Transcriptome Profiling of Circulating Tumor Cells to Predict Clinical Outcomes in Metastatic Castration-Resistant Prostate Cancer.

The clinical utility of circulating tumor cells (CTC) as a non-invasive multipurpose biomarker is broadly recognized. The earliest methods for enriching CTCs from whole blood rely on antibody-based positive selection. The prognostic utility of CTC enumeration using positive selection with the FDA-approved CellSearchTM system has been demonstrated in numerous studies. The capture of cells with specific protein phenotypes does not fully represent cancer heterogeneity and therefore does not realize the prognostic potential of CTC liquid biopsies. To avoid this selection bias, CTC enrichment based on size and deformability may provide better fidelity, i.e., facilitate the characterization of CTCs with any phenotype. In this study, the recently FDA-approved Parsortix® technology was used to enrich CTCs from prostate cancer (PCa) patients for transcriptome analysis using HyCEADTM technology. A tailored PCa gene panel allowed us to stratify metastatic castration-resistant prostate cancer (mCRPC) patients with clinical outcomes. In addition, our findings suggest that targeted CTC transcriptome profiling may be predictive of therapy response.

The Androgen Regulated lncRNA NAALADL2-AS2 Promotes Tumor Cell Survival in Prostate Cancer.

Castration resistance is the leading cause of death in men with prostate cancer. Recent studies indicate long noncoding RNAs (lncRNAs) to be important drivers of therapy resistance. The aim of this study was to identify differentially expressed lncRNAs in castration-resistant prostate cancer (CRPC) and to functionally characterize them in vitro. Tumor-derived RNA-sequencing data were used to quantify and compare the expression of 11,469 lncRNAs in benign, primary prostate cancer, and CRPC samples. CRPC-associated lncRNAs were selected for semi-quantitative PCR validation on 68 surgical tumor specimens. In vitro functional studies were performed by antisense-oligonucleotide-mediated lncRNA knockdown in hormone-sensitive prostate cancer (HSPC) and CRPC cell line models. Subsequently, cell proliferation, apoptosis, cell cycle, transcriptome and pathway analyses were performed using the appropriate assays. Transcriptome analysis of a prostate cancer tumor specimens unveiled NAALADL2-AS2 as a novel CRPC-upregulated lncRNA. The expression of NAALADL2-AS2 was found to be particularly high in HSPC in vitro models and to increase under androgen deprived conditions. NAALADL2-AS2 knockdown decreased cell viability and increased caspase activity and apoptotic cells. Cellular fractionization and RNA fluorescent in situ hybridization identified NAALADL2-AS2 as a nuclear transcript. Transcriptome and pathway analyses revealed that NAALADL2-AS2 modulates the expression of genes involved with cell cycle control and glycogen metabolism. We hypothesize that the nuclear lncRNA, NAALADL2-AS2, functions as a pro-survival signal in prostate cancer cells under pressure of targeted hormone therapy.

Liquid Biopsy for Prostate and Bladder Cancer: Progress and Pitfalls.

Urinary liquid biopsy (LB) tests have contributed to reducing overtreatment and improving the detection of bladder and prostate cancer. Circulating tumor cells, DNA, and RNA are the focus of next-generation blood-based LB tests. These tests aim to stratify risk according to the detection of resistance and recurrence markers in patients with metastatic disease. The costs, lack of standardization, reimbursement challenges, and specialized workflows associated with characterization of blood-derived biomarkers are the primary barriers to their clinical implementation. This review provides an overview of the successes and pitfalls of LB-based tests for bladder and prostate cancer. PATIENT SUMMARY: Urinary and blood-based liquid biopsy tests are minimally invasive and highly efficacious in detecting clinically significant cancer.

RNA Biomarkers as a Response Measure for Survival in Patients with Metastatic Castration-Resistant Prostate Cancer.

Treatment evaluation in metastatic castration-resistant prostate cancer is challenging. There is an urgent need for biomarkers to discriminate short-term survivors from long-term survivors, shortly after treatment initiation. Thereto, the added value of early RNA biomarkers on predicting progression-free survival (PFS) and overall survival (OS) were explored. The RNA biomarkers: KLK3 mRNA, miR-375, miR-3687, and NAALADL2-AS2 were measured in 93 patients with mCRPC, before and 1 month after start of first-line abiraterone acetate or enzalutamide treatment, in two prospective clinical trials. The added value of the biomarkers to standard clinical parameters in predicting PFS and OS was tested by Harell's C-index. To test whether the biomarkers were independent markers of PFS and OS, multivariate Cox regression was used. The best prediction model for PFS and OS was formed by adding miR-375 and KLK3 (at baseline and 1 month) to standard clinical parameters. Baseline miR-375 and detectable KLK3 after 1 month of therapy were independently related to shorter PFS, which was not observed for OS. In conclusion, the addition of KLK3 and miR-375 (at baseline and 1 month) to standard clinical parameters resulted in the best prediction model for survival assessment.

Liquid biopsy reveals KLK3 mRNA as a prognostic marker for progression free survival in patients with metastatic castration-resistant prostate cancer undergoing first-line abiraterone acetate and prednisone treatment.

Circulating RNAs extracted from liquid biopsies represent a promising source of cancer- and therapy-related biomarkers. We screened whole blood from patients with metastatic castration-resistant prostate cancer (mCRPC) following their first-line treatment with abiraterone acetate and prednisone (AA-P) to identify circulating RNAs that may correlate with progression-free survival (PFS). In a prospective multicenter observational study, 53 patients with mCRPC were included after they started first-line AA-P treatment. Blood was drawn at baseline, 1, 3, and 6 months after treatment initiation. The levels of predefined circulating RNAs earlier identified as being upregulated in patients with mCRPC (e.g., microRNAs, long noncoding RNAs, and mRNAs), were analyzed. Uni- and multivariable Cox regression and Kaplan-Meier analyses were used to analyze the prognostic value of the various circulating RNAs for PFS along treatment. Detectable levels of kallikrein-related peptidase 3 (KLK3) mRNA at baseline were demonstrated to be an independent prognostic marker for PFS (201 vs 501 days, P = 0.00054). Three months after AA-P treatment initiation, KLK3 could not be detected in the blood of responding patients, but was still detectable in 56% of the patients with early progression. Our study confirmed that KLK3 mRNA detection in whole blood is an independent prognostic marker in mCRPC patients receiving AA-P treatment. Furthermore, the levels of circulating KLK3 mRNA in patients receiving AA-P treatment might reflect treatment response or early signs of progression.

Prognostic Value of Novel Liquid Biomarkers in Patients with Metastatic Castration-Resistant Prostate Cancer Treated with Enzalutamide: A Prospective Observational Study.

BACKGROUND: Several treatment options were recently added for metastatic castration-resistant prostate cancer (mCRPC). However, response to therapy is variable, and biomarkers that can guide treatment selection and response evaluation are lacking. Circulating RNAs are a promising source of biomarkers. We explored messenger RNAs (mRNAs), microRNAs (miRNAs), and long noncoding RNAs (lncRNAs) as potential biomarkers in liquid biopsies of patients with mCRPC treated with enzalutamide. METHODS: Forty patients were included in this prospective multicenter observational study. Whole blood was drawn at baseline and 1, 3, and 6 months after start of therapy. Four mRNAs, 6 miRNAs, and 5 lncRNAs were analyzed by quantitative PCR. RNA levels in 30 healthy individuals were used as controls. RNA expression data were analyzed by Kaplan-Meier and Cox regression analyses, and the primary end point was progression-free survival. Clinical factors were included in the multivariable Cox regression analysis. RESULTS: Levels of 2 miRNAs, miR-375 and miR-3687, and 1 lncRNA, N-acetylated alpha-linked acidic dipeptidase like 2 antisense RNA 2 (NAALADL2-AS2), were more than 2-fold higher in patients with mCRPC compared with healthy volunteers. Patients with higher levels of miR-375 or miR-3687 showed a shorter time to progression. Patients with higher levels of NAALADL2-AS2 showed a longer time to progression. In the multivariable Cox regression analysis, higher miR-375, miR-3687 and serum prostate-specific antigen concentrations were shown to be independent predictors for shorter time to progression. CONCLUSIONS: We identified miR-3687 as a novel prognostic marker for response in patients with CRPC treated with enzalutamide, and we confirmed the prognostic value of miR-375.

Clinical utility of emerging biomarkers in prostate cancer liquid biopsies.

Introduction: Prostate cancer (PCa) is one of the most common malignancies in men and a major cause of cancer deaths among men worldwide. Prostate specific antigen (PSA) monitoring and histopathological examination of tumor biopsies remain gold standards in PCa diagnostics. These clinical parameters are not well suited for patient stratification, predicting and monitoring treatment response. On the other hand, liquid biopsies offer a unique opportunity to easily isolate tumor-derived material for longitudinal clinical assessment.Areas covered: In this review we focus on the clinical application of novel liquid biomarkers that have the potential to monitor and stratify patients in order to achieve better therapeutic effects and improve clinical outcomes. Enumeration and characterization of circulating tumor cells (CTCs), tumor-educated platelets, exosomes, and cell-free nucleic acids have been studied for their clinical utility in PCa diagnostics, prognostics, monitoring treatment response and guiding treatment choice.Expert opinion: Liquid biomarkers have high potential to be used for prognosis, monitoring treatment response and guiding treatment selection. Although there is a remarkable progress in PCa biomarker discovery, their clinical validation is very limited. Research should be focused on biomarker validation and the incorporation of these biomarkers in clinical practice.