Comparative analyses of chromosome alterations in soft-tissue metastases within and across patients with castration-resistant prostate cancer

Abstract

Androgen deprivation is the mainstay of therapy for progressive prostate cancer. Despite initial and dramatic tumor inhibition, most men eventually fail therapy and die of metastatic castration-resistant (CR) disease. Here, we characterize the profound degree of genomic alteration found in CR tumors using array comparative genomic hybridization (array CGH), gene expression arrays, and fluorescence in situ hybridization (FISH). Bycluster analysis, we show that the similarity of the genomic profiles from primary and metastatic tumors is driven by the patient. Using data adjusted for this similarity, we identify numerous high-frequency alterations in the CR tumors, such as 8p loss and chromosome 7 and 8q gain. By integrating array CGH and expression array data, we reveal genes whose correlated values suggest they are relevant to prostate cancer biology. We find alterations that are significantly associated with the metastases of specific organ sites, and others with CR tumors versus the tumors of patients with localized prostate cancer not treated with androgen deprivation. Within the high-frequency sites of loss in CR metastases, we find an overrepresentation of genes involved in cellular lipid metabolism, including PTEN. Finally, using FISH, we verify the presence of a gene fusion between TMPRSS2 and ERG suggested by chromosome 21 deletions detected by array CGH. We find the fusion in 54% of our CR tumors, and 81% of the fusion-positive tumors contain cells with multiple copies of the fusion. Our investigation lays the foundation for a better understanding of and possible therapeutic targets for CR disease, the poorly responsive and final stage of prostate cancer.

Figure 1

Similarity of the tumors of a given patient. Trees from hierarchical clustering of segmented data from (A) the CR tumors and (B) the CR tumors, LocPCs, and normal prostate stromal tissue. In both trees, the patient number precedes the organ description, and numerical suffixes indicate multiple tumors from the same organ. Each color in A indicates tumors from a different patient; in B, the three sample types are each shaded with a different color. The third panel (C) shows heat maps by chromosome of the CBS segment data for each of the 7 tumors from patient 10. Red denotes negative segment values (regions of copy-number loss), and blue denotes positive segment values (regions of copy-number gain). Note the expected relative loss of X-chromosome sequences and gain of Y chromosome in this male:female comparison. For each chromosome, the vertical black line within each box indicates centromere position, and the large gaps without data indicate unsurveyed repetitive regions. The y-axis indicates the sample: A, adrenal metastasis; Li, liver metastasis; 1–4, the four lymph-node metastases; and S, spleen metastasis. The color bar below the figure indicates the range of colors representing the segment values shown.

Figure 2

Deviations in CR disease. The y-axis indicates the adjusted frequency with which each BAC clone on the array was included in a deviant segment across the 54 CR tumors from 14 individuals. Gains are plotted in blue, and losses are plotted in red. The hatched grey lines are drawn at a frequency of 0.3 (i.e., the minimum frequency for significance p < 0.01). The x-axis is the genomic position of each BAC clone. Note that loss in X- and gain in Y-chromosome sequences are expected in these male:female comparisons.

Figure 3

Deviations in CR tumors stratified by organ of origin. (A) The adjusted frequencies of deviation (y-axis) for gain (blue) or loss (red) for prostate tumors (n=15), lymph-node (n=19), and liver (n=9) metastasis, respectively. (B) Zoom in for gains on chromosomes 5, 6, 8, 11, and 16 and losses on chromosome 22. Alterations significantly associated with lymph node (brown) and liver (green) metastases are indicated by arrows.

Figure 4

Experimental designs to detect the presence of the TMPRSS2:ERG fusion by FISH. In Experiment 1, three probes were used to detect the fusion: a probe 5′ of TMPRSS2 (blue, probe 1), one encompassing the 5′ exons of ERG (red, probe 2), and one encompassing the 3′ exons of ERG (green, probe 3). In the normal configuration, the signals of all three probes overlap. The fusion is indicated by overlapping signals of probes 1 and 3 with loss or dissociation of probe 2. A CR tumor nucleus with two fusions and one normal probe configuration is shown to the right. In Experiment 2, we confirmed the presence of fusion with probe 1 and a second probe encompassing the 3′ exons of TMPRSS2 (red, probe 4) in adjacent tissue sections. Lone probe 1 signals indicate a deletion consistent with a TMPRSS2:ERG fusion. The panel to the right shows a positive nucleus from a section adjacent to the section used to capture the nucleus shown for Experiment 1. DAPI staining is indicated in gray; the hybridization signals are pseudocolored to correspond to the experimental schematics.