Microsatellite instability and expression of DNA mismatch repair genes in malignant astrocytic tumors from adult and pediatric patients

Microsatellite instability (MSI) is used as a molecular marker for defective DNA mismatch repair (MMR) genes.We report here alterations of MSI in 15 malignant astrocytomas (WHO grade Ⅲ) and glioblastomas (GBM; WHO grade Ⅳ) of pediatric patients (2-21 years) and 12 GBM from adults (44-68 years) by comparative analysis of BAT25/BAT26 loci and 10 other microsatellite markers. High-level microsatellite instability (MSI-H) occurred in 4 of the 15 pediatric cases (26.7%) and in 1 of the 12 adult GBM cases (8.3%). Low-level mi-

Breast cancer and rectal cancer associated with Lynch syndrome:A case report

BACKGROUND The development mechanisms of Lynch syndrome(LS)-related breast cancer(BC)and rectal cancer are complex and variable,leading to personalized variations in diagnosis and treatment plans.CASE SUMMARY This paper presents a comprehensive review of clinical diagnosis and treatment data from a patient with LS-associated BC and rectal cancer.Moreover,screening data and management guidelines,as well as relevant literature on LS,are included in this report.This study summarizes the molecular pathogenesis,clinicopathological features,and screening and management protocols for LS-associated BC and rectal cancer.CONCLUSION Implementing early screening,prevention,and timely diagnosis and treatment measures is expected to reduce mitigate the incidence and mortality of LS-related BC and rectal cancer.

Causes and consequences of microsatellite instability in gastric carcinogenesis

Loss of DNA mismatch repair(MMR) function, due to somatic or germline epi/genetic alterations of MMR genes leads to the accumulation of numerous mutations across the genome, creating a molecular phenotype known as microsatellite instability(MSI). In gastric cancer(g C), MSI occurs in about 15% to 30% of the cases. This review summarizes the current knowledge on the molecular mechanisms underlying the acquisition of MSI in g C as well as on the clinic, pathologic and molecular consequences of the MSI phenotype. Additionally, current therapeutic strategies for g C and their applicability in the MSI subset are also discussed.

Mismatch repair gene hMSH2 protein as predictive maker for gastric carcinoma

Objective: To study the possibility of mismatch repair gene hMSH2 as a marker to predict the occurrence of gastric carcinoma. Methods: Immunohistochemical method was used to detect hMSH2 protein expressions of gastric carci- nomas (carcinoma group) and their surrounding epithelia (surrounding epithelium group) in patients and the epithelia (normal epithelium group) in persons without carcinoma. Results: The positive rates of hMSH2 protein expression in nucleus of carcinoma, surrounding epithelium and normal epithelium groups were 44.94% (71/158), 28.48% (45/158) and 11.76% (4/34), respectively (P=0.000); the positive rates of hMSH2 protein expression in plasma of the 3 groups were 37.97% (60/158), 27.85% (44/158) and 23.53% (8/34), respectively (P=0.084); the positive rates of hMSH2 protein expression in both nucleus and plasma of the 3 groups were 20.89% (33/158), 17.72% (28/158) and 2.94% (1/34), respectively (P=0.045). Although the difference of positive rates between carcinoma and surrounding epithelium groups was not significant (P=0.476), both of them were higher than that of normal epithelium group (P=0.018). Conclusion: Our findings show that the detection of hMSH2 protein expression in gastric epithelia may help to predict and diagnose gastric carcinoma.

Clinical and molecular analysis of hereditary non-polyposis colorectal cancer in Chinese colorectal cancer patients

AIM： To analyze the frequency of hereditary non-polyposis colorectal cancer （HNPCC） in Chinese colorectal cancer （CRC） patients, and to discuss the value of microsatellite instability （MSI） and/or immunohistochemistry （IHC） for MSH2/MLH1 protein analysis as pre-screening tests in China. METHODS： The Amsterdam criteria Ⅰ and Ⅱ （clinical diagnosis） and/or germline hMLHI/hMSH2 mutations （genetic diagnosis） were used to classify HNPCC families. Genetic tests, including microsatellite instability, immunohistochemistry for MSH2/MLH1 proteins and hMSH2/hMLH1 genes, were performed in each proband. RESULTS： From July 2000 to June 2004, 1988 patients with colorectal cancer were analysed and 114 CRC patients （5.7%） from 48 families were categorized as having HNPCC, including 76 from 26 families diagnosed clinically and 38 from the other 22 families diagnosed genetically. The sensitivity and specificity of high MSI and IHC for predicting mutations were 100% and 54%, and 79% and 77%, respectively. CONCLUSION： The frequency of HNPCC is approximately 10% among all Chinese CRC cases. The MSI and IHC detections for hMSH2/hMLH1 proteins are reliable prescreening tests for hMLHI/hMSH2 germline mutations in families suspected of having HNPCC.

Promoter methylation status of hMLH1,MGMT,and CDKN2A/p16 in colorectal adenomas

AIM:To investigate aberrant DNA methylation of CpG islands and subsequent low-or high-level DNA microsatellite instability(MSI)which is assumed to drive colon carcinogenesis. METHODS:DNA of healthy individuals,adenoma(tu-bular or villous/tubulovillous)patients,and colorectal carcinoma patients who underwent colonoscopy was used for assessing the prevalence of aberrant DNA methylation of human DNA mismatch repair gene mutator L homologue 1(hMLH1),Cyclin-dependent kinase inhibitor 2A(CDKN2A/p16),and O-6-methylguanine DNA methyltransferase(MGMT),as well as their rela- tion to MSI. RESULTS:The frequency of promoter methylation for each locus increased in the sequence healthy tissue/adenoma/carcinoma.MGMT showed the highest frequency in each group.MGMT and CDKN2A/p16 presented a statistically significant increase in promoter methylation between the less and more tumorigenic forms of colorectal adenomas(tubular vs tubullovillous and villous adenomas).All patients with tubulovillous/villous adenomas,as well as all colorectal cancer patients,showed promoter methylation in at least one of the examined loci.These findings suggest a potentially crucial role for methylation in the polyp/adenoma to cancer progres- sion in colorectal carcinogenesis.MSI and methylation seem to be interdependent,as simultaneous hMLH1, CDKN2A/p16,and MGMT promoter methylation was present in 8/9 colorectal cancer patients showing the MSI phenotype. CONCLUSION:Methylation analysis of hMLH1,CD- KN2A/p16,and MGMT revealed specific methylation profiles for tubular adenomas,tubulovillous/villous adenomas,and colorectal cancers,supporting the use of these alterations in assessment of colorectal tumorigenesis.

Germline mutation analysis of MLH1 and MSH2 in Malaysian Lynch syndrome patients

AIM: To investigate the protein expression profi le of mismatch repair (MMR) genes in suspected cases of Lynch syndrome and to characterize the associated germline mutations. METHODS: Immunohistochemical analysis of tumor samples was performed to determine the protein expression profile of MMR protein. Germline mutation screening was carried out on peripheral blood samples. The entire exon regions of MLH1 and MSH2 geneswere amplifi ed by polymerase chain reaction, screened by denaturing high performance liquid chromatography (dHPLC) and analyzed by DNA sequencing to characterize the germline mutations. RESULTS: Three out of 34 tissue samples (8.8%) and four out of 34 tissue samples (11.8%) showed loss of nuclear staining by immunohistochemistry, indicating the absence of MLH1 and MSH2 protein expression in carcinoma cells, respectively. dHPLC analysis followed by DNA sequencing showed these samples to have germline mutations of MSH2 gene. However, no deleterious mutations were identifi ed in any of the 19 exons or coding regions of MLH1 gene, but we were able to identify MLH1 promoter polymorphism, -93G > A (rs1800734), in 21 out of 34 patients (61.8%). We identified one novel mutation, transversion mutation c.2005G > C, which resulted in a missense mutation (Gly669Arg), a transversion mutation in exon 1, c.142G > T, which resulted in a nonsense mutation (Glu48Stop) and splice-site mutation, c.2006-6T > C, which was adjacent to exon 13 of MSH2 gene. CONCLUSION: Germline mutations were identified in four Malaysian Lynch syndrome patients. Immunohistochemical analysis of tumor tissue proved to be a good pre-screening test before proceeding to germline mutation analysis of DNA MMR genes.

Epithelial-mesenchymal transition in colorectal cancer tissue of patients with Lynch syndrome

AIM: To explore the epithelial-mesenchymal transition (EMT) in tissue from patients with Lynch syndrome, and to interpret biological behaviour of Lynch syndrome.

Expression of hMSH2 gene and mutant p53 in sporadic digestive tract tumors

Objective To investigate the role of mutated mismatch repair gene hMSH2 and mutant p53 gene in the carcinogenesis and development of sporadic digestive tract tumors. Methods hMSH2 gene in normal and tumor tissue of 30 digestive tract tumor specimens was examined using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) silver staining. The PCR product with an abnormal strand was sequenced directly. Mutant p53 protein in the tumor tissue was analyzed immunohistochemically. Results Six patients were identified as having mutated strands, three on hMSH2 exon 1 and three on hMSH2 exon 5. DNA sequencing revealed that all 6 patients had mutated basic groups that led to decrease in function of the hMSH2 protein. Forty percent (12/30) of patients were p53 positive. The frequency of mutated hMSH2 in p53 positive patients (41.7%) was significantly higher than in p53 negative patients (5.6%, P<0.05). Conclusion The mutation of hMSH2 plays an important role in the carcinogenesis and development of digestive tract tumors through stimulating p53 mutation.

Comparison of screening strategies for Lynch syndrome in patients with newly diagnosed endometrial cancer:a prospective cohort study in China

Background: The prevalence of Lynch syndrome and screening strategies for this disorder in Chinese patients with endometrial cancer have seldom been investigated. Such data would be essential for the screening, prevention, genetic counseling, and treatment of Lynch syndrome. The purpose of this prospective study was to determine the accuracy of the mismatch repair (MMR) protein immunohistochemistry (IHC), microsatellite instability (MSI) test, and clinical diagnostic criteria in screening for Lynch syndrome-associated endometrial cancer (LS-EC) in a prospective Chinese cohort. Methods: All patients with newly diagnosed endometrial cancer (EC) were evaluated using clinical diagnostic criteria (Amsterdam II criteria and the revised Bethesda guidelines), MSI test, and IHC of MMR proteins in tumor tissues. For all patients, the screening results were compared with results of germline sequencing for pathogenic variants of MMR genes. Results: Between December 2017 and August 2018, a total of 111 unselected patients with newly diagnosed EC were enrolled. Six patients (5.4%) harbored a pathogenic germline mutation of MMR genes: 1 had a mutation in MutL homolog 1 (MLH1), 2 in MutS homolog 2 (MSH2), and 3 in MutS homolog 6 (MSH6). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for identifying LS-EC were 33.3%, 88.6%, 14.3%, and 95.9%, for the clinical criteria, 66.7%, 75.0%, 14.3%, and 97.3% for IHC of MMR proteins, 100%, 89.9%, 33.3%, and 100% for MSI test, and 100%, 72.4%, 20.0% and 100% for combined IHC and MSI test, respectively. The combination of IHC and MSI test had higher sensitivity and PPV than the clinical criteria (p = 0.030). MSI test and IHC were highly concordant for LS-EC screening (73/77, 94.8%). Conclusion: The accuracy of the combination of IHC of MMR proteins and MSI test for screening LS among Chinese patients with EC was superior to that of the clinical criteria.

Clinical and genetic features of International Collaborative Group-hereditary nonpolyposis colorectal cancer families and suspected hereditary nonpolyposis colorectal cancer families

Background Hereditary nonpolyposis colorectal cancer (HNPPC) is one of the most common genetic syndrome related with mutation of human mismatch repair genes. This study was to evaluate the clinical significance of suspected hereditary nonpolyposis colorectal cancer (sHNPCC) criteria I and the clinical and genetic features of International Collaborative Group-HNPCC (ICG-HNPCC) and sHNPCC families Methods Twenty-nine ICG-HNPCC families fulfilling the Amsterdam criteria and 34 sHNPCC families fulfilling the sHNPCC criteria I were collected PCR-SSCP and DNA sequencing analysis were employed to screen the germline mutations of the hMLH1 and hMSH2 genes in these families Results The ICG group had more colorectal cancer (CRC) patients per family than did the suspected group ( P <0 05) No statistical difference was observed in Lynch classification and familial tumor spectrum In both groups of families, colorectal cancer was the most frequent malignancy, and carcinomas of the stomach, pancreas and uterus were the three most common extracolonic malignancies Mutation screening showed that ICG-HNPCC and sHNPCC families had a similar mutation rate (31 0% vs 29 4%, P >0 05), mutation type, and mutation distribution Comparison of the families with and without mutation showed no significant difference in CRC patients per family, Lynch classification, and tumor spectrum Conclusions ICG-HNPCC and sHNPCC families that have similar clinical manifestations and genetic basis indicate a similar nature for cancer development The application of sHNPCC criteria I will facilitate clinical diagnosis and treatment of small families

Mutation profiles of classic myeloproliferative neoplasms detected by a customized next-generation sequencing-based 50-gene panel

Objective:A sequencing panel consisting of 50 genes was customized to reveal the potential molecular land-scapes of essential thrombocytosis,polycythemia vera,and primary myelofibrosis in Chinese patients with myeloproliferative neoplasm(MPN).Methods:Sixty-five MPN patients(38 with essential thrombocytosis,21 with polycythemia vera,and 6 with primary myelofibrosis),including 12 triple-negative patients,were recruited and were screened for their mutational spectrum using next-generation sequencing technology in this retrospective observational study.This study was approved by the Institutional Review Board of Changhai Hospital,Naval Military Medical University,China.Results:In addition to the typical driver mutations in JAK2,CALR,and MPL,pathogenic mutations in 15 other genes were frequently detected among the 65 patients with MPN.The 15 mutated genes were TET2,EZH2,ASXL1,MIR662,MLH1,MLH3,SF3B1,MSH6,BARD1,DNMT3A,KIT,MSH2,RUNX1,TP53,and NRAS in this order according to the mutational frequency detected.The average number of mutated genes was 1.2 genes per patient,while in the 12 triple-negative patients with MPN(ie,patients that lack the JAK2,CALR,or MPL mutations),at least one of the 15 pathogenic mutations was detected for each patient.Interestingly,4 single nucleotide polymorphisms(rs4858647,rs9376092,rs58270997,rs621940)that might be correlated to individual susceptibility to myeloproliferative neoplasm were identified among the 65 patients.We also found that single nucleotide polymorphism and/or single nucleotide variation mutations occurred in multiple loci of mismatch repair-related genes,which might contribute to the development of MPN.Conclusion:Our study confirms the importance of the previously known MPN relative genes and,more importantly,provides some new and potentially valuable information about mutations associated with MPNs.