The Research On Genetic Heterogeneity Of Colorectal Cancer In Yunnan

Colorectal cancer(CRC)is one of the most common malignant tumors in human.With the development of economy and the improvement of people's living standard,the change of lifestyle and dietary structure in our country,the incidence of CRC is increasing year by year and threating to the people's physical and mental health.CRC patients from Yunnan Province are usually exposed to a variety of ethnic,geographical environment and climate.Because the unique characteristics of intestinal microenvironment in Yunnan area,it may affect the incidence of CRC,tumor heterogeneity and gene mutation spectrum.EGFR Targeted therapy can prolong the survival time and is currently the best treatment for metastatic CRC.However,tumor genetic heterogeneity is one of the important features of human malignant tumors.The tumor heterogeneity often leads to drug resistance.The tumor heterogeneity is determined by the pH,oxygen and glucose concentrations in the tumor microenvironment.The result may be false negative due to the heterogeneity.Thereby,it affects the clinic assessment of the patients.It is important to understand the heterogeneity of CRC for the diagnosis and treatment of CRC.Gene sequencing is the major method for detecting genetic diseases.However,this kind of method is time-consuming,laborious and expensive.Next-generation semiconductor sequencing technology(Ion torrent PGM)can simultaneously sequence multiple target genes with high throughput and low cost.In this study,we analyzed the mutation of 329 genes in the 10 samples(different spatial locations)and normal tissue from 3 cases of Yunnan CRC patients based on the Illumina NGS sequencing platform.There was the pathological heterogeneity in primary CRC and liver metastatic tumors.70.4-75%of the intra-tumor gene mutations were same in 2 cases of CRC patients.For the case with liver metastasis,87.9%of the intra-tumor gene mutations between the primary CRC and the liver metastasis lesion.These findings suggest that there are some differences in gene mutation in different spatial locations.The differences may be caused by microenvironment factors,such as the pH,oxygen,nutriation,and cytokines in different positions.While 59 gene mutations were detected in 3 cases of CRC patients,only 9 mutations were the same.The results suggest that the gene mutation differences are large among different patients,providing a theoretical basis for personized treatment.To measure the KRAS gene mutation rate in the Yunnan CRC patients,we analyzed mutations of codon 12 and 13 in KRAS gene and identified 59 cases of mutations in 247 CRC patients.The mutation rate was 23.9%.The mutations were G12D,G12V,G12A,G12C,G12R,and G13D.There was no significant correlation between these mutations and the clinical features,including sex,age,primary tumor site,differentiation degree,clinical stage and TNM stage.These findings suggest that KRAS gene mutation may be a very early event in the pathogenesis of CRC.Following that,we analyzed mutations and the heterogeneity of KRAS,BRAF,and PIK3CA genes in 144 samples from 26 CRC patients(2 patients with liver metastases).The KRAS gene mutation was detected in 7 patients(26.9%).The PIK3CA gene mutation was detected in 2 cases(7.7%).Both KRAS and PIK3CA gene were mutated in 1 case.No BRAF gene mutations were detected in all samples.No mutations of these genes were detected in the liver metastases of 2 cases.There was no KRAS gene mutation heterogeneity among 5 lesions in 4 patients.Only three patients showed intra-tumor heterogeneity.Of the 2 patients with PIK3CA mutations,one case showed heterogeneity.These findings laid a foundation for clinical diagnosis and treatment of CRC patients.We deduced that mitochondria DNA(mtDNA)may be more susceptible to the tumor microenvironment.To further characterize the heterogeneity of CRC patients,mutations of mitochondria DNA and the heterogeneity were also studied.We analyzed 88 samples from 22 patients with CRC in Yunnan and found that 10 cases(45.5%)had mutations in the mtDNA D-loop region.The mutation sites were HVSI 16114,16166,16248,16278,16304,16362,and 16390 as well as HVS? and ? 71,309,and 573.The most polular mutation site was 309.Simultaneous mutations in HVSI,? and ? were identified in 2 cases.Only one mutation was detected in other 8 patients(4 cases in HVSI and 4 cases in HVS ?/?).Among 10 patients with mtDNA D-loop mutations,9 cases showed intra-tumor heterogeneity.Compared to chromosome DNA mutations,mtDNA showed a higher degree of genetic heterogeneity.However,functional consequences of these mtDNA mutations need further investigation.In conclusion,The CRC patients have not only the inter-heterogeneity of gene mutation,but also the intra-heterogeneity in Yunnan area.The most of the genes mutations are not consistent in individuals,however the same mutation genes was also found the difference in the mutation site.At the same time,the heterogeneity of mutations was 9.7%-25%in different locations,which provided theoretical support for the individualized treatment.The of KRAS gene mutation was 26.9%in Yunnan area.The rate was 11.5%of in intra-heterogeneity.However,The reports found that the rate was 8%and 36%of.These findings may explain EGFR targeted therapy failed sometimes even although KRAS and PIK3CA genes were detected as wild type.We found that mtDNA showed a higher degree of genetic heterogeneity compared to chromosome DNA mutations.It may be more sensitive to the tumor microenvironment.There are genetic heterogeneity in CRC patients in terms of gene mutations of KRAS,PIK3CA.Genetic heterogeneity occurs among not only patients but also tumors in the same patient.These findings may explain EGFR targeted therapy failed sometimes even although KRAS and PIK3CA genes were detected as wild type.It is strongly recommended to take multiple samples from the same tumor for genetic screening in order to accurately predict drug response in CRC patients.