Abstract 3322: Chronology and risk-dependence of age-related remodelling of oesophageal epithelia

Clonal expansion in aged normal tissues has been implicated in cancer development. However, its chronology and risk-dependence are poorly understood. Esophageal squamous cell carcinoma (ESCC) is a predominant esophageal cancer among Asian populations and substantially affected by heavy smoking and drinking, likely through a ‘field effect’. To elucidate the role of these lifestyle risks on ESCC development, we investigated clonal expansion in physiologically normal esophageal epithelia (PNE) using multiple microscale sampling, as small as 0.2 mm2 in size, followed by an unbiased detection of somatic mutations with whole exome sequencing. Mutations were detected in most of PNE samples (151/157), where none of the mutations were shared between samples collected >10 mm apart. The number of mutations and their allele frequency increased with age, suggesting age-related clonal expansion in PNE (ARCE), which was significantly promoted by heavy smoking and drinking. Mutations were dominated by age-related patterns and a still poorly-defined, ‘esophagus-specific signature, as well as a COSMIC 16-like signature. The latter has recently been related to alcohol drinking and was enriched in high-risk samples, which was confirmed by whole genome sequencing of single cell-derived colonies. As many as 10 genes were significantly mutated or positively selected in ARCE. Among most commonly affected genes were NOTCH1, TP53, FAT1, PPM1D, NOTCH2, and NOTCH3, which substantially differed from those in ESCC, showing prominent over-representation of NOTCH1, PPM1D, FAT1 and NOTCH2, and significant underrepresentation of TP53, NFE2L2, and CDKN2A were significantly underrepresented, suggesting different mechanisms of positive selection between ARCE and ESCC. Driver mutations were detected more frequently and in higher numbers in high-risk PNE samples than low-risk ones, with accentuated NOTCH1, TP53 and PPM1D mutations. Analyses of densely collected micro-scale samples (0.2 mm2) disclosed fine structure of ARCE with its chronological history. Driver-mutated clones emerge multifocally from early childhood as early as