Seminal plasma as a source of prostate cancer peptide biomarker candidates for detection of indolent and advanced disease

Background:Extensive prostate specific antigen screening for prostate cancer generates a high number of unnecessary biopsies and over-treatment due to insufficient differentiation between indolent and aggressive tumours. We hypothesized that seminal plasma is a robust source of novel prostate cancer (PCa) biomarkers with the potential to improve primary diagnosis of and to distinguish advanced from indolent disease. <br>Methodology/Principal Findings: In an open-label case/control study 125 patients (70 PCa, 21 benign prostate hyperplasia, 25 chronic prostatitis, 9 healthy controls) were enrolled in 3 centres. Biomarker panels a) for PCa diagnosis (comparison of PCa patients versus benign controls) and b) for advanced disease (comparison of patients with post surgery Gleason score <7 versus Gleason score >>7) were sought. Independent cohorts were used for proteomic biomarker discovery and testing the performance of the identified biomarker profiles. Seminal plasma was profiled using capillary electrophoresis mass spectrometry. Pre-analytical stability and analytical precision of the proteome analysis were determined. Support vector machine learning was used for classification. Stepwise application of two biomarker signatures with 21 and 5 biomarkers provided 83% sensitivity and 67% specificity for PCa detection in a test set of samples. A panel of 11 biomarkers for advanced disease discriminated between patients with Gleason score 7 and organ-confined (<pT3a) or advanced (≥pT3a) disease with 80% sensitivity and 82% specificity in a preliminary validation setting. Seminal profiles showed excellent pre-analytical stability. Eight biomarkers were identified as fragments of N-acetyllactosaminide beta-1,3-N-acetylglucosaminyltransferase​,prostatic acid phosphatase, stabilin-2, GTPase IMAP family member 6, semenogelin-1 and -2. Restricted sample size was the major limitation of the study.</br> <br>Conclusions/Significance: Seminal plasma represents a robust source of potential peptide makers for primary PCa diagnosis. Our findings warrant further prospective validation to confirm the diagnostic potential of identified seminal biomarker candidates.</br&gt

Flow chart of study design.

<p>For biomarker discovery in total 125 seminal plasma samples were used from 70 patients with PCa, 21 patients with benign prostate hyperplasia (BPH), 25 patients with chronic prostatitis (CP) and 9 healthy control (HC). This pool of available samples was used in varying composition in three study arms. In study A “Diagnostic Markers” 50/125 patients with and without prostate cancer (22 PCa, 14 CP; 9 BPH and 5 HC) were used for biomarker discovery and the remaining 75/125 patients (48 PCa, 12 BPH, 11 CP, and 4HC) were used for diagnostic performance tests. In Study B “Advanced Disease Markers” available PCa samples (n = 70) were stratified according to Gleason score. For biomarker discovery patients with Gleason score <7 (n = 21) and Gleason score >7 (n = 16) were compared. The remaining 33/70 patients with Gleason score 7 (28 indolent disease </p

Polypeptides constituting the biomarker signatures 21PP, 5PP, and 11PP, respectively.

<p>ID: polypeptide identifier annotated by the SQL database (ID).</p>+<p>: upregulated biomarkers: mean(case)/mean(control);</p><p>downregulated biomarkers: -mean(control)/mean(case).</p>#<p>: Biomarker of 21PP and 11PP.</p

Assessment of biomarker stability and reproducibility.

<p>(<b>A</b>) An average of 1887±202 polypeptides was detected in each of the 14 measurements stored for different times at RT. The mean is marked with a bold line; standard deviation is highlighted in grey. (<b>B</b>) Beyond this qualitative assessment biomarker signatures were applied to the 14 stability replicates to obtain quantitative data of time-dependent stability of seminal plasma. For 21PP ranked correlation analysis revealed a significant decrease of SVM scores over time with Spearman’s rho of –0.576 (95% CI –0.854 to –0.07, P = 0.0379). Regression analysis unveiled a decrease rate of –0.05 a.u. (<2%) per hour. 5PP and 11PP displayed no significant time dependency. (<b>C</b>) Analytical precision of the established SVM classifiers was assessed by applying it to 15 CE-MS data sets obtained from independent replicates of a sample of a 57 years old patient with significant BPH. Mean classification scores were 0.619±0.07, 2.290±0.81, and -1.239±0.18 for 21PP, 5PP, and 11PP respectively. Coefficients of variations were calculated by dividing standard deviations by the observed overall range of SVM scores [highlighted in grey, 21PP from –1.50 to +1.50 (3.0 a.u.), 5PP from 4.50 to +3.0 (7.5 a.u.), and 11PP from –1.50 to +1.50 (3.0 a.u.)]. Coefficients of variations were 2.2%, 10.8%, and 6.1%, respectively. Classification cut offs are represented by horizontal lines. The boxes depict means and standard deviation as whiskers.</p

Biomarker signatures.

<p>Normalized molecular weight (700–25.000 Da) in logarithmic scale is plotted against normalized migration time (15–45 min). The mean signal intensity of the polypeptide peak is given in 3D-depiction. Averaged data sets of the training set are shown.</p

Patient descriptive statistics.

<p>CP  =  chronic prostatitis; HC  =  healthy control; BPH  =  benign prostata hyperplasia; PCa  =  prostate carcinoma; n.a.  =  not applicable or not available; §  =  sign. vs. BPH; $  =  sign. vs. PCa (two-tailed Kruskal-Wallis parameter free ANOVA with Dunn’s Multiple Comparison Test); D’Amico <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067514#pone.0067514-DAmico1" target="_blank">[28]</a> adopted by the AUA  =  American Urology Association <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067514#pone.0067514-Hanno1" target="_blank">[47]</a>; NCCN  =  National Comprehensive Cancer Network <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067514#pone.0067514-Mohler1" target="_blank">[29]</a>; EAU  =  European Association of Urology <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067514#pone.0067514-Heidenreich1" target="_blank">[31]</a>; RTOG  =  Radiation Therapy Oncology Group <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067514#pone.0067514-Roach1" target="_blank">[30]</a>; CAPRA  =  Cancer of the Prostate Risk Assessment Score <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067514#pone.0067514-Cooperberg2" target="_blank">[48]</a>.</p

Biomarker sequence data.

<p>ID: polypeptide identifier annotated by the SQL database (ID); Theo. Mass: theoretical mass of the peptide sequence; ▵M: Mass difference between experimental and theoretical mass normalized to theoretical mass in parts per million [ppm]. m: oxidized Methionine.</p

Biomarker performance validation.

<p>(<b>A</b>).Box and whisker plots of obtained 11PP results in the test cohort of PCa patients with GS 7 stratified according to TNM, (<b>B</b>) EAU, and (<b>C</b>) NCCN classification systems. Black squares indicate medians and whiskers 1.5-times the interquartile ranges. Rank correlation coefficients rho, the respective 95% CI and P-values are given above. (<b>D</b>) ROC curve (black lines) for 11PP classification of the independent validation cohort of PCa patients with GS 7 with either indolent (N = 28) or advanced (N = 5) disease according to EAU classification as reference standard. 95% confidence intervals are plotted as dashed lines. Diagonal line represents guessing probability with an area under the curve of 0.5. 95% confidence intervals (CI) are displayed as dashed lines.</p