Rucaparib

A total of 3,334 liquid biopsy samples and 2,621 tissue samples were assayed with hybrid-capture based comprehensive genomic profiling (CGP). CGP was performed in a Clinical Laboratory Improvement Amendments-certified (CLIA), College of American Pathologists-accredited (CAP), New York State‐regulated reference laboratory (Foundation Medicine, Inc., Cambridge, MA, USA). Patients who submitted screening samples for TRITON2 or TRITON3 provided written informed consent before participation. Approval for the study of the FMI dataset, including a waiver of informed consent and Health Insurance Portability and Accountability Act waiver of authorization, was obtained from the Western Institutional Review Board (protocol 20152817). Studies were conducted in accordance with the Declaration of Helsinki.
Liquid biopsy specimens were obtained from three cohorts: screening samples from the TRITON2 (NCT02952534) and TRITON3 (NCT02975934) trials of rucaparib (collected Nov 2016-March 2019), and samples submitted to Foundation Medicine (FMI) for routine clinical testing (December 2013-March 2019). 20–100 ng of cell-free DNA (cfDNA) was extracted to create adapted sequencing libraries before hybrid capture and sample-multiplexed sequencing (FoundationACT^™, FoundationOne^® Liquid) as described previously (32 ). Two versions of the plasma assay were used, with 62 (FoundationACT^™) or 70 genes (FoundationOne^® Liquid). Genomic regions baited in the two different liquid biopsy assays are depicted in Table S1. Genomic alterations detected by both assays included base substitutions, insertions and deletions (short variants), rearrangements, and copy number changes. This study did not evaluate gene deletions. Table S2 depicts frequencies of all GAs assessed by liquid biopsy in the three different datasets.
Tissue specimens from metastatic sites submitted for routine clinical testing (December 2013-March 2019) were used for global comparisons of liquid biopsy to metastatic tissue (N = 2,006). Additional tissue specimens used only in the concordance analysis were screening samples collected from the TRITON2 (N = 337) and TRITON3 (N = 277). At least 50 ng of DNA was isolated from PCaA formalin-fixed, paraffin-embedded tumor specimens and sequenced to high, uniform ≥500X coverage, with larger gene panels inclusive of all 70 genes in liquid assays.
Microsatellite instability (MSI) status was determined in samples screened with the 70 gene panel, as described previously (33 (link)). For tumor specimens, zygosity and somatic/germline status for mutations was computationally predicted without matched normal tissue as previously described (34 (link)); in validation testing of 480 tumor-only predictions against matched normal specimens, accuracy was 95% for somatic and 99% for germline predictions (35 (link)).
Quantification of the ctDNA fraction was measured using two complementary methods: the proprietary tumor fraction estimator (TFE) and the maximum somatic allele frequency (MSAF) method. TFE is based on a measure of tumor aneuploidy that incorporates observed deviations in coverage across the genome for a given sample. Calculated values for this metric are calibrated against a training set based on samples with well-defined tumor fractions to generate an estimate of the tumor fraction. When lack of tumor aneuploidy limits the TFE’s ability to return an informative estimate, MSAF is used. MSAF calculates the allele fraction of all known somatic, likely somatic, and variant of unknown significance (VUS) substitution alterations detected at >2000X median unique coverage by non-PCR duplicate read pairs, excluding germline variants, and variants associated with clonal hematopoiesis.