Microsatellite Instability and hMLH1 and hMSH2 expression analysis in familial and sporadic colorectal cancer

Immunohistochemical expression analysis of mismatch repair gene products has been suggested for the prediction of hereditary nonpolyposis colorectal cancer (HNPCC) carrier status in cancer families and the selection of microsatellite instability (MSI)-positive tumors in sporadic colorectal cancer. In this study, we aimed to evaluate hMSH2 and hMLH1 immunohistochemistry in familial and sporadic colorectal cancer. We found that immunohistochemistry allowed us to identify patients with germline mutations in hMSH2 and many cases with germline mutations in hMLH1. However, some missense and truncating mutations may be missed. In addition, hMLH1 promoter methylation, commonly occurring in familial and sporadic MSI-positive colorectal cancer, can complicate the interpretation of immunohistochemical expression analyses. Our results suggest that immunohistochemistry cannot replace testing for MSI to predict HNPCC carrier status or identify MSI-positive sporadic colorectal cancer.     

Low mutation rate of hMSH2 and hMLH1 in Taiwanese hereditary non-polyposis colorectal cancer

Hereditary non-polyposis colorectal cancer (HNPCC), the most common type of hereditary colorectal cancer, is thought to be a simple Mendelian disease involving DNA mismatch repair genes. The majority of mutations associated with HNPCC occur in the hMSH2 and hMLH1 genes. The reported incidence of mismatch repair gene mutations in HNPCC kindreds varies considerably (from 22 to 86%), and most mutations are unique. This study aimed to determine the genetic basis of Taiwanese HNPCC kindreds, focusing on the two major genes involved in this disease. A total of 15 Taiwanese HNPCC kindreds meeting the Amsterdam criteria, including 72 affected individuals among a total of 266 individuals, were analyzed using both RNA- and DNA-based methods. The mutation rate of hMSH2 and hMLH1 in these 15 kindreds was 0% and 20%, respectively, which is lower than that reported in other countries. Two novel mutations were discovered in hMLH1: one was an allelic loss of a 5.2-kb genomic fragment causing exon 16 deletion; and the other was a two-nucleotide deletion that resulted in a frameshift mutation of exon 3. We also identified one hMLH1 exon 4 mutation (a C to T transition in codon 117), which had been reported previously in western countries. This is the first genetic study of HNPCC from Taiwan.     

Immunohistochemical pattern of hMSH2/hMLH1 in familial and sporadic colorectal, gastric, endometrial and ovarian carcinomas with instability in microsatellite sequences

Alterations of DNA mismatch repair (MMR) genes are involved in carcinogenesis of sporadic and inherited human cancers characterised by instability of DNA microsatellite sequences (MSI). MSI tumours are usually identified using molecular analysis. In the present investigation, hMLH1 and hMSH2 immunohistochemistry was tested in order to evaluate the utility of this method in predicting MMR deficiency. Colorectal (72), gastric (68), endometrial (44) and ovarian (17) carcinomas were independently evaluated for familial history, histological type of tumour, MSI status and immunohistochemical results. Loss of expression of either hMLH1 or hMSH2 was observed in 51 of 55 (92.8%) MSI tumours, while 145 of 146 microsatellite stable (MSS) tumours expressed both the hMLH1 and hMSH2 gene products. Independently of tumour site, an overall agreement between immunohistochemical and molecular results was observed in 15 hereditary non-polyposis colorectal cancer-related tumours. Among sporadic tumours, only 2 of 60 colorectal and 2 of 66 gastric carcinomas, displaying MSI, expressed both hMLH1 and hMSH2 gene products. All 39 endometrial and 16 ovarian tumours presented a concordant molecular and immunohistochemical profile. These data show that immunohistochemistry is an accurate and rapid method to predict the presence of defective DNA MMR genes and to identify both sporadic and familial MSI tumours.     

中国人遗传性非腺瘤病性结直肠癌家系hMSH2和hMLH1基因突变分析

目的 分析符合不同临床标准的中国遗传性非腺瘤病性结直肠癌(HNPCC)家系hMSH2和hMLH1基因种系突变状况,评价不同临床标准预示突变检测的敏感性。方法应用DNA直接测序对24个符合Amsterdam标准、15个符合日本标准家系先证者和19个符合Bethesda指导纲要患者(字系中仅1例患者)进行hMSH2和hMLH1基因种系突变检测。对检出突变的家系进行家庭成员的突变筛选。并对检出突变患者进行肿瘤组织突变的检测。结果在16例家系先证者中检测到6个hMSH2突变和11个hMLHl种系突变,其中12个突变是国际上尚未报道过的新突变。突变位于不同外显子中,其中6个突变位于hMLHl第14-16外显子。Amsterdam标准家系突变阳性率为50％(12／24),以日本标准所筛家系突变阳性率为3／15,以上两组家系以外的Bethesda指导纲要患者突变阳性率为1／19。突变类型包括移码突变、无义突变、剪接异常、框架内插入或缺失以及错义突变。基因突变与疾病共分离,检出突变家系先证者的肿瘤组织错配修复基因表现出3种不同基因型：(1)野生型等位基因丢失;(2)肿瘤组织基因型与生殖细胞一致;(3)突变型等位基因丢失。结论中国人HNPCC家系hMSH2和hMLHl突变谱广泛,突变类型多样,hMLHl突变较hMSH2突变多见,突变较为集中于hMLHl外显子14-16。不同临床标准预示突变的敏感性不同。突变基因型与疾病表现型共分离。家系成员中尚未发病的突变携带者应予密切监测。     

Microsatellite instability and expression of hMLH1 and hMSH2 proteins in ovarian endometrioid cancer.

Microsatellite instability and loss of heterozygosity has been implicated in ovarian carcinogenesis. The reported frequency of microsatellite instability in human ovarian cancer varies significantly owing to the use of heterogeneous tumor histotypes and various microsatellite markers in different laboratories. In this study, we determined the frequency of microsatellite instability in 74 ovarian endometrioid carcinomas using four microsatellite markers (BAT25, BAT26, D5S346, D17S250), and examined hMLH1 and hMSH2 protein expression. In all, 20% of the tumors were microsatellite instability high (two or more markers showing instability) and 12% were microsatellite instability low (one marker showed instability). Loss of hMLH1 and/or hMSH2 expression was found in nine of 15 microsatellite instability-high tumors. The microsatellite instability-high phenotype tended to occur more frequently in low-grade tumors (P=0.053), but did not correlate with clinical stage. Totally, 38% of cases also displayed loss of heterozygosity at D17S250; this loss of heterozygosity was associated with high clinical stage (P=0.097). Our results indicate that both microsatellite and loss of heterozygosity at D17S250 are involved in the development of ovarian endometrioid carcinoma.     

Microsatellite instability in tumor and nonneoplastic colorectal cells from hereditary non-polyposis colorectal cancer and sporadic high microsatellite-instable tumor patients.

Genetic alterations such as loss of heterozygosity (LOH) and microsatellite instability (MSI) have been frequently studied in various tumor types. Genetic heterogeneity of nonneoplastic cells has not yet been sufficiently investigated. However, genomic instability in normal cells could be a potentially important issue, in particular when these cells are used as reference in LOH and MSI analyses of tumor samples. In order to investigate possible genetic abnormalities in normal colorectal cells of tumor patients, MSI analyses of normal colonic mucosa were performed. Up to 15 different laser-microdissected normal regions containing 50-150 cells were investigated in each of 15 individual microsatellite-stable, sporadic high microsatellite-instable (MSI-H) and hereditary non-polyposis coli cancer (HNPCC) colorectal cancer patients. Frequent MSI and heterogeneity in the MSI pattern were found both in normal and tumor cells from 10 HNPCC and sporadic MSI-H tumor patients whose tumors had defect mismatch repair protein expressions. This observation shows that MSI can also occur in nonneoplastic cells which has to be considered in MSI analyses for molecular HNPCC screening. In addition, considerable genetic heterogeneity was detected in all MSI-H (sporadic and HNPCC) tumors when analyzing five different regions with less than 150 cells, respectively. These differences were not detectable in larger tumor regions containing about 10,000 cells. Thus, heterogeneity of the MSI pattern (e.g. intratumoral MSI) is an important feature of tumors with the MSI-H phenotype.     

Microsatellite instability, loss of heterozygosity, and loss of hMLH1 and hMSH2 protein expression in endometrial carcinoma

Microsatellite instability (MSI) due to replication errors occurs frequently in hereditary tumors. Association with functional inactivation of the mismatch repair (MMR) genes and lack of protein expression has been described. In endometrial carcinoma (EC), the prevalence and clinical significance of these phenomena are not well known. Therefore, DNA samples from 89 EC and 5 metachronous tumors were analyzed with polymerase chain reaction, using 5 microsatellite markers and a DNA sequencer for amplicon detection. The results were correlated with immunohistochemistry of hMLH1 and hMSH2. MSI at >or=2 loci (MSI-H) was detected in 10/89 EC (11%); 1 of 10 showed loss of both hMLH1 and hMSH2, and 5 of 10 showed loss of hMLH1 (P < 0.0001). MSI-H was observed frequently in tumors with mucinous differentiation (P = 0.048), >10% of solid-cribriform pattern (P = 0.037), International Federation of Obstetrics and Gynecology (FIGO) stage III to IV (4 of 13; P = 0.016), and necrosis >5% (P = 0.07). Loss of heterozygosity (LOH) in >or=1 loci was found in 17 of 156 (11%). Survival (Kaplan-Meier) was longer for patients with endometrioid tumors with predominant glandular pattern, <5% necrosis, low FIGO stage and grade, superficial myometrial infiltration, no lymph-vascular invasion (LVI), and loss of hMLH1 expression (all P 相似文献   

散发性结直肠癌hMLH1和hMSH2蛋白表达

目的　研究hMLH1及hMSH2两种错配修复 (mismatchrepair,MMR)蛋白在散发性结直肠癌中的表达变化并评估其可能的临床意义。方法　应用EnVision免疫组化两步法检测 1 1 1例散发性结直肠癌中hMLH1和hMSH2的蛋白表达变化 ,采用Kaplan Meier曲线、Log rank检验分析hMLH1蛋白表达变化与患者生存率之间的关系。 结果　 1 1 1例散发性结直肠癌中 ,hMLH1失表达有 1 9例 ,占 1 7 1 % (1 9/ 1 1 1 ) ,hMSH2失表达有 2例 ,占 1 8% (2 / 1 1 1 ) ,两者之和占总散发性结直肠癌病例的 1 8 9% (2 1 / 1 1 1 )。hMLH1或hMSH2蛋白失表达与患者肿瘤部位、组织学类型密切相关。近端结肠、低 -未分化腺癌及黏液腺癌中MMR异常表达比例高 (P <0 0 5 ) ,而与患者年龄、性别、肿瘤大体类型、肿块大小、浸润深度、淋巴结转移与否以及患者的Dukes分期均无显著性相关 (P >0 0 5 )。癌组织中hMLH1正常表达及失表达患者的 5年生存率分别为 6 9 5 7%及73 6 8% ,8年生存率分别为 5 3 5 8%及 73 6 8% ,8年生存率差别较明显 ,然差别无统计学显著性 (P >0 0 5 )。结论　一定比例的散发性结直肠癌中存在MMR基因的缺陷 ,其中hMLH1所起的作用远远大于hMSH2 ,hMLH1失表达与否可能成为有意义的远期生存预后指标     

Microsatellite instability and alteration of the expression of hMLH1 and hMSH2 in ovarian clear cell carcinoma

Microsatellite instability (MSI) is commonly seen in tumors associated with the hereditary nonpolyposis colorectal cancer syndrome and is caused by defects in the DNA mismatch repair genes. MSI has also been observed in various sporadic cancers, including colorectal, gastric, and endometrial. The role and incidence of MSI in ovarian clear cell carcinoma remain unknown. This study was conducted to evaluate the frequency of MSI in ovarian clear cell carcinomas and to evaluate the sensitivity and specificity of immunohistochemistry in predicting mismatch-repair gene deficiency. A total of 42 ovarian clear cell carcinomas were analyzed for MSI using a panel of 5 microsatellite markers (BAT25, BAT26, D5S346, D2S123, and D17S250). Alterations in the expression of hMLH1 and hMSH2 proteins in these tumors were examined. Of the 42 ovarian clear cell tumors analyzed, 6 demonstrated a high level of MSI (MSI-H), 3 demonstrated a low level of MSI (MSI-L), and the remaining 33 exhibited microsatellite stability (MSS). No correlation was found between MSI level and patient age or tumor stage or size (P >0.05). Loss of expression of either hMLH1 or hMSH2 was observed in 4 of the 6 (67.7%) MSI-H tumors, whereas 34 of the 36 (94.4%) MSI-L or MSS tumors expressed both the hMLH1 and hMSH2 gene products. Our results indicate that MSI-H is involved in the development of a subset of ovarian clear cell carcinomas. A strong correlation exists between alterations in the expression of hMLH1 and hMSH2 and the presence of MSI-H in these tumors. However, immunohistochemical testing alone may miss a small fraction of cases with MSI-H.     

Microsatellite instability in young patients with colorectal cancer

Genetic instability Is closely correlated to the pathogenesis of hereditary non-palyposis colon cancer (HNPCC), which is clinicaily characterized by a family history and early onset. To investigate the role of genetic instabllity in young patients with colorectal cancer (CRC), 22 CRC patients, who were aged younger than 30 at the time of diagnosis, were studied. Patients with famllial adenomatous polyposis were excluded. Among the 22 cases, seven were identifled as microsateillte instability posltive (MI+), and more than five microsatellite markers among the 15 tested markers showed an additlonal band pattern in the tumor tissue. None of the remaining 15 cases showed instability in any microsatellite marker. Two of seven MI+ cases were classic HNPCC. While all MI+ cases had one or no metastatic lymph node, 53.3% of MI- cases showed metastasls in two or more reglonal lymph nodes. Allelic deletion of the 17p12–13 chromosome around the p53 locus occurred in 16.7% of MI+ cases, and 80.0% of MI- cases showed loss of het-erozygosity at that locus. hMSH2 Protein expression, assessed by immunohistochemistry, was absent in two cases, both of which were MI+. When we tested two to four sites of MI+ tumors, transforming growth factor β receptor type II was mutated in a homogeneous pattern in five MI+ cases. in addition, frame-shift mutations of BAX , insulin-like growth factor II receptor, hMSH3 and hMSH6 were found in three cases, five cases, five cases and one case, respectively. In contrast to the consistent mutatlon of the transforming growth factor-β receptor type II gene, mutations of other genes varied in different portions of the tumors.     

Microsatellite instability in early onset and familial colorectal cancer.

Hereditary non-polyposis colorectal cancer syndrome (HNPCC) is often considered to be the most common form of inherited colorectal cancer, although its precise incidence is unknown. The clinical diagnosis of HNPCC relies on a combination of family history and young age of onset of colorectal cancer, but as many familial aggregations of colorectal cancer do not fulfil the strict diagnostic criteria, HNPCC might be underdiagnosed. The majority of HNPCC families have germline mutations in mismatch repair (MMR) genes, such as MSH2 or MLH1, so that HNPCC cancers characteristically exhibit DNA replication errors (RERs) at microsatellite loci. Although an RER positive phenotype in tumours can also result from somatic mutations in an MMR gene, the prevalence of RER + tumours should provide a maximum estimate of the incidence of germline MMR gene mutations in patients with early onset and familial colorectal cancer. We investigated colorectal cancers for RERs from (1) a population based study of 33 patients with colorectal cancer aged 45 years or less, (2) 65 kindreds with familial colorectal cancer which only partially fulfilled the criteria for the diagnosis of HNPCC, and (3) 18 cancers from 12 HNPCC kindreds. Seven of 33 patients (21%) with colorectal cancer aged 45 years or less had an RER + cancer, with only two of these having a clear family history of HNPCC. A greater proportion of RER + tumours (5/7) occurred proximal to the splenic flexure than RER - tumours (4/26; chi2 = 6.14, p < 0.025). RERs were detected in all 18 cancers from HNPCC patients but in only six of 65 non-HNPCC familial colorectal cancer kindreds (9%; chi2 = 52.2, p < 0.0005). These findings suggest that most cancers in patients diagnosed at 45 years of age or less and familial aggregations of colorectal cancer which do not fulfil HNPCC diagnostic criteria do not have germline mutations in MSH2 and MLH1. Hence population screening for germline mutations in these genes is unlikely to be an efficient strategy for identifying people at high risk of developing colorectal cancer.     

Genomic structure of human mismatch repair gene, hMLH1, and its mutation analysis in patients with hereditary non-polyposis colorectal cancer (HNPCC)

Mutation of hMLH1, a gene involved in DNA mismatch repair, isresponsible for some families carrying the hereditary non-polypoticcolorectal cancer (HNPCC) syndrome. To establish a basis forpresymptomatic diagnosis of HNPCC patients who carry germlinemutations in this gene, we determined the exon—intronorganization of hMLH1. The results indicated that hMLH1 consistsof 19 coding exons spanning approximately 100 kb, and that exons1–7 contain a region that is highly conserved in the MLH1and PMS1 genes of yeast. We used PCR—SSCP analysis andDNA sequencing to examine the entire coding region of the MLH1gene in DNAs of 34 unrelated cancer patients who belong to HNPCCpedigrees. Germline mutations were detectable in eight (24%)of these patients; four of them were missense mutations, onehad occurred in an Intron where it would affect splicing, andthe remaining three were frameshift mutations resulting in truncationof the gene product downstream of the mutation site.     

散发性胆囊癌nm23-H1基因遗传不稳定性与错配修复基因hMLH1和hMSH2蛋白表达的研究

目的:研究人类17号染色体D17S396位点微卫星不稳定性和杂合性缺失,对nm23-H1蛋白表达的影响,同时检测错配修复基因hMLH1和hMSH2蛋白的表达,为揭示nm23-H1基因、hMLH1和hMSH2基因与肿瘤发生和转移机制提供实验依据.方法:采用石蜡包埋组织抽提DNA、PCR-SSCP、常规银染、Envision免疫组织化学等方法,对50例胆囊癌及其相应的正常组织,进行D17S396位点MSI、LOH的检测和nm23-H1、hMLH1和hMSH2蛋白表达研究.结果:①原发性胆囊癌D17S396位点遗传不稳定发生率为42.55%,LOH的发生率与肿瘤组织分化程度差异显著(P＜0.05);在肝脏侵润和淋巴转移组高于无肝脏侵润和无淋巴转移组(P＜0.01),在NevinⅣ+Ⅴ期高于Ⅰ+Ⅱ+Ⅲ期(P＜0.01);而MSI发生率则相反;②nm23-H1蛋白阳性率为46.81%,在淋巴转移组低于无淋巴转移组(P＜0.01);NevinⅣ+Ⅴ期低于Ⅰ+Ⅱ+Ⅲ期(P＜0.05);③hMLH1和hMSH2蛋白阳性率分别为51.06%和42.55%,hMLH1蛋白表达在有无淋巴转移组和Nevin分期有显著差异(P＜0.01),肝脏侵润组低于无肝脏侵润组(P＜0.05);④MSI阳性组中hMLH1蛋白阳性率显著高于MSI阴性组(P＜0.05).LOH阳性组中nm23-H1和hMSH2蛋白阳性率显著低于LOH阴性组(P＜0.05);⑤hMSH2蛋白阳性组中nm23-H1蛋白表达明显高于hMSH2蛋白阴性组(P＜0.05).结论:nm23-H1基因的遗传不稳定性可能是胆囊癌发生、发展的一个重要分子机制.nm23-H1基因的MSI和LOH,通过相互独立的途径调控胆囊癌的发生和转移.hMLH1/hMSH2表达异常可能是胆囊癌的早期分子事件.提高胆囊癌局部nm23-H1、hMLH1和hMSH2蛋白的表达,可减缓肿瘤的侵润转移并提高预后率.     

Family cancer histories predictive of a high risk of hereditary non-polyposis colorectal cancer associate significantly with a genomic rearrangement in hMSH2 or hMLH1

Hereditary non-polyposis colorectal cancer (HNPCC) results from inactivating germline mutations in a set of DNA-mismatch-repair genes, of which the most clinically relevant are hMSH2 and hMLH1. Computer-assisted pedigree risk assessment tools are available to assist in the calculation of an individual's likelihood of bearing such a deleterious mutation. One such tool, cancergene version 3.4 (http://www3.utsouthwestern.edu/cancergene) was used to assess the risk of a deleterious mutation in the genes hMSH2 and/or hMLH1 in a series of probands selected from a panel of 67 South-western Ontario kindred previously identified as likely candidates for HNPCC by established clinical criteria. A DNA sample isolated from peripheral blood leukocytes obtained from each of these probands was examined for genomic rearrangement using the multiplex ligation-dependent probe amplification (MLPA) method. Of the individuals calculated to have a risk of >50% of a hMSH2 or hMLH1 gene mutation by the CancerGene risk assessment tool, 69% (9/13) were shown to have a genomic rearrangement resulting in the deletion of one or more exons of one of these two genes. Family cancer histories predictive of a high risk of HNPCC significantly associate with a genomic rearrangement in hMSH2 or hMLH1.     

Altered expression of hMLH1 and hMSH2 protein in endometrial carcinomas with microsatellite instability

Microsatellite instability (MI) has been observed in approximately 20% of presumably sporadic cases of uterine endometrioid carcinoma (UEC). A previous mutational analysis of the 4 known DNA mismatch repair genes (hMSH2, hMLHI, hPMS1, and hPMS2) on a small number of Ml-positive tumors detected mutations in only 2 of 8 cases, both in hMSH2. To further explore the underlying cause of MI in UEC, we analyzed the protein expression of hMSH2 and hMLHI in UEC of known MI status. Formalin-fixed, paraffin-embedded archival tissue from 21 UECs was analyzed by immunoperoxidase staining with monodonal antibodies against hMLH1 and hMSH2 protein. Tumors were evaluated for presence of nuclear staining by 3 investigators. Lack of nuclear hMLHI staining was found in 7 of 13 carcinomas with MI, but in none of 8 carcinomas without MI (Fischer's exact, 0.018). Lack of nuclear hMSH2 staining was found in 3 of the MI-positive cases, but none of the MI-negative cases (not statistically significant). Taken together, lack of nuclear staining of either hMLH1 or hMSH2 was found in 9 of 13 cases with MI and in none of 8 cases without MI (Fischer's exact, 0.005). We conclude that MI in sporadic UEC appears to be associated with lack of expression of either hMLH1 or hMSH2, suggesting that inactivation of these genes may be responsible for MI in most MI-positive sporadic UECs.