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

Alexa Goldstein, Patricia Valda Toro, Justin Lee, John L. Silberstein, Mary Nakazawa, Ian Waters, Karen Cravero, David Chu, Rory L. Cochran, Minsoo Kim, Daniel Shinn, Samantha Torquato, Robert M. Hughes, Aparna Pallavajjala, Michael A. Carducci, Channing J. Paller, Samuel R. Denmeade, Bruce Kressel, Bruce J. Trock, Mario A. EisenbergerEmmanuel S. Antonarakis, Ben H. Park, Paula J. Hurley

Research output: Contribution to journalArticle

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 plasmaderived 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.

Original languageEnglish (US)
Pages (from-to)15651-15662
Number of pages12
JournalOncotarget
Volume8
Issue number9
DOIs
StatePublished - Jan 1 2017

Keywords

  • Androgen receptor
  • Circulating tumor DNA
  • DNA polymerase
  • Droplet digital PCR
  • Next generation sequencing

ASJC Scopus subject areas

  • Oncology

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    Goldstein, A., Toro, P. V., Lee, J., Silberstein, J. L., Nakazawa, M., Waters, I., Cravero, K., Chu, D., Cochran, R. L., Kim, M., Shinn, D., Torquato, S., Hughes, R. M., Pallavajjala, A., Carducci, M. A., Paller, C. J., Denmeade, S. R., Kressel, B., Trock, B. J., ... Hurley, P. J. (2017). Detection fidelity of AR mutations in plasma derived cell-free DNA. Oncotarget, 8(9), 15651-15662. https://doi.org/10.18632/oncotarget.14926