Clinical significance of MLH1/MSH2 for stage II/III sporadic colorectal cancer.
ABSTRACT: BACKGROUND:The development of colorectal cancer (CRC) is a complicated multistep process that involves an accumulation of mutations in tumor suppressor genes and oncogenes. In the process of DNA replication, base mismatch often occurs due to various factors leading to abnormal expression of mismatch repair genes (MMR), among which MLH1 and MSH2 are the most important. Recently, numerous studies indicated that MLH1/MSH2 phenotype is associated with CRC. We wanted to elucidate the role of MLH1/MSH2 in the prediction and prognosis of CRC through long-term clinical observation. AIM:To evaluate the prognostic and predictive significance of MLH1/MSH2 in patients with stage II-III CRC using immunohistochemical analysis and GeneScan. METHODS:Specimens from 681 patients with CRC (395 stage II and 286 stage III, 387 males and 294 females) who underwent curative surgical resection from 2013 to 2016 were tested. Immunohistochemistry was used to analyze MMR status and the microsatellite status of 133 patients was determined by GeneScan analysis. RESULTS:Five hundred and fifty (80.76%) patients were MLH1/MSH2 positive and 131 (19.24%) were negative by immunohistochemistry. MLH1/MSH2-positive tumors were significantly more frequent in the colon than in the rectum, and had poor differentiation and less mucin production (P < 0.05). Patients of different groups did not differ in terms of age, gender, tumor size, tumor stage, lymphocytic infiltration, or circumscribed margin. MLH1/MSH2-negative patients had a more favorable OS than MLH1/MSH2-positive patients (P < 0.001). Univariate and multivariate analyses demonstrated MLH1/MSH2 expression as an independent prognostic and predictive factor for stage II/III CRC. MLH1/MSH2 expression was a strong prognostic factor in all patients [P < 0.001, hazard ratio (HR) = 4.064, 95%CI: 2.241-7.369]. Adjuvant chemotherapy had a greater correlation with survival advantage in MLH1/MSH2-negative patients with stage III disease (P < 0.001, HR = 7.660, 95%CI: 2.974-15.883). However, patients with stage II disease or MLH1/MSH2-positive patients with stage III disease did not benefit from adjuvant chemotherapy. GeneScan analysis demonstrated that among 133 patients, 105 (78.95%) were microsatellite stable, and 28 (21.05%) had microsatellite instability (MSI), including 18 (13.53%) with high MSI and 10 (7.52%) with low MSI. This is consistent with the immunohistochemical results. CONCLUSION:MLH1/MSH2 phenotype constitutes a pathologically and clinically distinct subtype of sporadic CRC. MLH1/MSH2 is an independent prognostic and predictive factor for outcome of stage II-III CRC.
Project description:Micro-satellite instability (MSI) is relevant in the management of colorectal cancers (CRC) and relies on analysis of gene mutations, or production of the proteins involved in DNA mismatch repair (e.g. MLH1, MSH2). p53 mutation is also relevant in MSI, but high-level CRC (MSI-H) demonstrate fewer mutations than low-level (MSI-L) or stable (MSS) cancers. Recently, the importance of gene activity (transcription) in MSI has been identified, where rather than being mutated genes have been downregulated. In this study, 67 sporadic CRC and eight samples of normal bowel were analysed for MSI status (by SSCP) and levels of MLH1, MSH2 and p53 gene transcription (by RT-PCR and scanning densitometry). Micro-satellite instability correlated with gender and site, with more MSI-H CRC in females (P<0.02) and in the right colon (P<0.04). In MSI-H, p53 transcription was markedly reduced (P<0.003). Compared to normal bowel, MLH1 transcription was elevated in all cancers (P<0.01), while MSH2 transcription was elevated only in MSI-H (P<0.04). There was a direct correlation between MLH1 and MSH2 transcription (P<0.001). Although fewer mutations are reported in MSI-H than MSI-L/MSS, these results suggest that reduced p53 transcription might account for decreased tumour suppression in MSI-H. The direct correlation between MLH1 and MSH2 transcription suggests that control of these genes might be coordinated.
Project description:Microsatellite instability (MSI) is the molecular fingerprint of a deficient mismatch repair system. Approximately 15% of colorectal cancers (CRC) display MSI owing either to epigenetic silencing of MLH1 or a germline mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2. Methods to detect MSI are well established and routinely incorporated into clinical practice. A clinical and molecular profile of MSI tumors has been described, leading to the concept of an MSI phenotype in CRC. Studies have confirmed that MSI tumors have a better prognosis than microsatellite stable CRC, but MSI cancers do not necessarily have the same response to the chemotherapeutic strategies used to treat microsatellite stable tumors. Specifically, stage II MSI tumors might not benefit from 5-fluorouracil-based adjuvant chemotherapy regimens. New data suggest possible advantages of irinotecan-based regimens, but these findings require further clarification. Characterization of the molecular basis of MSI in CRC is underway and initial results show that mutations in genes encoding kinases and candidate genes with microsatellite tracts are over-represented in MSI tumors. Transcriptome expression profiles of MSI tumors and systems biology approaches are providing the opportunity to develop targeted therapeutics for MSI CRC.
Project description:Colorectal cancer (CRC) is uncommon in individuals <50 years old. Lynch syndrome is caused by germline mutations in DNA mismatch repair (MMR) genes and associated with early-onset CRC, but little is known about the proportion of young patients with apparently sporadic CRC who actually have Lynch syndrome. We examined patterns of microsatellite instability (MSI) and MMR genes among patients <50 years old with non-familial CRC (patients with not more than 1 family member with CRC).Tissue specimens were collected from 75 CRC patients <50 years old (mean age, 34.5 years) and analyzed using immunohistochemical analyses of MLH1, MSH2, MSH6, and PMS2. MSI and mutations in BRAF and KRAS were also analyzed.Most cancers (72%) arose in the distal colon. MSI was detected in 21% of the samples, and loss of 1 or more MMR proteins was observed in 21%. Interestingly, only 38% of the MMR-deficient CRCs lost either MLH1 or MSH2, whereas 63% of the MMR-deficient CRC samples lost either PMS2 or MSH6. All 11 CRC samples that had lost MSH2, MLH1, or PMS2 had MSI, but only 2 of the 5 tumors that lost only MSH6 had MSI. There were no BRAF mutations in any tumor.In young patients with apparently sporadic CRC, most tumors arise in the distal colon; only 21% have features of Lynch syndrome. Loss of MSH6 or PMS2 occurred in 13.3% of these tumors. Most tumors that lose MSH6 will not be detected in screens for MSI; CRC screening might be modified to identify more patients with Lynch syndrome.
Project description:Deficient mismatch repair (MMR) and microsatellite instability (MSI) contribute to ~15% of colorectal cancer (CRCs). We hypothesized MSI leads to mutations in DNA repair proteins including BRCA2 and cancer drivers including EGFR. We analyzed mutations among a discovery cohort of 26 MSI-High (MSI-H) and 558 non-MSI-H CRCs profiled at Caris Life Sciences. Caris-profiled MSI-H CRCs had high mutation rates (50% vs 14% in non-MSI-H, P < 0.0001) in BRCA2. Of 1104 profiled CRCs from a second cohort (COSMIC), MSH2/MLH1-mutant CRCs showed higher mutation rates in BRCA2 compared to non-MSH2/MLH1-mutant tumors (38% vs 6%, P < 0.0000001). BRCA2 mutations in MSH2/MLH1-mutant CRCs included 75 unique mutations not known to occur in breast or pancreatic cancer per COSMIC v73. Only 5 deleterious BRCA2 mutations in CRC were previously reported in the BIC database as germ-line mutations in breast cancer. Some BRCA2 mutations were predicted to disrupt interactions with partner proteins DSS1 and RAD51. Some CRCs harbored multiple BRCA2 mutations. EGFR was mutated in 45.5% of MSH2/MLH1-mutant and 6.5% of non-MSH2/MLH1-mutant tumors (P < 0.0000001). Approximately 15% of EGFR mutations found may be actionable through TKI therapy, including N700D, G719D, T725M, T790M, and E884K. NTRK gene mutations were identified in MSH2/MLH1-mutant CRC including NTRK1 I699V, NTRK2 P716S, and NTRK3 R745L. Our findings have clinical relevance regarding therapeutic targeting of BRCA2 vulnerabilities, EGFR mutations or other identified oncogenic drivers such as NTRK in MSH2/MLH1-mutant CRCs or other tumors with mismatch repair deficiency.
Project description:Early-onset colorectal cancer (CRC) is suggestive of a hereditary predisposition. Lynch syndrome is the most frequent CRC hereditary cause. The MUTYH gene has also been related to hereditary CRC. A systematic characterization of these two diseases has not been reported previously in this population.We studied a retrospectively collected series of 140 patients ≤50 years old diagnosed with nonpolyposis CRC. Demographic, clinical, and familial features were obtained. Mismatch repair (MMR) deficiency was determined by microsatellite instability (MSI) analysis, and immunostaining for MLH1, MSH2, MSH6, and PMS2 proteins. Germline MMR mutations were evaluated in all MMR-deficient cases. Tumor samples with loss of MLH1 or MSH2 protein expression were analyzed for somatic methylation. Germline MUTYH mutations were evaluated in all cases. BRAF V600E and KRAS somatic mutational status was also determined.Fifteen tumors (11.4%) were MSI, and 20 (14.3%) showed loss of protein expression (7 for MLH1/PMS2, 2 for isolated MLH1, 3 for MSH2/MSH6, 7 for isolated MSH6, and 1 for MSH6/PMS2). We identified 11 (7.8%) germline MMR mutations, 4 in MLH1, 1 in MSH2, and 6 in MSH6. Methylation analysis revealed one case with somatic MLH1 methylation. Biallelic MUTYH mutations were detected in four (2.8%) cases. KRAS and BRAF V600E mutations were present in 39 (27.9%) and 5 (3.6%) cases, respectively.Loss of MSH6 expression is the predominant cause of MMR deficiency in early-onset CRC. Our findings prompt the inclusion of MSH6 and MUTYH screening as part of the genetic counseling of these patients and their relatives.
Project description:Patients with early-onset colorectal cancer (CRC) or those with multiple tumours associated with hereditary non-polyposis colorectal cancer (HNPCC) raise suspicion of the presence of germline DNA mismatch repair (MMR) gene mutations.To analyse the value of family history, microsatellite instability (MSI) analysis and MMR protein staining in the tumour to predict the presence of an MMR gene mutation in such patients.In 281 patients diagnosed with CRC before the age of 50 years or with CRC and at least one additional HNPCC-associated cancer, germline mutation analysis in MLH1, MSH2 and MSH6 was carried out with denaturing gradient gel electrophoresis and multiplex ligation-dependent probe amplification. MSI analysis with five consensus markers and MMR protein staining for MLH1, MSH2 and MSH6 were carried out in the tumours.25 pathogenic mutations (8 in MLH1, 9 in MSH2 and 8 in MSH6) were found. MSI analysis missed three and immunohistochemistry (IHC) missed two mutation carriers. Sensitivities of family history, MSI analysis and IHC for the presence of a mutation were 76%, 82% and 88%, specificities were 64%, 70% and 84%, and positive predictive values were 19%, 23% and 38%, respectively. Multivariate analysis showed the highest odds ratio for IHC (38.3, 95% confidence interval 9.0 to 184). Prevalence of pathogenic germline MMR gene mutations in patients with CRC before the age of 50 years was 6% and in those with > or =2 HNPCC-associated tumours was 22%. In the second group, no mutation carriers were found among the 29 patients who were diagnosed with their first tumour after the age of 60 years.Family history, MSI analysis and IHC are indicative parameters to select patients with CRC for MMR gene mutation analysis. The data show that IHC is the best single selection criterion.
Project description:To evaluate the prognostic value of E-cadherin, CD44, and MSH2 expression for colorectal cancer (CRC) and construct nomograms that can predict prognosis.We retrospectively analyzed the expression of E-cadherin, CD44, and MSH2 in 223 paraffin-embedded stage II and III CRC specimens using immunohistochemistry in the training cohort. Their prognostic values were assessed using Kaplan-Meier curves and univariate and multivariate COX regression models. Moreover, a number of risk factors were used to form nomograms to evaluate survival, and Harrell's concordance index (C-index) was used to evaluate the predictive accuracy. Further validation of the nomograms was performed in an independent cohort of 115 cases.Low E-cadherin expression and low CD44 expression were significantly associated with diminished overall survival (OS) and disease-free survival (DFS) in stage II and III CRC patients and patients with negative MSH2 expression had better clinical outcomes. Moreover, the multivariate COX analysis identified E-cadherin, CD44 and MSH2 expression as independent prognostic factors for DFS and OS. Using these three markers and three clinicopathological risk variables, two nomograms were constructed and externally validated for predicting OS and DFS (C-index: training cohort, 0.779 (95% CI 0.722-0.835) and 0.771 (0.720-0.822), respectively; validation cohort, 0.773 (0.709-0.837) and 0.670 (0.594-0.747), respectively).The expression levels of E-cadherin, CD44 and MSH2 were independent prognostic factors for stage II and III CRC patients. By incorporating clinicopathological features and these biomarkers, we have established two nomograms that could be used to make individualized predictions for OS and DFS.
Project description:There is evidence that colorectal cancers (CRC) with DNA mismatch repair deficiency (MMR-D) are associated with a better prognosis than the generality of large bowel malignancies. Since an active immune surveillance process has been demonstrated to influence CRC outcome, we investigated whether MMR-D can enhance the immune response in CRC.A group of 113 consecutive patients operated for CRC (42 stage I or II and 71 with stage III or IV) was retrospectively analyzed. The expression of MMR genes (MSH2, MLH1, MSH6 and PSM2) and co-stimulatory molecule CD80 was assessed by tissue microarray immunohistochemistry. In addition, tumor infiltrating mononuclear cells (TIMC) and T cell subpopulations (CD4, CD8, T-bet and FoxP-3) were quantified. The effect of specific siRNA (siMSH2, siMLH1, siMSH6 and siPSM2) transfection in HT29 on CD80 expression was quantified by flow cytometry. Non parametric statistics and survival analysis were used.Patients with MMR-D showed a higher T-bet/CD4 ratio (p = 0.02), a higher rate of CD80 expression and CD8 lymphocyte infiltration compared to those with no MMR-D. Moreover, in the MMR-D group, the Treg marker FoxP-3 was not expressed (p = 0.05). MMR-D patients with stage I or II and T-bet expression had a significant better survival (p = 0.009). Silencing of MSH2, MLH1 and MSH6, but not PSM2, significantly increased the rate of CD80+ HT29 cells (p = 0.007, p = 0.023 and p = 0.015, respectively).CRC with MMR-D showed a higher CD80 expression, and CD8+ and Th1 T-cell infiltration. In vitro silencing of MSH2, MLH1 and MSH6 significantly increased CD80+ cell rate. These results suggest an enhanced immune surveillance mechanism in presence of MMR-D.
Project description:Colorectal cancer (CRC) represents a major malignancy globally, with microsatellite instability as its second top molecular mechanism of carcinogenesis. Immunohistochemical (IHC), whose sensitivity and specificity exceed 90%, is used routinely to detect 4 MMR proteins (MLH1, PMS2, MSH2, and MSH6) for screening mismatch repair system defects. We aimed to assess associations of clinicopathologic characteristics with MMR status in resectable CRC patients.Stage I-III CRC cases administered surgical resection in Zhejiang Cancer Hospital in 2013 to 2015 were retrospectively analyzed. MLH1, MSH2, MSH6, and PMS2 protein amounts were evaluated immunohistochemically. Clinicopathological information, including age, sex, tumor location, histological subclass, disease stage, regional lymph node (LN) metastasis, American Joint Committee on Cancer (AJCC) 8th edition stage, and survival data were retrospectively reviewed.A total of 133 CRC cases were assessed, including 74 (55.6%), 45 (33.8%), 55 (41.4%), and 77 (57.9%) not expressing MLH1, MSH2, MSH6, and PMS2, respectively. There were significant associations of MLH1, MSH2, MSH6, and PMS2 proteins with age and sex (P?<?.05). MLH1, MSH2, and MSH6 (but not PMS2) showed positive associations with primary tumor location (P?<?.05). Of the 133 patients, 70 and 63 cases were affected on the right and left sides, respectively; significant associations of primary site with age and sex were observed (P?<?.05). Regarding the MMR status, MLH1, MSH2, and MSH6 protein expression levels were positively associated with primary site (P?<?.05). Five-year overall survival (OS) rates were 84.2% and 79.2% in left-side and right-side cases, respectively; 5-year disease-free survival (DFS) rates were 74.0% and 69.8%, respectively. Survival had no differences between left- and right-side patients in terms of OS (P?=?.318) and DFS (P?=?.481).These data demonstrate that 4 major dMMR proteins are expressed differently in left- and right-side CRCs, and survival is comparable in right- and left-side resectable CRC cases with dMMR.
Project description:Mismatch repair defective (MMRd) colorectal carcinoma (CRC) is a distinct molecular phenotype of colorectal cancer, including 12% of sporadic CRC and 3% of Lynch Syndrome. In order to investigate the clinicopathological characteristics of MMRd colorectal carcinoma, and to find the most effective method for preliminary screening, 296 CRC fulfilled revised Bethesda Guideline (RB) were selected from 1450 CRCs to perform both IHC staining for MLH1, MSH2, MSH6, PMS2 and MSI analysis. Sixty-eight tumors were classified as MSI-H by MSI test. Colorectal carcinomas with MSI-H were prone to be proximal located, poorly differentiated, and relatively early staged, with infrequent metastasis to lymph node as well as to distant organs, compared with MSS ones. All of the 68 MMRd CRCs presented abnormal expression of at least one mismatch repair protein (MMRP), with 48 concurrent negative of MLH1 and PMS2, 14 concurrent negative of MSH2 and MSH6, 4 isolated negative of MSH6, 1 isolated negative of PMS2, and 1 concurrent negative of 4 MMRPs. All of the MLH1 negative tumors also showed abnormal expression of PMS2. All of the MSH2 negative cases also presented negative expression of MSH6. The sensitivity and specificity of the 2-antibody IHC test contained only PMS2 and MSH6 for screening for MMRd CRC were 100% and 98.2% respectively, exactly the same as that of the 4-antibody IHC test with all of the 4 MMRPs. The diagnostic accordance rate of the 2-antibody approach and MSI analysis was 98.6%. In conclusion, MMRd CRC has characteristic clinicopathological features different from MSS CRCs. The 2-antibody IHC approach containing MSH6 and PMS2 is the most easy and effective way to detecting MMR deficiency in CRC.