Intra-tumor Heterogeneity in Localized Lung Adenocarcinomas Delineated by Multi-region Sequencing

Cancers are composed of populations of cells with distinct molecular and phenotypic features, a phenomenon termed intra-tumor heterogeneity (ITH). ITH in lung cancers has not been well studied. We applied multi-region whole exome sequencing (WES) on 11 localized lung adenocarcinomas. All tumors showed clear evidence of ITH. On average, 76% of all mutations and 20/21 known cancer gene mutations were identified in all regions of individual tumors suggesting single-region sequencing may be adequate to identify the majority of known cancer gene mutations in localized lung adenocarcinomas. With a median follow-up of 21 months postsurgery, 3 patients have relapsed and all 3 patients had significantly larger fractions of subclonal mutations in their primary tumors than patients without relapse. These data indicate larger subclonal mutation fraction may be associated with increased likelihood of postsurgical relapse in patients with localized lung adenocarcinomas. Intra-tumor heterogeneity may have impacts on tumor biopsy strategy, characterization of actionable targets, treatment planning, and drug resistance (1–6). ITH has recently been elucidated in substantial detail in several cancer types using next-generation sequencing (NGS) approaches (7–14). Recent evidence supports a model of branched evolution leading to variable ITH in different tumors (9, 13, 15, 16). Studies in clear cell renal carcinoma (ccRCC) have demonstrated substantial ITH, with the majority of mutations in known cancer genes confined to spatially separated tumor regions except for VHL loss being a ubiquitous event (16, 17). These data suggest that a single biopsy may be inadequate for identifying all cancer gene mutations from a tumor, thus presenting an incomplete view of potential targets for therapy. Critically, the extent to which these observations in ccRCC apply to other solid tumors is currently not clear. To characterize ITH in localized lung adenocarcinomas, we applied multi-region WES on 48 tumor regions from 11 resected lung adenocarcinomas (8 stage I, 2 stage II and one stage III tumors, tumor size 2 – 4.6 cm), who had surgery with curative intent (Fig. 1 and Table S1). WES was conducted at mean depth of 277×. In total, 7,269 mutations were identified and 7,026 (97%) somatic mutations were validated by a separate bespoke capture sequencing experiment at mean depth of 863x (Table S2). The numbers of mutations varied substantially between tumors (Fig. S1), but no significant correlations were identified between mutation burden and age, gender, tumor size, lymph node status or smoking status. A useful approach when considering ITH is to depict a given tumor as a tree structure with the trunk representing ubiquitous mutations present in all regions of the tumor, branches representing heterogeneous mutations present in only some regions of the tumor and private branches representing mutations that are present only in one region of the tumor – analogous to a phylogenetic tree. Placement of mutations on trunks versus branches reflects relative molecular time of acquisition, with branch mutations occurring, by definition, subsequent to trunk mutations. We applied this approach to multi-region sequencing data from these 11 Zhang et al. Page 2 Science. Author manuscript; available in PMC 2015 October 10. A uhor M anscript