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Analytical Validation of Androgen Receptor Splice Variant 7 Detection in a Clinical Laboratory Improvement Amendments
Parvez M Lokhandwala1, Stacy L Riel1, Lisa Haley1
1Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
Abstract:
Patients with castration-resistant prostate cancer (CRPC) often are treated with drugs that target the androgen receptor (AR) ligand-binding domain. Constitutively active AR splice variant 7 (AR-V7) lacks the ligand-binding domain and, if detected in circulating tumor cells, may be associated with resistance to these agents. We validated an AR-V7 assay in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory. Circulating tumor cells were isolated, and mRNA was reverse-transcribed into cDNA. Real-time quantitative PCR amplification of reference transcripts (beta-actin and glyceraldehyde-3-phosphate dehydrogenase), prostate-specific transcripts (prostate-specific membrane antigen, prostate-specific antigen, and AR-full length), and AR-V7 was performed. Specimens for validation included an AR-V7 expressing prostate cancer (LNCaP95), 38 peripheral blood controls, and 21 blood samples from CRPC patients. The assay detected as few as five LNCaP95 cells spiked into peripheral blood, showing high analytical sensitivity. Multiple inter-run and intrarun replicates of LNCaP95 cell line experiments yielded similar cycle threshold values for all genes, showing high analytical precision (AR-V7 cycle threshold CV of 0.67%). All 38 healthy control samples were negative for AR-V7, showing high diagnostic specificity (100%). The diagnostic accuracy was confirmed by concurrent testing of 21 CRPC samples in the research laboratory and the clinical diagnostic laboratory: concordance in AR-V7 status was achieved in all cases (positive in 4, negative in 17) (100% accuracy). This first validated clinical assay detects the AR-V7 with high analytical sensitivity, precision, specificity, and accuracy.
Insights
A new assay accurately detects androgen receptor splice variant 7 (AR-V7) in circulating tumor cells from prostate cancer patients. This validated clinical test shows high sensitivity and specificity for predicting treatment resistance.
Area of Science:
- Oncology
- Molecular Biology
- Clinical Diagnostics
Background:
- Castration-resistant prostate cancer (CRPC) treatments targeting the androgen receptor (AR) ligand-binding domain may be ineffective in patients with AR splice variant 7 (AR-V7).
- AR-V7 lacks the ligand-binding domain and its presence in circulating tumor cells (CTCs) can indicate resistance to AR-targeted therapies.
Purpose of the Study:
- To validate a novel assay for detecting AR-V7 in CTCs within a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory setting.
- To establish the analytical sensitivity, precision, specificity, and accuracy of the AR-V7 assay for clinical use.
Main Methods:
- Isolation of CTCs from patient blood samples.
- Reverse transcription of mRNA to cDNA.
- Real-time quantitative PCR amplification of AR-V7 and control transcripts (e.g., beta-actin, PSA).
- Assay validation using spiked cell lines, healthy controls, and CRPC patient samples.
Main Results:
- The assay demonstrated high analytical sensitivity, detecting as few as five AR-V7 expressing cells spiked into blood.
- High analytical precision was observed with low coefficient of variation (CV) for AR-V7 cycle threshold values (0.67%).
- The assay showed 100% diagnostic specificity, with all 38 healthy controls testing negative for AR-V7.
- 100% diagnostic accuracy was confirmed by 100% concordance with a research laboratory assay in 21 CRPC patient samples (4 positive, 17 negative for AR-V7).
Conclusions:
- This represents the first validated clinical assay for detecting AR-V7 in CTCs.
- The assay provides high analytical sensitivity, precision, specificity, and accuracy for AR-V7 detection.
- This validated assay can aid in identifying CRPC patients likely to be resistant to AR-targeted therapies.
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