Microarray-based method for detecting methylation changes of p16^Ink4a gene 5＇-CpG islands in gastric carcinomas

AIM: Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation of the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. However, large-scale analysis of candidate genes has so far been hampered by the lack of high-throughput approach for analyzing DNA methylation. The aim of this study was to describe a microarray-based method for detecting changes of DNA methylation in cancer. METHODS: This method used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. Therefore, the amplified product might contain a pool of DNA fragments with altered nucleotide sequences due to differential methylation status. Nine sets of oligonucleotide probes were designed to fabricate a DNA microarray to detect the methylation changes of p16 gene CpG islands in gastric carcinomas. The results were further validated by methylation-specific PCR (MSP). RESULTS: The experimental results showed that the microarray assay could successfully detect methylation changes of p16 gene in 18 gastric tumor samples. Moreover, it could also potentially increase the frequency of detecting p16 methylation from tumor samples than MSP. CONCLUSION: Microarray assay could be applied as a useful tool for mapping methylation changes in multiple CpG loci and for generating epigenetic profiles in cancer.     

DNA methylation in gastric cancer,related to Helicobacter pylori and Epstein-Barr virus

Gastric cancer is a leading cause of cancer death worldwide,and significant effort has been focused on clarifying the pathology of gastric cancer.In particular,the development of genome-wide analysis tools has enabled the detection of genetic and epigenetic alterations in gastric cancer;for example,aberrant DNA methylation in gene promoter regions is thought to play a crucial role in gastric carcinogenesis.The etiological viewpoint is also essential for the study of gastric cancers,and two distinct pathogens,Helicobacter pylori(H.pylori)and Epstein-Barr virus(EBV),are known to participate in gastric carcinogenesis.Chronic inflammation of the gastric epithelium due to H.pylori infection induces aberrant polyclonal methylation that may lead to an increased risk of gastric cancer.In addition,EBV infection is known to cause extensive methylation,and EBV-positive gastric cancers display a high methylation epigenotype,in which aberrant methylation extends to not only Polycomb repressive complex(PRC)-target genes in embryonic stem cells but also non-PRC-target genes.Here,we review aberrant DNA methylation in gastric cancer and the association between methylation and infection with H.pylori and EBV.     

Interplay among epigenetic alterations and crosstalk between genetic and epigenetic alterations in esophageal squamous cell carcinoma

Epigenetic alterations that do not involve a change in the DNA sequence have been increasingly recognized to be important key events in the regulation of the gene expression and carcinogenesis. Major epigenetic mechanisms include the methylation of cytosine in DNA, changes in the histone and chromatin structure due to covalent posttranslational modification of histone proteins and the RNA-mediated regulation of the gene expression. Esophageal squamous cell carcinoma (ESCC) continues to be associated with a very poor prognosis, indicating that obtaining a clear understanding of the pathogenesis of ESCC is desired for improving clinical outcomes. In this review, we discuss the recent progress in research on epigenetic alterations in ESCC, with respect to the following points: (1) DNA methylation, including global hypomethylation and DNA hypermethylation at CpG islands in the promoters of tumor suppressor genes, (2) histone acetylation/deacetylation and histone methylation with the alteration of histone-modifying enzymes and (3) alterations in the expression of microRNA and the recently emerging long non-coding RNA. We then discuss the interplay among these epigenetic events and the crosstalk between epigenetic and genetic changes in ESCC. It is therefore important to understand the molecular mechanisms underlying the development and progression of ESCC to improve the treatment outcome of this devastating disease, although this information is quite complicated and confusing.     

Differential methylation landscape of pancreatic ductal adenocarcinoma and its precancerous lesions

Background: Pancreatic cancer is one of the most lethal diseases with an incidence almost equal to the mortality. In addition to having genetic causes, cancer can also be considered an epigenetic disease. DNA methylation is the premier epigenetic modification and patterns of aberrant DNA methylation are recognized to be a common hallmark of human tumor. In the multistage carcinogenesis of pancreas starting from precancerous lesions to pancreatic ductal adenocarcinoma(PDAC), the epigenetic changes play a significant role. Data sources: Relevant studies for this review were derived via an extensive literature search in Pub Med via using various keywords such as pancreatic ductal adenocarcinoma, precancerous lesions, methylation profile, epigenetic biomarkers that are relevant directly or closely associated with the concerned area of our interest. The literature search was intensively done considering a time frame of 20 years(1998–2018). Result: In this review we have highlighted the hypermethylation and hypomethylation of the precancerous PDAC lesions(pancreatic intra-epithelial neoplasia, intraductal papillary mucinous neoplasm, mucinous cystic neoplasm and chronic pancreatitis) and PDAC along with the potential biomarkers. We have also achieved the early epigenetic driver that leads to progression from precancerous lesions to PDAC. A bunch of epigenetic driver genes leads to progression of precancerous lesions to PDAC( pp ENK, APC, p14/5/16/17, h MLH1 and MGMT) are also documented. We summarized the importance of these observations in therapeutics and diagnosis of PDAC hence identifying the potential use of epigenetic biomarkers in epigenetic targeted therapy. Epigenetic inactivation occurs by hypermethylation of Cp G islands in the promoter regions of tumor suppressor genes. We listed all hyper-and hypomethylation of Cp G islands of several genes in PDAC including its precancerous lesions. Conclusions: The concept of the review would help to understand their biological effects, and to determine whether they may be successfully combined with other epigenetic drugs. However, we need to continue our research to develop more specific DNA-demethylating agents, which are the targets for hypermethylated Cp G methylation sites.     

Novel risk markers for gastric cancer screening: Present status and future prospects

Initial identification of populations at high risk of gastric cancer (GC) is important for endoscopic screening of GC. As serum pepsinogen (PG) test-positive subjects with progression of chronic atrophic gastritis (CAG) show a high likelihood of future cancer development, this population warrants careful follow-up observation as a high-risk GC group. By combining the PG test with Helicobacter pylori (HP) antibody titers, the HP-related chronic gastritis stage can be classified, thus identifying not only a GC high-risk group but also a low-risk group. Among PG test-negative patients without CAG, those with high serum PG II levels and HP antibody titers are thought to have severe gastric mucosal inflammation and the risk of diffuse-type GC is also high. Meanwhile, in gastric mucosae obtained by endoscopic biopsy, HP infection induces aberrant DNA methylation in CpG islands in multiple gene regions and the extent of methylation clearly correlates with GC risk. By quantifying aberrant DNA methylation in suitable gene markers, we can determine the extent of the epigenetic field for cancerization. These novel concepts and risk markers will have many clinical applications in gastrointestinal endoscopy, including more efficient endoscopic GC screening and a strategic approach to metachronous multiple GCs after endoscopic treatment.     

DNA methylation as a marker for the past and future

Aberrant methylation of CpG islands in promoter regions can permanently inactivate tumor-suppressor genes, as mutations and chromosomal abnormalities do. In gastric cancers, CDKN2A, CDH1, and MLH1 are inactivated more frequently by aberrant methylation than by mutations, and novel tumor-suppressor genes inactivated by promoter methylation are being identified. We recently found that Helicobacter pylori (HP), a potent gastric carcinogen, induces aberrant methylation in gastric mucosae. When a panel of CpG islands was examined, some CpG islands were consistently methylated in gastric mucosae of individuals with HP infection, while others were resistant. The amount of methylated DNA molecules in the gastric mucosae (methylation level) fluctuated while active HP infection was present, but decreased after it was no longer present. Among individuals without active HP infection, methylation levels in the gastric mucosae were higher in individuals with gastric cancers than in those without. DNA methylation is emerging as a promising marker for past exposure to carcinogens and future risk of cancers.     

Hedgehog通路中Ptchl基因在人胃癌中高甲基化状态分析

目的 研究Hedgehog通路中Ptchl基因表达及其甲基化状态在胃癌发生中的变化.方法 分别抽提10例胃癌组织及其癌旁＞3 cm组织和胃癌细胞株AGS的总RNA和基因组DNA.实时定量逆转录多聚酶链反应(QRT-PCR)检测Ptch基因的mRNA表达.应用软件分析Ptchl基因5'调控序列的CpG岛状态,亚硫酸氢盐测序PCR(BSP)分析甲基化水平.结果 对Ptchl mRNAla转录体转录起始位点(计为0点)上游-3950 bp和下游+2050 bp进行CpG岛分析,发现存在两个CpG岛,第1个为-1139 bp～+860 bp,第2个为+875 bp～+1692 bp.以第1个CpG岛的-870 bp～+229 bp区段内的19个CpG位点的BSP测序结果显示,胃癌细胞株AGS全部发生甲基化,胃癌组织中甲基化程度为16%～100%,平均64%±32%,癌旁组织甲基化程度为0%～42%,平均13%±14%,两组问差异有统计学意义(P＜0.05).Spearman秩相关分析发现,Ptchl基因甲基化同其表达呈负相关(r=-0.558,P=0.011).结论 Ptchl基因高甲基化参与胃癌的发生,可能为胃癌新的肿瘤标志物.     

New advances in targeted gastric cancer treatment

Despite a decrease in incidence over past decades,gastric cancer remains a major global health problem. In the more recent period, survival has shown only minor improvement, despite significant advances in diagnostic techniques, surgical and chemotherapeutic approaches, the development of novel therapeutic agents and treatment by multidisciplinary teams. Because multiple genetic mutations, epigenetic alterations, and aberrant molecular signalling pathways are involved in the development of gastric cancers, recent research has attempted to determine the molecular heterogeneity responsible for the processes of carcinogenesis, spread and metastasis. Currently, some novel agents targeting a part of these dysfunctional molecular signalling pathways have already been integrated into the standard treatment of gastric cancer, whereas others remain in phases of investigation within clinical trials. It is essential to identify the unique molecular patterns of tumours and specific biomarkers to develop treatments targeted to the individual tumour behaviour. This review analyses the global impact of gastric cancer, as well as the role of Helicobacter pylori infection and the efficacy of bacterial eradication in preventing gastric cancer development. Furthermore, the paper discusses the currently available targeted treatments and future directions of research using promising novel classes of molecular agents for advanced tumours.     

Role of Helicobacter pylori in the pathogenesis of gastric carcinoma and progression of lymphoid nodules to lymphoma.

The pathology of gastritis associated with Helicobacter pylori infection is summarized. The literature is reviewed regarding the role of H pylori in the pathogenesis of gastric carcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. The potential mechanisms of gastric carcinogenesis include transformation of the gastric mucosa by metabolic products of H pylori, transformation of the host cell by incorporation of H pylori DNA and genotoxic effects of the inflammatory response to the organism. A model for gastric carcinogenesis is proposed in which H pylori causes cell proliferation, and the risk of DNA damage is increased, leading to inadequate repair and malignant transformation. Investigation of early gastric carcinomas concluded that two main pathways operated in gastric carcinogenesis, both starting from H pylori gastritis and leading to phenotypically variable gastric or intestinal tumour growth. The histological features and molecular genetics of MALT lymphoma are briefly reviewed. There is evidence that tumour cells of low grade B cell MALT lymphoma proliferate specifically in response to H pylori. This response is dependent on T cell activation by H pylori. A proposed model for the pathogenesis of MALT lymphoma postulates that B lymphocytes with a genetic change acquire a growth advantage resulting in a monoclonal proliferation in response to H pylori-activated T cells. Further genetic changes may result in escape from T cell dependency.     

Association between multiple gene promoter hypermethylation and the risk of gastric cancer: A systematic review and meta-analysis

BackgroundSeveral studies have demonstrated an association between multiple gene hypermethylation and gastric cancer. However, the intrinsic mechanisms remain elusive and highly debatable. To this end, our study aims to investigate the correlation between the methylation status of multiple gene promoters and gastric cancer.MethodsPubMed, EMBASE, CNKI, WanFang, Cqvip, and Cochrane Library were queried from inception to May 2021, and the relationship between the methylation status of the CpG islands and gastric cancer risk was systematically assessed under the inclusion and exclusion criteria. The incidence of DNA methylation between tumor and non-tumor tissues was compared, and the clinicopathological significance of DNA methylation in gastric carcinoma was further evaluated. The odds ratio (OR) was estimated with a 95% confidence interval (CI), and forest plots were generated using the fixed-effects or random-effects model.ResultsIn total, 201 studies were enrolled, and a higher frequency of CpG islands methylation was identified in gastric cancer tissues than in non-neoplastic tissues. This suggests that aberrant polygene methylation might be associated with the initial onset and progression of gastric cancer.ConclusionThis study sheds light on the significance of polygene methylation status in gastric cancer. The DNA methylation of these genes may serve as underlying epigenetic biomarkers, providing a promising molecular diagnostic approach for human gastric cancer clinical diagnosis. More large randomized trials are needed to confirm the findings.     

Causal role of Helicobacter pylori infection and eradication therapy in gastric carcinogenesis

Many epidemiological reports indicate that Helicobacterpylori(H pylori)infection plays an important role ingastric carcinogenesis.Several genetic and epigeneticalterations contribute to the initiation,promotion,andprogression of the cancer cells in a multi-step manner.H pylori is known to induce chronic inflammation in thegastric mucosa.Its products,including superoxides,participate in the DNA damage followed by initiation,andthe inflammation-derived cytokines and growth factorscontribute to the promotion of gastric carcinogenesis.By eradicating H pylori,gastric inflammation can becured; the therapy diminishes the levels not onlyof inflammatory cell infiltration,but also atrophy/intestinal metaplasia in part.A randomized controlledtrial revealed that the eradication therapy diminishedthe gastric cancer prevalence in cases without pre-cancerous conditions.In addition,recent epidemiologicalstudies from Japanese groups demonstrated thatthe development of gastric cancer,especially of theintestinal type,was decreased by successful eradicationtherapy,although these were designed in a non-randomized manner.However,it should be mentionedthat endoscopic detection is the only way to evaluate thedegree of gastric carcinogenesis.We have reported thatthe endoscopic and histological morphologies could bemodified by eradication therapy and it might contributeto the prevalence of gastric cancer development.Considering the biological nature of cancer cellproliferation,it is considered that a sufficiently long-termfollow-up would be essential to discuss the anticancereffect of eradication therapy.     

Epigenetics and colorectal cancer

Colorectal cancer (CRC) is a leading cause of cancer deaths worldwide. It results from an accumulation of genetic and epigenetic changes in colon epithelial cells, which transforms them into adenocarcinomas. Over the past decade, major advances have been made in understanding cancer epigenetics, particularly regarding aberrant DNA methylation. Assessment of the colon cancer epigenome has revealed that virtually all CRCs have aberrantly methylated genes and that the average CRC methylome has hundreds to thousands of abnormally methylated genes. As with gene mutations in the cancer genome, a subset of these methylated genes, called driver genes, is presumed to have a functional role in CRC. The assessment of methylated genes in CRCs has also revealed a unique molecular subgroup of CRCs called CpG island methylator phenotype (CIMP) cancers; these tumors have a particularly high frequency of methylated genes. These advances in our understanding of aberrant methylation in CRC have led to epigenetic alterations being developed as clinical biomarkers for diagnostic, prognostic and therapeutic applications. Progress in this field suggests that these epigenetic alterations will be commonly used in the near future to direct the prevention and treatment of CRC.     

Accumulation of aberrant DNA methylation during colorectal cancer development

Despite the recent advances in the therapeutic modalities,colorectal cancer(CRC)remains to be one of the most common causes of cancer-related death.CRC arises through accumulation of multiple genetic and epigenetic alterations that transform normal colonic epithelium into adenocarcinomas.Among crucial roles of epigenetic alterations,gene silencing by aberrant DNA methylation of promoter regions is one of the most important epigenetic mechanisms.Recent comprehensive methylation analyses on genome-wide scale revealed that sporadic CRC can be classified into distinct epigenotypes.Each epigenotype cooperates with specific genetic alterations,suggesting that they represent different molecular carcinogenic pathways.Precursor lesions of CRC,such as conventional and serrated adenomas,already show similar methylation accumulation to CRC,and can therefore be classified into those epigenotypes of CRC.In addition,specific DNA methylation already occurs in the normal colonic mucosa,which might be utilized for prediction of the personal CRC risk.DNA methylation is suggested to occur at an earlier stage than carcinoma formation,and may predict the molecular basis for future development of CRC.Here,we review DNA methylation and CRC classification,and discuss the possible clinical usefulness of DNA methylation as biomarkers for the diagnosis,prediction of the prognosis and the response to therapy of CRC.     

Distinct methylated profiles in Helicobacter pylori dependent and independent gastric MALT lymphomas

BACKGROUND: Gastric mucosa associated lymphoid tissue (MALT) lymphomas are clinically subdivided into Helicobacter pylori dependent and independent, according to H. pylori infection and the therapeutic course. In previous reports it has been suggested that H. pylori independent cases develop from H. pylori dependent cases, and sometimes transform into high grade diffuse large B cell lymphomas (DLBCLs). METHODS: To better understand the pathogenesis of H. pylori dependent and independent MALT lymphomas, we analysed the methylation profiles of eight independent CpG islands, including p15, p16, p73, hMLH1, death associated protein kinase, MINT1, MINT2, and MINT31 in H. pylori dependent and independent MALT lymphomas, DLBCLs, and H. pylori associated chronic gastritis. RESULTS: We first confirmed that H. pylori independent cases had a high incidence of t(11;18)(q21;q21) (4/8 cases) and aberrant BCL10 expression (7/8 cases) compared with H. pylori dependent cases and gastric DLBCLs. In the methylation pattern study, all 13 H. pylori dependent MALT lymphomas had more than four methylated loci while H. pylori independent cases had less than two. According to the previous criterion, all H. pylori dependent MALT lymphomas (13/13, 100%) and five of 10 (50%) DLBCLs were classified as CpG island methylator phenotype positive (CIMP+). In contrast, all H. pylori independent MALT lymphomas were CIMP-. CONCLUSION: The distinct methylation pattern together with lack of chromosomal translocation in H. pylori dependent MALT lymphomas suggest that H. pylori dependent and independent MALT lymphomas have a different pathogenesis.     

Epigenetic modifications in cardiovascular disease

Epigenetics represents a phenomenon of altered heritable phenotypic expression of genetic information occurring without changes in DNA sequence. Epigenetic modifications control embryonic development, differentiation and stem cell (re)programming. These modifications can be affected by exogenous stimuli (e.g., diabetic milieu, smoking) and oftentimes culminate in disease initiation. DNA methylation has been studied extensively and represents a well-understood epigenetic mechanism. During this process cytosine residues preceding a guanosine in the DNA sequence are methylated. CpG-islands are short-interspersed DNA sequences with clusters of CG sequences. The abnormal methylation of CpG islands in the promoter region of genes leads to a silencing of genetic information and finally to alteration of biological function. Emerging data suggest that these epigenetic modifications also impact on the development of cardiovascular disease. Histone modifications lead to the modulation of the expression of genetic information through modification of DNA accessibility. In addition, RNA-based mechanisms (e.g., microRNAs and long non-coding RNAs) influence the development of disease. We here outline the recent work pertaining to epigenetic changes in a cardiovascular disease setting.