Ideatification Method Of CpG Island Methylation Phenotype (CIMP) Of Evaluation And Improvement In The Colorectal Cancer

Objective:CpG Island methylation in colorectal cancer phenotype (CpG Island methylator phenotype, CIMP) is one of the types of colorectal cancer molecular classification,it refers to have happened to the high proportion of genes in the genome DNA methylation in the colorectal cancer.Type CIMP colorectal cancer pathogenesis and its effect on treatment response and prognosis are greater differences with other types of colorectal cancer, therefore, has an important scientific research CIMP and clinical significance.However, there is no an effective, simple operation technology can be used to identify CIMP, thanks in large part blocking the CIMP pathogenesis research and its application in clinic. Now reported that the two main methods for the identification of CIMP, respectively using five gene promoter region of DNA methylation biomarkers. The first method (CIMP-1) biomarkers for CACNA1G, CDKN2A, CRABP1, MLH1 and NEUROG1;The second method (CIMP-2) biomarkers for CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1.The two methods are based on five genes have three or more than three gene DNA methylation to decide CIMP positive standard.But now the two methods in which one or which several biomarkers is most suitable for as a standard for the identification of CIMP did not form a broad consensus. Methylight methods are used in this study to detect the cancer tissue of patients with colorectal cancer CACNA1G, CDKN2A, CRABP1, MLH1, NEUROG1, IGF2, RUNX3 and SOCS1 the eight genes (two genes are two methods of the communist party of China) the promoter region of DNA methylation, and then according to the appraisal standard CIMP-1 and CIMP-2 respectively to CIMP classification of colorectal cancer samples, CIMP-1 and CIMP-2 are as CIMP positive, as a final standard decide CIMP positive (CIMP1+2, this should be the most reliable standard, but need to detect the most genes are not suitable for widely available), and standard to evaluate the eight gene identification CIMP specificity and sensitivity.In comprehensive consideration on the basis of the specificity and sensitivity of each gene, select the most suitable as CIMP identification of marker genes, to establish a more reliable CIMP appraisal method, and study CIMP appraisal results with with the relationship between the clinical data, such as the age, gender, location, differentiation degree, tumor staging,the tissue types.In order to promote CIMP related scientific research and clinical application.Method:Collecting the first affiliated hospital of kunming medical university resection of colorectal cancer tissue samples in April 2014 to December 2014,the sample after extracting DNA, through transformation of sulfite processing.Using Methylight method detecting the cancer tissue of patients with colorectal cancer CACNA1G, CDKN2A, CRABP1, MLH1, NEUROG1, IGF2, RUNX3 and SOCS1 the eight genes (two genes are two methods of the communist party of China) the promoter region of DNA methylation, and then according to the appraisal standard CIMP-1 and CIMP-2 respectively to CIMP classification of colorectal cancer samples, CIMP-1 and CIMP-2 are as CIMP positive, as a final standard decide CIMP positive, and the standard to evaluate the eight gene identification CIMP specificity and sensitivity, in comprehensive consideration on the basis of the specificity and sensitivity of each gene, select the most suitable as CIMP identification of marker genes, establish a more reliable CIMP appraisal method.Result:(1) According to CIMP-1, CIMP-2 and CIMP 1+2 as the standard respectively CIMP parting on colorectal cancerThis study tested 100 samples of colorectal cancer in this eight gene methylation status, CIMP-1 for the appraisal standard,51 patients (51%) were identified CIMP positive, negative and 49 (49%); CIMP-2 for the appraisal standard,37 cases (37%) were identified CIMP positive, negative,63 cases (63%). Identify CIMP-1 is a the significant higher positive rate CIMP-2(p<0.05).If CIMP1+2 as the appraisal standard, CIMP positive for 29 cases (29%), and negative for 71 cases (71%).(2) Classification standard for CIMP 1+2, according to the sensitivity and specificity of each gene screening out the most suitable CIMP identification tagsCIMP 1+2 as the standard, the sensitivity and specificity of each gene, the result shows: the MLH1, CRABP1, CDKN2A, CACNA1G, NEUROG1, IGF2, RUNX3 and SOCS1 sensitivity was 17.3%(5/29),93.1%(27/29),100%(29/29),93.1%(27/29),75.9%(22/29), 86.2%(25/29),24.1%(7/29) and 82.8%(24/29), specificity of 93.0%(66/71),39.4%(28/71), 40.8%(29/71),71.8%(51/71),67.6%(48/71),50.7%(36/71),95.8%(68/71) and 62.0% (44/71).Considering the sensitivity and specificity of each gene, found CACNA1G (sensitivity was 93.1%, specificity of 71.8%), NEUROG1 (sensitivity was 75.9%, specificity of 67.6%), IGF2 (sensitivity was 86.2%, specificity of 50.7%) and SOCS1 (sensitivity was 82.8%, specificity of 62.0%) the four genes is most suitable for as a biomarker of CIMP identification.(3) CIMP classification using filtered biomarkersWith CACNA1G, NEUROG1, IGF2 and SOCS1 as biomarkers, with at least three of the four genes of DNA methylation as CIMP positive judgment standard (CIMP-3), for colorectal cancer CIMP classification samples, the results showed positive CIMP is 34 cases (34%), negative for 66 cases (66%).With the original method CIMP-1 (51%) and CIMP-2 (37%) compared to the new method CIMP-3 positive rate (34%) of the appraisal result is the most close to the CIMP 1+2 (29%).(4) CIMP-1, CIMP-2 and CIMP-3 type classification results with CIMP1+2 points between the results of the analysisCIMP-1,CIMP-2 and CIMP-3 of the classification results were compared with CIMP1+2 of the classification results, found that CIMP-1 compared with CIMP1+2, consistent with 29, inconsistent with 22; CIMP-2 compared with CIMP 1+2, consistent with 29, inconsistent with 8.CIMP-3 compared with CIMP 1+2, consistent with 28, inconsistent with 7. Thus it can be seen that with the original method CIMP-1 and CIMP-2, compared to the new method on the consistency of CIMP-3 in CIMP identification is also the most close to CIMP1+2. In addition, the number of genes from the need to detect, CIMP-1 and CIMP-2 need to detect all five genes, and the new method CIMP-3 only need to detect four genes. That is to say, with the original method CIMP-land CIMP-2, compared the accuracy of the new method CIMP-3 appraisal CIMP is higher, and is more simple in experimental operation, costs are lower.(5) CIMP-1、CIMP-2、CIMP-3 and CIMP1+2 classification results with the correlation analysis of clinical informationAnalysis CIMP-1,CIMP-2,CIMP-3 and CIMP1+2 classification results with the relationship between the clinical data, such as the age, gender, location, differentiation degree, tumor staging,the tissue types. Found all CIMP parting with no correlation between age gender, location, degree of differentiation, tumor staging (p>0.05); But all the CIMP classification results and significant correlation between the differentiation degree.Conclusion:Through the above of CIMP parting biomarkers after comparative analysis, found CACNA1G, NEUROG1, IGF2, SOCS1 four biomarkers is more suitable for as a biomarker of CIMP parting; And based on these four biomarkers of new method for the identification of CIMP (CIMP-3) and compared to the original method CIMP-1 and CIMP-2,identification accuracy is higher, and is more simple in experimental operation, costs are lower and more suitable for widely in scientific research and clinical application.