Elucidating acquired PARP inhibitor resistance in advanced prostate cancer

Abstract

PARP inhibition (PARPi) has anti-tumor activity against castration-resistant prostate cancer (CRPC) with homologous recombination repair (HRR) defects. However, mechanisms underlying PARPi resistance are not fully understood. While acquired mutations restoring BRCA genes are well documented, their clinical relevance, frequency, and mechanism of generation remain unclear. Moreover, how resistance emerges in BRCA2 homozygously deleted (HomDel) CRPC is unknown. Evaluating samples from patients with metastatic CRPC treated in the TOPARP-B trial, we identify reversion mutations in most BRCA2/PALB2-mutated tumors (79%) by end of treatment. Among reversions mediated by frameshift deletions, 60% are flanked by DNA microhomologies, implicating POLQ-mediated repair. The number of reversions and time of their detection associate with radiological progression-free survival and overall survival (p < 0.01). For BRCA2 HomDels, selection for rare subclones without BRCA2-HomDel is observed following PARPi, confirmed by single circulating-tumor-cell genomics, biopsy fluorescence in situ hybridization (FISH), and RNAish. These data support the need for restored HRR function in PARPi resistance.

Keywords: DNA repair; PARP inhibition; cfDNA; genomics; prostate cancer; reversion.

Graphical abstract

Figure 1. Reversion mutations detectable in the blood of longitudinal mCRPC cfDNA samples

(A and B) Example patients (p22 with BRCA2 frameshift mutation and p35 with BRCA2 stop-gain mutation) showing changes in somatic pathogenic mutation allele frequency (black) across multiple time points alongside the number of detectable putative reversion variants (red). (C) Schematic of insertion/deletion variants from patient p22 with the capacity to restore reading frame in the context of the pathogenic frameshift variant (black), by time point (C4, cycle 4; C5, cycle 5 etc.; EOT, end of treatment). Bars with asterisks (*) indicate private variants only observed at one time point, other bars are reproduced across multiple time points. Nucleotide sequence shown (dark blue = T, orange = C, red = G, light-blue A). (D) Schematic of alternative codons detectable longitudinally in samples from patient p35 bearing BRCA2 stop-gain mutation. Amino acids shown along with variant codons in brackets. Initial pathogenic substitution in red, subsequent putative reversion nucleotide substitutions shown in blue. (E) Longitudinal tracking of reversion counts in ctDNA panel sequencing, lines colored by individual patients (n = 19, 128 samples). See also Figures S2 and S3.

Figure 2. Statistical modeling of emerging reversion count and survival

(A) Bar plot of estimated rate of reversion derived from linear regression slope line. (B) Maximum number of reversions observed across all studied time points. (C) Color bar indicating mutation details, red = stop-gains, tan = frameshift, pale blue = germline, mid green = somatic. (D) Swimmer plot of survival time including both radiological progression-free survival (rPFS) (orange) and overall survival (OS) (dark gray), censoring shown with “+” symbol. (A–D) Each bar represents a different patient. (E) Forest plot of results of univariable rPFS mixed-effect time-varying Cox regression, hazard ratios (HRs) with confidence intervals (CIs), and p values (Wald test) shown across multiple mutation count cut-points at 16 weeks (C4D1), all patients (n = 19) evaluated for reversions (n = 38 samples cycle 4 and earlier). (F) Forest plot of results of univariable OS mixed-effect time-varying Cox regression, HRs with CIs, and p values (Wald test) shown across multiple mutation count cut-points at 16 weeks (C4D1), all patients (n = 19) evaluated for reversions (n = 38 samples cycle 4 and earlier). (G) Kaplan-Meier plots of rPFS split by mutation count ≥4 at C4D1, risk table and confidence intervals shown, all patients (n = 19) included. (H) Kaplan-Meier plots of OS split by mutation count ≥4 at C4D1, all patients (n = 19) included. See also Figure S4, Table S2, and STAR Methods.

Figure 3. Subclonal shifts observable at the BRCA2 locus in homozygous-deleted samples through WGS of cfDNA samples

(A and B) Example results for patient p23, illustrating changes on chromosome 13. Phased germline B-allele frequency (BAF) and log2ratio (LogR) results for the BRCA2 locus and surrounding areas shown. Initial homozygous deleted segment indicated using dashed green lines. (C) Changes in log2ratio of BRCA2-affecting segment at baseline (BL) and end-of-treatment (EOT). (D) Changes in BAF of BRCA2 segment pre- and post-PARPi treatment (at BL and EOT). (E and F) Predictions of allele-specific copy-number aberration (CNA) state and associated clonality. All evaluated patients (n = 6) bearing a homozygous deletion at baseline could be classified as clonal. By end of study, however, 5 out of 6 showed subclonal events at this locus. Loss of heterozygosity, LoH. See also Figures S5–S8, and Tables S3 and S4.

Figure 4. Identification of PARPi-induced longitudinal selection against BRCA2 homozygous-deleted clones through single-cell assays

(A) Genome-wide copy-number aberration (CNA) heatmap of patient p23 low-pass whole-genome sequencing (lpWGS). Each row relates to one cell, each column a genomic position. Chromosomes indicated with black/white bar. Colors mapped to segment log2(coverage ratio). Rows grouped by collection time point (baseline, BL; on-treatment, OT; and end-of-treatment, EOT) and subsequently clustered with hierarchical clustering. Baseline, on-treatment, and end-of-treatment time points are marked, along with major subclone cluster. (B) Zoomed chromosome 13 copy-number heatmap, rows again grouped by collection time point and clustered. (C) Dot plot of BRCA2 locus showing segment log2(coverage ratio) values. Point shape indicates cells belonging to one of two major clones (c1, clone 1; c2, clone 2). (D) Proportion of circulating tumor cells (CTCs) at different time points split by proposed subclone cluster and the presence of a deep deletion at the BRCA2 locus (segment log2 ratio < −2), showing longitudinal changes in clone inclusion across time points. (E) Stacked barplots of BRCA2 FISH copies of pre- and post-treatment biopsy samples, tallied across 100 cells per sample, in individuals bearing homozygous deletions, pre- and post-treatment. Dark blue indicates complete loss; other colors indicate tumor cells bearing copies of BRCA2. See also Figures S4, S9, and S10.