Analysis Of Genomic Variants And Single-Cell Transcriptomic Alterations Of Adenocarcinoma At The Gastroesophageal Junction

Adenocarcinoma at the gastroesophageal junction is normally mentioned as ACGEJ.In the past fifty years,the incidence of adenocarcinoma at the gastroesophageal junction has increased significantly worldwide.Compared with western countries,Japan,South Korea,and China in East Asia are the areas with a high incidence of ACGEJ,Genomic changes and transcriptomic abnormalities are important factors affecting tumorigenesis and prognosis.With the development of genomic sequencing,researchers have performed mufti-omics sequencing analysis on various types of tumor samples,but there are still gaps of genomic and transcriptomic studies of ACGEJ,lacking for description of functional characteristics of tumor cells and stromal cells in tumor microenvironment of ACGEJ.Therefore,it is the key point to elucidate related molecular mechanisms of tumorigenesis and prognosis in ACGEJ,using a variety of methods to systematically explore the genomic changes and transcriptomic abnormalities.In this study,we firstly performed the whole-genome sequencing and bulk RNA-sequencing of 124 ACGEJ tumor tissues and paired adjacent normal tissues.Then we compared ACGEJ data from Chinese population with genomic and transcriptomic data of 105 samples from TCGA database.We conducted genomic analysis on multiple dimensions such as clinically characteristics of ACGEJ patients,tumor mutation burden,driver genes of somatic mutations and copy number variation from the whole genome,as well as altered gene signaling pathways.The results showed that ACGEJ patients from our Chinese population and TCGA database has substantially similar genomic alterations.However,there were significant differences in activation frequencies of somatic driver mutations such as CDKN2A and KRAS,copy number variation driver genes such as CCNE1 and BCL2L1,as well as MARK pathways,homologous recombination pathways,and chromatin histone modification related pathways.Although studies of the whole-genome sequencing have identified molecular characteristics of ACGEJ at the multicellular level,tumor genomic alterations obtained by next-generation sequencing were not enough to fully elucidate biological mechanism of cancer development and progression of ACGEJ.Therefore,we have performed single-cell RNA sequencing of 39 ACGEJ tumor tissues and 2 cases of normal tissues adjacent to tumor,to explore the characteristics of ACGEJ tumor cells and tumor microenvironment at single-cell level.We obtained a total of 85,160 single cells including 23,715 CD45-cells and 56,865 CD45+cells.We identified 9 essential co-expression programs from malignant epithelial cells.Among them,two co-expression programs,growth program and digestion program,might be related to normal characteristics and functions of gastroesophageal epithelial cells.In addition,we have further classified T cells,B cells and fibroblasts into cell subtypes.Totally,we have obtained 7 CD8+T cell subtypes,6 CD4+T cell subtypes,6 B cell subtypes and 3 fibroblast types.We applied exhaustion score and Treg score to characterize T cells.Finally,as it was reported that patients with higher expression of IFI44 have better prognosis,we explored the relationship between IFI44 expression of ACGEJ malignant epithelial cells and epithelial co-expression programs as well as stromal cells in microenvironment.We have detected that patients with higher expression of IFI44 not only had more activated growth program and digestion program,but had significantly lower exhaustion score and Treg score of CD8+T cells and CD4+T cells respectively.Furthermore,normal mucosal fibroblasts(NMF)were enriched in patients with higher expression of IFI44,suggesting that there was a positive and activated immune microenvironment in ACGEJ patients with high expression of IFI44.In conclusion,this study has further filled in blanks of single-cell sequencing analysis at ACGEJ.It was the first time to describe ACGEJ characteristics at single-cell dimension and deepen our understanding of tumor microenvironment at ACGEJ,which could also provide valuable resource and theoretical support for ACGEJ studies of prognostic biomarkers and clinical treatments.