Mutations in mismatch repair genes are involved in the neoplastic transformation of human breast epithelial cells

Mismatch repair (MMR) genes play a fundamental role in the correction of replication errors, leading to cancer development when mutated. In order to test the hypothesis that the MMR system was compromised in the initiation and progression of breast cancer, we used an in vitro-in vivo model for analyzing the mRNA levels of the MMR genes hMLH1, hMSH2, hPMS1, hPMS2 and hMSH6. MCF-10F, immortalized human breast epithelial cells, BP-1, benz(a)pyrene (BP)-transformed cells, BP-1Tras, a tumorigenic cell line derived from c-Ha-ras transfected BP-1 cells, and seven tumor-derived cell (TDC) lines obtained from BP-1Tras-induced tumors were tested. hMLH1, hMSH2, hPMS1, hPMS2 and hMSH6 mRNA expression were similar in MCF-10F, BP-1, and BP-1Tras cells; hMLH1 and hPMS1 were also equally expressed in TDC. An exception was hPMS2, whose mRNA level was decreased from BP-1Tras and from all TDC. hMSH2 and hMSH6 mRNA were also decreased in most TDC. DNA sequencing revealed mutations in hMSH2, which in MCF-10F cells had one frameshift mutation and one polymorphism in exons 12 and 13, respectively. Two mutations in exon 13, and three in exon 14 were detected in BP-1 and TDC, which had, in addition, three missense mutations in exon 14. hPMS2 had four mutations in exon 10 in MCF-10F cells, and BP-1 cells had three missense mutations in exon 9, four missense and one non-sense mutations in exon 10, codon 675 (Arg x Stop signal). BP-1Tras and TDC shared three missense mutations with BP-1 cells, and in addition had seven missense and one non-sense mutations in exon 9. hMSH6 had three frameshift and three missense mutations in exons 4 and 5 in BP-1, and 12 mutations in the same exons in BP-1Tras and TDC, which had three additional mutations in exons 4 and 5. This is the first report demonstrating mutations of MMR genes during the neoplastic transformation of human breast epithelial cells.     

Expression of the MutL homologue hMLH3 in human cells and its role in DNA mismatch repair

The human mismatch repair (MMR) proteins hMLH1 and hPMS2 function in MMR as a heterodimer. Cells lacking either protein have a strong mutator phenotype and display microsatellite instability, yet mutations in the hMLH1 gene account for approximately 50% of hereditary nonpolyposis colon cancer families, whereas hPMS2 mutations are substantially less frequent and less penetrant. Similarly, in the mouse model, Mlh1-/- animals are highly cancer prone and present with gastrointestinal tumors at an early age, whereas Pms2-/- mice succumb to cancer much later in life and do not present with gastrointestinal tumors. This evidence suggested that MLH1 might functionally interact with another MutL homologue, which compensates, at least in part, for a deficiency in PMS2. Sterility of Mlh1-/-, Pms2-/-, and Mlh3-/- mice implicated the Mlh1/Pms2 and Mlh1/Mlh3 heterodimers in meiotic recombination. We now show that the hMLH1/hMLH3 heterodimer, hMutLgamma, can also assist in the repair of base-base mismatches and single extrahelical nucleotides in vitro. Analysis of hMLH3 expression in colon cancer cell lines indicated that the protein levels vary substantially and independently of hMLH1. If hMLH3 participates in MMR in vivo, its partial redundancy with hPMS2, coupled with the fluctuating expression levels of hMLH3, may help explain the low penetrance of hPMS2 mutations in hereditary nonpolyposis colon cancer families.     

Mismatch repair deficiency in hematological malignancies with microsatellite instability

Mutations in human mismatch repair (MMR) genes are the genetic basis for certain types of solid tumors displaying microsatellite instability (MSI). MSI has also been observed in hematological malignancies, but whether these hematological malignancies are associated with MMR deficiency is still unclear. Using both biochemical and genetic approaches, this study analysed MMR proficiency in 11 cell lines derived from patients with hematological malignancies and demonstrated that six out of seven hematological cancer cell lines with MSI were defective in strand-specific MMR. In vitro complementation experiments, using characterized MMR mutant extracts or purified proteins, showed that these hematological cancer cells were defective in either hMutS(alpha) (a heterodimer of hMSH2 and hMSH6) or hMutL(alpha) (a heterodimer of hMLH1 and hPMS2). Furthermore, cell lines deficient in hMutS(alpha) showed large deletions or point mutations in hMSH2, while those deficient in hMutL(alpha) exhibited point mutations in hMLH1 or a lack of expression of hPMS2. From these results, we conclude that, as in solid tumors, hematological malignancies with MSI are also associated with MMR deficiency, and that the cause of MMR deficiency in these cell lines is due to a defective MutS(alpha) or MutL(alpha). We also report here, for the first time, that an MSI-positive cell line derived from Burkitt's lymphoma is proficient in MMR.     

Human colon cancer cells surviving high doses of cisplatin or oxaliplatin in vitro are not defective in DNA mismatch repair proteins

PURPOSE: Alterations in the DNA mismatch repair (MMR) proteins have been associated with an increased resistance of many cancer cell lines to cisplatin. The aim of this work was to investigate whether defects in DNA MMR proteins are involved in the survival of human colorectal cancer cells in the presence of high concentrations of cisplatin and oxaliplatin, a diaminocyclohexane (DACH) platinum compound whose adducts are not recognized by the MMR system. METHODS: Six unselected human colon cancer cell lines (HT29, HCT15, HCT116, Caco2, SW480 and SW620) were treated with a single 3-h exposure to cisplatin or oxaliplatin at suprapharmacological concentrations, ranging from 50 to 200 microg/ml. The microsatellite stability and the expression of MMR proteins in the parental cell lines and in the drug-selected subpopulations were studied. RESULTS: Most cells underwent apoptosis in the days following the cisplatin or oxaliplatin treatment, but some colonies expanded 3 to 4 weeks after, suggesting the presence of innately resistant cells in the six parental cell lines. Microsatellite instability (MIN), which reflects genetic defects in the DNA MMR system, was detected only in the HCT116 parental cell line and its drug-selected counterparts, due to a known mutation in the hMLH1 gene. No acquired MIN was observed in the other cisplatin-selected sublines derived from the HT29, HCT15, Caco2, SW480 or SW620 parental cells. In the same way, Western blot analysis showed that expression of the DNA MMR proteins hMLH1, hPMS1, hPMS2, hMSH2 and hMSH6 did not differ between the parental and the drug-surviving cells. CONCLUSIONS: These results indicate that high-level resistance of human colon cancer cells to high doses of cisplatin and oxaliplatin does not seem to be related to acquired defects in the DNA MMR proteins.     

Conditional nuclear localization of hMLH3 suggests a minor activity in mismatch repair and supports its role as a low-risk gene in HNPCC

DNA mismatch repair (MMR) mechanism contributes to the maintenance of genomic stability. Loss of MMR function predisposes to a mutator cell phenotype, microsatellite instability (MSI) and cancer, especially hereditary non-polyposis colorectal cancer (HNPCC). To date, five MMR genes, hMSH2, hMSH6, hMLH1, hPMS2, and hMLH3 are associated with HNPCC. Although, hMLH3 is suggested to be causative in HNPCC, its relevance to MMR needs to be confirmed to reliably assess significance of the inherited sequence variations in it. Recently, a human heterodimer hMLH1/hMLH3 (hMutLgamma) was shown to be able to assist hMLH1/hPMS2 (hMutLalpha) in the repair of mismatches in vitro. To repair mismatches in vivo, hMLH3 ought to localize in the nucleus. Our immunofluorescence analyses indicated that when all the three MutL homologues are natively expressed in human cells, endogenous hMLH1 and hPMS2 localize in the nucleus, whereas hMLH3 stays in the cytoplasm. Absence of hPMS2 and co-expression of hMLH3 with hMLH1 results in its partial nuclear localization. Our results are clinically relevant since they show that in the nuclear localization hMLH3 is dependent on hMLH1 and competitive with hPMS2. The continuous nuclear localization of hMLH1 and hPMS2 suggests that in vivo, hPMS2 (hMutLalpha) has a major activity in MMR. In absence of hPMS2, hMLH3 (hMutLgamma) is located in the nucleus, suggesting a conditional activity in MMR and supporting its role as a low-risk gene in HNPCC.     

Characterization of the nuclear import of human MutLalpha

DNA mismatch repair (MMR) is essential for the maintenance of replication fidelity. Its major task is to recognize mismatches as well as insertion/deletion loops of newly synthesized DNA strands. Although different players of human MMR have been identified, the regulation of essential steps of MMR is poorly understood. Because MMR is initiated in the nucleus, nuclear import might be a mechanism to regulate MMR. Nuclear targeting is accomplished by conserved signal sequences called nuclear localization signals (NLS), which represent clusters of positively charged amino acids (aa). hMLH1 contains two clusters of positively charged amino acids, which are candidate NLS sequences (aa 469-472 and 496-499), while hPMS2 contains one (aa 574-580). To study the effect of these clusters on nuclear import, NLS mutants of hMLH1 and hPMS2 were generated and expressed in 293T cells. The subcellular localization of the mutant constructs was monitored by confocal laser microscopy. We demonstrated that missense mutations of two signal sequences, one in hMLH1 and one in hPMS2, lead to impaired nuclear import, which was especially prominent for mutants of the hMLH1 residues K471 and R472; and hPMS2 residues K577 and R578.     

The role of mismatch repair in small-cell lung cancer cells

The role of mismatch repair (MMR) in small-cell lung cancer (SCLC) is controversial, as the phenotype of a MMR-deficiency, microsatellite instability (MSI), has been reported to range from 0 to 76%. We studied the MMR pathway in a panel of 21 SCLC cell lines and observed a highly heterogeneous pattern of MMR gene expression. A significant correlation between the mRNA and protein levels was found. We demonstrate that low hMLH1 gene expression was not linked to promoter CpG methylation. One cell line (86MI) was found to be deficient in MMR and exhibited resistance to the alkylating agent MNNG. Surprisingly, MSI was not detected in 86MI and it appears to express all the major MMR components hMSH2, hMSH6, hMLH1, hPMS2, hMSH3, hMLH3, MBD4 (MED1) and hExo1. These data are consistent with at least two possibilities: (1) A missense mutation in one of the MMR genes, which dissociates MSI from drug resistance, or (2) inactivation of a second pathway that leads to MMR-deficiency and MNNG resistance, but induces negligible levels of MSI. We conclude that MMR deficiency is largely not associated with the pathogenesis of SCLC.     

Mutational analysis of transforming growth factor beta receptor type II and DNA mismatch repair genes in sporadic endometrial carcinomas with microsatellite instability

To clarify how microsatellite instability (MI) is involved in carcinogenesis of sporadic endometrial carcinoma, we examined mutations of the transforming growth factor beta receptor type II (TGF beta RII) gene in 32 patients with MI-positive sporadic endometrial carcinoma. Moreover, mutations of 4 DNA mismatch repair (MMR) genes (hPMS1, hPMS2, hMLH1, hMSH2), which are considered to cause MI, were investigated as well. With respect to the TGF beta RII gene, mutations in the 10-bp polyadenine repeat sequence were observed in 7 of 29 informative cases (24%). Concerning MMR genes, a T to C point mutation at the -6 intronic splice acceptor site of exon 13 of hMSH2 was detected in 43% (6/14). However, there was no mutation in any exon of these 4 MMR genes. These results suggest that there is a carcinogenic mechanism via mutation of the TGF beta RII gene in some cases of MI-positive sporadic endometrial carcinoma. It seems unlikely that the unknown MMR genes are responsible for MI. The implication of the mutation at the intronic splice acceptor site in hMSH2 remains to be clarified.     

Spontaneous mutation rates of tumorigenic and nontumorigenic Chinese hamster embryo fibroblast cell lines

The genomic stability of a series of nontumorigenic, tumorigenic, and tumor-derived Chinese hamster embryo fibroblastic (CHEF) cell lines was compared by examining their rates of spontaneous mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus, using thioguanine resistance for selection of mutants. The spontaneous mutation rates were 1.1 x 10(-6) mutations/cell/generation in the non-tumor-forming CHEF/18 cell line and 4.9 x 10(-6) in the tumorigenic CHEF/16 cells. Three tumorigenic and tumor-derived CHEF cell lines derived from CHEF/18 (J132 3-2 T3L, focus 2, focus 3) and two lines (16-2 Tuk 4 and 204 Bu50 Tuk 2) derived from CHEF/16 were chosen on the basis of their karyotypes, which demonstrated a considerable level of chromosomal rearrangement. Mutation rates of four of these five lines ranged from 1.2 x 10(-6) to 8.9 x 10(-6) mutations per cell per generation. Only the fifth line, 16-2 Tuk 4, showed a significantly elevated rate of mutation as compared with the nontumorigenic CHEF/18 cell line. Thus, we have found no simple correlation between spontaneous mutation rate and the malignant phenotype, and we conclude that mutation rate per se is not a sensitive index of malignancy. In addition, we have compared three methods of calculating mutation rate and find that they rank the cell lines in the same order, but each stresses a different aspect of the distribution and therefore produces different estimates of the mutation rate.     

Microsatellite Instability Assessment by Immunohistochemistry in Acute Myeloid Leukemia: A Reappraisal and Review of the Literature

BackgroundMicrosatellite instability (MSI) is caused by defects in DNA mismatch repair (MMR) components. Inactivation of any MMR gene(s), including hMLH1, hMSH2, hMSH6, and hPMS2, can result in MSI. Immunohistochemistry (IHC) is a sensitive and specific screening tool for MSI that can detect loss of expression of one or more MMR components. Of the four MMR markers, hMLH1 and hMSH2 are considered most informative of MSI status. There has been renewed interest in MSI status in view of its favorable association with response to immune checkpoint inhibitors in some cancers. MMR expression patterns in acute myeloid leukemia (AML) have not been evaluated systematically.MethodsWe used clinically-validated IHC assays to assess the expression of hMLH1, hMSH2, hMSH6, and/or hPMS2 in formalin-fixed paraffin-embedded tissue sections of bone marrow core biopsies from patients diagnosed with AML. Mutation profiling was performed using next-generation sequencing to assess for mutations in MMR genes.ResultsThe study group included 236 patients with AML, including a cohort treated on a clinical trial of azacitidine and nivolumab (NCT02397720). In addition, hMSH6, and/or hPMS2 expression was assessed in 99 AML patients with diploid karyotype. All patients, except two, had retained expression of all MMR markers assessed: One patient from the azacytidine+nivolumab group had zonal patchy loss of staining of hMLH1 and, to a lesser extent, a similar staining pattern of hMSH2; and one patient from the AML with diploid karyotype group had loss of hMSH2 but retained expression of hMLH1, hMSH6 and hPMS2. In addition, a retrospective analysis on a separate cohort of 139 patients with primary AML, on which next generation sequencing profiling was performed, identified 14 cases with alterations in MMR genes.Conclusion and remarksMMR loss is a rare event in AML, thus does not appear to underlie response patterns to anti-PD1 therapy.     

Persistent mismatch repair deficiency following targeted correction of hMLH1

The use of gene therapy to correct mutated or lost gene function for the treatment of human cancers has been an active, yet problematic area of biomedical research. Many technical difficulties, including efficient tissue-specific delivery, integration site specificity and general toxicity, are being addressed. Little is known, however, about the genetic and phenotypic stability that accompanies a successful gene-specific targeting event in a cancer cell. This question was addressed following the creation of a colon cancer cell line in which a mutated hMLH1 gene was corrected via targeted homologous recombination. This correction resulted in the expression of wild-type hMLH1 protein, restoration of the hPMS2 protein and mismatch repair (MMR) proficiency. One of two hMLH1-corrected clones, however, was found to retain defects in MMR activity. These cells continued to express the corrected hMLH1 protein, but had lost expression of another MMR protein, hMSH6. DNA sequence analysis of the hMSH6 gene revealed biallelic expansions of a cytosine repeat region in exon 5 that result in frameshifts leading to premature stop codons. These findings suggest that, similar to acquired drug resistance, the presence of genetically heterogeneous cancer cell populations or acquisition of compensatory mutations can result in 'resistance' to gene replacement therapy.     

Infrequent widespread microsatellite instability in hepatocellular carcinomas

Widespread or high-frequency microsatellite instability (MSI) due to the defective DNA mismatch repair (MMR) occurs in the majority of hereditary non-polyposis colorectal cancer and a subset of sporadic malignant tumors. The incidence of MSI and underlying DNA MMR defects have been well characterized in gastrointestinal carcinogenesis, but not in hepatocarcinogenesis. To address the issue, we analyzed 55 Japanese hepatocellular carcinomas using several indicators of DNA MMR defects, such as microsatellite analysis, loss of heterozygosity (LOH) and mutation analysis of MMR genes, methylation of hMLH1 promoter, and frameshift mutations of mononucleotide repeat sequences within possible target genes. Mutation of beta2-microglobulin gene, which is presumably involved in MSI-positive tumor cell escape from immune surveillance was also examined. Some of these analyses were also carried out in 9 human liver cancer cell lines. None of the 3 quasi-monomorphic mononucleotide markers sensitive for MSI, BAT26, BAT25, and BAT34C4 presented shortened unstable alleles in any of the carcinoma, cirrhosis, chronic hepatitis tissues, or cell lines. LOH at MMR genes was infrequent (4.4 approximately 7.1%), and no mutations were detected. Neither hMLH1 hypermethylation nor frameshift mutation in the target genes was detected. No mutations were found in beta2-microglobulin. Widespread MSI due to the defective DNA MMR appears to play little if any part in Japanese hepatocarcinogenesis.     

Differential radiosensitization in DNA mismatch repair-proficient and -deficient human colon cancer xenografts with 5-iodo-2-pyrimidinone-2'-deoxyribose.

PURPOSE: 5-iodo-2-pyrimidinone-2'-deoxyribose (IPdR) is a pyrimidinone nucleoside prodrug of 5-iododeoxyuridine (IUdR) under investigation as an orally administered radiosensitizer. We previously reported that the mismatch repair (MMR) proteins (both hMSH2 and hMLH1) impact on the extent (percentage) of IUdR-DNA incorporation and subsequent in vitro IUdR-mediated radiosensitization in human tumor cell lines. In this study, we used oral IPdR to assess in vivo radiosensitization in MMR-proficient (MMR+) and -deficient (MMR-) human colon cancer xenografts. EXPERIMENTAL DESIGN: We tested whether oral IPdR treatment (1 g/kg/d for 14 days) can result in differential IUdR incorporation in tumor cell DNA and subsequent radiosensitization after a short course (every day for 4 days) of fractionated radiation therapy, by using athymic nude mice with an isogenic pair of human colon cancer xenografts, HCT116 (MMR-, hMLH1-) and HCT116/3-6 (MMR+, hMLH1+). A tumor regrowth assay was used to assess radiosensitization. Systemic toxicity was assessed by daily body weights and by percentage of IUdR-DNA incorporation in normal bone marrow and intestine. RESULTS: After a 14-day once-daily IPdR treatment by gastric gavage, significantly higher IUdR-DNA incorporation was found in HCT116 (MMR-) tumor xenografts compared with HCT116/3-6 (MMR+) tumor xenografts. Using a tumor regrowth assay after the 14-day drug treatment and a 4-day radiation therapy course (days 11-14 of IPdR), we found substantial radiosensitization in both HCT116 and HCT116/3-6 tumor xenografts. However, the sensitizer enhancement ratio (SER) was substantially higher in HCT116 (MMR-) tumor xenografts (1.48 at 2 Gy per fraction, 1.41 at 4 Gy per fraction), compared with HCT116/3-6 (MMR+) tumor xenografts (1.21 at 2 Gy per fraction, 1.20 at 4 Gy per fraction). No substantial systemic toxicity was found in the treatment groups. CONCLUSIONS: These results suggest that IPdR-mediated radiosensitization can be an effective in vivo approach to treat "drug-resistant" MMR-deficient tumors as well as MMR-proficient tumors.     

Somatic mutation of hPMS2 as a possible cause of sporadic human colon cancer with microsatellite instability

Inactivation of DNA-mismatch repair underlies the genesis of microsatellite unstable (MSI) colon cancers. hPMS2 is one of several genes encoding components of the DNA-mismatch repair complex, and germline hPMS2 mutations have been found in a few kindreds with hereditary nonpolyposis colorectal carcinoma (HNPCC), in whom hereditary MSI colon cancers develop. However, mice bearing null hPMS2 genes do not develop colon cancers and hPMS2 mutations in sporadic human colon cancers have not been described. Here we report that in Vaco481 colon cancer the hPMS2 gene is inactivated by somatic mutations of both hPMS2 alleles. The cell line derived from this tumor is functionally deficient in DNA mismatch repair. This deficiency can be biochemically complemented by addition of a purified hMLH1-hPMS2 (hMutLalpha) complex. The hPMS2 deficient Vaco481 cancer cell line demonstrates microsatellite instability, an elevated HPRT gene mutation rate, and resistance to the cytotoxicity of the alkylator MNNG. We conclude that somatic inactivation of hPMS2 can play a role in development of sporadic MSI colon cancer expressing the full range of cancer phenotypes associated with inactivation of the mismatch repair system.     

Microsatellite instability in colorectal-cancer patients with suspected genetic predisposition

Hereditary non-polyposis colorectal cancer (HNPCC) is a dominantly inherited syndrome linked to DNA-mismatch-repair (MMR) gene defects, which also account for microsatellite instability (MSI) in tumour tissues. Diagnosis is based mainly on family history, according to widely accepted criteria (Amsterdam Criteria: AC). Aim of this work was to assess MSI in colorectal-cancer patients with suspected genetic predisposition, and to verify whether MSI represents a tool to manage MMR gene (hMSH2 and hMLH1) mutation analysis. We investigated 13 microsatellites (including the 5 NCI/ICG-HNPCC markers) in 45 patients with suspected hereditary predisposition (including 16 subjects from HNPCC families fulfilling the AC). We found MSI-H (high frequency of instability, i.e., in > or =30% of the markers) in 85% of the HNPCC patients and in 16% of the non-HNPCC subjects. The 5 NCI/ICG-HNPCC microsatellites proved to be the most effective in detecting MSI, being mononucleotide repeats the most unstable markers. We investigated the association between hMSH2- and hMLH1 gene mutations and MSI. Our results indicate that AC are highly predictive both of tumour instability and of MMR-gene mutations. Therefore, as the most likely mutation carriers, HNPCC subjects might be directly analyzed for gene mutations, while to test for MSI in selected non-HNPCC patients and to further investigate MMR genes in MSI-H cases, appears to be a cost-effective way to identify subjects, other than those from kindred fulfilling AC, who might benefit from genetic testing.     

Overexpression of the DNA mismatch repair factor, PMS2, confers hypermutability and DNA damage tolerance

Inherited defects in genes associated with DNA mismatch repair (MMR) have been linked to familial colorectal cancer. Cells deficient in MMR are genetically unstable and demonstrate a tolerance phenotype in response to certain classes of DNA damage. Some sporadic human cancers also show abnormalities in MMR gene function, typically due to diminished expression of one of the MutL homologs, MLH1. Here, we report that overexpression of the MutL homolog, human PMS2, can also cause a disruption of the MMR pathway in mammalian cells, resulting in hypermutability and DNA damage tolerance. A mouse fibroblast cell line carrying a recoverable lambda phage shuttle vector for mutation detection was transfected with either a vector designed to express hPMS2 or with an empty vector control. Cells overexpressing hPMS2 were found to have elevated spontaneous mutation frequencies at the cII reporter gene locus. They also showed an increase in the level of mutations induced by the alkylating agent, methynitrosourea (MNU). Clonogenic survival assays demonstrated increased survival of the PMS2-overexpressing cells following exposure to MNU, consistent with the induction of a damage tolerance phenotype. Similar results were seen in cells expressing a mutant PMS2 gene, containing a premature stop codon at position 134 and representing a variant found in an individual with familial colon cancer. These results show that dysregulation of PMS2 gene expression can disrupt MMR function in mammalian cells and establish an additional carcinogenic mechanism by which cells can develop genetic instability and acquire resistance to cytotoxic cancer therapies.     

食管癌和贲门癌组织中错配修复基因表达的研究

目的　研究错配修复基因 (MMR)表达与食管癌和贲门癌癌变的关系和甲基苄基亚硝胺 (NMBzA)对其影响的研究。方法　采用多重逆转录聚合酶链反应 (RT -PCR)。结果　 31例食管癌hMSH2基因表达明显降低占 2 2 .6 % ;hMLH1基因表达明显降低占 6 .45 % ;未发现hPMS2 ,GTBP和hPMS1基因表达降低。 1 1例食管癌旁组织分析 :hMSH2 ,hPMS2 ,GTBP ,hMLH1基因表达明显降低占 9.0 9% ;未发现hPMS1基因表达明显降低。 1 2例贲门癌组织分析 :hMSH2基因表达明显降低占 41 .7% ;GTBP ,hMLH1 ,hPMS1基因表达明显降低占 8.3 % ;未发现hPMS2基因表达明显降低。 8例贲门癌旁组织分析 :3例hMSH2基因表达明显降低 ;1例hPMS2基因表达明显降低 ;3例hMLH1基因表达明显降低 ;未发现GTBP ,hPMS1基因表达明显降低。对NMBzA致癌物诱发的人胎儿食管上皮癌细胞株分析发现 :hMLH1和hPMS1基因低表达 ;hMSH2 ,hPMS2 ,GTBP基因不表达。结论　食管癌和贲门癌癌变可能与错配修复基因失活有关 ;甲基苄基亚硝胺能引起错配修复基因表达异常。     

Use of molecular tumor characteristics to prioritize mismatch repair gene testing in early-onset colorectal cancer.

PURPOSE: The relationships between mismatch repair (MMR) protein expression, microsatellite instability (MSI), family history, and germline MMR gene mutation status have not been studied on a population basis. METHODS: We studied 131 unselected patients with colorectal cancer diagnosed younger than age 45 years. For the 105 available tumors, MLH1, MSH2, MSH6, and PMS2 protein expression using immunohistochemistry (IHC) and MSI were measured. Germline DNA was screened for hMLH1, hMSH2, hMSH6, and hPMS2 mutations for the following patients: all from families fulfilling the Amsterdam Criteria for hereditary nonpolyposis colorectal cancer (HNPCC); all with tumors that were high MSI, low MSI, or that lacked expression of any MMR protein; and a random sample of 23 with MS-stable tumors expressing all MMR proteins. RESULTS: Germline mutations were found in 18 patients (nine hMLH1, four hMSH2, four hMSH6, and one hPMS2); all tumors exhibited loss of MMR protein expression, all but one were high MSI or low MSI, and nine were from a family fulfilling Amsterdam Criteria. Sensitivities of IHC testing, MSI (high or low), and Amsterdam Criteria for MMR gene mutation were 100%, 94%, and 50%, respectively. Corresponding positive predictive values were 69%, 50%, and 75%. CONCLUSIONS: Tumor IHC analysis of four MMR proteins and MSI testing provide a highly sensitive strategy for identifying MMR gene mutation-carrying, early-onset colorectal cancer patients, half of whom would have been missed using Amsterdam Criteria alone. Tumor-based approaches for triaging early-onset colorectal cancer patients for MMR gene mutation testing, irrespective of family history, appear to be an efficient screening strategy for HNPCC.     

Optimising methods for determining RER status in colorectal cancers

Approximately 13% of colorectal cancers display microsatellite instability (MSI), a form of replication error repair. Colorectal cancers developing in individuals with constitutional defects in the mismatch repair (MMR) genes hMLH1, hMSH2, hPMS1 and hPMS2 consistently show evidence of this phenomenon. Since MSI is indicative of MMR deficiency, testing colorectal cancers for MSI provides a method of refining the identification of carriers of germline MMR mutations. To assess which microsatellites represent the best reporters of replication error (RER) status we have examined 116 early onset colorectal cancers for MSI. MSI was assessed using eight dinucleotide- and two mononucleotide-repeat fluorescently labelled polymerase chain reaction (PCR) markers. The two mononucleotide repeat markers (BAT25 and BAT26) were highly sensitive and typing of either represents an efficient strategy for defining RER status of colorectal cancers and obviates the requirement of typing numerous microsatellite markers.