The Clinical Values And Molecular Biological Features Of Screening The Chinese Hereditary Nonpolyposis Colorectal Cancer With NCCN Recommended Protocol

Background: Colorectal cancer (CRC) is one of the most common malignant tumors occurring in the gut with an increasing mobidity in China these years. Hereditary nonpolyposis colorectal carcinoma (HNPCC, Lynch syndrome) is the most common hereditary form of colorectal carcinoma and may account for 5-10% of the total CRC burden. HNPCC is characterized genetically by pathogenic germline mutations in one of the DNA mismatch repair (MMR) genes, usually MLH1, MSH2.Clinically, affected individuals display a predisposition for the development of early-onset colorectal cancers and various second primary tumours. In response to the criticism that Amsterdam II criteria (AC-II) were too stringent, NCCN(V.1.2006) recommended the protocol to screen the HNPCC patients. It was recently known that MSH2 played important role not only in mismatch repair but also in cell's proliferation, apoptosis and cell cycle control.Objective: To investigate the effects of NCCN V.1.2006 recommended protocol: recognition of clinical syndrome characterized with revised Bethesda guidelines, genetic counseling with immunohistochemistry and finally genetic testing to diagnose HNPCC. Evaluate the clinical feartures'diferences between the MMR deficient group and MMR proficient group. Study the role of MMR played in the cell's proliferation, cell cycle control and cell invasiveness.Methods: Newly diagnosed 419 patients with colorectal cancer from January 1, 2002 to Febrary 28, 2006. Nighty patients fulfilled the revised Bethesda guidelines. Immunohistochemistry was used to detect the expression of MLH1 and MSH2 in the ninety patients.The frozen tissues were collected from the patients who showed loss of MLH1 or MSH2 protein expression, RNA was extracted, and RT-PCR and cDNA sequencing were used to detect the germline mutations of MLH1 and MSH2. Cell proliferation, cell cycle and invasiveness were quantified by MTT, flow cytometry andTranswell respectively.Results:1. Twenty one patients were diagnosed as HNPCC by NCCN recommended protocol.(1) Nighty patients fulfilled the revised Bethesda guidelines.(2) Eighteen patients'tumors exhibiting loss expression of MLH1 or MSH2 protein.(3) Three cases of MLH1 or MSH2 mutation were detected in the patients whose tumors exhibiting loss expression of MLH1 or MSH2 protein. The 3 cases whose cDNA were detected mutations were not fulfilled the Amsterdam II criteria, and finally were diagnosed as HNPCC.(4)Three cases who exhibiting normal expression of MLH1 and MSH2 proteins were diagnosed as HNPCC by the Amsterdam II criteria.(5) Finally 21 patients (18 patients who showed loss expression of MMR protein and 3 patients who fulfilled the Amsterdam II criteria ) were diagnosed as HNPCC.2. Eight patients were diagnosed as HNPCC by Amsterdam II criteria.3. The expression of MSH2 and MLH1 were associated with the tumors'location, size and status of lymph node metastasis. It may be a factor to predict the patients'prognosis.4. A simulated HNPCC cell model was established by knocking down the MSH2 expression in colorectal cancer cell line-SW480 using small interference RNA (SiRNA).5. Proliferation were inhibited, cell cycle was arrested at G0/G1 phase and cell invasiveness was reduced after the colon cancer cell line-SW480 has been knocked down the MSH2 expression by SiRNA.Conclusion:(1) The NCCN(V.1.2006) recommended protocol constitutes a useful approach to identify Chinese patients at risk for HNPCC.(2) Mismatch repair gene-MSH2 play important roles in the cell's proliferation , cell cycle control and invasiveness.