Study On Microsatellite Instability In Colorectal Cancer Patients With Familial Predisposition

IntroductionColorectal cancer is a common malignant tumor in the world. The incidence of colorectal cancer shows a rising trendency. Accumulated evidence has established that carcinogenesis is associated with alterations in cellular oncogenes and in tumor suppressor genes necessary for malignant transformation. About twenty percent of colorectal cancer is characterized by a familial aggregation. Hereditar-y non - polyposis colorectal cancer (HNPCC) is thought to be the most common form of inherited colorectal cancer. HNPCC is an autosomal dominant disorder which accounts for 5% - 15% of colorectal cancer cases. Most cases of HNPCC and part of sporadic colorectal cancer are caused by a defect in the DNA mismatch repair gene ( MMR) but not in oncogenes or tumor suppressor genes. Tumors which have defective mismatch repair gene show a characteristic molecular phenotype, termed microsatellite instability ( MSI).Microsatellites are polymorphic, short, tandem repeat segments dispersed throughout the human genome. MSI is a form of genetic instability characterized by expansions and contractions of simple sequence repeats in DNA. It occurs because of a defect in DNA MMR. Nowadays, MSI has been paid more attention to because of its key role in the initiation and development of carcinoma. MSI is considered a new mechanism of colorectal cancer.In our experiment, polymerase chain reaction - single strand conformation polymorphism (PCR - SSCP) and sliver - staining methods were performed to detect incidence of MSI in 29 cases of CRC with familial predisposition and in 30 sporadic CRC. The goal is to discuss the molecular mechanisms of CRC with familial predisposition and the possibility to discover disease in early stage, directclinical treatment and judge the prognosis.Materials and MethodsI. Materials1. Samples All samples used were obtained from paraffin - embedded tissues of the surgical resected specimens of patients admitted to the 1 st Affiliated Hospital of China Medical University during 1997 -2003.2. Reagents Primer; BAT25, BAT26, D5S346, D17S250 and D2S123. (GIAGEN company); TaKaRa Taq?, Proteinase K, Agarose Regular, DNA Marker DL2000 ( TaKaRa Biotechnology company ) ; the other reagents made in China were all analytical pure.II. MethodsSerial five 8 — jxm thick sections of selected tissue blocks were obtained. Corresponding samples of colorectal mucosa that were free of tumor and metaplasia , were obtained from each case as control tissue. Sections were dewaxed. Then genomic DNA was purified by proteinase K digestion and phenol - chloroform extraction. The ratio of A260 and A280 was measured by ultraviolet spec-troscopic photometer. The concentration of DNA was calculated. PCRs were performed to amplify colorectal lesions and corresponding normal DNA. After amplification, PCR products were separated by 2% Agarose gels to detect DNA. Then PCR products were electrophoresed on non - denaturing 8% ( 29 :1) poly-acrylamide gels, and bands were visualized by silver staining.Alterations in the microsatellites in the form of changes in the size of DNA strands were detected by comparing tumor DNA and normal DNA. It was judged negative if their strands have no difference. MSI was defined as a band shift in either of the two alleles or the appearance of a band with different sizes in the tumor or normal sample. Using a panel of five microsatellites, a specimen was defined as high - level MSI ( MSI - H) if two or more of the five markers displayed instability,as low -level (MSI - L) if one marker displayed instability, and as microsatellite stable (MSS) if none of the markers exhibited MSI.III. Statistical analysisStatistical analysis was performed using the t - test and Fishers exact test. P <0.05 was considered statistically significant.Result1. In 29 cases of CRC with familial predisposition, 75. 86% (22/29) of the cases showed MSI - positive at one or more than one locus. All the 5 loci showed MSI at different sensitivity. BAT26 was the most sensitive, then the D2S123, D5S346, BAT25 and D17S250 the least sensitive.2. The incidence of MSI -positive was higher in CRC with familial predisposition 48. 28% (14/29) ,than in sporadic CRC 13.33% (4/30). The result had statistic significance ( P < 0.05 ).3. The average age of MSI - positive cancer onset in CRC with familial predisposition ( 44. 6 ) was lower than that of MSI - negative cancer onset in sporadic CRC (57.9) and had obvious statistic significance (P <0.01). The average age of MSI -negative cancer onset in CRC with familial predisposition (50. 1) was lower than in sporadic CRC (57.9) and had statistic significance (P < 0.05).4. Familial predisposition and MSI - positive related strongly with the proclivity for proximal colon ( P <0. 05 ). No association was observed between MSI status and the factors of sex, tumor size, invasive depth and lymph node metasta-ses.DiscussionMSI is a form of genetic instability characterized by expansions and contractions of simple sequence repeats in DNA. It occurs because of a defect in DNA mismatch repair genes. While microsatellite instability is seen in about 70% ~ 90% of all HNPCC and at the majority of microsatellite loci, it is only seen in 10% -15% of sporadic colorectal cancers. In 1990, the International Collaborative Group ( ICG - HNPCC ) proposed Amsterdam criteria to be used for selection of families for research collaborative study for the identification of theHNPCC predisposing genes: at least three relatives should have histologically verified CRC and one of them should be a first - degree relative to the other two; at least two successive generations should be affected; in one of the relatives, CRC should be diagnosed under 50 years of age; familial adenomatous polyposis must be excluded. Nevertheless, the clinical diagnosis of HNPCC is difficult to establish because the criteria is too strict and rigid for small families. We have screened a group of CRC with familial predisposition and sporadic CRC, with the reference set of 5 markers and criteria established by National Cancer Institute Workshop for identification of tumors as MSI.In our experiment, dependent on PCR - SSCP, we found the total incidence of MSI was 75. 86% in CRC with familial predisposition, whereas 26. 67% in sporadic CRC. It shows that MSI is a common molecular biological characteristic in CRC with familial predisposition.The association between MSI arid clinicopathologic chatacteristics is still unknown. Our findings suggest CRC displaying MSI is only associated with early age of cancer onset and the proclivity for proximal colon. Previous reports showed that MSI was found to be connected with poor differentiation. In our experiment , the incidence of MSI in poorly differentiated and mucoid adenocarci-noma was higher than in well and moderately differentiated adenocarcinoma. But there is no statistic difference (P > 0. 05 ).MSI incidence rates in different reports vary. We therefore postulate that MSI rates may be associated with markers, the criteria for definition of MSI, the number of samples, race and different areas.Our results demonstrate that MSI plays an important role in CRC with familial predisposition. MSI analysis is very useful in detecting tumors with mismatch repair genes as the first step. The verticle relatives of MSI carriers have a higher risk of HNPCC. We expect to discover HNPCC in early stage among those people by surveillance and have the possibility of early surgical intervention that could be curative.