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 Pallavajjalla, Michael A Carducci, Channing Paller, Samuel R Denmeade, Bruce Kressel, Bruce Trock, Mario EisenbergerEmmanuel Antonarakis, Ben H. Park, Paula 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 - 2017

Fingerprint

Androgen Receptors
Plasma Cells
Mutation
Castration
DNA
Prostatic Neoplasms
Polymerase Chain Reaction
High-Throughput Nucleotide Sequencing
Genetic Testing
Point Mutation
Androgens

Keywords

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

ASJC Scopus subject areas

  • Oncology

Cite this

Goldstein, A., Toro, P. V., Lee, J., Silberstein, J. L., Nakazawa, M., Waters, I., ... Hurley, P. (2017). Detection fidelity of AR mutations in plasma derived cell-free DNA. Oncotarget, 8(9), 15651-15662. https://doi.org/10.18632/oncotarget.14926

Detection fidelity of AR mutations in plasma derived cell-free DNA. / Goldstein, Alexa; Toro, Patricia Valda; Lee, Justin; Silberstein, John L.; Nakazawa, Mary; Waters, Ian; Cravero, Karen; Chu, David; Cochran, Rory L.; Kim, Minsoo; Shinn, Daniel; Torquato, Samantha; Hughes, Robert M.; Pallavajjalla, Aparna; Carducci, Michael A; Paller, Channing; Denmeade, Samuel R; Kressel, Bruce; Trock, Bruce; Eisenberger, Mario; Antonarakis, Emmanuel; Park, Ben H.; Hurley, Paula.

In: Oncotarget, Vol. 8, No. 9, 2017, p. 15651-15662.

Research output: Contribution to journalArticle

Goldstein, A, Toro, PV, Lee, J, Silberstein, JL, Nakazawa, M, Waters, I, Cravero, K, Chu, D, Cochran, RL, Kim, M, Shinn, D, Torquato, S, Hughes, RM, Pallavajjalla, A, Carducci, MA, Paller, C, Denmeade, SR, Kressel, B, Trock, B, Eisenberger, M, Antonarakis, E, Park, BH & Hurley, P 2017, 'Detection fidelity of AR mutations in plasma derived cell-free DNA', Oncotarget, vol. 8, no. 9, pp. 15651-15662. https://doi.org/10.18632/oncotarget.14926
Goldstein A, Toro PV, Lee J, Silberstein JL, Nakazawa M, Waters I et al. Detection fidelity of AR mutations in plasma derived cell-free DNA. Oncotarget. 2017;8(9):15651-15662. https://doi.org/10.18632/oncotarget.14926
Goldstein, Alexa ; Toro, Patricia Valda ; Lee, Justin ; Silberstein, John L. ; Nakazawa, Mary ; Waters, Ian ; Cravero, Karen ; Chu, David ; Cochran, Rory L. ; Kim, Minsoo ; Shinn, Daniel ; Torquato, Samantha ; Hughes, Robert M. ; Pallavajjalla, Aparna ; Carducci, Michael A ; Paller, Channing ; Denmeade, Samuel R ; Kressel, Bruce ; Trock, Bruce ; Eisenberger, Mario ; Antonarakis, Emmanuel ; Park, Ben H. ; Hurley, Paula. / Detection fidelity of AR mutations in plasma derived cell-free DNA. In: Oncotarget. 2017 ; Vol. 8, No. 9. pp. 15651-15662.
@article{66a4cf9db3d14474b0b39f956099afa4,
title = "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 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.",
keywords = "Androgen receptor, Circulating tumor DNA, DNA polymerase, Droplet digital PCR, Next generation sequencing",
author = "Alexa Goldstein and Toro, {Patricia Valda} and Justin Lee and Silberstein, {John L.} and Mary Nakazawa and Ian Waters and Karen Cravero and David Chu and Cochran, {Rory L.} and Minsoo Kim and Daniel Shinn and Samantha Torquato and Hughes, {Robert M.} and Aparna Pallavajjalla and Carducci, {Michael A} and Channing Paller and Denmeade, {Samuel R} and Bruce Kressel and Bruce Trock and Mario Eisenberger and Emmanuel Antonarakis and Park, {Ben H.} and Paula Hurley",
year = "2017",
doi = "10.18632/oncotarget.14926",
language = "English (US)",
volume = "8",
pages = "15651--15662",
journal = "Oncotarget",
issn = "1949-2553",
publisher = "Impact Journals",
number = "9",

}

TY - JOUR

T1 - Detection fidelity of AR mutations in plasma derived cell-free DNA

AU - Goldstein, Alexa

AU - Toro, Patricia Valda

AU - Lee, Justin

AU - Silberstein, John L.

AU - Nakazawa, Mary

AU - Waters, Ian

AU - Cravero, Karen

AU - Chu, David

AU - Cochran, Rory L.

AU - Kim, Minsoo

AU - Shinn, Daniel

AU - Torquato, Samantha

AU - Hughes, Robert M.

AU - Pallavajjalla, Aparna

AU - Carducci, Michael A

AU - Paller, Channing

AU - Denmeade, Samuel R

AU - Kressel, Bruce

AU - Trock, Bruce

AU - Eisenberger, Mario

AU - Antonarakis, Emmanuel

AU - Park, Ben H.

AU - Hurley, Paula

PY - 2017

Y1 - 2017

N2 - 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.

AB - 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.

KW - Androgen receptor

KW - Circulating tumor DNA

KW - DNA polymerase

KW - Droplet digital PCR

KW - Next generation sequencing

UR - http://www.scopus.com/inward/record.url?scp=85014114939&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85014114939&partnerID=8YFLogxK

U2 - 10.18632/oncotarget.14926

DO - 10.18632/oncotarget.14926

M3 - Article

C2 - 28152506

AN - SCOPUS:85014114939

VL - 8

SP - 15651

EP - 15662

JO - Oncotarget

JF - Oncotarget

SN - 1949-2553

IS - 9

ER -