Optimising Extracellular Vesicle Metabolomic Methodology for Prostate Cancer Biomarker Discovery.

Conventional diagnostic tools for prostate cancer (PCa), such as prostate-specific antigen (PSA), transrectal ultrasound (TRUS), digital rectal examination (DRE), and tissue biopsy face, limitations in individual risk stratification due to invasiveness or reliability issues. Liquid biopsy is a less invasive and more accurate alternative. Metabolomic analysis of extracellular vesicles (EVs) holds a promise for detecting non-genetic alterations and biomarkers in PCa diagnosis and risk assessment. The current research gap in PCa lies in the lack of accurate biomarkers for early diagnosis and real-time monitoring of cancer progression or metastasis. Establishing a suitable approach for observing dynamic EV metabolic alterations that often occur earlier than being detectable by other omics technologies makes metabolomics valuable for early diagnosis and monitoring of PCa. Using four distinct metabolite extraction approaches, the metabolite cargo of PC3-derived large extracellular vesicles (lEVs) was evaluated using a combination of methanol, cell shearing using microbeads, and size exclusion filtration, as well as two fractionation chemistries (pHILIC and C18 chromatography) that are also examined. The unfiltered methanol-microbeads approach (MB-UF), followed by pHILIC LC-MS/MS for EV metabolite extraction and analysis, is effective. Identified metabolites such as L-glutamic acid, pyruvic acid, lactic acid, and methylmalonic acid have important links to PCa and are discussed. Our study, for the first time, has comprehensively evaluated the extraction and separation methods with a view to downstream sample integrity across omics platforms, and it presents an optimised protocol for EV metabolomics in PCa biomarker discovery.

HDR brachytherapy afterloader quality assurance optimization using monolithic silicon strip detectors.

BACKGROUND: There currently exists no widespread high dose-rate (HDR) brachytherapy afterloader quality assurance (QA) tool for simultaneously assessing the afterloader's positional, temporal, transit velocity and air kerma strength accuracy. PURPOSE: The purpose of this study was to develop a precise and rigorous technique for performing daily QA of HDR brachytherapy afterloaders, incorporating QA of: dwell position accuracy, dwell time accuracy, transit velocity consistency and relative air kerma strength (AKS) of an Ir-192 source. METHOD: A Sharp ProGuide 240 mm catheter (Elekta Brachytherapy, Veenendaal, The Netherlands) was fixed 5 mm above a 256 channel epitaxial diode array 'dose magnifying glass' (DMG256) (Centre for Medical and Radiation Physics, University of Wollongong). Three dwell positions, each of 5.0 s dwell times, were spaced 13.0 mm apart along the array with the Flexitron HDR afterloader (Elekta Brachytherapy, Veenendaal, The Netherlands). The DMG256 was connected to a data acquisition system (DAQ) and a computer via USB2.0 link for live readout and post-processing. The outputted data files were analyzed using a Python script to provide positional and temporal localization of the Ir-192 source by tracking the centroid of the detected response. Measurements were repeated on a weekly basis, for a period of 5 weeks to determine the consistency of the measured parameters over an extended period. RESULTS: Using the DMG256 for relative AKS measurements resulted in measured values within 0.6%-3.0% of the expected activity over a 7-week period. The sub-millisecond temporal accuracy of the device allowed for measurements of the transit velocity with an average of (10.88 ± 1.01) cm/s for 13 mm steps. The dwell position localization for 1, 2, 3, 5, and 10 mm steps had an accuracy between 0.1 and 0.3 mm (3σ), with a fixed temporal accuracy of 10 ms. CONCLUSION: The DMG256 silicon strip detector allows for clinics to perform rigorous daily QA of HDR afterloader dwell position and dwell time accuracy with greater precision than the current standard methodology using closed circuit television and a stopwatch. Additionally, DMG256 unlocks the ability to perform measurements of transit velocity/time and relative AKS, which are not possible using current standard techniques.

Prostate cancer-derived extracellular vesicles metabolic biomarkers: Emerging roles for diagnosis and prognosis.

Prostate cancer (PCa) is a global health concern, ranking as the most common cancer among men in Western countries. Traditional diagnostic methods are invasive with adverse effects on patients. Due to the heterogeneous nature of PCa and their multifocality, tissue biopsies often yield false-negative results. To address these challenges, researchers are exploring innovative approaches, particularly in the realms of proteomics and metabolomics, to identify more reliable biomarkers and improve PCa diagnosis. Liquid biopsy (LB) has emerged as a promising non-invasive strategy for PCa early detection, biopsy selection, active surveillance for low-risk cases, and post-treatment and progression monitoring. Extracellular vesicles (EVs) are lipid-bilayer nanovesicles released by all cell types and play an important role in intercellular communication. EVs have garnered attention as a valuable biomarker resource in LB for PCa-specific biomarkers, enhancing diagnosis, prognostication, and treatment guidance. Metabolomics provides insight into the body's metabolic response to both internal and external stimuli, offering quantitative measurements of biochemical alterations. It excels at detecting non-genetic influences, aiding in the discovery of more accurate cancer biomarkers for early detection and disease progression monitoring. This review delves into the potential of EVs as a resource for LB in PCa across various clinical applications. It also explores cancer-related metabolic biomarkers, both within and outside EVs in PCa, and summarises previous metabolomic findings in PCa diagnosis and risk assessment. Finally, the article addresses the challenges and future directions in the evolving field of EV-based metabolomic analysis, offering a comprehensive overview of its potential in advancing PCa management.

Proteomic Identification of Small Extracellular Vesicle Proteins LAMB1 and Histone H4 for Prostate Cancer Diagnosis and Risk Stratification.

Diagnosis and stratification of prostate cancer (PCa) patients using the prostate-specific antigen (PSA) test is challenging. Extracellular vesicles (EVs), as a new star of liquid biopsy, has attracted interest to complement inaccurate PSA screening and invasiveness of tissue biopsy. In this study, a panel of potential small EV (sEV) protein biomarkers is identified from PCa cell lines using label-free LC-MS/MS proteomics. These biomarkers underwent further validation with plasma and urine samples from different PCa stages through parallel reaction monitoring-based targeted proteomics, western blotting, and ELISA. Additionally, a tissue microarray containing cancerous and noncancerous tissues is screened to provide additional evidence of selected sEV proteins associated with cancer origin. Results indicate that sEV protein LAMB1 is highly expressed in human plasma of metastatic PCa patients compared with localised PCa patients and control subjects, while sEV protein Histone H4 is highly expressed in human urine of high-risk PCa patients compared to low-risk PCa patients and control subjects. These two sEV proteins demonstrate higher specificity and sensitivity than the PSA test and show promise for metastatic PCa diagnosis, progression monitoring, and risk stratification.

Development of patient and catheter specific error thresholds for high dose rate prostate brachytherapy.

BACKGROUND: In-vivo source tracking has been an active topic of research in the field of high-dose rate brachytherapy in recent years to verify accuracy in treatment delivery. Although detection systems for source tracking are being developed, the allowable threshold of treatment error is still unknown and is likely patient-specific due to anatomy and planning variation. PURPOSE: The purpose of this study was to determine patient and catheter-specific shift error thresholds for in-vivo source tracking during high-dose-rate prostate brachytherapy (HDRPBT). METHODS: A module was developed in the previously described graphical processor unit multi-criteria optimization (gMCO) algorithm. The module generates systematic catheter shift errors retrospectively into HDRPBT treatment plans, performed on 50 patients. The catheter shift model iterates through the number of catheters shifted in the plan (from 1 to all catheters), the direction of shift (superior, inferior, medial, lateral, cranial, and caudal), and the magnitude of catheter shift (1-6 mm). For each combination of these parameters, 200 error plans were generated, randomly selecting the catheters in the plan to shift. After shifts were applied, dose volume histogram (DVH) parameters were re-calculated. Catheter shift thresholds were then derived based on plans where DVH parameters were clinically unacceptable (prostate V100 < 95%, urethra D0.1cc > 118%, and rectum Dmax > 80%). Catheter thresholds were also Pearson correlated to catheter robustness values. RESULTS: Patient-specific thresholds varied between 1 to 6 mm for all organs, in all shift directions. Overall, patient-specific thresholds typically decrease with an increasing number of catheters shifted. Anterior and inferior directions were less sensitive than other directions. Pearson's correlation test showed a strong correlation between catheter robustness and catheter thresholds for the rectum and urethra, with correlation values of -0.81 and -0.74, respectively (p < 0.01), but no correlation was found for the prostate. CONCLUSIONS: It was possible to determine thresholds for each patient, with thresholds showing dependence on shift direction, and number of catheters shifted. Not every catheter combination is explorable, however, this study shows the feasibility to determine patient-specific thresholds for clinical application. The correlation of patient-specific thresholds with the equivalent robustness value indicated the need for robustness consideration during plan optimization and treatment planning.

Prostate Virtual High-dose-rate Brachytherapy Boost: 5-Year Results from the PROMETHEUS Prospective Multicentre Trial.

BACKGROUND AND OBJECTIVE: Despite the high efficacy of high-dose-rate brachytherapy boost (HDRB) in the management of prostate cancer (PC), use of this approach is declining. Similar dosimetry can be achieved using stereotactic body radiotherapy or "virtual HDRB" (vHDRB). The aim of the multicentre, single-arm, phase 2 PROMETHEUS trial (ACTRN12615000223538) was to evaluate the safety and efficacy of vHDRB in patients with PC. METHODS: Patients with intermediate-risk PC or selected patients with high-risk PC were eligible for inclusion. vHDRB was given as 19-20 Gy in two fractions, delivered 1 wk apart, followed by conventionally fractionated external beam radiotherapy (EBRT) at 46 Gy in 23 fractions or 36 Gy in 12 fractions. The primary endpoint was the biochemical/clinical relapse-free rate (bcRFR). Toxicity was graded using Common Terminology Criteria for Adverse Events version 4 and quality of life (QoL) data were collected used the Expanded Prostate Cancer Index Composite-26 questionnaire. KEY FINDINGS AND LIMITATIONS: From March 2014 to December 2018, 151 patients (74% intermediate risk, 26% high risk) with a median age of 69 yr were treated across five centres. Median follow-up was 60 mo. The 5-yr bcRFR was 94.1% (95% confidence interval [CI] 90-98%) and the local control rate was 98.7%. Acute grade 2 gastrointestinal (GI) and genitourinary (GU) toxicity occurred in 6.6% and 23.2% of patients, respectively, with no acute grade 3 toxicity. At 60 mo after treatment, the prevalence of late grade ≥2 GI toxicity was 1.7% (95% CI 0.3-6.5%) and the prevalence of late grade ≥2 GU toxicity was 3.3% (95% CI 1.1-8.8%). Between baseline and 60 mo, QoL improved for urinary obstructive and hormonal domains, was stable for the bowel domain, and deteriorated slightly for the sexual and urinary incontinence domains. CONCLUSIONS: Delivery of gantry-based vHDRB followed by conventionally fractionated EBRT is feasible in a multicentre setting, with high 5-yr bcRFR and low toxicity. This approach is being compared with prostate ultrahypofractionated radiotherapy in the TROG 18.01 NINJA randomised trial (ACTRN12618001806257). PATIENT SUMMARY: The PROMETHEUS trial investigated noninvasive high-dose precision radiotherapy combined with conventional radiotherapy in patients with prostate cancer. We found that this new technique was well tolerated and resulted in better cancer control outcomes than historically reported.

Preliminary Characterization of an Active CMOS Pad Detector for Tracking and Dosimetry in HDR Brachytherapy.

We assessed the accuracy of a prototype radiation detector with a built in CMOS amplifier for use in dosimetry for high dose rate brachytherapy. The detectors were fabricated on two substrates of epitaxial high resistivity silicon. The radiation detection performance of prototypes has been tested by ion beam induced charge (IBIC) microscopy using a 5.5 MeV alpha particle microbeam. We also carried out the HDR Ir-192 radiation source tracking at different depths and angular dose dependence in a water equivalent phantom. The detectors show sensitivities spanning from (5.8 ± 0.021) × 10-8 to (3.6 ± 0.14) × 10-8 nC Gy-1 mCi-1 mm-2. The depth variation of the dose is within 5% with that calculated by TG-43. Higher discrepancies are recorded for 2 mm and 7 mm depths due to the scattering of secondary particles and the perturbation of the radiation field induced in the ceramic/golden package. Dwell positions and dwell time are reconstructed within ±1 mm and 20 ms, respectively. The prototype detectors provide an unprecedented sensitivity thanks to its monolithic amplification stage. Future investigation of this technology will include the optimisation of the packaging technique.