Spatial intratumor heterogeneity of genetic, epigenetic alterations and temporal clonal evolution in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is among the most common malignancies, but little is known about the spatial intratumor heterogeneity (ITH) and the temporal clonal evolutionary processes in this cancer. To address this, we performed multiregion whole-exome sequencing on 51 tumor regions from 13 ESCCs, and multiregion global methylation profiling on three of these 13 cases. We found an average of 37.1% heterogeneous somatic mutations, with strong evidence of spatial ITH. Nearly half of driver mutations located on the branches (late events) targeted oncogenes, including PIK3CA, NFE2L2, MTOR and FAM135B. Notably, a number of driver variants detected as clonally dominant within some individual tumor regions, such as PIK3CA (M1043I) and KIT (E601K), were absent in others from the same individual, indicating that they might not be suitable as drug targets inhibiting the growth of all the tumor cells in the entire tumor. By contrast, the majority of truncal and clonal driver mutations (early events) occurred in tumor suppressor genes, including TP53, NOTCH1, KMT2D, ZNF750, etc. Genes affected by early events, such as ERBB4, FGFR2, BRCA2, ATM and TP53, might potentially be utilized for targeted therapy in ESCC. In addition, we detected that the APOBEC signature was more prominent in truncal mutations compared with branched ones, demonstrating the temporal dynamics of the mutagenic processes in ESCC. Interestingly, the epigenetic profiling also showed strong evidence of spatial ITH, and the phyloepigenetic trees were extremely similar with the phylogenetic ones, indicating the interplay and co-dependency of genetic and epigenetic alterations in ESCC. We found that several genes were both mutated and hypermethylated at their promoters, such as ASXL1 and EPHA7. Our integrated investigations of the spatial ITH and the temporal clonal evolution might provide insights into developing biomarkers for early diagnosis of ESCC, as well as personalized therapeutic targets for treating this malignancy.