Chinese Hereditary Nonpolyposis Colorectal Cancer And Microsatellite Instability In Gastrointestinal Cancer Clinical Pathology And Mismatch Repair Gene Mutation Studies

Hereditary nonpolyposis colorectal cancer is one of the commonest hereditary cancer . It is characterized by dysfunction of mismatch repair genes, autochromosome dominant transmission and increased risk of several malignant tumors. There are several clinical diagnostic criteria being used to define HNPCC, the classic Amsterdam criteria I and II, Korea suspected criteria, Japanese Criteria and Chinese Criteria. According to reports, incidence of HNPCC which is defined by Amsterdam criteria is about 1% to 5%. However, diagnostic incidence of HNPCC in China could be lower than that in western countries, because western diagnostic criterias for HNPCC are too strict to define HNPCC families for Chinese.HNPCC has been described as autochromosome dominant disease with high risk of colorectal and several other specific cancers and it is caused by mutation of mismatch repair genes. In this description, mutation of mismatch repair genes is the driving factor of HNPCC and the main manifestation of HNPCC is increased risk of cancer. As a matter of fact, HNPCC is not as simple as that has been regarded. For instance, it is not that all clinically diagnosed HNPCC carry mutant mismatch repair genes, it is not that all the mutant gene carriers grow cancer. There arestill many problems like that how mutant MMR genes cause HNPCC, are there relations between cancergenesis mechanisms and clinopathological manifestations of HNPCC. Moreover, we need to find appropriate diagnostic method for relatively small Chinese families.One of the most complicated problem of sporadic cancer is the driving factor of cancergenesis. However, HNPCC has more apparent driving factor of cancergenesis than sporadic cancer. Most of HNPCC belong to microsatellite instability cancer, whereas microsatellite instability is caused by dysfunction of mismatch repair genes. In a condition that large amount of HNPCC tumor samples are unavailable. Research on microsatellite instability gastrointestinal cancer may help to look insight into HNPCC.The purpose of this study is to obtain a systematic knowledge of clinicopathological features and genetic alterations of Chinese HNPCC and To set up appropriate methods for genetic detection and classification of Chinese HNPCC.PART 1 Establish Methods and Techniques of Detecting Gene Mutation/Methylation/MSI by DHPLCObjective: to set up reliable detecting methods and techniques of hMSH2/hMLH1 gene mutation, hMLH1 promoter methylation and DNA microsatellite instability, to standardize detecting procedure. Method:design PCR primers by bioinformatics software, PCR amplify DNA samples which have known mutation, methylation and microsatellite instability. Analyze PCR products by DHPLC, adjust parameters of DHPLC for better results. Result: successfully established methods and techniques of gene mutation/methylation/microsatellite instability detection. Conclusion:detecting gene mutation/methylation/ microsatellite instability is simple, effective, automatic and economic.PART2 Detecting hMSH2/hMLH1 Mutation in Clinically Diagnosed Chinese HNPCCObjective: To identify germline mutations of hMLH1 and hMSH2 in HNPCC kindred fulfilling Chinese HNPCC criteria. Method: Fourteen peripheral blood DNA samples respectively from 14 unrelated HNPCC probands fulfilling Chinese HNPCC criteria were obtained. PCR amplified 35 exons of two main mismatch repair genes, hMLH1 and hMSH2. DHPLC followed by DNA sequencing was used to detect and confirm mutations. fluoresent labeled quantitative mutiplex PCR was used to detect large genomic rearrangement of hMSH2/hMLH1. Result: 12 single nucleotide changes were identified by DHPLC in 14 probands. Among them, 3 were germline pathological mutations. one of the 14 probands was to found has a deletion of hMSH2 exon 1-7. 5/7-6/7 relatives of mutation carriers carry the same mutation . Conclusion:Valid mutations of hMLH1 and hMSH2 genes were identified in about one-third HNPCC kindreds fulfilling Chinese HNPCC criteria. Incidence of mutation carriers in family members is more than 1/2.PART3 Screening Hereditary Nonpolyposis Colorectal Cancer from Microsatellite Instability Colorectal CancerObjective: many studies have evaluated mismatch repair gene germline mutations in clinically diagnosed hereditary nonpolyposis colorectal cancer (HNPCC), the purpose of this study is to screen germline mutation positive HNPCC from microsatellite instability (MSI) colorectal cancer in unselected Chinese colorectal cancer cases.Method: During 2002-10-1 to 2004-11-1 , 303 tumor and paired normal samples of unselected colorectal adenocarcinoma from patients having their surgery done in the second affiliated hospital, School of Medicine, Zhejiang University, were collected, blood samples were also collected if necessary, two NCI recommended microsatellite marker, BAT25 and BAT26, were used to verify MSI status by denaturing high performance liquid chromatography (DHPLC).both of the markers positive defines a microsatellite highly instability (MSI-H) tumor. For MSI-H positive tumor, hMLH1 promoter methylation was detected by methylation specific PCR and PCR product sequencing , then Mutations in MSH2 and MLH1 gene were detected by DHPLC and DNA sequencing . Result: 7out of 303 (2.3%) patients meet Chinese HNPCC criteria. 31(10.2%) MSI-H positive tumor was found. 6 (19%) of them were highly methylated in hMLH1 gene promoter region. 4 out of 303(1.3%) patients were detected having pathological mutations in their tumor, normal control and blood samples. 1 of them does not meet any HNPCC criteria. The entire methylated tumor was mutation negative. Conclusion: incidence of germline mutation positive HNPCC in China is approximately 1.3%, which is significantly lower than that diagnosed by clinical criteria.PART4 Similarities and Differences of Microsatellite Instability Subtype Cancers between Colorectal Cancer and Gastric Cancer in Chinese PopulationObjective: The purpose of this study is to discover clinicopathological features as well as the genetic and epigenetic causes of both MSI-H CRC and MSI-H GC in Chinese population. Method: A total of 303 CRC and 288 GC unselected patients were involved in this study. Instability of both BAT25 and BAT26 were used to define MSI-H tumor. Mutation of hMSH2/hMLHl and methylation of hMLH1 promoter region were detected in every MSI-H tumors. Result: MSI-H CRC and MSI-H GC account for 10.2% and 6.6% of unselected CRC and GC patients respectively. MSI-H CRC are strongly featured as early onset,right side location, low differentiation, mucinous tumor, less infiltration , less lymphatic metastasis and better survival. But MSI-H GC only tend to be located at antrum , to be with less numbers of lymphatic metastasis. Genetic and epigenetic analysis resulted in 6/31 MSI-H CRC & 0/19 MSI-H GC with pathological mutation and 6/31 MSI-H CRC & 15/19 MSI-H GC with methylated hMLHl promoter. Conclusion: most of MSI-H GC in Chinese population is caused by methylation of hMLH1. But neither methylation or somatic mutation is main cause of MSI-H CRC. Clinicopathological features of MSI-H GC is relatively indefinite. However, the common feature of both MSI-H CRC and MSI-H GC is less local lymphatic metastasis involvement.PART5 Criteria Specific Overall Survival of 102 Chinese Colorectal Family CancersObjective: hereditary nonpolyposis colorectal cancer (HNPCC) patients were reported having better survival than sporadic colorectal cancer, the aim of this study is to study survival of Chinese colorectal family cancer according to specific clinical diagnostic criteria. Method: 10 year overall survival of 102 Chinese colorectal family cancers with reliable following up data was investigated. For each family, conditions as at least 2 first degree relatives with 1 individual with colorectal cancer and another with HNPCC-related cancer are guaranteed. Result : 102families completed regular follow up. Among them, 7 meet Amsterdam criteria I and 8 meet criteria II, 25 meet Chinese suspect HNPCC criteria ,59 meet Bethesda guidelines. 10 year survival for Amsterdam I HNPCC (71.4%), Amsterdam II (100%), Chinese HNPCC (88%) and Bethesda guideline (71.2%) is significantly higher than sporadic colorectal cancer (50.7%). Conclusion: long term survival of typical HNPCC cancer is better than sporadic colorectal cancer; there are no differences in long term survival of HNPCC which are diagnosed by different HNPCC criteria.