Detection fidelity of AR mutations in plasma derived cell-free DNA.
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.
The James Buchanan Brady Urological Institute, Department of Urology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Alexa
A
Goldstein
The James Buchanan Brady Urological Institute, Department of Urology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Patricia Valda
PV
Toro
The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Justin
J
Lee
The James Buchanan Brady Urological Institute, Department of Urology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
John L
JL
Silberstein
The James Buchanan Brady Urological Institute, Department of Urology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Mary
M
Nakazawa
The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Ian
I
Waters
The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Karen
K
Cravero
The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
David
D
Chu
The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Rory L
RL
Cochran
The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Minsoo
M
Kim
The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Daniel
D
Shinn
The James Buchanan Brady Urological Institute, Department of Urology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Samantha
S
Torquato
The James Buchanan Brady Urological Institute, Department of Urology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Robert M
RM
Hughes
The Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Aparna
A
Pallavajjala
The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Michael A
MA
Carducci
The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Channing J
CJ
Paller
The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Samuel R
SR
Denmeade
The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Bruce
B
Kressel
The James Buchanan Brady Urological Institute, Department of Urology, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Bruce J
BJ
Trock
The James Buchanan Brady Urological Institute, Department of Urology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Mario A
MA
Eisenberger
The James Buchanan Brady Urological Institute, Department of Urology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Emmanuel S
ES
Antonarakis
The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Whiting School of Engineering, Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA.
Ben H
BH
Park
The James Buchanan Brady Urological Institute, Department of Urology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA.|The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Paula J
PJ
Hurley
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000097:blood / Q000235:genetics; D004273:DNA, Neoplasm / Q000097:blood / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D064129:Prostatic Neoplasms, Castration-Resistant / Q000097:blood / Q000175:diagnosis / Q000235:genetics*; D011944:Receptors, Androgen / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
C410798:AR protein, human; D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; D011944:Receptors, Androgen
DNA polymerase; androgen receptor; circulating tumor DNA; droplet digital PCR; next generation sequencing