title: Analytical Validation of Androgen Receptor Splice Variant 7 Detection in a Clinical Laboratory Improvement Amendments (CLIA) Laboratory Setting.
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.
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Parvez M
authors.initials: PM
authors.lastname: Lokhandwala
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Stacy L
authors.initials: SL
authors.lastname: Riel
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Lisa
authors.initials: L
authors.lastname: Haley
authors.affiliation: Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland; James Buchanan Brady Urological Institute, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Changxue
authors.initials: C
authors.lastname: Lu
authors.affiliation: Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland; James Buchanan Brady Urological Institute, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland; James Buchanan Brady Urological Institute, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: John
authors.initials: J
authors.lastname: Silberstein
authors.affiliation: Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland; James Buchanan Brady Urological Institute, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Yezi
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Gang
authors.initials: G
authors.lastname: Zheng
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Ming-Tseh
authors.initials: MT
authors.lastname: Lin
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland; Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Gocke
authors.affiliation: Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland; James Buchanan Brady Urological Institute, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Alan W
authors.initials: AW
authors.lastname: Partin
authors.affiliation: Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland; James Buchanan Brady Urological Institute, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland; Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Emmanuel S
authors.initials: ES
authors.lastname: Antonarakis
authors.affiliation: Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland; James Buchanan Brady Urological Institute, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland; Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland. Electronic address: jluo2@jhmi.edu.
authors.forename: Jun
authors.initials: J
authors.lastname: Luo
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland; Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland. Electronic address: jeshlem@jhmi.edu.
authors.forename: James R
authors.initials: JR
authors.lastname: Eshleman
mesh_terms: D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D005260:Female; D015870:Gene Expression; D006801:Humans; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques; D064129:Prostatic Neoplasms, Castration-Resistant / Q000175:diagnosis / Q000235:genetics*; D020033:Protein Isoforms / Q000235:genetics; D058996:Quality Improvement; D011944:Receptors, Androgen / Q000235:genetics* / Q000378:metabolism; D055815:Young Adult
chemical_list: C410798:AR protein, human; D014408:Biomarkers, Tumor; D020033:Protein Isoforms; D011944:Receptors, Androgen