Tumor-associated copy number changes in the circulation of patients with prostate cancer identified through whole-genome sequencing

Abstract

Background: Patients with prostate cancer may present with metastatic or recurrent disease despite initial curative treatment. The propensity of metastatic prostate cancer to spread to the bone has limited repeated sampling of tumor deposits. Hence, considerably less is understood about this lethal metastatic disease, as it is not commonly studied. Here we explored whole-genome sequencing of plasma DNA to scan the tumor genomes of these patients non-invasively.

Methods: We wanted to make whole-genome analysis from plasma DNA amenable to clinical routine applications and developed an approach based on a benchtop high-throughput platform, that is, Illuminas MiSeq instrument. We performed whole-genome sequencing from plasma at a shallow sequencing depth to establish a genome-wide copy number profile of the tumor at low costs within 2 days. In parallel, we sequenced a panel of 55 high-interest genes and 38 introns with frequent fusion breakpoints such as the TMPRSS2-ERG fusion with high coverage. After intensive testing of our approach with samples from 25 individuals without cancer we analyzed 13 plasma samples derived from five patients with castration resistant (CRPC) and four patients with castration sensitive prostate cancer (CSPC).

Results: The genome-wide profiling in the plasma of our patients revealed multiple copy number aberrations including those previously reported in prostate tumors, such as losses in 8p and gains in 8q. High-level copy number gains in the AR locus were observed in patients with CRPC but not with CSPC disease. We identified the TMPRSS2-ERG rearrangement associated 3-Mbp deletion on chromosome 21 and found corresponding fusion plasma fragments in these cases. In an index case multiregional sequencing of the primary tumor identified different copy number changes in each sector, suggesting multifocal disease. Our plasma analyses of this index case, performed 13 years after resection of the primary tumor, revealed novel chromosomal rearrangements, which were stable in serial plasma analyses over a 9-month period, which is consistent with the presence of one metastatic clone.

Conclusions: The genomic landscape of prostate cancer can be established by non-invasive means from plasma DNA. Our approach provides specific genomic signatures within 2 days which may therefore serve as 'liquid biopsy'.

Figures

Figure 1

Implementation of our approach using plasma DNA samples from individuals without cancer and simulations. (a) Z-scores calculated for sequential 1-Mbp windows for 10 male (upper panel) and 9 female (lower panel) individuals without malignant disease. (b) Detection of tumor DNA in plasma from patients with prostate cancer using simulated copy-number analyses. ROC analyses of simulated mixtures of prostate cancer DNA with normal plasma DNA using the genome-wide z-score. Detection of 10% circulating tumor DNA could be achieved with a sensitivity of >80% and specificity of >80%. (c) Hierarchical cluster analysis (Manhattan distances of chromosomal z-scores) with normal female controls and the HT29 serial dilution series. One percent of tumor DNA still had an increased genome-wide z-score and did not cluster together with the controls (for details see text).

Figure 2

Outline of our whole-genome plasma analysis strategy. After blood draw, plasma preparation, and DNA-isolation we start our analysis, which is two-fold: first (left side of the panel), an Illumina shotgun library is prepared (time required, approximately 24 h). Single-read whole genome plasma sequencing is performed with a shallow sequencing depth of approximately 0.1x (approximately 12 h). After alignment we calculate several z-scores: a genome-wide z-score, segments with identical log2-ratios required to establish corresponding segmental z-scores, and gene-specific z-scores, for example, for the AR-gene. Each of these z-scores calculations takes approximately 2 h so that these analyses are completed within 48 h and the material costs are only approximately €300. Second (right side of the panel), we prepare a library using the SureSelect Kit (Agilent) and perform sequence enrichment with our GB-panel (approximately 48-72 h), consisting of 55 high-interest genes and 38 introns with frequent fusion breakpoints. The GB-panel is sequenced by paired-end sequencing with an approximately 50x coverage (around 26 h). The evaluation of the sequencing results may take several hours, the confirmation by Sanger sequencing several days. Hence, complete analysis of the entire GB-panel analysis will normally require around 7 days.

Figure 3

Copy number analyses of plasma samples from men with prostate cancer. (a) Z-scores calculated for 1-Mbp windows from the 13 plasma samples of patients with prostate cancer showed a high variability (compare with same calculations from men without malignant disease in Figure 1a, upper panel). (b) Hierarchical clustering (Manhattan distances of chromosomal z-scores) separates samples from men without cancer and with prostate cancer. (c) Copy number analyses, based on segmental z-scores, of an unmatched normal male plasma sample and five plasma samples from patients with prostate cancer (CRPC2, CRPC3, CRPC5, CSPC2, and CSPC4). The Y-axis indicates log2-ratios.

Figure 4

Identification of the TMPRSS2-ERG associated 3-Mb deletion on chromosome 21 and mapping of the breakpoints. Exemplary log2 ratio plots of chromosome 21 from plasma DNA of several patients (regions with log2 ratios >0.2 are shown in red and those with log2 ratios <-0.2 in blue). A deletion with size of 3 Mbp located at the TMPRSS2-ERG region was visible in patients CRPC1, CRPC5, CSPC4, and CSPC1. For comparison we also included chromosome 21 plots from CSPC2 and CRPC2 without this deletion. Mapping of the exact breakpoints was based on fusion transcripts identified with our GB-panel. In CRPC1, CRPC5, and CSPC4 the breakpoints were in exon 1 of the TMPRSS2 gene and exon 3 of the ERG gene, respectively (center panel). In CSPC1 the proximal breakpoint was approximately 24 Kb upstream of the ERG gene.

Figure 5

Analyses of tumor and serial plasma samples from patient CRPC1. DNA was extracted from six different regions (designated as T2-T7) from the primary tumor and separately analyzed by our whole-genome sequencing approach (corresponding histology images are in Additional file 5). The first plasma sample (CRPC1) was obtained 13 years after resection of the primary tumor, the interval between the first and second (CRPC1_2) sample was 7 months and between the second and third (CRPC1_3) 2 months. The patient had stable disease under AD and chemotherapy. Hierarchical clustering (Manhattan distances of chromosomal z-scores) of the plasma samples and the sectors of the primary tumor is shown on the left side, the samples are shown in the corresponding order.

Figure 6

Analyses of serial plasma samples from patient CSPC1. The first plasma sample (CSPC1) was collected 12 months after initial diagnosis, only a biopsy had been taken from the primary tumor to confirm diagnosis. The interval between the first and second (CSPC1_2) sample was 5 months and 1 month between the second and third (CSPC1_3). The patient was clinically responding to castration therapy.