胃癌的微卫星不稳定性与hMLH1基因启动子甲基化的关系

背景与目的:由于细胞错配修复功能缺陷而导致基因组微卫星序列高度不稳定是遗传性非息肉性大肠癌发生的主要原因。以往的研究表明胃癌组织也有错配修复蛋白表达的缺失,但错配修复基因突变频率却很低;而启动子的甲基化是肿瘤抑癌基因失活的主要途径,也可能是错配修复基因功能丧失的主要原因。本研究拟通过对胃癌组织的微卫星不稳定性的分析及对hMLH1基因启动子甲基化和蛋白表达的检测,对胃癌发病的分子机制进行探讨。方法:从52例胃癌患者的癌组织及其周围组织提取DNA,PCR扩增基因组的5个微卫星位点BAT-26、D17S261、D3S1283、D2S123和D3S1611,毛细管电泳后,判定胃癌组织的微卫星不稳定性;免疫组织化学方法检测hMLH1蛋白的表达;酶切法检测hMLH1基因启动子甲基化。结果:52例胃癌标本中微卫星高度不稳定13例,低度不稳定2例,稳定37例。微卫星高度不稳定的13例胃癌组织中,均检测到hMLH1基因启动子甲基化(100.0%);微卫星低度不稳定和微卫星稳定的39例胃癌组织中,hMLH1基因启动子甲基化仅1例(2.6%),前者发生率高于后者,两者之间有显著性差异(P<0.01)。微卫星高度不稳定的13例胃癌的癌旁组织中,hMLH1基因启动子甲基化6例(46.2%),而微卫星低度不稳定和微卫星稳定39例胃癌的癌旁肿瘤组织标本中,hMLH1基因启动子甲基     

hMLH1和hMSH2蛋白表达与散发性大肠癌

目的通过对散发性大肠癌中错配修复基因表达的检测,探讨错配修复基因与大肠癌发生和演进的关系.方法采用蛋白提取技术提取肿瘤组织蛋白,利用免疫印记技术对hMSH2和hMLH1蛋白表达情况进行检测,应用方差分析和t检验对数据进行统计学分析.结果 70岁以上组hMLH1蛋白表达水平显著低于70岁以下两组,在不同分化各组间差异有显著性;右半结肠癌hMHL1蛋白表达显著低于除直肠以外其他部位的肿瘤;无转移肿瘤hMLH1蛋白的表达显著低于有转移肿瘤;其表达与性别无显著相关.hMSH2蛋白表达水平与患者性别、年龄、肿瘤部位、分化程度及转移间均无显著相关.结论高龄所造成的hMLH1基因蛋白表达降低可能是导致散发性大肠癌的相关因素之一;hMLH1基因表达降低与右半结肠癌的发生关系密切并影响其分化;hMLH1基因表达较高的肿瘤可能易出现转移.hMSH2基因与大肠癌临床病理间无密切联系.     

散发性大肠癌中hMLH1蛋白表达及意义

[目的]探讨散发性大肠癌中错配修复基因hMLH1蛋白表达及其与临床病理特征的关系.[方法]采用免疫组织化学SP法检测50例散发性大肠癌标本hMLH1蛋白表达情况.[结果]hMLH1蛋白表达阳性率在70岁以上组(64.3%),低分化组(52.6%),无转移组(74.1%),右半结肠(68.2%)分别低于70岁以下组(80.6%),高分化组(90%),有转移组(82.6%)和左半结肠(82.1%)(P＜0.05).[结论]散发性大肠癌中hMLH1蛋白表达与患者年龄及肿瘤发生的部位、分化程度、有无转移密切相关.     

微卫星不稳定散发性大肠癌的临床病理特征和DNA倍体研究

目的:探讨微卫星不稳定的散发性大肠癌的临床病理特征及微卫星不稳定表型和DNA倍体类型的关系。方法:对71例散发性大肠癌行BAT25和BAT26两个位点的微卫星不稳定检测和流式细胞术倍体分析,探讨微卫星不稳定状态和临床病理特征及DNA倍体类型的关系。结果:微卫星不稳定的阳性率为9.86%(7/71),微卫星不稳定表型和发病部位、组织学类型及分化程度相关(P<0.05),而与性别、年龄、淋巴结转移和分期无关。微卫星不稳定的散发性大肠癌中右半结肠癌和低分化腺癌的比例高于微卫星稳定者。68例患者检出二倍体和异倍体分别为18和50例,微卫星不稳定表型者5例为二倍体,因此和DNA倍体类型显著相关(P=0.012)。结论:微卫星不稳定的散发性大肠癌好发于右半结肠,具有低分化腺癌的倾向,多为二倍体。     

胃癌MSI、hMLH1基因甲基化及其蛋白表达研究

目的：通过研究散发性胃癌中错配修复基因hMLH1启动子区5’CpG岛甲基化及蛋白表达、微卫星不稳定性(MSI)发生的情况及各指标之间的关系，探讨hMLH1基因、MSI在胃癌发生过程中的作用，揭示胃癌发生的分子机制。方法：取标本55例，其中胃癌41例，正常组织(包括正常及浅表性胃炎)14例。酚／氯仿法提取DNA，PCR扩增一变性聚丙烯酰胺凝胶电泳-硝酸银染法检测BAT-26、D2S123两个位点的MSI，酶切产物特异性扩增法检测hMLH1基因启动子区5’CpG岛甲基化异常情况，免疫组化SP法检测hMLH1蛋白的表达。结果：胃癌组MSI发生率为51.2％(21／41)，显著高于正常组(P(0．05)胃癌组hMLH1基因启动子区5’CpG岛甲基化发生率为41．5％，其中88．5％表现为蛋白表达缺失或降低，正常组织中未发现甲基化。胃癌组MSI( )中，甲基化发生率81．0％。结论：hMLH1基因甲基化是导致胃癌组织出现MSI的重要因素，MSI在散发性胃癌发生、发展过程中发挥一定作用。由于错配修复基因甲基化而丧失修复功能，引起MSI的产生，促进肿瘤的发生。对胃癌组织中hMLH1基因、MSI的深入研究，可以为肿瘤早期发现、早期诊断提供信息。     

食管鳞癌中hMLH1、E-cadherin、p16^INK4a基因启动子甲基化及其意义

背景与目的:目前认为抑癌基因启动子甲基化导致转录抑制是恶性肿瘤发生的重要机制之一,hgLH1、E-cadherin及p16INK4a基因在多种恶性肿瘤中都已被证实存在较高频率的甲基化.本研究通过检测食管鳞癌组织及癌旁组织中hMLH1、E-cadherin,p16INK4a基因启动子甲基化的发生情况,探讨hMLH1、E-cadherin、p16INK4a基因启动子甲基化在食管鳞癌发生发展中的作用.方法:采用酚-氯仿法提取105例食管鳞癌组织及癌旁组织的基因组DNA,应用甲基化特异性PCR对所提DNA进行hMLH1、E-cadherin、p16INK4a基因甲基化检测.采用EnVison免疫组织化学二步法对癌组织中上述3种基因蛋白表达进行检测.结果:癌组织中E-cadherin、hMLH1、p16INK4a基因启动子甲基化的阳性率分别为57.1%(60/105)、20.9%(22/105)和50.5%(53/105),而癌旁食管组织中相应的3个基因的甲基化率分别为10.5%(11/105)、1.9%(2/105)和7.6%(8/105),均显著低于癌组织.E-cadherin(P=0.021)及p16INK4a(P=0.026)基因甲基化与蛋白表达缺失密切相关,而hMLH1基因甲基化与蛋白表达无显著相关性.E-cadherin基因启动子甲基化与淋巴结转移有关(P=0.016),p16INK4a基因启动子甲基化与低分化癌有关性(P=0.024).hMLH1基因甲基化与各项临床病理特征均无关.结论:食管鳞癌中p16INK4a基因启动子甲基化与相应蛋白表达缺失密切相关,且在低分化癌中更多见;E-cadherin基因启动子甲基化与相应蛋白质表达缺失有相关性,并且有淋巴结转移多见的显著特征,这2个基因的甲基化位点与食管鳞癌密切相关.hMLH1基因甲基化可能并不直接参与食管鳞癌的发生、发展.     

散发性子宫内膜癌组织中错配修复基因hMLH1表达与微卫星不稳定性的相关性分析

目的：分析散发性子宫内膜癌组织中微卫星不稳定性（microsatellites instability,MSI）与错配修复基因hMLH1表达之间是否存在相关性。方法：应用免疫组化SP法检测40例子宫内膜癌、22例子宫内膜不典型增生、23例正常子宫内膜组织中错配修复基因hMLH1的表达;应用PCR方法检测子宫内膜癌中5个微卫星位点（D2S123、D10S197、D13S175、D10S215和D10S541）的微卫星不稳定性。结果：错配修复基因hMLH1在正常子宫内膜、子宫内膜不典型增生、子宫内膜癌组织中的表达逐渐下降,差异有统计学意义,χ^2=33.34,P=0.00;错配修复基因hMLH1蛋白的表达与内膜癌组织分化程度有关,χ^=7.98,P=0.02;hMLH1蛋白在内膜癌G1和G2期表达均为阳性;子宫内膜癌中微卫星不稳定性的发生与错配修复基因hMLH1的失表达密切相关,Pearson相关系数r=1,P=0.00。MSI在内膜癌组织中发生率显著高于正常子宫内膜,χ^2=5.26,P=0.02。结论：MSI是子宫内膜癌发生过程中重要的分子水平改变。hMLH1蛋白的表达有望成为筛查子宫内膜不典型增生向内膜癌发展的高危人群的一种手段,同时也可能成为判断内膜癌患者预后的指标。     

5-氮杂-2'-脱氧胞苷和曲古抑菌素A对卵巢癌顺铂耐药细胞中hMLH1表达及DNA甲基化的影响

背景与目的:顺铂能造成细胞内DNA的损伤,而DNA错配修复蛋白能发现顺铂导致的DNA损伤,产生损伤信号,诱导细胞凋亡,从而摧毁肿瘤细胞.hMLH1是DNA错配修复系统的一个重要成员,其缺失能导致肿瘤对顺铂的耐受.本研究目的在于探讨卵巢癌顺铂耐药细胞中hMLH1表达及其基因启动子区DNA甲基化状态,同时探讨去甲基化制剂——5-氮杂-2'-脱氧胞苷(5-aza-2'-deoxycytidine,5-Aza-dC)和组蛋白脱乙酰化酶抑制剂——曲古抑菌素A(trichostatin A,TSA)对hMLH1表达及DNA甲基化的逆转作用.方法:应用5-Aza-dC和TSA作用于卵巢癌细胞COC1与其顺铂耐药细胞COC1/DDP,应用甲基化特异性PCR、RT-PCR和Western blot检测上述两种细胞中hMLH1基因启动子区DNA甲基化、hMLH1 mRNA和hMLH1蛋白表达的变化.MTT法检测药物对细胞增殖的影响.结果:COC1细胞存在hMLH1 mRNA和蛋白的表达,其启动子区域表现为DNA非甲基化.单独应用5-Aza-dC、TSA及联合应用5-Aza-dC和TSA对COC1细胞中hMLH1 mRNA、hMLH1蛋白表达和DNA甲基化均没有影响(P>0.05),细胞形态变化不明显.COC1/DDP细胞中hMLH1 mRNA和蛋白表达缺失,启动子区域表现为DNA甲基化.5-Aza-dC不但能使COC1/DDP细胞中hMLH1基因发生DNA去甲基化,而且能使表达缺失的hMLH1 mRNA和hMLH1蛋白重新表达.TSA对COC1/DDP细胞中hMLH1 mRNA、hMLH1蛋白表达和DNA甲基化均没有影响(P>0.05).联合应用5-Aza-dC和TSA不但能使hMLH1基因发生DNA去甲基化,而且与单独应用5-Aza-dC相比较,对hMLH1 mRNA和hMLH1蛋白表达的影响更明显(P<0.05).5-Aza-dC单用及联合应用TSA对COC1/DDP细胞生长抑制率明显高于COC1组、阴性对照组(P<0.05).结论:COC1/DDP细胞对顺铂的耐药性与hMLH1mRNA和蛋白的表达缺失有关,而hMLH1基因和蛋白的表达缺失与其基因启动子区DNA甲基化相关,5-Aza-dC单用及联合TSA使hMLH1基因发生DNA去甲基化,促进hMLH1的表达,逆转COC1/DDP细胞对DDP的耐药性.     

散发性结直肠癌、腺瘤组织中hMLH1和突变型p53表达

[目的]探讨hMLH1和p53基因在散发性结直肠癌发生机制中所起的作用。[方法]采用PV-6000二步法免疫组化检测技术对60例散发性结直肠癌，45例大肠腺瘤和26例正常大肠黏膜石蜡切片进行hMLH1和p53表达的检测。[结果]（1）60例散发性结直肠癌hMLH1蛋白阴性表达和p53蛋白阳性表达分别为11例（18．3％）和32例（53.3％），hMLH1蛋白阴性表达和p53蛋白阳性表达与患者年龄、肿瘤部位、组织学类型和分化程度密切相关（P〈0．05或P〈0．01），而与患者性别、肿瘤大体类型、肿块大小、浸润深度、淋巴结及远处转移与否和患者的Duke’s分期均无显著相关性（P〉0．05）。（2）大肠腺瘤患者p53蛋白阳性表达与患者腺瘤部位、组织学类型和不典型增生程度密切相关（P〈0．05），而与患者性别、年龄无显著相关性（P〉0．05）。（3）散发性结直肠癌中hMLH1蛋白阴性表达组p53蛋白阳性表达率（18.2％，2/11）低于阳性表达组（61．2％，30／49）（P〈O．05）。[结论]（1）大部分散发性结直肠癌是沿染色体不稳定途径发生，其中抑癌基因p53的突变起重要作用。同时，一定比例的散发性结直肠癌中存在错配修复基因的缺陷，并具有相对特殊的临床病理特征。（2）大肠腺瘤组织中有一定比例的hMLH1蛋白阴性表达和p53蛋白阳性表达，提示hMLH1和p53基因的突变可能是散发性结直肠癌发生的早期事件之一。     

Microsatellite instability and mismatch repair gene inactivation in sporadic pancreatic and colon tumours.

Genomic instability has been proposed as a new mechanism of carcinogenesis involved in hereditary non-polyposis colorectal cancer (HNPCC) and in a large number of sporadic cancers like pancreatic and colon tumours. Mutations in human mismatch repair genes have been found in HNPCC patients, but their involvement in sporadic cancer has not been clarified yet. In this study we screened 21 pancreatic and 23 colorectal sporadic cancers for microsatellite instability by ten and six different microsatellite markers respectively. Microsatellite alterations were observed at one or more loci in 66.6% (14/21) of pancreatic cancers and in 26% (6/23) colon tumours, but all the pancreatic and half of the colon samples showed a low rate of microsatellite instability. All the unstable samples were further analysed for mutations in the hMLH1 and hMSH2 genes and for hypermethylation of the hMLH1 promoter region. Alterations in the hMLH1 gene were found only in colorectal tumours with a large presence of microsatellite instability. None of the pancreatic tumours showed any alteration in the two genes analysed. Our results demonstrate that microsatellite instability is unlikely to play a role in the tumorigenesis of sporadic pancreatic cancers and confirm the presence of mismatch repair gene alterations only in sporadic colon tumours with a highly unstable phenotype.     

Microsatellite instability and intratumoural heterogeneity in 100 right-sided sporadic colon carcinomas

Microsatellite instability has been proposed as an alternative pathway of colorectal carcinogenesis. The aim of this study was to evaluate the interest of immunohistochemistry as a new tool for highlighting mismatch repair deficiency and to compare the results with a PCR-based microsatellite assay. A total of 100 sporadic proximal colon adenocarcinomas were analysed. The expression of hMLH1, hMSH2 and hMSH6 proteins evaluated by immunohistochemistry was altered in 39% of the cancers, whereas microsatellite instability assessed by PCR was detected in 43%. There was discordance between the two methods in eight cases. After further analyses performed on other tumoural areas for these eight cases, total concordance between the two techniques was observed (Kappa=100%). Our results demonstrate that immunohistochemistry may be as efficient as microsatellite amplification in the detection of unstable phenotype provided that at least two samples of each carcinoma are screened, because of intratumoural heterogeneity.     

hMLH1 and hMSH2 expression in human hepatocellular carcinoma

The role of microsatellite instability (MSI) in the pathogenesis of hepatocellular carcinoma (HCC) is incompletely defined. Although high-frequency MSI (MSI-H) is infrequently seen in HCC, some studies have suggested a role for MSI in HCC development. While MSI has been clearly defined for a subset of tumors, in particular colorectal, gastric and endometrial cancers, generally accepted criteria have not been developed for other tumors. Colorectal cancers (CRC) are classified as MSI-H if >30-40% of >5 microsatellite loci analyzed show instability. The MSI-H phenotype is associated with defective DNA mismatch repair (MMR) and is observed in the majority of tumors from patients with hereditary non-polyposis colon cancer (HNPCC) and also in 15% of sporadic CRCs. Inactivating mutations of the hMLH1 or hMSH2 genes lead to defects in MMR in HNPCC. In sporadic CRCs, MMR is usually due to hypermethylation of the hMLH-1 promoter. The role of defective MMR in hepatocellular carcinogenesis is controversial. Immunohistochemistry for hMLH1 and hMSH2 reliably indicates hMLH1 or hMSH2 loss in MSI-H CRC tumors. To investigate the role of defective MMR in HCC carcinogenesis, we performed immunohistochemistry for hMLH1 and hMSH2 on 36 HCCs. BAT26, a microsatellite marker that reliably predicts MSI-H was also examined. All 36 of the tumors stained positively for both hMLH1 and hMSH2, strongly suggesting an absence of either inactivating mutations of hMLH1 and hMSH2 or promoter hypermethylation of hMLH1. None of the tumors showed MSI at the BAT26 locus. These findings suggest that defective MMR does not contribute significantly to hepatocellular carcinogenesis.     

Involvement of hMSH6 in the development of hereditary and sporadic colorectal cancer revealed by immunostaining is based on germline mutations, but rarely on somatic inactivation.

Germline mutations in human mismatch repair (MMR) genes yield a predisposition for the hereditary nonpolyposis colon cancer (HNPCC) syndrome. In contrast to hMLH1 and hMSH2, little is known about the overall involvement of hMSH6 in colorectal cancer. We investigated 82 tumors from patients who fulfilled the Bethesda guidelines for HNPCC as well as 146 sporadic tumors, analyzing microsatellite instability and expression of the 4 MMR proteins hMSH6, hMSH2, hMLH1 and hPMS2. Four tumors with lost expression and 1 tumor with cytoplasmic expression of hMSH6 were identified. Sequence analysis revealed germline mutations in 4 of the 5 patients, including 1 patient with sporadic disease. The lost or reduced expression of hMSH2 and hMLH1 was always identical to its heterodimerization partners, hMSH6 and hPMS2, respectively. Furthermore, hMSH2 expression was reduced upon hMSH6 deficiency. Abnormal expression of 1 or more of the 4 proteins was always associated with a high level of microsatellite instability (MSI-H). Conversely, all but 1 of the 44 MSI-H tumors had abnormal expression of 1 or more of the proteins, basically excluding additional genes associated with the MSI-H phenotype. We conclude that the involvement of somatic or epigenetic hMSH6 inactivation in colorectal cancer is rare.     

Methylation of the hMLH1 promoter but no hMLH1 mutations in sporadic gastric carcinomas with high-level microsatellite instability

Microsatellite instability (MSI) in tumors from patients with hereditary non-polyposis colorectal cancer (HNPCC) is caused by germline mutations in mismatch repair (MMR) genes, principally hMSH2 and hMLH1. In contrast, somatic mutations in MMR genes are relatively rare in sporadic MSI(+) colon cancers. Rather, the majority of mutation-negative, MSI(+) cases involve hypermethylation of the hMLH1 promoter and subsequent lack of expression of hMLH1. The details of the mechanisms of this epigenetic gene silencing remain to be elucidated. In some colon cancer cell lines, hMLH1 promoter methylation is accompanied by mutation of 1 of the 2 alleles, whereas in other cell lines and tumors, such combinations have not been reported. To contribute to the characterization of MSI in gastric cancer and to directly investigate whether hMLH1 promoter methylation is accompanied by gene mutation in these cancers, we have analyzed 42 gastric tumors and corresponding normal tissue for MSI, hypermethylation of the hMLH1 promoter, and mutations in hMLH1 as well as hMSH2. We found that 10 (23.8%) of 42 cases of sporadic gastric cancer were MSI(+) and that 8 had at least 2 of 12 altered microsatellite loci. All samples with at least 2 altered loci exhibited methylation of the hMLH1 promoter region, but none had detectable mutations in hMLH1 or hMSH2. Our results confirm the importance of methylation of the hMLH1 promoter region in MSI(+) gastric tumors and suggest that methylation takes place in the absence of hMLH1 mutations in these tumors.     

BAT-26 microsatellite instability does not correlate with the loss of hMLH1 and hMSH2 protein expression in sporadic endometrial cancers

Microsatellite instability (MSI) is detected in about 20-25% of endometrial cancers (ECs). Incidence of this alteration correlates with lack of expression of certain mismatch repair genes such as hMLH1 and hMSH2. Although assessment of several markers has been proposed for identification of microsatellite unstable tumours, BAT-26, a mononucleotide microsatellite repeat, has been shown to be highly efficient when used as a single marker. The aim of the study was to evaluate instability within BAT-26 and expression of hMLH1 and hMSH2 proteins in sporadic endometrial cancer as well as to correlate these findings with histopathologic and clinical characteristics of tumours. Samples of 88 (74 endometrioid and 14 non-endometrioid) ECs were investigated for instability within BAT-26 by means of PCR and expression of hMLH1 and hMSH2 proteins using immunohistochemistry. BAT-26 MIS was discovered in 23.9% of endometrial cancers. Incidence of MSI did not correlated with grade, stage or depth of invasion. BAT-26 MSI was more frequent in non-endometrioid compared to endometrioid tumours (35.7% vs. 21.6%, respectively), but the difference was not statistically significant. Lack of hMLH1 and hMSH2 protein expression was detected in 21.6 and 15.9% of ECs, respectively, and did not correlate with clinicopathologic features of tumours. Loss of both hMLH1 and hMSH2 protein expression was similar in BAT-26 stable and unstable cancers. All cases of non-endometrioid tumours with BAT-26 MSI were positive for hMLH1. We can conclude that BAT-26 used alone may not be a reliable marker for identification of sporadic ECs with microsatellite instability induced by deficient expression of hMLH1 and hMSH2.     

Not hMSH2 but hMLH1 is frequently silenced by hypermethylation in endometrial cancer but rarely silenced in pancreatic cancer with microsatellite instability

Disruption of the DNA mismatch repair (MMR) system has been found to play an important role in sporadic human cancers of several organs such as colorectum, stomach, endometrium, and pancreas. In cancers of the former three organs, disruption of the MMR system is mainly caused by hypermethylation of the hMLH1 gene. We investigated the expression of the hMLH1 and hMSH2 proteins immunohistochemically in pancreatic and endometrial cancers with high frequency microsatellite instability (MSI-H). Loss of expression of hMLH1 was found in none of seven pancreatic cancer, whereas eight (57%) of 14 endometrial cancer showed loss of expression of hMLH1. On the other hand, one (14%) of seven pancreatic cancers and two (14%) of 14 endometrial cancers showed loss of hMSH2 expression. We further analyzed the methylation status at the promoter region of the hMLH1 and hMSH2 genes and found hypermethylation of hMLH1 at the promoter region in the great majority of endometrial cancers with loss of expression. However, no pancreatic cancer showed hypermethylation. We then further analyzed 22 pancreatic cancer cell lines and obtained similar results. These results suggested that MSI-H in pancreatic cancer is probably caused by different mechanisms from those of other sporadic cancers with MSI-H.     

Microsatellite instability in hereditary and sporadic breast cancers

Sporadic cancers and familial breast cancers are characterized by an increase in genetic instability. Little is known about whether mismatch repair defects accompany this genetic instability. We investigated invasive and/or in situ breast cancers from 30 women with deleterious BRCA1/2 mutations and unclassified variant BRCA1/2 alterations. Forty cases of sporadic breast cancers were also investigated, including 7 medullary carcinomas. Malignant and benign lesions were examined from all cases to better understand tumor progression. Automated immunohistochemistry, with antibodies directed against hMLH1 and hMSH2, was used to screen cases for possible mismatch repair defects. When loss of expression was noted, DNA ploidy was performed by cytomorphometry. DNA, after laser microdissection, was extracted from a majority of familial cases and their corresponding controls, and microsatellite instability analysis was performed. None of the familial or sporadic cases had loss of hMSH2 expression. All but one lesion, a DCIS arising in a deleterious BRCA2 mutation carrier, had loss of hMLH1 expression and a tetraploid profile by image cytomorphometry. There was no MSI in any explored lesions (n = 34), as determined by molecular analysis, including the DCIS with loss of hMLH1 expression. We conclude that DNA mismatch repair defects involving hMLH1 and hMSH2 underexpression are extremely rare events in sporadic and familial breast cancer. Mismatch repair gene mutations may be secondary random events in breast cancer progression.