Plasma Of Prostate Cancer Patients Research Articles

MP29-19 PRE-OPERATIVE PLASMA METABOLITES CAN PREDICT THE RISK OF BONE METASTASIS IN PROSTATE CANCER

You have accessJournal of UrologyCME1 Apr 2023MP29-19 PRE-OPERATIVE PLASMA METABOLITES CAN PREDICT THE RISK OF BONE METASTASIS IN PROSTATE CANCER Saurav Subedi, Federico La Manna, Katja Ovchinnikova, Eugenio Zoni, George N. Thalmann, and Marianna Kruithof-de Julio Saurav SubediSaurav Subedi More articles by this author , Federico La MannaFederico La Manna More articles by this author , Katja OvchinnikovaKatja Ovchinnikova More articles by this author , Eugenio ZoniEugenio Zoni More articles by this author , George N. ThalmannGeorge N. Thalmann More articles by this author , and Marianna Kruithof-de JulioMarianna Kruithof-de Julio More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003257.19AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Early identification of prostate cancer (PCa) patients that are most likely to develop a lethal disease is still a major hurdle. While for most patients, a radical treatment will result in the eradication of the disease, about 70% of patients with advanced PCa are diagnosed with bone metastasis, a lethal feature with limited treatment options. There is growing evidence suggesting that lipid metabolism cooperates with the genetic landscape of prostate cancer. Currently, several studies have shown that the metabolome of PCa could be used as non-invasive diagnostic and prognostic tool. In this study, we analyzed the plasma of 73 prostate cancer patients to identify metabolites with diagnostic or prognostic value. METHODS: The plasma was collected during fasting and shortly before the patient received radical prostatectomy. The patients were then followed up for up to 10 years, to monitor the development of bone metastases. Targeted metabolomics was run on the pre-operative plasma. 10 different machine learning models were tested on the available metabolomics data-set. All patients provided informed consent for participation in this study. RESULTS: By using machine learning algorithms, we identified a signature of 41 metabolites either positively or negatively associated with the development of bone metastatic PCa. Neural network and support vector machine yielded the best results for classification of metastasis based on the metabolites. Furthermore, linear support vector machine and Gaussian process generated the best regression models for determination of time-to-bone metastasis using absolute Spearman`s R correlation. Determination of both the classification of bone metastasis and time-to-metastasis were identified based on 41 metabolites in the Neural network and support vector machine algorithms. The top-four metabolites associated with bone metastatic progression were lysophosphatidylethanolamine(LPE 22:5/0:0)b, butyrylcarnitine (C4), pyruvic acid and dodecanoylcarnitine (C12), whereas monoacylglycerol (MAG 14:0/0:0), MAG (0:0/22:5), acetylornithine and MAG (0:0/14:0) were the bottom-four metabolites. CONCLUSIONS: In this study, we evaluated metabolites in the plasma of prostate cancer patients to determine if we could identify a predictive signature of metastatic progression. Our studies show that metabolic phenotypes have substantial clinical value. Given the context of precision medicine, we show the feasibility of a simple metabolomics test on the plasma of patients that could help stratify patient`s risk of lethal disease based on the presence/absence of signature metabolites. Further studies in a larger cohort will be required in order to test the signature metabolites found in our study to test the reliability for patient stratification. Source of Funding: Swiss Cancer Research Foundation (KFS-5510-02-2022-R) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e389 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Saurav Subedi More articles by this author Federico La Manna More articles by this author Katja Ovchinnikova More articles by this author Eugenio Zoni More articles by this author George N. Thalmann More articles by this author Marianna Kruithof-de Julio More articles by this author Expand All Advertisement PDF downloadLoading ...

Diagnostic potential value of circulating PCA3 mRNA in plasma and urine of prostate cancer patients

Current reports show that most of the routinely available prostate specific antigen (PSA) derived biomarkers of prostate cancer (PCa) have failed to demonstrate sufficient sensitivity and specificity in clinical use for diagnosis and prognosis of PCa. Consequently, a number of molecular markers including the prostate cancer gene 3 (PCA3) are being considered for clinical use. This study was therefore designed to determine the levels of PCA3 mRNA expression in urine and plasma of prostate cancer patients and to assess its diagnostic characteristics in Nigerian population with prostate cancer. A total of 90 participants comprising 30 participants each with PCa, BPH and 30 apparently healthy adults as controls were enrolled into this case-control study. Blood and urine samples were collected from study participants and dispensed into PaxGene RNA protecting fluid, lithium heparin bottles and nuclease free bottle as appropriate. Anthropometric indices were obtained using standard methods, while the plasma levels of PSA, testosterone and estrogen were determined using enzyme linked immunosorbent assay (ELISA). The PCA3 mRNA expression in urine and plasma was determined using Quantitative Polymerase Chain Reaction. Comparison of groups was done using analysis of variance (ANOVA) followed by Tukey Post-Hoc test. The sensitivity and specificity of PCA3 mRNA in the diagnosis of PCa was evaluated through estimation of the area under the receiver operating characteristic (ROC) curve. 'The mean waist circumference (WC) and (HC) was significantly higher in the BPH group (43.94 ± 2.32) compared to both the control (36.04 ± 0.80) and the PCa group (38.50 ± 0.94) (p < 0.05). The prostate cancer group had a significantly higher levels (p < 0.05) of tPSA (47.58 ± 12.23) and an increased expression of PCA3 mRNA in urine (10.46 ± 1.68) and plasma (14.89 ± 1.37) when compared with control values [tPSA (0.67 ± 0.27), urine (2.22 ± 0.22) and plasma (1.45 ± 0.13) expression of PCA3 mRNA]. A negative correlation was found to exist between Plasma PCa3 mRNA and plasma levels of estradiol (r=-0.425, p = 0.024) and testosterone (r=-0.423, p = 0.025) in PCa group. A similar trend was observed between urine PCA3 mRNA, estrogen (r=-0.459, p = 0.014) and testosterone (r=-0.407, p = 0.031). The ROC curve shows the tPSA (p = 0.03) is the most sensitive marker of detection followed by plasma PCA3 mRNA and subsequently the urine PCA3 mRNA. The curve further shows the urine PCA3 mRNA (p = 0.006) is the most specific for detection of prostate cancer and the less and least sensitive are the plasma PCA3 mRNA and tPSA. It could be concluded from this study that there is reduced levels of estradiol and testosterone and elevated levels of tPSA and PCA3 mRNA expression in urine and blood in patients with benign prostatic hyperplasia and prostate cancer. Hence, determination of plasma or urine expression of PCA3 mRNA is valueable in the diagnosis and treatment monitoring of prostate cancer.

Small Extracellular Vesicle-Derived microRNAs Stratify Prostate Cancer Patients According to Gleason Score, Race and Associate with Survival of African American and Caucasian Men.

Simple SummaryNovel biomarkers are needed to guide prognosis and treatment of aggressive forms of prostate cancer (PCa). In this study, small extracellular vesicles (sEVs)-derived microRNAs (miRs) are used to predict aggressive phenotypes and ancestral background of PCa patients. Two cohorts was used to study the diagnostic and prognostic utility of sEV-associated miRs in predicting aggressive forms of PCa in African American (AA) and Caucasian (CA) men. In training cohort, miR profiling was performed and top-ranked sEV-associated miRs were then validated in two independent confirmatory cohorts comprising 150 plasma samples. Results revealed that sEV-associated miR-6068 and miR-1915-3p were enriched in PCa patients compared to healthy subjects. sEV-associated miR-6716-5p and miR-3692-3p distinguished AA from CA men and low from high Gleason score. However, miR-1915-3p was the only studied miR associated with longer recurrence-free survival as independent prognostic marker. The utility of small extracellular vesicles (sEVs)-derived microRNAs (miRs) to segregate prostate cancer (PCa) patients according to tumor aggressiveness and ancestral background has not been fully investigated. Thus, we aimed to determine the diagnostic and prognostic utility of sEV-associated miRs in identifying aggressive PCa in African American (AA) and Caucasian (CA) men. Using a training cohort, miR profiling was performed on sEVs isolated from plasma of PCa patients. Top-ranked sEV-associated miRs were then validated in 150 plasma samples (75 AA and 75 CA) collected from two independent cohorts; NIH (n = 90) and Washington University (n = 60) cohorts. Receiver operating characteristic (ROC) curve, Kaplan–Meier and Cox proportional hazards regression were used to assess these miRs as clinical biomarkers. Among nine top-ranked sEV-associated miRs, miR-6068 and miR-1915-3p were enriched in sEVs collected from PCa patients compared to healthy volunteers. Moreover, miR-6716-5p and miR-3692-3p segregated AA from CA men and low from high Gleason score (GS), respectively. Upregulation of sEV-associated miR-1915-3p, miR-3692-3p and miR-5001-5p was associated with improved survival time, and only miR-1915-3p was associated with longer recurrence-free survival (RFS) as an independent prognostic marker. Taken together, we identified novel sEV-associated miRs that can differentiate PCa patients from normal, AA from CA and high from low GS and predicts RFS.

Lipidomic Analysis Demonstrates that 36:1 Phosphatidylcholine is Enriched in Cell Models of Drug Resistance Prostate Cancer

A lack of understanding exists between the association between circulating lipids and clinical outcomes of drug‐resistant castration‐resistant prostate cancer (CRPC). Although an increase in select circulating lipids correlates to decreased patient survival, neither the mechanisms mediating alterations in these lipids, nor the correlation to drug resistance are well characterized. This gap‐in‐knowledge was addressed using in vitro models of non‐cancerous, hormone‐sensitive, CRPC and drug‐resistant cell lines combined with quantitative HPLC‐ESI‐Orbitrap‐MS lipidomic analysis.Lipid extracts were obtained using Bligh‐Dyer (methanol/chloroform/water) extraction from non‐cancerous, hormone‐sensitive, CRPC and drug‐resistant prostate cell lines. Lipids present in both cells and media were initially analyzed using an untargeted shotgun approach (ESI‐MS), followed by a targeted‐based quantitative HPLC‐ESI‐Orbitrap‐MS approach. Shotgun experiments were performed using commercially available standards. For target‐based lipidomics, LC separation yielded specific lipid class separation prior to ESI/MS/MS enabling enhanced detection of lipids suppressed in the shotgun approach. Post data acquisition for both shotgun and HPLC‐ESI/MS/MS lipidomics were based on multiple methods including isotope, carbon number (to internal standards) and ionization efficiency‐based corrections. Pathway analysis was also performed on the various prostate cell lines. Online resources and software’s utilized included MetaboAnalyst, LIPIDMATCH LIPID MAPS, XCMS, and MZmine.Principle component analysis (PCA) generated scores plots of non‐cancerous, hormone‐sensitive, CRPC parent cell lines and drug‐resistant CRPC cell lines in the positive mode (ESI+) had a total variance of 66%. Additionally, a distinct separation between drug‐resistant and parent control cell lines was shown. Several discriminatory ion features were identified between prostate cell lines, especially between drug‐resistant and parent control neuroendocrine cell lines. Of these discriminatory features, 83 showed significant alterations. Two of the identified lipids had alterations matching those measured in plasma from prostate cancer patients.Distinct phospholipid classes were observed to have increased levels in prostate cancer cells, when compared to non‐cancer cells. Lipidomic analysis identified phosphatidylcholines (PC 36:1), whose levels were increased in Docetaxel resistant CRPC. These data also show that the lipidomic profiles of prostate cancer cell lines mirror that seen in the plasma of prostate cancer patients. These data may lead to new early detection strategies and more personalized treatment options for patients.Support or Funding InformationDepartment of Defense Prostate Cancer Research Program Idea Development Award (PC150431 GRANT11996600)Phospholipids in Prostate Cancer(A. Scores Plot) PCA scores plot comparing drug‐resistant CRPC cells to DU145 control parent cells in the positive mode (ESI+). ~80% of the total variance is explained by Component 1 and Component 2. (B. MTT Assay) MTT assay shows decrease in MTT staining in parent cell lines as compared to docetaxel‐resistant cell lines. This suggests DU145‐DR (DR) maintain resistance to Docetaxel as compared to DU145 parent cell line. (C. Heat Map) Each row represents a metabolite feature and each column represents a sample. Metabolite features whose levels vary significantly (p&lt; 0.01) are projected on the heat map and used for sample clustering displayed above. (D. Cloud Plot) Differential feature plot: Only features that are dysregulated in both DU145‐DR cells and DU145‐DR media (p‐value&lt;= 0.05 threshold, fold change &gt;= 1.5 threshold) are displayed above. Up‐regulated features (features that have a positive fold change) are graphed above the x‐axis while down‐regulated features (features that have a negative fold change) are graphed below the x‐axis.Figure 1PC 36‐1 in Prostate Cancer(A) Cloud plot showing 7460 discriminatory ion features (positive ion data) in human cell lines. Only dysregulated features are displayed above (p‐value &lt;= 0.05 threshold, fold change &gt;= 1.5 threshold). The radius of each bubble corresponds to the log fold change of that feature. Features with higher fold change (or higher intensity) will have larger radii. The shade of bubbles corresponds to the magnitude of the p‐value (the darker the color, the smaller the p‐value). The statistical significance of the fold change, as calculated by a Welch t‐test with unequal variances.Figure 6. (B) Box‐plot identified 788.6 feature in human cell lines. Of the lipids identified by the cloud plot, PC(36:1) was one of the few lipid species enriched in all drug resistant prostate cancer cell lines as compared to control cells. This was previously observed in castration‐resistant prostate cancer patient‐derived plasma samples.Figure 2

Circulating MicroRNAs as Biomarkers for Prostate Cancer Detection and Metastasis Development Prediction.

Prostate Cancer (PCa) overdiagnosis and overtreatment, as a consequence of the limited specificity of current detection and prognostication methods, remains a major challenge in clinical practice. Therefore, development and validation of new molecular biomarkers amenable of detecting clinically significant disease is crucial. MicroRNAs (miRNA) deregulation is common in cancer, constituting potential non-invasive biomarkers for PCa detection and prognostication. Herein, we evaluated the screening and prognostic biomarker potential of two onco-microRNAs (miR-182-5p and miR-375-3p) in liquid biopsies (plasma) of PCa patients with clinically localized disease undergoing curative-intent treatment. A first cohort of 98 PCa and 15 normal prostates were used to assess PCa-specificity of miR-182-5p in tissues. A cohort composed of PCa 252 patients and 52 asymptomatic controls allowed for assessment of diagnostic and prognostic value in plasmas. After RNA extraction from tissue and plasma samples, cDNA synthesis specific for miRNAs was performed followed by measurement of miR-182-5p and miR-375-3p relative expression by RT-qPCR, using U6 snRNA gene as reference. MiR-182-5p was significantly overexpressed in PCa tissues (p < 0.0001) and in plasma of PCa patients (p = 0.0020), compared to respective controls. Moreover, miR-182-5p expression identified PCa with AUC = 0.81 (95% CI: 0.725–0.892, p = 0.0001) in tissue and with 77% specificity and 99% NPV (AUC = 0.64, 95% CI: 0.561–0.709, p = 0.0021) in plasma. Both circulating miR-182-5p and miR-375-3p levels associated with more advanced pathologic stage and the former was significantly higher in patients that developed metastasis (p = 0.0145). Indeed, at the time of diagnosis, circulating miR-375-3p levels predicted which patients would develop metastasis, with almost 50% sensitivity, 76% specificity, and a NPV of 89% (AUC = 0.62, 95% CI: 0.529–0.713, p = 0.0149). We conclude that these two circulating miRNAs might be clinical useful as non-invasive biomarkers for detection and prediction of metastasis development at the diagnosis together with clinical variables used in routine practice.

Multi-omics Biomarker Pipeline Reveals Elevated Levels of Protein-glutamine Gamma-glutamyltransferase 4 in Seminal Plasma of Prostate Cancer Patients

Seminal plasma, because of its proximity to prostate, is a promising fluid for biomarker discovery and noninvasive diagnostics. In this study, we investigated if seminal plasma proteins could increase diagnostic specificity of detecting primary prostate cancer and discriminate between high- and low-grade cancers. To select 147 most promising biomarker candidates, we combined proteins identified through five independent experimental or data mining approaches: tissue transcriptomics, seminal plasma proteomics, cell line secretomics, tissue specificity, and androgen regulation. A rigorous biomarker development pipeline based on selected reaction monitoring assays was designed to evaluate the most promising candidates. As a result, we qualified 76, and verified 19 proteins in seminal plasma of 67 negative biopsy and 152 prostate cancer patients. Verification revealed a prostate-specific, secreted and androgen-regulated protein-glutamine gamma-glutamyltransferase 4 (TGM4), which predicted prostate cancer on biopsy and outperformed age and serum Prostate-Specific Antigen (PSA). A machine-learning approach for data analysis provided improved multi-marker combinations for diagnosis and prognosis. In the independent verification set measured by an in-house immunoassay, TGM4 protein was upregulated 3.7-fold (p = 0.006) and revealed AUC = 0.66 for detecting prostate cancer on biopsy for patients with serum PSA ≥4 ng/ml and age ≥50. Very low levels of TGM4 (120 pg/ml) were detected in blood serum. Collectively, our study demonstrated rigorous evaluation of one of the remaining and not well-explored prostate-specific proteins within the medium-abundance proteome of seminal plasma. Performance of TGM4 warrants its further investigation within the distinct genomic subtypes and evaluation for the inclusion into emerging multi-biomarker panels.

Circulating microRNAs in plasma before and after radical prostatectomy

PurposeMicroRNAs (miRNAs/miRs) as circulating biomarkers for prostate cancer have yet to be determined. We examined whether circulating miRNAs in plasma could be employed as biomarkers of disease among men treated for prostate cancer by radical prostatectomy (RP). MethodsThe expression of 17 preselected circulating miRNAs associated with prostate cancer (miR-381, -34a, -365, -122, -375, -1255b, -34b, -450b-5p, -885-5p, -1260, -150, -378, -671-3p, -148a, and -224) or high-grade prostate cancer (miR-28 and -100) in plasma at prostate biopsy was examined in pre- and post-RP plasma of prostate cancer patients using real-time PCR and compared using Wilcoxon signed-ranked test. Wilcoxon rank sum test was used to compare the expression of miRNAs in pre-RP plasma between pathologic tumor stage (T2 vs. T3) and Gleason score (6–7 [3 + 4] vs. ≥ 7 [4 + 3]) groups. Partial correlation coefficient between the expression of miRNAs in pre-RP plasma and serum prostate-specific antigen (PSA) level at RP, adjusting for age, was calculated. ResultsTwenty-nine men, aged 43 to 77 years, were included. Median follow-up time after RP was 55 days. There was no significant change in the expression of miRNAs in plasma from before to after RP. However, higher expression of miR-34a, -378, and 450b-5p in pre-RP plasma was observed among T3 compared to T2 patients (P values = 0.01). Overall, there were no statistically significant relationships observed between the expression of these circulating miRNAs and Gleason score and serum PSA at RP. ConclusionsThere was no significant change in the expression of circulating miRNAs in plasma from before to approximately 2 months after RP. This finding may be due to the lack of immediate effect RP may have on the expression of circulating miRNAs. However, higher expression of miR-34a, -378, and -450b-5p in plasma was found among patients with more advanced disease at RP. A longer follow-up time after RP is warranted to investigate RP's possible influence on circulating miRNAs among men treated for prostate cancer and to evaluate miRNAs’ diagnostic potential for prostate cancer.

Ultrasensitive detection of cancer biomarkers by nickel-based isolation of polydisperse extracellular vesicles from blood.

BackgroundExtracellular vesicles (EVs) are secreted membranous particles intensively studied for their potential cargo of diagnostic markers. Efficient and cost-effective isolation methods need to be established for the reproducible and high-throughput study of EVs in the clinical practice.MethodsWe designed the nickel-based isolation (NBI) to rapidly isolate EVs and combined it with newly-designed amplified luminescent proximity homogeneous assay or digital PCR to detect biomarkers of clinical utility.FindingsFrom plasma of 46 healthy donors, we systematically recovered small EV (~250 nm of mean diameter; ~3 × 1010/ml) and large EV (~560 nm of mean diameter; ~5 × 108/ml) lineages ranging from 50 to 700 nm, which displayed hematopoietic/endothelial cell markers that were also used in spike-in experiments using EVs from tumor cell lines. In retrospective studies, we detected picomolar concentrations of prostate-specific membrane antigen (PSMA) in fractions of EVs isolated from the plasma of prostate cancer patients, discriminating them from control subjects. Directly from oil-encapsulated EVs for digital PCR, we identified somatic BRAF and KRAS mutations circulating in the plasma of metastatic colorectal cancer (CRC) patients, matching 100% of concordance with tissue diagnostics. Importantly, with higher sensitivity and specificity compared with immuno-isolated EVs, we revealed additional somatic alterations in 7% of wild-type CRC cases that were subsequently validated by further inspections in the matched tissue biopsies.InterpretationWe propose NBI-combined approaches as simple, fast, and robust strategies to probe the tumor heterogeneity and contribute to the development of EV-based liquid biopsy studies.FundAssociazione Italiana per la Ricerca sul Cancro (AIRC), Fondazione Cassa di Risparmio Trento e Rovereto (CARITRO), and the Italian Ministero Istruzione, Università e Ricerca (Miur).

Lipidomic Analysis of Media from Castration and Drug Resistant Prostate Cancer Cells

Castration‐resistant prostate cancer (CRPC) is a severe, metastatic form of prostate cancer (PCa). Several treatment modalities have been approved during the past decade by the FDA for treating CRPC patients. Unfortunately, a high percentage of patients fail to respond to these treatments for several reasons, including developing resistance. One reason for these failures is inadequate methods that allow for accurate staging of PCa. This poses a diagnostic problem that limits our ability to create a patient‐centered treatment plan. We addressed this problem by testing the hypothesis that an association exists between circulating lipids and clinical outcomes of CRPC. This hypothesis is based on recent studies demonstrating a correlation between plasma lipids and CRPC prognosis. To understand this correlation, we used in vitro models of non‐cancerous, hormone‐sensitive, and drug‐resistant CRPC cell lines combined with quantitative lipidomic analysis to study the correlation between CRPC and lipid profiles. An untargeted approach indicated that media from hormone sensitive cells (LNCaP), and CRPC cells (DU145) had lower relative abundance of glycerophospholipids, as compared to a non‐cancer cell model (PNT2). However, drug resistant cells (DU145 DR) and their media had a higher abundance of glycerophospholipids such as phosphatidylinositol (PI), phosphatidic acid (PA), and phosphatidylserine (PS) as compared to parent control (DU145). These data confirm previous in vivo findings and suggest that the lipidomic profiles of PCa cells lines mirror lipidomic profiles in the plasma of PCa patients. Further, these data provide an in vitro model to investigate the molecular mechanisms mediating lipidomic changes during the progression of drug resistant PCa.Support or Funding InformationNational Institute of Biomedical Imaging and Bioengineering NIBIB (EB0160100), Department of Defense Prostate Cancer Research Program Idea Development Award (PC150431 GRANT11996600)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phospholipids as Indicators of Docetaxel‐Resistant Castration‐Resistant Prostate Cancer

The association of circulating lipids with clinical outcomes of drug‐resistant castration‐resistant prostate cancer (CRPC) is not fully understood. While it is known that increases in plasma lipids correlates to decreased survival, neither the mechanisms mediating alterations in these lipids nor the correlation of resistance to treatment are well characterized. We addressed this gap‐in‐knowledge using in vitro models of non‐cancerous, hormone sensitive, CRPC and drug‐resistant cell lines combined with quantitative HPLC‐ESI‐Orbitrap‐MS lipidomic analysis. HPLC‐MS based metabolomic analysis identified 83 features that were significantly altered in the drug‐resistant cell lines as compared to non‐resistant control group. Within the 83 species, two were identified as individual plasma lipid biomarkers for diagnosis of prostate cancer from previous studies. Principal component analysis (PCA) demonstrated that drug‐resistant and control cell‐lines were visually separated by these identified lipid biomarkers. Two out of the 83 lipid classes were identified as phosphatidylcholine (PC) 18:0/18:1 and PC 18:0/18:2), both of which had high level of abundance in Docetaxel‐resistant CRPC. An untargeted approach indicated that lipids corresponding to PC, phosphatidylglycerol (PG), phosphatidic acid (PA) and phosphatidylserine (PS) all had higher relative abundance in DR‐CRPC cells (DU145‐DR) compared to parent control (DU145). These data suggest that the lipidomic profiles of prostate cancer cells lines recapitulate lipidomic profiles in the plasma of prostate cancer patients. As such, these metastatic dependent and drug‐resistant cell lines may prove useful for understanding the molecular mechanisms mediating lipid changes in prostate cancer patients.Support or Funding InformationNational Institute of Biomedical Imaging and Bioengineering NIBIB (EB0160100) Department of Defense Prostate Cancer Research Program Idea Development Award (PC150431 GRANT11996600)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hydrolysis of ATP, ADP, and AMP is increased in blood plasma of prostate cancer patients.

Prostate cancer is among the major malignancies that affect men around the world. Adenine nucleotides are important signaling molecules that mediate innumerous biological functions in pathophysiological conditions, including cancer. These molecules are degraded by several ectoenzymes named ectonucleotidases that produce adenosine in the extracellular medium. Some of these ecto-enzymes can be found in soluble in the blood stream. Thus, the present study aimed to evaluate the hydrolysis of adenine nucleotides (ATP, ADP, and AMP) in the plasma blood of patients with prostate cancer. Peripheral blood samples were collected, and questionnaires were filled based on the clinical data of the medical records. The nucleotide hydrolysis was performed by Malachite Green method using ATP, ADP, and AMP as substrates. Plasma from prostate cancer patients presented an elevated hydrolysis of all nucleotides evaluated when compared to healthy individuals. NTPDase inhibitor (ARL67156) and the alkaline phosphatase inhibitor (levamisole) did not alter ATP hydrolysis. However, AMP hydrolysis was reduced by the CD73 inhibitor, APCP, and by levamisole, suggesting the action of a soluble form of CD73 and alkaline phosphatase. On microvesicles, it was observed that there was a low expression and activity of CD39 and almost absent of CD73. The correlation of ATP, ADP, and AMP hydrolysis with clinic pathological data demonstrated that patients who received radiotherapy showed a higher AMP hydrolysis than those who did not, and patients with lower clinical stage (CS-IIA) presented an elevated ATP hydrolysis when compared to those with more advanced clinical stages (CS-IIB and CS-III). Patients of all clinical stages presented an elevated AMPase activity. Therefore, we can suggest that the nucleotide hydrolysis might be attributed to soluble ecto-enzymes present in the plasma, which, in a coordinate manner, produce adenosine in the blood stream, favoring prostate cancer progression.

Long non-coding RNA SAP30L-AS1 promotes prostate cancer growth through repressing SAP30L

Accumulating evidences have demonstrated the importance of long non-coding RNAs (lncRNAs) in initiation and progression of various cancers, including prostate cancer. LncRNA SAP30L-AS1 is previously identified in the plasma of prostate cancer patients. In this study, we further investigated the expression of SAP30L-AS1 in prostate cancer tissues and cell lines. Moreover, we explored the biological roles and mechanisms of action of SAP30L-AS1 in prostate cancer. The expression of SAP30L-AS1 is found to be increased in prostate cancer tissues and cell lines compared with adjacent noncancerous tissues and normal prostate epithelial cell line, respectively. Increased expression of SAP30L-AS1 is associated with greater Gleason score, advanced pathological T stage, and poor over survival of prostate cancer patients. Functional assays demonstrated that ectopic expression of SAP30L-AS1 promotes prostate cancer proliferation and inhibits prostate cancer apoptosis. SAP30L-AS1 knockdown represses prostate cancer proliferation and induces prostate cancer apoptosis. Furthermore, SAP30L-AS1 knockdown represses prostate cancer xenograft growth in vivo. Mechanistic investigation revealed that SAP30L-AS1 physically binds to the promoter of SAP30L and represses SAP30L expression. The expression of SAP30L is negatively associated with that of SAP30L-AS1 in prostate cancer tissues. Rescue assays demonstrated that overexpression of SAP30L attenuated the roles of SAP30L-AS1 in promoting prostate cancer proliferation and inhibiting prostate cancer apoptosis. In conclusion, SAP30L-AS1 is upregulated and has oncogenic roles in prostate cancer via repressing SAP30L. Our data suggest that SAP30L-AS1 may be a promising prognostic biomarker and therapeutic target for prostate cancer.

Abstract B047: Biochemical parameters of prostate cancer patients in Nigeria

Abstract The screening of biochemical parameters of cancer patients and their development into reliable and specific diagnostic and prognostic biomarkers has been reported in several studies. The biochemical signature of prostate cancer patients in Nigeria may vary from others and can serve as important leads in discovery of new biomarkers for the disease. This will ultimately provide more information on the specific causes and prognosis of the disease as well as improve its diagnosis and management. This study examined some biochemical parameters including alanine transaminase (ALT), alkaline phosphatase (ALP), low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglycerides (TRIG), reduced glutathione (GSH), lipid peroxidation and peroxidase (POD) in the plasma of prostate cancer patients and controls. The data obtained indicate that there were no significant differences in the ALT, LDL, HDL, and POD while ALP and TRIG were significantly increased. Also, there was a significant reduction in GSH. This indicates the possible implication of ALP, TRIG, and GSH in prostate cancer disease in Nigeria and their potential to be further developed into diagnostic and prognostic biomarkers. Citation Format: Emeka Iweala, Solomon Rotimi, Faith Kanu, Chidiebere Ogo. Biochemical parameters of prostate cancer patients in Nigeria [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B047.

Prostate cancer sheds the αvβ3 integrin in vivo through exosomes

The αvβ3 integrin has been shown to promote aggressive phenotypes in many types of cancers, including prostate cancer. We show that GFP-labeled αvβ3 derived from cancer cells circulates in the blood and is detected in distant lesions in NOD scid gamma (NSG) mice. We, therefore, hypothesized that αvβ3 travels through exosomes and tested its levels in pools of vesicles, which we designate extracellular vesicles highly enriched in exosomes (ExVs), and in exosomes isolated from the plasma of prostate cancer patients. Here, we show that the αvβ3 integrin is found in patient blood exosomes purified by sucrose or iodixanol density gradients. In addition, we provide evidence that the αvβ3 integrin is transferred through ExVs isolated from prostate cancer patient plasma to β3-negative recipient cells. We also demonstrate the intracellular localization of β3-GFP transferred via cancer cell-derived ExVs. We show that the ExVs present in plasma from prostate cancer patients contain higher levels of αvβ3 and CD9 as compared to plasma ExVs from age-matched subjects who are not affected by cancer. Furthermore, using PSMA antibody-bead mediated immunocapture, we show that the αvβ3 integrin is expressed in a subset of exosomes characterized by PSMA, CD9, CD63, and an epithelial-specific marker, Trop-2. Finally, we present evidence that the levels of αvβ3, CD63, and CD9 remain unaltered in ExVs isolated from the blood of prostate cancer patients treated with enzalutamide. Our results suggest that detecting exosomal αvβ3 integrin in prostate cancer patients could be a clinically useful and non-invasive biomarker to follow prostate cancer progression. Moreover, the ability of αvβ3 integrin to be transferred from ExVs to recipient cells provides a strong rationale for further investigating the role of αvβ3 integrin in the pathogenesis of prostate cancer and as a potential therapeutic target.

Abstract 1618: Simultaneous isolation of exosomes and cfDNA from liquid biopsies using universal kit based on SubX-MatrixTM technology

Abstract In 2016 we have developed novel approach for isolation of circulating cell-free DNA employing the proprietary bi-functional substance SubX® that tightly binds DNA and forms sandwich [cfDNA- SubX®-magnetic particles]. These sandwich aggregates are easily separated from bio-liquid with magnet followed by cfDNA isolation by speedy protocol. Our approach does not require Proteinase K digestion and 2-3-fold dilution of bio-liquid with lysis/binding buffer in contrast to all commercially available kits. Our kit has been evaluated in various research and clinical labs for cfDNA isolation from serum, plasma and urine of patients with different forms of cancer. SubX®-approach also allows for isolation of cfDNA from large volumes of urine samples without its concentration. We demonstrated linear increase of the DNA yield with liquid biopsy volume. Another unique property of the SubX® is that both ends of the molecule display lipid binding groups. This feature allows each molecule of SubX® to anchor two exosomes (i.e. dimerize). Excess of SubX® molecules in the solution results in aggregation of up to 10-15 exosomes and formation of micron-size particles that are easily precipitated in a brief 14K x g (or 30 min 2K x g) centrifugation step. A specially designed buffer allows for reconstitution of the pelleted exosomes back to monomer format for downstream applications. Flow cytometry with specific surface marker CD9 confirmed that SubX®-aggregated vesicles are exosomes. Analysis of membrane-bound protein markers and microRNA profile of exosomes isolated from plasma of prostate cancer patients will be presented. Difference in SubX® binding strength for cfDNA and exosomes allows for consequent isolation from the [cfDNA/exosomes- SubX®-magnetic particles] pellet first exosomes, then cfDNA. Eventually, we combined both approaches in one kit for isolation of cfDNA and exosomes from the same sample. This simultaneous isolation protocol is simple and fast, it allows to employ minimal volume of valuable bio samples as well as to reduce labor and cost. The kit was validated in serum and urine samples from patients with ovarian and pancreatic cancer. Exosome nature of isolated vesicles was confirmed by three independents methods - light scattering, immunological and electron microscopy. Thus, one could analyze both genetic (DNA and RNA) as well as protein markers present in the same liquid biopsy sample. Citation Format: Andrei G. Malykh, Anastasia Malek, Anna Lokshin, Vladimir Evtushenko. Simultaneous isolation of exosomes and cfDNA from liquid biopsies using universal kit based on SubX-MatrixTM technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1618.

Circulating microRNA expression in connection with prostate cancer lymphogenous metastasis

Background. Lymph node metastases in prostate cancer (PC) are a negative prognostic factor. Non-invasive methods for their diagnostics are of primary importance. Objectives are identification of miRNA markers of lymph node metastases in plasma of PC patients and investigation of changes in primary tumors transcriptomes and plasma miRNA profiles during metastasis. Materials and methods. Plasma of 20 PC patients (10 with pN0M0 and 10 with pN1M0 stage) were collected and plasma miRNA expression was profiled on GeneChip miRNA 4.0 arrays (Affymetrix, USA). Target genes were searched for miRNAs with significant expression difference between pN0M0 and pN1M0 groups (fold change ≥2; p &lt;0,05). In addition, bioinformatic analysis of 392 PC primary tumors transcriptomes from PRAD collection (ТCGA Research Network: http://cancergenome.nih.gov/) was done (318 for pN0M0 stage and 74 for pN1M0 stage). Results. The level of 17 miRNAs were significantly lower in plasma of pN1M0 group. Analysis of primary tumors expression profiles revealed 88 genes with significantly different expression between pN0M0 and pN1M0 groups (fold change ≥1,5; p &lt;0,05). 11 of these genes are the potential targets of 17 miRNAs with lower levels in plasma of pN1M0 group. Interestingly, in most cases (8 out of 11) expression of these genes in primary tumor is elevated. Conclusion. The level of 17 miRNAs were significantly lower in plasma of PC patients with lymph nodes metastases (pN1M0). Analysis of primary tumor transcriptomes revealed a possible connection between miRNAs and their target genes levels in primary tumor and plasma. 17 plasma miRNAs found in this work could be a novel non-invasive markers of lymph nodes metastases in PC.

Phospholipids as Indicators of Castration Resistant Prostate Cancer

The association of circulating lipids with clinical outcomes of metastatic castration‐resistant prostate cancer (CRPC) is not fully understood. Interestingly, recent studies demonstrated a correlation between plasma lipids and CRPC prognosis, resulting in a proposed prognostic four‐lipid signature of (ceramide, sphingomyelin, phosphatidylcholine (PC) and phosphatidylserine (PS)). While the increase in these lipids correlated to decreased survival, the mechanisms mediating alterations in these lipids, and the correlation to resistance to treatment are not known. We addressed this gap‐in‐knowledge using in vitro models of non‐cancerous, hormone sensitive and CRPC cell lines and quantitative lipidomic analysis. An initial untargeted approach assessing both cells and media indicated that ceramides, sterols and glycerophospholipids had higher relative abundance in CRPC cells (PC‐3 and DU145), as well as hormone sensitive cells (LNCaP), as compared to a non‐cancer cell model (PNT2). Similar data was seen in media. A targeted approach focusing on lipid species shown to correlate to survival in recent publications showed significantly higher levels of 36:2 phosphatidylserine (PS) in all prostate cancer cell lines tested, and lower levels of 36:2 PC in the media of these same cells. Thus, both untargeted and targeted analysis of prostate cancer cells showed significant differences in lipidomic profiles as compared to non‐cancer prostate cells. These data suggest that the lipidomic profiles of prostate cancer cells lines mirror lipidomic profiles in the plasma of prostate cancer patients. As such, these cells may prove useful for understanding the molecular mechanism mediating lipid changes in prostate cancer patients and yield insight into the role of lipids in mediating CRPC.Support or Funding InformationNational Institute of Biomedical Imaging and Bioengineering NIBIB (EB0160100)Department of Defense Prostate Cancer Research Program Idea Development Award (PC150431 GRANT11996600)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Immune checkpoint molecule expression measured using circulating cell-free RNA isolated from the blood of metastatic prostate cancer patients.

380 Background: Recent studies have identified panels of immune-related genes linked to progression and survival. Expressions usually are measured using RNA from tissue samples. The aim of the present study was to utilize circulating RNA (cfRNA) isolated from hormone-naïve (HN) and castrate-resistant prostate cancer (CRPC) patients to measure gene expressions of PD-L1, cytotoxic T-lymphocyte associated protein 4 (CTLA-4), T-cell immunoglobulin and mucin domain 3 (TIM-3) and lymphocyte activated gene 3 (LAG-3). Androgen receptor (AR) and AR-variant 7 (AR-v7) expressions were also measured in these samples to test for any possible relationships with the immune checkpoint genes. Methods: Blood samples were collected from 53 metastatic prostate cancer patients. cfRNA was extracted from patients’ plasma and reverse transcribed into complementary DNA. The gene expressions (mRNA levels) of the above genes were determined by quantitative reverse transcription-PCR. Beta-actin was used to normalize gene expressions to total RNA content. Results: Each of the immune signatures tested (PD-L1, TIM-3, LAG-3, CTLA-4) was expressed in over 50% (29/53) of the patients’ blood samples. AR was expressed in 25/53 (47%) samples. Four of these patients (8%) expressed AR-v7, indicating resistance to anti-AR drugs. Patient disease status included: 25% (13/53) CRPC, 74% (39/53) HN, and 1 unknown. Within the CRPC group, a significant negative correlation was found between TIM-3 and CTLA-4 expression (-0.833), and CTLA-4 also correlated with disease progression (0.719). Within the HN group, a significant negative correlation was measured between TIM-3 and PD-L1 (0.589). AR expression did not correlate with other targets measured among all patients or within the CRPC or HN groups. Conclusions: This study demonstrates successful quantitation of immune checkpoint gene expressions as well the AR and AR-v7 genes in plasma of prostate cancer patients. This result opens the possibility of the use of a noninvasive method to measure, monitor and track the dynamic interplay of these genes with changing disease status. Clinical trial information: NCT02853097.

Nanoscale flow cytometry of patient plasma for the detection of prostate cancer-associated extracellular vesicles

Introduction Prostate cancer is the predominant cancer in men, affecting one in seven men during their lifetime. Current tests for prostate cancer include the digital rectal exam and the prostate-specific antigen (PSA) test. Extracellular vesicles (EVs) are submicron particles that participate in intercellular cross-talk by releasing cell mediators such as microRNA, carbohydrates and proteins. While they are known to express the broad tetraspanin family of proteins, i.e. CD9/CD63, prostate cancer-derived EVs have also been found to express PSA and six transmembrane epithelial antigen of the prostate (STEAP1). Traditionally, scientists have purified these EVs through ultracentrifugation. Here we propose a tandem purification of patient plasma, followed by nanoscale flow cytometry (A50+) as a novel method to detect tumour-derived EVs. Materials and Methods Plasma was obtained from healthy volunteers, patients with benign prostatic hyperplasia (BPH), and patients with metastatic prostate cancer. CD9, CD63, PSA and STEAP1 were used as primary antibodies for the purification of EVs from neat plasma. To perform the purification in tandem, Protein G immunoprecipitation using CD9 and PSA was carried out first, followed by immunoaffinity purification with biotinylated CD63 and STEAP1. First and second elutions were collected for the enumeration of dual positive events by A50+. Initial histogram overlays and bivariate plots of neat and purified plasma were computed, then exported as comma-separated values for mathematical modelling by MATLAB. Results Strong dual positive EV populations from patient plasma were optimised, demonstrating that the method enriches tumour-derived EVs from neat samples of patient plasma. This was observed for HVs, patients with benign prostatic hyperplasia, and prostate cancer patients with Gleason 4+4. Enrichment in these purified samples was measured by A50+ and demonstrated by overlaying purified and non-purified histoplots by MATLAB. Additional results show that PSA and STEAP1 can be adapted to detect single or dual positive populations of tumour-associated EVs in prostate cancer patients. Discussions and Conclusions This study suggests that tandem purification of tumour-associated EVs and A50+ analysis from plasma of prostate cancer patients can lead to earlier diagnosis and risk stratification, compared to traditional screening tests and aspiration cytology. Future studies will be directed toward optimizing this detection method for markers of other cancer types to achieve better outcomes in cancer detection and prognosis.

Detection of circulating miRNAs: comparative analysis of extracellular vesicle-incorporated miRNAs and cell-free miRNAs in whole plasma of prostate cancer patients

BackgroundCirculating cell-free miRNAs have emerged as promising minimally-invasive biomarkers for early detection, prognosis and monitoring of cancer. They can exist in the bloodstream incorporated into extracellular vesicles (EVs) and ribonucleoprotein complexes. However, it is still debated if EVs contain biologically meaningful amounts of miRNAs and may provide a better source of miRNA biomarkers than whole plasma. The aim of this study was to systematically compare the diagnostic potential of prostate cancer-associated miRNAs in whole plasma and in plasma EVs.MethodsRNA was isolated from whole plasma and plasma EV samples from a well characterised cohort of 50 patient with prostate cancer (PC) and 22 patients with benign prostatic hyperplasia (BPH). Nine miRNAs known to have a diagnostic potential for PC in cell-free blood were quantified by RT-qPCR and the relative quantities were compared between patients with PC and BPH and between PC patients with Gleason score ≥ 8 and ≤6.ResultsOnly a small fraction of the total cell-free miRNA was recovered from the plasma EVs, however the EV-incorporated and whole plasma cell-free miRNA profiles were clearly different. Four of the miRNAs analysed showed a diagnostic potential in our patient cohort. MiR-375 could differentiate between PC and BPH patients when analysed in the whole plasma, while miR-200c-3p and miR-21-5p performed better when analysed in plasma EVs. EV-incorporated but not whole plasma Let-7a-5p level could distinguish PC patients with Gleason score ≥ 8 vs ≤6.ConclusionsThis study demonstrates that for some miRNA biomarkers EVs provide a more consistent source of RNA than whole plasma, while other miRNAs show better diagnostic performance when tested in the whole plasma.