Intratumor heterogeneity in localized lung adenocarcinomas delineated by multiregion sequencing

Abstract

Cancers are composed of populations of cells with distinct molecular and phenotypic features, a phenomenon termed intratumor heterogeneity (ITH). ITH in lung cancers has not been well studied. We applied multiregion whole-exome sequencing (WES) on 11 localized lung adenocarcinomas. All tumors showed clear evidence of ITH. On average, 76% of all mutations and 20 out of 21 known cancer gene mutations were identified in all regions of individual tumors, which suggested that 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 after surgery, three patients have relapsed, and all three patients had significantly larger fractions of subclonal mutations in their primary tumors than patients without relapse. These data indicate that a larger subclonal mutation fraction may be associated with increased likelihood of postsurgical relapse in patients with localized lung adenocarcinomas.

Fig. 1

Assessment of ITH of 11 lung adenocarcinomas by multi-region sequencing. An example of regional mutation distribution (case 330) of resected tumors (represented by the ellipsis) is shown at the up left corner. Mutated cancer genes are indicated to the right of representative H&E image of each sequenced tumor region. Numbers of trunk, branch and private branch mutations for each region are indicated in associated windows. A phylogenetic tree was generated from all validated mutations utilizing Wagner parsimony method in PHYLIP. Blue, yellow and red lines represent trunk, branch and private branches respectively. Trees are anchored at germline DNA sequence obtained from peripheral blood of the relevant patients. Known cancer gene mutations are mapped to the trunks and branches as indicated. Point mutations, amplifications and deletions of known cancer genes are presented as black, +red and −green respectively. Trunk and branch lengths are proportional to the numbers of mutations acquired on the corresponding trunk or branch. Note: 5 tumors have their trunk lengths reduced to 10%^S or 2%^SS of original length for visualization purposes.

Fig. 2

Distribution of trunk, branch and private branch mutations defined by exome sequencing (average sequencing depth of 277×) versus deep sequencing (average sequencing depth of 863×). Only validated mutations meeting the following criteria are included: total counts in tumor DNA≥100; total counts in germ line DNA≥50; variant allele frequency (VAF) ≥5% in tumor DNA and VAF=0 in germ line DNA.

Fig. 3

Mutation spectrum of the 11 lung adenocarcinomas. (A) Mutation spectrum of all validated mutations. (B) Mutation spectrum of trunk mutations. (C) Mutation spectrum of non-trunk mutations. The difference of mutation spectrum between trunk and non-trunk mutations in each patient was evaluated with Fisher’s exact test and significant p values are shown as *(P<0.05) and **(P<0.01). (D) APOBEC mutation signature enrichment odds ratio for trunk and non-trunk mutations. 95% confidence intervals for Fisher’s exact test are indicated. PY: pack year. ¶: Patient had cut down to 2 cigarettes a day at the time of cancer diagnosis.