High Levels of Chromosomal Copy Number Alterations and TP53 Mutations Correlate with Poor Outcome in Younger Breast Cancer Patients

Abstract

Prognosis in young patients with breast cancer is generally poor, yet considerable differences in clinical outcomes between individual patients exist. To understand the genetic basis of the disparate clinical courses, tumors were collected from 34 younger women, 17 with good and 17 with poor outcomes, as determined by disease-specific survival during a follow-up period of 17 years. The clinicopathologic parameters of the tumors were complemented with DNA image cytometry profiles, enumeration of copy numbers of eight breast cancer genes by multicolor fluorescence in situ hybridization, and targeted sequence analysis of 563 cancer genes. Both groups included diploid and aneuploid tumors. The degree of intratumor heterogeneity was significantly higher in aneuploid versus diploid cases, and so were gains of the oncogenes MYC and ZNF217. Significantly more copy number alterations were observed in the group with poor outcome. Almost all tumors in the group with long survival were classified as luminal A, whereas triple-negative tumors predominantly occurred in the short survival group. Mutations in PIK3CA were more common in the group with good outcome, whereas TP53 mutations were more frequent in patients with poor outcomes. This study shows that TP53 mutations and the extent of genomic imbalances are associated with poor outcome in younger breast cancer patients and thus emphasize the central role of genomic instability vis-a-vis tumor aggressiveness.

Figure 1

Mutation analysis and multiplex interphase fluorescence in situ hybridization (miFISH) results presented for the entire cohort, with corresponding color codes. A: Summary of the clinicopathologic features (rows), plotted per individual case (columns). The patients were divided into two groups characterized by short and long survival. B: Distribution of mutations that affected genes in at least two cases. The color indicates the type of mutations. C: miFISH analysis of eight breast cancer–associated genes. Dark hues indicate gains and losses seen in a major clone, light hues indicate copy number alterations observed only in a minor clone.

Figure 2

Patients with short survival. Multiplex interphase fluorescence in situ hybridization (miFISH) results (A and E), histology (B and F), image cytometry (C and G), and imbalance clone plots (D and H) for case A6 (A–D) and case A14 (E–H). A and E: Color display of miFISH analysis with eight gene-specific probes. Copy number counts for each nucleus are displayed as gains (green), amplifications (dark green), losses (red) and neutral (blue). Markers are plotted horizontally with the Locus column depicting the specific chromosome arm for each probe on the left of the plot, and the corresponding gene name on the right. Nuclei are plotted vertically by pattern frequency. Each vertical line discerns specific gain and loss patterns and the prevalence of these clones in the population. The Gain and Loss columns refer to the percentage of nuclei in which a gain or loss was observed. The ASN column denotes the mean signal number. Color-labeled percentages indicate a 15% threshold of gain or loss. The Instability Index and the mean ploidy were calculated as described in Materials and Methods. B and F: Hematoxylin and eosin–stained sections showing the histomorphology of the respective breast cancers. C and G: Histograms of the quantitative measurements of the nuclear DNA content. The DNA histograms show the quantitative measurements of the nuclear DNA content (x axis) of the tumor cells given in c units. Based on the 2c stemline, the percentages of cells in the G1, S phase and exceeding the G2/M phase of the cell cycle are determined. The brackets visualize the area of the corresponding cells in the G2/M phase. The y axis represents the total cell count; between 100 and 200 tumor cells were measured per case. D and H: Imbalance clone plots visualizing the clonal composition and their putative evolutionary trajectory. The areas of the circles reflect the percentages with which these clones are present. Clones derived by a single gain or loss change are connected by arrows. The arrows indicate the clonal evolution according to gain and loss patterns in the color charts. Clones that are not connected by arrows must have undergone > one gain or loss. Color coding allows for the assignment of the individual clones to the corresponding clones in the color displays in A and E. Scale bars = 200 μm (B and F).

Figure 3

Patients with long survival. Multiplex interphase fluorescence in situ hybridization (miFISH) results (A and E), histology (B and F), image cytometry (C and G), and imbalance clone plots (D and H) for case B3 (A–D) and case B15 (E–H). A and E: Color display of miFISH analysis with eight gene-specific probes. Copy number counts for each nucleus are displayed as gains (green), losses (red), and neutral (blue). Markers are plotted horizontally with the Locus column depicting the specific chromosome arm for each probe on the left of the plot, and the corresponding gene name on the right. Nuclei are plotted vertically by pattern frequency. Each vertical line discerns specific gain and loss patterns and the prevalence of these clones in the population. The Gain and Loss columns refer to the percentage of nuclei in which a gain or loss was observed. The ASN column denotes the mean signal number. Color-labeled percentages indicate a 15% threshold of gain or loss. The Instability Index and the mean ploidy were calculated as described in Materials and Methods. B and F: Hematoxylin and eosin–stained sections showing the histomorphology of the respective breast cancer that has been analyzed. C and G: Histograms of the quantitative measurements of the nuclear DNA content. The DNA histograms show the quantitative measurements of the nuclear DNA content (x axis) of the tumor cells given in c units. Based on the 2c stemline, the percentages of cells in the G1, S phase and exceeding the G2/M phase of the cell cycle are determined. The brackets visualize the area of the corresponding cells in the G2/M phase. The y axis represents the total cell count: between 100 and 200 tumor cells were measured per case. D and H: Imbalance clone plots visualizing the clonal composition and their putative evolutionary trajectory. The areas of the circles reflect the percentages with which these clones are present. Clones derived by a single gain or loss change are connected by arrows. The arrows indicate the clonal evolution according to gain and loss patterns in the color charts. Clones that are not connected by arrows must have undergone > one gain or loss. Color coding allows for the assignment of the individual clones to the corresponding clones in the color displays in A and E. Scale bars = 200 μm (B and F).

Figure 4

FISHtrees analysis of case A6. The FISHtrees result shows the clonal evolution. Details of the analysis are described in Materials and Methods. FISH patterns are depicted in the following gene order: MT-CO2 (COX2), RHOBTB2 (DBC2), MYC, CCND1, CDH1, TP53, ERBB2 (HER2), and ZNF217. The size of the nodes reflects, but is not proportional to, the frequency of the patterns in the cell population. The blue-labeled node indicates the presence of the major tetraploid clone (pattern 6-2-13-3-3-1-4-4) and a smaller tetraploid clone (pattern 6-2-13-3-3-1-4-3). The pink clone (pattern 12-4-20-6-6-2-8-8) reflects a whole-genome duplication event.

Figure 5

Results of the multiplex interphase fluorescence in situ hybridization (miFISH) analysis showing the frequency of copy number alterations (CNAs) and Instability Indices. A: Total number of CNAs of the short and long survival groups. Note the significant difference between the subgroups. B: Instability indices of the ploidy subgroups, calculated as described in Materials and Methods. Note the significant difference between aneuploid and diploid cases. C: Percentage of cases showing a gain or loss in the eight targeted genes. The differences in MYC, CCND1, and ZNF217 are significant between the aneuploid and diploid subgroups. Data are expressed as medians (ranges) and outliers. ∗P < 0.05.