Tumor clone dynamics in lethal prostate cancer

Abstract

It is unclear whether a single clone metastasizes and remains dominant over the course of lethal prostate cancer. We describe the clonal architectural heterogeneity at different stages of disease progression by sequencing serial plasma and tumor samples from 16 ERG-positive patients. By characterizing the clonality of commonly occurring deletions at 21q22, 8p21, and 10q23, we identified multiple independent clones in metastatic disease that are differentially represented in tissue and circulation. To exemplify the clinical utility of our studies, we then showed a temporal association between clinical progression and emergence of androgen receptor (AR) mutations activated by glucocorticoids in about 20% of patients progressing on abiraterone and prednisolone or dexamethasone. Resistant clones showed a complex dynamic with temporal and spatial heterogeneity, suggesting distinct mechanisms of resistance at different sites that emerged and regressed depending on treatment selection pressure. This introduces a management paradigm requiring sequential monitoring of advanced prostate cancer patients with plasma and tumor biopsies to ensure early discontinuation of agents when they become potential disease drivers.

Conflict of interest statement

Competing interests: The ICR developed abiraterone and therefore has a commercial interest in this agent. G.A. is on the ICR list of rewards to inventors for abiraterone. J.S.d.B. has received consulting fees and travel support from Amgen, Astellas, AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Dendreon, Enzon, Exelixis, Genentech, GlaxoSmithKline, Mediation, Merck, Novartis, Pfizer, Roche, Sanofi-Aventis, Supergen, and Takeda and grant support from AstraZeneca and Genentech. G.A. has received honoraria, consulting fees or travel support from Astellas, Medivation, Janssen, Millennium Pharmaceuticals, Ipsen, Takeda, and Sanofi-Aventis and grant support from Janssen, AstraZeneca, and Genentech. A.O. serves on advisory boards for Janssen, Astellas, Bayer, and AstraZeneca. F.D. is co-inventor of the patent on the detection of gene fusions in prostate cancer, filed by the University of Michigan and the Brigham and Women's Hospital. The diagnostic field of use for ETS gene fusions has been licensed to Hologic Gen-Probe.

Copyright © 2014, American Association for the Advancement of Science.

Figures

Fig. 1. Strategy for serial evaluation of plasma and tumor samples from prostate cancer patients

Schematic showing change in tumor volume and activity with different treatments and time points at which samples were collected for sequencing. TRBP, transrectal biopsy of the prostate; BV, blood vessel; LN, lymph nodes; LHRH, luteinizing hormone releasing hormone.

Fig. 2. Detection of genomic lesions in ERG-positive CRPC

(A) In silico sensitivity and specificity estimations for monoallelic deletion with aberrant tumor reads at 10%. Each cell in each panel represents a combination of the number of informative SNPs and local mean coverage used to generate the simulated data. (B) Distribution of 21q22, 8p21, and 10q23 deletions across patients and samples. Only samples with detected tumor DNA are considered. Gray lines represent patients with an ERG gene fusion secondary to rearrangement detected by FISH on tissue. Left panel shows the proportion of patients in whom evidence of loss was detected in at least one sample, grouped by sample type and main genomic lesion. Right panel is the equivalent on a per sample basis. AF, allele frequency. (C) For each of the three main deletions, the Venn diagrams indicate the number of patients with evidence of loss in precastration (PreC) samples only, CRPC only, or both. (D) Point mutations detected. Cases where no sample was available are identified by “-.” (E and F) Tumor content estimated using the dominant lesion compared to circulating total double-strand DNA (E) and CTC count per 7.5 ml of blood (F). Samples collected on or off treatment with abiraterone and from patients who did or did not have a response to abiraterone are distinguished as shown in the figure.

Fig. 3. Changes in clonality of deletions in precastration and sequential CRPC plasma and tumor samples

(A) Allelic frequency of heterozygous SNPs across 21q22 and 8p21 detected in precastration (PreC) tumor (left column) and in two plasma samples collected 17 days (middle column) and 36 days (right column) after discontinuation of abiraterone. The mono-allelic deletion at 21q, initially not detected, is subsequently present at an allelic frequency similar to the deletion at 8p. (B) Dominance heat maps for four patients showing clonal changes in deletions at 21q22, 8p21, and 10q23 in precastration and sequential plasma and CRPC samples. SNP allelic frequency plots in (A) and dominance heat maps in (B) connected by dotted lines are the same samples. (C) Temporal representation of clonal changes for deletions of 21q22, 8p21, and 10q23 in days from the first CRPC sample. Patients are divided by response to abiraterone. Horizontal bars represent time on treatment, with colored bars representing treatment with abirater-one. The chart includes 15 patients in whom tumor lesions were detected in a minimum of two CRPC samples. The panel on the right specifies the sequence of treatments administered for CRPC, divided by the “|” symbol and letters representing different drugs. Concomitant glucocorticoid administered with enzalutamide or abiraterone is indicated (for example, A + P indicates abiraterone and prednisolone). Samples with touching borders were collected within 3 days of each other. Synchronous CRPC plasma and tumor biopsies are surrounded by a gray rectangle if a clonal change is observed. A sample is underlined by a red dot if a clonal change is observed with respect to the previous sample. For detected loss, different dominance heat map colors represent clonality status, and different color shades represent different methods used to call the loss.

Fig. 4. AR point mutations associated with treatment resistance in patients receiving exogenous glucocorticoids

(A) These graphs show the fraction of detected tumor content (TC, estimated from the dominant lesion) containing AR-L702H mutation relative to coexisting genomic aberrations (upper panel), AR copy number (CN) state (middle panel; dashed line represents AR neutral copy number state), and estimated tumor content asa proportionof total circulating double-stranded DNA in sequential plasma samples, as well as PSA (lower panel) from patient V4012. Treatment with enzalutamide and dexamethasone followed by abiraterone and prednisolone is shown. Diagonal lines link bar chart representations to the time points when samples were collected, measured in days from collection of the first sample. (B) Inset shows changes in PSA and circulating tumor DNA (ctDNA) fractions at the start of and on treatment with abiraterone and prednisolone. (C) PC-3 cells cotransfected with an androgen-response element (ARE3) bound to luciferase and wild-type AR or AR-L702H were treated as shown. R1881, a synthetic androgen, was used at a concentration of 0.1 nM; enzalutamide was used at 10 μM; and prednisolone was used at 50, 100, and 500 nM. Data are from three independent experiments and represent mean and SD of 21 replicates. *P = 0.05 to 0.01, ***P < 0.001 [one-way analysis of variance (ANOVA) with Bonferroni correction]. Dotted lines represent no difference. (D) Fraction of detected tumor content containing H875Y and T878A AR mutations as a proportion of coexisting genomic aberrations, AR copy number, estimated tumor content, and PSA in sequential samples from patient V5074.

Fig. 5. Quantitation of tumor AR copy number in circulating tumor DNA

(A) Comparison of AR CN state inferred from target sequencing (seq) data and from digital droplet PCR (dPCR). Black dots represent combinations of CN states, and gray bars represent SDs in copy number (CN) calculation. Inset panel magnifies the highlighted area. Blue lines highlight the threshold at copy number 1.5 used for AR gain identification. Pearson correlation analysis shows evidence of statistically significant concordance between target sequencing and digital droplet PCR (n = 128, P < 2.2 × 10−16). In particular, 40 of 41 (97%) AR gain calls from target sequencing were confirmed by digital droplet PCR. (B) AR copy number calculated from observed AR copy number and corrected for tumor content. (C) AR copy number state of CRPC plasma samples collected during treatment with abiraterone from responders and nonresponders. Statistical difference between the two distributions was determined by Kruskal-Wallis statistical test (n = 53, P = 0.003).

Fig. 6. Regression of independent clones with treatment-specific resistant aberrations

Top: Fraction of tumor content (TC) containing detected genomic lesions in sequential plasma and tumor samples collected after development of resistance to castration and bicalutamide for patient V5086. Diagonal lines link bar chart representations to time points when respective samples were collected, measured in days from collection of the first sample. Middle: Estimated real AR copy number (CN) status. Bottom: PSA and estimated tumor content as a proportion of total circulating DNA. An AR-W742C mutation was detected on biopsy of a liver metastasis but not in synchronously collected plasma, which showed AR copy number gain. AR-W742C was not detected after progression on docetaxel.

Fig. 7. Translational relevance of targeted sequencing of plasma from CRPC patients

(A) Schematic showing how three different tumor clones [dark gray, harboring deletion of lesion 1 (L1); gray, deletion of lesions 2 and 3 (L2 and L3); light gray, deletion of lesion 3 (L3)] released into circulation or present in metastases would be represented in dominance heat maps of copy number changes. The signal compatible with clonal mono-allelic deletion of L1 at time point 1 decreases, becoming first subclonal and then undetectable. Signal for L2 is subject to the inverse trend. L3 is identified as a dominant lesion at time points 2 and 3 because of the presence of multiple clones harboring this lesion. (B) Schematic showing inhibition of wild-type AR signaling by enzalutamide or abiraterone with bypass activation of mutant AR by glucocorticoids, such as exogenous prednisolone.