PROGNOSIS OF THE EARLY DISEASE RECURRENCE IN PATIENTS WITH LOCALISED PROSTATE CANCER AFTER RADICAL SURGERY Omelchenko

Aims. Aim: to improve the effectiveness of prognosis of early recurrence in patients with localised prostate cancer (PC) after radical surgery by assessment of expression of prostate-specific antigen РCА3 [prostate cancer associated 3] in the urine sediment and exosomes. Materials and methods. The study included 148 patients with localised PC. The serum level of prostate-specific antigen (PSA) was determined for all patients by enzyme immunoassay at the baseline and every 3 months after radical prostatectomy (RPE) for two years. Patients were divided into two subgroups depending on the presence or absence of biochemical recurrence (BR). Expression of PCA3 gene was determined in the urine sediment and exosomes by PCR [polymerase chain reaction] in real time, relative to the reference human kallikrein gene KLK3 [kallikrein related peptidase 3]. Results. PCA3 gene expression level in urine exosomes of patients with prostate cancer and prostatic intraepithelial neoplasia grade 2 (PIN2) was higher in the subsequent recurrence compared with a favourable course of the disease. With a decrease of ∆Ct РСА3–KLK3 to less than 1.86 inclusive in urine sediment, BR in patients with PC and PIN2 in the peritumoral area occurred more frequently (83% vs. 45%, p=0.008). Conclusions. Prognostic significance of the evaluation of РСА3 gene expression in the postmassage urine sediment and exosomes for determining BR risk after RPE in patients with localised PC is higher than the determination of PSA in the blood serum. With an increase in the PCA3 gene expression in the postmassage urine sediment and exosomes in patients with localised PC, the BR risk within two years after RPE increases.

microenvironment [7]. Search for new biological media for non-invasive quantification of PCA3 expression in patients will enhance cancer diagnosis and prognosis.
The goal hereof was to estimate PCA3 expression in urine sediments and exosomes to improve the prognosis of early recurrence in localized PC patients having undergone radical surgical treatment.

MATERIALS AND METHODS.
The study was carried out by the Center for Urology, Nephrology, and Hemodialysis of the Anatomic Pathology Unit, Rostov Regional Hospital No. 2, in 2015-2017.
The study protocol followed guidelines for experimental investigation with human subjects in accordance with the Declaration of Helsinki and was approved by the ethics committee. Written informed consent was obtained from each patient (or an official representative) before the study.
Histologically, PC was adenocarcinoma in all patients.
Upon entry and then every 3 months after RPE, patients' blood serum was tested for PSA by immunoassay using a Multiscan-P 2 photometer (Thermo Fisher Scientific Inc., Finland).
Biochemical recurrence (BR) was diagnosed if PSA exceeded 0.2 ng/ml of blood in three consecutive samples spaced by a fortnight or more.
For genetic testing, the first step of preparing the samples was to collect 70 ml of urine after prostate massage (pressing each lobe thrice). Once collected, 20 ml of urine was centrifuged over 15 minutes at 3,000 rpm; the supernatant was further removed, and the sediment was resuspended and sampled in an Eppendorf tube, 1.5 ml a sample. The remaining urine was preserved by adding 1 ml of the RNA Medium (InterLabService LLC, Russia). To extract exosomes, 50 ml of the postmassage sample was centrifuged over 15 minutes at 10,000 rpm; the resulting supernatant was centrifuged over 3 hours at 100,000 rpm. The sediment was washed by adding 3 ml of phosphate- RESULTS. PC may recur within months or years after conservative or radical treatment (which comprises surgery). Recurrence can be local, systemic, or biochemical, which only manifests itself as an increased serum PSA level. Thirty (20.3%) of 148 examined patients were found to have biochemical recurrence within two years after RPE. Literature suggests that five-year post-RPE BR rates range from 15% to 30% in PC patients [4]. Thus, this study showcased trends consistent with earlier epidemiological data. The preoperative results of genetic tests were analyzed in retrospect in the context of two-year recurrence history and BR development, see Table 1. frequency analysis and crosstabulation to study the association of these processes in detail, see Table 2. Thus, urine sediment ΔCt PCA3-KLK3 reduction to below 1.86 (which Apolikhin et al. [8] state is the differential point that separates PC and benign hyperplasia) was more frequent (83% vs. 45%, p=0.008). Therefore, comparing PCA3 expression in urine sediment is informative when it comes to evaluating the tumor recurrence risk.
Since Apolikhin et al. [8] proposed urine exosome ΔCt PCA3-KLK3 = 1.48 as the threshold for concluding PC development risk when differentiating it from benign processes, the authors used ROC analysis to further find such Ct threshold that would effectively isolate patients at a high risk of recurrence. ROC analysis identified the separation threshold of ΔCt PCA3 -KLK3 ≤ -2.9. That is, if a prostate adenocarcinoma patient had ΔCtPCA3-KLK3 of -2.9 or below in urine exosomes, a high risk of BR with 24 months of RPE could be expected at a diagnostic sensitivity of 90% and a diagnostic specificity of 86%. There were 10% false negatives and 14% false positives. The overall diagnostic effectiveness of quantifying urine exosome PCA3 expression to predict early post-PRE BR was 87%. The area under ROC was 0.896±0.0007 (p<0.0001), suggesting that the method was highly informative.
For urine sediment, the Ct threshold that separated patients at a high risk of recurrence was -0.51. That is, if a prostate adenocarcinoma patient had ΔCtPCA3-KLK3 of -0.51 or below in urine sediments, a high risk of BR with 24 months of RPE could be expected at a diagnostic sensitivity of 87% and a diagnostic specificity of 85%. There were 13% false negatives and 15% false positives.
The overall diagnostic effectiveness of quantifying urine sediment PCA3 expression to predict early post-PRE BR was 85%. The area under ROC was 0.802±0.0004 (p<0.0001), suggesting that the method was highly informative.
Detecting PC recurrence by monitoring the serum PSA levels in lab tests is a common clinical practice. However, serum-only PSA tests result in overdiagnosing PC progression in 1.7% to 67% of all cases [4]. Despite the developed tactics and additional strategies for monitoring postoperative PSA levels in the serum of PC patients, which include adjustments for age, PSA level growth and doubling rates, marker isoforms, and PSA "density" [2], a wide range of biochemical and molecular genetic markers in various biological fluids and tissues must be tested for more accurate prediction of postoperative disease course.
The results of this research into PCA3 expression are in line with the data of other authors that analyzed the expression of PCA3 and that of other genes of prostate-specific expression in whole urine, urine sediment, and urine exosomes [5; 8]. The authors underlined the informativeness of testing these biological fluids for differential diagnosis of benign and malignant prostate tumors.
Apolikhin et al. [6] proposed estimating PCA3 expression in urine sediment and exosomes as a promising test for PC diagnosis. Applying this method improved early PC detection while avoiding unnecessary biopsies. Continuing the development of a non-invasive PC patient monitoring system, the authors hereof estimated PCA3 expression in urine sediment and exosomes sampled from BR and non-recurrent PC patients before surgery. This approach helped assess the prognostic value of estimating PCA3 expression in urine sediment and exosomes to evaluate the early recurrence risks before surgery. The research effort identified urine sediment and exosome PCA3 expression levels, exceeding which could indicate a risk of early disease progression. Thus, the key finding is that estimating PCA3 expression in urine sediment and exosomes is informative for predicting BR within two years of RPE, albeit with some constraints.

1.
PCA3 expression in post-massage urine sediment and exosomes sampled from localized-PC patients has a higher prognostic value for predicting post-RPE BR risks than serum PSA.

2.
A higher PCA3 expression is associated with a higher risk of BR within two years of RPE. Evaluating the risk of localized PC progression by PCA3 expression as found by real-time polymerase chain reaction has a diagnostic effectiveness of 85% for urine sediment, 87% for urine exosomes.

FINANCIAL SUPPORT AND SPONSORSHIP
Nil.