Genomic Alterations In Multistep Development Of Esophageal Squamous-Cell Carcinoma

Researchers have found that somatic mutations are prevalent in morphologically normal human tissues and organs and accumulate with age.Among them are cancerassociated driver mutant genes,suggesting that cancer may derive from the driver mutant clones which evolve under the interaction of intra and extra environments,become positively selected and invasively proliferate without control.Accompanied with the process are more driver mutations,genome instabilities and large-scale chromosomal structure aberrations,leading to malignant phenotype with subsequent poor prognosis.Even the intervention strategies are applied as soon as possible,the transformation cannot stop and go ahead irreversibly.Therefore,we need to focus on the earlier stage of carcinogenesis and eliminate the clones with malignant potential as soon as possible.China is the hardest hit area for esophageal squamous cell carcinoma(ESCC),and about 50%of the new cases of ESCC in the world occur in China every year.The development of invasive ESCC usually takes multiple precancerous steps,recognized as intraepithelial neoplasia(IN).Based on the proportion of dysplastic cells in mucosa,IN is further categorized into low-grade intraepithelial neoplasia(LGIN)and high-grade intraepithelial neoplasia(HGIN).The multi-step process renders itself an ideal model to study cancer clone evolution.Disclosure of the key molecular events in this progress will help us deepen the understanding of tumorigenesis and shed new light on the early diagnosis and prevention of ESCC.Through a cancer screening program in a high-incidence area of China,we collected 42 esophageal biopsy samples and matched peripheral blood from 28 individuals with high risk for ESCC.Each fresh frozen tissue was consecutively dissected into 2-5 cryosections and the histopathological features were identified under microscopy.On each slide,microbiopies containing～600 cells were densely collected based on the histological structure by using laser-capture microdissection(LCM)technique.After conducting whole-exome sequencing(WES,mean coverage 36.1 x),low-depth whole-genome sequencing(WGS,mean coverage 0.8 x)and subsequent quality control,a total of 1,275 micro-biopsies at normal,LGIN,HGIN and ESCC stages were included in downstream analysis.We detected potential cancer driving events including single nucleotide variations(SNVs),small insertions and deletions(indels)and genome-wide copy-number alterations(CNAs),thus depicting genomic alteration landscape of the multi-stage ESCC initiation process.We found that mutation burden accumulates with age in normal esophagus,while CNA burden does not show correlation with age.Somatic mutation and CNA burden increase extensively by pathological stage,similar with the pattern of TP53 mutation and loss-ofheterozygosity(LOH)frequencies.After the reconstruction of the phylogeny in the same individual,we demonstrated that clones with TP53 biallelic loss occur at the very early stage of ESCC initiation,accompanied with higher mutation burden and more extensive CNAs.Furthermore,we focused on the specific amplified genes in clones with multi-hit TP53(biallelic loss or multiple mutations).Integrating our previous ESCC single-cell and bulk RNA-seq data.we identified 605 amplified genes which were also upregulated in ESCC than normal esophagus.These genes enriched in cell cycle and DNA damage repair pathways and were further validated by CRISPR screening experiments.We also identified 195 deleted genes which were also downregulated in ESCC than normal esophagus.Gene pathway analysis show that these genes enriched in P53 and cell apoptosis pathways.indicating an aberrant cell status after TP53 loss.In summary,we have conducted WES and WGS analysis and depicted a complete genomic alteration landscape of multi-step ESCC transition at clone-level resolution.We demonstrate that TP53 biallelic loss and the correlated CNA patterns are the early events of ESCC.We elucidate the dynamics of cancer clone evolution in both precancer and cancer,which provides crucial clues to the discovery of molecular markers for ESCC early diagnosis.