Background:The dysregulation of Isocitrate dehydrogenase(IDH)and the subsequent production of 2-Hydroxyglutrate(2HG)may alter the expression of epigenetic proteins in Grade 4 astrocytoma.The interplay mechanism betwee...Background:The dysregulation of Isocitrate dehydrogenase(IDH)and the subsequent production of 2-Hydroxyglutrate(2HG)may alter the expression of epigenetic proteins in Grade 4 astrocytoma.The interplay mechanism between IDH,O-6-methylguanine-DNA methyltransferase(MGMT)-promoter methylation,and protein methyltransferase proteins-5(PRMT5)activity,with tumor progression has never been described.Methods:A retrospective cohort of 34 patients with G4 astrocytoma is classified into IDH-mutant and IDH-wildtype tumors.Both groups were tested for MGMT-promoter methylation and PRMT5 through methylation-specific and gene expression PCR analysis.Inter-cohort statistical significance was evaluated.Results:Both IDH-mutant WHO grade 4 astrocytomas(n=22,64.7%)and IDH-wildtype glioblastomas(n=12,35.3%)had upregulated PRMT5 gene expression except in one case.Out of the 22 IDH-mutant tumors,10(45.5%)tumors showed MGMT-promoter methylation and 12(54.5%)tumors had unmethylated MGMT.All IDH-wildtype tumors had unmethylated MGMT.There was a statistically significant relationship between MGMT-promoter methylation and IDH in G4 astrocytoma(p-value=0.006).Statistically significant differences in progression-free survival(PFS)were also observed among all G4 astrocytomas that expressed PRMT5 and received either temozolomide(TMZ)or TMZ plus other chemotherapies,regardless of their IDH or MGMT-methylation status(p-value=0.0014).Specifically,IDH-mutant tumors that had upregulated PRMT5 activity and MGMT-promoter methylation,who received only TMZ,have exhibited longer PFS.Conclusions:The relationship between PRMT5,MGMT-promoter,and IDH is not tridirectional.However,accumulation of D2-hydroxyglutarate(2-HG),which partially activates 2-OG-dependent deoxygenase,may not affect their activities.In IDH-wildtype glioblastomas,the 2HG-2OG pathway is typically inactive,leading to PRMT5 upregulation.TMZ alone,compared to TMZ-plus,can increase PFS in upregulated PRMT5 tumors.Thus,using a PRMT5 inhibitor in G4 astrocytomas may help in tumor regression.展开更多
Background:Colorectal cancer(CRC)is one of the most frequently diagnosed cancers.In many cases,the poor prognosis of advanced CRC is associated with resistance to treatment with chemotherapeutic drugs such as 5-Fluoro...Background:Colorectal cancer(CRC)is one of the most frequently diagnosed cancers.In many cases,the poor prognosis of advanced CRC is associated with resistance to treatment with chemotherapeutic drugs such as 5-Fluorouracil(5-FU).The epithelial-to-mesenchymal transition(EMT)and dysregulation in protein methylation are two mechanisms associated with chemoresistance in many cancers.This study looked into the effect of 5-FU dose escalation on EMT and protein methylation in CRC.Materials and Methods:HCT-116,Caco-2,and DLD-1 CRC cell lines were exposed to dose escalation treatment of 5-FU.The motility and invasive potentials of the cells before and after treatment with 5-FU were investigated through wound healing and invasion assays.This was followed by aWestern blot which analyzed the protein expressions of the epithelial marker E-cadherin,mesenchymal marker vimentin,and the EMT transcription factor(EMTTF),the snail family transcriptional repressor 1(Snail)in the parental and desensitized cells.Western blotting was also conducted to study the protein expressions of the protein methyltransferases(PMTs),Euchromatic histone lysine methyltransferase 2(EHMT2/G9A),protein arginine methyltransferase(PRMT5),and SET domain containing 7/9(SETD7/9)along with the global lysine and arginine methylation profiles.Results:The dose escalation method generated 5-FU desensitized CRC cells with distinct morphological features and increased tolerance to high doses of 5-FU.The 5-FU desensitized cells experienced a decrease in migration and invasion when compared to the parental cells.This was reflected in the observed reduction in E-cadherin,vimentin,and Snail in the desensitized cell lines.Additionally,the protein expressions of EHMT2/G9A,PRMT5,and SETD7/9 also decreased in the desensitized cells and global protein lysine and arginine methylation became dysregulated with 5-FU treatment.Conclusion:This study showed that continuous,dose-escalation treatment of 5-FU in CRC cells generated 5-FU desensitized cancer cells that seemed to be less aggressive than parental cells.展开更多
Endocrine therapy using estrogen receptor-u （ER-α） antagonists for attenuating horm2one-driven cell proliferation is a major treatment modality for breast cancers. To exploit any DNA repair deficiencies associated ...Endocrine therapy using estrogen receptor-u （ER-α） antagonists for attenuating horm2one-driven cell proliferation is a major treatment modality for breast cancers. To exploit any DNA repair deficiencies associated with endocrine therapy, we investigated the functional and physical interactions of ER-α with O^6-methylguanine DNA methyltransferase （MGMT）, a unique DNA repair protein that confers tumor resistance to various anticancer alkylating agents. The ER-α -positive breast cancer cell lines （MCF-7, T47D） and ER- negative cell lines （MDAMB- 468, MDAMB-231）, and established inhibitors of ER-α and MGMT, namely, ICI-182,780 （Faslodex） and O^6- benzylguanine, respectively, were used to study MGMT- ER interactions. The MGMT gene promoter was found to harbor one full and two half estrogen-responsive elements （EREs） and two antioxidant-responsive elements （AREs）. MGMT expression was upregulated by estrogen, downregulated by tamoxifen in Western blot and promoter-linked reporter assays. Similarly, both transient and stable transfections of Nrf-2 （nuclear factor-erythroid 2-related factor-2） increased the levels of MGMT protein and activity 3 to 4-fold reflecting novel regulatory nodes for this dragresistance determinant. Of the different ER-α antagonists tested, the pure anti-estrogen fulvestrant was most potent in inhibiting the MGMT activity in a dose, time and ER-α dependent manner, similar to O^6-benzylguanine. Interestingly, fulvestrant exposure led to a degradation of both ER-α and MGMT proteins and O^6-benzylguanine also induced a specific loss of ER-a and MGMT proteins in MCF-7 and T47D breast cancer cells with similar kinetics. Immunoprecipitation revealed a specific association of ER-a and MGMT proteins in breast cancer cells. Furthermore, silencing of MGMT gene expression triggered a decrease in the levels of both MGMT and ER-a proteins. The involvement of proteasome in the drug-induced degradation of both proteins was also demonstrated. Fulvestrant enhanced the cytotoxicity of MGMT-targeted alkylating agents, namely, temozolomide and BCNU by 3 to 4-fold in ER-α positive cells, but not in ER-negative cells. We conclude that MGMT and ER-α proteins exist as a complex and are co-targeted for ubiquitin-conjugation and subsequent proteasomal degradation. The findings offer a clear rationale for combining alkylating agents with endocrine therapy.展开更多
目的了解我国实验室开展甲基化相关肿瘤标志物检测的总体情况,调查对象为Septin9基因、MGMT基因以及其他相关基因,调查内容包括检测方法和范围、检测试剂和样本,以及分析性能等。方法国家卫生健康委临床检验中心设计Septin9基因、MGMT...目的了解我国实验室开展甲基化相关肿瘤标志物检测的总体情况,调查对象为Septin9基因、MGMT基因以及其他相关基因,调查内容包括检测方法和范围、检测试剂和样本,以及分析性能等。方法国家卫生健康委临床检验中心设计Septin9基因、MGMT基因及其他相关基因甲基化检测的调查问卷,调查实验室开展检测的范围及方法、试剂及厂家、样本类型及要求、性能验证和性能确认的情况。2022年5月向全国24个省(自治区、直辖市)的125家实验室发放了调查问卷,并对回报结果进行统计分析。结果125家实验室包括68家医疗单位和57家独立医学实验室。67家实验室填写了Septin9基因,18家实验室填写了MGMT基因,54家实验室填写了其他相关基因,包括SDC2、SHOX2、RASSF1A、GSTP1、RNF180、MLH1、PAX1基因。基于PCR的方法是甲基化检测的主要手段,分别占Septin9基因、MGMT基因和其他基因甲基化检测的97.0%(65/67)、66.7%(12/18)和100.0%(54/54)。Septin9基因甲基化检测范围在启动子V2转录本CGI3区域,且涵盖3个以上CpG位点;MGMT基因甲基化检测范围在启动子DMR1和DMR2区域,涵盖4个以上CpG位点。Septin9基因甲基化检测试剂包括3种中国国家药品监督管理局(National medical products administration,NMPA)批准的商品化试剂(54/67,80.6%)和12种实验室自建试剂(Laboratory-developed tests,LDTs)(13/67,19.4%);MGMT基因甲基化检测试剂包括1种NMPA批准的试剂盒和14种LDTs,主要为实验室自建方法。97.0%(65/67)的Septin9基因甲基化检测采用血浆样本(2~10 ml);88.9%(16/18)的MGMT基因甲基化检测采用新鲜组织或福尔马林固定石蜡包埋组织切片样本(肿瘤细胞含量>10%)。有89.6%的Septin9基因甲基化检测实验室采用磁珠法以及61.1%的MGMT基因甲基化检测实验室采用离心柱法进行DNA提取。所有参与实验室均进行了DNA亚硫酸氢盐转化及纯化。有88.1%的Septin9基因甲基化检测实验室和61.1%的MGMT基因甲基化检测实验室在检测过程中使用了阴性/阳性质控品,有82.1%的Septin9基因甲基化检测实验室和72.2%的MGMT基因甲基化检测实验室做过试剂的性能验证或性能确认。98.5%的Septin9基因甲基化检测实验室的最低检出限(The limit of detection,LOD)为0.1%~10%的甲基化DNA水平或35~80pg/ml的甲基化DNA浓度;72.2%的MGMT基因甲基化检测实验室的LOD为1%~20%的甲基化DNA水平。结论我国实验室在Septin9基因和MGMT基因甲基化的检测方法及范围、检测试剂及样本方面存在差异,为了保证检测结果的准确可靠,各实验室应规范开展性能验证和性能确认工作,积极优化检测流程,进一步完善质量保证措施。展开更多
背景与目的:胶质瘤是中枢神经系统常见且预后较差的恶性肿瘤,手术后联合替莫唑胺(temozolomide,TMZ)同步放化疗是胶质瘤的主要治疗方案。O6-甲基鸟嘌呤DNA甲基转移酶(O6-methylguanine DNA methyltranferase,MGMT)基因启动子甲基化(MGMT...背景与目的:胶质瘤是中枢神经系统常见且预后较差的恶性肿瘤,手术后联合替莫唑胺(temozolomide,TMZ)同步放化疗是胶质瘤的主要治疗方案。O6-甲基鸟嘌呤DNA甲基转移酶(O6-methylguanine DNA methyltranferase,MGMT)基因启动子甲基化(MGMT promoter methylation,MGMTmet)状态可预测胶质瘤患者对TMZ治疗的敏感性,但其与临床病理学特征的关系及如何更好地预测治疗效果及患者预后尚需深入研究。本研究旨在分析胶质瘤中MGMTmet状态及其与临床病理学特征和其他常见分子异常的相关性,探讨MGMTmet与其他分子异常联合分析对胶质瘤患者预后和TMZ治疗效果判断的价值。方法:回顾性收集复旦大学附属肿瘤医院病理科2019年7月—2022年9月诊断的205例胶质瘤患者的临床病理学资料,采用实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQPCR)法检测MGMTmet状态;采用Sanger测序法检测异柠檬酸脱氢酶(isocitrate dehydrogenase,IDH)1和IDH2端粒酶反转录酶(telomerase reverse transcriptase,TERT)基因突变情况;采用荧光原位杂交(fluorescence in situ hybridization,FISH)检测1号染色体短臂和19号染色体长臂(the short arm of chromosome 1 and the long arm of chromosome 19,1p19q)缺失情况。结果:205例患者中,女性患者的MGMTmet发生率高于男性。相比于胶质母细胞瘤(47.3%),星形细胞瘤(74.1%)和少突胶质细胞瘤(100.0%)中MGMT基因启动子更易发生甲基化(P<0.05)。在MGMTmet组中,IDH1突变(mutant,mut)率和1p19q共缺失(co-deletion,co-del)率明显提高,且MGMTmet与IDH1mut和1p19qco-del具有相关性(P<0.05)。MGMTmet、年龄小于55岁、少突胶质细胞瘤及世界卫生组织(World Health Organization,WHO)1~3级的患者均表现出较长的总生存期(overall survival,OS),差异有统计学意义(P<0.05)。相比于单个影响因素,双/三基因联合分析[MGMTmet/IDH1mut、MGMTmet/1p19q co-del或MGMTmet/IDH1mut/1p19q co-del]预测患者预后的效果更好(P<0.05),后两者是独立预后因素。在TMZ治疗患者中,MGMTmet(MGMTmet/TMZ+)患者比其他组预后好,如果患者联合存在IDH1mut,患者预后得到进一步提高(P<0.05)。结论:MGMTmet好发于女性和少突胶质细胞瘤患者中;其与IDH1mut及1p19 qco-del呈正相关。MGMTmet的患者与TMZ治疗效果及预后较好有关,且MGMTmet联合IDH突变和1p19q co-del分析可能具有更好的TMZ治疗效果和预后提示价值。展开更多
The reversible and precise temporal and spatial regulation of histone lysine methyltransferases(KMTs)is essential for epigenome homeostasis.The dysregulation of KMTs is associated with tumor initiation,metastasis,chem...The reversible and precise temporal and spatial regulation of histone lysine methyltransferases(KMTs)is essential for epigenome homeostasis.The dysregulation of KMTs is associated with tumor initiation,metastasis,chemoresistance,invasiveness,and the immune microenvironment.Therapeutically,their promising effects are being evaluated in diversified preclinical and clinical trials,demonstrating encouraging outcomes in multiple malignancies.In this review,we have updated recent understandings of KMTs'functions and the development of their targeted inhibitors.First,we provide an updated overview of the regulatory roles of several KMT activities in oncogenesis,tumor suppression,and immune regulation.In addition,we summarize the current targeting strategies in different cancer types and multiple ongoing clinical trials of combination therapies with KMT inhibitors.In summary,we endeavor to depict the regulation of KMT-mediated epigenetic landscape and provide potential epigenetic targets in the treatment of cancers.展开更多
文摘Background:The dysregulation of Isocitrate dehydrogenase(IDH)and the subsequent production of 2-Hydroxyglutrate(2HG)may alter the expression of epigenetic proteins in Grade 4 astrocytoma.The interplay mechanism between IDH,O-6-methylguanine-DNA methyltransferase(MGMT)-promoter methylation,and protein methyltransferase proteins-5(PRMT5)activity,with tumor progression has never been described.Methods:A retrospective cohort of 34 patients with G4 astrocytoma is classified into IDH-mutant and IDH-wildtype tumors.Both groups were tested for MGMT-promoter methylation and PRMT5 through methylation-specific and gene expression PCR analysis.Inter-cohort statistical significance was evaluated.Results:Both IDH-mutant WHO grade 4 astrocytomas(n=22,64.7%)and IDH-wildtype glioblastomas(n=12,35.3%)had upregulated PRMT5 gene expression except in one case.Out of the 22 IDH-mutant tumors,10(45.5%)tumors showed MGMT-promoter methylation and 12(54.5%)tumors had unmethylated MGMT.All IDH-wildtype tumors had unmethylated MGMT.There was a statistically significant relationship between MGMT-promoter methylation and IDH in G4 astrocytoma(p-value=0.006).Statistically significant differences in progression-free survival(PFS)were also observed among all G4 astrocytomas that expressed PRMT5 and received either temozolomide(TMZ)or TMZ plus other chemotherapies,regardless of their IDH or MGMT-methylation status(p-value=0.0014).Specifically,IDH-mutant tumors that had upregulated PRMT5 activity and MGMT-promoter methylation,who received only TMZ,have exhibited longer PFS.Conclusions:The relationship between PRMT5,MGMT-promoter,and IDH is not tridirectional.However,accumulation of D2-hydroxyglutarate(2-HG),which partially activates 2-OG-dependent deoxygenase,may not affect their activities.In IDH-wildtype glioblastomas,the 2HG-2OG pathway is typically inactive,leading to PRMT5 upregulation.TMZ alone,compared to TMZ-plus,can increase PFS in upregulated PRMT5 tumors.Thus,using a PRMT5 inhibitor in G4 astrocytomas may help in tumor regression.
基金supported through the Faculty of Medicine and Surgery Award 2021 University of Malta(awarded to K.F).
文摘Background:Colorectal cancer(CRC)is one of the most frequently diagnosed cancers.In many cases,the poor prognosis of advanced CRC is associated with resistance to treatment with chemotherapeutic drugs such as 5-Fluorouracil(5-FU).The epithelial-to-mesenchymal transition(EMT)and dysregulation in protein methylation are two mechanisms associated with chemoresistance in many cancers.This study looked into the effect of 5-FU dose escalation on EMT and protein methylation in CRC.Materials and Methods:HCT-116,Caco-2,and DLD-1 CRC cell lines were exposed to dose escalation treatment of 5-FU.The motility and invasive potentials of the cells before and after treatment with 5-FU were investigated through wound healing and invasion assays.This was followed by aWestern blot which analyzed the protein expressions of the epithelial marker E-cadherin,mesenchymal marker vimentin,and the EMT transcription factor(EMTTF),the snail family transcriptional repressor 1(Snail)in the parental and desensitized cells.Western blotting was also conducted to study the protein expressions of the protein methyltransferases(PMTs),Euchromatic histone lysine methyltransferase 2(EHMT2/G9A),protein arginine methyltransferase(PRMT5),and SET domain containing 7/9(SETD7/9)along with the global lysine and arginine methylation profiles.Results:The dose escalation method generated 5-FU desensitized CRC cells with distinct morphological features and increased tolerance to high doses of 5-FU.The 5-FU desensitized cells experienced a decrease in migration and invasion when compared to the parental cells.This was reflected in the observed reduction in E-cadherin,vimentin,and Snail in the desensitized cell lines.Additionally,the protein expressions of EHMT2/G9A,PRMT5,and SETD7/9 also decreased in the desensitized cells and global protein lysine and arginine methylation became dysregulated with 5-FU treatment.Conclusion:This study showed that continuous,dose-escalation treatment of 5-FU in CRC cells generated 5-FU desensitized cancer cells that seemed to be less aggressive than parental cells.
基金supported by grants from the Cancer Prevention Research Institute of Texas(RP130266)the Carson-Leslie Foundation and the Association for Research of Childhood Cancer
文摘Endocrine therapy using estrogen receptor-u （ER-α） antagonists for attenuating horm2one-driven cell proliferation is a major treatment modality for breast cancers. To exploit any DNA repair deficiencies associated with endocrine therapy, we investigated the functional and physical interactions of ER-α with O^6-methylguanine DNA methyltransferase （MGMT）, a unique DNA repair protein that confers tumor resistance to various anticancer alkylating agents. The ER-α -positive breast cancer cell lines （MCF-7, T47D） and ER- negative cell lines （MDAMB- 468, MDAMB-231）, and established inhibitors of ER-α and MGMT, namely, ICI-182,780 （Faslodex） and O^6- benzylguanine, respectively, were used to study MGMT- ER interactions. The MGMT gene promoter was found to harbor one full and two half estrogen-responsive elements （EREs） and two antioxidant-responsive elements （AREs）. MGMT expression was upregulated by estrogen, downregulated by tamoxifen in Western blot and promoter-linked reporter assays. Similarly, both transient and stable transfections of Nrf-2 （nuclear factor-erythroid 2-related factor-2） increased the levels of MGMT protein and activity 3 to 4-fold reflecting novel regulatory nodes for this dragresistance determinant. Of the different ER-α antagonists tested, the pure anti-estrogen fulvestrant was most potent in inhibiting the MGMT activity in a dose, time and ER-α dependent manner, similar to O^6-benzylguanine. Interestingly, fulvestrant exposure led to a degradation of both ER-α and MGMT proteins and O^6-benzylguanine also induced a specific loss of ER-a and MGMT proteins in MCF-7 and T47D breast cancer cells with similar kinetics. Immunoprecipitation revealed a specific association of ER-a and MGMT proteins in breast cancer cells. Furthermore, silencing of MGMT gene expression triggered a decrease in the levels of both MGMT and ER-a proteins. The involvement of proteasome in the drug-induced degradation of both proteins was also demonstrated. Fulvestrant enhanced the cytotoxicity of MGMT-targeted alkylating agents, namely, temozolomide and BCNU by 3 to 4-fold in ER-α positive cells, but not in ER-negative cells. We conclude that MGMT and ER-α proteins exist as a complex and are co-targeted for ubiquitin-conjugation and subsequent proteasomal degradation. The findings offer a clear rationale for combining alkylating agents with endocrine therapy.
文摘目的 探讨应用平均表观传播子MRI(mean apparent propagator-magnetic resonance imaging, MAP-MRI)及动态对比增强MRI(dynamic contrast enhanced magnetic resonance imaging, DCE-MRI)预测3、4级胶质瘤患者O6-甲基鸟嘌呤-DNA-甲基转移酶(O6-methylguanine-DNA methyhransferase, MGMT)启动子甲基化状态的可行性。材料与方法 前瞻性纳入本院自2018年6月至2022年1月经病理证实的14例MGMT启动子甲基化和17例MGMT启动子非甲基化胶质瘤患者,进行了术前常规MRI、DCE-MRI及MAP-MRI扫描。通过手动勾画肿瘤实质区域为感兴趣区(region of interest, ROI)并提取ROI定量参数特征,测量DCE-MRI参数及MAP-MRI参数。参数与MGMT甲基化间的相关性采用Pearson相关分析。所有参数均采用两独立样本t检验,比较DCE-MRI和MAP-MRI对预测3、4级胶质瘤MGMT启动子甲基化状态的诊断效能。进一步建立单因素和多因素logistic回归模型,分析构建受试者工作特征(receiver operating characteristic, ROC)曲线,以DeLong检验比较DCE-MRI参数、MAP-MRI参数与多参数联合模型预测MGMT甲基化的诊断效果。结果 DCE-MRI参数容积转运常数(volume transfer constant,Ktrans)、血管外细胞外容积分数(fractional volume of the extravascular-extracellular space, Ve)以及MAP-MRI参数非高斯(non-Gaussianity, NG)、非高斯轴向(non-Gaussianity axial, NGAx)、Q空间逆方差(Q-space inverse variance,QIV)、返回原点概率(return to the origin probability, RTOP)、返回轴线概率(return to the axis probability,RTAP)与MGMT启动子甲基化间呈中等相关性,对预测3、4级胶质瘤MGMT启动子甲基化与非甲基化组间差异具有统计学意义(P<0.05);ROC曲线下面积(area under the curve, AUC)分别为0.803、0.815、0.807、0.803、0.765、0.790、0.739。多因素logistic分析显示Ve是预测MGMT启动子甲基化的最佳预测因子,其准确性最高,AUC为0.815(95%CI:0.659~0.971),比值比(odds ratio, OR)为0.891(95%CI:0.815~0.975)。DeLong检验结果表明DCE-MRI和MAP-MRI多参数联合后预测胶质瘤MGMT启动子甲基化状态的诊断效能最高,AUC为0.992。结论 DCE-MRI和MAP-MRI对于预测高级别胶质瘤MGMT启动子甲基化状态具有一定的应用价值,同时应用两者联合诊断将有助于进一步提高诊断的效能。
文摘目的了解我国实验室开展甲基化相关肿瘤标志物检测的总体情况,调查对象为Septin9基因、MGMT基因以及其他相关基因,调查内容包括检测方法和范围、检测试剂和样本,以及分析性能等。方法国家卫生健康委临床检验中心设计Septin9基因、MGMT基因及其他相关基因甲基化检测的调查问卷,调查实验室开展检测的范围及方法、试剂及厂家、样本类型及要求、性能验证和性能确认的情况。2022年5月向全国24个省(自治区、直辖市)的125家实验室发放了调查问卷,并对回报结果进行统计分析。结果125家实验室包括68家医疗单位和57家独立医学实验室。67家实验室填写了Septin9基因,18家实验室填写了MGMT基因,54家实验室填写了其他相关基因,包括SDC2、SHOX2、RASSF1A、GSTP1、RNF180、MLH1、PAX1基因。基于PCR的方法是甲基化检测的主要手段,分别占Septin9基因、MGMT基因和其他基因甲基化检测的97.0%(65/67)、66.7%(12/18)和100.0%(54/54)。Septin9基因甲基化检测范围在启动子V2转录本CGI3区域,且涵盖3个以上CpG位点;MGMT基因甲基化检测范围在启动子DMR1和DMR2区域,涵盖4个以上CpG位点。Septin9基因甲基化检测试剂包括3种中国国家药品监督管理局(National medical products administration,NMPA)批准的商品化试剂(54/67,80.6%)和12种实验室自建试剂(Laboratory-developed tests,LDTs)(13/67,19.4%);MGMT基因甲基化检测试剂包括1种NMPA批准的试剂盒和14种LDTs,主要为实验室自建方法。97.0%(65/67)的Septin9基因甲基化检测采用血浆样本(2~10 ml);88.9%(16/18)的MGMT基因甲基化检测采用新鲜组织或福尔马林固定石蜡包埋组织切片样本(肿瘤细胞含量>10%)。有89.6%的Septin9基因甲基化检测实验室采用磁珠法以及61.1%的MGMT基因甲基化检测实验室采用离心柱法进行DNA提取。所有参与实验室均进行了DNA亚硫酸氢盐转化及纯化。有88.1%的Septin9基因甲基化检测实验室和61.1%的MGMT基因甲基化检测实验室在检测过程中使用了阴性/阳性质控品,有82.1%的Septin9基因甲基化检测实验室和72.2%的MGMT基因甲基化检测实验室做过试剂的性能验证或性能确认。98.5%的Septin9基因甲基化检测实验室的最低检出限(The limit of detection,LOD)为0.1%~10%的甲基化DNA水平或35~80pg/ml的甲基化DNA浓度;72.2%的MGMT基因甲基化检测实验室的LOD为1%~20%的甲基化DNA水平。结论我国实验室在Septin9基因和MGMT基因甲基化的检测方法及范围、检测试剂及样本方面存在差异,为了保证检测结果的准确可靠,各实验室应规范开展性能验证和性能确认工作,积极优化检测流程,进一步完善质量保证措施。
文摘背景与目的:胶质瘤是中枢神经系统常见且预后较差的恶性肿瘤,手术后联合替莫唑胺(temozolomide,TMZ)同步放化疗是胶质瘤的主要治疗方案。O6-甲基鸟嘌呤DNA甲基转移酶(O6-methylguanine DNA methyltranferase,MGMT)基因启动子甲基化(MGMT promoter methylation,MGMTmet)状态可预测胶质瘤患者对TMZ治疗的敏感性,但其与临床病理学特征的关系及如何更好地预测治疗效果及患者预后尚需深入研究。本研究旨在分析胶质瘤中MGMTmet状态及其与临床病理学特征和其他常见分子异常的相关性,探讨MGMTmet与其他分子异常联合分析对胶质瘤患者预后和TMZ治疗效果判断的价值。方法:回顾性收集复旦大学附属肿瘤医院病理科2019年7月—2022年9月诊断的205例胶质瘤患者的临床病理学资料,采用实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQPCR)法检测MGMTmet状态;采用Sanger测序法检测异柠檬酸脱氢酶(isocitrate dehydrogenase,IDH)1和IDH2端粒酶反转录酶(telomerase reverse transcriptase,TERT)基因突变情况;采用荧光原位杂交(fluorescence in situ hybridization,FISH)检测1号染色体短臂和19号染色体长臂(the short arm of chromosome 1 and the long arm of chromosome 19,1p19q)缺失情况。结果:205例患者中,女性患者的MGMTmet发生率高于男性。相比于胶质母细胞瘤(47.3%),星形细胞瘤(74.1%)和少突胶质细胞瘤(100.0%)中MGMT基因启动子更易发生甲基化(P<0.05)。在MGMTmet组中,IDH1突变(mutant,mut)率和1p19q共缺失(co-deletion,co-del)率明显提高,且MGMTmet与IDH1mut和1p19qco-del具有相关性(P<0.05)。MGMTmet、年龄小于55岁、少突胶质细胞瘤及世界卫生组织(World Health Organization,WHO)1~3级的患者均表现出较长的总生存期(overall survival,OS),差异有统计学意义(P<0.05)。相比于单个影响因素,双/三基因联合分析[MGMTmet/IDH1mut、MGMTmet/1p19q co-del或MGMTmet/IDH1mut/1p19q co-del]预测患者预后的效果更好(P<0.05),后两者是独立预后因素。在TMZ治疗患者中,MGMTmet(MGMTmet/TMZ+)患者比其他组预后好,如果患者联合存在IDH1mut,患者预后得到进一步提高(P<0.05)。结论:MGMTmet好发于女性和少突胶质细胞瘤患者中;其与IDH1mut及1p19 qco-del呈正相关。MGMTmet的患者与TMZ治疗效果及预后较好有关,且MGMTmet联合IDH突变和1p19q co-del分析可能具有更好的TMZ治疗效果和预后提示价值。
基金the Science and Technology Commission of Shanghai,China(Grant Nos.:20DZ2270800 and 19JC1410200)Innovative Research Team of High-Level Local Universities in Shanghai,China(Grant No.:SHSMU-ZDCX20210900)the National Natural Science Foundation of China(Grant No.:82073889).
文摘The reversible and precise temporal and spatial regulation of histone lysine methyltransferases(KMTs)is essential for epigenome homeostasis.The dysregulation of KMTs is associated with tumor initiation,metastasis,chemoresistance,invasiveness,and the immune microenvironment.Therapeutically,their promising effects are being evaluated in diversified preclinical and clinical trials,demonstrating encouraging outcomes in multiple malignancies.In this review,we have updated recent understandings of KMTs'functions and the development of their targeted inhibitors.First,we provide an updated overview of the regulatory roles of several KMT activities in oncogenesis,tumor suppression,and immune regulation.In addition,we summarize the current targeting strategies in different cancer types and multiple ongoing clinical trials of combination therapies with KMT inhibitors.In summary,we endeavor to depict the regulation of KMT-mediated epigenetic landscape and provide potential epigenetic targets in the treatment of cancers.
