Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Sep 1;112(35):10995-1000.
doi: 10.1073/pnas.1508074112. Epub 2015 Aug 18.

Phylogenetic Analyses of Melanoma Reveal Complex Patterns of Metastatic Dissemination

Affiliations
Free PMC article

Phylogenetic Analyses of Melanoma Reveal Complex Patterns of Metastatic Dissemination

J Zachary Sanborn et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Melanoma is difficult to treat once it becomes metastatic. However, the precise ancestral relationship between primary tumors and their metastases is not well understood. We performed whole-exome sequencing of primary melanomas and multiple matched metastases from eight patients to elucidate their phylogenetic relationships. In six of eight patients, we found that genetically distinct cell populations in the primary tumor metastasized in parallel to different anatomic sites, rather than sequentially from one site to the next. In five of these six patients, the metastasizing cells had themselves arisen from a common parental subpopulation in the primary, indicating that the ability to establish metastases is a late-evolving trait. Interestingly, we discovered that individual metastases were sometimes founded by multiple cell populations of the primary that were genetically distinct. Such establishment of metastases by multiple tumor subpopulations could help explain why identical resistance variants are identified in different sites after initial response to systemic therapy. One primary tumor harbored two subclones with different oncogenic mutations in CTNNB1, which were both propagated to the same metastasis, raising the possibility that activation of wingless-type mouse mammary tumor virus integration site (WNT) signaling may be involved, as has been suggested by experimental models.

Keywords: genetics; melanoma; metastasis.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Patterns of somatic mutations (SSNVs) in primary and metastases illustrate evolutionary divergence during metastatic dissemination. For the seven of eight patients also yielding high-confidence copy number estimates, the number of SSNVs in each tumor tissue is shown. (A) The number of fully shared SSNVs present in all tumors is displayed in Lower (black). (B) The number of SSNVs not present in all samples is shown in the upper column graph. The SSNVs not present in all samples are further subdivided for each sample into tiers of SSNVs: those that are partially shared with other tumors (red), those that are private to the sample (present only in one tumor of a patient, gray), and those not detected in the sample (white). Tiers with fewer than three SSNVs are not displayed. The SSNVs within each tier are listed in Dataset S3 and their interpretation is annotated in Dataset S7.
Fig. 2.
Fig. 2.
Metastases depart the primary melanoma in parallel, from genetically divergent cell subpopulations. For patients A, C, D, E, F, and H, phylogenetic history of the metastasizing cells is reconstructed based on sequencing of fresh and formalin-fixed, paraffin-embedded (FFPE) portions of each tumor (Datasets S3, S6, and S8). Solid arrows denote probable dissemination routes, and dotted arrows denote multiple possible paths. Numbers in squares denote partially shared SSNVs. Instances of SSNVs deduced to be in the primary, but not detected directly by sequencing, are color coded by line of reasoning. The patterns of dissemination demonstrate that metastases in each patient derived from distinct cells in the primary, which often demonstrate extensive genetic divergence from each other.
Fig. 3.
Fig. 3.
Identical subclonal deletions in different metastases reveal multiple founding populations during metastatic dissemination. The scatter graphs chart show on the y axis, for tumors of patient E, the allelic fraction of heterozygous SNPs along chromosomes 3, 20, and 14. Shown are the primary tumor, three locoregional metastases, and a lymph node metastasis, ordered from top to bottom according to time of clinical presentation. Divergence of the allelic fractions from 0.5 indicates a copy number change. The resulting allelic states are shown in the red numbers underneath each segment. The red lines depict the expected allelic fraction, if the CNA were present in all cells of the tumor, taking into account the normal cell contamination. Blue lines indicate the observed average copy number level for each CNA. The 33.9-Mb region on chromosome 3 represented by the red circle shows a subclonal deletion in locoregional metastases 1 (TVP = 32.1%, 99%CI = 28.0–35.6%) and 2 (TVP = 91.9%, 99%CI = 90.4–93.2%) and fully clonal deletion in metastasis 3 (TVP = 100%) (Supporting Information and Datasets S5 and S9). Chromosome 20 shows two separate deletions reaching from 0–25.53 Mb and from 40.39–50.93 Mb, respectively, represented by the green diamond, which are present at fully clonal levels in all metastases but absent in the primary tumor. One entire copy of chromosome 14 is deleted at fully clonal levels in all tumors and is thus considered fully shared (yellow triangle). The presence of the deletion from 9.72–43.6 Mb of chromosome 3 at subclonal levels in locoregional metastases 1 and 2 suggests at least one of these tumors was founded by two distinct cell populations: one harboring the chromosome 3 deletion and one without.
Fig. 4.
Fig. 4.
Identical subclones in different metastases, as defined by SSNVs, reveal multiple founding populations during metastatic dissemination. The scatter graphs show, for patient C, the TVPs for all SSNVs in genomic regions not affected by copy number changes (Dataset S10). Shown are the TVPs for the primary tumor on the x axes and the locoregional metastases 1 (upper graph) and 2 (lower graph) on the respective y axes. Fully shared SSNVs are depicted as yellow triangles and are present in all tumors at fully clonal levels. A subclone present in ∼30% of the cells of the primary tumor (blue ×) is present at close to fully clonal levels (TVP = 100%) in both metastases. A second subclone with TVP of 25% in the primary (red diamonds) is fully clonal in metastasis 1 but at a TVP of 25% in metastasis 2, suggesting that metastasis 2 was seeded by at least two genetically distinct founding cells, one containing the SSNVs depicted as red diamonds and one without. A third subclone present at 3% in the primary melanoma, (green circles) is absent in metastasis 1 but present at ∼75% abundance in metastasis 2, indicating that it has contributed partly, but not entirely, to the cells of metastasis 2. This third subclone therefore also indicates that metastasis 2 was founded by two genetically distinct populations.
Fig. 5.
Fig. 5.
An integrated portrait of metastatic subclone formation, departure, and arrival for patient C. The ancestral cell harboring 855 SSNVs proliferated, generating the primary tumor. During expansion into the primary, a specific cell acquired 142 more SSNVs and then two cells from that subpopulation subsequently acquired 15 more (red) and 20 more (green) SSNVs. Intriguingly, each of these later-evolving subpopulations (red and green, identical to those seen in metastases) each acquires a different known oncogenic CTNNB1 mutation. Both subclones are seen in locoregional metastasis 2, suggesting that once the ability to metastasize is acquired, the competent subclone can either reach existing metastases or travel with other metastatic subclones simultaneously.
Fig. S1.
Fig. S1.
Median exome sequencing coverage. On-target median fold coverage for each sample in each patient.

Similar articles

See all similar articles

Cited by 40 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback