Aneuploidy, TP53 mutation, and amplification of MYC correlate with increased intratumor heterogeneity and poor prognosis of breast cancer patients

Abstract

The clinical course of breast cancer varies from one patient to another. Currently, the choice of therapy relies on clinical parameters and histological and molecular tumor features. Alas, these markers are informative in only a subset of patients. Therefore, additional predictors of disease outcome would be valuable for treatment stratification. Extensive studies showed that the degree of variation of the nuclear DNA content, i.e., aneuploidy, determines prognosis. Our aim was to further elucidate the molecular basis of aneuploidy. We analyzed five diploid and six aneuploid tumors with more than 20 years of follow-up. By performing FISH with a multiplexed panel of 10 probes to enumerate copy numbers in individual cells, and by sequencing 563 cancer-related genes, we analyzed how aneuploidy is linked to intratumor heterogeneity. In our cohort, none of the patients with diploid tumors died of breast cancer during follow-up in contrast to four of six patients with aneuploid tumors (mean survival 86.4 months). The FISH analysis showed markedly increased genomic instability and intratumor heterogeneity in aneuploid tumors. MYC gain was observed in only 20% of the diploid cancers, while all aneuploid cases showed a gain. The mutation burden was similar in diploid and aneuploid tumors, however, TP53 mutations were not observed in diploid tumors, but in all aneuploid tumors in our collective. We conclude that quantitative measurements of intratumor heterogeneity by multiplex FISH, detection of MYC amplification and TP53 mutation could augment prognostication in breast cancer patients.

Keywords: FISH; MYC amplification; TP53 mutations; aneuploidy; breast cancer; intratumor heterogeneity.

Figure 1

Quantitative DNA content measurements, histology, and multiplex FISH results for the diploid case D4 (A) and the aneuploid cases A5 (B) and A3 (C). The DNA histograms show the quantitative measurements of the nuclear DNA content, and the percentage of cells in G1, S, and G2/M phases of the cell cycle. The histology of each case is based on H&E staining. Color display of miFISH analysis with eight gene-specific probes. Copy number counts for each nucleus are displayed as gains (green), losses (red) and unchanged (blue). Markers are plotted vertically with the “Locus” column depicting the specific chromosome arm for each probe. Nuclei are plotted horizontally by pattern frequency. Each vertical line discerns specific gain and loss patterns and how prevalent these clones are in the population. The instability index and the average ploidy were calculated as described in Material and Methods.

Figure 2

Patterns of clonal evolution of cases D4 (A), A5 (B) and A3 (C). We show the percentage of dominant imbalance clones and their derivations. Clones derived by a single gain or loss change are connected by arrows. In (C) we also denote specific gain and loss patterns to the right of each clone. Clones that are not connected by arrows must have undergone more than one gain or loss change. Note the greatly increased genomic instability in aneuploid cases (B, C).

Figure 3

FISHtrees analysis of case A3. The FISHtrees analysis shows the clonal evolution of tumor A3. Details of the analysis are described in Materials & Methods. FISH patterns are depicted in the following gene order COX2, DBC2, MYC, CCND1, CDH1, TP53, HER2, ZNF217. The size of the nodes reflects the frequency of the patterns in the cell population. The blue labeled nodes indicate the presence of the major diploid clone (pattern 22221222) and a smaller diploid clone (pattern 22212222) from which a genome duplication (labeled in pink, pattern 44422444) might have possibly originated leading in turn to the emergence of a triploid clone (pattern 44422224) and its progeny, labeled in green.