Integration of Liquid Biopsies in Clinical Management of Metastatic Prostate Cancer.

PURPOSE OF REVIEW: The field of liquid biopsies is constantly evolving through novel technologies. This review outlines current data on liquid biopsies and application to clinical management of metastatic prostate cancer. RECENT FINDINGS: To date, there are three platforms with FDA approval for use in the setting of metastatic prostate cancer and others which have been clinically validated. There is substantial evidence supporting the use of circulating tumor cell (CTC) enumeration to guide prognosis in metastatic castration-resistant prostate cancer (mCRPC). Additional evidence supports targeted sequencing of CTC and cell-free DNA (cfDNA) to guide androgren-directed therapy, identify candidates for treatment with PARP inhibitors, and monitor development of resistance. As a real-time and minimally invasive approach, utilization of liquid biopsies has the potential to drastically impact the treatment of metastatic prostate cancer and improve overall survival. With further clinical validation, additional liquid biopsy is likely to enter standard clinical practice.

Baseline Circulating Tumor Cell Count as a Prognostic Marker of PSA Response and Disease Progression in Metastatic Castrate-Sensitive Prostate Cancer (SWOG S1216).

PURPOSE: In metastatic castrate-sensitive prostate cancer (mCSPC), combined androgen axis inhibition is a standard of care. Noninvasive biomarkers that guide initial therapy decisions are needed. We hypothesized that CellSearch circulating tumor cell (CTC) count, an FDA-cleared assay in metastatic castrate-resistant prostate cancer (mCRPC), is a relevant biomarker in mCSPC. EXPERIMENTAL DESIGN: SWOG S1216 is a phase III prospective randomized trial of androgen deprivation therapy (ADT) combined with orteronel or bicalutamide for mCSPC. CellSearch CTC count was measured at registration (baseline). Prespecified CTC cut-off points of 0, 1-4, and ≥5 were correlated with baseline patient characteristics and, in a stratified subsample, were also correlated with two prespecified trial secondary endpoints: 7-month PSA ≤0.2 ng/mL versus 0.2-4.0 versus >4.0 (intermediate endpoint for overall survival); and progression-free survival (PFS) ≤ versus >2 years. RESULTS: A total of 523 patients submitted baseline samples, and CTCs were detected (median 3) in 33%. Adjusting for two trial stratification factors (disease burden and timing of ADT initiation), men with undetectable CTCs had nearly nine times the odds of attaining 7-month PSA ≤ 0.2 versus > 4.0 [OR 8.8, 95% confidence interval (CI), 2.7-28.6, P < 0.001, N = 264] and four times the odds of achieving > 2 years PFS (OR 4.0, 95% CI, 1.9-8.5, P < 0.001, N = 336) compared with men with baseline CTCs ≥5. CONCLUSIONS: Baseline CTC count in mCSPC is highly prognostic of 7-month PSA and 2-year PFS after adjusting for disease burden and discriminates men who are likely to experience poor survival outcomes.

Cancer transcriptomic profiling from rapidly enriched circulating tumor cells.

Transcriptomic profiling of metastatic cancer can illuminate mechanisms of progression and lead to new therapies, but standard biopsy is invasive and reflects only a single metastatic site. In contrast, circulating tumor cell (CTC) profiling is noninvasive and repeatable, reflecting the dynamic and systemic nature of advanced disease. To date, transcriptomic profiling of CTCs has not delivered on its full potential, because white blood cells (WBCs) vastly outnumber CTCs. Current profiling strategies either lack cancer sensitivity and specificity or require specialized CTC capture protocols that are not readily scalable to large patient cohorts. Here, we describe a new strategy for rapid CTC enrichment and transcriptomic profiling using commercially available WBC depletion, microfluidic enrichment and RNA sequencing. When applied to blood samples from patients with advanced prostate cancer (PC), transcriptomes from enriched samples cluster with cancer positive controls and previously undetectable prostate-specific transcripts become readily measurable. Gene set enrichment analysis reveals multiple significantly enriched signaling pathways associated with PC, as well as novel pathways that merit further study. This accessible and scalable approach yields cancer-specific transcriptomic data and can be applied repeatedly and noninvasively in large cancer patient cohorts to discover new therapeutic targets in advanced disease.

Development and Application of Liquid Biopsies in Metastatic Prostate Cancer.

PURPOSE OF REVIEW: Metastatic prostate cancer is a lethal and highly heterogeneous malignancy, associated with a broad spectrum of potentially actionable molecular alterations. In the past decade, disease profiling has expanded to include not only traditional tumor tissue, but also liquid biopsies of cells and genetic material circulating in the blood. These liquid biopsies offer a minimally invasive, repeatable source of tumor material for longitudinal disease profiling but also raise new technical and biological challenges. Here we will summarize recent advances in liquid biopsy strategies and the role they have played in biomarker development and disease management. RECENT FINDINGS: Technologies for analysis of circulating tumor cells (CTCs) continue to evolve rapidly, and the latest high content scanning platforms have underscored the phenotypic heterogeneity of CTC populations. Among liquid biopsies, CTC enumeration remains the most extensively validated prognostic marker to date, but other clinically relevant phenotypes like androgen receptor (AR) localization or presence of AR-V7 splice variant are important new predictors of therapy response. Serial genomic profiling of CTCs or circulating tumor DNA (ctDNA) is helping to define primary and acquired resistance mechanisms and helping to guide patient selection for targeted therapies such as poly(adenosine diphosphate [ADP] ribose) polymerase (PARP) inhibition. The era of liquid biopsy-based biomarkers has arrived, driven by powerful new enrichment and analysis techniques. As new blood-based markers are identified, their biological significance as disease drivers must be elucidated to advance new therapeutic strategies, and their clinical impact must be translated through assay standardization, followed by analytical and clinical validation. These efforts, already ongoing on multiple fronts, constitute the critical steps toward more effective precision management of advanced prostate cancer.

Considerations in the identification of endogenous substrates for protein L-isoaspartyl methyltransferase: the case of synuclein.

Protein L-isoaspartyl methyltransferase (PIMT) repairs abnormal isoaspartyl peptide bonds in age-damaged proteins. It has been reported that synuclein, a protein implicated in neurodegenerative diseases, is a major target of PIMT in mouse brain. To extend this finding and explore its possible relevance to neurodegenerative diseases, we attempted to determine the stoichiometry of isoaspartate accumulation in synuclein in vivo and in vitro. Brain proteins from PIMT knockout mice were separated by 2D electrophoresis followed by on-blot [(3)H]-methylation to label isoaspartyl proteins, and by immunoblotting to confirm the coincident presence of synuclein. On-blot (3)H-methylation revealed numerous isoaspartyl proteins, but no signal in the position of synuclein. This finding was corroborated by immunoprecipitation of synuclein followed by on-blot (3)H-methylation. To assess the propensity of synuclein to form isoaspartyl sites in vitro, samples of recombinant mouse and human α-synucleins were aged for two weeks by incubation at pH 7.5 and 37 °C. The stoichiometries of isoaspartate accumulation were extremely low at 0.02 and 0.07 mol of isoaspartate per mol of protein respectively. Using a simple mathematical model based on the first order kinetics of isoaspartyl protein methyl ester hydrolysis, we ascribe the discrepancy between our results and the previous report to methodological limitations of the latter stemming from an inherent, and somewhat counterintuitive, relationship between the propensity of proteins to form isoaspartyl sites and the instability of the (3)H-methyl esters used to tag them. The results presented here indicate that synuclein is not a major target of PIMT in vivo, and emphasize the need to minimize methyl ester hydrolysis when using methylation to assess the abundance of isoaspartyl sites in proteins.

Efficient production of human beta-defensin 2 (HBD2) in Escherichia coli.

Human beta-defensin 2 (HBD2) has been shown to interact with pathogenic bacteria and components of the mammalian innate and adaptive immune response. We describe a quick and reliable method for the production of HBD2 in Escherichia coli. HBD2 was expressed as an insoluble fusion, chemically cleaved and oxidised to give a single, folded HBD2 beta-isoform. The purified peptide was analysed by high resolution mass spectrometry, displayed a well-dispersed (1)H NMR spectrum, was a chemoattractant to HEK293 cells expressing CCR6 and acted as an antimicrobial agent against E. coli, P. aeruginosa, C. albicans and S. aureus.