Screening, Validation And Application Of Genes Associated With Sensitivity To Neoadjuvant Chemoradiotherapy In Rectal Cancer Patients

ObjectiveThis study aimed to initially discover novel genes associated with neoadjuvant chemoradiosensitivity in rectal cancer by using microarray analysis. After identifying the mRNA and protein expression of those selected genes by real-time quantitative PCR and immunohistochemistry respectively, we expected to find valuable markers for predicting response to neoadjuvant chemoradiotherapy (NCRT) in rectal cancer.MethodsPart 1. We first enrolled sixteen rectal cancer patients receiving NCRT, dividing them into two groups according to tumor pathological regression. There were 8 patients in the complete pathological regression (pCR) group and 8 in the non-complete pathological regression (npCR) group. Then we carried out an exploratory screening of candidate genes by using Human Genome GeneChip plus U133 2.0 Array with pretreatment biopsies. Significance Analysis of Microarrays (SAM) software was utilized to select differential expression genes between pCR and npCR groups. Finally, we applied DAVID Bioinformatics Resources 6.7 online database to perform KEGG pathway and GO analysis.Part 2. We chose another ninety-five patients, including 27 patients in the pCR group and 68 in the npCR group. Sixty-three pretreatment biopsies from these patients were using for RT-qPCR test, and fifty-three formalin-fixed paraffinembedded (FFPE) biopsies for immunohistochemistry test. Data were analyzed by spss 19.0 software. Results Part 1. Through microarray analysis,839 genes were found differentially expressed between groups, involving 362 high expression genes and 477 low expression genes in the pCR group. We obtained 9 associated pathways and 95 associated biological processes of the differentially expressed genes from KEGG analysis and GO analysis. The former ones included Antigen processing and presentation, Insulin signaling pathway, Ribosome and so on. The latter ones included translation, immune response, apoptosis, protein transportation, inflammatory response and so on. Furthermore, by limiting the P value and fold change value, CHFR、CXCL10、CXCL9、GBP1、HOXB8、HPGD and PLA2G7 were selected from the most differentially expressed genes.Part 2. RT-qPCR were performed to test the differential expression of the above 7 genes between pCR and npCR groups in a large sample size. The results of student t-test showed that the mean mRNA expression of CHFR、CXCL9、HOXB8、 HPGD and PLA2G7 were not statistically significant between groups (p>0.05), except for CXCL10 and GBP1 (p<0.05). Then, the data were ranked based on percentile groups:low expression for cases below the 50th percentile and high expression for cases above the 50th percentile. By chi-square test, the mRNA expression CHFR.. CXCL10 and GBP1 were identified statistically significant in the pCR and npCR groups. This result indicates that patients with high expression CHFR、CXCL10 or GBP1 are more likely to respond to NCRT (OR:3.13, 95% CI:1.00-9.77; OR:4.45,95% CI:1.36-14.59; OR:3.90,95% CI: 1.19-12.84, respectively). The diagnostic performances of CHFR、CXCL10 and GBP1, as assessed by the ROC curve, showed area under the curve of 0.68, 0.72 and 0.69, respectively. There were no statistically significant differences of CXCL10 and GBP1 protein expression between pCR and npCR groups.Conclusion1. It is meaningful for finding valuable genes to predict sensitivity to NCRT in rectal cancer patients and acknowledging their biological function and signaling pathway by using microarray technology with subsequent RT-qPCR test. Moreover, the new discovery may be a cornerstone for having knowledge of the function and molecular mechanisms of those genes in rectal cancer patients with NCRT.2. It is the first time to report the newly found 7 genes associated with response to NCRT in rectal cancers. Most importantly, the mRNA expressions of CHFR、 CXCL10 and GBP1 may be potential markers to predict sensitivity to NCRT in rectal cancers.3. Compared with protein expression, the mRNA expressions of CXCL10 and GBP1 may be more sensitive to predict response to NCRT in rectal cancers.