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DNA-Sequencing of Baseline and Progression Plasma from the Phase III Alliance A031201 Trial

Circulating tumor DNA (ctDNA) in plasma cell free DNA (cfDNA) of cancer patients is associated with poor prognosis, but is challenging to detect from low plasma volumes. In metastatic castration-resistant prostate cancer (mCRPC), ctDNA assays are needed to prognosticate outcomes of patients treated with androgen receptor (AR) inhibitors. We developed a custom targeted cfDNA sequencing assay, named AR-ctDETECT, to detect ctDNA in limiting plasma cfDNA available from mCRPC patients in the Alliance A031201 randomized phase 3 trial of enzalutamide with or without abiraterone.

For the AR ctDETECT assay, DNA-seq libraries were constructed from cfDNA using Takara ThruPLEX Tag-Seq or Plasma-Seq kits, and subjected to hybridization enrichment using a custom-designed Agilent SureSelect bait panel. Post-capture DNA-seq libraries were pooled and sequenced on an Illumina HiSeq 2500 or an Illumina NovaSeq 6000 SP using 2x150 bp settings. ctDNA positivity was defined by samples harboring high ctDNA aneuploidy as measured by a modified ichorCNA algorithm, or samples harboring low ctDNA aneuploidy but displaying AR gain or genomic structural rearrangement (AR-GSR), MYC/MYCN gain, or a pathogenic mutation.

We used the AR-ctDETECT assay to analyze plasma collected at baseline from 776 patients, and found that 59% were ctDNA-positive. Patients who were ctDNA-positive at baseline had significantly worse median overall survival than patients who were ctDNA-negative at baseline. We also used the AR-ctDETECT assay to analyze plasma collected at progression from 431 patients. Relative to baseline cfDNA, progression cfDNA exhibited enrichment of features associated with poor prognosis, including elevated cfDNA concentrations and increased ctDNA positivity. AR alterations were enriched at progression relative to baseline, primarily driven by AR-GSRs, including those predicted to truncate the AR ligand binding domain and originate from AR amplification on extrachromosomal DNA. Stratification by radiographic progression-free survival revealed that patients with rapid progression were enriched for non-AR gene alterations, while patients with initially durable responses showed selective enrichment of AR alterations. These results indicate that non-AR genetic alterations in ctDNA are associated with primary resistance to AR pathway inhibitors in first-line mCRPC, while AR alterations are associated with secondary resistance, even in patients who initially respond.

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