Breast tumor copy number aberration phenotypes and genomic instability

Abstract

Background: Genomic DNA copy number aberrations are frequent in solid tumors, although the underlying causes of chromosomal instability in tumors remain obscure. Genes likely to have genomic instability phenotypes when mutated (e.g. those involved in mitosis, replication, repair, and telomeres) are rarely mutated in chromosomally unstable sporadic tumors, even though such mutations are associated with some heritable cancer prone syndromes.

Methods: We applied array comparative genomic hybridization (CGH) to the analysis of breast tumors. The variation in the levels of genomic instability amongst tumors prompted us to investigate whether alterations in processes/genes involved in maintenance and/or manipulation of the genome were associated with particular types of genomic instability.

Results: We discriminated three breast tumor subtypes based on genomic DNA copy number alterations. The subtypes varied with respect to level of genomic instability. We find that shorter telomeres and altered telomere related gene expression are associated with amplification, implicating telomere attrition as a promoter of this type of aberration in breast cancer. On the other hand, the numbers of chromosomal alterations, particularly low level changes, are associated with altered expression of genes in other functional classes (mitosis, cell cycle, DNA replication and repair). Further, although loss of function instability phenotypes have been demonstrated for many of the genes in model systems, we observed enhanced expression of most genes in tumors, indicating that over expression, rather than deficiency underlies instability.

Conclusion: Many of the genes associated with higher frequency of copy number aberrations are direct targets of E2F, supporting the hypothesis that deregulation of the Rb pathway is a major contributor to chromosomal instability in breast tumors. These observations are consistent with failure to find mutations in sporadic tumors in genes that have roles in maintenance or manipulation of the genome.

Figure 1

Frequency plot of copy number alterations in ER positive and negative tumors. The top two panels show the frequency of gains, indicated by the green bars ranging from 0 to 1, and losses, indicated by the red bars ranging from 0 to -1, in 62 sporadic breast tumors for each clone. The bottom panel displays the magnitude of the t-statistic for each clone computed based on the smoothed data as described in the Methods. The horizontal dotted lines indicate the statistic cut-off corresponding to the FDR-adjusted p-value of 0.05 (blue) and 0.1 (green).

Figure 2

Analysis of TP53 mutation in breast tumors. Frequency plot of copy number changes in TP53 mutant and wild type tumors. The top two panels show the frequency of gains, indicated by the green bars ranging from 0 to 1, and losses, indicated by the red bars ranging from 0 to -1, in 62 sporadic breast tumors for each clone. The bottom panel displays the magnitude of the t-statistic for each clone computed based on the smoothed data as described in the Methods. The horizontal dotted lines indicate the statistic cut-off corresponding to the FDR-adjusted p-value of 0.01 (red), 0.05 (blue) and 0.1 (green). Thus, copy number alterations occurring more frequently in TP53 mutant tumors included losses of regions on 3p, 4q, 5q, 15q, 17q and gain of a small region on distal 8q.

Figure 3

Genomic analysis of breast tumors reveals three subtypes. A. Hierarchical clustering of 62 ductal invasive breast tumors and five BRCA1 mutant tumors based on their genome-wide DNA copy number profiles. Individual clones are represented as rows, ordered by chromosome and genome position according to the July 2003 freeze of the human genome. Clones on the p-arm and q-arm of chromosomes are indicated in shades of dark gray (odd numbered chromosomes) or light gray (even numbered chromosomes). Acrocentric chromosomes are shown in dark or light gray. Columns represent individual tumor samples. The estrogen receptor status of the tumors is shown in shades of blue (dark blue = ER negative, light blue = ER positive), BRCA1 mutant tumors are indicated in orange, and TP53 mutation status is indicated with a maroon box for TP53 mutant tumors, a gray box for tumors with no detected mutation and a black box if the TP53 status is unknown. Copy number losses are indicated in red, gains in green and amplifications as yellow dots. Three main clusters are evident. B-F. Genome-wide copy number aberrations profiles of sporadic and hereditary (BRCA1) breast tumors are plotted as the normalized log₂ratio for each clone sorted by chromosome and ordered according to genome position from the p-arm to the q-arm. Normalized copy number ratios of genomic DNA are shown for a tumor from the 1q/16q cluster with few copy number changes including gain of 1q and loss of 16q (B), a tumor from the ER negative, complex cluster showing many low level chromosome changes and few amplifications (C), a tumor from the amplifier cluster with low level gains and losses and amplifications (D) and tumors from patients with mutations in BRCA1 (E and F). G. Numbers and types of copy number aberrations in breast tumor subtypes. The mean numbers of whole chromosome copy number changes, copy number transitions and amplifications were determined for the tumors within each subtype. H. Numbers and types of copy number aberrations in breast tumor subtypes. The mean numbers of chromosomes showing no copy number change, whole chromosome copy number changes, copy number transitions, copy number transitions at centromeres and amplifications were determined for the tumors within each subtype. I. Association with disease-specific survival. Significance of the log-rank test was used to assess the association between a genomic subclass and survival phenotypes. The significance was declared at p < 0.05. Patients with complex tumors experienced significantly worse outcome compared to the other groups.

Figure 4

Copy number changes more frequently associated with one subtype. Frequency for each clone of gains and losses, which were uniquely present in more than 50% of samples of one subtype and in less than 30% of samples in other subtypes. Gains are indicated by green bars, ranging from 0 to 1, and losses, by the red bars ranging from 0 to -1 for each clone.

Figure 5

Overview of most frequent DNA amplifications on chromosomes 8, 11, 17 and 20 in 62 breast tumors determined by genome-wide array CGH. A. Heat map depiction of aberrations on chromosomes 8, 11, 17 and 20 and typical chromosome copy number profiles showing amplifications of 8p (including FGFR1), 11q13 (including CCND1), 17q (ERBB2) and regions on 20q (including ZNF217). Note that the chromosome 11 copy number profiles vary depending on whether amplification of chromosome 8 is also present. In both cases copy number losses distal to the amplicon are observed, however in the absence of chromosome 8 amplification (right), the region of loss extends distally from the amplified region, whereas, when chromosome 8 was amplified (left), the copy number loss includes regions proximal as well as distal to CCND1. B. For each tumor, the numbers of copy number transitions is compared to the number of chromosome arms with at least one amplification.

Figure 6

Telomere length measurements in 28 breast tumor samples. Plotted is the telomere length determined by Southern blotting relative to the number of chromosome arms with amplification. A significant inverse correlation is evident.

Figure 7

Signaling pathway and E2F responsive genes associated with numbers of copy number transitions and amplifications. The receptor signaling cascade impinging on Rb includes a number of up-stream genes that are frequently altered in cancers and result in deregulation of the E2F family of transcription factors through inhibition or loss of Rb function. Here, we show genes enriched for association with numbers of copy number transitions (FDR < 0.05). Genes, which have been identified as E2F targets (asterisk) and/or are correlated with E2F1 expression levels (|Pearson correlation| > 0.3) are shown in the top box. Genes which showed less correlation with E2F1 are shown in the bottom box. Genes also associated with numbers of amplifications (FDR < 0.05) are highlighted in yellow. Four additional genes associated with amplifications (FDR < 0.05) are ASCIZ, MSH5, RAE1 and TDG. Red, increased expression. Blue, decreased expression.