CpG island methylation in gastroenterologic neoplasia: a maturing field

Fifteen years after the first demonstration of epigenetic tumor-suppressor gene inactivation associated with promoter methylation, the field has reached a level of understanding that threatens a re-writing of established biologic concepts. In gastrointestinal malignancies, epigenetic analysis has led to novel hypotheses regarding the etiology of age-associated cancer susceptibility and the interactions between environmental exposures and neoplasia. Methylation profiling has uncovered a distinct pathway to colorectal neoplasia that may arise from a hitherto underestimated precursor lesion, the proximal hyperplastic polyp-serrated adenoma pathway. Epigenetic information has shown promise in clarifying susceptibility to cancer and defining poor prognosis groups in gastrointestinal cancers. Finally, the field has engendered renewed interest in therapeutic targeting of epigenetic regulatory molecules, and several such drugs are currently in clinical trials. It is likely that epigenetic pathways will be integrated in the routine management of gastrointestinal malignancies over the next decade.     

CpG island methylator phenotype and its relationship with prognosis in adult acute leukemia patients

Objective

To investigated the relationship between CpG island methylator phenotype (CIMP) and prognosis in adults with acute leukemia.

Methods

Bone marrow samples from 53 acute myeloid leukemia and 50 acute lymphoblastic leukemia patients were collected. The methylation status of 18 tumor suppressor genes was determined using methylation-specific polymerase chain reaction.

Results

Greater than 30% of acute leukemia patients had methylated p15, p16, CDH1, CDH13, RUNX3, sFRP1, ID4, and DLC-1 genes; methylation of ≥4 were defined as CIMP positive. Age, type of leukemia, white blood cell count, and CIMP status were significantly associated with recurrence-free survival (RFS) and overall survival (OS) (P < 0.05). CIMP status was an independent prognostic factor for OS (hazard ratio: 2.07, 95% confidence interval: 1.03–4.15, P = 0.040). CIMP-negative patients had significantly improved RFS and OS (P < 0.05). p16 and DLC1 methylation was significantly associated with RFS and OS (P < 0.05).

Conclusions

CIMP may serve as an independent risk factor for evaluating the prognosis of patients with acute leukemia.     

Molecular correlates with MGMT promoter methylation and silencing support CpG island methylator phenotype-low (CIMP-low) in colorectal cancer

Background

The CpG island methylator phenotype (CIMP or CIMP‐high) with widespread promoter methylation is a distinct epigenetic phenotype in colorectal cancer. In contrast, a phenotype with less widespread promoter methylation (CIMP‐low) has not been well characterised. O‐6‐methylguanine‐DNA methyltransferase (MGMT) promoter methylation and silencing have been associated with G>A mutations and microsatellite instability‐low (MSI‐low).

Aim

To examine molecular correlates with MGMT methylation/silencing in colorectal cancer.

Methods

Utilising MethyLight technology, we quantified DNA methylation in MGMT and eight other markers (a CIMP‐diagnostic panel; CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1) in 920 population‐based colorectal cancers.

Results

Tumours with both MGMT methylation and loss were correlated positively with MSI‐low (p = 0.02), CIMP‐high (⩾6/8 methylated CIMP markers, p = 0.005), CIMP‐low (1/8–5/8 methylated CIMP markers, p = 0.002, compared to CIMP‐0 with 0/8 methylated markers), KRAS G>A mutation (p = 0.02), and inversely with 18q loss of heterozygosity (p = 0.0002). Tumours were classified into nine MSI/CIMP subtypes. Among the CIMP‐low group, tumours with both MGMT methylation and loss were far more frequent in MSI‐low tumours (67%, 12/18) than MSI‐high tumours (5.6%, 1/18; p = 0.0003) and microsatellite stable (MSS) tumours (33%, 52/160; p = 0.008). However, no such relationship was observed among the CIMP‐high or CIMP‐0 groups.

Conclusion

The relationship between MGMT methylation/silencing and MSI‐low is limited to only CIMP‐low tumours, supporting the suggestion that CIMP‐low in colorectal cancer may be a different molecular phenotype from CIMP‐high and CIMP‐0. Our data support a molecular difference between MSI‐low and MSS in colorectal cancer, and a possible link between CIMP‐low, MSI‐low, MGMT methylation/loss and KRAS mutation.     

The Role of the CpG Island Methylator Phenotype on Survival Outcome in Colon Cancer

Background/AimsCpG island methylator phenotype (CIMP)- high colorectal cancers (CRCs) have distinct clinicopathological features from their CIMP-low/negative CRC counterparts. However, controversy exists regarding the prognosis of CRC according to the CIMP status. Therefore, this study examined the prognosis of Korean patients with colon cancer according to the CIMP status.MethodsAmong a previous cohort population with CRC, a total of 154 patients with colon cancer who had available tissue for DNA extraction were included in the study. CIMP-high was defined as 3/5 methylated markers using the five-marker panel (CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1).ResultsCIMP-high and CIMP-low/negative cancers were observed in 27 patients (17.5%) and 127 patients (82.5%), respectively. Multivariate analysis adjusting for age, gender, tumor location, tumor stage and CIMP and microsatellite instability (MSI) statuses indicated that CIMP-high colon cancers were associated with a significant increase in colon cancer-specific mortality (hazard ratio [HR], 3.23; 95% confidence interval [CI], 1.20 to 8.69; p=0.02). In microsatellite stable cancers, CIMP-high cancer had a poor survival outcome compared to CIMP-low/negative cancer (HR, 2.91; 95% CI, 1.02 to 8.27; p=0.04).ConclusionsRegardless of the MSI status, CIMP-high cancers had poor survival outcomes in Korean patients.     

CpG岛甲基子表型阳性的散发性大肠癌的临床病理特征

目的 探讨CpG岛甲基子表型阳性的散发性大肠癌的临床病理特征.方法 采用甲基化特异性PCR方法对71例散发性大肠癌患者行p14、hMLH1、p16、MGMT和MINT1共5个基因启动子甲基化检测,分析CpG岛甲基子表型阳性的散发性大肠癌的临床病理特征.结果 71例散发性大肠癌患者中共检出CpG岛甲基子表型阳性者15例,阳性率为21.1%.CpG岛甲基子表型阳性的散发性大肠癌中右半结肠癌（40.0%比12.5%,P＜0.05）、低分化癌（46.7%比14.3%,P＜0.05）、淋巴结转移（86.7%比48.2%,x2=7.112,P＜0.05）、Dukes C期或D期（86.7%比50.0%,x2=6.519,P＜0.05）所占比例均显著高于CpG岛甲基子表型阴性者.结论 CpG岛甲基子表型阳性的散发性大肠癌具有右半结肠癌多发、低分化多见、常有淋巴结转移和分期较晚的特点.     

急性白血病患者抑癌基因p27的表达及其启动子甲基化状态的研究

目的研究急性白血病(AL)患者抑癌基因p27的表达及其启动子的甲基化状态,探讨其在AL发生发展中的作用。方法采用逆转录—聚合酶链反应(RT-PCR)及甲基化特异性聚合酶链反应(MSP)技术研究AL患者骨髓单个核细胞p27 mRNA的表达水平及其启动子甲基化发生率。结果 AL组和对照组p27 mRNA阳性率分别为36.9%和87.8%(P<0.01),p27 mRNA的表达缺失与AL患者外周血白细胞计数、髓外浸润呈负相关(P均<0.05)。AL患者的p27基因启动子甲基化阳性率较低(4.62%)。结论 p27基因的表达缺失是AL发生发展的机制之一,p27基因启动子甲基化是否为AL患者p27基因表达缺失的机制之一尚需进一步研究证实。     

Aberrant methylation of multiple tumor suppressor genes in aging liver, chronic hepatitis, and hepatocellular carcinoma

Aberrant DNA methylation is an important epigenetic alteration in hepatocellular carcinoma (HCC). However, the molecular processes underlying the methylator phenotype and the contribution of hepatitis viruses are poorly understood. The current study is a comprehensive methylation analysis of human liver tissue specimens. A total of 176 liver tissues, including 77 pairs of HCCs and matching noncancerous liver and 22 normal livers, were analyzed for methylation. Methylation of 19 epigenetic markers was quantified, and the results were correlated with different disease states and the presence or absence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. Based on methylation profiles, the 19 loci were categorized into 3 groups. Normal liver tissues showed methylation primarily in group 1 loci (HIC-1, CASP8, GSTP1, SOCS1, RASSF1A, p16, APC), which was significantly higher than group 2 (CDH1, RUNX3, RIZ1, SFRP2, MINT31) and group 3 markers (COX2, MINT1, CACNA1G, RASSF2, MINT2, Reprimo, DCC) (P < 0.0001). Noncancerous livers demonstrated increased methylation in both group 1 and group 2 loci. Methylation was significantly more abundant in HCV-positive livers compared with normal liver tissues. Conversely, HCC showed frequent methylation at each locus investigated in all 3 groups. However, the group 3 loci showed more dense and frequent methylation in HCV-positive cancers compared with both HBV-positive cancers and virus-negative cancers (P < 0.0001). CONCLUSION: Methylation in HCC is frequent but occurs in a gene-specific and disease-specific manner. Methylation profiling allowed us to determine that aberrant methylation is commonly present in normal aging livers, and sequentially progresses with advancing stages of chronic viral infection. Finally, our data provide evidence that HCV infection may accelerate the methylation process and suggests a continuum of increasing methylation with persistent viral infection and carcinogenesis in the liver.     

Aberrant methylation of DAP-kinase in therapy-related acute myeloid leukemia and myelodysplastic syndromes

Death-associated protein kinase (DAP-kinase), a proapoptotic serine/threonine kinase, is a candidate tumor suppressor gene. We studied the methylation status of DAP-kinase of 194 bone marrow samples from 160 patients with acute myeloid leukemia (AML) and 34 with a myelodysplastic syndrome (MDS) at the time of initial diagnosis by polymerase chain reaction (PCR). Hypermethylation of DAP-kinase was present in 27.5% (44 of 160) of AML and in 47% (16 of 34) of MDS specimens and significantly correlated to loss of DAP-kinase expression (P =.008). It was significantly more frequent in AML secondary to therapy for other malignancies (s-AML; 14 of 29, 48.3%), as compared to de novo AML (30 of 131, 22.9%, P =.01). DAP-kinase hypermethylation in AML was associated with myelodysplastic changes in the bone marrow at the time of the initial diagnosis (P =.002) and with the presence of cytogenetic abnormalities (P =.02). Alteration in the apoptotic response due to the loss of DAP-kinase function may be an early event in the transformation pathway to secondary leukemia via myelodysplasia.     

CpG island methylator phenotype in plasma is associated with hepatocellular carcinoma prognosis

AIM:To evaluate the clinical significance of CpG island methylator phenotype(CIMP) in plasma and its asso-ciation with hepatocellular carcinoma(HCC) progress.METHODS:CIMP status of 108 HCC patients wasanalyzed using a methylation marker panel in tumortissues and plasma with methylation-specific poly-merase chain reaction. Fifteen samples of non-neo-plastic liver tissues and 60 of plasma from healthy persons were examined simultaneously. Examinedgenes included APC,WIF-1,RUNX-3,DLC-1,SFRP-1,DKK and E-cad .RES...     

Loss of CDX2 expression is associated with poor prognosis in colorectal cancer patients

AIM:To investigate the clinicopathologic characteristics and prognostic implications associated with loss of CDX2 expression in colorectal cancers(CRCs).METHODS:We immunohistochemically evaluated CDX2 expression in 713 CRCs and paired our findings to clinicopathologic and molecular characteristics of each individual.Endpoints included cytokeratin 7 and CK20 expression,microsatellite instability,Cp G island methylator phenotype,and KRAS and BRAF mutation statuses.Univariate and multivariate survival analysis was performed to reveal the prognostic value of CDX2 downregulation.RESULTS:CDX2 expression was lost in 42(5.9%) patients.Moreover,loss of CDX2 expression was associated with proximal location,infiltrative growth,advanced T,N,M and overall stage.On microscopic examination,loss of CDX2 expression was associated with poor differentiation,increased number of tumor-infiltrating lymphocytes,luminal serration and mucin production.Loss of CDX2 expression was also associated with increased CK7 expression,decreased CK20 expression,Cp G island methylator phenotype,microsatellite instability and BRAF mutation.In a univariate survival analysis,patients with loss of CDX2 expression showed worse overall survival(P < 0.001) and progression-free survival(P < 0.001).In a multivariate survival analysis,loss of CDX2 expression was an independent poor prognostic factor of overall survival [hazard ratio(HR) = 1.72,95%CI:1.04-2.85,P = 0.034] and progression-free survival(HR = 1.94,95%CI:1.22-3.07,P = 0.005).CONCLUSION:Loss of CDX2 expression is associated with aggressive clinical behavior and can be used as a prognostic marker in CRCs.     

Serrated pathway in colorectal carcinogenesis

Serrated adenocarcinoma is a recently described subset of colorectal cancer(CRC),which account for about10%of all CRCs and follows an alternative pathway in which serrated polyps replace the traditional adenoma as the precursor lesion to CRC.Serrated polyps form a heterogeneous group of colorectal lesions that includes hyperplastic polyps(HPs),sessile serrated adenoma(SSA),traditional serrated adenoma(TSA)and mixed polyps.HPs are the most common serrated polyp followed by SSA and TSA.This distinct histogenesis is believed to have a major influence in prevention strategies,patient prognosis and therapeutic impact.Genetically,serrated polyps exhibited also a distinct pattern,with KRAS and BRAF having an important contribution to its development.Two other molecular changes that have been implicated in the serrated pathway include microsatellite instability and the CpG island methylator phenotype.In the present review we will address the current knowledge of serrated polyps,clinical pathological features and will update the most recent findings of its molecular pathways.The understanding of their biology and malignancy potential is imperative to implement a surveillance approach in order to prevent colorectal cancer development.     

New approaches in acute myeloid leukemia

Today, treatment of patients with acute myeloid leukemia (AML) remains predominantly a "one-fits-all" approach with intensive cytarabine-based chemotherapy as the mainstay, but we are finally beginning to reap the rewards of decades of basic, translational, and clinical research. Developing individualized, "targeted" therapy for each AML patient based on unique molecular features of disease remains a daunting goal, yet one that we can now begin to envision. Hypothesis-based study designs--from preclinical/laboratory experiments to phase 1 and subsequent efficacy trials--provide the foundation for advances in the diagnosis, risk stratification, and treatment of patients with AML. The following is an outline of several key areas of ongoing AML research.     

Aberrant methylation frequency of TNFRSF10C promoter in pancreatic cancer cell lines

BACKGROUND:A growing body of evidence suggests that many tumors are initiated by both epigenetic abnormalities and gene mutations,which promote tumor progression. Epigenetic abnormalities include changes in DNA methylation and in the modification of histones.This study aimed to assess the status of methylation in the CpG island(CGI)of the tumor necrosis factor receptor superfamily member 10c(TNFRSF10C) with combined bisulfite restriction analysis(COBRA)and to evaluate its role in the progression of pancreat...     

胰腺癌细胞系BxPC3及胰腺癌组织Ras相关区域家族1A启动子CpG岛的甲基化状态

目的 检测Ras相关区域家族1A(Ras association domain family 1A,RASSF1A)在胰腺癌细胞株BxPC3及胰腺癌组织中的甲基化状态,探讨其启动子异常甲基化在胰腺癌发病机制中的可能作用.方法 采用结合重亚硫酸盐的限制性内切酶法(combined bisulfite restriction analysis,COBRA)检测胰腺癌细胞株BxPC3、5例正常胰腺组织、13对胰腺癌及相应癌旁正常胰腺组织中RASSF1A启动子CpG岛的甲基化状态,计算其甲基化率.以甲基化酶抑制剂5-Aza-dC(5-Aza-2-deoxycitydine)处理BxPC3,观察处理前后甲基化率变化情况及RASSF1A mRNA表达变化.结果 在BxPC3细胞株中,RASSF1A启动子的CpG岛甲基化率为62.90%;正常胰腺、癌旁及癌组织中平均分别为9.14%、53.79%和55.82%.与正常胰腺组织相比,胰腺癌旁及癌组织的RASSF1A启动子甲基化率明显增高(P值<0.01),而癌旁及癌组织之间无明显差异(P>0.05).BxPC3经5-Aza-dC处理后,RASSF1A的CpG岛甲基化率显著下降至42.50%(P<0.05),同时RASSF1A mRNA表达增强.结论 RASSF1A启动子CpG岛异常甲基化是胰腺癌发生发展中的早期事件,可能参与胰腺癌的发病过程.     

胃腺癌凝血栓蛋白1基因异常甲基化及其临床意义

目的　探讨凝血栓蛋白 1(THBS1)基因CpG岛异常甲基化与胃腺癌 (GAC)发生的关联。方法　应用甲基化特异性PCR检测技术 ,检测 82例GAC患者肿瘤组织 ,30例十二指肠球部溃疡 (对照组 )、30例慢性胃炎伴肠上皮化生或异型增生患者胃窦黏膜组织中 ,THBS1基因启动子CpG岛甲基化分布情况。结果　THBS1基因启动子CpG岛甲基化在对照组 (6 .7% )中的频率明显低于胃炎组(2 6 .7% ,χ2 =4 .32 ,P =0 .0 38)和胃癌组 (4 7.6 % ,χ2 =16 .2 ,P <0 .0 0 1) ,在胃炎组与胃癌组之间的频率差异也有显著性 (χ2 =4 .14 ,P =0 .0 4 2 ) ,老年胃癌患者 (6 3.6 % )中的频率明显高于非老年 (36 .7% ,χ2 =5 .72 ,P =0 .0 17) ,在TNMⅢ和Ⅳ期肿瘤 (6 6 .7% )组织中的频率明显高于Ⅰ期 (36 .8% )或Ⅱ期(36 .4 % ,χ2 =6 .93,v =2 ,P =0 .0 31) ,而肿瘤部位之间、Lauren分型肿瘤之间及不同分化程度肿瘤之间 ,THBS1基因甲基化率的差异无显著性。结论　THBS1基因启动子CpG岛甲基化可能与胃腺癌的发生有关 ,且以老年患者及Ⅲ和Ⅳ期肿瘤多见。     

Serrated polyposis syndrome: molecular, pathological and clinical aspects

Hyperplastic polyps have traditionally been considered not to have malignant potential. New pathological classification of serrated polyps and recent discoveries about the serrated pathway of carcinogenesis have revolutionized the concepts and revitalized the research in this area. Until recently, it has been thought that most colorectal cancers arise from conventional adenomas via the traditional tumor suppressor pathway initiated by a mutation of the APC gene, but it has been found that this pathway accounts for only approximately 70%-80% of colorectal cancer (CRC) cases. The majority of the remaining colorectal cancer cases follow an alternative pathway leading to CpG island methylator phenotype carcinoma with BRAF mutation and with or without microsatellite instability. The mechanism of carcinomas arising from this alternative pathway seems to begin with an activating mutation of the BRAF oncogene. Serrated polyposis syndrome is a relatively rare condition characterized by multiple and/or large serrated polyps of the colon. Clinical characteristics, etiology and relationship of serrated polyposis syndrome to CRC have not been clarified yet. Patients with this syndrome show a high risk of CRC and both sporadic and hereditary cases have been described. Clinical criteria have been used for diagnosis and frequent colonoscopy surveillance should be performed in order to prevent colorectal cancer. In this review, we try to gather new insights into the molecular pathogenesis of serrated polyps in order to understand their possible clinical implications and to make an approach to the management of this syndrome.