Fifth-generation digital immunoassay for prostate-specific antigen by single molecule array technology.

BACKGROUND Measurement of prostate-specific antigen (PSA) in prostate cancer patients following radical prostatectomy (RP) has been hindered by the limit of quantification of available assays. Because radical prostatectomy removes the tissue responsible for PSA production, postsurgical PSA is typically undetectable with current assay methods. Evidence suggests, however, that more sensitive determination of PSA status following RP could improve assessment of patient prognosis and response to treatment and better target secondary therapy for those who may benefit most. We developed an investigational digital immunoassay with a limit of quantification 2 logs lower than current ultrasensitive third-generation PSA assays. METHODS We developed reagents for a bead-based ELISA for use with high-density arrays of femtoliter-volume wells. Anti-PSA capture beads with immunocomplexes and associated enzyme labels were singulated within the wells of the arrays and interrogated for the presence of enzymatic product. We characterized analytical performance, compared its accuracy with a commercially available test, and analyzed longitudinal serum samples from a pilot study of 33 RP patients. RESULTS The assay exhibited a functional sensitivity (20% interassay CV) <0.05 pg/mL, total imprecision <10% from 1 to 50 pg/mL, and excellent agreement with the comparator method. All RP samples were well within the assay measurement capability. PSA concentrations following surgery were found to be predictive of prostate cancer recurrence risk over 5 years. CONCLUSIONS The robust 2-log improvement in limit of quantification relative to current ultrasensitive assays and the validated analytical performance of the assay allow for accurate assessment of PSA status after RP.

[1]  G. Aus.  Second-line therapy after radical prostatectomy failure: for whom? When? How? , 2007, European urology.

[2]  E. Messing,et al.  Adjuvant radiotherapy for pathologically advanced prostate cancer: a randomized clinical trial. , 2006, JAMA.

[3]  M. Cooperberg,et al.  Prognostic implications of an undetectable ultrasensitive prostate-specific antigen level after radical prostatectomy. , 2010, European urology.

[4]  G. Klee,et al.  How sensitive is a prostate-specific antigen measurement? How sensitive does it need to be? , 2004, Archives of pathology & laboratory medicine.

[5]  A. Partin,et al.  Long-term biochemical disease-free and cancer-specific survival following anatomic radical retropubic prostatectomy. The 15-year Johns Hopkins experience. , 2001, The Urologic clinics of North America.

[6]  J. Moul,et al.  Prostate specific antigen recurrence after definitive therapy. , 2007, The Journal of urology.

[7]  E. Diamandis,et al.  Prostate-specific Antigen: Its Usefulness in Clinical Medicine , 1998, Trends in Endocrinology & Metabolism.

[8]  M. Terris,et al.  Postoperative prostate‐specific antigen nadir improves accuracy for predicting biochemical recurrence after radical prostatectomy: Results from the Shared Equal Access Regional Cancer Hospital (SEARCH) and Duke Prostate Center databases , 2010, International journal of urology : official journal of the Japanese Urological Association.

[9]  Samir S Taneja,et al.  Ultrasensitive serum prostate specific antigen nadir accurately predicts the risk of early relapse after radical prostatectomy. , 2005, The Journal of urology.

[10]  Misop Han,et al.  Prostate cancer-specific survival following salvage radiotherapy vs observation in men with biochemical recurrence after radical prostatectomy. , 2008, JAMA.

[11]  David R Walt,et al.  Digital concentration readout of single enzyme molecules using femtoliter arrays and Poisson statistics. , 2006, Nano letters.

[12]  J. Trachtenberg,et al.  Relapse and cure rates of prostate cancer patients after radical prostatectomy and 5 years of follow-up. , 2000, Clinical biochemistry.

[13]  David H Wilson,et al.  Simultaneous detection of single molecules and singulated ensembles of molecules enables immunoassays with broad dynamic range. , 2011, Analytical chemistry.

[14]  P. Scardino,et al.  Radical prostatectomy: long-term cancer control and recovery of sexual and urinary function ("trifecta"). , 2005, Urology.

[15]  David M. Rissin,et al.  Single-Molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations , 2010, Nature Biotechnology.

[16]  A W Partin,et al.  Natural history of progression after PSA elevation following radical prostatectomy. , 1999, JAMA.

[17]  A. Haese*,et al.  Ultrasensitive detection of prostate specific antigen in the followup of 422 patients after radical prostatectomy. , 1999, The Journal of urology.

[18]  A. Kibel.  Identification of Patients With Prostate Cancer Who Benefit From Immediate Postoperative Radiotherapy: EORTC 22911 , 2008 .

[19]  S. Byun,et al.  Prognostic significance of undetectable ultrasensitive prostate-specific antigen nadir after radical prostatectomy. , 2010, Urology.

[20]  D. Chan,et al.  Natural History of Progression After PSA Elevation Following Radical Prostatectomy , 1999 .

[21]  J. Oesterling,et al.  Prostate specific antigen: a critical assessment of the most useful tumor marker for adenocarcinoma of the prostate. , 1991, The Journal of urology.

[22]  T. Christmas,et al.  Undetectable ultrasensitive PSA after radical prostatectomy for prostate cancer predicts relapse-free survival , 2000, British Journal of Cancer.

[23]  H. Klocker,et al.  Nanoparticle-based bio-barcode assay redefines “undetectable” PSA and biochemical recurrence after radical prostatectomy , 2009, Proceedings of the National Academy of Sciences.

[24]  J. Trachtenberg,et al.  Detection of prostate cancer relapse with prostate specific antigen monitoring at levels of 0.001 to 0.1 microG./L. , 1997, The Journal of urology.

[25]  P. Carroll.  Salvage radiotherapy for recurrent prostate cancer after radical prostatectomy , 2004 .

[26]  W. Catalona,et al.  5-year tumor recurrence rates after anatomical radical retropubic prostatectomy for prostate cancer. , 1994, The Journal of urology.

[27]  Lori J Sokoll,et al.  Clinical evaluation of a novel method for the measurement of prostate‐specific antigen, AccuPSATM, as a predictor of 5‐year biochemical recurrence‐free survival after radical prostatectomy: results of a pilot study , 2012, BJU international.

[28]  K. Harada,et al.  Usefulness of the Nadir Value of Serum Prostate-Specific Antigen Measured by an Ultrasensitive Assay as a Predictor of Biochemical Recurrence after Radical Prostatectomy for Clinically Localized Prostate Cancer , 2006, Urologia Internationalis.

[29]  C. Lawton.  Adjuvant Radiotherapy for Pathological T3N0M0 Prostate Cancer Significantly Reduces Risk of Metastases and Improves Survival: Long-Term Followup of a Randomized Clinical Trial , 2010 .

[30]  P. Warde,et al.  Salvage radiotherapy following biochemical relapse after radical prostatectomy: proceedings of the Genito-Urinary Radiation Oncologists of Canada consensus meeting. , 2013, Canadian Urological Association journal = Journal de l'Association des urologues du Canada.

[31]  Richard K Valicenti,et al.  Predicting the outcome of salvage radiation therapy for recurrent prostate cancer after radical prostatectomy. , 2007, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[32]  E. Diamandis,et al.  Ultrasensitive time-resolved immunofluorometric assay of prostate-specific antigen in serum and preliminary clinical studies. , 1993, Clinical chemistry.