Materials and Methods
Study Design and Recruitment
A nonexperimental comparison-group design comprising 3 groups was performed. Recruitment was done from May 2017 to September 2017. Patients were recruited at the Research Institute of Urology and Reproductive Health, Sechenov University. Individuals were classified as noncancerous control (BPH), PIN, or PCa based on biopsy results, as follows: the control group comprised individuals diagnosed with BPH, defined as the absence of caverns, poorly differentiated cells, or areas with bad differentiation; the PIN group showed some areas of poorly differentiated cells with several caverns; and the PCa group showed multiple areas of badly differentiated cells with multiple caverns.[23,24]
This research was approved by the Ethics Committee at the I.M. Sechenov First Moscow State Medical University, Moscow, Russia (Document #05–17). Written signed informed consent was obtained from each volunteer before entry into the study. The study was performed in conformity with the ethical principles for medical research involving humans as stated in the Declaration of Helsinki.
Prostate-specific Antigen, Biopsy, and Gleason Score
Total PSA levels were measured by a chemiluminescent immunoassay method (Department of Uronephrology, Sechenov University). The histological material from biopsy specimens was used for grading according to the Gleason classification system. Patients with PCa were grouped according to Gleason scores of <7 or ≥7.
Venous blood specimens (5 ml) were collected in heparin-treated tubes in the morning after an overnight fast of at least 8 hours. Immediately after blood collection, specimens were centrifuged at 5000 rpm for 10 minutes at 4°C to obtain plasma and were stored at −80°C until laboratory analysis.
Plasma Sarcosine Determination
Plasma sarcosine was analyzed as a methyl chloroformate (MCF) derivative according to the procedures published by Windelberg et al. and Midttun et al., with modifications. Plasma (100 μL) was mixed with 1 μL of 1 mM 4-chloro-L-phenylalanine solution (internal standard) and with 25 μl of 500 mM D,L-dithiothreitol (DTT), followed by incubation at room temperature for 20 min. Next, the specimen was deproteinized by adding 450 μl of ethanol. Three hundred eighty microliters of the supernatant obtained after centrifugation (5 min at 16,900 relative centrifugal force) was transferred into an empty Eppendorf tube and mixed with 300 μl of water, 50 μl of pyridine, and 250 μl of 20% (v/v) MCF in toluene. Mixing was achieved by repeated pipetting. After incubation at room temperature for 10 min to obtain phase separation, 600 μl of the aqueous phase was replaced by water (500 μl), and the specimen was mixed again. After centrifugation, 100 μl of toluene layer was placed in an autosampler vial, and 1 μl was used for gas chromatography-mass spectrometry (GC-MS) analysis.
GC-MS analysis was performed using an Agilent 6890 (Santa Clara, CA) equipped with a 30-m-long, 0.25-mm-inner-diameter Rtx5Sil-MS column (Restek Corporation, Bellefonte, PA). Specimens were injected in the splitless mode; the oven temperature was ramped up from 75°C to 85°C at 45°C/min, with a hold for 1 min, and then increased at a rate of 45°C/min to 125°C, followed by a further increase at 60°C/min to 260°C. Helium was used as a carrier gas at a constant flow rate of 1 mL/min. The inlet temperature was 270°C. Detection was done using an Agilent 5850 single quadrupole mass spectrometer (St. Joseph, MI) with a 280°C transfer line temperature and electron ionization at 70 eV. The scanning mass range was 50 Da to 750 Da.
Chemicals and Reagents
Amino acid standards, DTT, phosphate-buffered saline (PBS), and 4-chloro-L-phenylalanine were purchased from Sigma-Aldrich. Purified water was obtained from the EMD Millipore Milli-Q reference ultrapure water purification system, and ethanol was from J.T. Baker, pyridine was from Biochem Chemopharma, methyl chloroformate was from Fluka, and toluene was from ACROS Organic (Fisher Scientific).
Calibration and Quality Controls
Stock solutions were prepared in PBS solution and stored in brown glass vials at −80°C. For calibration and quality control specimens, appropriate volumes of working solutions were added to PBS buffer (blank specimen) and prepared according to specimen preparation procedures.
Diagnostic Accuracy Assessment
The diagnostic accuracy of plasma PSA and plasma sarcosine was assessed considering the PIN and the PCa groups as the target conditions and the noncancerous control group (BPH) as the reference standard. The rationale to choose this control group as a reference standard was based on choosing an available group of patients with a nononcological alteration of the prostate gland tissues. Youden's index ([sensitivity + specificity] – 1) was used to identify the most appropriate cut points for both markers. This index was obtained from receiver operating characteristic (ROC) curve analysis. The area under the curve (AUC) values obtained from ROC curve analysis were classified for diagnostic accuracy as "test not useful," "bad," "sufficient," "good," "very good," or "excellent" if the AUC was <0.5, 0.5 to 0.6, 0.6 to 0.7, 0.7 to 0.8, 0.8 to 0.9, or 0.9 to 1.0, respectively. Sensitivity, specificity, predictive values, and diagnostic accuracy were used as methods to assess diagnostic accuracy. Sensitivity expresses the proportion of true-positive (TP) patients with the disease divided by the total number of patients diagnosed with the disease. Specificity was expressed as the proportion of true-negative (TN) patients without the disease divided by the total number of patients without diagnosis of the disease. The probability of having or not having PCa was estimated by calculation of the positive and negative predictive values (PPV and NPV, respectively), as follows: PPV equals the number of TP patients divided by the total number of individuals with positive results, and NPV equals the number of TN patients divided by the total number of individuals with negative results. Overall diagnostic accuracy was expressed as the proportion of correctly classified individuals (TP + TN) versus all participants (TP + TN + false positive + false negative).
The distribution of the variables was checked with the Shapiro-Wilk test. The Kruskal-Wallis test was used to compare differences across groups with Dunn's correction for multiple comparisons for crude analyses. Plasma PSA and sarcosine levels were log transformed for analysis of covariance (ANCOVA). Age and prostate volume were considered as covariates based on the well-known influence of these parameters as risk factors for cancer.[30,31] Sidak post hoc correction was used for comparison between groups. For subgroup analyses, the Mann-Whitney U test and ANCOVAs were used to compare the groups with a Gleason score of <7 versus ≥7 in the PCa group. Statistical analyses were performed with STATISTICA 8.0, SPSS Statistics 17.0, and plots were improved with Adobe Illustrator 14.0.
Lab Med. 2020;51(6):566-573. © 2020 American Society for Clinical Pathology