Allele-informed copy number evaluation of plasma DNA samples from metastatic prostate cancer patients: the PCF_SELECT consortium assay

Abstract

Sequencing of cell-free DNA (cfDNA) in cancer patients' plasma offers a minimally-invasive solution to detect tumor cell genomic alterations to aid real-time clinical decision-making. The reliability of copy number detection decreases at lower cfDNA tumor fractions, limiting utility at earlier stages of the disease. To test a novel strategy for detection of allelic imbalance, we developed a prostate cancer bespoke assay, PCF_SELECT, that includes an innovative sequencing panel covering ∼25 000 high minor allele frequency SNPs and tailored analytical solutions to enable allele-informed evaluation. First, we assessed it on plasma samples from 50 advanced prostate cancer patients. We then confirmed improved detection of genomic alterations in samples with <10% tumor fractions when compared against an independent assay. Finally, we applied PCF_SELECT to serial plasma samples intensively collected from three patients previously characterized as harboring alterations involving DNA repair genes and consequently offered PARP inhibition. We identified more extensive pan-genome allelic imbalance than previously recognized in prostate cancer. We confirmed high sensitivity detection of BRCA2 allelic imbalance with decreasing tumor fractions resultant from treatment and identified complex ATM genomic states that may be incongruent with protein losses. Overall, we present a framework for sensitive detection of allele-specific copy number changes in cfDNA.

Figure 1.

The PCF_SELECT assay. (A) Schematic for selecting gene regions for inclusion in panel. (B) Summary of panel statistics. Genomic sizes and number of high MAF SNPs are reported for target and control gene-regions. (C) Schematics summarizing the main components of the pipeline. Copy-number assessment and gene-region allelic imbalance detection are independently performed; results are integrated and corrected by tumor ploidy and tumor content to generate allele-specific copy number status for each gene-region. (D) Integration of read-depth estimation and allelic imbalance. Top: distribution of Log2R colored by allele-specific copy-number. Bottom: Log2R-beta space obtained by integration of read-depth estimations and allelic imbalance. Each point represents a gene-region. Clusters of points are annotated with their expected allele-specific copy numbers.

Figure 2.

Assessment of allelic imbalance (AI) calls using high density iSNPs achieves increased accuracy and sensitivity. (A) Percentage of AI calls as a function of number of informative SNPs (iSNPs) available for that gene-region and sequential bars showing detection at decreasing percentage of iSNPs (80%, 60%, 40%, 20%) and using only iSNPs spanning exonic region simulating whole-exome sequencing (WES) data (ranges of SNPs used are reported). Values are shown for both control germline and cfDNA samples (green and red boxes at the bottom of each bar, respectively). Reported proportions are relative to the number of AI calls obtained using all the iSNPs available for each gene in the PCF_SELECT panel (i.e. 904). The number of AI calls is shown on top of each bar. AI calls are stratified for the number of iSNPs used (bar colors). (B) Evidence of allelic imbalance (E(AI_T), y-axis) for representative gene-regions at varying percentages of iSNPs (WCM cohort, N = 66). Lines color shade indicates ctDNA level of the sample. Donuts show proportion of evidence of imbalance per percentage of iSNPs used. Data for all gene-regions are shown in Supplementary Figure S10A. (C) Proportion of recovered AI calls in synthetically diluted samples. Tumor fraction levels are reported on x-axis. Calls are stratified by allele-specific CN call as established in the real cfDNA samples subjected to dilution.

Figure 3.

Comparative overview of somatic copy-number aberrations (SCNA) calls on three serial samples from two CRPC patients using two independent assays (A). SCNA and SNVs calls detected by the two assays (Standard Log2ratio assay from Annala et al. upper row; PCF_SELECT, lower row) are reported for serial samples with varying ctDNA levels as determined by PCF_SELECT. Gene-regions are grouped by pathways and sorted alphabetically. (B). Log2R-beta spaces for patient #110. CHD1 and TP53 are highlighted in red and blue, respectively. Bottom panels show the mirrored allelic fraction distribution of the informative SNPs (iSNPs) spanning CHD1 and TP53 for both cfDNA (red) and matched control (green) samples. (C) Log2R-beta spaces for patient #134. PTEN and RB1 are highlighted in red and blue, respectively. Bottom panels show the mirrored allelic fraction distribution of the iSNPs spanning PTEN and RB1 for both cfDNA (red) and matched control (green) samples.

Figure 4.

In vivo serial sampling in a BRCA2-mutant CRPC patient (TR029) treated with niraparib confirms enhanced sensitivity of detection of BRCA2 allelic imbalance using PCF_SELECT. (A) Changes in serum prostate specific antigen (PSA) and ctDNA levels over 250-day period of niraparib treatment for mCRPC. – dotted line refers to treatment interruption for toxicity. (B) BRCA2 copy number (CN) status. The allele-specific CN space of plasma samples with estimated ctDNA level > 15% is shown; BRCA2 gene-region corresponding status is highlighted. (C) Mirrored allelic fractions (AF) distribution of informative SNPs (iSNPs) within the BRCA2 gene-region. Dashed lines represent the mirrored AF median. Left panel corresponds to germline signal derived from peripheral blood mononuclear cells (green); subsequent panels show signal in sequential plasma samples and are ordered by time from first to last. (D) Variant allele frequency (VAF) of BRCA2 p.E2663V germline mutation from plasma samples. Dotted green line represents the VAF observed in the matched control sample. *Indicates manually imputed CN-corrected VAF.

Figure 5.

PCF_SELECT design identifies allelic imbalance secondary to gain of ATM wild-type allele in ATM-mutant CRPC patients. (A, E) Changes in serum prostate specific antigen (PSA) and ctDNA levels over a 550-day period of treatment with docetaxel followed by niraparib (TR067 and TR081) and radium223 (TR081); black diamond indicates time of death. The inset in A shows the summary of the tissue samples collected after death and profiled with PCF_SELECT from TR067 who participated in the PEACE trial. (B) ATM copy number (CN) status in patient TR067. The allele-specific CN space of plasma and tissue samples is shown; ATM gene-region corresponding status is highlighted; for plasma samples average CN and standard deviations are reported. (C) Mirrored allelic fractions (AF) distribution of patient's ATM gene-region informative SNPs (iSNPs). Dashed lines represent the mirrored AF median. Allele-specific ploidy and ctDNA level/TC of each sample are reported on top of the plot. TC: tumor content. (D) CN-corrected variant allele frequency (VAF) of ATM p.R2763* mutation. (F) ATM and TP53 copy number (CN) status in patient TR081. The allele-specific CN space of plasma samples is shown; ATM and TP53 gene-regions corresponding statuses are highlighted; average CN and standard deviations are reported. (G) CN-corrected variant allele frequency (VAF) of ATM p.R2763* and TP53 p.R175G mutations. (H) Observed events in TR081 plasma samples using bespoke droplet digital PCR multiplex assays with probes for ATM p.R250* mutation and wild type and two control genes (AP3B1, top panel or NSUN3, bottom panel).