Association between Helicobacter pylori infection,mismatch repair,HER2 and tumor-infiltrating lymphocytes in gastric cancer

BACKGROUND The influence of Helicobacter-pylori(H.pylori)infection and the characteristics of gastric cancer(GC)on tumor-infiltrating lymphocyte(TIL)levels has not been extensively studied.Analysis of infiltrating-immune-cell subtypes as well as survival is necessary to obtain comprehensive information.AIM To determine the rates of deficient mismatch-repair(dMMR),HER2-status and H.pylori infection and their association with TIL levels in GC.METHODS Samples from 503 resected GC tumors were included and TIL levels were evaluated following the international-TILs-working-group recommendations with assessment of the intratumoral(IT),stromal(ST)and invasive-border(IB)compartments.The density of CD3,CD8 and CD163 immune cells,and dMMR and HER2-status were determined by immunohistochemistry(IHC).H.pylori infection was evaluated by routine histology and quantitative PCR(qPCR)in a subset of samples.RESULTS dMMR was found in 34.4%,HER2+in 5%and H.pylori-positive in 55.7%of samples.High IT-TIL was associated with grade-3(P=0.038),while ST-TIL with grade-1(P<0.001),intestinal-histology(P<0.001)and no-recurrence(P=0.003).dMMR was associated with high TIL levels in the ST(P=0.019)and IB(P=0.01)compartments,and STCD3(P=0.049)and ST-CD8(P=0.05)densities.HER2-was associated with high IT-CD8(P=0.009).H.pylorinegative was associated with high IT-TIL levels(P=0.009)when assessed by routine-histology,and with high TIL levels in the 3 compartments(P=0.002-0.047)and CD8 density in the IT and ST compartments(P=0.001)when assessed by qPCR.A longer overall survival was associated with low IT-CD163(P=0.003)and CD8/CD3(P=0.001 in IT and P=0.002 in ST)and high IT-CD3(P=0.021),ST-CD3(P=0.003)and CD3/CD163(P=0.002).CONCLUSION TIL levels were related to dMMR and H.pylori-negativity.Low CD8/CD3 and high CD163/CD3 were associated with lower recurrence and longer survival.

Comprehensive analysis of gene mutations and mismatch repair in Chinese colorectal cancer patients

BACKGROUND RAS,BRAF,and mismatch repair(MMR)/microsatellite instability(MSI)are crucial biomarkers recommended by clinical practice guidelines for colorectal cancer(CRC).However,their characteristics and influencing factors in Chinese patients have not been thoroughly described.AIM To analyze the clinicopathological features of KRAS,NRAS,BRAF,and PIK3CA mutations and the DNA MMR status in CRC.METHODS We enrolled 2271 Chinese CRC patients at the China-Japan Friendship Hospital.MMR proteins were tested using immunohistochemical analysis,and the KRAS/NRAS/BRAF/PIK3CA mutations were determined using quantitative polymerase chain reaction.Microsatellite status was determined using an MSI detection kit.Statistical analyses were conducted using SPSS software and logistic regression.RESULTS The KRAS,NRAS,BRAF,and PIK3CA mutations were detected in 44.6%,3.4%,3.7%,and 3.9% of CRC patients,respectively.KRAS mutations were more likely to occur in patients with moderate-to-high differentiation.BRAF mutations were more likely to occur in patients with right-sided CRC,poorly differentiated,or no perineural invasion.Deficient MMR(dMMR)was detected in 7.9% of all patients and 16.8% of those with mucinous adenocarcinomas.KRAS,NRAS,BRAF,and PIK3CA mutations were detected in 29.6%,1.1%,8.1%,and 22.3% of patients with dMMR,respectively.The dMMR was more likely to occur in patients with a family history of CRC,aged<50 years,right-sided CRC,poorly differentiated histology,no perineural invasion,and with carcinoma in situ,stage I,or stage II tumors.CONCLUSION This study analyzed the molecular profiles of KRAS,NRAS,BRAF,PIK3CA,and MMR/MSI in CRC,identifying key influencing factors,with implications for clinical management of CRC.

Tislelizumab in previously treated,locally advanced unresectable/metastatic microsatellite instability-high/mismatch repair-deficient solid tumors

Objective:The open-label,phase II RATIONALE-209 study evaluated tislelizumab(anti-programmed cell death protein 1 antibody)as a tissue-agnostic monotherapy for microsatellite instability-high(MSI-H)/mismatch repair-deficient(dMMR)tumors.Methods:Adults with previously treated,locally advanced unresectable or metastatic MSI-H/dMMR solid tumors were enrolled.Patients received tislelizumab 200 mg intravenously every 3 weeks.Objective response rate(ORR;primary endpoint),duration of response(DoR),and progression-free survival(PFS)were assessed by independent review committee(Response Evaluation Criteria in Solid Tumors v1.1).Results:Eighty patients were enrolled and treated;75(93.8%)patients had measurable disease at baseline.Most had metastatic disease and received at least one prior therapy for advanced/metastatic disease(n=79;98.8%).At primary analysis(data cutoff July 8,2021;median follow-up 15.2 months),overall ORR[46.7%;95%confidence interval(95%CI),35.1−58.6;one-sided P<0.0001]and ORR across tumor-specific subgroups[colorectal(n=46):39.1%(95%CI,25.1–54.6);gastric/gastroesophageal junction(n=9):55.6%(95%CI,21.2−86.3);others(n=20):60.0%(95%CI,36.1−80.9)]were significantly greater with tislelizumab vs.a prespecified historical control ORR of 10%;five(6.7%)patients had complete responses.Median DoR,PFS,and overall survival were not reached with long-term follow-up(data cutoff December 5,2022;median follow-up 28.9 months).Tislelizumab was well tolerated with no unexpected safety signals.Treatment-related adverse events(TRAEs)of grade≥3 occurred in 53.8%of patients;7.5%of patients discontinued treatment due to TRAEs.Conclusions:Tislelizumab demonstrated a significant ORR improvement in patients with previously treated,locally advanced unresectable or metastatic MSI-H/dMMR tumors and was generally well tolerated.

子宫内膜癌组织中dMMR蛋白和miRNA Let-7表达水平及临床价值的研究

目的探讨子宫内膜癌(endometrial carcinoma,EC)中微小RNA Let-7(miRNA Let-7)表达水平与DNA错配修复(DNA mismatch repair,dMMR)蛋白表达缺失的关系及意义。方法选取2016年5月~2022年12月在江苏省连云港市妇幼保健院行根治性手术切除的子宫内膜癌患者74例,分别用免疫组织化学法检测dMMR蛋白(包括MLH1,PMS2,MSH2,MSH6)的表达、实时荧光定量聚合酶链反应(qRT-PCR)检测miRNA Let-7的相对表达量,按照dMMR蛋白表达情况,将EC患者分为表达完整组(n=43)和表达缺失组(n=31),采用Logistic多因素回归分析与dMMR蛋白缺失相关的危险因素、绘制受试者工作特征(receiver operating characteristic,ROC)曲线评估相关因素的预测价值。结果74例EC病例中,miRNA Let-7的表达水平在肌层浸润<1/2组显著高于肌层浸润≥1/2组,差异具有统计学意义(t=1.79,P=0.04);dMMR蛋白表达的缺失率为41.89%,且年龄<55岁组和miRNA Let-7低表达组(<0.715)患者中的dMMR蛋白缺失率高于≥55岁组和miRNA Let-7高表达组(≥0.715),差异具有统计学意义(χ2=3.92,4.50,均P<0.05);Logistic多因素回归分析结果显示miRNA Let-7表达水平是发生dMMR表达缺失的独立危险因素(P=0.012);Spearman相关分析表明,miRNA Let-7的表达水平与dMMR蛋白缺失呈明显负相关(r=-0.247,P=0.034);ROC曲线分析结果显示,miRNA Let-7表达水平对于预测EC患者中发生dMMR蛋白的缺失具有一定的价值,其AUC为0.737,最佳临界值为0.77,敏感度和特异度分别为0.651,0.806。结论子宫内膜癌患者中miRNA Let-7的表达水平与dMMR蛋白缺失具有相关性,也是发生dMMR蛋白缺失的危险因素,有望为预测dMMR的表达缺失提供帮助。

结直肠癌组织中HMGB2、MAGE-A9、MMR蛋白表达及其临床意义

目的探讨结直肠癌组织中高迁移率族蛋白B2(HMGB2)、黑色素瘤相关抗原-A9(MAGE-A9)、DNA错配修复基因(MMR)蛋白表达情况,以及其在评估患者预后中的临床价值。方法选取52例结直肠癌患者作为研究对象,术中采集所有患者肿瘤标本及癌旁组织标本,检测HMGB2、MAGE-A9、MMR蛋白表达水平,并分析各指标表达情况与病理特征、预后间的关系。结果肿瘤组织内HMGB2、MAGE-A9、MMR阳性表达高于癌旁组织,差异有统计学意义(P<0.05)。肿瘤分期Ⅲ期、分化程度低分化、淋巴结转移患者HMGB2、MAGE-A9、MMR阳性表达率高于Ⅰ期、Ⅱ期、中高分化、无淋巴结转移患者,差异有统计学意义(P<0.05)。随访3年,52例患者存活率为71.15%(37/52);HMGB2阳性组存活率[61.76%(21/34)]低于HMGB2阴性组[88.89%(16/18)],差异有统计学意义(χ^(2)=4.219,P=0.040);MAGE-A9阳性组存活率[58.62%(17/29)]低于MAGE-A9阴性组[86.96%(20/23)],差异有统计学意义(χ^(2)=5.018,P=0.025);MMR阳性组存活率[52.00%(13/25)]低于MMR阴性组[88.89%(24/27)],差异有统计学意义(χ^(2)=8.606,P=0.003)。结论HMGB2、MAGE-A9、MMR在结直肠癌中呈高表达状态,其表达与肿瘤分期、分化程度、淋巴结转移关系密切,且不同表达患者存活率存在较大差异,可作为评估预后的重要指标。

绝经后子宫内膜癌患者MMR蛋白表达缺失的影响因素分析

目的 探讨绝经后子宫内膜癌患者错误配对修复(MMR)蛋白表达缺失的影响因素。方法 选取62例绝经后子宫内膜癌手术患者为研究对象,以错误配对修复蛋白的表达为依据,将1种及以上错误配对修复蛋白表达缺失的22例患者纳入微卫星不稳定性(MSI)组,而4种错误配对修复蛋白表达不缺失的40例患者则纳入微卫星稳定型(MSS)组。检测统计并分析两组的临床资料[年龄、绝经年龄、体质量指数(BMI)、子宫内膜厚度、孕次、血清癌胚抗原(CEA)、睾酮(T)],采用Logistic回归分析绝经后子宫内膜癌患者错误配对修复蛋白表达的影响因素,并分析血清T、CEA水平对绝经后子宫内膜癌患者错误配对修复蛋白表达缺失的预测价值。结果 两组的年龄、绝经年龄、子宫内膜厚度、孕次均无显著性差异(P>0.05)。MSI组的血清CEA和T水平及BMI分别为(13.81±1.06)ng/ml、(59.94±9.16)ng/dl、(27.18±2.59)kg/m^(2), MSS组分别为(10.98±1.84)ng/ml、(52.16±11.31)ng/dl、(24.75±0.86)kg/m^(2);与MSS组相比, MSI组的血清CEA和T水平及BMI均较高,差异显著(P<0.05)。将绝经后子宫内膜癌患者血清CEA、T、BMI水平分别作为协变量,错误配对修复蛋白表达情况作为因变量(MSS=1, MSI=0),经Logistic回归分析结果显示,血清CEA、T、BMI水平升高是绝经后子宫内膜癌患者错误配对修复蛋白表达缺失的影响因素[OR(95%置信区间)=0.217(0.093, 0.507)、0.860(0.773, 0.956)、1.326(1.036, 1.697),P<0.05]。将子宫内膜癌患者错误配对修复蛋白表达缺失作为状态变量,血清T、CEA水平作为检验变量,绘制受试者工作特征曲线(ROC曲线),结果示血清T、CEA水平预测绝经后子宫内膜癌患者错误配对修复蛋白表达缺失的曲线下面积(AUC)分别为0.750、0.893,均具有预测价值。结论 血清CEA、T水平与绝经后子宫内膜癌患者错误配对修复蛋白表达缺失密切相关,其水平升高是患者MMR蛋白表达缺失的重要影响因素,临床可通过检测血清CEA、T水平,以评价错误配对修复蛋白表达缺失的可能性,并推测预后及优化治疗方法。

MMR、PD-L1在子宫内膜样癌中的表达及其与预后的相关性

目的探讨错配修复蛋白(MMR)、程序性死亡配体-1(PD-L1)在子宫内膜样癌(EEC)中的表达及其与预后的关系。方法收集2015年1月-2020年9月潍坊市人民医院病理科诊断的EEC组织标本90例、子宫内膜不典型增生组织30例、子宫内膜样癌旁组织30例及子宫内膜不典型增生旁组织10例,对不同子宫内膜组织中的MMR、PD-L1表达情况采用免疫组化法进行检测,分析MMR、PD-L1表达与患者临床病理特征关系及与预后的相关性。结果MMR缺失(dMMR)、PD-L1在EEC组织中表达高于不典型增生组织、子宫内膜不典型增生旁组织及EEC旁组织(检验水准<0.017);dMMR、PD-L1与EEC的年龄及淋巴结转移、FIGO分期、肌层浸润深度无关(P>0.05);PD-L1的表达在G3期较G1期表达上调(检验水准<0.017);PD-L1与dMMR呈正相关(P<0.05);dMMR、PD-L1与患者预后有关,dMMR、PD-L1阳性的患者预后更差,MMR可以作为影响EEC患者生存的独立因素(P<0.05)。结论dMMR、PD-L1在EEC发生发展过程中起协同作用。dMMR、PD-L1与患者预后有关。dMMR是影响EEC预后的独立因素,联合检测MMR、PD-L1可能为EEC预后评估提供理论依据。

结直肠癌组织中MMR蛋白表达、MSI、RAS和BRAF基因突变与临床病理特征的关系

目的 探讨结直肠癌患者癌组织中错配修复(MMR)蛋白表达、微卫星不稳定性(MSI)、大鼠肉瘤(RAS)基因和致癌同源体B1(BRAF)基因突变与临床病理特征的关系。方法 选取该院2022年1-12月接受根治手术治疗的352例结直肠癌患者的肿瘤组织和血液标本、42例非肠癌患者的实体瘤组织和血液标本,采用免疫组化法检测MMR蛋白表达,一代测序片段分析法检测MSI,实时荧光定量聚合酶链反应检测KRAS、NRAS和BRAF基因突变状态,分析MMR蛋白表达、MSI和3种基因突变状态与结直肠癌临床病理特征的关系。结果 352例CRC患者肿瘤组织中检出MMR缺陷(dMMR)29例(8.2%),高度微卫星不稳定(MSI-H)26例(7.4%),KRAS基因突变161例(45.7%),NRAS基因突变13例(3.7%),BRAF基因突变11例(3.1%)。与dMMR有关因素为低龄、黏液腺癌、原发于右半结肠癌(P<0.05);与MSI-H相关因素包括低龄、肿瘤家族史、原发于右半结肠癌(P<0.05);KRAS和NRAS基因高突变率分别与黏液腺癌和淋巴结转移有关(P<0.05);BRAF基因高突变率与低分化和原发于右半结肠癌有关(P<0.05)。BRAF基因突变与dMMR和MSI-H有关(P<0.05)。结论 MMR蛋白表达,MSI,以及KRAS、NRAS和BRAF基因突变与不同的临床病理特征有关。通过这5种分子标志物的联合检测可以对肿瘤进行分子分型,进一步为结直肠癌患者的个体化精准诊疗提供理论依据。

The Effect of Curcumin on Mismatch Repair(MMR) Proteins hMSH2 and hMLH1 after Ultraviolet(UV) Irradiation on HL-60 Cells

To understand the expression and effect of mismatch repair genes, hMSH2 and hMLH1, and to investigate anti-leukemic cell proliferation mechanism of curcumin, the levels of both genes were detected by multiple comparative RT-PCR. The protein of hMSH2 was determined by flow cy-tometry (FCM) and the gene mutation of hMSH2 and hMLH1 were detected by PCR-SSCP and mi-crosatellite instability assay. After UV irradiation, the gene expression of hMSH2 and hMLHl was not increased and showed no response. This phenomenon was not ascribed to gene mutation, because PCP-SSCP and microsatellite instability assay revealed no abnormal gel-shift band in both genes. After irradiation and addition of curcumin, the expression of hMSH2 mRNA increased and the cellular apoptotic rate also increased at the same time. The difference was statistically significant as compared with groups without addition of curcumin and control groups (P<0. 05). Our results suggested that when MMR system was inhibited by the same agents, curcumin can remove this suppression and switch to cellular apoptosis.

结直肠癌MMR表达与微卫星病灶状态及预后水平的关联研究

目的分析结直肠癌错配修复(MMR)基因表达情况与微卫星病灶状态及预后水平的关联。方法本次研究以2020年7月至2022年7月河南省濮阳惠民医院收治的102例结直肠癌患者为研究对象,根据免疫组化检测结果,将MMR表达缺失的42例患者列为dMMR组,将其余60例MMR完全表达的患者列为pMMR组,收集、对比两组患者的一般资料、临床资料,经统计学单因素分析、Logistic多因素回归分析归纳结直肠癌MMR表达缺失的危险因素;所有患者均行含奥沙利铂的一线化疗,并开展为期1年随访,比较两组治疗后的生存情况,应用Spearman相关性系数验证MMR表达与微卫星不稳定(MSI)及预后水平的关联。结果统计学单因素分析结果显示,两组患者的年龄、病灶直径、肿瘤分化程度、肿瘤区域淋巴结受累情况、肿瘤远处转移情况及微卫星病灶状态均差异有统计学意义(P<0.05);Logistic多因素回归分析结果显示,年龄≥60岁、病灶直径≥5 cm、肿瘤低分化、区域淋巴结受累N0、肿瘤远处转移M0、微卫星病灶检测标志物<40%或≥40%(MSI-L或MSI-H)为导致结直肠癌MMR表达缺失的主要影响因素。经含奥沙利铂一线化疗治疗后,pMMR组的客观缓解率(ORR)为75.00%(45/60),疾病控制率(DCR)为80.00%(48/60),中位无进展生存期(PFS)为(10.24±3.35)个月,中位总生存期(OS)为(11.46±2.49)个月,均高于dMMR组[52.38%(22/42)、59.52%(25/42)、(8.28±3.14)个月、(10.33±2.02)个月],MSI为(32.44±5.18)%,低于dMMR组(35.45±5.26)%,差异有统计学意义(P<0.05)。经Spearman相关性系数验证,MMR表达与结直肠癌的ORR、DCR、中位PFS、中位OS负相关,与MSI表达正相关。结论区域淋巴结未受累且无发生远处转移,及MSI为导致结直肠癌MMR表达缺失的主要影响因素,MMR表达缺失可导致结直肠癌患者MSI,若患者存在dMMR或MSI不宜应用含奥沙利铂一线化疗治疗。

YAP1、PD-L1及MMR蛋白在消化道神经内分泌癌中的表达及意义

目的探讨Yes相关蛋白1(YAP1)、程序性死亡-配体1(PD-L1)及错配修复(MMR)蛋白在消化道神经内分泌癌组织中的表达及临床意义。方法应用组织芯片及免疫组化法,检测122例消化道神经内分泌癌组织中YAP1、PD-L1及MMR蛋白的表达情况,并结合消化道神经内分泌癌不同临床病理参数和患者预后进行比较分析。结果YAP1核、YAP1质、PD-L1及MMR蛋白缺失的阳性率分别为27.1%、35.3%、22.1%、2.5%。YAP1在神经内分泌癌组织中的表达显著高于正常组织或神经内分泌瘤(均P<0.05),YAP1核表达与脉管浸润、淋巴结转移及TNM分期呈显著正相关(均P<0.05)。PD-L1表达与临床病理特征无明显相关性(均P>0.05)。相关性分析显示YAP1核表达与PD-L1表达呈负相关(P<0.05)。生存分析未发现YAP1及PD-L1的预后意义。结论YAP1核表达可能与消化道神经内分泌癌的发生发展有关;消化道神经内分泌癌表达PD-L1及错配修复缺陷(dMMR);针对YAP1的靶向治疗及抗PD-1/PD-L1治疗可能成为消化道神经内分泌癌的潜在新疗法。

KRAS and BRAF gene mutations and DNA mismatch repair status in Chinese colorectal carcinoma patients

AIM:To investigate gene mutations and DNA mismatch repair(MMR) protein abnormality in Chinese colorectalcarcinoma(CRC) patients and their correlations with clinicopathologic features.METHODS:Clinical and pathological information for 535 patients including 538 tumors was reviewed and recorded.Mutation analyses for exon 2 of KRAS gene and exon 15 of BRAF gene were performed by Sanger sequencing except that in 9 tumors amplification refractory mutation system PCR was used.Expression of MMR proteins including MHL1,MSH2,MSH6 and PMS2 was evaluated by immunohistochemistry.Correlations of KRAS and BRAF mutation status and the expression status of MMR proteins with age,gender,cancer stage,location,and histology were analyzed.Correlations between KRAS or BRAF mutations and MMR protein expression were also explored.RESULTS:The overall frequencies of KRAS and BRAF mutations were 37.9% and 4.4%,respectively.KRAS mutations were more common in patients ≥ 50 years old(39.8% vs 22% in patients < 50 years old,P < 0.05).The frequencies of BRAF mutants were higher in tumors from females(6.6% vs males 2.8%,P < 0.05),located in the right colon(9.6% vs 2.1% in the left colon,1.8% in the rectum,P < 0.01),with mucinous differentiation(9.8% vs 2.8% without mucinous differentiation,P < 0.01),or being poorly differentiated(9.5% vs 3.4% well/moderately differentiated,P < 0.05).MMR deficiency was strongly associated with proximal location(20.5% in the right colon vs 9.2% in the left colon and 5.1% in the rectum,P < 0.001),early cancer stage(15.0% in stages Ⅰ-Ⅱ vs 7.7% in stages Ⅲ-Ⅳ,P < 0.05),and mucinous differentiation(20.2% vs 9.2% without mucin,P < 0.01).A higher frequency of MLH1/PMS2 loss was found in females(9.2% vs 4.4% in males,P < 0.05),and MSH2/MSH6 loss tended to be seen in younger(<50 years old) patients(12.0% vs 4.0% ≥ 50 years old,P < 0.05).MMR deficient tumors were less likely to have KRAS mutations(18.8% vs 41.7% in MMR proficient tumors,P < 0.05) and tumorswith abnormal MLH1/PMS2 tended to harbor BRAF mutations(15.4% vs 4.2% in MMR proficient tumors,P < 0.05).CONCLUSION:The frequency of sporadic CRCs having BRAF mutation,MLH1 deficiency and MSI in Chinese population may be lower than that in the Western population.

ARID1A expression in gastric adenocarcinoma:Clinicopathological significance and correlation with DNA mismatch repair status

AIM:To analyze the mismatch repair(MMR)status and the ARID1A expression as well as their clinicopathological significance in gastric adenocarcinomas.METHODS:We examined the expressions of MMR proteins and ARID1A by immunohistochemistry in consecutive 489 primary gastric adenocarcinomas.The results were further correlated with clinicopathological variables.RESULTS:The loss of any MMR protein expression,indicative of MMR deficiency,was observed in 38cases(7.8%)and was significantly associated with an older age(68.6±9.2 vs 60.4±11.7,P<0.001),a female sex(55.3%vs 31.3%,P=0.004),an antral location(44.7%vs 25.7%,P=0.021),and a differentiated histology(57.9%vs 39.7%,P=0.023).Abnormal ARID1A expression,including reduced or loss of ARID1A expression,was observed in 109 cases(22.3%)and was significantly correlated with lymphatic invasion(80.7%vs 69.5%,P=0.022)and lymph node metastasis(83.5%vs 73.7%,P=0.042).The tumors with abnormal ARID1A expression more frequently indicated MMR deficiency(47.4%vs 20.2%,P<0.001).A multivariate analysis identified abnormal ARID1A expression as an independent poor prognostic factor(HR=1.36,95%CI:1.01-1.84;P=0.040).CONCLUSION:Our observations suggest that the AIRD1A inactivation is associated with lymphatic invasion,lymph node metastasis,poor prognosis,and MMR deficiency in gastric adenocarcinomas.

Deficient DNA mismatch repair is associated with favorable prognosis in Thai patients with sporadic colorectal cancer

AIM:To determine the prognostic significance of deficient mismatch repair(d MMR) and BRAF V600 E in Thai sporadic colorectal cancer(CRC) patients.METHODS:We studied a total of 211 out of 405 specimens obtained from newly diagnosed CRC patients between October 1,2006 and December 31,2007 at Siriraj Hospital,Mahidol University.Formalinfixed paraffin-embedded blocks of CRC tissue samples w e re a n a l y ze d fo r d M M R b y d e t e c t i o n o f M M R protein expression loss by immunohistochemistry or microsatellite instability using polymerase chain reaction(PCR)-DHPLC.BRAF V600 E mutational analysis was performed in DNA extracted from the same archival tissues by two-round allele-specific PCR and analyzed by high sensitivity DHPLC.Associations between patient characteristics,MMR and BRAF status with diseasefree survival(DFS) and overall survival(OS) were determined by Kaplan-Meier survival plots and log-rank test together with Cox's proportional hazard regression.RESULTS:d MMR and BRAF V600 E mutations were identified in 31 of 208(14.9%) and 23 of 211(10.9%) tumors,respectively.d MMR was more commonly found in patients with primary colon tumors rather than rectal cancer(20.4% vs 7.6%,P =0.01),but there was no difference in MMR status between the right-sided and left-sided colon tumors(20.8% vs 34.6%,P = 0.24).d MMR was associated with early-stage rather than metastatic disease(17.3% vs 0%,P = 0.015).No clinicopathological features such primary site or tumor differentiation were associated with the BRAF mutation.Six of 31(19.3%) samples with d MMR carried the BRAFmutation,while 17 of 177(9.6%) with proficient MMR(p MMR) harbored the mutation(P = 0.11).Notably,patients with d MMR tumors had significantly superior DFS(HR = 0.30,95%CI:0.15-0.77; P = 0.01) and OS(HR = 0.29,95%CI:0.10-0.84; P = 0.02) compared with patients with p MMR tumors.By contrast,the BRAF V600 E mutation had no prognostic impact on DFS and OS.CONCLUSION:The prevalence of d MMR and BRAF V600 E in Thai sporadic CRC patients was 15% and 11%,respectively.The d MMR phenotype was associated with a favorable outcome.

Preliminary Studies on Base Substitutions and Repair of DNA Mismatch Damage Stimulated by Low Energy N^+ Ion Beam Implantation in Escherichia coli

Ever since the low energy N+ ion beam has been accepted that the mutation effects of ionizing radiation are attributed mainly to direct or indirect damage to DNA. Evidences based on naked DNA irradiation in support of a mutation spectrum appears to be consistent, but direct proof of such results in vivo are limited. Using mutS, dam and/or dcm defective Eschericha coli imitator strains, an preliminary experimental system on induction of in vivo mutation spectra of low energy N+ ion beam has been established in this study. It was observed that the mutation rates of rifampicin resistance induced by N+ implantation were quite high, ranging from 9.2 x 10~8 to 4.9× 10~5 at the dosage of 5.2×1014 ions/cm2. Strains all had more than 90-fold higher mutation rate than its spontaneous mutation rate determined by this method. It reveals that base substitutions involve in induction of mutation of low energy nitrogen ion beam implantation. The mutation rates of mutator strains were nearly 500-fold (GM2929), 400-fold (GM5864) and 6-fold larger than that of AB1157. The GM2929 and GM5864 both lose the ability of repair DNA mismatch damage by virtue of both dam and dcm pathways defective (GM2929) or failing to assemble the repair complex (GM5864) respectively. It may explain the both strains had a similar higher mutation rate than GM124 did. It indicated that DNA cytosine methylase might play an important role in mismatch repair of DNA damage induced by N+ implantation. The further related research were also discussed.

Mismatch repair genes (hMLH1, hPMS1, hPMS2, GTBP/hMSH6,HMSH2) in the pathogenesis of hepatocellular carcinoma

AIM: DMA mismatch repair (MMR) is an important mechanism for maintaining fidelity of genomic DNA. Abnormalities in one or more MMR genes are implicated in the development of many cancers. We investigated the role of expression of MMR genes (hMLH1, hPMS1, hPMS2, GTBP/hMSH6, hMSH2) in hepatocellular carcinogenesis. METHODS: We evaluated the expression level of MMR genes in 33 hepatocellular carcinoma (HCC) cases using the multiplex reverse transcription (RT) PCR assays, as well as in 16 cases of normal adjacent hepatic tissues. β-actin gene was used as an internal control and calibrator for quantification of gene expression. RESULTS: Out of the 33 studied cases, 25 were HCV positive and 30 (90.9%) showed reduced expression in one or more of the studied MMR genes. Reduced expression was found in hMSH2(71.9%), hMLH1 (53.3%), GTBP(51.1%), hPMS2 (33.3%) and hPMS1 (6%). A significant correlation was found between reduced expression of hPMS2(P= 0.0069) and GTBP(P= 0.0034), hPMS2 and non-cirrhosis (P= 0.0197), hMLH1 and high grade. On the other hand, 57.1%, 50%, 20%, 18.8%, and 6% of the normal tissues distant to tumors showed reduced expression of hMSH2, hMLH1, GTBP, hPMS2, and hPMS1 respectively. Multivariate analysis revealed a significant correlation between the expression level of hMSH2(P= 0.008), hMLH1 (P= 0.001) and GTBP (P= 0.032) and HCC, between hPMS2, GTBP and HCV-associated HCC (P<0.001, 0.002). CONCLUSION: Reduced expression of MMR genes seems to play an important role in HCV-associated HCC. hPMS2 is likely involved at an early stage of hepatocarcinogenesis since it was detected in normal adjacent tissues. Reduced expression of hPMS2 provides a growth advantage and stimulates proliferation which encourages malignant transformation in non-cirrhotic HCV-infected patients via acquisition of more genetic damages.

Helicobacter pylori infection and expression of DNA mismatch repair proteins

AIM： TO determine the expression of DNA （MMR） proteins, including hMLH1 and hMSH2, in gastric epithelial cells in the patients with or without Helicobacter pylori （H pylori）-infected gastritis. METHODS： Fifty Hpylori-positive patients and 50 H pylori-negative patients were enrolled in the study. During endoscopy of patients with non-ulcer dyspepsia, two antral and two corpus biopsies were taken for histological examination （Giemsa stain） and for immunohistochemical staining of hMLH1 and hMSH2. RESULTS： The percentage of epithelial cell nuclei that demonstrated positivity for hMLH1 staining was 84.14 ± 7.32% in Hpylori-negative patients, while it was 73.34 ±10.10% in Hpylori-positive patients （P 〈 0.0001）. No significant difference was seen between the two groups regarding the percentage of epithelial cell nuclei that demonstrated positivity for hMSH2 staining （81.16±8.32% in H pylori-negative versus 78.24 ± 8.71% in Hpylori-positive patients; P = 0.09）. CONCLUSION： This study indicates that Hpylori might promote development of gastric carcinoma at least in part through its ability to affect the DNA MMR system

Relationship between mismatch repair protein, RAS, BRAF, PIK3CA gene expression and clinicopathological characteristics in elderly colorectal cancer patients

BACKGROUND Colorectal cancer(CRC)is common in elderly patients.Mismatch repair(MMR)protein deletion is one of the causes of CRC.The RAS(KRAS/NRAS),BRAF,and PIK3CA genes are important gene targets in CRC treatment and are closely related to the prognosis and survival of patients.However,little is known regarding the relationship between the expression of MMR,RAS,BRAF,PIK3CA and the clinicopathological features in CRC patients.AIM To analyze the relationship between the expression of MMR,RAS,BRAF,PIK3CA and the clinicopathological features in CRC.METHODS A total of 327 elderly patients with CRC were enrolled,and immunohistochemistry was used to detect the MMR protein.Real-time quantitative polymerase chain reaction was used to detect the RAS(KRAS/NRAS),BRAF,and PIK3CA genes.The clinicopathological data of the patients were recorded and analyzed by SPSS 19.0 statistical software.RESULTS In 327 elderly patients with CRC,the rate of MMR protein loss was 9.79%(32/327),and the deletion rate of four MMR proteins(MSH2,MSH6,MLH1,PMS2)was 1.83%(6/327),3.06%(10/327),7.65%(25/327),and 7.65%(25/327),respectively.There were no significant differences between MMR protein deletion and sex,pathological type,tumor morphology,differentiation degree or lymph node metastasis(P>0.05),but there was a significant difference between MMR protein deletion and tumor diameter and tumor location(P=0.048/P=0.000).The mutation rates of the KRAS,NRAS,BRAF and PIK3CA genes in elderly CRC patients were 44.95%(147/327),2.45%(8/327),3.36%(11/327)and 2.75%(9/327),respectively;the KRAS gene mutation was closely related to tumor morphology(P=0.002)but not to other clinicopathological features(P>0.05),and there were no significant differences between NRAS gene mutation and clinicopathological features(P>0.05).The BRAF gene mutation showed a significant difference in pathological type,tumor location,differentiation degree and lymph node metastasis(P<0.05),but was not correlated with sex,tumor size and tumor morphology(P>0.05).The PIK3CA gene mutation showed no significant differences in the above clinicopathological characteristics(P>0.05).Significant differences were observed between MMR protein deletion and KRAS,BRAF,and PIK3CA gene mutations in elderly CRC patients(P=0.044,P=0.000,P=0.003,respectively),but there was no significant difference between MMR protein deletion and NRAS mutation(P>0.05).CONCLUSION In elderly CRC patients,the tumor is mainly located in the right colon,and the deletion rate of MMR protein is higher when the tumor diameter is greater than or equal to 5 cm;the deletion rate of MLH1 and PMS2 is more common;the mutation rate of KRAS gene is higher than that of the NRAS,BRAF and PIK3CA genes,the BRAF gene mutation has different degrees of correlation with clinicopathological characteristics;when the MMR protein is deleted,the BRAF and PIK3CA gene mutations are often present,and the KRAS gene mutation rate is low.

Complementary analysis of microsatellite tumor profile and mismatch repair defects in colorectal carcinomas

Microsatellite instability (MSI) is a prognostic factor and a marker of defi cient mismatch repair (MMR) in colorectal adenocarcinomas (CRC). However, a proper application of this marker requires understanding the following: (1) The MSI concept: The PCR approach must amplify the correct locus and accurately identify the microsatellite pattern in the patient’s normal tissue. MSI is demonstrat- ed when the length of DNA sequences in a tumor differs from that of nontumor tissue. Any anomalous expansion or reduction of tandem repeats results in extra-bands normally located in the expected size range (100 bp, above or below the expected product), differ from the germline pattern by some multiple of the repeating unit, and must show appropriate stutter. (2) MSI mechanisms: MMR gene inactivation (by either mutation or protein down-regulation as frequently present in deep CRC com- partments) leads to mutation accumulation in a cell with every cellular division, resulting in malignant transforma- tion. These mechanisms can express tumor progression and result in a decreased prevalence of aneuploid cells and loss of the physiologic cell kinetic correlations in the deep CRC compartments. MSI molecular mechanisms are not necessarily independent from chromosomal in- stability and may coexist in a given CRC. (3) Because of intratumoural heterogeneity, at least two samples from each CRC should be screened, preferably from the su- perfi cial (tumor cells above the muscularis propria) and deep (tumor cells infi ltrating the muscularis propria) CRC compartments to cover the topographic tumor hetero- geneity. (4) Pathologists play a critical role in identify- ing microsatellite-unstable CRC, such as occur in young patients with synchronous or metachronous tumors or with tumors showing classic histologic features. In these cases, MSI testing and/or MMR immunohistochemistry are advisable, along with gene sequencing and genetic counseling if appropriate. MSI is an excellent functional and prognostically useful marker, whereas MMR immuno- histochemistry can guide gene sequencing.

Expression of Cyclooxygenase-2 and Its Relationship with Mismatch Repair and Microsatellite Instability in Hereditary Nonpolyposis Colorectal Cancer

Objective To investigate cyclooxygenase-2 (COX-2) expression and its relationship with mismatch repair (MMR) protein expression and microsatellite instability (MSI) in hereditary nonpolyposis colorectal cancer (HNPCC). Methods A total of 28 cases of colorectal adenoma and 14 cases of colorectal carcinoma were collected between July 2003 and July 2007 from 33 HNPCC families. Sporadic colorectal adenoma (n=32) and carcinoma patients (n=24) served as controls. With samples of tumor tissues and normal colonic mucosa collected from the patients, the protein expressions of COX-2 and MMR (hMLH1, hMSH2, and hMSH6) were examined with immunohistochemical assay. Frequency of MSI in five standard MSI loci BAT25, BAT26, D2S123, D5S346, and D17S250 were analyzed by means of polymerase chain reaction. Results The rate of COX-2 high-expression was 53.6% (15/28) and 42.9% (6/14) in HNPCC adenoma and carcinoma; 62.5% (20/32) and 91.7% (22/24) in sporadic adenoma and carcinoma, respectively. That rate was lower in HNPCC carcinoma than in sporadic carcinoma (P<0.05). MMR-deletion rate and percentage of high-frequency MSI (MSI-H) in HNPCC carcinoma were higher than those in sporadic colorectal carcinoma [both 71.4% (10/14) vs. 12.5% (3/24), both P<0.01]. Among the 10 MMR-deficient HNPCC carcinoma patients, COX-2 low-expression was observed in 8 cases (80.0%), while COX-2 high-expression was observed in all of the 4 MMR-positive HNPCC carcinoma cases (P<0.05). In comparison to MMR positive HNPCC carcinoma, HNPCC adenoma, and sporadic carcinoma, COX-2 expression was significantly lower in corresponding MMR-deficient cases (all P<0.05). The rates of COX-2 low-expression in HNPCC adenoma, HNPCC carcinoma, and sporadic carcinoma with MSI-H were significantly higher than those in the cases with microsatellite stability (all P<0.05). Conclusion COX-2 is expressed at a low level in HNPCC carcinoma, different from the high COX-2 expression in sporadic carcinoma.