Detection fidelity of AR mutations in plasma derived cell-free DNA

Abstract

Somatic genetic alterations including copy number and point mutations in the androgen receptor (AR) are associated with resistance to therapies targeting the androgen/AR axis in patients with metastatic castration resistant prostate cancer (mCRPC). Due to limitations associated with biopsying metastatic lesions, plasma derived cell-free DNA (cfDNA) is increasingly being used as substrate for genetic testing. AR mutations detected by deep next generation sequencing (NGS) of cfDNA from patients with mCRPC have been reported at allelic fractions ranging from over 25% to below 1%. The lower bound threshold for accurate mutation detection by deep sequencing of cfDNA has not been comprehensively determined and may have locus specific variability. Herein, we used NGS for AR mutation discovery in plasma-derived cfDNA from patients with mCRPC and then used droplet digital polymerase chain reaction (ddPCR) for validation. Our findings show the AR (tTC>cTC) F877L hotspot was prone to false positive mutations during NGS. The rate of error at AR (tTC>cTC) F877L during amplification prior to ddPCR was variable among high fidelity polymerases. These results highlight the importance of validating low-abundant mutations detected by NGS and optimizing and controlling for amplification conditions prior to ddPCR.

An optimized targeted Next-Generation Sequencing approach for sensitive detection of single nucleotide variants

“…The importance of suitable enzymes, i.e. proofreading DNA-polymerases (containing 3′ → 5′ exonuclease activity) for high-fidelity requirements like the amplification for high-throughput sequencing and rare mutation detection, confirms previous reports [ 24 , 25 ]. Due to its particular relevance as the most frequent somatic change in myeloproliferative neoplasms [ 26 ], several assays (mainly qPCR-based) have already been described for the detection of JAK2 p.Val617Phe with only some achieving a sufficient sensitivity for MRD detection [ [27] , [28] , [29] , [30] , [31] , [32] ].…”

Cross-platform comparison for the detection of RAS mutations in cfDNA (ddPCR Biorad detection assay, BEAMing assay, and NGS strategy)

“…By contrast, Goldstein et al . showed that cfDNA mutation detection in prostate cancers by NGS in the androgen receptor axis may lead to false positive cases that are not observed with ddPCR assays [ 22 ]. This highlighted the importance of setting the proper clinical cut-off values specific for the platform being used to detect mutations in cfDNA with particular attention to establishing margins of security that are assay-specific.…”

Circulating oestrogen receptor mutations and splice variants in advanced prostate cancer

“…In comparison, we detected D538G in 24.5% of our prostate cancer samples. To contrast with other hormone receptors investigated in men with advanced prostate cancer, reported AR mutations range from 8.2% to 45%, where they have been associated with poor outcomes . In the present study we used RNA samples derived from blood as an indirect measure of cfRNA and, in turn, circulating tumour RNA, to identify somatic mutations associated with prostate cancer and to analyse RNA splice variants.…”