| Objective:CIMP is a subset of CRC with an exceptionally high frequency of methylated genes. The study found that the pathogenesis and its response to treatment and prognosis of CIMP colorectal cancer are greater differences with other types of colorectal cancer, therefore, researchs of CIMP have important value. Current researches show that CIMP colorectal cancer was significantly associated with microsatellite instability, BRAF and KARS gene mutations. Microsatellite are simple repeat sequences composition of 1-6 bases in DNA sequences, and these regions are easy to occur mismatch or restructure error. During DNA replication.When MMR system function is normal, microsatellite’s error replication are repaired by MMR system. So, MSI is defined as a change of any length due to either insertion or deletion of repeating units.That is to say, MSI is usually caused by dysfunction of MMR.The MMR mainly include four genes:MLH1, MSH2, MSH6 and PMS2.Any one of the four genes loss of expression leads to dMMR. Since previous studies have shown that CIMP was significantly associated with MSI, MSI is often caused by the abnormal function of MMR, so we speculated that the loss of MMR system gene expression and CIMP status should also be linked. This study will detect loss of the four mismatch repair gene expression in colorectal cancer by immunohistochemical method, at the same time Methylight method are used to determine which belongs to CIMP types, and then through statistical analysis to determine the relevance between loss of mismatch repair gene expression and CIMP status, provide evidences to pathogenesis and clinical treatment for CIMP colorectal cancer.Methods:Patients with a new diagnosis of colorectal adenocarcinoma at the First Affiliated Hospital of Kunming Medical University in Yunnan.. Sodium bisulfite treatment on genomic DNA and subsequent real-time PCR (MethyLight) were validated and done as previously described. We quantified DNA methylation in five CIMP-specific promoters [CACNA1G, IGF2, NEUROG1,RUNX3, and SOCS1] and ALU repeats. PMR values were derived exclusively using the ALU normalization control reaction.Methylation positivity at each locus was defined as the PMR values >4, as previously validated.CIMP-positive was defined as the presence of>3 methylated promoters,And CIMP-negative as he presence of< 2 methylated promoters,according to the previously established criteria.We searched for staining for mismatch-repair proteins in the MLHl,MSH2,MSH6,and PMS2 genes with the use of immunohistochemical. In this study, we examined the relation between tumoral MMR expression and CIMP in colorectal cancers.Results:(l)CIMP classification resultsThis study examined 216 cases of CIMP status in colorectal cancer,56% (120/216) of patients were rectal cancer,44%(96/216) of patients were colon cancer. The CIMP-positive of the 216 CRC patients was 12.5%(27/216). The CIMP-negative of the 216 CRC patients was 87.5%(189/216). In colon cancer,16.6%(16/96) of patients were CIMP-positive,74.4%(80/96) of patients were CIMP-negative. In rectal cancer,9.2%(11/120) of patients were CIMP-positive,90.8%(109/120) of patients were CIMP-negative.(2) Immunohistochemistry resultsSamples of 216 eligible patients were available for IHC analysis for the MLH1, MSH2and MSH6 proteins..MLH1:Of the 216 CRC patients,44 patients (20.4%)showed loss of MLH1 gene expression,172 patients (79.6%) showed no loss of MLH1 gene expression. Of the 96 CC patients,27 patients (28.1%)showed loss of MLH1 gene expression,69 patients (71.8%) showed no loss of MLH1 gene expression. Of the 120 RC patients,17 patients (14.2%) showed loss of MLH1 gene expression,103 patients (85.8%) showed no loss of MLH1 gene expression, Loss of MLH1 gene expression was significantly associated with the CC patients (P=0.017,28.1%VS 14.2%)PMS2:Of the 216 CRC patients,103 patients (47.7%)showed loss of gene expression,113 patients (52.3%) showed no loss of gene expression. Of the 96 CC patients,47 patients (49.0%)showed loss of gene expression,49 patients (51.0%) showed no loss of gene expression. Of the 120 RC patients,56 patients (46.7%)showed loss of gene expression,64 patients (53.3%) showed no loss of gene expression,MSH2:Of the 216 CRC patients,12 patients (5.6%)showed loss of gene expression,204 patients (94.4%) showed no loss of gene expression. Of the 96 CC patients,6 patients (6.2%)showed loss of gene expression,90 patients (93.8%) showed no loss of gene expression. Of the 120 RC patients,6 patients (5%)showed loss of gene expression,114 patients (95.0%) showed no loss of gene expression,MSH6:Of the 216 CRC patients,8 patients (3.7%)showed loss of gene expression,208 patients (92.3%) showed no loss of gene expression. Of the 96 CC patients,6 patients (6.3%)showed loss of gene expression,90patients (93.7%) showed no loss of gene expression. Of the 120 RC patients,2 patients (1.7%)showed loss of gene expression,118 patients (98.3%) showed no loss of gene expression,incidence of dMMR was 51.9%(112\216)n CRC,49.2%(53\120) in the RC, 55.2%(53,96) in CC.(3)CIMP status and clinicopathologic featuresClinical and pathologic features according to tumoral CIMP status in 216 colorectal cancer patients,age(p=0.021, CIMP-positive tumor<60 was 66.7%, CIMP-negative tumor<60 was 41.4%),tumor location (p<0.05,0.000, CIMP-positive distal tumor was 44.4%, CIMP-negative distal tumor was 9%) and tumor grade (p=0.049, CIMP-positive low grade tumor was 18.5%, CIMP-negative low grade tumor was 6.3%) was significantly associated with the CIMP-positive tumour. Of the 96 CC patients, gender (p=0.021, CIMP-positive women patients was 72.7%, CIMP-negative women patients was 30.3%) was significantly associated with the CIMP-positive tumour. Of the 120 RC patients, clinicopathologic features has no significantly associated with the CIMP-positive tumour (p>0.05)(4)Loss of MMR gene expression and clinicopathologic featuresClinical and pathologic features according to loss of MMR gene expression in 216 colorectal cancer patients,tumor location (p=0.037, no staining for PMS2 in proximal tumor was 30.1%, no staining for PMS2 in distal tumor was 69.9%) and tumor grade (p=0.021, no staining for PMS2 in low grade tumor was 12.6%, no staining for PMS2 in middle or high grade tumor was 85.4%) was significantly associated with no staining for PMS2. Tumor location (p=0.016, no staining for MLH1 in proximal tumor was 38.6%, no staining for MLH1 in distal tumor was 61.4%) was significantly associated with no staining for MLH1.(5) Loss of MMR gene expression and CIMP statusLoss of MMR gene expression according to CIMP status in 216 colorectal cancer patients, no staining for MMR gene was no significantly associated with CIMP status in CRC or RC (p>0.05). However, loss of PMS2 gene expression was significantly associated with CIMP status in CC (p=0.029, no staining for PMS2 in CIMP-positive CC was 75%, no staining for MLH1 in CIMP-negative CC was 43.8%). No staining for MLH1,MSH2 and MSH6 was no significantly associated with CIMP status in CC.DMMR was significantly associated with tumor location in CRC (p=0.038, dMMR in proximal tumor was 29.5%, dMMR in distal tumor was 70.5%)(6) Comparative molecular characteristics in RC and CCStatistical analysis found that loss of MLH1 gene expression was statistically difference between RC and CC(p=0.017, loss of MLH1 gene expression was 14.2% in RC, loss of MLH1 gene expression was 28.1% in CC) (6) comparative analysis of the molecular characteristics of RC and CC showed that the expression of CC protein in RC and MLH1 (P<0.05) was statistically different, PMS2, MSH and MSHConclusion:According to CIMP status and loss of MMR gene protein expression analysis, we found loss of PMS2 gene expression was significantly associated with CIMP status in CC.Loss of MMR gene expression was no significantly associated with CIMP status. |
