错配修复基因蛋白在散发性结直肠癌中表达的临床意义

目的探究散发性结直肠癌错配修复基因蛋白检的表达及其临床意义。方法收集150例散发性结直肠癌患者的肿瘤标本,通过免疫组织化学染色法测定错配修复基因蛋白(hPSm^2、hMLH1、hMSH6、hMSH2)水平,并分析其影响因素。结果 150例标本中有42例存在hPSm^2、hMLH1、hMSH6、hMSH2表达缺失,缺失率为28.00%,根据错配修复基因蛋白表达是否缺失分为C组(42例)和D组(108例)。经单因素分析,肿瘤部位、分化程度、BMI与错配修复基因蛋白缺失表达有关(P<0.05);经Logistic多因素回归分析,肿瘤分化程度低、位于右半结肠、BMI≥28.0与错配修复基因蛋白缺失表达有关(P<0.05)。结论散发性结直肠癌患者存在错配修复基因蛋白表达缺失现象,且表达缺失与肿瘤部位、分化程度及BMI相关,可为病情及预后评估提供依据。     

散发性大肠癌患者中错配修复基因胚系突变筛查策略

目的探讨在散发性大肠癌患者中筛查错配修复(MMR)基因胚系突变的可行性和策略。方法以150例散发性大肠癌患者为研究对象,以微卫星不稳定性(MSI)检测和免疫组化(IHC)检测作为初筛方法,对MSI-H表型或IHC检测MMR蛋白缺失的患者行hMLH1和hMSH2基因测序,检测MMR基因胚系突变。结果150例散发性大肠癌中MSI-H表型20例,IHC示22例MMR蛋白缺失。共发现3例MMR基因突变。MSI检测和IHC检测结果具有很好的一致性。结论散发性大肠癌患者中分子生物学筛查方法能够有效鉴别出MMR基因胚系突变。     

散发性结直肠癌中hMSH2与PTEN的表达及临床意义

目的:研究错配修复蛋白hMSH2与PTEN在散发性结直肠癌的表达变化及两者之间的关系,以进一步探讨其临床意义.方法:采用SP免疫组织化学两步法对42例散发性结直肠癌、相应近端癌旁组织(距癌组织3 cm)和远端癌旁组织(距癌组织>10 cm)及15例正常结直肠组织进行hMSH2与PTEN蛋白袁达检测.同时Western blot法检测42例散发性结直肠癌、相应远端对照组织hMSH2与PTEN蛋白表达.分析散发性结直肠癌发病机制及hMSH2与PTEN蛋白表达与临床病理之间的关系.结果:结直肠癌组织中,hMSH2与PTEN蛋白失表达率(阴性率)均低于正常结直肠组织及近端癌旁组织、远端癌旁组织(X2hMSH2=7.967,X2PTEN=11.67,均P<0.05).PTEN蛋白表达与肿瘤分化程度呈正相关(rs=0.727,P<0.05),与Dukes分期、侵润深度、淋巴结转移、肝转移均呈负相关(rs=-0.727,-0.718,-0.718,-0.535,均P<0.05).hMSH2蛋白表达与上述临床病理特征未见明显相关.hMSH2与PTEN蛋白表达在散发性结直肠癌中呈正相关(rs=0.679,P<0.05).与远端对照组织相比42例胃癌组织中hMSH2蛋白低表达率为59.52%(25/42),PTEN低表达率为45.24%(19/42).结论:PTEN表达与其临床病理特征相关;在散发性结直肠癌发生、发展过程中错配修复蛋白hMSH2表达缺失伴有PTEN蛋白表达下调.     

HNPCC大肠腺瘤及癌组织微卫星不稳定与TGFβRRII表达的关系

目的观察遗传性非息肉病性大肠癌（HNPCC）和大肠腺瘤癌组织中TGFβRRII、错配修复基因（hMLH1、hMSH2、hMSH6）蛋白表达和微卫星不稳定（MSI）的关系。方法以来源于33个HNPCC家系的大肠腺瘤28例和大肠腺癌14例为观察对象，以32例散发性大肠腺瘤和24例散发性大肠癌作为对照。采用免疫组织化学技术，检测大肠腺瘤及大肠腺癌组织中TGFβRRII、hMLH1、hMSH2、hMSH6蛋白表达。从活检组织中提取DNA，选择BAT-25、BAT-26、D2S123、D5S346、D17S250五个微卫星位点行荧光标记聚合酶链反应（PCR），以GeneMapper软件分析PCR产物。通过与正常黏膜微卫星序列PCR片断长度进行比较，判定腺瘤和癌组织的MSI情况。结果64．29％的HNPCC大肠腺瘤和71．43％的HNPCC大肠癌表现为MSI—H，明显高于散发性大肠腺瘤9．38％和散发性大肠癌12，5％。分别有67．86％的HNPCC大肠腺瘤和71.43％的HNPCC大肠癌表现为MMR蛋白表达缺失。明最高于散发性大肠腺瘤3．13％和散发性大肠癌12．5％。分别有57．14％的HNPCC大肠腺瘤和78.57％的HNPCC大肠癌表现为TGFβRRII低表达，明显高于散发性大肠腺瘤9．38％和散发性大肠癌41．67％。在MSI．H的HNPCC大肠腺瘤中，TGFl3RII低表达者占77．78％，MSI—H的HNPCC大肠癌中90％出现TGFβRRII的低表达。MSI—H的散发性大肠腺瘤中TGFβRRII的低表达率为66．67％，MSI—H的散发性大肠癌TGFβRRII的低表达率为100％。结论大部分HNPCC大肠腺瘤和大肠癌出现MSI—H，大部分MSI-H大肠腺瘤和大肠癌表现为TGFβRRII低表达。MSI、MMR、TGFβRRII的检测对腺瘤癌变风险的估计具有重要意义。     

hMLH1和hMSH2蛋白表达在遗传性非息肉病性大肠癌诊断中的应用

目的　评价错配修复基因hMLH1和hMSH2蛋白表达在筛选遗传性非息肉病性大肠癌(HNPCC)中的价值。方法　收集大肠癌患者 6 6例 ,分为HNPCC患者 (A组 ,n =19)、高度可疑HNPCC患者 (B组 ,n =2 0 )、符合Bethesda指导标准的可疑HNPCC患者 (C组 ,n =14 )及散发性大肠癌患者 (D组 ,n =13)四组 ,用免疫组化方法检测各组错配修复基因hMLH1和hMSH2的蛋白表达。结果　A组hMLH1和hMSH2蛋白表达减低或缺失达 72 .8% ;B组为 6 0 .0 % ;C组为 2 8.4 % ;D组为 7.7%。hMLH1和hMSH2蛋白表达减低或缺失与HNPCC显著相关 (P =0 .0 0 0 8)。此外 ,hMLH1蛋白表达减低或缺失率显著高于hMSH2 (P <0 .0 1)。结论　hMLH1和hMSH2蛋白表达减低或缺失与HNPCC的可能性显著相关 ,免疫组化检测此二种蛋白表达能快速、有效地帮助临床医生评估患者HNPCC的可能性 ,同时提示相应错配修复基因存在突变。中国HNPCC患者中hMLH1基因发生突变的机会可能高于hMSH2基因。     

HNPCC大肠腺瘤及癌组织微卫星不稳定与TGFβRII表达的关系

目的观察遗传性非息肉病性大肠癌（HNPCC）和大肠腺瘤癌组织中TGFβRRII、错配修复基因（hMLH1、hMSH2、hMSH6）蛋白表达和微卫星不稳定（MSI）的关系。方法以来源于33个HNPCC家系的大肠腺瘤28例和大肠腺癌14例为观察对象，以32例散发性大肠腺瘤和24例散发性大肠癌作为对照。采用免疫组织化学技术，检测大肠腺瘤及大肠腺癌组织中TGFβRRII、hMLH1、hMSH2、hMSH6蛋白表达。从活检组织中提取DNA，选择BAT-25、BAT-26、D2S123、D5S346、D17S250五个微卫星位点行荧光标记聚合酶链反应（PCR），以GeneMapper软件分析PCR产物。通过与正常黏膜微卫星序列PCR片断长度进行比较，判定腺瘤和癌组织的MSI情况。结果64．29％的HNPCC大肠腺瘤和71．43％的HNPCC大肠癌表现为MSI—H，明显高于散发性大肠腺瘤9．38％和散发性大肠癌12，5％。分别有67．86％的HNPCC大肠腺瘤和71.43％的HNPCC大肠癌表现为MMR蛋白表达缺失。明最高于散发性大肠腺瘤3．13％和散发性大肠癌12．5％。分别有57．14％的HNPCC大肠腺瘤和78.57％的HNPCC大肠癌表现为TGFβRRII低表达，明显高于散发性大肠腺瘤9．38％和散发性大肠癌41．67％。在MSI．H的HNPCC大肠腺瘤中，TGFl3RII低表达者占77．78％，MSI—H的HNPCC大肠癌中90％出现TGFβRRII的低表达。MSI—H的散发性大肠腺瘤中TGFβRRII的低表达率为66．67％，MSI—H的散发性大肠癌TGFβRRII的低表达率为100％。结论大部分HNPCC大肠腺瘤和大肠癌出现MSI—H，大部分MSI-H大肠腺瘤和大肠癌表现为TGFβRRII低表达。MSI、MMR、TGFβRRII的检测对腺瘤癌变风险的估计具有重要意义。     

幽门螺杆菌与DNA错配修复系统

H pylori感染导致了真核细胞错配修复(mismatch repair,MMR)系统的缺陷,在胃上皮细胞中引起微卫星不稳定(micrsatellite instable,MSI),使相关基因自发突变率增加,最终可导致机体对肿瘤的易感.DNA错配修复功能通过纠正复制或重组过程中出现的错配碱基、诱导DNA严重受损的细胞发生凋亡,从而阻止突变细胞过度增殖以及肿瘤的发生.在错配修复基因突变中,绝大部分是点突变,以单个碱基的替代大于碱基的缺失或插入,其中hMLH1和hMSH2基因是DNA错配修复系统的主要控制基因.研究表明H pylori的感染及其导致的错配修复系统功能缺陷在胃癌的发生、发展中起着主导作用.     

错配修复基因hMSH2、hMLH1在非霍奇金

目的 探讨错配修复基因hMSH2、hMLH1在非霍奇金淋巴瘤中的表达及意义.方法 运用免疫组化SP法检测22例B细胞性非霍奇金淋巴瘤及18例NK/T细胞性非霍奇金淋巴瘤中hMSH2、hMLH1蛋白表达情况,并探讨其与非霍奇金淋巴瘤临床病理间的关系.结果 NK/T细胞非霍奇金淋巴瘤hMSH2、hMLH1蛋白表达缺失率分别为55.56%、44.44%;B细胞非霍奇金淋巴瘤分别为31.82%、50.00%,两种肿瘤间缺失率比较均无统计学意义.hMSH2、hMLH1蛋白表达缺失与患者性别及肿瘤是否发生于淋巴结无关.结论 错配修复基因hMSH2、hMLH1在非霍奇金淋巴瘤肿瘤组织中存在蛋白表达缺失,可导致基因组不稳定,该肿瘤易感.     

遗传性非息肉病性结直肠癌临床分子诊断的研究进展

遗传性非息肉病性结直肠癌(hereditary nonpolyposis colorectal cancer,HNPCC)是一种由于错配修复基因(mismatch repair gene,MMR)突变导致的常染色体显性遗传性疾病,占结直肠癌的5%-15%.研究显示,与HNPCC发生相关的错配修复基因有hMSH2、hMLH1、hMSH6、hPSM1和hPSM2.HNPCC肿瘤具有发病早、近段结肠多见、原发癌多见、肠外肿瘤多见、病理以黏液腺癌为主的特点.到目前为止,HNPCC的诊断主要依赖病史及相关遗传检测结果.对于符合Amsterdam Ⅱ或Bethesda标准的结直肠癌患者应进行微卫星不稳定(microsatellite instability,MSI)和免疫组织化学错配修复蛋白的检测,继而进行错配修复基因等种系突变检测.     

遗传性非息肉病性结直肠癌的微卫星不稳定研究

目的　探讨国人北方人群HNPCC的微卫星不稳定 (microsatelliteinstability ,MSI)发生情况及其意义。方法　 44例患者来源于 3 0个HNPCC (hereditarynonpolyposiscolorectalcancer)家系 ,这些家系主要分布于北方 5省市。所有患者均符合BGl 3 (Bethes dal 3 )HNPCC诊断标准。以荧光标记法检测 44例患者的石蜡包埋组织微卫星稳定性。结果　 44例患者中高度微卫星不稳定 (highfrequencymicrosatelliteinstability ,MSI H)为 81.81( 3 6/ 44 ) ,低度微卫星不稳定 (lowfrequencymicrosatelliteinstability ,MSI L)为 6.82 ( 3 / 44 ) ,微卫星稳定 (microsatellitestable ,MSS)为 11.3 ( 5 / 44 ) ;所选择的 5个微卫星位点中Bat2 5和Bat2 62个位点MSI H的表达率较高 ,分别为 10 0 %和 97.2 2 %。符合AmsterdamⅡ和符合BGl 3标准的HNPCC患者的MSI H表达率分别为 85 .2 9%和 81,81% ,仅符合BGl 3标准 ,而不符合AmsterdamII的 10个患者中 ,7个发现MSI H。结论　HNPCC肿瘤的MSI H发生率高 ,MSI检测方法简便、易行 ,可作为错配修复基因种系突变初筛方法 ,Bethesdal 3标准可更多地收集到可疑的HNPCC患者     

Immunohistochemical detection of the hMLH1 and hMSH2 proteins in hereditary non-polyposis colon cancer and sporadic colon cancer

Defects in DNA mismatch repair system are involved in carcinogenesis of sporadic and inherited human cancers. We assessed the feasibility of using immunohistochemistry to detect tumors with DNA mismatch repair deficiency. We analyzed 81 samples (74 colon cancers (CC), 1 colon dysplasia and 6 extracolonic cancers) for hMLH1 and hMSH2 protein expression, microsatellite instability (MSI) and/or mutational analysis. A meta-analysis of the published data on immunohistochemistry of hMLH1/hMSH2 proteins was performed. Sensitivity and specificity of the method was calculated. Twenty four of 29 tumors from hMLH1/hMSH2 mutation carriers and 10 of 13 sporadic high frequency MSI tumors lost one of the proteins. None of the 42 tumors with stable microsatellites or low frequency MSI lost the proteins. Based on literature review of 49 publications on colorectal cancer, hMLH1 immunohistochemistry was able to detect 136 of 154 tumors from hMLH1 germline mutation carriers (the sensitivity of 88.3% [95%CI, 85.8-90.8%]), hMSH2 immunohistochemistry detected 99 of 109 tumors from hMSH2 mutation carriers (the sensitivity of 90.8% [95%CI, 88.5-93.1%]), and hMLH1/hMSH2 immunohistochemistry identified 1262 of 1382 tumors with high-frequency microsatellite instability not correlated with mutational analysis (the sensitivity of 91.3% [95%CI, 90.4-92.2%]). The specificity of the method was 99.4% (95%CI, 99.2-99.6%). In conclusion, immunohistochemistry of hMLH1 and hMSH2 proteins is a useful method to predict the presence of mismatch repair deficiency, although its sensitivity is lower than that of MSI analysis.     

Telomere erosion is independent of microsatellite instability but related to loss of heterozygosity in gastric cancer

AIM: To correlate the length of the telomere to microsatellite instability (MSI) and loss of heterozygosity (LOH) of APC, MCC and DCC genes in gastric carcinomas. METHODS: Telomeric restriction fragment (TRF) length of gastric cancer was measured with Southern blot. LOH of APC, MCC and DCC genes, microsatellite instability (MSI) and frameshift mutation of hMSH6, TGF-betaRII and BAX genes were analyzed by PCR-based methods. RESULTS: Sixty-eight cases of sporadic gastric carcinoma were studied for MSI using five microsatellite markers. MSI in at least one locus was detected in 17 (25%) of 68 tumors analyzed. Frameshift mutations of hMSH6, TGF-betaRII and BAX were detected in 2,6 and 3 of gastric carcinomas respectively showing high MSI (> or = 2 loci, n = 8), but none was found in those showing low MSI (only one locus, n = 9) or MSS (tumor lacking MSI or stable, n = 51). Thirty-five cases, including all high MSI and low MSI, were studied for TRF. The mean TRF length was not correlated with clinicopathological parameters. No association was observed between TRF length and MSI or frameshift mutation. On the contrary, LOH at the DCC locus was related to telomere shortening (P<0.01). This tendency was also observed in APC and MCC genes, although there was no statistical significance. CONCLUSION: The development of gastric cancer can arise through two different genetic pathways. In high MSI gastric cancers, defective mismatch repair allows mutations to accumulate and generate the high MSI phenotype. In gastric cancers showing either low MSI or MSS, multiple deletions may represent the LOH pathway. Telomere erosion is independent of high MSI phenotype but related to the LOH pathway in gastric cancer.     

A novel insulinoma tumor suppressor gene locus on chromosome 22q with potential prognostic implications.

The molecular mechanisms contributing to the tumorigenesis of insulinomas are still poorly understood. As moderate to high rates of LOH have been found on chromosome 22q in gastrinomas, we performed a finer deletion mapping study of chromosome 22q with 8 microsatellite markers in 15 insulinomas (4 malignant and 11 benign). Fourteen of 15 (93%) insulinomas revealed LOH on chromosome 22q, whereas the shortest region of overlap implicated a deletion of approximately 700 kb at 22q12.1-q12.2 with an LOH rate of up to 57% (8 of 14). Although the expressed sequence tag marker A006E25 that is localized in the hSNF5/INI1 gene on 22q11.2 revealed LOH in 50% of informative cases (7 of 14), no alterations in this gene could be identified by single strand conformational polymorphism analysis, direct DNA sequencing, or RNA expression analysis. Remarkably, the four malignant tumors showed a common deleted region between markers D22S345 and D22S1144 compared with none of the 11 benign insulinomas. The observed high frequency of chromosome 22q12 deletions in insulinomas is suggestive for a region compatible with harboring a tumor suppressor gene. The hSNF5/INI1 gene is most likely not the candidate gene, because no alterations could be identified. The distinct pattern of allelic loss identified in this chromosomal region appears to be an attractive candidate marker for further evaluation with regard to the discrimination between benign and malignant insulinomas.     

Early genetic instability of both epithelial and stromal cells in esophageal squamous cell carcinomas, contrasted with Barrett's adenocarcinomas

Background We have shown previously that stromal genetic alteration may make a greater contribution to early genesis of ulcerative colitis-associated tumors than sporadic colon cancers. We assessed whether similar differences in genetic alteration might exist between squamous cell carcinomas (SCCs) and Barrett's adenocarcinomas (BACs) of the esophagus. Methods We investigated epithelial and stromal genetic instability with five National Cancer Institute standard (NCI), four chromosome 17 (Chr. 17), and six tumor suppressor gene (TSG) microsatellite markers in 26 SCC and 12 BAC cases and in 11 normal controls, using a novel combination of microdissection, polymerase chain reaction, and GeneScan. Results Frequency of epithelial loss of heterozygosity (LOH) increased in the order background mucosa, to precursor lesions, to tumors with both types of carcinoma, especially for the Chr. 17 and TSG markers, while stromal LOH was relatively high but consistent from background mucosa to carcinoma. Epithelial LOH of D17S796 demonstrated a significantly higher frequency in SCCs than in BACs, without significant variation in p53 overexpression. The frequency of microsatellite instability (MSI) showed constant high levels in both epithelium and stroma of background, dysplasia, and carcinomas in the SCC series, and rather low frequencies in the BAC series. Although epithelial hMLH1 and hMSH2 expression decreased with tumor progression, no correlation was found with the individual MSI status. Conclusions Although epithelial LOH exists similarly in both lesion types, whereas epithelial and stromal MSI may occur in a relatively early phase of SCC development, stromal MSI is rare in BACs, strongly suggesting differences in tumorigenesis between the two types.     

Defective DNA mismatch repair in long-term (> or =3 years) survivors with pancreatic cancer.

BACKGROUND/AIMS: Defective DNA mismatch repair (MMR) in pancreatic cancer, reported in up to 13% of sporadic pancreatic cancers, may predict a good prognosis. To determine if long-term survival in pancreatic cancer could be attributed to defective DNA MMR, we ascertained its prevalence in 35 pancreatic cancer patients who survived > or =3 years after surgery. METHODS: We performed immunohistochemistry (IHC) for MMR proteins hMLH1, hMSH2, and hMSH6 in all 35 tumors and microsatellite instability (MSI) studies in 34/35 tumors using 10 microsatellite markers in paired normal and tumor DNA. Defective DNA MMR was defined as absence of protein expression on IHC and/or MSI in > or =30% of markers studied. RESULTS: On IHC, 3/35 (8.6%) tumors had defective DNA MMR. All 3 had absent expression of a DNA MMR protein (hMLH1 in 2 and hMSH2) and 2/3 also had MSI; the third could not be tested. Definitely 2, and probably all 3 patients had hereditary nonpolyposis colon cancer as determined by clinical and genetic profiles. CONCLUSION: Defective DNA MMR is uncommon in long-term survivors of pancreatic cancer and does not account for the survival benefit in those with sporadic pancreatic cancer.     

Screening for defective DNA mismatch repair in stage II and III colorectal cancer patients.

BACKGROUND & AIMS: Colorectal cancers associated with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome usually present in younger patients, show loss of mismatch repair (MMR) gene expression, and exhibit microsatellite instability (MSI). About 12% of sporadic colorectal cancers also show MMR loss and MSI. The aims of this study were to evaluate MMR loss and MSI in relation to patient age, sex, tumor stage, and site in the large bowel. METHODS: Tissue microarrays were created from 1020 stage II and III colorectal cancer cases and immunohistochemical staining performed to detect expression of the 2 major MMR proteins, hMLH1 and hMSH2. MSI was determined using the BAT-26 mononucleotide repeat. RESULTS: Ten percent of tumors showed loss of hMLH1 expression and 1.2% showed loss of hMSH2 expression. hMLH1 loss was more frequent in women (P < .001), older patients (P = .004), earlier stage tumors (P = .0001), and proximal colon tumors ( P < .0001). In contrast, tumors showing hMSH2 loss were more frequent in younger (P < .001), male (P = .05) patients and were distributed evenly between the proximal colon and distal colon/rectum. Eleven percent of tumors were MSI+ and these showed similar age, sex, stage, and site characteristics as tumors with hMLH1 loss. Discordance between MMR loss and MSI+ was found in 24 of 983 (2.4%) tumors. Of the 231 patients aged <60 years at diagnosis, 12 (5.2%) showed loss of hMLH1 and 8 (3.5%) showed loss of hMSH2. CONCLUSIONS: Routine immunohistochemical screening for MMR loss in younger colorectal cancer patients may provide a useful, first-step screening tool for the population-based detection of HNPCC.     

Hyperplastic polyps in hereditary nonpolyposis colorectal cancer

OBJECTIVES: Hereditary nonpolyposis colorectal cancer (HNPCC) is a genetic syndrome caused by germline mutations in DNA mismatch repair (MMR) genes, in particular hMLH1, hMSH2, and hMSH6. Dysfunction of MMR genes leads to loss of MMR protein expression and to microsatellite instability (MSI). MSI is also detected in 10-20% of sporadic colorectal cancers. Hyperplastic polyps (HP) may serve as precursor for these MSI+ sporadic colorectal cancers. The aim of this study was to examine whether hyperplastic polyps are also possible premalignant lesions in HNPCC. METHODS: All HPs resected from (suspected) mismatch repair gene mutation carriers were retrieved from a screening program database. Clinical information on patient age at colonoscopy and location of the HP was collected. MLH1, MSH2, and MLH6 protein expression was evaluated using immunohistochemistry. RESULTS: A total of 90 HPs were resected from 21 men and 19 women. The mean patient age at resection was 45.7 yr (44.7 yr in men and 46.6 yr in women). In all patients, 19 (21%) HPs were resected from the proximal colon, 23 (26%) from the distal colon, and 48 (53%) from the rectum. None of the HPs demonstrated loss of MMR protein expression. CONCLUSIONS: Mismatch repair dysfunction in HPs of HNPCC patients is apparently very rare. It seems unlikely that HPs in HNPCC patients are precursors for (MSI+) cancers in these patients.     

Low-level microsatellite instability phenotype in sporadic glioblastoma multiforme

Purpose Genetic instability is a hallmark of glioblastoma multiforme (GBM). Microsatellite instability (MSI) is a significant event in the tumorigenesis of many sporadic malignancies. The aim of our investigation was to study microsatellite instability in newly diagnosed glioblastomas.Methods MSI was investigated in 109 GBMs with 15 microsatellite markers. Immunohistochemistry was performed for the mismatch repair (MMR) proteins hMLH1, hMSH2, hPMS2, and hMSH6 in cases showing MSI. Sequence and promoter methylation status of hMLH1 were analyzed in the case of a decreased hMLH1 protein expression as well. To further investigate MSI(+) GBMs we carried out studies of LOH at selected chromosome regions, EGFR amplification, and sequence of p53 and PTEN.Results MSI was observed in six GBMs (5.5%) and it was more frequent in GBMs with a previous lower grade astrocytoma (18.8% vs. 3.2%). MMR protein staining was positive in all MSI(+) GBMs except in one case, which showed an aberrant expression of hMLH1 and hPMS2 without hMLH1 inactivation. Among MSI(+) GBMs, one tumor corresponded to the GBM molecular type 1 (p53 mutation, no EGFR amplification), another tumor to type 2 (wild-type p53, EGFR amplification), and four tumors to neither type (wild-type p53, no EGFR amplification). None of the six tumors carried a PTEN mutation.Conclusions MSI in GBM might be caused by inactivation of minor MMR genes rather than by a deficiency of hMLH1 or hMSH2 and it appears not to play a decisive role in the pathogenesis of these tumors. MSI(+) GBMs predominantly showed a profile which included wild-type of p53 and PTEN and absence of EGFR amplification but MSI occurred in all GBM molecular subtypes.Grant sponsor: supported by a grant from the Deutsche Forschungsgemeinschaft, DFG (MA 2448/1)     

Mutation and methylation of hMLH1 in gastric carcinomas with microsatellite instability

AIM: To appraise the correlation of mutation and methylation of hMSH1 with microsatellite instability (MSI) in gastric cancers.METHODS: Mutation of hMLH1 was detected by Twodimensional electrophoresis (Two-D) and DNA sequencing;Methylation of hMLH1 promoter was measured with methylation-specific PCR; MSI was analyzed by PCR-based methods.RESULTS: Sixty-eight cases of sporadic gastric carcinoma were studied for mutation and methylation of hMLH1 promoter and MSI. Three mutations were found, two of them were caused by a single bp substitution and one was caused by a 2 bp substitution, which displayed similar Two-D band pattern.Methylation of hMLH1 promoter was detected in 11(16.2%)gastric cancer. By using five MSI markers, MSI in at least one locus was detected in 17/68(25%) of the tumors analyzed.Three hMLH1 mutations were all detected in MSI-H (≥2 loci,n=8), but no mutation was found in MSI-L (only one locus,n=9) or MSS (tumor lacking MSI or stable, n=51). Methylation frequency of hMLH1 in MSI-H (87.5%, 7/8) was significantly higher than that in MSI-L (11.1%, 1/9) or MSS (5.9%, 3/51)(P&lt;0.01-0.001), but no difference was found between MSI-L and MSS (P&gt;0.05).CONCLUSION: Both mutation and methylation of hMLH1 are involved in the MSI pathway but not related to the LOH pathway in gastric carcinogenesis.     

Clinicopathological significance of loss of heterozygosity and microsatellite instability in hepatocellular carcinoma in China

AIM: To determine the features of microsatellite alterations and their association with clinicopathological characteristics of hepatocellular carcinoma (HCC). METHODS: Loss of heterozygosity (LOH) and microsatellite instability (MSI) of 55 microsatellite loci were detected with PCR-based microsatellite polymorphism analyses in tumors and corresponding noncancerous liver tissues of 56 surgically resected HCCs using the MegaBACE 500 automatic DNA analysis system. RESULTS: LOH was found in 44 of 56 HCCs (78.6%) at one or several loci. Frequencies of LOH on 1p, 4q, 8p, 16q, and 17p were 69.6% (39/56), 71.4% (40/56), 66.1% (37/56), 66.1% (37/56), and 64.3% (36/56), respectively. MSI was found in 18 of 56 HCCs (32.1%) at one or several loci. Ten of fifty-six (17.9%) HCCs had MSI-H. Serum HBV infection, alpha-fetoprotein concentration, tumor size, cirrhosis, histological grade, tumor capsule, as well as tumor intrahepatic metastasis, might be correlated with LOH on certain chromosome regions. CONCLUSION: Frequent microsatellite alterations exist in HCC. LOH, which represents a tumor suppressor gene pathway, plays a more important role in hepatocarcin-ogenesis. MSI, which represents a mismatch repair gene pathway, is a rare event during liver carcinogenesis. Furthermore, LOH on certain chromosome regions may be correlated with clinicopathological characteristics in HCC.