Inferring tumor progression from genomic heterogeneity

Abstract

Cancer progression in humans is difficult to infer because we do not routinely sample patients at multiple stages of their disease. However, heterogeneous breast tumors provide a unique opportunity to study human tumor progression because they still contain evidence of early and intermediate subpopulations in the form of the phylogenetic relationships. We have developed a method we call Sector-Ploidy-Profiling (SPP) to study the clonal composition of breast tumors. SPP involves macro-dissecting tumors, flow-sorting genomic subpopulations by DNA content, and profiling genomes using comparative genomic hybridization (CGH). Breast carcinomas display two classes of genomic structural variation: (1) monogenomic and (2) polygenomic. Monogenomic tumors appear to contain a single major clonal subpopulation with a highly stable chromosome structure. Polygenomic tumors contain multiple clonal tumor subpopulations, which may occupy the same sectors, or separate anatomic locations. In polygenomic tumors, we show that heterogeneity can be ascribed to a few clonal subpopulations, rather than a series of gradual intermediates. By comparing multiple subpopulations from different anatomic locations, we have inferred pathways of cancer progression and the organization of tumor growth.

Figure 1.

Sector-Ploidy-Profiling (SPP) approach. The SPP approach separates tumor subpopulations by macro-dissection and cell sorting by ploidy. (A) Macro-dissection of tumor sectors. (B) Sorting of DAPI-stained nuclei using FACS by differences in total genomic DNA content. (C) Profiling of chromosome breakpoints across the genome by ROMA CGH. (D) Calculation of neighbor-joining trees using copy number profiles. (E) Coalescence of highly similar copy number profiles. (F) Topography of subpopulations in the tumor. Tumor sectors S7–S12 are colored according to the adjacent subpopulations in S1–S6.

Figure 2.

Summary of Sector-Ploidy-Profiling (SPP) results for tumors T5–T20. (A) Monogenomic tumors. (B) Polygenomic tumors. Tumors were cut into four to six sectors. Nuclei were isolated from each sector and sorted by FACS according to differences in total genomic DNA content. DNA content is plotted on the x-axis (calibrated with a normal diploid control with a DNA index 1.0). Tumor sectors are plotted on the y-axis (S1–S6). Filled blocks indicate FACS peaks. Colors represent different subpopulations as distinguished by their CGH profiles: (blue) hypodiploid; (green) normal diploid; (orange, red, purple) distinguishable aneuploid tumor subpopulations. The total number of colors used in the schematic of a given tumor is the same as the total number of subpopulations distinguished in that tumor. For example, tumor T12 contains four subpopulations: one diploid subpopulations present in all sectors, one hypodiploid subpopulation present only in sectors 1–3, one aneuploid subpopulation present only in sectors 4–6, and a second aneuploid subpopulation present only in sectors 5–6.

Figure 3.

Distance trees of copy number profiles. Neighbor-joining trees were constructed from distance trees by calculating 1-correlation matrices of all copy number profiles in a single tumor. (Green) The trees were rooted with a single coalesced diploid profile. (Green) Monogenomic tumors; (red) polygenomic tumors. (Red, yellow, blue) The leaves are colored to show different subpopulations as determined by comparing ROMA copy number profiles. (A) Tumor trees with a minimum correlation coefficient > 0.9. (B) Tumor trees with a minimum correlation coefficient < 0.9. (C) Distance trees of all tumor profiles without a diploid root node. Two trees were calculated separately: one from 85K experiments (T4–T14) and one from the 390K experiments (T15–T20).

Figure 4.

Focal lesions that differ between subpopulations in single tumors. Segmented log ratio CGH data from coalesced tumor profiles are plotted in genome order. (A) Tumor T8 contains three focal amplifications, including the amplification of the PPP1R12A locus on Chr12q21, which is present in the A2 tumor subpopulation (red), but absent in A1 (yellow). (B) Tumor T10 contains a focal amplification of the KRAS locus on Chr12p12.1, which is present in the A2 tumor subpopulation (red), but absent in A1 (yellow). T8 also contains a homozygous deletion of the EFNA5 and FER locus on Chr5q21.3 in the (red) A2 subpopulations that is hemizygously deleted in A1 (yellow). (C) Tumor T19 contains a focal amplification of the PTPN2 locus on Chr18p11.21, which is present in the A2 subpopulation (red), but absent in A1 (yellow). T19 also contains a focal amplification of the MCM10 locus on Chr10p13 in the A1 tumor subpopulation that is absent in A2.

Figure 5.

Genomic progression from hypodiploid to hyperaneuploid. Coalesced, segmented copy number profiles are ordered in increasing numbers of chromosome breakpoints. The topography of the subpopulations in the tumor sectors is shown with a white vector to indicate the direction of progression. FACS histograms are shown with the gated subpopulation highlighted in color. (A) Tumor T10 progresses from diploid (D) (green) to hypodiploid (H) (blue), to hyperaneuploid (A1) (yellow), to hyperaneuploid (A2) (red), as the number of chromosome breakpoints increases. (B) Tumor T12 progresses from diploid (D) (green) to hypodiploid (H) (blue) to hyperaneuploid (A1) (yellow). (C) Illustration of the clonal expansion of subpopulations that occur as the tumor grows.

Figure 6.

Regional amplification of the KRAS locus. Tissue sections from sectors 1–6 from tumor T10 are hybridized with a single FISH probe specific to the KRAS locus. (B–G, left) The topography of each tumor sector from which the tissues sections are cut. The log ratio and segmented copy number data of the KRAS amplification are also shown for each tumor sector. (A) Ideogram showing the cytobands and location of the KRAS FISH probe on chromosome 12p12.1. (B–D) Tissue sections from sectors 1–3 show two or three copies of the KRAS locus in the stromal and tumor cells. (E) Sector 4 contains a majority of tumor and stromal cells with two or three copies of the KRAS locus; however, one tumor cell shows a massive amplification of the KRAS locus. (F–G) Sectors 5 and 6 show numerous tumor cells with a high copy number of KRAS as a homologous staining region intermixed with other stromal and tumor cells that contain two or three copies of the KRAS locus.

Figure 7.

Intermixing of tumor subpopulations in tissue sections. A FISH probe strategy was used to mark chromosomes that are differentially amplified in two tumor subpopulations (A1 and A2) in tissue sections from sector 5 and sector 6 of T10. (A) Tumor T10 contains four sectors (S11, S12, S5, S6) with similar FACS histograms. The FACS histogram from sector 5 is shown and contains one diploid peak (green) and two aneuploid peaks that were gated and analyzed by CGH (yellow and red). (B) Segmented copy number data are plotted with FISH probes annotated to show the strategy for distinguishing the diploid cells from the A1 and A2 tumor subpopulations. The MYC probe on chromosome 8q24.21 (orange) detects two copies in the diploid cells and three copies in both of the tumor subpopulations (A2 and A3). LCON (purple) and RCON (blue) are control FISH probes on Chr12p12.1 that report two copies in all of the subpopulations. The KRAS (red) and ETNK (green) probes report six to 10 copies in the A2 subpopulation, but not in A1. (C,D) Tissue sections from T10 sector 5 show three types of cells: D diploid, A1 tumor cells, and A2 tumor cells. Diploid cells contain two copies of all of the probes. A1 tumor cells contain three copies of MYC and two copies of the other probes. The A2 tumor cells display a bright yellow signal resulting from the colocalization of the KRAS and ETNK probes, which are present in high copy number. (E,F) DAPI channels are false-colored to show the location of the three cell types: D (green), A1 (yellow), and A2 (red) in the tissue sections from panels C and D. The three cell types are stochastically intermixed in the tissues.