Temporal dissection of tumorigenesis in primary cancers

Abstract

Timely intervention for cancer requires knowledge of its earliest genetic aberrations. Sequencing of tumors and their metastases reveals numerous abnormalities occurring late in progression. A means to temporally order aberrations in a single cancer, rather than inferring them from serially acquired samples, would define changes preceding even clinically evident disease. We integrate DNA sequence and copy number information to reconstruct the order of abnormalities as individual tumors evolve for 2 separate cancer types. We detect vast, unreported expansion of simple mutations sharply demarcated by recombinative loss of the second copy of TP53 in cutaneous squamous cell carcinomas (cSCC) and serous ovarian adenocarcinomas, in the former surpassing 50 mutations per megabase. In cSCCs, we also report diverse secondary mutations in known and novel oncogenic pathways, illustrating how such expanded mutagenesis directly promotes malignant progression. These results reframe paradigms in which TP53 mutation is required later, to bypass senescence induced by driver oncogenes.

Keywords: Notch; cancer genetics; genomic; mutation; p53.

Figure 1

Loss of second TP53 wild-type allele precedes simple mutations in cutaneous squamous cell carcinomas and ovarian cancers. (A) Schematic illustrating acquisition of discretely higher copy number for mutations preceding a regional duplication, enabling temporal ordering of mutations during tumorigenesis. For representative samples, mutant allele frequency (y-axis) shown plotted against physical position on chromosome 17 (x-axis) for cutaneous squamous cell (B,C) and ovarian cancers (D). More than 50 independent reads were required for inclusion of a mutation. Estimated allele frequencies are shown as lines for heterozygous (solid) and homozygous (dotted) mutations (See Supplementary Methods and Appendix for details). Regions without CN-LOH in ovarian cancers often demonstrate complex ploidy, generating greater variance in mutation frequency; these simple frequency estimates were therefore not applied. Mutation frequencies are centered below 1.0 for homozygotes and 0.5 for heterozygotes because of the effects of partial dilution with non-tumorous cells (see Supplementary Methods and Appendix for more details).

Figure 2

Conceptual framework for temporal ordering of chromosomal aberrations. (A) Schematic for differential mutant allele frequencies in areas of copy-neutral loss-of-heterozygosity, based on relative age of chromosomal event during tumorigenesis. Earlier events allow greater time for accumulation of heterozygous mutations, whereas later events are predominated by homozygous mutations. Three events are ordered in a single sample, earliest on chromosome 17 (B), later on chromosome 2 (C), and last on chromosome 14 (D). Relative mutant allele frequency (y-axis) is plotted against chromosomal physical position (x-axis) for cutaneous squamous cell cancers. To the right of each panel, a histogram of mutant allele frequencies for each CN-LOH event shows density (y-axis) plotted against allele frequency (x-axis). A modified binomial distribution function (Supplementary Methods and Appendix) distinguishes the proportion of heterozygote and homozygote mutant frequencies ρ, ordering duplications in chromosome 17 (0.00–0.12), chromosome 2 (0.17–0.45), and 14 (0.65–0.94).

Figure 3

For two regional chromosomal aberrations, mutant allele frequencies reveal distinct evolutionary histories. On chromosome 11, two peaks in mutation frequency are seen, one for heterozygous and one for homozygous mutations, reflect a simple regional gain, including for a COSMIC mutation in WT1. In contrast, three separate frequencies are detected on chromosome 9, culminating in a triploid state for mutant CDKN2A and loss of all wild-type copies. Three discrete allele frequencies can only be achieved by two gains and one loss, with the highest frequency (triploid) peak representing the first gain, and the diploid peak containing mutations from the interval preceding the second gain. The disproportionate size of the triploid peak (p < 0.03) indicates that the first gain occurred more than halfway through in tumor evolution, as measured by the evolutionary clock, and the second gain even later (see Supplementary Methods and Appendix for more detail).