Multi-region Sequencing Revealed That TP53 Drives The Intratumoral Heterogeneity Of Somatic Mutations In Patients With Non-small Cell Lung Cancer

Lung cancer remains the most common in modality and the leading cause of cancer death so far the world[1],of which more than 80% were Non-small cell lung cancer(non-smallcell lung cancer,NSCLC)[2].Most patients with NSCLC will relapse,metastasis,and show drug resistance following initial treatment.The current view is that the tumor cell heterogeneity is the cause of treatment failure[3].In order to confirm the presence of clonal heterogeneity of NSCLC,we chose NSCLC patients wearing a recognized pathogenic mutations of the TP53 to study intratumor heterogeneity.We selected 12 patients with a known TP53 mutation,and performed whole genome or whole exome sequencing.With bioinformatics analysis,a total of 223 significantly mutated genes were found.We then performed laser capture microdissection,and get 61 cutting portion.Then we conducted multiplex PCR amplification and sequencing using a secondgeneration sequence for the mutation points of 223 genes on 61 cutting tissues.After different part of the VAF(variant allele frequency)on the same mutation points were analyzed by phylogenetic tree and hierarchical clustering analysis,we found the prevalence of tumor heterogeneity in patients with non-small cell lung cancer.And we found that,generally accepted TP53 gene that caused NSCLC did not exist in every part of microdissection.This suggests that TP53 gene mutation is not an early molecular events.In addition,our research found that many mutations VAF are greater than 75%,suggesting a homozygous mutation in tumor.In order to explore the reasons of homozygous mutation,we selected two known tumor suppressor gene TP53,CDKN2 A,and performed loss of heterozygosity(LOH,loss of heterozygosity)verification in one patient,the results showed that the presence of loss of heterozygosity difference in different parts of the same tumor sample,and the allele deletions likely occur earlier than mutation events.This result provides strong evidence for Professor Knudsen's "two-hit" theory[4].