Related Experiment Video
Updated: Mar 8, 2026
Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids
Published on: June 8, 2019
Detection fidelity of AR mutations in plasma derived cell-free DNA
Alexa Goldstein1,2,3, Patricia Valda Toro1,2,3, Justin Lee2,3
1The James Buchanan Brady Urological Institute, Department of Urology, Johns Hopkins School of Medicine, Baltimore, MD, USA.
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.
Insights
Detecting androgen receptor (AR) mutations in cell-free DNA (cfDNA) for prostate cancer resistance is challenging. Next-generation sequencing (NGS) can yield false positives for low-abundance AR mutations, requiring validation with methods like droplet digital PCR (ddPCR).
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Somatic genetic alterations in the androgen receptor (AR) are linked to treatment resistance in metastatic castration-resistant prostate cancer (mCRPC).
- Plasma-derived cell-free DNA (cfDNA) is a valuable tool for genetic testing in mCRPC due to limitations of tissue biopsies.
- Accurate detection of low-frequency AR mutations in cfDNA using next-generation sequencing (NGS) requires careful validation.
Purpose of the Study:
- To determine the lower bound threshold for accurate AR mutation detection in cfDNA using NGS.
- To investigate locus-specific variability in AR mutation detection sensitivity.
- To validate NGS findings for AR mutations in mCRPC patients using droplet digital PCR (ddPCR).
Main Methods:
- Next-generation sequencing (NGS) was employed for AR mutation discovery in plasma cfDNA from mCRPC patients.
- Droplet digital PCR (ddPCR) was utilized for the validation of detected AR mutations.
- Amplification conditions and polymerase fidelity were assessed for potential impact on mutation detection.
Main Results:
- The AR (tTC>cTC) F877L hotspot was identified as prone to false positive mutations during NGS.
- Variable error rates were observed at the AR (tTC>cTC) F877L locus during amplification prior to ddPCR, depending on the polymerase used.
- Low-abundant AR mutations detected by NGS necessitate rigorous validation due to potential locus-specific variability and amplification biases.
Conclusions:
- Validation of low-abundant mutations detected by NGS is crucial for accurate genetic testing in mCRPC.
- Optimizing and controlling amplification conditions before ddPCR is essential to mitigate false positive results.
- These findings underscore the importance of robust methodologies for cfDNA-based mutation detection in clinical oncology.
