OS in OSCC cases (p=0.006, 0.006, and 0.012, respectively). Cases with methylation of CMTM3 and RBP4 showed a trend toward poor prognosis (p=0.052 and 0.059, respectively). Methylation profiles of the five genes indicated the presence of the CpG island methylator phenotype (CIMP), and the CIMP-positive cases showed poor prognosis (HR=20.5, p&amp;lt;0.001). In conclusion, CIMP was closely associated with the prognosis of OSCC cases. Citation Format: Masanobu Abe, Satoshi Yamashita, Toshikazu Ushijima, Kazuto Hoshi. The CpG island methylator phenotype is closely associated with prognosis in patients with oral squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4410.

Abstract 3241: Elevated H3K27 trimethylation mediates adaptation to DNA demethylation in BRAFV600E-mutated colorectal cancer

Abstract Background: BRAFV600E in colorectal cancer (CRC) defines a unique subgroup of patients with poor prognosis and scarcely benefit from BRAF inhibitor-based treatment. Interestingly, BRAFV600E tumors demonstrated profound CpG island methylator phenotype (CIMP), suggesting extensive epigenetic dysregulation. Thus, we investigated epigenomic regulations in this subset of tumors with treatment-induced DNA demethylation. Methods: We treated six cell lines and two patient-derived xenograft (PDX) models of BRAFV600E CRC with vehicle, 5-azacitidine (DNMTi), vemurafenib (BRAFi), and combination treatment. PDX tumors treated with either vehicle or 5-azacitidine were collected on day 21 and sequenced for DNA methylation (Infinium HumanMethylation 450k BeadChip array), histone marks (ChIP-seq), chromatin accessibility (ATAC-seq), and gene expression (RNA-seq). Comprehensive post-translational modifications (PTMs) in histones with DNMTi or BRAFi treatment were assessed by mass spectrometry (MS). The combinational effect of 5-azacitidine with EZH2 or RNF2 inhibitors (Polycomb repressive complex activity (PRC) inhibition) was assessed through cell viability assays and colony formation assays in BRAFV600E CRC cell lines. Results: Analysis of TCGA COAD/READ tumors demonstrated a higher DNA methylation profile in BRAFV600E tumors than wild-type tumors even among the CIMP population (FDR &amp;lt; 1e-4). The combination of DNMTi with BRAFi showed selective additivity in vitro; however, did not improve efficacies in PDXs. The 5-azacitidine treatment induced profound demethylation in PDXs compared to control samples, predominantly attributed to promoter regions (48%). However, DNA demethylation failed to reactivate classic CIMP markers or tumor suppressor genes in CRC (e.g., CDKN2A, MGMT). Interestingly, demethylated genomic regions gained H3K27 trimethylation, repressive histone mark, in azacitidine-treated samples compared to control (9,431 vs 5,147 peaks respectively). We validated the elevation of H3K27 trimethylation as well as additional histone PTMs following DNMTi in MS-based histone analysis. Finally, combining a demethylating agent with EZH2i or RNF2i, which either deplete H3K27 trimethylation or block the PRC activity respectively, has demonstrated additive growth inhibitory effects in BRAFV600E CRC cell lines (p &amp;lt;0.05). Conclusions: This study unveiled intriguing aspects of demethylation agent treatment, which led to the compensatory engagement of other epigenomic markers, particularly repressive histone marks, resulting in limited efficacies to azacitidine treatment in preclinical BRAFV600E CRC models. This finding suggests the existence of adaptive interactions between epigenetic modifiers and potential clinical strategies in combinational epigenetic therapeutics. Citation Format: Hey Min Lee, Ajay Kumar Saw, Van Karlyle Morris, Anand Kamal Singh, Stefania Napolitano, Alexey Sorokin, Preeti Kanikarla Marie, Oluwadara Coker, Oscar Eduardo Villarreal, Nazanin Esmaeili Anvar, Kunal Rai, Scott Kopetz. Elevated H3K27 trimethylation mediates adaptation to DNA demethylation in BRAFV600E-mutated colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3241.

Epigenetic MLH1 silencing concurs with mismatch repair deficiency in sporadic, naturally occurring colorectal cancer in rhesus macaques

BackgroundNaturally occurring colorectal cancers (CRC) in rhesus macaques share many features with their human counterparts and are useful models for cancer immunotherapy; but mechanistic data are lacking regarding the comparative molecular pathogenesis of these cancers.MethodsWe conducted state-of-the-art imaging including CT and PET, clinical assessments, and pathological review of 24 rhesus macaques with naturally occurring CRC. Additionally, we molecularly characterized these tumors utilizing immunohistochemistry (IHC), microsatellite instability assays, DNAseq, transcriptomics, and developed a DNA methylation-specific qPCR assay for MLH1, CACNA1G, CDKN2A, CRABP1, and NEUROG1, human markers for CpG island methylator phenotype (CIMP). We furthermore employed Monte-Carlo simulations to in-silico model alterations in DNA topology in transcription-factor binding site-rich promoter regions upon experimentally demonstrated DNA methylation.ResultsSimilar cancer histology, progression patterns, and co-morbidities could be observed in rhesus as reported for human CRC patients. IHC identified loss of MLH1 and PMS2 in all cases, with functional microsatellite instability. DNA sequencing revealed the close genetic relatedness to human CRCs, including a similar mutational signature, chromosomal instability, and functionally-relevant mutations affecting KRAS (G12D), TP53 (R175H, R273*), APC, AMER1, ALK, and ARID1A. Interestingly, MLH1 mutations were rarely identified on a somatic or germline level. Transcriptomics not only corroborated the similarities of rhesus and human CRCs, but also demonstrated the significant downregulation of MLH1 but not MSH2, MSH6, or PMS2 in rhesus CRCs. Methylation-specific qPCR suggested CIMP-positivity in 9/16 rhesus CRCs, but all 16/16 exhibited significant MLH1 promoter hypermethylation. DNA hypermethylation was modelled to affect DNA topology, particularly propeller twist and roll profiles. Modelling the DNA topology of a transcription factor binding motif (TFAP2A) in the MLH1 promoter that overlapped with a methylation-specific probe, we observed significant differences in DNA topology upon experimentally shown DNA methylation. This suggests a role of transcription factor binding interference in epigenetic silencing of MLH1 in rhesus CRCs.ConclusionsThese data indicate that epigenetic silencing suppresses MLH1 transcription, induces the loss of MLH1 protein, abrogates mismatch repair, and drives genomic instability in naturally occurring CRC in rhesus macaques. We consider this spontaneous, uninduced CRC in immunocompetent, treatment-naïve rhesus macaques to be a uniquely informative model for human CRC.Graphical abstract

Prognostic and predictive role of immune microenvironment in colorectal cancer.

Colorectal cancer (CRC) represents a molecularly heterogeneous disease and one of the most frequent causes of cancer-related death worldwide. The traditional classification of CRC is based on pathomorphological and molecular characteristics of tumor cells (mucinous, ring-cell carcinomas, etc.), analysis of mechanisms of carcinogenesis involved (chromosomal instability, microsatellite instability, CpG island methylator phenotype) and mutational statuses of commonly altered genes (KRAS, NRAS, BRAF, APC, etc.), as well as expression signatures (CMS 1-4). It is also suggested that the tumor microenvironment is a key player in tumor progression and metastasis in CRC. According to the latest data, the immune microenvironment can also be predictive of the response to immune checkpoint inhibitors. In this review, we highlight how the immune environment influences CRC prognosis and sensitivity to systemic therapy.

Molecular pathological approach to cancer epigenomics and its clinical application.

Careful microscopic observation of histopathological specimens, accumulation of large numbers of high-quality tissue specimens, and analysis of molecular pathology in relation to morphological features are considered to yield realistic data on the nature of multistage carcinogenesis. Since the morphological hallmark of cancer is disruption of the normal histological structure maintained through cell-cell adhesiveness and cellular polarity, attempts have been made to investigate abnormalities of the cadherin-catenin cell adhesion system in human cancer cells. It has been shown that the CDH1 tumor suppressor gene encoding E-cadherin is silenced by DNA methylation, suggesting that a "double hit" involving DNA methylation and loss of heterozygosity leads to carcinogenesis. Therefore, in the 1990s, we focused on epigenomic mechanisms, which until then had not received much attention. In chronic hepatitis and liver cirrhosis associated with hepatitis virus infection, DNA methylation abnormalities were found to occur frequently, being one of the earliest indications that such abnormalities are present even in precancerous tissue. Aberrant expression and splicing of DNA methyltransferases, such as DNMT1 and DNMT3B, was found to underlie the mechanism of DNA methylation alterations in various organs. The CpG island methylator phenotype in renal cell carcinomawas identified for the first time, and its therapeutic targets were identified by multilayer omics analysis. Furthermore, the DNA methylation profile of nonalcoholic steatohepatitis (NASH)-related hepatocellular carcinomawas clarified in groundbreaking studies. Since then, we have developed diagnostic markers for carcinogenesis risk in NASH patients and noninvasive diagnostic markers for upper urinary tract cancer, as well as developing a new high-performance liquid chromatography-based diagnostic system for DNA methylation diagnosis. Research on the cancer epigenome has revealed that DNA methylation alterations occur from the precancerous stage as a result of exposure to carcinogenic factors such as inflammation, smoking, and viral infections, and continuously contribute to multistage carcinogenesis through aberrant expression of cancer-related genes and genomic instability. DNA methylation alterations at the precancerous stages are inherited by or strengthened in cancers themselves and determine the clinicopathological aggressiveness of cancers as well as patient outcome. DNA methylation alterations have applications as biomarkers, and are expected to contribute to diagnosis, as well as preventive and preemptive medicine.

The histologic features, molecular features, detection and management of serrated polyps: a review.

The serrated pathway to colorectal cancers (CRCs) is a significant pathway encompassing five distinct types of lesions, namely hyperplastic polyps (HPs), sessile serrated lesions (SSLs), sessile serrated lesions with dysplasia (SSL-Ds), traditional serrated adenomas (TSAs), and serrated adenoma unclassified. In contrast to the conventional adenoma-carcinoma pathway, the serrated pathway primarily involves two mechanisms: BRAF/KRAS mutations and CpG island methylator phenotype (CIMP). HPs are the most prevalent non-malignant lesions, while SSLs play a crucial role as precursors to CRCs, On the other hand, traditional serrated adenomas (TSAs) are the least frequently encountered subtype, also serving as precursors to CRCs. It is crucial to differentiate these lesions based on their unique morphological characteristics observed in histology and colonoscopy, as the identification and management of these serrated lesions significantly impact colorectal cancer screening programs. The management of these lesions necessitates the crucial steps of removing premalignant lesions and implementing regular surveillance. This article provides a comprehensive summary of the epidemiology, histologic features, molecular features, and detection methods for various serrated polyps, along with recommendations for their management and surveillance.

IDH mutation, glioma immunogenicity, and therapeutic challenge of primary mismatch repair deficient IDH‐mutant astrocytoma PMMRDIA: a systematic review

In 2021, Suwala et al. described Primary Mismatch Repair Deficient IDH‐mutant Astrocytoma (PMMRDIA) as a distinct group of gliomas. In unsupervised clustering, PMMRDIA forms distinct cluster, separate from other IDH‐mutant gliomas, including IDH‐mutant gliomas with secondary mismatch repair (MMR) deficiency. In the published cohort, three patients received treatment with an immune checkpoint blocker (ICB), yet none exhibited a response, which aligns with existing knowledge about the decreased immunogenicity of IDH‐mutant gliomas in comparison to IDH‐wildtype. In the case of PMMRDIA, the inherent resistance to the standard‐of‐care temozolomide caused by MMR deficiency is an additional challenge. It is known that a gain‐of‐function mutation of IDH1/2 genes produces the oncometabolite R‐2‐hydroxyglutarate (R‐2‐HG), which increases DNA and histone methylation contributing to the characteristic glioma‐associated CpG island methylator phenotype (G‐CIMP). While other factors could be involved in remodeling the tumor microenvironment (TME) of IDH‐mutant gliomas, this systematic review emphasizes the role of R‐2‐HG and the subsequent G‐CIMP in immune suppression. This highlights a potential actionable pathway to enhance the response of ICB, which might be relevant for addressing the unmet therapeutic challenge of PMMRDIA.