| Objective:Lung cancer is the most common malignancy worldwide.Radiotherapy is one of the important means of comprehensive treatment of lung cancer.How to evaluate the curative effect of radiotherapy in time and accurately is of great significance to the formulation of individualized treatment strategy for lung cancer.It is difficult to distinguish the active tumor tissues from the fibrosis and internal hemorrhage and cystic changes after radiotherapy by traditional imaging examination,and it is also difficult to respond to the small residual lesions,so it is difficult to respond to the tumor treatment effect at an early stage.Tumor biomarkers are of limited value in evaluating efficacy due to their low sensitivity and specificity.As one of the three major means of liquid biopsy,circulating tumor DNA contains all the information gene mutations,tumor cell has overcome defects biopsy tumor heterogeneity,real-time monitoring,can the advantages of the high frequency monitoring,peripheral blood circulating tumor DNA tests can radiation sensitivity in patients with tumor and tumor mutation load monitoring,at the same time can be to drive tumor gene and high frequency gene screening and analysis.The purpose of this study was to preliminarily explore the correlation between circulating tumor DNA mutations and radiotherapy sensitivity in peripheral blood of patients with non-small cell lung cancer(NSCLC)by detecting the characteristics and gene spectrum of circulating tumor DNA mutations.Methods:Prospectively,some patients with NSCLC who were initially treated in sichuan tumor hospital from 2017 to 2019 and had evaluable primary lesions and required radiotherapy were included.Fasting elbow venous blood was collected in the morning at 10 ml,centrifuged at 4 °5000rpm for 10 min,and supernatant was collected to enrich serum circulating tumor DNA.Using Agilent Sure Select Human All Exon V6 kit circulating tumor DNA separation,preparation of DNA library,the specificity of the index of library with the cords labeled probe for liquid-phase hybridization,streptomycin magnetic beads capture exons,linear amplification by PCR library after quality inspection qualified after Illumina Hi Seq PE150 sequenced,than their reference genome,detect the variation information,obtain mutation characteristic spectrum,and screening for cancer drive gene and the high frequency spectrum genes.At the same time,the tumor volume shrinkage rate before and after radiotherapy was evaluated,and then the normalized radiotherapy dose was used to obtain the bioequivalent shrinkage rate(tumor shrinkage rate/bioequivalent dose).The correlation between mutation characteristics and gene spectrum and bioequivalent shrinkage rate was further analyzed.Results:1.Clinical into groups: A total of 35 patients into the group,Among them,7 patients chose to give up treatment before treatment for various reasons,and 5 patients chose to give up treatment during treatment but did not complete the radiotherapy plan,finally complete a total of 23 patients with radiation therapy plan,1 cases in the treatment of severe lung infection,appeared in the process of pleural effusion,radiotherapy was suspended,after waiting for infection control intermittent finish radiotherapy,total radiation duration > 8 weeks,the other 2 cases within 1 week before the start of radiotherapy completed platinum based dual drug combination chemotherapy.All patients signed the informed consent before into research,in order to avoid the platinum chemotherapy drugs to tumor mutation load,gene encoding and potential influence of driver mutations,and severe infection radiotherapy interruption time is too long influence on tumor regression,when we proceed analysis of treatment,screen except 2patients who underwent chemotherapy of sample and 1 case of radiation therapy interruption time is too long,for the rest of the samples of 20 cases were analyzed,and all the samples are calculated separately,and the equivalent withdrawal rate,tumor indirectly evaluate tumor sensitivity to radiation therapy.2.General characteristics of mutations: the sequencing results showed that the mutations of peripheral blood circulating tumor DNA mainly included Single nucleotide variant,small fragment insertion/deletion mutations and copy number variations.Among the Single nucleotide variant,the majority were missense mutations in the exon coding region,accounting for about 70%,while synonymous mutations accounted for about 25%.The non-synonymous mutations and unknown mutations caused by insertion,deletion or continuous base substitution that resulted in a new termination codon or loss of termination codon at the mutation site accounted for only about 5%.Some intronic and intergnic were mutated except the mutation of the gene exon region.However,the UTR region,the non-coding RNA region and the 4b P region around the splicing site had relatively few mutations.The insertion/deletion of small gene fragments is very rare,mainly manifested as exon coding region shift/or non-shift insertion/deletion,some intronic mutations are also seen,and other mutations are rare.There are two types of copy number variation: duplication and deletion.The sequencing results showed that both types of copy number variation were present,and duplication and deletion mostly occurred at different sites in the same sample,but there was no absolute positive correlation between the number of variation and the size of the region.From the overall evaluation of tumor mutation load,it was found that with the radiotherapy,tumor mutation load also appeared or increased or decreased.In addition to screening for cancer gene expression profile found six susceptibility genes,15 drive gene and high-frequency 16 genes,at the same time to all gene mutation frequency and mutation characteristic analysis,found that point mutations are given priority to with C>T/G>A,further line loss of heterozygosity analysis,enrichment of genetic correlation analysis,path analysis and high frequency CNV analysis,etc.3.Radiotherapy sensitivity analysis:Samples,20 patients with tumor equivalent withdrawal rate sensitive point(average 30% /biological equivalent dose)was 0.43%,the lowest of 0.19%(including representative tumor progression),the highest is 1.52%,the sample is divided into sensitive and resistance groups,respectively to observe the gene mutation characteristics of two groups of samples,and radiation sensitivity analysis,found that the total number of single nucleotide mutation,the number of non-synonymous mutations,the number of mutations,non-coding RNA UTR region splice site mutation,four bp number of mutations,cancer mutation,gene copy number variation load type and number of no statistical differences were found in the group of sensitive and resistance,However,the size of the total region of gene copy number variation,especially the size of the region of copy number duplication,was statistically different in the sensitive group and the resistance group(P<0.05),and showed a negative correlation.At the same time,radiosensitivity analysis was also carried out on the screening susceptibility genes,driver genes and high-frequency genes,and it was found that there was no statistical difference in the expression of these genes in the sensitive group and the resistance group.Conclusion:1.The mutation feature of circulating tumor DNA is mainly represented by the missense mutation in the exon coding region.Gene copy number variation is another important mutation feature,and small fragment insertion/deletion is rare.2.The total number of mononucleotide mutations,missense mutations,non-synonymous mutations,non-coding RNA mutations,UTR regional mutations,4bp regional mutations at the shear site and tumor mutation load were all independent of radiotherapy sensitivity.None of the known tumor driver genes and high-frequency genes showed any correlation with radiotherapy.The size of the region of gene copy number variation,especially the size of the repetitive region,was negatively correlated with the radiation sensitivity of tumor,that is,the larger the region of copy number duplication was,the lower the sensitivity of radiotherapy was. |
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