Deciphering the promotional determinants of esophageal cancer in countries with varying incidence

Esophageal squamous cell carcinoma (ESCC) incidence varies widely across the world, correlating with exposure to known risk factors like alcohol, smoking, and hot liquids. It has traditionally been assumed that such exposures lead to malignant cell transformation by directly or indirectly causing DNA mutations. However, recent evidence revealed that most ESCC risk factors do not elicit distinct mutation profiles, suggesting that these carcinogens may act as non-mutagenic tumor-promoting agents. This study aims to investigate the role of exogenous exposures in promoting the clonal expansion of pre-initiated cells in normal esophageal tissue, leading to ESCC. To address this, we are examining the clonal structure of 200 esophageal epithelium samples from ESCC patients and non-cancer donors across nine geographical regions in China, Iran, Malawi, South Africa, Russia, Brazil, and Canada, with age-standardized ESCC incidence rates ranging from 1 to 84 per 100,000. Detailed demographic, lifestyle, and exposure information, including tobacco, alcohol, and hot liquids, is available. Esophageal epithelial tissues are dissected and analyzed by NanoSeq for mutational signature and mutation burden evaluation, and by 500x whole-exome sequencing to identify mutant genes under positive selection. Spatial proteomics and transcriptomics are ongoing to profile the phenotype of premalignant cells and their microenvironment niches. Preliminary analyses of 97 cases from high- and intermediate-risk regions revealed a strong mutagenic effect of alcohol and tobacco on normal esophageal tissue. Alcohol-related mutational signatures SBS16 and ID11 were detected in 73% of drinkers (22/30), and were enriched in cases exposed to both alcohol and tobacco. In contrast, hot liquid consumption was not related to distinct mutation profiles. dN/dS analysis in 47 cases with available WES data identified 11 genes under positive selection, including NOTCH1 , TP53 , FAT1 , and PPM1D . Cases exposed to tobacco presented higher fractions of mutated epithelia in several of these cancer genes. Notably, the fraction of NOTCH1 -mutant epithelia was inversely associated with ESCC incidence, suggesting a reduction of clones harboring this protective mutation in high-risk populations. These findings shed light on the complex interplay between mutagenic and promotional processes in ESCC development and provide insights into the role of known and suspected risk factors in clonal selection. This work may ultimately guide preventive strategies targeting the earliest stages of esophageal carcinogenesis.