DNA错配修复与肿瘤的发生及治疗

DNA错配修复系统能够识别和纠正错配的DNA碱基对,确保DNA复制过程的保真性.若是错配修复系统存在缺陷将导致基因突变或基因组不稳定性,最终会导致肿瘤的产生.错配修复系统不仅可以通过修复在DNA复制和重组过程中产生的碱基错配来维持基因组的稳定性,还可以通过识别DNA损伤介导细胞的凋亡,所以细胞的错配修复功能与化疗疗效也有密切相关性.错配修复系统对肿瘤诊断、治疗及预后的重要价值决定了它在肿瘤研究中的重要性.     

DNA错配修复蛋白多功能性的研究进展

DNA错配修复(mismatch repair,MMR)系统是DNA损伤修复的多种途径之一,存在于从细菌、酵母到人体的所有生物体,由一组高保守性酶蛋白组成.其通过校正DNA复制及重组中产生的碱基错配与插入/缺失环,维持所有生物基因组稳定性的功能已研究比较清楚.越来越多的研究还揭示了错配修复蛋白的其他功能:参与调控DNA损伤应答,同源重组,减数分裂的染色体配对和分离,抗体多样性产生及三核苷酸重复序列扩增等过程.本文将对错配修复蛋白多功能性的研究进展作一综述.     

Frequency of mismatch repair deficiency in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) has an ominous prognosis and there are only few treatment options. It is therefore crucial to investigate possible predictive markers that may improve the treatment of this disease. Mismatch repair (MMR) deficiency (d-MMR), meaning MMR protein loss (l-MMR) and/or microsatellite instability (MSI), is predictive of response to immunotherapy, but its frequency has to our knowledge not been elucidated in Scandinavian PDACs. Our aims were to examine the frequency of d-MMR in a Danish cohort of PDACs. We constructed multi-punch tissue microarrays (TMAs) using primary tumor tissue. Immunohistochemistry (IHC) for the DNA MMR proteins MLH1, MSH2, MSH6 and PMS2 was performed, and their expression was evaluated using a scoring system from 0 to 4. If the overall score was between 0–2 or if IHC was inconclusive for technical reasons, IHC on whole-tissue sections and MSI using PCR was performed. A final score of 0, 1–2 or 3–4 defined the tumor as l-MMR, MMR reduced (r-MMR) or MMR proficient. In total, 4/164 (2.4 %), 2/164 (1.2 %) and 3/164 (1.8 %) were l-MMR, r-MMR, or inconclusive based on IHC. MSI testing of these specimens showed that two of the four l-MMR tumors were MSI-high, while the remaining cases were microsatellite stable (MSS). In conclusion, in this study of Danish PDACss, d-MMR was found in a small proportion of the tumors. For these patients, individualized treatment using immunotherapy could be considered.     

DNA mismatch repair protein expression and microsatellite instability in primary mucosal melanomas of the head and neck

AIMS: To examine the expression of DNA mismatch repair (MMR) proteins and the presence of microsatellite instability (MSI) in seven primary mucosal melanomas of the head and neck (MMHN). METHODS AND RESULTS: Haematoxylin and eosin staining and immunohistochemical analysis for routine diagnostic markers and for MMR proteins were performed. Six cases were examined for MSI. Four cases were monomorphous and three cases were pleomorphic type MMHN. Melanocytic markers were positive in all cases. Immunoreactivity for MMR proteins was weak in normal epithelium. The neoplastic tissue in six cases showed positivity for all MMR proteins with different percentages. One case showed weak positivity for hMSH2 and hMSH6 and no immunoreactivity for hMLH1 or hPMS2. Staining intensity was higher in tumour cells than in matched normal mucosa in three cases for hMSH2 and hMLH1 and in two cases for hPMS2. None of the examined cases showed MSI. CONCLUSIONS: Expression of hMSH2 and hMLH1 proteins was up-regulated in three cases, whereas in two cases that of hPMS2 was increased. hMSH6 expression was comparable to that of normal cells in all cases. The percentage of positive neoplastic cells and the intensity of staining seemed to be greater in pleomorphic melanomas. Six cases were MMR-proficient and microsatellite stable.     

Constitutive deficiency in DNA mismatch repair

Mutations in the DNA mismatch repair (MMR) genes are associated with the inheritance of hereditary non-polyposis colorectal cancer, also known as Lynch syndrome, a cancer syndrome with an average age at onset of 44. Individuals presenting with colorectal cancer are diagnosed with Lynch I, whereas individuals who present with extra-colonic tumors (such as endometrial, stomach, etc.) are identified as patients with Lynch syndrome II. Recently, 30 families have been reported with inheritance of biallelic mutations in the MMR genes. Here we summarize the phenotype of individuals with inheritance of homozygous or compound heterozygous mutations in the MMR genes that result in a complete lack of protein or greatly compromised protein function. In contrast to individuals with Lynch syndrome I and II, individuals with no MMR function present with childhood onset of hematological and brain malignancies, whereas residual MMR function can also result in gastrointestinal cancers and an age of onset in the second to fourth decade. Individuals with biallelic MMR mutations often present with café-au-lait spots, regardless of the level of MMR function remaining. Thus, the inheritance of two MMR gene mutations is a separate entity from Lynch I or II or the subtypes Turcot and Muir-Torre.     

Medullary carcinoma of the pancreas in a man with hereditary nonpolyposis colorectal cancer due to a mutation of the MSH2 mismatch repair gene

Pancreatic adenocarcinoma has been reported in kindreds with hereditary nonpolyposis colorectal cancer (HNPCC). Medullary carcinoma of the pancreas is a recently described rare variant of pancreatic adenocarcinoma. We describe a man with colorectal carcinoma who subsequently developed pancreatic medullary carcinoma. The tumor displayed microsatellite instability and loss of expression of the mismatch repair proteins MSH2 and MSH6. Mutational analysis of the mismatch repair genes MLH1 and MSH2 demonstrated a pathogenic nonsense mutation within the MSH2 gene, which is consistent with a diagnosis of HNPCC. This report adds support to an association between HNPCC and pancreatic adenocarcinoma displaying the medullary phenotype, suggesting that medullary features in a pancreatic carcinoma may point toward a genetic cancer predisposition. To our knowledge, this is the first reported case of medullary carcinoma of the pancreas in a patient with HNPCC due to a mutation of the MSH2 gene.     

Extensive gene conversion at the PMS2 DNA mismatch repair locus

Mutations of the PMS2 DNA repair gene predispose to a characteristic range of malignancies, with either childhood onset (when both alleles are mutated) or a partially penetrant adult onset (if heterozygous). These mutations have been difficult to detect, due to interference from a family of pseudogenes located on chromosome 7. One of these, the PMS2CL pseudogene, lies within a 100-kb inverted duplication (inv dup), 700 kb centromeric to PMS2 itself on 7p22. Here, we show that the reference genomic sequences cannot be relied upon to distinguish PMS2 from PMS2CL, because of sequence transfer between the two loci. The 7p22 inv dup occurred prior to the divergence of modern ape species (15 million years ago [Mya]), but has undergone extensive sequence homogenization. This process appears to be ongoing, since there is considerable allelic diversity within the duplicated region, much of it derived from sequence exchange between PMS2 and PMS2CL. This sequence diversity can result in both false-positive and false-negative mutation analysis at this locus. Great caution is still needed in the design and interpretation of PMS2 mutation screens.     

Four new mutations in the DNA mismatch repair gene MLH1 in colorectal cancers with microsatellite instability

Hereditary nonpolyposis colorectal cancer (HNPCC) is frequently associated with inherited mutation in one of four DNA mismatch repair genes. Somatic mutations in the same genes are also found in a subset of sporadic colorectal cancers. A defect in DNA mismatch repair results in an RER (replication error) tumor phenotype. We screened 110 archival and 11 prospectively acquired colorectal cancers for the RER phenotype. A total of 22 cancers were RER-positive. RER-positive tumors were investigated for mutations in the DNA mismatch repair gene MLH1 using single-strand-conformation-polymorphism (SSCP) analysis. We identified four previously undescribed mutations in four different samples. Three mutations were exonic: a point mutation at codon 69 (AGG→AAG [arg→lys]); a single base pair deletion at codon 42/43 (GCAAAATCC→GCAAATCC) leading to a new stop codon downstream; and a point mutation at codon 757 (TAA→TAT) [termination→tyr] which extend the MLH1 peptide by 36 amino acids. The fourth mutation was a 1 base pair insertion six base pairs 5′ to the start of exon 14 (tttgtttt→tttggtttt). The mutations were not seen in the patients' constitutional DNA. The somatic MLH1 mutations identified appear to be causally associated with the RER phenotype. Hum Mutat 12:73, 1998. © 1998 Wiley-Liss, Inc.     

Microsatellite instability and defects in mismatch repair proteins: a new aetiology for Sertoli cell-only syndrome

Microsatellite instability is characteristic of certain types of cancer, and is present in rodents lacking specific DNA mismatch repair proteins. These azoospermic mice exhibit spermatogenic defects similar to some human testicular failure patients. Therefore, we hypothesized that microsatellite instability due to deficiencies in mismatch repair genes might be an unrecognized aetiology of human testicular failure. Because these azoospermic patients are candidates for testicular sperm extraction and ICSI, transmission of mismatch repair defects to the offspring is possible. Seven microsatellite loci were analysed for instability in specimens from 41 testicular failure patients and 20 controls. Blood and testicular DNA were extracted from patient and control specimens, and amplified by PCR targeting seven microsatellite loci. DNA fragment length was analysed with an ABI Prism 310 Genotyping Machine and GeneScan software. Immunohistochemistry was performed on paraffinized testis biopsy sections and cultured testicular fibroblasts from each patient to determine if expression of the mismatch repair proteins hMSH2 and hMLH1 was normal in both somatic and germline cells. Results demonstrate that microsatellite instability and DNA mismatch repair protein defects are present in some azoospermic men, predominantly in Sertoli cell-only patients (P < 0.01 and P < 0.05 respectively). This provides evidence of a previously unrecognized aetiology of testicular failure that may be associated with cancer predisposition.     

DNA mismatch repair deficiency in sporadic colorectal cancer and Lynch syndrome

Poulogiannis G, Frayling I M & Arends M J
(2010) Histopathology 56, 167–179 DNA mismatch repair deficiency in sporadic colorectal cancer and Lynch syndrome DNA mismatch repair (MMR) deficiency is one of the best understood forms of genetic instability in colorectal cancer (CRC), and is characterized by the loss of function of the MMR pathway. Failure to repair replication‐associated errors due to a defective MMR system allows persistence of mismatch mutations all over the genome, but especially in regions of repetitive DNA known as microsatellites, giving rise to the phenomenon of microsatellite instability (MSI). A high frequency of instability at microsatellites (MSI‐H) is the hallmark of the most common form of hereditary susceptibility to CRC, known as Lynch syndrome (LS) (previously known as hereditary non‐polyposis colorectal cancer syndrome), but is also observed in ～15–20% of sporadic colonic cancers (and rarely in rectal cancers). Tumour analysis by both MMR protein immunohistochemistry and DNA testing for MSI is necessary to provide a comprehensive picture of molecular abnormality, for use in conjunction with family history data and other clinicopathological features, in order to distinguish LS from sporadic MMR‐deficient CRC. Identification of the gene targets that become mutated in MMR‐deficient tumours may explain, at least in part, some of the clinical, pathological and biological features of MSI‐H CRCs and holds promise for developing novel therapeutics.     

敲低错配修复基因hMLH1可导致人胚肾细胞株293t的微卫星不稳定

 目的：探讨人胚胎肾细胞株293t中错配修复基因hMLH1的表达量与微卫星稳定性的关系。方法：构建针对hMLH1 基因的shRNA 表达载体,并干扰293t细胞hMLH1的表达,评价敲低hMLH1前后293t细胞微卫星状态的变化。结果：干扰组293t细胞相比对照组hMLH1表达明显敲低,微卫星状态由稳定变为不稳定。结论：293t细胞的hMLH1表达量与其微卫星状态密切相关,敲低hMLH1可导致293t细胞的微卫星不稳定。     

Role of DNA mismatch repair in apoptotic responses to therapeutic agents

Deficiencies in DNA mismatch repair (MMR) have been found in both hereditary cancer (i.e., hereditary nonpolyposis colorectal cancer) and sporadic cancers of various tissues. In addition to its primary roles in the correction of DNA replication errors and suppression of recombination, research in the last 10 years has shown that MMR is involved in many other processes, such as interaction with other DNA repair pathways, cell cycle checkpoint regulation, and apoptosis. Indeed, a cell's MMR status can influence its response to a wide variety of chemotherapeutic agents, such as temozolomide (and many other methylating agents), 6-thioguanine, cisplatin, ionizing radiation, etoposide, and 5-fluorouracil. For this reason, identification of a tumor's MMR deficiency (as indicated by the presence of microsatellite instability) is being utilized more and more as a prognostic indicator in the clinic. Here, we describe the basic mechanisms of MMR and apoptosis and investigate the literature examining the influence of MMR status on the apoptotic response following treatment with various therapeutic agents. Furthermore, using isogenic MMR-deficient (HCT116) and MMR-proficient (HCT116 3-6) cells, we demonstrate that there is no enhanced apoptosis in MMR-proficient cells following treatment with 5-fluoro-2'-deoxyuridine. In fact, apoptosis accounts for only a small portion of the induced cell death response.     

散发性错配修复缺陷大肠癌的临床及分子生物学特征

目的：探讨散发性大肠癌中表现微卫星不稳定性（ｍｉｃｒｏｓａｔｅｌｌｉｔｅ ｉｎｓｔａｂｉｌｉｔｙ，ＭＳＩ）大肠癌的临床病理和分子生物学特征。方法：７９例未接受术前抗肿瘤治疗的大肠癌根治的大肠癌根治术组织收入研究，分析１４个微卫星位点，Ｋ－ｒａｓ基因和ＴＧＦβ－ＲⅡ基因异常。结果：３０／７９例（３８％）大肠癌显示不同程度ＭＳＩ。１１／７９（１４％）显示高度ＭＳＩ（≥３个位点），其中９例（８１％）显示     

Semiquantitative assessment of immunohistochemistry for mismatch repair proteins in Lynch syndrome

Barrow E, Jagger E, Brierley J, Wallace A, Evans G, Hill J & McMahon R
(2010) Histopathology 56, 331–344 Semiquantitative assessment of immunohistochemistry for mismatch repair proteins in Lynch syndrome Aims: To assess semiquantitative immunohistochemistry as used in the diagnosis of Lynch syndrome. Methods and Results: Tumour sections from 51 mutation carriers and 17 controls were stained with antibodies against MLH1, MSH2, MSH6 and PMS2. Intensity of immunoreactivity and percentage positivity were recorded on scales of 0–3 and 0–4, respectively. These scores were multiplied for a score of 0–12 per slide. Receiver–operator characteristic (ROC) curves of staining performance for the identification of mutation carriers were evaluated, and optimum cut‐offs calculated. The area under the MLH1 ROC curve was 0.981 [95% confidence interval (CI) 0.952, 1.000]. The area under the MSH2 ROC curve was 0.899 (95% CI 0.796, 1.000). For MLH1 staining, a score ≤4 gives a sensitivity of 100.0% (95% CI 84.0, 100.0) and a specificity of 91.5% (95% CI 79.6, 97.6) for identifying MLH1 mutation carriers. For MSH2 staining, a score ≤4 gives a sensitivity of 87.5% (95% CI 61.7, 98.4) and specificity of 88.5% (95% CI 76.5, 95.6) for identifying MSH2 mutation carriers. Conclusions: This study supports a semiquantitative slide assessment method. Protein expression may occur in the context of known pathogenic mutations, a potential pitfall in the screening process.     

Prognostic and predictive role of DNA mismatch repair status in stage II-III colorectal cancer: A systematic review and meta-analysis

DNA mismatch repair (MMR) status was considered to be a potential prognostic factor for colorectal cancer (CRC) but with conflicting reports, and varied in terms of TNM stages. Its relationship with prognosis in stage II-III CRC had not yet been systematically established. Therefore, we retrieved eligible studies published through May 2019, and screened out 51 studies that reported survival data (overall survival [OS] and/or disease-free survival [DFS]) in 28 331 CRC patients at stage II-III, totally 16.4% of whom were characterized as deficient MMR (dMMR). Significant associations of dMMR status were observed with longer OS (Hazard Ratio [HR] = 0.74, 95% CI: 0.68-0.82; P < .001), as well as DFS (HR = 0.67, 95% CI: 0.59-0.75, P < .001). However, dMMR patients received no statistically significant benefit from fluoropyrimidine-based treatment for either OS (HR = 0.84, 95%CI: 0.60-1.17; P = .31) or DFS (HR = 0.83, 95%CI: 0.60-1.15; P = .27), compared with that in proficient MMR (pMMR) patients for both OS (HR = 0.55, 95% CI: 0.43-0.71; P < .001) and DFS (HR = 0.60, 95% CI: 0.50-0.73; P < .001). Our analysis indicate that dMMR CRC patients at stage II-III had higher OS and DFS than pMMR ones, and fluoropyrimidine-based chemotherapy could improve survival in pMMR patients rather than dMMR ones.     

Expression of DNA mismatch repair gene MSH2 in cytological material from lung cancer patients

Mismatch repair genes encode for proteins responsible for the correction of bases incorrectly paired in the DNA. Loss of DNA mismatch repair activity has been associated with various cancers including tumors of the lung. In the present study, we have analyzed by immunocytochemistry the expression of MSH2 DNA repair gene in cytological material obtained by fine needle aspiration from a panel of 42 primary lung cancer patients. Specimens included 13 adenocarcinomas, 11 small cell carcinomas and 18 squamous cell carcinomas. Loss of expression or low expression was detected in 6 out of 13 (46%) adenocarcinomas and in 7 out of 18 (39%) of squamous cell carcinomas, although all 11 small cell carcinomas expressed MSH2. Our results suggest that loss of MSH2 expression is frequent in nonsmall cell carcinomas of the lung (P < 0.01, chi2 test). Evaluation of MSH2 expression can be applied for the screening of cytological material from fine needle aspirations from the lung.     

Granular dot-like staining with MLH1 immunohistochemistry is a clone-dependent artefact

Immunohistochemistry (IHC) for DNA mismatch repair proteins MLH1, PMS2, MSH2, and MSH6 is used for microsatellite instability (MSI) screening in colorectal carcinoma (CRC) and endometrial carcinoma (EC). Loss of PMS2, with retained MLH1 staining occurs in germline mutations of PMS2 gene, and is an indication for genetic testing. We report a pitfall of immunohistochemical interpretation in an EC, initially regarded as MLH1-positive and PMS2-negative. Review of the MLH1-IHC (M1-clone) revealed a granular, dot-like, nuclear staining. On repeating the MLH1-IHC with a different clone (ES05-clone), complete negativity was noted, and on molecular testing, MLH1 promotor methylation was detected. The dot-like pattern was therefore adjudged a clone-dependent artefact. On reviewing the archived MLH1-IHC slides, we observed the same dot-like pattern in two CRCs; in both cases the M1-clone had been used. Awareness of this artefact may prevent reporting errors, and unnecessary referrals for germline mutation testing.     

Unusual DNA mismatch repair-deficient tumors in Lynch syndrome: a report of new cases and review of the literature

Immunohistochemical detection of DNA mismatch repair proteins and polymerase chain reaction detection of microsatellite instability have enhanced the recognition of mismatch repair-deficient neoplasms in patients with Lynch syndrome and, consequently, led to the identification of tumors that have not been included in the currently known Lynch syndrome tumor spectrum. Here, we report 4 such unusual tumors. Three of the 4, a peritoneal mesothelioma, a pancreatic acinar cell carcinoma, and a pancreatic well-differentiated neuroendocrine tumor, represented tumor types that, to the best of our knowledge, have not been previously reported in Lynch syndrome. The fourth tumor was an adrenocortical carcinoma, which has rarely been reported previously in Lynch syndrome. Three of our 4 patients carried a pathogenic germ-line mutation in a mismatch repair gene. The unusual tumor in each of the 3 patients showed loss of the mismatch repair protein corresponding to the mutation. The fourth patient did not have mutation information but had a history of colonic and endometrial carcinomas; both lacked MSH2 and MSH6 proteins. Interestingly, none of the 4 unusual tumors revealed microsatellite instability on polymerase chain reaction testing, whereas an appendiceal carcinoma from 1 of the study patients who was tested simultaneously did. The recognition of such tumors expands the repertoire of usable test samples for the workup of high-risk families. As yet, however, there are no data to support the inclusion of these tumors into general screening guidelines for detecting Lynch syndrome, nor are there data to warrant surveillance for these tumors in patients with Lynch syndrome.