结直肠癌中的微卫星不稳定现象

微卫星不稳定DNA标记目前已被广泛用于基因作图的遗传标记、遗传疾病致病基因的连锁分析及基因位点缺失或杂合性缺失的研究.因微卫星不稳定高频型结直肠癌有其特殊的临床特征,因此微卫星不稳定现象对临床有重要的指导意义.对微卫星不稳定现象可能的原因、微卫星不稳定高频型结直肠癌的临床特征及微卫星不稳定现象与抑癌基因的关系作一简单综述.     

结直肠癌中的微卫星不稳定现象

微卫星不稳定DNA标记目前已被广泛用于基因作图的遗传标记、遗传疾病致病基因的连锁分析及基因位点缺失或杂合性缺失的研究。因微卫星不稳定高频型结直肠癌有其特殊的临床特征 ,因此微卫星不稳定现象对临床有重要的指导意义。对微卫星不稳定现象可能的原因、微卫星不稳定高频型结直肠癌的临床特征及微卫星不稳定现象与抑癌基因的关系作一简单综述     

结直肠癌中的微卫生不稳定现象

微卫星不稳定DNA标记目前已被广泛用于基因作图的遗传标记、遗传疾病致病基因的连锁分析及基因位点缺失或杂合性缺失的研究。因微卫星不稳定高频型结直肠癌有其特殊的临床特征，因此微卫星不稳定现象对临床有重要的指导意义。对微卫星不稳定现象可能的原因、微卫星不稳定高频型结直肠癌的临床特征及微卫星不稳定现象与抑癌基因的关系作一简单综述。     

微卫星不稳定性:肿瘤的分子标志

微卫星不稳定性是指基因组中简单重复序列的增加或丢失。许多肿瘤中存在微卫星不稳定性现象。特别是微卫星不稳定性仅在肿瘤细胞中发现,未见于与肿瘤相邻的正常组织,提示与肿瘤的发生发展有关。人类遗传性非息肉样结肠直肠癌(HNPCC)的微卫星不稳定是DNA复制过程中负责碱基对错配修复的基因突变所致,它们的突变使DNA复制的精确性降低,是HNPCC发生的原因,这是继癌基因与抑癌基因之后发现的又一类肿瘤相关基因。     

MMR/MSI在Ⅱ/Ⅲ期结直肠癌辅助化疗

微卫星稳定性作为评价DNA错配修复系统(MMR)的功能标记,可作为Ⅱ期和Ⅲ期结直肠癌患者辅助治疗的风险评估有效工具.全文从错配修复基因/微卫星不稳定性与结直肠癌发生及其在Ⅱ/Ⅲ期结肠癌化疗辅助治疗中的应用方面进行综述.     

结直肠癌分子分型的研究进展

结直肠癌（CRC）分子分型的方法与研究进展主要从基因组学、转录组学和蛋白质组学3个方面展开。依据基因突变 和基因组的细胞遗传学改变进行分型,可以将CRC分为错配修复缺陷导致微卫星不稳定的超突变肿瘤、具有DNA聚合酶epsilon 或Delta 1核酸外切酶结构域突变的超突变肿瘤、具有高频率DNA体细胞拷贝数改变的染色体不稳定性肿瘤等。基于肿瘤的上 皮间质转化、错配修复基因缺陷导致的微卫星不稳定性和细胞增殖相关的高突变频率,可分为MMR缺陷型上皮亚型（A型）、增 殖性上皮亚型（B型）和间充质亚型（C型）。根据基因表达谱的差异分型,可基于临床和组织病理学参数及基因特征、CRC基因表 达谱、全基因组mRNA表达亚型、对表皮生长因子受体靶向药物西妥昔单抗的治疗反应、共识分子等进行CRC亚型分类。基于 生物标志物研究的蛋白质组学分型,可分为亚型A—E等5种不同蛋白质组学亚型。分子分型有助于CRC个体化和精确化的治 疗策略。     

错配修复系统hMutS、hMutL的变异与结直肠癌的相关性研究进展

DNA错配修复系统（mismatch repair system,MMR）是重要的复制后修复机制,修复各种类型的碱基错配和插入/缺失环。hMutS、hMutL是MMR中重要的两个子系统,它们的功能缺陷与多种肿瘤,尤其结直肠癌密切相关。hMutS、hMutL系统关键基因SNPs、启动子甲基化水平的变化,都会直接或间接影响MMR的功能,以及由此引起微卫星不稳定性增加,进而影响结直肠癌的发生、发展和预后。本文主要从hMutS、hMutL系统关键基因的SNPs、启动子甲基化和微卫星不稳定三个方面综述了hMutS、hMutL系统关键基因变异与结直肠癌相关性研究的进展。     

MSS型结直肠癌免疫联合治疗研究进展

近年来,免疫治疗在恶性肿瘤治疗方面取得了很大的进展,已经丰富了多种恶性肿瘤的治疗模式。结直肠癌是全球三大常见的恶性肿瘤之一,免疫检查点抑制剂治疗对DNA错配修复缺陷（dMMR）/高微卫星不稳定性（MSI-H）转移性结直肠癌（mCRC）患者有显著临床获益,但约95%mCRC患者是错配修复完整（pMMR）/微卫星稳定（MSS）型,这类患者对单药免疫治疗的反应差,如何提高该类患者的免疫治疗疗效一直备受关注。本文就MSS型mCRC的免疫联合治疗研究进展进行综述。     

p53与结直肠癌的行为

约50％的结直肠腺癌发生p53基因位点突变,并往往伴有17号染色体的等位基因缺失。p53基因位点突变常导致最具稳定性的蛋白堆积于恶性细胞中,并可用免疫组化方法测定。在正常细胞中,采用标准方法测不到这种蛋白。在结直肠癌时40％～70％的肿瘤存在p53过度表达。新加坡总医院肛肠外科应用Sakai等人的技术,对187例p53位点突变的结直肠肿瘤进行了检查分析。 由Duke＇s C期肿瘤的位点突变率(72％)显著高于Duke＇s A,B,D期可知,淋巴道播散     

结直肠癌的微卫星不稳定与染色体不稳定

结直肠癌中存在微卫星不稳定（MSI）和染色体不稳定（CIN）。微卫星不稳定指微卫星序列中重复单位的获得或丢失，多由于DNA错配修复系统改变所致。染色体不稳定包括整条染色体的获得或缺失，染色体易位、重排等，结直肠癌中常见有1p和8p的删除、17p和18q的杂合性缺失以及20q的扩增。两种类型的结直肠癌具有不同的临床病理特征，在基因表达、疗效和预后等方面的差异性有待进一步研究。     

Genetic and clinical features of human pancreatic ductal adenocarcinomas with widespread microsatellite instability

The incidences of microsatellite instability (MSI) and underlying DNA mismatch repair (MMR) defects in pancreatic carcinogenesis have not been well established. We analyzed 100 sporadic and 3 hereditary pancreatic ductal adenocarcinomas for MSI, and high-frequency MSI (MSI-H) and low-frequency MSI (MSI-L) tumors were further analyzed for frameshift mutations of possible target genes and for promoter methylation and mutation of DNA MMR genes, including hMLH1, hMSH2, hMSH3, and hMSH6 genes. Among the 100 sporadic tumors, 13 (13%) were MSI-H, 13 (13%) were MSI-L, and 74 (74%) were microsatellite stable (MSS) tumors. All of the three hereditary tumors from hereditary nonpolyposis colorectal cancer (HNPCC) patients were MSI-H. MSI-H tumors were significantly associated with poor differentiation and the presence of wild-type K-RAS and p53 genes. Patients with MSI-H tumors had a significantly longer overall survival time than did those with MSI-L or MSS tumors (P = 0.0057). Frameshift mutations of hMSH3, hMLH3, BRCA-2, TGF-beta type II receptor, and BAX genes were detected in MSI-H tumors. Hypermethylation of the hMLH1 promoter was observed in 6 (46%) of the 13 sporadic MSI-H tumors but not in any of the 3 hereditary MSI-H tumors or 13 MSI-L tumors. All of the 3 HNPCC cases had germ-line hMLH1 mutation accompanied by loss of heterogeneity or other mutation in the tumor. Our results suggest that pancreatic carcinomas with MSI-H represent a distinctive oncogenic pathway because they exhibit peculiar clinical, pathological, and molecular characteristics. Our results also suggest the principal involvement of epigenetic or genetic inactivation of the hMLH1 gene in the pathogenesis of pancreatic carcinoma with MSI-H.     

Genetic alterations of sporadic colorectal cancer with microsatellite instability, especially characteristics of primary multiple colorectal cancers

BACKGROUND AND OBJECTIVES: The purpose of this study was to elucidate genetic alterations of sporadic colorectal cancers with Microsatellite instability (MSI). METHODS: One hundred and ten patients with sporadic colorectal cancer were examined. The MSI was assessed using eight microsatellite markers. In addition, mutation analysis was performed for Transforming growth-beta type II receptor (TGF beta R II), bcl-2 associated X protein (BAX), Insulin-like growth factor II receptor (IGF II R), human MutS homolog 3 (hMSH3), human MutS homolog 6 (hMSH6), human MutS homolog 2 (hMSH2), and human MutL homolog 1 (hMLH1) genes. Tumors with three or more positive loci have been determined to be MSI-H (high-frequency MSI), tumors with one or two positive loci were designated as MSI-L (low-frequency MSI) and tumors lacking apparent instability were designated as MSS (microsatellite stable). RESULTS: There were 11 cases with MSI-H (MSI-H group) and 99 cases with MSI-L/MSS (MSI-L/MSS group). The frequency of cases with multiple colorectal cancer in the MSI-H group was significantly higher than that of MSI-L and MSS group (81.8% vs. 34.3%, p = 0.0022). The frequency of cases with multiple cancers increased as the number of locus with microsatellite instability increased. Among 11 tumors with MSI-H, 7 indicated mutation of the TGF beta R II (63.6%), whereas no tumor had a mutation of the TGF beta R II among 20 tumors with MSI-L (p = 0.0001). Regarding the BAX, hMSH3, hMSH6 gene, the same trend was obtained as the TGF beta R II gene (18.2% vs. 0%, p = 0.0487, 36.4% vs. 0%, p = 0.0039, 27.3% vs. 0%, p = 0.0140, respectively). Only two cases indicated a somatic point mutation of the hMSH2 gene. CONCLUSIONS: The multiple occurrence of the colorectal carcinoma may be related to MSI-H as well as the mutation of genes possessing repetitive mononucleotide tracts. Furthermore, we recommend strict follow-up in sporadic colorectal patients that indicate MSI-H.     

Microsatellite instability testing in colorectal carcinoma: choice of markers affects sensitivity of detection of mismatch repair-deficient tumors.

PURPOSE: Microsatellite instability (MSI) is found in 10% to 15% of sporadic colorectal tumors and is usually caused by defects in DNA mismatch repair (MMR). In 1997, a panel of microsatellite markers including mononucleotide and dinucleotide repeats was recommended by a National Cancer Institute workshop on MSI. We investigated the relationship between instability of these markers and MMR protein expression in a cohort of sporadic colorectal cancer patients. EXPERIMENTAL DESIGN: Paraffin sections of normal and tumor tissue from 262 colorectal cancer patients were examined for MSI status by PCR amplification and for MMR protein expression using antibodies against hMLH1, hPMS2, hMSH2, and hMSH6. RESULTS: Twenty-six (10%) of the patients studied had tumors with a high level of MSI (MSI-H). The frequencies of MSI were the same in African-American and Caucasian patients. Each of the MSI-H tumors had mutations in both mononucleotide and dinucleotide repeats and had loss of MMR protein expression, as did two tumors that had low levels of MSI (MSI-L). These two MSI-L tumors exhibited mutations in mononucleotide repeats only, whereas eight of the other nine MSI-L tumors had mutations in just a single dinucleotide repeat. There was not a statistically significant difference in outcomes between patients whose tumors were MMR-positive or MMR-negative, although there was a slight trend toward improved survival among those with MMR-deficient tumors. CONCLUSIONS: The choice of microsatellite markers is important for MSI testing. Examination of mononucleotide repeats is sufficient for detection of tumors with MMR defects, whereas instability only in dinucleotides is characteristic of MSI-L/MMR-positive tumors.     

Biological features of sporadic colorectal carcinoma with high-frequency microsatellite instability: special reference to tumor proliferation and apoptosis

Background We evaluated the relationship between biological behavior and microsatellite instability (MSI) status, with or without p53 status, in sporadic colorectal carcinoma.Methods MSI was analyzed with regard to biological features such as cellular proliferation and apoptotic cell death, in addition to clinicopathological features, in 87 patients with sporadic colorectal carcinoma.Results Fourteen (16.1%) of 87 tumors showed instability at two or more of the five loci examined (high-frequency MSI [MSI-H]). Four demonstrated instability at one locus (low-frequency MSI [MSI-L]), and 69 showed no instability (microsatellite-stable [MSS]). The MSI-H tumors tended to be located in the proximal colon and more often were mucinous carcinoma. The MSI-H tumors also tended to be in patients with multiple colorectal carcinomas and to demonstrate, rarely, an infiltrating growth pattern or venous invasion. The incidence of p53 protein overexpression in the MSI-H tumors was significantly lower than that in the MSI-L/MSS tumors (21% vs 54%). There was no significant difference in the proliferating-cell nuclear antigen (PCNA) labeling index (PI) or apoptotic index (AI) between the MSI-H and MSI-L/MSS tumors. The AI in the MSI-H tumors with p53 overexpression was significantly lower than that in the MSI-H tumors without p53 overexpression, and was also significantly lower than that in the MSI-L/MSS tumors with p53 overexpression. In the MSI-H tumors with p53 overexpression, no expression of BAX protein was found, and there was high expression of bcl-2 protein, resulting in a low BAX/bcl-2 ratio.Conclusion In sporadic colorectal carcinoma, an MSI-H tumor with p53 protein overexpression may display aggressive biological features.     

Microsatellite instability of each tumor in sporadic synchronous multiple colorectal cancers

The purpose of this study was to elucidate microsatellite instability (MSI) and p53 expression for each tumor in cases with sporadic synchronous multiple colorectal cancers. Twenty-nine patients with sporadic synchronous multiple colorectal cancer were examined. There were sixty-five tumors, all of which indicated adenocarcinoma histopathologically. The MSI was assessed using six microsatellite markers (BAT26, BAT40, D2S136, D5S346, D11S922, D17S250). Tumors with two or more positive loci were determined to be MSI-H (high-frequency MSI), tumors with one positive locus were designated as MSI-L (low-frequency MSI) and tumors lacking apparent instability were designated as MSS (microsatellite stable). In addition, overexpression of p53 protein was examined using immunohistochemical (IHC) methods for each tumor. The DO-7 monoclonal antibody was used in the IHC assessments. The following results were obtained: i) there were nine patients who indicated MSI-H at the first tumor (1-H group) and 20 patients who had MSI-L or MSS at the first tumor (1-LS group). ii) The ratio of cases that indicated MSI-H at the second tumor and beyond in the 1-H group was 88.9% (8/9), which was significantly higher than that of the 1-LS group (30.0%, 6/20) (p=0.0021). iii) The frequency of cases with the right-sided colon in the 1-H group (61.9%) was significantly higher than that of the 1-LS group (27.3%) (p=0.0073). In addition, a significant difference was noted in terms of the ratio of cases with poorly differentiated adenocarcinoma or mucinous carcinoma between the two groups [1-H group (19.0%) vs 1-LS group (0%), p=0.0028]. Furthermore, no distinct relationship between MSI status and p53 overexpression was obtained. In conclusion, we think that sporadic synchronous multiple colorectal cancers should be divided into two types; one type that indicates multiple occurrence of MSI-H consecutive tumors and another type that shows multiple occurrence irrespective of MSI.     

Sporadic colorectal adenocarcinomas with high-frequency microsatellite instability

BACKGROUND: Widespread microsatellite instability (MSI) occurs in nearly 15% of sporadic colorectal cancers. Large bowel carcinomas with high-frequency MSI (MSI-H) (instability at > or = 30% of microsatellite loci) are believed to display distinctive pathologic features and to behave less aggressively than microsatellite-stable (MSS) tumors and carcinomas with low-frequency MSI (MSI-L) (instability at < 30% of microsatellite loci). The aim of the current study was to accurately define the clinicopathologic and biologic features of MSI-H sporadic colorectal carcinomas. METHODS: MSI status was evaluated in 216 large bowel adenocarcinomas using polymerase chain reaction (PCR) and 6 microsatellite markers. Tumors that showed instability with at least two microsatellite markers were classified as MSI-H, whereas the other tumors were classified as MSI-L (instability at one locus) or MSS (no instability). Expression of p53, hMLH1, and hMSH2 gene products was determined by immunohistochemistry, and DNA ploidy pattern was determined by flow cytometry. The prognostic significance of MSI status was assessed by univariate and multivariate survival analyses. RESULTS: The significantly different pathologic features of MSI-H carcinomas were proximal location; large size; mucinous and medullary histotype; poor differentiation; expanding pattern of growth; more frequent Crohn-like conspicuous lymphoid reaction; and low incidence of extramural vein invasion. Most MSI-H tumors were DNA diploid (33 of 40 tumors; 82.5%) and p53 negative (34 of 44 tumors; 77.3%). Conversely, DNA aneuploidy and p53 overexpression were observed in 82.3% (130 of 158 tumors; P < 0.0001) and 54.1% (93 of 172 tumors; P = 0.0002) of MSI-L/MSS tumors, respectively. Loss of hMLH1 or hMSH2 expression was detected in a high fraction of MSI-H carcinomas (86. 0%). Patients with MSI-H tumors showed a better clinical outcome than patients with MSI-L/MSS tumors (P = 0.0017). Furthermore, in multivariate analysis that included conventional clinicopathologic parameters, MSI status, and p53 expression as covariates, MSI status was a significant independent prognostic indicator of disease specific survival. CONCLUSIONS: Assessment of MSI status is an essential step in the genetic characterization of large bowel carcinomas and identifies a subset of tumors with distinct clinical, pathologic, and biologic features.     

Frequent Bax frameshift mutations in gastric cancer with high but not low microsatellite instability

Widespread microsatellite instability (MSI) due to the defective DNA mismatch repair underlies the pathogenesis of the majority of hereditary non-polyposis colorectal cancer and a subset of various sporadic malignant tumors. Using 5 microsatellite markers and the criteria of MSI proposed by the National Cancer Institute (NCI) workshop, we analyzed 205 gastric adenocarcinomas for MSI. Based on the number of markers showing instability per tumor, the tumors were divided into three groups; those with two or more of the five markers displaying instability (high MSI, MSI-H), those with one of five markers displaying instability (low MSI, MSI-L), and those with no instability (microsatellite stable, MSS). Among 205 tumors, 30 (15%) were MSI-H, 15 (7%) were MSI-L, and 160 (78%) were MSS. All of the 30 MSI-H tumors demonstrated instability at BAT26, a sensitive marker for the widespread MSI, while none of the 15 MSI-L tumors did. MSI-H tumors were significantly associated with distal location and well or moderate differentiation, but MSI-L tumors were indistinguishable from MSS tumors. Bax frameshift mutations were detected in 60% of the 30 MSI-H tumors, while not in any of the 15 MSI-L tumors. These results suggest that microsatellite analysis using the criteria proposed by the NCI workshop may be appropriate for gastric cancers because it unveils real differences in genotype and phenotype.     

Thymidylate synthase expression in colon carcinomas with microsatellite instability.

PURPOSE: Colon cancer cells with high-frequency microsatellite instability (MSI-H) display resistance to 5-fluorouracil (5-FU) that can be reversed by restoring DNA mismatch repair (MMR) proficiency. Given that thymidylate synthase (TS) is inhibited by 5-FU, we studied the relationship between MSI and TS expression, and the prognostic effect of these and other markers (i.e., p53 and 17p allelic imbalance). EXPERIMENTAL DESIGN: Dukes' stage B2 and C colon carcinomas (n = 320) from participants in 5-FU-based adjuvant therapy trials were analyzed for MSI and 17p allelic imbalance. Expression of MMR (hMLH1, hMSH2), TS, and p53 proteins were analyzed by immunohistochemistry. Correlations between markers and associations with overall survival were determined. RESULTS: Of 320 cancers studied, 60 (19%) were MSI-H. TS expression variables were similar in MSI-H and microsatellite stable/low-frequency MSI (MSS/MSI-L) cancers, and unrelated to MMR proteins. MSI-H tumors had lower stage (P = 0.0007), fewer metastatic lymph nodes (P = 0.004), and improved overall survival (P = 0.01). Loss of MMR proteins was also associated with better overall survival (P = 0.006). None of the TS variables were prognostic. Histologic grade (P = 0.0008) and nodal status (P = 0.0002) were associated with overall survival, in contrast to 17p allelic imbalance or p53. Only MSI status or loss of MMR proteins, histologic grade, and tumor stage were independent markers for overall survival. CONCLUSIONS: MSI-H tumors show earlier stage at presentation and better stage-adjusted survival rates. MSI status and TS expression were unrelated and TS was not prognostic, suggesting that TS levels cannot explain therapeutic resistance to 5-FU reported in MSI-H colon cancers.     

Loci for efficient detection of microsatellite instability in hereditary non-polyposis colorectal cancer

Most hereditary non-polyposis colorectal cancer (HNPCC) is due to germline mutations in DNA mismatch repair genes. Tumors arising as a result of these mutations display instability in microsatellites, which are short tandem repeats of DNA that are distributed throughout the genome. Although a subset of sporadic colorectal carcinomas also have microsatellite instability (MSI), the phenotype is a useful screening test in identifying patients with HNPCC caused by mutations in mismatch repair (MMR) genes. Studies have shown that some microsatellite markers are more efficient than others in identifying tumors with MSI. Furthermore, the frequency of instability can be assessed by categorizing patients into high (MSI-H, >/= 30-40% positive markers), low (MSI-L), and microsatellite stable (MSS) groups. Using a panel of 28 microsatellite markers, tumor and normal DNA from 10 HNPCC patients was used to identify the five most efficient markers for detecting MSI (BAT26, D2S123, FGA, D18S35, and TP53-DI). Each of the five markers detected MSI in 80-100% of the cases examined. We then expanded the sample size to 17 tumors from HNPCC patients. Each case had evidence for a mutation in either hMSH2 or hMLH1. We compared the efficiency of our panel of five best markers with another panel of five markers (BAT25, BAT26, D2S123, D17S250, and D5S346) identified as being efficient markers for detection of MSI at a recent NCI workshop. Our five selected markers were more efficient (85% vs. 79%) in detecting MSI. However, using either panel, 100% of the cases fell into the MSI-H category and the probability of misclassifying an MSI-H case as MSI-L is very low (0.002-0.008). We also examined four cases meeting the Amsterdam criteria for HNPCC, but with no evidence for mutation in either the hMSH2 or hMLH1 gene. With our panel, three were classified as MSI-H, while only two were classified as such with the NCI reference panel. The probability of misclassifying an MSI-L case as an MSI-H, using a panel of five markers is high (0.263).