外周血GP5、APC、GSTP1基因甲基化联合检测与乳腺癌相关性的研究

目的探索外周血GP5、APC、GSTP1基因甲基化联合检测与乳腺癌相关性。方法收集了2020年5月—2020年11月同济大学附属东方医院就诊的172例女性患者,其中有35例乳腺癌患者,90例乳腺良性疾病患者和47例其他组织来源的恶性肿瘤患者。实时荧光定量PCR法测定外周血中GP5、APC、GSTP1的基因甲基化表达水平,并与癌胚抗原（carcinoembryonic antigen, CEA）、糖类抗原153（carbohydrate antigen 153, CA153）、糖类抗原125（carbohydrate antigen 125, CA125）及乳腺钼靶进行比较。通过一致性检验和受试者工作特征（receiver operating characteristic, ROC）曲线分析并比较不同检测方法对乳腺癌的评价指标。分析三种基因联合检测的甲基化阳性率与乳腺癌临床病理特征相关性。结果35例乳腺癌患者和90例乳腺良性疾病患者外周血GP5、APC、GSTP1基因甲基化联合检测与组织病理结果的Kappa一致性为0.770。ROC曲线分析外周血基因甲基化联合检测的曲线下面积（area under the curve, AUC）为0.913(95%CI： 0.849~0.956),其中灵敏度为88.57%,特异度为92.22%,高于乳腺钼靶（AUC=0.765）,高于血清CEA、CA125、CA153联合检测（AUC=0.761）。外周血基因甲基化与3种血清肿瘤指标联合检测的AUC最高为0.949（95%CI： 0.894~0.980)。35例乳腺癌患者外周血GP5、APC、GSTP1基因甲基化联合检测表达阳性率（88.6%）高于3种血清肿瘤标志物联合检测阳性率（28.6%）;术前外周血基因甲基化表达的阳性率与年龄、淋巴结转移、肿瘤分期、肿瘤类型及脉管侵犯均无关（P>0.05）。47例其他组织来源恶性肿瘤与35例乳腺癌患者比较分析结果显示,外周血GP5、APC、GSTP1基因甲基化检测与病理的Kappa一致性为0.849。ROC曲线分析外周血基因甲基化检测的AUC为0.936(95%CI： 0.859~0.978),灵敏度为88.57%,特异度高达95.74%。结论外周血GP5、APC、GSTP1基因甲基化联合检测具有较高的灵敏度和特异度。外周血GP5、APC、GSTP1基因甲基化有望成为诊断乳腺癌的非侵入性生物标志物。     

DNA methylation profile in patients with indolent systemic mastocytosis

BackgroundMastocytosis is a clinically heterogeneous, usually acquired disease of the mast cells with a survival time that depends on the onset of the disease and ranges from skin‐limited to systemic disease, including indolent and more aggressive variants. The crucial element in pathogenesis is the presence of oncogenic KIT somatic mutation D816V. Further epigenetic alterations are responsible for regulating the expression of genes. It is essential to identify indicators of disease progression, and the specific clinical picture to establish an appropriate therapeutic strategy.ObjectiveThe aim of this study was to analyze the relation of mastocytosis symptoms and epigenetic changes, and to identify epigenetic predictors of the disease.MethodsGlobal DNA methylation profile analysis was performed in peripheral blood collected from 73 patients with indolent systemic mastocytosis (ISM) and 43 healthy adult volunteers. Levels of 5‐methylcytosine (5‐mC) and 5‐hydroxymethylcytosine (5‐hmC) were determined using an ELISA‐based method, while the methylation of the Alu and LINE‐1 repeats were assayed with the quantitative methylation‐specific PCR technique. A questionnaire interview was conducted among the study participants to collect data on possible epigenetic modifiers. Additionally, the methylation profile was compared between three human mast cell lines: ROSA KIT D816V, ROSA KIT WT, and HMC‐1.1 KIT V560G, in order to assess the association between KIT mutations and methylation profile.ResultsA significantly lower level of DNA hydroxymethylation (5‐hmC) in the blood was found in patients with ISM as compared to the controls (0.022% vs. 0.042%, p = 0.0001). Differences in the markers of global DNA methylation (5‐mC, Alu, LINE‐1) were not statistically significant, although they did indicate generally higher DNA methylation in patients with mastocytosis. The 5‐hmC level was significantly associated with allergy (p = 0.011) in patients with ISM, showing a higher level of 5‐hmC in patients with allergy as compared to patients without allergy. The in vitro study revealed significant differences between the studied cell lines at the level of 5‐mC, Alu, and LINE‐1.ConclusionsThis study confirms that epigenetic changes are involved in mastocytosis, and suggests that allergy may be an important epigenetic modifier of the disease. A possible association between KIT mutations and methylation status observed in human mast cell lines requires further investigation in human studies.Clinical ImplicationsEpigenetic alterations are involved in mastocytosis pathology. The possible role of allergy as an important epigenetic modifier suggests the more impaired function of mast cells in ISM patients without allergy.Capsule summaryDecreased DNA demethylation in the blood DNA of patients with ISM confirms that epigenetic alterations are involved in mastocytosis pathology. We observed a possible role of allergy as an important epigenetic modifier. There is a possible association between KIT mutations and the methylation status observed in human mast cell lines.     

DNA methylation markers have universal prognostic value for anal cancer risk in HIV‐negative and HIV‐positive individuals

Anal cancer has increasing incidence and is preceded by high‐grade anal intraepithelial neoplasia (HGAIN; AIN2–3). Previously, we identified and validated several methylation markers for accurate detection of anal cancer and HGAIN with cancer risk in HIV‐positive (HIV+) men who have sex with men (MSM). This study aimed to evaluate these markers in HIV‐negative risk groups. A cross‐sectional series of 176 tissue samples of anal cancer, AIN3, AIN2, AIN1 and control biopsies obtained in HIV‐negative women and men was tested for six methylation markers (ASCL1, LHX8, SST, WDR17, ZIC1 and ZNF582). Accuracy for detection of AIN3 and cancer (AIN3+) was determined by univariable and multivariable mixed‐effect ordinal logistic regression. Methylation levels of all markers increased with increasing severity of disease (P < 0.0001) and were comparable to results in HIV+ MSM. All markers showed high accuracy for AIN3+ detection [area under the curve (AUC): 0.83–0.86]. The optimal marker panel (ASCL1 and ZIC1; AUC = 0.85 for AIN3+) detected 98% of cancers at 79% specificity. In conclusion, DNA methylation markers show a high diagnostic performance for AIN3+ detection in HIV+ and HIV‐negative risk groups, justifying broad application of methylation analysis for anal cancer prevention programmes.     

A panel of DNA methylation markers for the classification of consensus molecular subtypes 2 and 3 in patients with colorectal cancer

Consensus molecular subtypes (CMSs) can guide precision treatment of colorectal cancer (CRC). We aim to identify methylation markers to distinguish between CMS2 and CMS3 in patients with CRC, for which an easy test is currently lacking. To this aim, fresh‐frozen tumor tissue of 239 patients with stage I‐III CRC was analyzed. Methylation profiles were obtained using the Infinium HumanMethylation450 BeadChip. We performed adaptive group‐regularized logistic ridge regression with post hoc group‐weighted elastic net marker selection to build prediction models for classification of CMS2 and CMS3. The Cancer Genome Atlas (TCGA) data were used for validation. Group regularization of the probes was done based on their location either relative to a CpG island or relative to a gene present in the CMS classifier, resulting in two different prediction models and subsequently different marker panels. For both panels, even when using only five markers, accuracies were > 90% in our cohort and in the TCGA validation set. Our methylation marker panel accurately distinguishes between CMS2 and CMS3. This enables development of a targeted assay to provide a robust and clinically relevant classification tool for CRC patients.     

Downregulation of Waf1, C2, C3, and major histocompatibility complex class I loci within an 18-cM region of chromosome 17 in adenovirus-transformed mouse cells

In this study, the expression of the p53 tumor suppressor gene and the p53-regulated Mdm2 and Waf1 genes was evaluated in adenovirus (Ad)-transformed mouse cells. The expected levels of p53 mRNA and protein and Mdm2 mRNA were detected in all transformed cells. However, the level of Waf1 mRNA was markedly reduced in Ad12-transformed cells and in some Ad5-transformed cells. Waf1 expression was not reduced in untransformed mouse cells infected with Ad12 or Ad5. Expression of the class 1 major histocompatibility complex (MHC) locus was downregulated in 13 Ad-transformed cell lines (derived from four different strains of mice) that exhibited reduced expression of Waf1. Waf1 is located in mouse chromosome 17 proximal to the MHC class I locus. To determine whether other chromosome 17 genes were downregulated, the cells were examined for expression of other genetic loci. Of those tested, only the C2 and C3 complement loci were expressed in mouse fibroblasts. Expression of C2 (which is within the MHC) and expression of C3 (which is 15 cM distal to the MHC) were downregulated in those transformed cells in which Waf1 and MHC class I were downregulated. The Ad12- and Ad5-transformed cells that expressed low levels of Waf1, MHC class I, C2, and C3 formed tumors in syngeneic adult mice. These data suggest that the downregulation of multiple genes within the 32 Mb of mouse chromosome 17 that includes the Waf1 locus to the C3 locus occurs in Ad mouse-cell transformation and may contribute to the tumorigenicity of transformed cells. Mol. Carcinog. 18:213–220, 1997. © 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Histone methyltransferase SUV420H1/KMT5B contributes to poor prognosis in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) has a high rate of recurrence and poor prognosis, even after curative surgery. Multikinase inhibitors have been applied for HCC patients, but their effect has been restricted. This study aims to clarify the clinical impact of SUV420H1/KMT5B, one of the methyltransferases for histone H4 at lysine 20, and elucidate the novel mechanisms of HCC progression. We retrospectively investigated SUV420H1 expression using HCC clinical tissue samples employing immunohistochemical analysis (n = 350). We then performed loss-of-function analysis of SUV420H1 with cell cycle analysis, migration assay, invasion assay and RNA sequence for Gene Ontology (GO) pathway analysis in vitro, and animal experiments with xenograft mice in vivo. The SUV420H1-high-score group (n = 154) had significantly poorer prognosis for both 5-year overall and 2-year/5-year disease-free survival than the SUV420H1-low-score group (n = 196) (p < 0.001 and p < 0.05, respectively). The SUV420H1-high-score group had pathologically larger tumor size, more tumors, poorer differentiation, and more positive vascular invasion than the SUV420H1-low-score group. Multivariate analysis demonstrated that SUV420H1 high score was the poorest independent factor for overall survival. SUV420H1 knockdown could suppress cell cycle from G1 to S phase and cell invasion. GO pathway analysis showed that SUV420H1 contributed to cell proliferation, cell invasion, and/or metastasis. Overexpression of SUV420H1 clinically contributed to poor prognosis in HCC, and the inhibition of SUV420H1 could repress tumor progression and invasion both in vitro and in vivo; thus, further analyses of SUV420H1 are necessary for the discovery of future molecularly targeted drugs.     

Unexpected genetic toxicity to rodents of the N′, N′-dimethyl analogues of MNU andENU

Lijinsky and his colleagues have reported that the N′, N′-dimethyl analogues of ENU and MNU [N′, N′-dimethyl-N-ethyl-N-nitrosourea (DMENU) and trimethylnitrosourea (TMNU), respectively] are carcinogenic to rats despite their extreme hydrolytic stability which would reduce or preclude generation of alkylating species analogous to those formed upon hydrolysis of ENU and MNU. Lijinsky and his colleagues were unable to rationalize those activities of DMENU and TMNU despite extensive experimentation. We therefore decided to study this problem further. Whichever mode is accepted for the generation of electrophilic/mutagenic/carcinogenic reactive species from ENU and MNU, blocking of the free-NH₂ group with methyl groups (−NMe₂) should ablate or abolish activity. Consistent with this, DMENU and TMNU gave negative results in the NBP alkylation test while the parent compounds gave an instantaneous deep blue coloration. Studies of the rate of hydrolysis of these four compounds revealed ENU and MNU to have half-lives of 8 min, while the alkylated analogues (DMENU and TMNU) had half-lives of 25 and 41 days, respectively. Hydrolysis of ENU and MNU, to yield the alkylating species, proceeds either via proton abstraction from the −NH₂ group or by attack by water on the carbon of the carbonyl group. Methylation will inhibit both of these pathways, the first absolutely (no −NH₂ protons) and the second partially, via steric inhibition. The slow hydrolysis observed for DMENU and TMNU suggests that the latter route of hydrolysis is applicable. Studies with strain TA1535 of Salmonella typhimurium (without S9 mix) confirmed the potent mutagenic activity for ENU and MNU (∼300-fold increase in revertants at 2,000 μg/plate and ∼180-fold increase in revertants at 150 μg/plate respectively). In contrast, the methylated analogues showed only weak mutagenic activity (∼3-fold) at ∼100-fold higher dose-levels. Addition of S9 mix did not affect the mutagenicity of DMENU or TMNU. To this point, hypothesis and data coincide. ENU and MNU are potent micronucleus-inducing agents to the mouse bone marrow, and given the above data, it was expected that DMENU and TMNU would show weak or no activity in that assay. In fact, the methylated analogues were as effective as ENU and MNU as clastogens to the mouse bone marrow. Four possible reasons for this conflict of theory and data are explored. The speculative explanation we favour for these effects is that the net alkylation of bone marrow DNA is the same for all four chemicals. With ENU and MNU, most of the alkylating activity is dissipated by rapid hydrolysis. Thus, only a small fraction of the administered dose survives to alkylate the bone marrow. Due to the enhanced stability of the methyl analogues most of the delivered dose will reach the bone marrow. However, because of their lower intrinsic reactivity, only a small fraction of the target dose will alkylate the bone marrow DNA during the time window of the experiment. If these opposing influences happen to balance out, the essentially identical bone marrow genetic toxicity for the four chemicals could be explained. © 1996 Wiley-Liss, Inc.     

系统性红斑狼疮microRNA的异常表达及其与DNA甲基化的相互调控

系统性红斑狼疮是一种复杂的慢性、全身性自身免疫性疾病,其发病机制尚不明确.近年研究表明,microRNA和DNA甲基化在内的表观遗传修饰异常可能在系统性红斑狼疮的发生发展中起作用.MicroRNA是非编码单链小RNA,已有研究提示,microRNA参与了系统性红斑狼疮的发病和疾病进展,而其与DNA甲基化的相互作用可通过调控细胞内基因表达进而影响疾病的发生发展.概述了microRNA的异常表达及其与DNA甲基化之间的相互关系在系统性红斑狼疮发病机制中的作用,旨在为系统性红斑狼疮的研究提供新的思路.     

非小细胞肺癌表皮生长因子受体相关研究现状

近半个世纪,我国肺癌发病率和病死率均居全部恶性肿瘤之首。将来的20年,我国的肺癌病发数和病死数还在持续上升,传统的癌症治疗方法正面临着一个新的艰巨的挑战。近年来肺癌的分子病理指导下的个体化靶向治疗作为一项极具潜力的新方法已逐渐成为肺癌临床标准治疗的一部分,有着传统化疗及免疫治疗无可匹敌的优越性。肺癌分子靶向的诊断和治疗正逐渐成为常规治疗以及最佳个体化治疗的重要依据。表皮生长因子受体已成为炙手可热的临床肺癌治疗的分子靶点。     

Prognostic CpG Methylation Biomarkers Identified by Methylation Array in Esophageal Squamous Cell Carcinoma Patients

Background: Esophageal squamous cell carcinoma (ESCC) is an aggressive cancer with poor prognosis. We aimed to identify a panel of CpG methylation biomarkers for prognosis prediction of ESCC patients.Methods: Illumina''s GoldenGate methylation array, supervised principal components, Kaplan-Meier survival analyses and Cox regression model were conducted on dissected tumor tissues from a training cohort of 40 ESCC patients to identify potential CpG methylation biomarkers. Pyrosequencing quantitative methylation assay were performed to validate prognostic CpG methylation biomarkers in 61 ESCC patients. The correlation between DNA methylation and RNA expression of a validated marker, SOX17, was examined in a validation cohort of 61 ESCC patients.Results: We identified a panel of nine CpG methylation probes located at promoter or exon1 region of eight genes including DDIT3, FES, FLT3, NTRK3, SEPT5, SEPT9, SOX1, and SOX17, for prognosis prediction in ESCC patients. Risk score calculated using the eight-gene panel statistically predicted poor outcome for patients with high risk score. These eight-gene also showed a significantly higher methylation level in tumor tissues than their corresponding normal samples in all patients analyzed. In addition, we also detected an inverse correlation between CpG hypermethylation and the mRNA expression level of SOX17 gene in ESCC patients, indicating that DNA hypermethylation was responsible for decreased expression of SOX17.Conclusions: This study established a proof-of-concept CpG methylation biomarker panel for ESCC prognosis that can be further validated by multiple cohort studies. Functional characterization of the eight prognostic methylation genes in our biomarker panel could help to dissect the mechanism of ESCC tumorigenesis.