Comparative lesion sequencing provides insights into tumor evolution

Abstract

We show that the times separating the birth of benign, invasive, and metastatic tumor cells can be determined by analysis of the mutations they have in common. When combined with prior clinical observations, these analyses suggest the following general conclusions about colorectal tumorigenesis: (i) It takes approximately 17 years for a large benign tumor to evolve into an advanced cancer but <2 years for cells within that cancer to acquire the ability to metastasize; (ii) it requires few, if any, selective events to transform a highly invasive cancer cell into one with the capacity to metastasize; (iii) the process of cell culture ex vivo does not introduce new clonal mutations into colorectal tumor cell populations; and (iv) the rates at which point mutations develop in advanced cancers are similar to those of normal cells. These results have important implications for understanding human tumor pathogenesis, particularly those associated with metastasis.

Fig. 1.

Major genetic alterations associated with colorectal tumorigenesis. See SI Methods for further explanation.

Fig. 2.

Representative examples of sequencing chromatograms of DNA from a xenograft, from the metastatic lesion from which the xenograft was derived, and from the patient's normal cells. Note that the ratio of the mutant to wild-type allele in the xenograft is higher than that in the metastatic lesion because the latter represented a mixture of neoplastic and nonneoplastic cells (stroma, white blood cells, etc.). The arrow points to the mutated base.

Fig. 3.

Histopathology of representative lesions. (A) Primary invasive moderately differentiated adenocarcinoma (enclosed by black boundary) arising in a tubular adenoma (enclosed by red boundary) from patient 10. (B) Primary invasive moderately differentiated adenocarcinoma (enclosed by black boundary) with adjacent nonneoplastic colonic mucosa (enclosed by red boundary) from patient 2. (C) Metastatic adenocarcinoma (enclosed by black boundary) to liver (enclosed by red boundary) derived from primary colon adenocarcinoma of patient 2. All sections were stained with H&E, and the tissues within each boundary were separately microdissected.

Fig. 4.

Representative examples of BEAMing assays from the indicated patients and lesions. In patient 13, the mutation shown represents one that was present in a new metastasis that occurred 29 months after chemotherapy (see Application to Individual Patients). The red dots correspond to beads attached to mutant DNA fragments [labeled with phycoerythrin (PE)], the blue dots correspond to beads attached to WT DNA fragments [labeled with fluorescein (FITC)], and the black dots correspond to beads attached to both WT and mutant DNA fragments.

Fig. 5.

Evolution of a lethal cancer. Each cell-filled cone represents one or more clonal expansions (see SI Methods for details). The times required for the evolution of the large adenoma founder cell to an advanced carcinoma founder cell (Δ_LAd,ACa) and evolution of the advanced carcinoma founder cell to metastatic founder cell (Δ_ACa,Met) were determined by comparative lesion sequencing. Other intervals, such as the time (T_exp) required for the expansion of the metastasis founder cell FCell_Met to the size detected in our patients, were estimated as described in SI Methods. The model posits that there are at least two clonal expansions, denoted by question marks, that are not associated with any known genetic alterations.