Analysis of Circulating Cell-Free DNA Identifies Multiclonal Heterogeneity of BRCA2 Reversion Mutations Associated with Resistance to PARP Inhibitors

Results

We studied a patient (patient 1) who harbored a germline BRCA2 mutation (p.K1872X, chr13:32,914,106 A→T). This mutation introduces a premature stop codon in BRCA2 exon 11 by converting a lysine codon (AAA) to a stop codon (TAA), truncating the encoded protein at residue p.1872. Patient 1 was initially diagnosed with a Gleason 6 localized prostate cancer and was treated with radiotherapy in 2009. His disease subsequently recurred and progressed despite a series of androgen-directed therapies, including nilutamide, enzalutamide, and abiraterone. In June 2014, he underwent biopsy of a bone metastasis as part of a research protocol and was then treated with the PARPi talazoparib, to which his disease initially responded. The prostate-specific antigen (PSA) of patient 1 decreased from 16.52 at talazoparib initiation to a nadir of 0.66 after 3 months on treatment (Fig. 1A). However, after seven and a half months of treatment, radiographic and PSA testing showed disease progression. A second metastatic biopsy, this time from the liver, was obtained at this time.

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Figure 1.

PARPi resistance by multiple large deletions. A, Timeline of patient 1′s serum PSA level before and during treatment with talazoparib. B, Hematoxylin and eosin stains of the solid-tumor biopsy pre- and post-talazoparib were consistent with approximately 60% tumor purity. C, DNA read coverage of patient 1′s germline, pre- and post-talazoparib solid-tumor, and post-talazoparib liquid biopsy. Horizontal white lines indicate the span of deletions, numbered D1 through D7, with deletion frequency indicated at the right side. The location and frequency of the p.K1872X/wild-type allele are noted in gold/blue. D, Schematic protein models for BRCA2 wild-type (WT), p.K1872X, and reversion isoforms. E, PCR amplification of the region around deletions D1 and D2 in patient 1′s germline, pre-talazoparib, and post-talazoparib solid biopsy confirms the presence of deletions D1 and D2 in post-talazoparib DNA.

First, we performed mutation and DNA copy-number analysis on DNA extracted from the pre-talazoparib and resistant solid-tumor biopsies. Tumor content was estimated at greater than 60% by histopathologic review of tumor sections (Fig. 1B) and variant allele frequency analysis. We identified extensive genome-wide LOH and the presence of the BRCA-associated mutation signature three (6), both signature hallmarks of HRR deficiency (ref. 15; Supplementary Figs. S1–S3). The pathogenic p.K1872X BRCA2 allele frequency in this sample was 65%, compared with 50% in the germline DNA sample, and only a single copy of the BRCA2 locus was present in pre-talazoparib DNA, suggesting loss of the wild-type allele (Supplementary Fig. S2, Fig. 1C). Loss of wild-type BRCA2 would be expected to sensitize the tumor to talazoparib, consistent with the observed decrease in PSA after treatment initiation (Fig. 1A). The resistant biopsy bore two secondary mutant BRCA2 alleles that carried deletions of 177 and 66 nucleotides, respectively. Each allele eliminated the pathogenic p.K1872X mutation and removed 59 or 22 amino acids from the predicted encoded protein, while restoring the open reading frame to encode the critical C terminal region of BRCA2 (Fig. 1C, labeled D1 and D2, Supplementary Tables S1 and S2). Alleles D1 and D2 were predicted to produce proteins 3,359 and 3,396 amino acids in length that carried an altered BRC repeat domain 7 (Fig. 1D). Previous observations suggest that these altered proteins would highly likely restore HRR function and be resistant to talazoparib (12). Reversion deletions in BRCA2 that eliminate BRC repeats 5 to 8 have been shown in cell line models to produce BRCA2 proteins with sufficient HRR function to confer cisplatin and PARPi resistance (12, 13). We confirmed that alleles D1 and D2 were exclusively present in the resistant tumor biopsy and not the pre-talazoparib or germline DNA by PCR amplification (Fig. 1E). These observations suggest that these alleles were not present in these samples until after talazoparib therapy was initiated.

Targeted sequencing of circulating cell-free DNA (cfDNA) was performed on plasma that was collected from patient 1 when the resistant liver biopsy was performed. At a local read depth of 200x, 48 DNA reads (24%) bore the p.K1872X allele, whereas 38 reads (19%) bore the wild-type allele. We identified 7 DNA fragments corresponding to deletion D1, 37 fragments corresponding to D2, and 5 additional deletion alleles not observed in the solid tumor (Fig. 1C, labeled D3 through D7, Supplementary Tables S1 and S2). A total of 84 reads, when aligned to the genome, contained deletion alleles D1 through D7, accounting for the valley in read depth visible at this locus (Fig. 1C). All deletion alleles eliminated the pathogenic p.K1872X mutation and restored the open reading frame of BRCA2, producing predicted proteins of residue length 3,409 to 3,212 compared with the 3,418 amino acid full-length BRCA2 protein. Notably, deletion D4 was observed in cfDNA more than 4 times as frequently as deletion D1, but D4 was observed zero times in the solid biopsy. Targeted sequencing of a paraffin section isolated from a physically distinct portion of the resistant liver biopsy using an orthogonal sequencing technology (Ion Torrent) at 1,000x coverage depth identified deletion D1 but not deletion D4 (Supplementary Tables S1 and S2). Applying this approach to a paraffin-embedded biopsy of patient 1′s metastatic lesion that was obtained before talazoparib therapy did not identify any of the deletions in Fig. 1C. These observations raised the possibility that deletions other than D1 and D2 originated from a distinct metastasis that was not biopsied, or from a physically separate region of the same metastasis.

We next analyzed cfDNA isolated from a second prostate cancer case. Patient 2 was diagnosed with widespread metastatic prostate cancer in 2013 and progressed on numerous systemic therapies, including leuprolide, bicalutamide, enzalutamide, abiraterone, and eventually docetaxel. In late 2015, germline genetic testing demonstrated that he carried a germline heterozygous deletion of two nucleotides in BRCA2 (p.R259fs) that introduced a stop-gain prematurely truncating the protein at residue 274. The PSA of patient 2 decreased on olaparib treatment from 355 to 42 after 4 months of treatment, with a nadir of 40.63 (Fig. 2A). Scans at 4 months of treatment showed a radiographic response (decrease in size of bony lesions, visceral lesions, and lymphadenopathy) and functional improvement (Eastern Cooperative Oncology Group score improvement from 2 to 1; Fig. 2B and C). However, by 7 months of treatment, the patient's PSA had reached 96.10, and a repeat bone scan at that time demonstrated numerous new bone and hepatic metastases (Fig. 2C).

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Figure 2.

Olaparib resistance in patient 2 was associated with reversion mutations. A, Timeline of patient 2′s serum PSA level before and during treatment with olaparib, showing a drop in PSA from 355 to a nadir of 40, followed by increase associated with drug resistance. B, CT scans from patient 2 pre-olaparib, on treatment, and after resistance show a liver metastasis at 17 mm on February 8, 2016, decreased to 6 mm on June 16, 2016, after olaparib treatment began, and at 18 mm on September 13, 2016, respectively. C, Bone scans of patient 2 at times matching B, showing widespread metastasis, treatment response, and recurrence of metastatic lesions. D, Counts of indels observed exclusively in resistant cfDNA, binned into 50 bp intervals across BRCA2. Exon bounds shown at top. The pathogenic stop gain in p.R259fs allele is at nucleotide 1,050, noted with red arrow. E, Diagram of BRCA2 reversion mutation alleles observed in patient 2. Wild-type (WT) BRCA2 nucleotide and protein sequence, p.R259fs nucleotide and protein sequence, and BRCA2 reversion alleles present in patient 2′s post-olaparib DNA in all of exon 9 and the first 48 nucleotides of exon 10. Black versus gray bars indicate observed versus inferred nucleotide sequence. Deleted nucleotides shown as a thin black line with tics for each deleted nucleotide, insertions highlighted in blue, mutations highlighted in orange, and alternate splice acceptor highlighted in green. For clarity, nucleotides are drawn on reversion alleles where insertions are present; otherwise, nucleotide sequence corresponds to WT. Count of each allele is shown at right.

DNA copy-number analysis of cfDNA of patient 2 identified single copy loss at the BRCA2 locus (Supplementary Fig. S4). In cfDNA isolated from plasma before treatment resistance, at a local read depth of 950x, 258 reads (27%) bore the wild-type allele, whereas 711 reads (73%) bore the pathogenic p.R259fs allele (Supplementary Fig. S5). In cfDNA isolated from plasma after treatment resistance, at a local read depth of 968x, 459 reads (44%) bore the wild-type allele. The remaining 509 reads bore the pathogenic p.R259fs allele. Any combination of somatic insertions or deletions (indels) in the p.R259fs allele that restored the reading frame before the stop-gain at residue 274, and did not produce a new stop-gain, would produce an almost full-length BRCA2 protein. We identified 105 distinct reads bearing somatic alterations beginning upstream of the germline stop-gain mutation, with 82 of these in exons 9 and 10 immediately preceding the stop gain (Fig. 2D). Indel mutations were most frequent immediately upstream of the germline stop-gain mutation, the region a priori most likely to harbor reversion mutations. Each of the 34 distinct alleles resulting from indels in this region was predicted to restore the open reading frame of BRCA2 (Fig. 2E). Predicted protein products from these indels are listed in Supplementary Fig. S6. The number of distinct reads bearing each type of reversion allele ranged from 22 to 1, with deletion sizes ranging from 1 to 42 bp. Reversion indels on exon 9 were confirmed to be in cis with p.R259fs, as the tumor bore only one copy of BRCA2 (Supplementary Fig. S4) and the same DNA read contained both the germline p.R259fs frameshift and the somatic indel.

A total of 82 of the 968 reads in this region (8%) bore an indel. We performed two statistical analyses to support the genetic evidence that indels at this locus, including those observed at low frequency in cfDNA, were likely to represent bona fide reversion alleles. To produce a reversion at this locus, an indel of any size must, when combined with the pathogenic germline deletion of two bp, produce a nucleotide change divisible by three (add 2 bp, lose 1 bp, lose 4 bp, etc.). The probability that 82 randomly generated indels would all be of one of these lengths is (⅓)⁸² or 7.5 × 10⁻⁴⁰. We also compared the length of reversion indels with the length of all 10,652 indels observed in sequenced cfDNA across 73 genes. Indels that produced a predicted reversion were significantly longer than the overall distribution (mean length, 10.2 bp altered; s.d., 12.6 vs. mean length, 1.8 bp altered; s.d., 2.7, P < 3 × 10⁻¹⁶, Wilcoxon rank sum test, Supplementary Fig. S7). This analysis also held for reversion indels supported by a single read (mean length, 15.5; s.d., 15.9, P = 1.3 × 10⁻¹³).