Elevated levels of Dickkopf-related protein 3 in seminal plasma of prostate cancer patients

Eighty-one patients who were to undergo TRUS-guided biopsies because they had an abnormal serum PSA level (> 4 ng/mL) and/or abnormal DRE were recruited into the study from the outpatient department or from private rooms of urologists at Royal Brisbane and Women's Hospital (RBWH) between 1996 and 2009. Of this group, 40 men were confirmed to have cancer and 41 men had negative biopsies. Subsequently, patients with negative TRUS biopsy findings were followed with PSA and DREs and with further TRUS biopsies as indicated in selected cases to ensure the veracity of the TRUS-negative findings. A subgroup of 25 patients with negative and 21 with positive biopsy had a clinical follow-up of > 3 years (collected between 1996 and 08/2007). This subset of patients had been selected from our archive because we were as sure as we can be that those designated not to have prostate cancer were true negatives. All SP specimens were obtained before biopsy and treatment and after giving informed consent. Patients collected ejaculate via masturbation into urine collection jars containing 20 mL of Hanks balanced salt solution in the morning at home and brought them to the hospital within 2 hours of collection (without freezing or cooling, i.e. at temperatures from ~5-25°C) where they were processed within 15 minutes after arrival. Samples were layered over 10 mL of 64% isotonic Percoll (Amersham Biosciences) and centrifuged at 2200 rpm for 30 min at 4°C. The ejaculate supernatants (SP) were removed, aliquoted and stored at -70°C until further processing (as reported previously [16, 17]). The study had the approval of the ethics committees of the RBWH and University of Queensland.

A series of six monoclonal Antibodies (mAb) INN(sbruck)-Dkk3-1 to INN-Dkk3-6 as previously described [18] and an affinity purified polyclonal goat anti Dkk-3 antibody (R&D Systems Cat.#AF1118) were characterized by radioimmunoassays (RIA) according to affinity, molecular epitope localization and specificity with native (untagged) Dkk-3 and the commercially available homologous proteins Dkk-1, Dkk-4 and Sgy-1 (R&D Systems) as described elsewhere [18]. The antibodies with the highest affinity towards Dkk-3 (mAb INN-Dkk3-1 and the goat polyclonal antibody) showed no significant reactions towards the homologous proteins Dkk-1 and Dkk-4 however the polyclonal antibody when tested at a concentration of 5 μg/mL reacted slightly with the homologous protein Sgy-1 (Figure 1A). In competitive RIA to assess cross-reactivity increasing concentrations of Dkk-3 or Sgy-1 competed with ¹²⁵I labeled Dkk-3 (approximately 25,000 cpm) for binding to 30 ng of polyclonal goat anti Dkk-3 antibody in 300 μL total reaction volume (1% BSA/PBS) overnight at 4°C. Separation of bound from free tracer was performed as described [18], and cross-reactivity towards Sgy-1 was determined to be 0.025% (Figure 1B). Epitope mapping by polypeptide screening revealed localization of the INN-Dkk3-1 epitope in the N-terminal region (aminoacids 21-147) while the polyclonal antibody predominantly reacted with the C-terminal region (aminoacids 200-350; data not shown).

A sensitive sandwich immunoradiometric assay (IRMA) was established with mAb INN-Dkk3-1 (coating antibody) and the ¹²⁵l labeled goat polyclonal antibody (R&D Systems) as described previously [19]. All samples were run in duplicate and results calculated from specific mean signal (mean - zero standard). Native i.e. untagged Dkk-3 purified via HPLC (anion exchange followed by size exclusion chromatography) from conditioned media of stably transfected LNCaP cells [13] was used as standard. Cross-reactivity was shown to be < 0.1% for Sgy-1 and < 0.01% for Dkk-1 and Dkk-4 (Figure 1C).

To increase sensitivity and to allow higher sample throughput, the assay was commuted to a 96-well plate based indirect immunoenzymometric assay (IEMA) described previously [18] by replacement of the ¹²⁵l labeled antibody by a biotinylated variant of the polyclonal goat anti Dkk-3 antibody (R&D Systems Cat.#BAF1118). All samples were run in duplicate and results were calculated from specific mean signal (mean - zero standard). Recovery was 92% and sensitivity 1 pmol/L. The intra- and inter-assay variances (%CV) at a protein concentration of 40 pmol/L were 11% and 13%, respectively.

Serum PSA was assayed using a fluoroimmunoenzymetric two-site assay (Medics 1200 Dx AIA, Tosoh Corp.). SP PSA values were analyzed by the ADVIA Centaur PSA assay (Siemens Healthcare Diagnostics) at a dilution of 1:5000. SP Dkk-3 and PSA levels were normalized to total protein levels determined by the Pierce BCA Protein Assay Kit (Thermo Scientific).

Assay sensitivity was defined as the mean signal of non-specific binding (NSB = zero standard) plus three standard deviations (NSB + 3SD). Percent coefficient of variation (%CV) was used as a measure of dispersion about the mean and expressed SD as a percentage of the mean.

Statistical differences among groups were calculated by Mann-Whitney test. Multivariate logistic regression was used to analyze correlations between investigated markers and PCa diagnostic status and the quality of the obtained models were estimated by Hosmer-Lemeshow goodness-of-fit tests. The diagnostic performance of the tested variables and the multivariate models was evaluated by plotting the receiver-operating characteristics (ROC) curve and calculating the area under the ROC curve (AUC). Associations between the investigated markers, age at time of the ejaculation specimen and Gleason score were evaluated using Pearson correlation analysis or Spearman's rank correlation analysis when data belonged to categorial variables.

All statistical analyses were performed using PASW Statistics 18 (IBM).