Study On The Discovery And Detection Of Targets For Cancer Therapy And Drug Resistance Based On The Molecular Evolutionary Analysis

This study focuses on the study of pharmacogenomics in three aspects of cancer precision medicine,including cancer therapy,diagnostics and detection method.On the one hand,the somatic mutation characteristics of individul cancer patient was analysed by pharmacogenomic technology,which helps to get a deeper view of tumor heterogeneity so as to search for treatment countermeasure to overcome the challenge of tumor heterogeneity.On the other hand,it was also based on the study of pharmacogenomics,this study aims to find more potential biomarkers for cancer drug targets and diagnostic biomarkers by the functional analysis of the gene family which are important drug target genes.Finally,this study established the detection method of three drug resistant mutations based on the liquid biopsy,which shows a way to achieve clinical transformation from theoretical analysis for pharmacogenomic research.1.Analysis of evolutionary characteristics for the somatic mutations among a gastric carcinomaWith the development of sequencing technology and molecular biology as well as bioinformatics,we have a deeper view of the development of cancer,which drives cancer therapy into the era of precision medicine.Tumor heterogeneity is an urgent challenge for cancer precision therapy which usually leads to the fails of cancer treatment and causes drug resistance.Many studies have pointed out that trunk mutations are in all tumor cells which are better targets against tumor heterogeneity,thus,tumor cells can be killed more widely.In this study,we applied multiregional(n=6)whole-exome sequencing(WES)to investigate the genetic heterogeneity and homogeneity of a case of gastric carcinoma.Exonic nonsynonymous mutations and variant allele frequency(VAF)were mainly analyzed.This analysis helps to get further understanding of tumor heterogeneity and predict the trunk mutations.Furthermore,we conducted a secondary analysis of a published dataset on a different cancer type,which was a multiregional WES of 48 regions from eleven resected lung adenocarcinomas.The results show that each sample had different mutational landscape,while trunk mutations accounted for nearly half of all nonsynonymous somatic mutations for each sample.Private branch mutations show heterogeneity,whereas trunk mutations indicate homogeneity.The analysis of gastric carcinoma indicates that in any two samples,the proportion of trunk mutations among the shared mutations is approximately 83%on average.Approximately 89%of shared mutations is trunk mutations in any three samples.Besides,the VAFs of most trunk mutations are much higher than branch and private mutations.Similarly,the analysis of lung adenocarcinomas also gives out the consistent results that 86.29%of the exonic nonsynonymous SNVs were trunk mutations on average,more than 82%of the shared mutations of any two samples and more than 90%of the shared mutations of any three samples are trunk mutations.Our study suggests that small-scale multiregional sampling and subsequent screening of low-VAF somatic mutations might be a cost-effective strategy for identifying trunk mutations for individual cancer patient.And it helps to provide potential biomarkers as tumor biomarkers and therapeutic targets,which is of critical importance for diagnosis and treatment of cancer.It’s also helpful in the development of individualized therapy of cancer.2.Functional divergence analysis of ErbB gene family and prediction of functional sitesDrug resistance is a big problem in the clinical drug application,and the target mutations are one of the resistant mechanisms.It’s necessary to find functional sites like activating mutation sites and resistant mutation sites,which can be targets for cancer targeted therapy and biomarkers in cancer diagnostics.Searching for potential targets plays a key role in cancer precision therapy and overcoming the drug resistance.Epidermal growth factor gene family members are important targets in cancer therapy,for example,EGFR is the target of tyrosine-kinase inhibitors like gefitinib and erlotinib,and monoclonal antibodies for EGFR are cetuximab and panitumumab,besides,ERBB2/HER2 is the target of trastuzumab and pertuzumab.This study conducted the evolutionary functional divergence analysis of ErbB gene family accompanied with conservation analysis to predict important functional sites.We finally predicted 145 critical functional divergence amino acid sites,there are several functional sites have been reported.For example,EGFR Y275A and R748P influence the autophosphorylation of the kinase and then increase the activity.Besides,EGFR L833V and L858R were reported as activating mutations.ERBB2 P780ins is also an activating mutation,but ERBB2 K333A is a resistant mutation to pertuzumab.ERBB3 Y868E was reported to change the autophosphorylation of kinase,which is like mutations ERBB4 L864R and R927Q.Results of the conservation analysis indicate that there are 94 conserved amino acid sites in EGFR gene family and 345 conserved amino acid sites in individual gene averagely.Five conserved sites in gene family(the alignment sites are 1117,1119,1121,1122 and 1124)have been reported as ATP binding sites,which demonstrates that conserved sites are responsible for certain important common function.Among these conserved sites,EGFR G719 is conserved in gene family and G719S is activating mutation.What’s more,EGFR L858 and L833 are critical functional divergence sites,and sites like L858、L833、L861、T790 as well as K745 are conserved in EGFR,EGFR L858R、L833V and L861Q were reported as activating mutations while EGFR T790M and K745A were reported as resistance mutation and loss of function mutation respectively.Additionally,ERBB2 sites like S310,K333 and L755 are conserved in ERBB2.ERBB2 K333 is one of functional divergence sites,which was reported to largely reduce the binding of pertuzumab when substituted with alanine,and ERBB 2 S310F was also reported to be resistance mutation like K333A.Also,ERBB2 L755S and S310F were reported to be activating mutations.As for conserved sites in ERBB4,D861 is conserved in gene family,D861Y mutation has been reported to lead to loss of kinase activity.ERBB4 L864R and R927Q also change the autophosphorylation of kinase.Upon sites described above that have been reported,we can speculate that mutations on conserved sites may lead to activating mutations or resistance mutations.The reported sites,including critical functional divergence amino acid sites and conserved sites above,are functional sites,other sites predicted by our study,which have not been reported yet are also potential functional sites,and may also generate loss of function mutations,activating mutations or resistance mutations when mutated.This study is helpful to find more potential functional sites of ErbB gene family members.On the one hand,it gives a hint for drug design to reduce side effect,which results from lack of specificity.Broad spectrum drugs could also be designed according to the conserved sites among genes.On the other hand,our prediction of functional sites could provide potential targets for cancer precision therapy and potential biomarkers for diagnostics.3.Detection of EGFR drug resistant mutations based on simulated cfDNADrug resistance is inevitable during the cancer targeted treatment,which reduces the sensitivity to therapy and leads to fails of therapy or cancer relapse.It’s a big challenge in clinical cancer therapy and it’s urgent to overcome this problem.In recent years,many studies have found that EGFR S492R,G465E and G465R are resistance mutations to cetuximab and panitumumab in colorectal cancer treatment.Detection of these resistance mutations is necessary for the clinical medication guide of metastatic colorectal cancer treatment.This study constructed three plasmids with EGFR S492R,G465E and G465R mutations respectively,and then specific primers were designed based on the length of cfDNA for detection of three resistance mutations by ARMS-qPCR.PCR specificity was further increased by modifying the condition of annealing temperature.Results show that the optimal annealing temperature to detect EGFR S492R、G465E and G465R mutations are 620C、63 ℃ and 62 ℃ respectively.And the sensitivity to detect these three mutations at plasmid level is 0.1%.Moreover,we collected 7 plasma samples from cancer patients to extract the cfDNA and then conducted the fragment analysis of cfDNA to confirm the length again.The fragment analysis result indicates that the length of cfDNA is indeed in the range of 160-180bp.The genomic DNA of H460 cell line was taken as wild type template,and the mutated plasmids were taken as mutated type templates.Both of them were interrupted randomly to simulate cfDNA.At the simulated cfDNA level,the detection sensitivity of EGFR S492R、G465E and G465R reach 1%,0.5%and 0.5%,respectively.The detection method is more cost-effective than probe technology with a high sensitivity.