Designing a Simple Electrochemical Genosensor for the Detection of Urinary PCA3, a Prostate Cancer Biomarker.

This study investigates the feasibility of a simple electrochemical detection of Prostate Cancer Antigen 3 (PCA3) fragments extracted from patients' urine, using a thiolated single-strand DNA probe immobilized on a gold surface without using a redox probe. To enhance the PCA3 recognition process, we conducted a comparative analysis of the hybridization location using two thiolated DNA probes: Probe 1 targets the first 40 bases, while Probe 2 targets the fragment from bases 47 to 86. Hybridization with PCA3 followed, using square wave voltammetry. The limit of detection of the designed genosenors were of the order of (2.2 ng/mL), and (1.6 ng/mL) for Probes 1 and 2, respectively, and the subsequent sensitivities were of the order of (0.09 ± 0.01) µA-1 · µg-1 · mL and (0.10 ± 0.01) µA-1 · µg-1 · mL. Specificity tests were then conducted with the sensor functionalized with Probe 2, as it presents better analytical performances. The electrochemical results indicate that the designed sensor can clearly discriminate a complementary target from a non-complementary one. A further modeling of the calibration curves with the Power Law/Hill model indicates that the dissociation constant increases by one order of magnitude, confirming the ability of the designed sensor to perfectly discriminate complementary targets from non-complementary ones.

Biomarkers for Prostate Cancer: From Diagnosis to Treatment.

Prostate cancer (PCa) is a widespread malignancy with global significance, which substantially affects cancer-related mortality. Its spectrum varies widely, from slow-progressing cases to aggressive or even lethal forms. Effective patient stratification into risk groups is crucial to therapeutic decisions and clinical trials. This review examines a wide range of diagnostic and prognostic biomarkers, several of which are integrated into clinical guidelines, such as the PHI, the 4K score, PCA3, Decipher, and Prolaris. It also explores the emergence of novel biomarkers supported by robust preclinical evidence, including urinary miRNAs and isoprostanes. Genetic alterations frequently identified in PCa, including BRCA1/BRCA2, ETS gene fusions, and AR changes, are also discussed, offering insights into risk assessment and precision treatment strategies. By evaluating the latest developments and applications of PCa biomarkers, this review contributes to an enhanced understanding of their role in disease management.

Diagnostic value of total serum/free prostate specific antigen and prostate cancer antigen-3 levels in prostate cancer.

BACKGROUND: The purpose of this study was to compare serum total and free prostate specific antigen (PSA) levels and serum prostate cancer antigen-3 (PCA3) levels in patients with prostate cancer in 2018 and 2019. METHODS: This research was a prospective case-control study. The case group included all patients with suspected prostate cancer, and the control group included individuals without prostate disease who were referred to Ali Asghar and Nour Hospital in Isfahan, Iran, from October 2018 to October 2020. The serum total PSA, free PSA, and PCA3 levels in both groups were measured using the ELISA method with standard kits and compared between the groups. RESULTS: The two groups were matched in terms of age and body mass index (BMI). The results showed that the mean free PSA level in the control group was significantly higher than that in the case group (P<0.05). Conversely, the mean total PSA level in the case group was significantly higher than that in the control group (P<0.05). However, no significant difference was observed in the mean PCA3 levels between the case and control groups. In addition, the total PSA variable with a cutoff of ≤3.14 exhibited 93% sensitivity and 82% specificity, demonstrating the highest diagnostic accuracy in distinguishing between prostate cancer and healthy individuals. Similarly, the PCA3 value with a cutoff of ≤3.5 had a sensitivity and specificity of 70% and 72%, respectively. CONCLUSION: Overall, the study results indicated that total PSA and PCA3 levels have higher diagnostic accuracy in distinguishing patients with suspected prostate cancer from healthy individuals.

PCA3 and TMPRSS2: ERG Urine Level as Diagnostic Biomarker of Prostate Cancer.

Background: Prostate cancer is a highly prevalent urological carcinoma with an increasing incidence in Indonesia and all around the world. Early diagnosis can greatly affect treatment outcomes and increase life expectancy. Several biomarkers for detecting prostate cancer have been studied and showed great promise. Purpose: This study aims to analyze prostate cancer antigen 3 (PCA3) as well as transmembrane serine protease 2:ERG (TMPRSS2:ERG) for diagnosing and serving as urine biomarkers in predicting prostate cancer incidences. Methods: We conducted an analytical study to assess the utility of PCA3 and TMPRSS2:ERG for detecting prostate cancer. Thirty samples were included in this study to see the utilization of PCA3 and TMPRSS2:ERG as diagnostic biomarkers of prostate cancer. A urine sample was taken and the PCA3 test was performed using the PCA3 PROGENSA test, while the TMPRSS2:ERG was performed using the chemiluminescent DNA probe method with a hybridization protection test. Results: The average age of the subject was 61.07±8.3 years. Based on calculations using the Mann-Whitney test, there was a significant relationship between prostate-Specific Antigen (PSA) overexpression (p<0.001), TMPRSS2:ERG (p=0.001), and PCA3 (p=0.003) with prostate cancer incidence. The sensitivity of PCA3 and TMPRSS2:ERG in detecting prostate cancer was 76.9% and 92.3%, respectively. Hence, TMPRSS2:ERG and PCA3 can be used as biomarkers for the occurrence of prostate cancer. We also performed a Kruskal-Wallis test; however, there was no significant relationship between PSA (p=0.236), TMPRSS2:ERG (p=0.801), and PCA3 (p=0.091) with the Gleason score. Conclusion: There is a significant correlation between overexpression of PSA, TMPRSS2:ERG and PCA3 with the incidence of prostate cancer, and TMPRSS2:ERG and PCA3 can be used as biomarkers of prostate cancer.

The performance and limitations of PCA3, TMPRSS2:ERG, HOXC6 and DLX1 urinary markers combined in the improvement of prostate cancer diagnostics.

BACKGROUND: Prostate cancer (PCa) is the second most commonly diagnosed cancer in men. To date, the role of the combined application of long non-coding RNAs (PCA3, DLX1, HOXC6, TMPRSS2:ERG) for obtaining the most accurate method of detection of PCa has not yet been comprehensively investigated. METHODS: In total 240 persons were included in the retrospective study. Among them were 150 patients with confirmed PCa, 30 patients with benign prostatic hyperplasia, 30 patients with active chronic prostatitis and 30 healthy volunteers. In all patients, the urine samples were collected prior to biopsy or treatment. Polymerase chain reaction with reverse transcription was performed to detect the expression level of PCA3, HOXC6, DLX1 and the presence of the TMPRSS2:ERG transcript. RESULTS: PCA3 was detected in urine samples in all cases. Using a PCA3 score of 56 allowed the differentiation between PCa and all other cases with a sensitivity of 61% and specificity of 96% (p < 0.001) while a PCA3 score threshold value of 50 resulted in a differentiation between clinically significant PCa (ISUP grades 2-5) and all other cases with a sensitivity of 93% and specificity of 93% (p < 0.001). The TMPRSS2:ERG expression in urine was detected exclusively in the group of patients with PCa and only in 16% of all cases. CONCLUSIONS: PCA3 score detected in urine demonstrated moderate sensitivity and good specificity in differentiation between PCa and non-PCa and high sensitivity and specificity in differentiation between clinically significant PCa and non-PCa.

Dysregulation of the PRUNE2/PCA3 genetic axis in human prostate cancer: from experimental discovery to validation in two independent patient cohorts.

Background: We have previously shown that the long non-coding (lnc)RNA prostate cancer associated 3 (PCA3; formerly prostate cancer antigen 3) functions as a trans-dominant negative oncogene by targeting the previously unrecognized prostate cancer suppressor gene PRUNE2 (a homolog of the Drosophila prune gene), thereby forming a functional unit within a unique allelic locus in human cells. Here, we investigated the PCA3/PRUNE2 regulatory axis from early (tumorigenic) to late (biochemical recurrence) genetic events during human prostate cancer progression. Methods: The reciprocal PCA3 and PRUNE2 gene expression relationship in paired prostate cancer and adjacent normal prostate was analyzed in two independent retrospective cohorts of clinically annotated cases post-radical prostatectomy: a single-institutional discovery cohort (n=107) and a multi-institutional validation cohort (n=497). We compared the tumor gene expression of PCA3 and PRUNE2 to their corresponding expression in the normal prostate. We also serially examined clinical/pathological variables including time to disease recurrence. Results: We consistently observed increased expression of PCA3 and decreased expression of PRUNE2 in prostate cancer compared with the adjacent normal prostate across all tumor grades and stages. However, there was no association between the relative gene expression levels of PCA3 or PRUNE2 and time to disease recurrence, independent of tumor grades and stages. Conclusions: We concluded that upregulation of the lncRNA PCA3 and targeted downregulation of the protein-coding PRUNE2 gene in prostate cancer could be early (rather than late) molecular events in the progression of human prostate tumorigenesis but are not associated with biochemical recurrence. Further studies of PCA3/PRUNE2 dysregulation are warranted. Funding: We received support from the Human Tissue Repository and Tissue Analysis Shared Resource from the Department of Pathology of the University of New Mexico School of Medicine and a pilot award from the University of New Mexico Comprehensive Cancer Center. RP and WA were supported by awards from the Levy-Longenbaugh Donor-Advised Fund and the Prostate Cancer Foundation. EDN reports research fellowship support from the Brazilian National Council for Scientific and Technological Development (CNPq), Brazil, and the Associação Beneficente Alzira Denise Hertzog Silva (ABADHS), Brazil. This work has been funded in part by the NCI Cancer Center Support Grants (CCSG; P30) to the University of New Mexico Comprehensive Cancer Center (CA118100) and the Rutgers Cancer Institute of New Jersey (CA072720).

Distinct Expression of Surface and Genetic Biomarkers in Prostate Cancer Cell Lines.

BACKGROUND/AIM: Surface biomarkers, such as CD44 and CD133, have been demonstrated to be expressed in prostate cancer cells, and our previous study has shown that prostate cancer cell lines could be divided into three groups according to the single and combined expression pattern of CD44 and 133. In order to refine prognostication in prostate cancer cells, we further investigated genetic biomarkers, prostate cancer antigen 3 (PCA3), kallikrein 4 (KLK4), and KLK9 in different prostate cancer cell lines. MATERIALS AND METHODS: CWR22Rv1, PC3, and DU145 cell lines were cultured until 95% confluence. The single expression of CD44 or CD133 and their combined expression were analyzed by flow cytometry, and gene expression of b-actin, PCA3, KLK4, and KLK9 was analyzed by real-time polymerase chain reaction. RESULTS: The single expression of CD133 was less than 4% in all cell lines examined. PC3 and DU145 cells displayed a high expression of CD44 (>91%), whereas CWR22Rv1 was the only cell line that demonstrated a high co-expression of both CD44 and CD133 (>91%). In addition, PC3 and DU145 displayed low expression of PCA3, KLK4, and KLK9 when compared with their own b-actin expression. In contrast, CWR22Rva showed high expression of PCA3 and KLK4 although KLK9 expression was also low. CONCLUSION: Both surface and genetic biomarkers should be validated for a more accurate prognosis in prostate cancer.

Accurate diagnosis of prostate cancer with CRISPR-based nucleic acid test strip by simultaneously identifying PCA3 and KLK3 genes.

Although serum prostate specific antigen (PSA) testing could decrease the morality of prostate cancer (PCa), its low specificity usually led to misdiagnosis due to prostatitis or benign prostatic hyperplasia (BPH). Prostate cancer antigen 3 (PCA3) as an alternative prostate tumor-specificity biomarker could be used to increase the specificity of PCa diagnosis, however, it usually required sophisticated operation and expensive equipment for routine detection. Herein, we constructed an early detection platform for prostate cancer with reverse transcriptase-recombinase aided amplification (RT-RAA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 based nucleic acid test strip. The amplicons of PCA3 and kallikrein related peptidase 3 (KLK3) gene, which amplified simultaneously by single-amplification unit of RT-RAA were specifically recognized by Cas9-sgRNA and visual on the nucleic acid test strip by naked eyes without instruments. Simultaneously detection of PCA3 and KLK3 gene could improve specificity and accuracy of the diagnosis but avoid mutual interference. In addition, the platform presented a detection limit of 500 fg/µL and 50 fg/µL in PCA3 and KLK3 gene, respectively. Furthermore, the analysis result of signal ratio of PCA3 to KLK3 gene of urine and peripheral blood specimens from 32 men with suspected prostate cancer on test strips illustrated that the area under the curve values of urine and peripheral blood specimens were 0.998 and 1.0 respectively. In summary, our study highlighted a facile strategy to design an accurate prostate cancer gene detection platform which had the potential to conduct prostate cancer early detection in the resource-limited or other point-of-care testing (POCT) environments.

Evaluation and multi-institutional validation of a novel urine biomarker lncRNA546 to improve the diagnostic specificity of prostate cancer in PSA gray-zone.

Background and objectives: Prostate specific antigen (PSA) is currently the most commonly used biomarker for prostate cancer diagnosis. However, when PSA is in the gray area of 4-10 ng/ml, the diagnostic specificity of prostate cancer is extremely low, leading to overdiagnosis in many clinically false-positive patients. This study was trying to discover and evaluate a novel urine biomarker long non-coding RNA (lncRNA546) to improve the diagnostic accuracy of prostate cancer in PSA gray-zone. Methods: A cohort study including consecutive 440 participants with suspected prostate cancer was retrospectively conducted in multi-urology centers. LncRNA546 scores were calculated with quantitative real-time polymerase chain reaction. The area under the receiver operating characteristic curve (AUROC), decision curve analysis (DCA) and a biopsy-specific nomogram were utilized to evaluate the potential for clinical application. Logistic regression model was constructed to confirm the predictive power of lncRNA546. Results: LncRNA546 scores were sufficient to discriminate positive and negative biopsies. ROC analysis showed a higher AUC for lncRNA546 scores than prostate cancer antigen 3 (PCA3) scores (0.78 vs. 0.66, p<0.01) in the overall cohort. More importantly, the AUC of lncRNA546 (0.80) was significantly higher than the AUCs of total PSA (0.57, p=0.02), percentage of free PSA (%fPSA) (0.64, p=0.04) and PCA3 (0.63, p<0.01) in the PSA 4-10 ng/ml cohort. A base model constructed by multiple logistic regression analysis plus lncRNA546 scores improved the predictive accuracy (PA) from 79.8% to 86.3% and improved AUC results from 0.862 to 0.915. DCA showed that the base model plus lncRNA546 displayed greater net benefit at threshold probabilities beyond 15% in the PSA 4-10 ng/ml cohort. Conclusion: LncRNA546 is a promising novel biomarker for the early detection of prostate cancer, especially in the PSA 4-10 ng/ml cohort.

Long non-coding RNA in prostate cancer.

Prostate cancer is the most frequently diagnosed cancer in males and its development and progression remains an important area of study. Recently, long non-coding RNAs (lncRNAs) have been evidenced as key players in cancer pathogenesis. Specifically, dysregulation of long non-coding RNA (lncRNA) expression has shown to affect tumor proliferation and metastasis, acting as either tumor suppressors or oncogenes. However, its specific mechanisms and functions in prostate cancer remain unclear. This review provides an overview of currently available information on prostate cancer-related lncRNAs, including GAS5, GAS-007, MEG3, PCA3, PCAT14, PCAT1, PVT1, UCA1, SChLAP1, MALAT1, HOTAIR, and NEAT1. Notable tumor growth inhibitors include GAS5 and MEG3. GAS5 is evidenced to interfere with the AKT/MTOR signaling pathway through targeting microRNA mir-103. MEG3, however, is proposed to inhibit the cycle, sponge miR-9-5p, and induce gene silencing. PCAT1, PVT1, and UCA1 are important tumor growth promoters. PCAT1 is indicated to be a transcriptional repressor, a mir-145-5P sponge, and a P13K/AKT pathway activator. Studies suggest that PVT1 acts via microRNA targeting and regulating proliferating cell nuclear antigen. UCA1 may sponge miR-204 and miR-331-3p as well as regulate myosin VI. Thorough understanding of these lncRNAs may elucidate new aspects of prostate cancer pathology and serve a pivotal role in developing novel diagnostic and prognostic techniques.

Detection of Prostate Cancer Biomarker PCA3 by Using Aptasensors.

BACKGROUND: Prostate cancer cells have very high PCA3 messenger RNA levels, which turns them into one of the new biomarkers for prostate cancer prognosis and diagnosis. OBJECTIVE: Our goal here is to develop a new aptasensor to detect PCA3 release by the cancer cell. METHODS: DNA hairpin containing PCA3 aptamer was thiolated, conjugated to methylene blue (MB) redox probe, and immobilized on gold electrode through self-assembly to detect label-free cancer cells. RESULTS: Our data have evidenced stable and sensitive sensors presenting a wide linear detection range (0-150ng/mL). In addition, monitoring PCA3 released by different types of prostate cells can provide in-depth knowledge about prostate cancer dynamics; therefore, it is a powerful platform for earlier clinical diagnostic. The released PCA3 can vary depending on the type of adopted prostate cells. CONCLUSION: PCA3 release was monitored in a group of cells for 2 h; it showed significantly higher expression in both LNCaP and PC-3 cells. This strategy provides a unique and simple methodology to achieve more sensitive and specific PCA3 detection; thus, it emerged as a promising tool for early cost-effective diagnosis.

[Urinary biomarkers in the diagnosis of prostate cancer.] / Biomarcadores urinarios en el diagnóstico de cancer de próstata.

INTRODUCTION: The use of prostatespecific antigen (PSA) is useful for the diagnosis ofprostate cancer. Its main limitation is its low specificity,which has led to the search for new biomarkersin order to identify clinically significant prostatecancer and to reduce overdiagnosis and overtreatment.The aim of this article is to summarize the currentliterature on urinary biomarkers used in thediagnosis of prostate cancer.A PubMed-based literature search was conductedup to December 2020. We selected the most recentand relevant original articles, clinical trials and reviewsthat have provided relevant information onthe use of biomarkers.In this review, we have discussed four importanturinary biomarkers useful for prostate cancer diagnosis:PCA3, Select MDX, ExoDX, TMPRSS2:ERG. CONCLUSION: The use of urinary biomarkers hasimproved of clinically significant prostate cancerdiagnosis. Their use reduces the number of unnecessarybiopsies and avoids overtreatment of indolentprostate cancer.

INTRODUCCIÓN: El uso del antígenoprostático específico (PSA) es útil para el diagnósticodel cáncer de próstata. Su principal limitación es labaja especificidad, esto ha llevado a la búsqueda denuevos biomarcadores con el fin de identificar el cáncerde próstata clínicamente significativo y poder disminuirel sobrediagnóstico y sobretratamiento.El objetivo de este artículo es resumir la literaturaactual sobre los biomarcadores urinarios utilizados enel diagnóstico de cáncer de próstata.Se llevó a cabo una búsqueda bibliográfica en Pub-Med hasta diciembre del 2020. Hemos seleccionadolos artículos originales, ensayos clínicos y revisionesmás recientes que proporcionan información sobre eluso de biomarcadores.En esta revisión, hemos discutido cuatro importantesbiomarcadores urinarios útiles para el diagnósticodel cáncer de próstata: PCA3, Select MDX, ExoDX, TMPRSS2:ERG.CONCLUSIÓN: El uso de biomarcadores urinariosha mejorado del diagnóstico de cáncer de próstata clínicamentesignificativo. Su uso reduce el número debiopsias innecesarias y evita el sobretratamiento delcáncer de próstata indolente.

Biomarcadores urinarios en el diagnóstico de cáncer de próstata / Urinary biomarkers in the diagnosis of prostate cáncer

INTRODUCCIÓN: El uso del antígenoprostático específico (PSA) es útil para el diagnósticodel cáncer de próstata. Su principal limitación es labaja especificidad, esto ha llevado a la búsqueda denuevos biomarcadores con el fin de identificar el cáncer de próstata clínicamente significativo y poder disminuir el sobrediagnóstico y sobretratamiento.El objetivo de este artículo es resumir la literaturaactual sobre los biomarcadores urinarios utilizados enel diagnóstico de cáncer de próstata.Se llevó a cabo una búsqueda bibliográfica en PubMed hasta diciembre del 2020. Hemos seleccionadolos artículos originales, ensayos clínicos y revisionesmás recientes que proporcionan información sobre eluso de biomarcadores.En esta revisión, hemos discutido cuatro importantes biomarcadores urinarios útiles para el diagnósticodel cáncer de próstata: PCA3, Select MDX, ExoDX, TMPRSS2:ERG.CONCLUSIÓN: El uso de biomarcadores urinariosha mejorado del diagnóstico de cáncer de próstata clínicamente significativo. Su uso reduce el número debiopsias innecesarias y evita el sobretratamiento delcáncer de próstata indolente. (AU)

INTRODUCTION: The use of prostatespecific antigen (PSA) is useful for the diagnosis ofprostate cancer. Its main limitation is its low specificity, which has led to the search for new biomarkersin order to identify clinically significant prostatecancer and to reduce overdiagnosis and overtreatment.The aim of this article is to summarize the current literature on urinary biomarkers used in thediagnosis of prostate cáncer.A PubMed-based literature search was conductedup to December 2020. We selected the most recentand relevant original articles, clinical trials and reviews that have provided relevant information onthe use of biomarkers.In this review, we have discussed four importanturinary biomarkers useful for prostate cancer diagnosis: PCA3, Select MDX, ExoDX, TMPRSS2:ERG.CONCLUSION: The use of urinary biomarkers hasimproved of clinically significant prostate cancerdiagnosis. Their use reduces the number of unnecessary biopsies and avoids overtreatment of indolent prostate cancer. (AU)

A review on the role of PCA3 lncRNA in carcinogenesis with an especial focus on prostate cancer.

The prostate cancer antigen 3 (PCA3) is a long non-coding RNA (lncRNA) with high level of specificity for prostate cancer. This lncRNA has fundamental effects in the prostate carcinogenesis through modulation of expression of miR-132-3p, miR-1261, SREBP1, PRKD3 and LAP2α as well as regulation of p53 signaling. Expression of PCA3 in prostate cancer cells can be enhanced by Snail. Moreover, in vitro studies have documented up-regulation of PCA3 in three other types of neoplastic cells, namely those being originated from choriocarcinoma, ovarian carcinoma and thyroid carcinoma. The diagnostic value of PCA3 in differentiation of prostate cancer from benign prostate hyperplasia has been assessed in different studies. Studies aimed at identification of diagnostic power of PCA3 in prostate cancer using receiver operating characteristic curves have reported area under curve values ranging from 0.66 to 0.86. In the current review, we describe the role of PCA3 in the carcinogenesis particularly in the pathoetiology of prostate cancer. Moreover, we review the results of studies appraising diagnostic value of this lncRNA in prostate cancer.

miR-19a and miR-421 target PCA3 long non-coding RNA and restore PRUNE2 tumor suppressor activity in prostate cancer.

BACKGROUND: Prostate cancer antigen 3 (PCA3) is the most promising diagnostic biomarker for the differential diagnosis of prostate cancer identified to date. As a dominant-negative oncogene, PCA3 negatively regulates the expression of tumor suppressor PRUNE2 (a human homolog of the Drosophila prune gene) gene. Although interaction between PCA3-PRUNE2 was clearly reported, the precise mechanism how PCA3 is upregulated in prostate cancer remained highly elusive. Accordingly, here we aimed demonstrate the role of microRNAs in PCA3 upregulation and interplay between these miRNAs and PCA3-PRUNE2 axis. METHODS AND RESULTS: We evaluated expression of PCA3, PRUNE2 and miRNAs by quantitative reverse transcription polymerase chain reaction. Overexpression and silencing of miRNAs were achieved by synthetic miRNA mimics and inhibitors, respectively. Colony formation, migration, apoptosis, and cell cycle assays were performed to reveal the effects of miRNA modulation. We identified that PCA3 expression was significantly downregulated in both prostate cancer tissues and cells and inversely correlated with the expressions of miR-19a and miR-421. Restoring the functions of miR-19a and miR-421 by miRNA mimics significantly downregulated the expression of PCA3 and promoted apoptosis and cell cycle blockade and interfered with the proliferation and migration in prostate cancer cells. Conversely, silencing the expressions of these miRNAs yielded the opposite effect. CONCLUSIONS: Collectively, our results uncover a previously unrecognized novel mechanism on PCA3 upregulation in prostate cancer and proved that miR-19a and miR-421 might be responsible for the increased expression of PCA3, indicating that both miRNAs might be novel candidates for prostate cancer diagnosis and therapy.

Electrochemical LAMP-based assay for detection of RNA biomarkers in prostate cancer.

Current molecular diagnostics of prostate cancer relies on detection of elevated levels of PSA protein in serum, but its specificity has been questioned due to its higher levels also in non-malignant prostate diseases. A long non-coding RNA biomarker, PCA3, demonstrated excellent specificity for prostate cancer, and thus has become an interesting alternative to PSA monitoring. Its detection utilizes mostly reverse transcription PCR with optical detection, making the protocol longer and more expensive. To avoid PCR, we have developed an electrochemical assay coupled with LAMP, an isothermal amplification technique showing high sensitivities at constant temperatures and shorter reaction times. We amplified PCA3 RNA as well as PSA mRNA (serving as a control), hybridized LAMP products on magnetic beads and measured them with chronoamperometry at carbon electrode chips. We show good sensitivity and specificity for both biomarkers in prostate cancer cell lines, and successful detection of PCA3 in clinical samples, i.e., urine samples from 11 prostate cancer patients and 7 healthy controls, where we obtained excellent correlation with clinical data. This is to our knowledge a first such attempt to apply electrochemistry to determine two RNA biomarkers directly in urine samples of prostate cancer patients in a minimally invasive diagnostics format.

Optimization of Apta-Sensing Platform for Detection of Prostate Cancer Marker PCA3.

This work is a continuation of our research into the development of simple, reliable, and cost-effective methods for the early diagnosis of prostate cancer (PCa). The proposed method is based on the electrochemical detection of the PCA3 biomarker of PCa (long non-coded RNA transcript expressed in urine) using a specific aptamer labeled with a redox group (methylene blue). The electrochemical measurements (cyclic voltammograms) obtained from electrodes functionalized with the aptamer were complemented in this work by another biosensing technique: total internal reflection ellipsometry (TIRE). In addition to proving the concept of the detection of PCA3 in low concentrations down to 90 pM, this study improved our understanding of the processes by which PCA3 binds to its specific aptamer. The high specificity of the binding of PCA3 to the aptamer was assessed by studying the binding kinetics, which yielded an affinity constant (KD) of 2.58 × 10-9 M. Additional XPS measurements confirmed the strong covalent binding of aptamers to gold and showed spectral features associated with PCA3 to aptamer binding.

A Novel Urine Exosomal lncRNA Assay to Improve the Detection of Prostate Cancer at Initial Biopsy: A Retrospective Multicenter Diagnostic Feasibility Study.

PURPOSE: This study aimed at developing and validating a novel noninvasive urinary exosome-based post-DRE (digital rectal examination) lncRNA assay to diagnose PCa (prostate cancer) and clinically significant PCa (Gleason score ≥ 7) from the initial prostate biopsy. METHODS: A total of 602 urine samples from eligible participants were collected. The expression levels of urinary exosomal PCA3 (prostate cancer antigen 3) and MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) were detected by qPCR (quantitative real-time PCR). Receiver operating characteristic (ROC) analysis was applied to evaluate the diagnostic performance of PCA3, MALAT1 and the lncRNA assay. A decision curve analysis (DCA) and waterfall plots were used to assess the clinical value of the lncRNA assay. RESULTS: Urinary exosomal PCA3 and MALAT1 were overexpressed in PCa and clinically significant PCa (p < 0.001). The lncRNA assay combining PCA3 and MALAT1 had a better diagnostic performance (AUC 0.828) than the current clinical parameters in detecting PCa. More importantly, the lncRNA assay yielded an AUC of 0.831 to detect clinically significant PCa, which is much higher than that of the current clinical parameters. The lncRNA assay was superior to PSA, f/tPSA and the base model for detecting PCa and clinically significant PCa, with a higher net benefit for almost all threshold probabilities. At the cutoff value of 95% sensitivity, the lncRNA assay could avoid 24.2% unnecessary biopsies while only missing 1.2% of the cases of clinically significant PCa. CONCLUSION: We developed and validated a novel noninvasive post-DRE urine-based lncRNA assay that presented good diagnostic power and clinical utility for the early diagnosis of PCa and high-grade PCa.

Papel de PCA3 y SelectMDx en la optimización de la vigilancia activa en el cáncer de próstata / Role of PCA3 and SelectMDx in the optimization of active surveillance in prostate cancer

Introducción y objetivos: Un porcentaje no despreciable de pacientes incluidos en programas de vigilancia activa (VA) para el cáncer de próstata (CaP) de bajo y muy bajo riesgo son reclasificados en la biopsia confirmatoria o desarrollan progresión de la enfermedad durante el seguimiento. Nuestro objetivo es evaluar el papel del PCA3 y el SelectMDx, de manera individual y combinada, para predecir la progresión patológica (PP) en un programa habitual de VA.Materiales y métodosEstudio prospectivo y observacional que incluyó 86 pacientes inscritos en un protocolo de VA desde 2009 hasta 2019, con resultados de PCA3 y SelectMDx previos al diagnóstico de CaP o durante su periodo de confirmación. Se realizaron análisis univariantes y multivariantes para la correlación de las puntuaciones de PCA3 y SelectMDx, así como de las variables clinicopatológicas con la supervivencia libre de progresión patológica (SLPP). Se definieron los puntos de corte más fiables para ambos biomarcadores en el contexto de VA.ResultadosSelectMDx mostró diferencias estadísticamente significativas en relación con la SLPP (HR: 1,035; IC95%: 1,012-1,057) (p=0,002) con un índiceC de 0,670 (IC95%: 0,529-0,810) y un AUC de 0,714 (IC95%: 0,603-0,825) a 5años. En nuestra serie, el punto de corte más fiable para el SelectMDx fue 5, con una sensibilidad y una especificidad para la PP del 69,8 y del 67,4%, respectivamente. El punto de corte del test PCA3 fue de 65, con una sensibilidad y una especificidad para la PP del 51,16 y del 74,42%, respectivamente. La combinación de ambos biomarcadores no mejoró la predicción de la PP, con un índiceC de 0,630 (IC95%: 0,455-0,805).ConclusionesEn el contexto del CaP de bajo o muy bajo riesgo, SelectMDx >5 predijo una supervivencia libre de PP de 5años con una capacidad de discriminación moderada, superando al PCA3. La combinación de ambos no mejoró los resultados. (AU)

Introduction and objectives: A not negligible percentage of patients included in active surveillance (AS) for low and very low risk prostate cancer (PCa) are reclassified in the confirmatory biopsy or have disease progression during follow-up. Our aim is to evaluate the role of PCA3 and SelectMDx, in an individual and combined way, in the prediction of pathological progression (PP) in a standard AS program.Materials and methodsProspective and observational study comprised of 86 patients enrolled in an AS program from 2009 to 2019, with results for PCA3 and SelectMDx previous to PCa diagnosis or during their confirmatory period. Univariate and multivariate analysis were performed to correlate PCA3 and SelectMDx scores as well as clinical and pathological variables with PP-free survival (PPFS). The most reliable cut-offs for both biomarkers in the context of AS were defined.ResultsSelectMDx showed statistically significant differences related to PPFS (HR: 1.035; 95%CI: 1.012-1.057) (P=.002) with a C-index of 0.670 (95%CI: 0.529-0.810) and AUC of 0.714 (95%CI: 0.603-0.825) at 5years. In our series, the most reliable cut-off point for SelectMDx was 5, with a sensitivity and specificity for PP of 69.8% and 67.4%, respectively. Same figure for PCA3 was 65, with a sensitivity and specificity for PP of 51.16% and 74.42%, respectively. The combination of both biomarkers did not improve the prediction of PP, C-index 0.630 (95%CI: 0.455-0.805).ConclusionsIn the context of low or very low risk PCa, SelectMDx >5 predicted 5years PP free survival with a moderate discrimination ability outperforming PCA3. The combination of both tests did not improved outcomes. (AU)

Dual electrochemical genosensor for early diagnosis of prostate cancer through lncRNAs detection.

The prostate specific antigen (PSA) test is the gold standard for the screening of prostate cancer (PCa), despite its limited clinical specificity. Long noncoding RNAs are released from the tumor tissue to the urine and show great potential for improving specificity in PCa diagnosis. This work reports on a sandwich-type hybridization assay to detect both the urinary biomarker prostate cancer antigen 3 (PCA3) and an endogenous control, the PSA mRNA. Multiple fluorescein-tagged hybridization assistant probes are used to promote the selective capture of this long noncoding RNA, and sensitivity by incorporating multiple redox enzymes per target molecule, after addition of antifluorescein Fab fragment-peroxidase conjugate. This strategy alleviates the problems associated with the low natural abundance of this marker, its large size, and complex secondary structure. The individual genosensors exhibit good sensitivity (2.48 ± 0.01 µA nM-1 and 6.4 ± 0.3 µA nM-1 for PCA3 and PSA, respectively), with wide linear ranges (from 25 pM to 10 nM for PCA3 and 1 nM for PSA), and detection limits in the low picomolar range (4.4 pM and 1.5 pM for PCA3 and PSA, respectively). This analytical performance is retained in the dual configuration without significant cross-talk, despite using the same enzyme label. The usefulness of this dual platform was demonstrated by analyzing RNA extracts from the prostate cancer cell line LNCaP and from urine samples of prostate cancer patients.