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Ossoliński et al. J Cancer Metastasis Treat 2019;5:1 I http://dx.doi.org/10.20517/2394-4722.2018.63 Page 3 of 12

patients with PCa and 3000 controls and showed that elevated serum levels of sarcosine were associated with
decreased risk of PCa.

The difficulty in developing PCa biomarker results from the heterogeneity of this type of cancer. It has been
demonstrated that factors like obesity and hormonal profile may be associated with increased risk of high
[21]
grade PCa. de Cobelli et al. demonstrated that high body mass index (BMI) is associated with upgrading
and upstaging in patients with low-risk PCa. Moreover it has been shown that low testosterone level is a
[22]
predictor of upstaging and upgrading in patents with low-risk PCa . This results support inclusion of BMI
and testosterone level into selection criteria for active surveillance programs.

In this study we evaluate the role of mass spectrometry in the metabolomic study of PCa and the possibility
to develop potential novel PCa biomarker.

METHODS
Protocol of this study was approved by the ethics committee at the University of Rzeszów, Poland (no.
14/06/2016). Specimens and clinical data related to this study were collected after signing informed consents.
Data were prospectively collected from 10 patients with elevated PSA who underwent transrectal prostate
biopsy and 10 healthy volunteers between December 2016 and February 2017 at Department of Urology,
City Hospital in Rzeszów. Five out ten patients were diagnosed with PCa - 3 with Gleason 7, 1 with Gleason
8 and 1 with Gleason 9. Each biopsy was performed by the same urologist at our department with extensive
experience in prostate biopsies (over 10,000 performed procedures) and was examined by dedicated
genitourinary pathologist using 2005 International Society of Urological Pathology Gleason grading criteria.
Exclusion criteria were: active acute urinary tract infection, bleeding disorder and bowel malignancies
or inflammatory bowel diseases. Collected data included age, total PSA level, DRE examination result,
TRUS calculated prostate volume and full pathological report. Standard 12-core TRUS guided biopsy was
performed. Before procedure, single dose of i.v. ciprofloxacin was used as an antibiotic prophylaxis. Each
core was placed into a separate container.

Decision to perform prostate biopsy was based on either elevated PSA level (higher or equal to 4.0 ng/mL)
or positive DRE examination. For the purpose of metabolomic analysis, 10 mL of blood, 100 mL of urine
and 50 µL of interstitial fluid (IF) extracted from each biopsy core were collected from each patient who has
had prostate biopsy. Control group constituted of 10 young (< 40 years old, mean 28.7) healthy volunteers
with average PSA of 0.7 ng/mL and average estimated PCa risk (using multivariable ERSPC cohort PCa risk
calculator) of 2%. Each volunteer donated 10 mL of blood and 100 mL of urine, no biopsies were performed
in this group. Whole blood was collected and centrifuged at 3000 rpm at room temperature for 10 min.

Preparation of samples for mass spectrometry imaging
Urine, serum and IF samples were immediately stored at -80 °C until use. Urine and blood plasma samples
were diluted 600 times with water and placed on target plate (0.5 µL). Tissue samples were washed with 50 µL
of water and directly placed on target plate (0.5 µL). All samples of the same type (for example all urine
samples) were measured on the same target plate in one mass spectrometry imaging (MSI) experiment.
Extracts were placed on target plate with the aid of 3D precision translation stage system with spot diameter
ca. 1.5 mm.

Mass spectrometry measurements and data handling
Mass spectrometry (MS) analysis was performed using high-resolution laser desorption/ionization (LDI)-
[23]
time-of-flight (ToF)-MS based on gold nanoparticle-enhanced target (AuNPET) plate and is presented in
simplified form in flowchart in Figure 1. AuNPET target plates of 2.5 × 3.5 cm size and ca. 0.8 mm thickness
were used with Bruker NALDI adapter. AuNPET-based surface-assisted, LDI-ToF-MS experiments were