Development and clinical testing of individual immunoassays for the quantification of serum glycoproteins to diagnose prostate cancer

Abstract

Prostate Cancer (PCa) diagnosis is currently hampered by the high false-positive rate of PSA evaluations, which consequently may lead to overtreatment. Non-invasive methods with increased specificity and sensitivity are needed to improve diagnosis of significant PCa. We developed and technically validated four individual immunoassays for cathepsin D (CTSD), intercellular adhesion molecule 1 (ICAM1), olfactomedin 4 (OLFM4), and thrombospondin 1 (THBS1). These glycoproteins, previously identified by mass spectrometry using a Pten mouse model, were measured in clinical serum samples for testing the capability of discriminating PCa positive and negative samples. The development yielded 4 individual immunoassays with inter and intra-variability (CV) <15% and linearity on dilution of the analytes. In serum, ex vivo protein stability (<15% loss of analyte) was achieved for a duration of at least 24 hours at room temperature and 2 days at 4°C. The measurement of 359 serum samples from PCa positive (n = 167) and negative (n = 192) patients with elevated PSA (2–10 ng/ml) revealed a significantly improved accuracy (P <0.001) when two of the glycoproteins (CTSD and THBS1) were combined with %fPSA and age (AUC = 0.8109; P <0.0001; 95% CI = 0.7673–0.8545). Conclusively, the use of CTSD and THBS1 together with commonly used parameters for PCa diagnosis such as %fPSA and age has the potential to improve the diagnosis of PCa.

Thrombospondin 1 and cathepsin D improve prostate cancer diagnosis by avoiding potentially unnecessary prostate biopsies

“…The two proteins were previously tested in serum samples from 359 men from a combined cohort of two clinical centres: Martini‐Klinik, Hamburg‐Eppendorf, Germany and the Cantonal Hospital St. Gallen, Switzerland . In the present study, we further validated those findings in a larger cohort collected at the Martini‐Klinik, University Hospital Hamburg‐Eppendorf, Germany.…”

“…All serum samples were measured using the original Luminex‐based assays as well as the newly developed ELISAs. There was good correlation for both assays between Luminex and ELISA measurements.…”

“…In addition, all men with a positive biopsy outcome needed to have undergone subsequent in‐house radical prostatectomy, so that the Gleason score (GS) from both the biopsy and the prostatectomy specimen were available. Out of the 474 serum samples used in the present study, 67 samples were used in a previous study . The study was approved by the local ethics commission Medical Chamber Hamburg under the reference PV3652.…”

“…In brief, human THBS1 purified from platelets and recombinant human CTSD were used as calibrators; both capture and detection antibodies were mouse monoclonals generated against the human mature protein of THBS1 and the full‐length recombinant protein of CTSD. For verification purposes, THBS1 and CTSD assays were re‐measured using a Luminex MAGPIX system (Luminex Corp., Austin, TX, USA), as described by Endt et al . Serum tPSA and fPSA concentrations were re‐analysed for all samples using the ADVIA Centaur immunoassay system (Siemens Healthcare, Erlangen, Germany) to calculate the percentage of fPSA to tPSA ratio (%fPSA).…”

“…Additional studies also highlighted the prognostic potential of these biomarkers as well as their use in predicting response to therapy . More recently, we showed that a refined selection of two glycoprotein biomarkers, thrombospondin 1 (THBS1) and cathepsin D (CTSD) aids in the discrimination between PCa‐positive and PCa‐negative men .…”