Evaluation of microsatellite instability in routine examinations of surgical samples

Context: Approximately 20%–30% of colon cancer cases have a hereditary basis. The genetic defect may involve mismatch repair (MMR) genes, which results in microsatellite instability (MSI). MMR-deficient colorectal cancer may occur due to germline mutation (Lynch syndrome) or be a sporadic one. A tumor's histological features, supported by a panel of immunohistochemistry stains, enables pathologists to assess the MMR status, which in turn has beneficial effects on clinical management. Aims: We aimed to show the relations between histopathological features identified during routine examinations and MMR genes' mutations. Methods and Material: We reviewed retrospectively the material of the Department of Pathology fulfilling the revised Bethesda Guidelines. Statistical Analysis Used: We used Chi-square test, Spearman test, and epidemiological analysis. Results: For the PMS2 gene, the positive predictive value (PPV) indicates that 91% of cases neither present any histological lesions nor have genetic abnormalities. The negative predictive value (NPV) indicates that only 50% of cases have both histological and genetic changes. For the MSH6 gene, the PPV indicates that 85% of tumors without specific histological features do not have genetic abnormalities. Conclusions: We advise universal staining for MLH1, MSH2, MSH6, and PMS2 in every newly diagnosed colon cancer, but due to costly analyses we suggest a protocol for the selection of cases for MMR examinations.

Comprehensive application of various screening strategies of Lynch syndrome / 中华胃肠外科杂志

Lynch syndrome (LS), which is the most common hereditary colorectal cancer, accounts for about 3% of all colorectal cancers. However, due to its various clinical manifestations, it is difficult to be diagnosed. The diagnosis of LS requires comprehensive application of various screening criteria (such as the Amsterdam criteria, Bethesda criteria), predictive models, risk factors, immunohistochemistry test of mismatch repair (MMR) protein, microsatellite instability (MSI) detection, MLH1 methylation detection, BRAF gene mutation detection, germline gene mutation detection, and so on. LS can be diagnosed only after the identification of pathogenic germline mutation of MMR gene. The first-degree and second-degree relatives of LS patients are recommended to be tested for the identified mutant gene. For LS patients and gene mutation carriers, LS associated cancer can be detected early or even prevented by monitoring and preventive surgery. Reproductive techniques can be used to prevent this disease from being passed down to the next generation.

Identifying Common Mutations in Colorectal Cancer Using a 7-Gene Panel by Next Generation Sequencing

Abstract@#Introduction: Colorectal cancer (CRC) arises from the cumulative effects of genetic and epigenetic alterations. Current treatment of metastatic CRC relies on combination of chemotherapy and targeted therapies such as anti-EGFR therapies. The success of targeted therapies relies on the detection of actionable targets and predictive biomarkers of resistance. The study aims to determine mutations in common actionable targets and predictive biomarkers of resistance to anti-EGFR therapies in Malaysian CRC patients. Methods: Mutations in 10 CRC tissues were determined by next-generation sequencing with a panel of 7 cancer-related genes covering all exons in KRAS, BRAF, PIK3CA, PTEN, TP53, NRAS, and EGFR genes. Immunohistochemistry was used to determine mismatch repair (MMR) status. Results: Of the ten samples, 5 and 4 samples harboured two and one mutation, respectively and one had no mutation. All were missense mutations and were in five genes, namely, KRAS, PIK3CA, TP53, BRAF, and EGFR. They were, G12D, G12V, G12A, G13D, and V14I in KRAS, E545K, K733R, and D1056N in PIK3CA, G199V, D259Y, and R282W in TP53, V600E in BRAF and G696R in EGFR. Deficient mismatch repair (dMMR) was detected in three samples, of which two had KRAS mutation. Conclusion: Mutations in KRAS codon 12 and 13, BRAF and PIK3CA which predict resistance to anti-EGFR therapies and three TP53 mutations were found. This is the first report of EGFR mutation in Malaysian CRC patients. It is predicted to be a pathogenic variant. dMMR, one of the biomarkers for treatment with immune checkpoint inhibitor was also detected.

Identification of nonfamilial MSI-H colorectal cancer based on clinicopathological features / 基础医学与临床

Objective To identify clinicopathological features of high MSI (MSI-H).Methods We enrolled 150 patients,standard microsatellite loci (BA T25,BA T26,D2S123,D5S346,D17S250) were amplified by polymerase chain reaction(PCR) with fluorescent primers,and the PCR products were analyzed by GeneMapper software;age at diagnosis,gender and site were obtained;various pathological features were observed by light microscopy;the expression of tumor infiltrating lymphocytes (CD4~+ and CD8~+) was detected by immunohistochemistry.Using a stepwise logistic regression model,a formula was generated that could be used to calculate the probability of a colorectal carcinoma being MSI-H based on pathological features.Results Among 150 cancers,MSI-H was 13.33%.Independent identifiers inclucle poor differentiation,histologic heterogeneity,Crohn's-like reaction and tumor-infiltrating lymphocytes,logistic regression formula shows a sensitivity of 70.0% and a specificity of 99.2% and a accurate ratio of 95.3% for MSI-H.Conclusion MSI-H phenotype cancer is a type of nonfamilial colorectal cancer with specific pathological features,Clinicopathological features can efficiently identify MSI-H colorectal cancers.

The"modification"Type Microsatellite Change in High Frequency Microsatellite Instability Colorectal Cancer Closely Relating to MLH1 and KRAS Mutation / 现代生物医学进展

Microsatellite instability(MSI)was defined according to the frequency of positive findings in a panel of MSI markers.High frequency MSI(MSI-H)was the phenotype in which repeat sequences were extraordinarily unstable, and was considered to be the bona fide phenotype of DNA mismatch repair defection. However base substitutions in some well studied oncogenes or tumor suppressors were reported to be uncommon in MSI-H tumors. To explore this obvious contradiction, the relationship between MSI and KRAS gene mutations were studied in a panel of 76 human colorectal carcinomas, the whole exon of MLH1 and MSH2 were sequenced for MSI-H tumors. KRAS gene mutation was confirmed by similar frequencies in tumors of different MSI status. Intriguingly, all of the KRAS mutant MSI-H tumors harbored sequence alterations in MLH1gene, which was a key player in DNA mismatch repair system. This implied that in MSI-H tumors carrying MMR mutations, KRAS mutation were frequently and almost exclusively occurred. Furthermore, these MMR mutants were uniformly carrying a unique "modification" + "jumping" type MSI, which was different to MSI-H tumors without MLH1 or MSH2 gene mutations. This study shaded lights on the heterogeneity of MSI-H tumors, and implied the connection between "modification" type MSI and DNA mismatch defection.

The"modification"Type Microsatellite Change in High Frequency Microsatellite Instability Colorectal Cancer Closely Relating to MLH1 and KRAS Mutation / 现代生物医学进展

Microsatellite instability(MSI)was defined according to the frequency of positive findings in a panel of MSI markers.High frequency MSI(MSI-H)was the phenotype in which repeat sequences were extraordinarily unstable, and was considered to be the bona fide phenotype of DNA mismatch repair defection. However base substitutions in some well studied oncogenes or tumor suppressors were reported to be uncommon in MSI-H tumors. To explore this obvious contradiction, the relationship between MSI and KRAS gene mutations were studied in a panel of 76 human colorectal carcinomas, the whole exon of MLH1 and MSH2 were sequenced for MSI-H tumors. KRAS gene mutation was confirmed by similar frequencies in tumors of different MSI status. Intriguingly, all of the KRAS mutant MSI-H tumors harbored sequence alterations in MLH1gene, which was a key player in DNA mismatch repair system. This implied that in MSI-H tumors carrying MMR mutations, KRAS mutation were frequently and almost exclusively occurred. Furthermore, these MMR mutants were uniformly carrying a unique "modification" + "jumping" type MSI, which was different to MSI-H tumors without MLH1 or MSH2 gene mutations. This study shaded lights on the heterogeneity of MSI-H tumors, and implied the connection between "modification" type MSI and DNA mismatch defection.

"The""modification""Type Microsatellite Change in High Frequency Microsatellite Instability Colorectal Cancer Closely Relating to MLH1 and KRAS Mutation" / 现代生物医学进展

Microsatellite instability(MSI)was defined according to the frequency of positive findings in a panel of MSI markers.High frequency MSI(MSI-H)was the phenotype in which repeat sequences were extraordinarily unstable, and was considered to be the bona fide phenotype of DNA mismatch repair defection. However base substitutions in some well studied oncogenes or tumor suppressors were reported to be uncommon in MSI-H tumors. To explore this obvious contradiction, the relationship between MSI and KRAS gene mutations were studied in a panel of 76 human colorectal carcinomas, the whole exon of MLH1 and MSH2 were sequenced for MSI-H tumors. KRAS gene mutation was confirmed by similar frequencies in tumors of different MSI status. Intriguingly, all of the KRAS mutant MSI-H tumors harbored sequence alterations in MLH1gene, which was a key player in DNA mismatch repair system. This implied that in MSI-H tumors carrying MMR mutations, KRAS mutation were frequently and almost exclusively occurred. Furthermore, these MMR mutants were uniformly carrying a unique modification + jumping type MSI, which was different to MSI-H tumors without MLH1 or MSH2 gene mutations. This study shaded lights on the heterogeneity of MSI-H tumors, and implied the connection between modification type MSI and DNA mismatch defection.

Four Synchronous Colorectal Cancers and One Small Bowel Cancer in a Patient with HNPCC

The frequency of multiple synchronous carcinomas of the colon and rectum have varied in different reports from 3~4% to more than 10% of all tumors of the large bowel. Especially, the frequency is higher in hereditary non-polyposis colorectal cancer (HNPCC) patients. There are a few reported cases of five simultaneous cancers in a patient at the same time. We report here on a case of five synchronous cancers arising from the terminal ileum and colon in a patient with a strong familial tendency for colon cancer. The patient was a 43-year-old-female who presented with intermittent abdominal pain and diarrhea for one month. Colonoscopic examination revealed four adenocarcinomas at the proximal ascending, the proximal transverse, the distal descending and the sigmoid colon; the cancer in the sigmoid colon was at 30 cm above the anal verge. During the operation, another 3 cm sized ulcerative lesion was noted at the terminal ileum. Total colectomy, including the lesion of the terminal ileum, and ileorectal anastomosis were performed. Histologic evaluation revealed that all those lesions were adenocarcinomas invading the pericolic fat and three out of 126 lymph nodes were invaded by the cancer cells. It was a MSI-high cancer; 5 markers of MSI (BAT25, BAT26, D5S346, D17S250 and D2S123) were all unstable. We revealed a point mutation of the 67th base (GaT) of the 1st exon of hMLH1.

Study on Microsatellite Instability in Breast Cancer / 医学研究杂志

Objective To investigate the incidence and clinicopathologic significance of MSI in breast caner.Methods 40 paired sporadic invasive breast cancer were collected.Genomic DNA was extracted from live sample.Twelve microsatellites on chromosomes 2p,3p,5q,6q,16q,17q were amplified for MSI,respectively,by polymerase chain reaction(PCR)with designed primers and detecting after polyacrylamide gel electrophoresis.Results MSI in 15 out of 40(35%)of the carcinomas were observed.There was no MSI in benign hyperplasia.MSI was mainly located at D3S1766,D2S2739 and TP53 in the breast cancer.The incidence rate of MSI in breast cancer is associated with the degree of carcinoma differentiation.Conclusions Microsatellite instability might play a role in the early stage during multistep breast carcinogenesis.MSI indicated poor histologic differentiation in breast carcinoma.D3S1766,D2S2739 and TP53 might be the sensitive sites to detect MSI in breast carcinoma transformation.

MICROSATELLITE INSTABILITY AND p53 GENE MUTATION IN ORAL SQUAMOUS CELL CARCINOMA

Germ-line mutations at DNA repair loci confer susceptibility to colon cancer in hereditary non-polypopsis colorectal cancer. Somatic loss of DNA mismatch repair gene has been reported in a large variety of other tumor types. Replication errors(RERs) judged by microsatellite instability(MSI) and its associated mutations have been recognized as an important mechanism in various tumor types. To investigate associations between MSI and oral squamous cell carcinoma, the frequency of MSI using 12 microsatellite markers were analyzed for the series of oral tumors. Of 17 tumors, 8 cases(47%) did not show instability at any of the 12 loci; 5(29%) showed instability at 2~3 loci; and 4(24%) showed instability above 4 loci. The 4 cases showing widespread MSI did not differ from those without evidence of instability in terms of age at diagnosis, degree of differentiation, metastasis to lymph node, tumor location or the presence of mutations in the p53 tumor suppressor gene. DCC and D17S 796 were the most frequently detected in MSI analysis. There were no correlation between smoking and MSI frequency, instead, smoking was suggested to increase the mutation rate of p53 and development of oral carcinomas.

Microsatellite Instability in Korean Hepatocellular Carcinoma using Fluorescent - PCR / 대한암학회지

PURPOSE: Hepatocellular carcinoma (HCC) is one of the most common cancers in many parts of the world, however the molecular mechanisms underlying liver cell transformation remain obscure. The instability of microsatellite sequences dispersed in the genome has been linked to a deficiency in cellular mismatch repair. This phenotype has been frequently observed in various human neoplasms and is regarded as a major factor in tumorigenesis. To investigate cumulative genetic changes related with apoptosis during development and progression of HCC, we examined DNAs isolated from 12 Korean HCCs and their adjacent non-tumorous parts to look for evidence of microsatellite instability (MSI). MATERIALS AND METHODS: Twelve microsatellite loci (D6S271, D6S426, D13S153, D13S263, D17S849, D17S938, D17S945, D18S474, D18S64, D19S420, D.19S418 and D19S210) were amplified by PCR from 12 Korean HCCs, and analyzed using an automated DNA analyzer. RESULTS: The high percentages of the MSI were found for the loci of D6S426 (33.3%) and D17S945 (25.0%). The related genes with high frequency of MSI were noted in the wafl (41.7%) and p53 (25.0%). From this study, fifty eight percent of HCCs (7/12) showed MSI with at least one marker. CONCLUSION: This results suggest that the analysis of MSI in HCC might be useful for identifying genes whose loss of function contributes to the development of liver cancer. Furthennore, this method may give a more rapid and accurate sizing of the PCR products of microsatellite; making the routine assessment of MSI possible in many clinical fields.