CpG Island Methylator Phenotype Research Articles

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.

Single-cell chromatin accessibility analysis reveals the epigenetic basis and signature transcription factors for the molecular subtypes of colorectal cancers.

Colorectal cancer (CRC) is a highly heterogeneous disease, with well-characterized subtypes based on genome, DNA methylome, and transcriptome signatures. To chart the epigenetic landscape of CRCs, we generated a high-quality single-cell chromatin accessibility atlas of epithelial cells for 29 patients. Abnormal chromatin states acquired in adenomas were largely retained in CRCs, which were tightly accompanied by opposite changes of DNA methylation. Unsupervised analysis on malignant cells revealed two epigenetic subtypes, exactly matching iCMS classification, and key iCMS-specific transcription factors were identified, including HNF4A, PPARA for iCMS2 tumors, and FOXA3, MAFK for iCMS3 tumors. Notably, subtype-specific TFs bind to distinct target gene sets and contribute to both inter-patient similarities and diversities for both chromatin accessibilities and RNA expressions. Moreover, we identified CpG-island methylator phenotypes and pinpointed chromatin state signatures and TF regulators for CIMP-High subtype. Our work systematically revealed the epigenetic basis of the well-known iCMS and CIMP classifications of CRCs.

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<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 < 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 <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.

Intratumoral presence of the genotoxic gut bacteria pks+E. coli, Enterotoxigenic Bacteroides fragilis, and Fusobacterium nucleatum and their association with clinicopathological and molecular features of colorectal cancer

BackgroundThis study aimed to investigate clinicopathological and molecular tumour features associated with intratumoral pks+Escherichia coli (pks+E.coli+), pks+E.coli- (non-E.coli bacteria harbouring the pks island), Enterotoxigenic Bacteroides fragilis (ETBF) and Fusobacterium nucleatum (F. nucleatum).MethodsWe screened 1697 tumour-derived DNA samples from the Australasian Colorectal Cancer Family Registry, Melbourne Collaborative Cohort Study and the ANGELS study using targeted PCR.ResultsPks+E.coli+ was associated with male sex (P < 0.01) and APC:c.835-8 A > G somatic mutation (P = 0.03). The association between pks+E.coli+ and APC:c.835-8 A > G was specific to early-onset CRCs (diagnosed<45years, P = 0.02). The APC:c.835-A > G was not associated with pks+E.coli- (P = 0.36). F. nucleatum was associated with DNA mismatch repair deficiency (MMRd), BRAF:c.1799T>A p.V600E mutation, CpG island methylator phenotype, proximal tumour location, and high levels of tumour infiltrating lymphocytes (Ps < 0.01). In the stratified analysis by MMRd subgroups, F. nucleatum was associated with Lynch syndrome, MLH1 methylated and double MMR somatic mutated MMRd subgroups (Ps < 0.01).ConclusionIntratumoral pks+E.coli+ but not pks+E.coli- are associated with CRCs harbouring the APC:c.835-8 A > G somatic mutation, suggesting that this mutation is specifically related to DNA damage from colibactin-producing E.coli exposures. F. nucleatum was associated with both hereditary and sporadic MMRd subtypes, suggesting the MMRd tumour microenvironment is important for F. nucleatum colonisation irrespective of its cause.

Enrichment of Bacteroides fragilis and enterotoxigenic Bacteroides fragilis in CpG island methylator phenotype-high colorectal carcinoma

ObjectivesData support that enterotoxigenic Bacteroides fragilis (ETBF) harbouring the Bacteroides fragilis toxin (bft) gene may promote colorectal tumourigenesis through the serrated neoplasia pathway. We hypothesized that ETBF may be enriched in colorectal carcinoma subtypes with high-level CpG island methylator phenotype (CIMP-high), BRAF mutation, and high-level microsatellite instability (MSI-high). MethodsQuantitative PCR assays were designed to quantify DNA amounts of Bacteroides fragilis, ETBF, and each bft gene isotype (bft-1, bft-2, or bft-3) in colorectal carcinomas in the Health Professionals Follow-up Study and Nurses' Health Study. We used multivariable-adjusted logistic regression models with the inverse probability weighting method. ResultsWe documented 4476 colorectal cancer cases, including 1232 cases with available bacterial data. High DNA amounts of Bacteroides fragilis and ETBF were positively associated with BRAF mutation (p ≤ 0.0003), CIMP-high (p ≤ 0.0002), and MSI-high (p < 0.0001 and p = 0.01, respectively). Multivariable-adjusted odds ratios (with 95% confidence interval) for high Bacteroides fragilis were 1.40 (1.06–1.85) for CIMP-high and 2.14 (1.65–2.77) for MSI-high, but 1.02 (0.78–1.35) for BRAF mutation. Multivariable-adjusted odds ratios for high ETBF were 2.00 (1.16–3.45) for CIMP-high and 2.86 (1.64–5.00) for BRAF mutation, but 1.09 (0.67–1.76) for MSI-high. Neither Bacteroides fragilis nor ETBF was associated with colorectal cancer-specific or overall survival. DiscussionThe tissue abundance of Bacteroides fragilis is associated with CIMP-high and MSI-high, whereas ETBF abundance is associated with CIMP-high and BRAF mutation in colorectal carcinoma. Our findings support the aetiological relevance of Bacteroides fragilis and ETBF in the serrated neoplasia pathway.

Malignant glioma in L-2-Hydroxyglutaric Aciduria: thorough molecular characterization of a case and literature review.

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 inactivating homozygous 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, the stereoisomer of L-2-HD. These findings were against all our expectations given the inhibitory potential of 2-HG on DNA-demethylation enzymes. Our final integrated histomolecular diagnosis of the tumor 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.

Focusing on colorectal cancer in young adults (Review).

Colorectal cancer (CRC) ranks as the third leading cause of cancer-related mortality worldwide. Recent years have witnessed an increase in the incidence of CRC among adults <50 years old on a global scale. The increased incidence is associated with several modifiable risk factors, including obesity, type II diabetes, physical inactivity and frequent antibiotic use. In younger individuals, haematochezia and abdominal pain are the most common symptoms, predominantly affecting the left-side colon. While certain cases of early-onset CRC (eoCRC) are associated with a genetic predisposition, the majority result from sporadic mutations in the genes APC, KRAS, BRAF and TP53, which trigger uncontrolled cell proliferation and tumour formation. Colorectal carcinogenesis involves three major pathways: The chromosomal instability (CIN), microsatellite instability and CpG island methylator phenotype pathways. Dysregulation of the CIN pathway accounts for 85% of sporadic cases of eoCRC. Notably, eoCRC exhibits a distinctive molecular profile, characterized by a decreased prevalence of BRAF mutations, an increased prevalence of KRAS mutations and LINE-1 hypomethylation, and involvement of the Microsatellite and Chromosomal Stable pathway. Prevention strategies for eoCRC primarily centre on lifestyle modifications and the development of screening programs targeting younger populations. Further exploration into the molecular mechanisms involved in the identification of novel risk factors associated with eoCRC is imperative. These efforts, in conjunction with the development of specific screening strategies, hold the potential to reduce morbidity and mortality in the future.

CTNI-47. DOSE ESCALATION OF ASTX727 IN ADULT PATIENTS WITH RECURRENT/PROGRESSIVE NON-ENHANCING IDH MUTANT GLIOMAS

Abstract BACKGROUND IDH mutant gliomas typically display a hypermethylation pattern known as the glioma CpG island methylator phenotype (G-CIMP) which results in gene activation that promotes gliomagenesis. This study aims to reverse this methylation signature and disrupt tumor growth via treatment with an oral demethylating agent. METHODS This is 2-stage clinical trial investigating oral ASTX727 (decitabine + cedazuridine, a cytidine deaminase inhibitor, as a combination tablet) in patients with recurrent primarily non-enhancing IDH mutant gliomas. The Dose Escalation Stage consisted of a single arm 3 + 3 design. The primary objective was to establish the MTD and assess safety in this patient population. RESULTS Eleven pts (F = 45%, M = 55%) with a median age of 44 yrs (range 26-61 yrs) were enrolled. Two DLTs (grade 4 neutropenia for &amp;gt;72 hrs) were observed in 5 pts enrolled at the starting dose (35 mg decitabine/ 100 mg cedazuridine for 5 consecutive days). Six additional patients were enrolled at dose level -1 (35 mg decitabine/100 mg cedazuridine for 4 consecutive days). There were no DLTs at dose level -1 and this was declared the MTD. Grade ≥ 3 AEs included neutropenia (5 pts), leukopenia (1 pt), lymphopenia (1 pt), hyponatremia (1 pt) and increased ALT (1 pt). Best treatment response was SD in 11 patients. At the time of last follow-up (5/15/23) 10 pts have discontinued treatment (8 for PD, and 2 based on physician decision), 2 pts (18%) have died due to disease progression, 8 pts remain alive and in follow-up, and 1 patient remains on treatment. Median duration on treatment was 212 days (range 36 days – not reached). CONCLUSIONS ASTX727 was well tolerated in patients with recurrent non-enhancing IDH mutant glioma at dose level -1 with no DLTs . A surgical expansion cohort is currently enrolling to confirm drug target engagement and explore pharmacodynamics.

Helicobacter pylori infection associated DNA methylation in primary gastric cancer significantly correlates with specific molecular and clinicopathological features.

Helicobacter pyloriinduces DNA methylation in gastric mucosa, which links to gastric cancer (GC) risk. In contrast, CpG island methylator phenotype (CIMP) is defined as high levels of cancer-specific methylation and provides distinct molecular and clinicopathological features of GC. The association between those two types of methylation in GC remains unclear. We examined DNA methylation of well-validated H. pylori infection associated genes in GC and its adjacent mucosa and investigated its association with CIMP, various molecular subtypes and clinical features.We studied 50 candidate loci in 24 gastric samples to identify H. pylori infection associated genes. Identified loci were further examined in 624 gastric tissue from 217 primary GC, 217 adjacent mucosa, and 190 mucosae from cancer-free subjects.We identified fivegenes (IGF2, SLC16A2, SOX11, P2RX7, and MYOD1) as hypermethylated in H. pylori infected gastric mucosa. In non-neoplastic mucosa, methylation of H. pylori infection associated genes was higher in patients with GC than those without. In primary GC tissues, higher methylation of H. pylori infection associated genes correlated with CIMP-positive and its related features, such as MLH1 methylated cases. On the other hand, GC with lower methylation of these genes presented aggressive clinicopathological features including undifferentiated histopathology, advanced stage at diagnosis.H. pylori infection associated DNA methylation is correlated with CIMP, specific molecular and clinicopathological features in GC, supporting its utility as promising biomarker in this tumor type.

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.

Decitabine disrupts EBV genomic epiallele DNA methylation patterns around CTCF binding sites to increase chromatin accessibility and lytic transcription in gastric cancer.

Epstein-Barr virus (EBV) latency is controlled by epigenetic silencing by DNA methylation [5-methyl cytosine (5mC)], histone modifications, and chromatin looping. However, how they dictate the transcriptional program in EBV-associated gastric cancers remains incompletely understood. EBV-associated gastric cancer displays a 5mC hypermethylated phenotype. A potential treatment for this cancer subtype is the DNA hypomethylating agent, which induces EBV lytic reactivation and targets hypermethylation of the cellular DNA. In this study, we identified a heterogeneous pool of EBV epialleles within two tumor-derived gastric cancer cell lines that are disrupted with a hypomethylating agent. Stochastic DNA methylation patterning at critical regulatory regions may be an underlying mechanism for spontaneous reactivation. Our results highlight the critical role of epigenetic modulation on EBV latency and life cycle, which is maintained through the interaction between 5mC and the host protein CCCTC-binding factor.

Microbiota composition and its impact on DNA methylation in colorectal cancer.

Colorectal cancer is a complex disease resulting from the interaction of genetics, epigenetics, and environmental factors. DNA methylation is frequently found in tumor suppressor genes to promote cancer development. Several factors are associated with changes in the DNA methylation pattern, and recently, the gastrointestinal microbiota could be associated with this epigenetic change. The predominant phyla in gut microbiota are Firmicutes and Bacteroidetes; however, an enrichment of Bacteroides fragilis, Fusobacterium nucleatum, and Streptococcus bovis, among others, has been reported in colorectal cancer, although the composition could be influenced by several factors, including diet, age, sex, and cancer stage. Fusobacterium nucleatum, a gram-negative anaerobic bacillus, is mainly associated with colorectal cancer patients positive for the CpG island methylator phenotype, although hypermethylation in genes such as MLH1, CDKN2A, MTSS1, RBM38, PKD1, PTPRT, and EYA4 has also been described. Moreover, Hungatella hathewayi, a gram-positive, rod-shaped bacterium, is related to hypermethylation in SOX11, THBD, SFRP2, GATA5, ESR1, EYA4, CDX2, and APC genes. The underlying epigenetic mechanism is unclear, although it could be implicated in the regulation of DNA methyltransferases, enzymes that catalyze the transfer of a methyl group on cytosine of CpG sites. Since DNA methylation is a reversible event, changes in gut microbiota could modulate the gene expression through DNA methylation and improve the colorectal cancer prognosis.