Mutations of hMLH1 and hMSH2 in patients with suspected hereditary nonpolyposis colorectal cancer: correlation with microsatellite instability and abnormalities of mismatch repair protein expression.

PURPOSE: The relationship between germ-line mutations of hMSH2 and hMLH1, microsatellite instability (MSI), and loss of DNA mismatch repair (MMR) gene expression were studied to formulate an effective selection protocol for patients with suspected hereditary nonpolyposis colorectal cancer who should be offered genetic testing. PATIENTS AND METHODS: Patients eligible for germ-line analysis of hMLH1 and hMSH2 were selected. Tumor specimens were obtained to assess MSI and loss of MMR gene expression. RESULTS: Among 37 patients who participated in the study, two hMSH2 and two hMLH1 missense mutations (11%) were detected, none of which was found in a panel of 60 healthy volunteers. High MSI was found in five tumors (19%) and low MSI in 10 tumors (39%); 12 tumors (46%) were microsatellite stable. Four tumors demonstrated loss of hMLH1, and three tumors demonstrated loss of hMSH2 protein expression. CONCLUSION: No relationship was found between MMR gene mutations and MSI; low or no MSI was found in the four patients with germ-line mutations, and none of the five patients with high MSI demonstrated abnormalities of MMR genes. On the contrary, loss of hMLH1 or hMSH2 expression was found in the tumors from three of the four patients demonstrating germ-line mutations. These data suggest that germ-line mutations of the MMR gene can occur in people with MSI-negative tumors. Sensitive clinical criteria and the study of MMR gene expression may be useful to identify this subset of patients.     

hMLH1和hMSH2表达在中国人遗传性非息肉病性结直肠癌遴选中的价值

背景与目的:遗传性非息肉病性结直肠癌(hereditary non-polyposis colorectal cancer, HNPCC)的发病主要与遗传因素有关,呈常染色体显性遗传.目前错配修复(mismatch repair,MMR)基因突变检测为诊断HNPCC的金标准,但尚处于实验室阶段.本研究拟通过免疫组织化学检测的方法来观察错配修复基因中的hMLH1和hMSH2蛋白的表达情况,并评价其在HNPCC遴选中的价值及与临床病理学的关系,为临床筛检HRPCC提供了简单、快速及经济的检测手段.方法:采用免疫组织化学SP法分别对HNPCC组(A组)、普通遗传性结直肠痛组(B组)、散发性结直肠痛组(C组)及结直肠息肉组(D组)4组各20例(共80例)标本进行错配修复基因hMLH1和hMSH2蛋白表达检测,并与临床病理学行为作相关分析.结果:A组hMLH1和hMSH2蛋白总阳性表达率为35%(7/20),B组为70%(14/20),C组为95%(19/20),D组为100%(20/20).即随着D组至A组患HNPCC可能性的增加,hMLH1和hMSH2蛋白阳性表达率呈明显下降的趋势,各组之间相比,筹异有显著性(P<0.05).A组20例HNPCC患者中,右半结肠的发生率为76.9%(10/13),左半结肠的发生率为42.9%(3/7),两者相比,差异有显著性(P<0.05).高、中分化癌发生率为33.3%(2/6),而低分化或术分化癌的发生率为78.6%(11/14),两者相比有显著性差异(P<0.05).hMLH1和hMSH2蛋白的阴性表达与患者的发病年龄、性别、有无淋巴结转移、Dukes分期等尤明显相关性(P>0.05).结论:hMLH1和hMSH2蛋白阴性表达与HNPCC的可能性以及结肠癌的发生部位和分化程度之间显著相关,免疫组织化学检测可为临床筛检HNPCC提供依据.     

Predominant germ-line mutation of the hMSH2 gene in Japanese hereditary non-polyposis colorectal cancer kindreds.

By means of PCR-SSCP and direct sequencing, we detected 12 germ-line mutations of hMSH2 or hMLH1 in 37 Japanese hereditary non-polyposis colorectal cancer (HNPCC) kindreds, of whom 15 satisfied the Amsterdam and 22 the Japanese criteria. The germ-line mutation detection rate of hMSH2 was much higher than that of hMLH1 (11/37 vs. 1/37). The total mutation detection rate of hMSH2 and hMLH1 in the Amsterdam criteria group was significantly higher than that in the Japanese criteria group (9/15 vs. 3/22). Furthermore, the mean age of the HNPCC patients in the mutation-positive group was lower than that in the mutation-negative one; the rates of both vertical transmission and multiplicity of tumors in the mutation-positive group were higher than those in the mutation-negative one. In addition, the number of patients with microsatellite instability-positive cancers in the mutation-positive group was higher than that in the mutation-negative one. Our results suggest firstly that the hMSH2 gene plays a much more important role than hMLH1 in the carcinogenesis of Japanese HNPCC patients, secondly that the rate of hMSH2 and hMLH1 mutations is high in the kindreds satisfying the Amsterdam criteria and thirdly that both the clinical phenotypes (early onset, vertical transmission and multiplicity of tumors) and the microsatellite instability status are important for the genetic screening of HNPCC.     

hMLH1 and hMSH2 mutations in families with familial clustering of gastric cancer and hereditary non-polyposis colorectal cancer

The pattern of hMLHI and hMSH2 mutations was assessed to identify the genetic correlation between hereditary gastric and colorectal cancers. Four disease groups and their healthy family members were assembled according to the presentation of gastric cancer: FG, familial clustering of gastric cancer (n = 32); CG, family with one or more colorectal and gastric cancers in first-degree relatives (n = 22); HS, seven HNPCC families corresponding to the Amsterdam criteria (AMS+) and 12 suspected HNPCC families which did not satisfy one of the criteria (AMS-), but no gastric cancer among first- and second-degree relatives (n = 19); and SG, sporadic gastric cancer (n = 33). In the CG group, three were included in AMS + and six in AMS- criteria. Peripheral blood was obtained from them to detect hMLHI and hMLH2 mutations using PCR-SSCP analysis and direct sequencing. The incidence of mutations was 9.4% in the FG group, 54.5% in the CG group, 31.6% in the HS group, and none in the SG group. The incidence, type, and number of the mutation were not different between the CG and HS groups. Thirty-four different mutations included 19 in hMLH1 and 15 in hMSH2. Gastric cancer was the most common extracolonic malignancy in HNPCC and suspected HNPCC families (9/28, 32.1%). The hMLH1 or hMSH2 mutation occurred in seven of 10 families with AMS+, whereas it occurred in four of 18 with AMS- (70% vs. 22.2%, P = .013). Five mutations in the hMLH1 and six mutations in the hMSH2 were exclusively found in families with gastric cancer. All three mutations in the FG group were in hMLHI and there was no mutation in their healthy family members. This study demonstrates that some familial clustering type of gastric cancer appears to be associated with hMLHI mutations thereby indicating a difference from the hereditary gastric cancer studies previously reported. In addition, hMLHI and hMSH2 mutations may impact the gastric cancer carcinogenesis in HNPCC or suspected HNPCC.     

Analysis of hMLH1 missense mutations in East Asian patients with suspected hereditary nonpolyposis colorectal cancer.

PURPOSE: Germ line mutations in the DNA mismatch repair gene hMLH1 are a frequent cause of hereditary nonpolyposis colorectal cancer and about one-third of these are missense mutations. Several missense mutations in hMLH1 have frequently been detected in East Asian patients with suspected hereditary nonpolyposis colorectal cancer, but their pathogenic role has not been extensively assessed. The aim of this study was to perform functional analyses of these variants and their association with gastrointestinal cancer in East Asians. EXPERIMENTAL DESIGN: Altogether, 10 hMLH1 variants were analyzed by yeast two-hybrid and coimmunoprecipitation assays. RESULTS: The carboxyl-terminal replacements Q542L, L549P, L574P, and P581L in hMLH1 resulted in complete loss of activity in both yeast two-hybrid and coimmunoprecipitation tests and thus might be considered as pathogenic. The amino-terminal variants S46I, G65D, G67R, and R217C did not affect complex formation with hPMS2 in coimmunoprecipitation, but partly or fully lost their activity in yeast two-hybrid assay, and we suggested that these variants might reduce the efficiency of the heterodimer to go into the nucleus and thus the mismatch repair function might be blocked or reduced. The V384D and the Q701K variant resulted in the interaction of hMLH1 with hPMS2 at reduced efficiency and might raise the gastrointestinal cancer risk of the mutation carriers. CONCLUSIONS: This work availably evaluated the functional consequences of some missense mutations not previously determined in the hMLH1 gene and might be useful for the clinical diagnosis of hereditary gastrointestinal cancer, especially in East Asians.     

2例遗传性非息肉病性大肠癌家系分析

目的分析2例遗传性非息肉病性大肠癌（HNPCC）家系的临床病理特征。方法对2个HNPCC家系进行病例回顾及家系调查。结果HNPCC的主要特点是病灶好发于右半结肠，发病年龄较轻，预后好于散发性大肠癌。结论应对HNPCC患者及其家族严密监测、随访，以便早期诊断、及时治疗、改善预后。     

Combined deficiency of hMLH1, hMSH2, hMSH3 and hMSH6 is an independent prognostic factor in colorectal cancer

We examined biological and clinicopathological significance of individual and combined hMLH1, hMSH2, hMSH3 and hMSH6 expression with immunohistochemistry in 301 unselected colorectal cancers. Weak hMLH1 expression was correlated to microsatellite instability (P=0.04), negative p53 expression (P=0.005) and mucinous carcinomas (P=0.02). Weak hMSH2 expression was related to negative ras (P<0.001) and p53 expression (P=0.005), and better survival (P=0.03). hMSH2, hMSH3 and hMSH6, as well as hMLH1, hMSH2, hMSH3 and hMSH6, were combined into a 'functional' and a 'less-functional' group, respectively. Both 'less-functional' groups were/tended to be associated with microsatellite instability, negative ras and p53 expression, and better survival. In summary, hMLH1 and hMSH2 were more important when investigated individually, and the combined groups were more related to the mutator pathway, suggesting that combined deficiencies of the proteins are more efficiently involved in the mutator pathway. Our result from weak versus strong staining may suggest that the intensity of staining should be considered in future studies on mismatch repair proteins.     

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.     

幽门螺杆菌对胃癌hMLH1和hMSH2基因表达的影响

目的探讨幽门螺杆菌(Hp)对胃癌中hMLH1和hMSH2基因表达的影响和Hp的致癌机制.方法利用免疫组织化学S-P法检测胃癌、癌旁和胃炎黏膜中hMLH1、hMSH2 基因的表达情况.结果在全部被检组织中,Hp感染组hMLH1和hMSH2表达阳性率均低于相应的非感染组,其中胃癌组织中hMSH2表达阳性率在Hp感染组(54.7%)和非感染组(82.1%)差异有显著性(P<0.05).结论 Hp感染引起hMLH1和hMSH2基因表达降低,这可能是Hp导致胃癌的分子机制之一.     

Promoter methylation and immunohistochemical expression of hMLH1 and hMSH2 in sporadic colorectal cancer: a study from India

To determine the etiological factors of human colorectal cancer (CRC) we assessed the frequency and prognostic significance of hMLH1 and hMSH2 genes in conjunction with hMLH1 and hMSH2 protein expression in 30 Indian CRC patients. The protein expression and promoter methylation of hMLH1 and hMSH2; Mismatch Repair genes (MMR) were analyzed by immunohistochemistry and methylation-specific PCR (MSP), respectively. A loss of hMLH1 expression was recognized in 4(13.3 %) and loss of hMSH2 expression was recognized in 2(6.6 %) of 30 CRC cases whereas 50 % tumors showed reduced expression of hMLH1 and 33.3 % showed reduced expression of hMSH2 protein. One tumor showed a loss of both hMLH1 and hMSH2 expression. Normal nuclear staining pattern of hMLH1 and hMSH2 was observed in almost all the adjoining and normal mucosa. Promoter hypermethylation of the hMLH1 gene was detected in 15 of 30 CRC cases (50 %) and of hMSH2 gene was only in 3 of 30 CRC cases (10 %). No promoter methylation of hMLH1 and hMSH2 genes was observed in adjoining and normal mucosa. Combination of methylation of hMLH1 and hMSH2 gene was observed in two tumors (6.6 %). A significant correlation between histological grade of the tumor, methylation and expression of hMLH1 gene (p?<?0.05) was observed. Normal expression of hMLH1 and hMSH2 was seen in all of the unmethylated tumors (100 %). Nuclear staining and promoter methylation of hMLH1 and hMSH2 did not significantly influence survival. hMLH1 methylation was common and was significantly correlated with loss of hMLH1 protein expression. In contrast, hMSH2 methylation was infrequent. These findings suggest that the inactivation of MMR gene expression probably via hypermethylation may lead to inactivation of their functions which finally leads to tumor aggressiveness and the immunostaining of hMLH1 protein can be used as a prognostic factor for determining the grade of the tumor.     

Prolonged survival in hereditary nonpolyposis colorectal cancer

We compared the survival between 46 patients with hereditary nonpolyposis colorectal cancer (HNPCC) and 1185 cases with sporadic colorectal cancer, who underwent a resection for the disease between 1972 and 1995. In a univariate analysis, the survival correlated well with stage, curability, size and the presence of HNPCC. Patients with HNPCC had a longer survival than those with sporadic colorectal cancer (P=0.0277). A multivariate analysis suggested that stage, curability, age, and HNPCC were the effective combination of factors predictive of survival. The above findings are thus expected to have a major influence on the evaluation of clinical trials in patients with colorectal cancer.     

Current approaches in hereditary nonpolyposis colorectal cancer

This article emphasizes the central role of tumor-based testing for microsatellite instability followed by performance of genetic counselor-driven germline mutation testing in hereditary nonpolyposis colorectal cancer (HNPCC). Suitably aggressive colorectal neoplasm surveillance is shown to be critical. Limitations of the evidentiary base for extracolonic screening are conceded, with some cautious suggestions for possible strategies notwithstanding the lack of data. Advances in chemoprevention have been made in both familial adenomatous polyposis (clinical trial data favoring eicosapentaenoic acid) and HNPCC (controversial aspirin data). For various reasons, however, no agent or combination of agents has yet come into routine use in either condition, with further trials underway or being designed for both conditions.     

Altered expression of MLH1, MSH2, and MSH6 in predisposition to hereditary nonpolyposis colorectal cancer.

PURPOSE: A considerable fraction (30% to 70%) of families with verified or putative hereditary nonpolyposis colorectal cancer fails to show mutations in DNA mismatch repair (MMR) genes. Our purpose was to address the genetic etiology of such families. Materials and METHODS: We scrutinized a population-based cohort of 26 families from Finland that had screened mutation-negative by previous techniques. Blood was tested for allelic messenger RNA (mRNA) expression of MLH1, MSH2, and MSH6 by single nucleotide primer extension (SNuPE), and tumor tissue for MMR protein expression by immunohistochemistry (IHC) as well as for microsatellite instability (MSI). Full-length cDNAs of genes implicated by SNuPE or IHC were cloned and sequenced. RESULTS: Unbalanced mRNA expression of MLH1 alleles was evident in two families. An inherited nonsense mutation was subsequently identified in one family, and complete silencing of the mutated allele was identified in the other family. Extinct protein expression by IHC implicated MLH1 in these two and in four other families, MSH2 in four families, and MSH6 in one family. Although no unequivocal genomic mutations were detected in the latter families, haplotype and other findings provided support for heritable defects. With one exception, all tumors with IHC alterations showed MSI, in contrast to the remaining families, which showed neither IHC changes nor MSI. CONCLUSION: Our expression-based strategy stratified the present "mutation-negative" cohort into two discrete categories: families linked to the major MMR genes MLH1, MSH2, and MSH6 (11 [42%] of 26) and those likely to be associated with other, as yet unknown susceptibility genes (15 [58%] of 26).     

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.     

结直肠癌组织hMLH1和hMSH2蛋白缺失临床意义的分析

目的:探讨hMLH1和hMSH2蛋白缺失与Ⅱ和Ⅲ期结直肠癌患者临床病理特征及预后的关系。方法:选取162例行结直肠癌根治术的患者,采用免疫组化方法检测患者hMLH1和hMSH2蛋白表达情况,采用χ2检验分析hMLH1和hMSH2蛋白表达缺失与结直肠癌临床病理特征之间的关系;采用Kaplan-Meier生存曲线、Log-Rank检验、Cox风险回归模型分析不同因素与预后的关系。结果:1)hMLH1蛋白的表达缺失率为38.89%,hMSH2蛋白的表达缺失率为21.60%。2)hMLH1和(或)hMSH2蛋白表达缺失的患者较hMLH1和hMSH2蛋白表达无缺失的患者2年无瘤生存率(DFS)高,P=0.023。3)hMLH1和(或)hMSH2蛋白缺失的患者与hMLH1和hMSH2蛋白表达无缺失的患者相比,前者多发病在右半结肠(P=0.018)、淋巴结转移数目较少(P=0.007)、多存在家族史(P=0.013),且TS蛋白多为低表达(P=0.004)。4)单因素分析显示,hMLH1和hMSH2蛋白表达情况(P=0.010)、TNM分期(P=0.010)、淋巴结转移数目(P=0.002)为结直肠癌患者预后的影响因素;多因素分析显示,hMLH1和hMSH2蛋白表达情况、淋巴结转移数目为结直肠癌患者预后的独立影响因素。结论:hMLH1和hMSH2蛋白表达缺失的结直肠癌患者具有独特的临床病理特征,预后较好,hMLH1和hMSH2蛋白表达缺失是预后的独立影响因素。     

Risk of gastric cancer in hereditary nonpolyposis colorectal cancer in Korea.

After endometrial cancer, gastric cancer is the second most common extracolonic cancer in cases of hereditary nonpolyposis colorectal cancer (HNPCC), where the relative risk in HNPCC familial members is known to be 4-fold. However, it is not yet clear whether HNPCC families from Korea, an endemic area for gastric cancer, have the same relative risk or whether the incidence of gastric cancer is high enough to justify familial screening. To clarify these questions, we investigated 1011 individuals from 66 Korean HNPCC families (28 families fulfilled the Amsterdam criteria and 38 did not). The overall and age-specific relative risk of gastric cancer in HNPCC families when compared with the background population was calculated. Twenty-five patients with gastric cancer were identified from among 22 HNPCC families. The calculated risk of gastric cancer in patients with HNPCC and their first-degree relatives was 2.1-fold greater than in the general population (95% confidence interval; range, 1.4-3.2). However, the relative risk of gastric cancer in the younger generations was much greater (11.3-fold in the 30s and 5.5-fold in the 40s). Additionally, the relative risk was greater in mutation carrier families than noncarrier families (3.2-fold versus 1.6-fold). This study demonstrates that the risk of gastric cancer in members of HNPCC families in a gastric cancer endemic population, particularly in younger subjects and mutation carrier families, is high enough to justify careful screening.     

IGF1 gene polymorphism and risk for hereditary nonpolyposis colorectal cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disorder caused by germline mutations in DNA mismatch repair (MMR) genes. Insulin-like growth factor-I (IGF-I) is involved in colorectal carcinogenesis, and elevated plasma IGF-I levels are associated with sporadic colorectal cancer (CRC) risk. We investigated the relationship between IGF1 promoter cytosine-adenine (CA) dinucleotide-repeat polymorphism length and CRC risk in 121 MMR gene mutation carriers using Cox regression and Kaplan-Meier analysis. All statistical tests were two-sided. Time to onset for CRC increased for each decrease in CA-repeat number (median = 19 repeats, range = 12-22 repeats; hazard ratio [HR] = 1.17, 95% confidence interval [CI] = 1.05 to 1.31; P = .006). Patients carrying a CA(< or = 17) repeat allele had a statistically significantly higher CRC risk (HR = 2.36; 95% CI = 1.28 to 4.36; P = .006) than all others and were younger at onset (44 years versus 56.5 years; P = .023). These findings indicate a statistically significant association between shorter IGF1 CA-repeat lengths and increased risk for CRC in HNPCC. This is the first report, to our knowledge, to show that IGF1 variant genotypes modify risk of a hereditary form of cancer.     

N-acetyltransferase 2 influences cancer prevalence in hMLH1/hMSH2 mutation carriers.

Hereditary nonpolyposis colorectal cancer (HNPCC), an inherited cancer predisposition syndrome, has been associated with germline mutations in DNA mismatch repair (MMR) genes. Because a deficiency in MMR does not predict a specific cancer phenotype, modifying genes may account in part for the variation in disease expression. We determined the N-acetyltransferase 2 (NAT2) genotype in 26 unaffected and 52 cancer-affected hMLH1/hMSH2 mutation carriers coming from 21 Swiss HNPCC families. Slow acetylators were found to be significantly (P < 0.03) more prevalent in the group of affected mutation carriers. Our results suggest a protective effect of the NAT2 rapid acetylator phenotype, an observation that could have implications for genetic counseling and management of MMR gene mutation carriers.