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.

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.

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.

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.

Viability of focal dose escalation to prostate cancer intraprostatic lesions using HDR prostate brachytherapy.

PURPOSE: This study aimed to determine the viability of focal dose escalation to prostate cancer intraprostatic lesions (IPLs) from multiparametric magnetic resonance (mpMRI) and prostate-specific membrane antigen positron emission tomography (PSMA-PET) images using high-dose-rate (HDR) prostate brachytherapy (pBT). METHODS AND MATERIALS: Retrospective data from 20 patients treated with HDR pBT was utilized. The interobserver contouring variability of 5 observers was quantified using the dice similarity coefficient (DSC) and mean distance to agreement (MDA). Uncertainty in propagating IPL contours to trans-rectal ultrasound (TRUS) was quantified using a tissue equivalent prostate phantom. Feasibility of incorporating IPLs into HDR pBT planning was tested on retrospective patient data. RESULTS: The average observer DSC was 0.65 (PSMA-PET) and 0.52 (mpMRI). The uncertainty in propagating IPL contours was 0.6 mm (PSMA-PET), and 0.4 mm (mpMRI). Uncertainties could be accounted for by expanding IPL contours by 2 mm to create IPL PTVs. The mean D98% achieved using HDR pBT was 166% and 135% for the IPL and IPL PTV contours, respectively. CONCLUSIONS: Focal dose escalation to IPLs identified on either PSMA-PET or mpMRI is viable using TRUS-based HDR pBT. Utilizing HDR pBT allows dose escalation of up to 166% of the prescribed dose to the prostate.

Anti-cancer potential of synergistic phytochemical combinations is influenced by the genetic profile of prostate cancer cell lines.

Introduction: Despite strong epidemiological evidence that dietary factors modulate cancer risk, cancer control through dietary intervention has been a largely intractable goal for over sixty years. The effect of tumour genotype on synergy is largely unexplored. Methods: The effect of seven dietary phytochemicals, quercetin (0-100 µM), curcumin (0-80 µM), genistein, indole-3-carbinol (I3C), equol, resveratrol and epigallocatechin gallate (EGCG) (each 0-200 µM), alone and in all paired combinations om cell viability of the androgen-responsive, pTEN-null (LNCaP), androgen-independent, pTEN-null (PC-3) or androgen-independent, pTEN-positive (DU145) prostate cancer (PCa) cell lines was determined using a high throughput alamarBlue® assay. Synergy, additivity and antagonism were modelled using Bliss additivism and highest single agent equations. Patterns of maximum synergy were identified by polygonogram analysis. Network pharmacology approaches were used to identify interactions with known PCa protein targets. Results: Synergy was observed with all combinations. In LNCaP and PC-3 cells, I3C mediated maximum synergy with five phytochemicals, while genistein was maximally synergistic with EGCG. In contrast, DU145 cells showed resveratrol-mediated maximum synergy with equol, EGCG and genistein, with I3C mediating maximum synergy with only quercetin and curcumin. Knockdown of pTEN expression in DU145 cells abrogated the synergistic effect of resveratrol without affecting the synergy profile of I3C and quercetin. Discussion: Our study identifies patterns of synergy that are dependent on tumour cell genotype and are independent of androgen signaling but are dependent on pTEN. Despite evident cell-type specificity in both maximally-synergistic combinations and the pathways that phytochemicals modulate, these combinations interact with similar prostate cancer protein targets. Here, we identify an approach that, when coupled with advanced data analysis methods, may suggest optimal dietary phytochemical combinations for individual consumption based on tumour molecular profile.Graphical abstract.

Genitourinary Quality-of-Life Comparison Between Urethral Sparing Prostate Stereotactic Body Radiation Therapy Monotherapy and Virtual High-Dose-Rate Brachytherapy Boost.

PURPOSE: Although radiation dose escalation improves prostate cancer disease control, it can cause increased toxicity. Genitourinary (GU) symptoms after prostate radiation therapy affect patient health-related quality of life (QoL). We compared patient-reported GU QoL outcomes following 2 alternative urethral sparing stereotactic body radiation therapy regimens. METHODS AND MATERIALS: Expanded Prostate Cancer Index Composite (EPIC)-26 GU scores were compared between 2 urethral sparing stereotactic body radiation therapy trials. The SPARK trial prescribed a "Monotherapy" dose of 36.25 Gy in 5 fractions to the prostate. The PROMETHEUS trial prescribed 2 phases: a 19- to 21-Gy in 2 fractions "Boost" to the prostate, followed by 46 Gy in 23 fractions or 36 Gy in 12 fractions. The biological effective dose (BED) for urethral toxicity was 123.9 Gy for Monotherapy and 155.8 to 171.2 Gy for Boost. Mixed effects logistic regression models were utilized to estimate the difference in the odds of a minimal clinically important change from baseline EPIC-26 GU score between regimens at each follow-up. RESULTS: 46 Monotherapy and 149 Boost patients completed baseline EPIC-26 scoring. Mean EPIC-26 GU scores revealed statistically superior urinary incontinence outcomes for Monotherapy at 12 months (mean difference, 6.9; 95% confidence interval [CI], 1.6-12.1; P = .01) and 36 months (mean difference, 9.6; 95% CI, 4.1-15.1; P < .01). Monotherapy also revealed superior mean urinary irritative/obstructive outcomes at 12 months (mean difference, 6.9; 95% CI, 2.0-12.9; P < .01) and 36 months (mean difference, 6.3; 95% CI, 1.9-10.8; P < .01). For both domains and at all time points, the absolute differences were <10%. There were no significant differences in the odds of reporting a minimal clinically important change between regimens at any time point. CONCLUSIONS: Even in the presence of urethral sparing, the higher BED delivered in the Boost schedule may have a small adverse effect on GU QoL compared with Monotherapy. However, this did not translate to statistically significant differences in minimal clinically important changes. Whether the higher BED of the boost arm offers an efficacy advantage is being investigated in the Trans Tasman Radiation Oncology Group 18.01 NINJA randomized trial.

Urine exosomes as biomarkers in bladder cancer diagnosis and prognosis: From functional roles to clinical significance.

Bladder cancer is one of the top ten most common cancers and top ten causes of cancer death globally. 5-year survival rates have decreased in Australia from 66% to 55% in the past three decades. The current gold standard for diagnosis is cystoscopy. However, cystoscopies are an invasive and health-resource intensive procedure which has sub-optimal sensitivity for flat lesions such as CIS (carcinoma in situ) and low specificity for differentiating inflammation from cancer - hence requiring biopsies under anesthesia. Frequent and life-long surveillance cystoscopy is required for most patients since there are high rates of progression and local recurrence in high-risk non-muscle invasive cancer (NMIBC) as well as poor outcomes associated with delayed detection of muscle-invasive bladder cancer (MIBC). There is an unmet need for a non-invasive test to provide better discrimination and risk-stratification of bladder cancer which could aid clinicians by improving patient selection for cystoscopy; enhanced risk stratification methods may guide the frequency of surveillance cystoscopies and inform treatment choices. Exosomes, which are nano-sized extracellular vesicles containing genetic material and proteins, have been shown to have functional roles in the development and progression of bladder cancer. Exosomes have also been demonstrated to be a robust source of potential biomarkers for bladder cancer diagnosis and prognosis and may also have roles as therapeutic agents. In this review, we summarize the latest evidence of biological roles of exosomes in bladder cancer and highlight their clinical significance in bladder cancer diagnosis, surveillance and treatment.

Feasibility of online adaptive HDR prostate brachytherapy: A novel treatment concept.

PURPOSE: The purpose of this study was to determine the feasibility of online adaptive transrectal ultrasound (TRUS)-based high-dose-rate prostate brachytherapy (HDRPBT) through retrospective simulation of source positioning and catheter swap errors on patient treatment plans. METHOD: Source positioning errors (catheter shifts in 1 mm increments in the cranial/caudal, anterior/posterior, and medial/lateral directions up to ±6 mm) and catheter swap errors (between the most and least heavily weighted) were introduced retrospectively into DICOM treatment plans of 20 patients that previously received TRUS HDRPBT. Dose volume histogram (DVH) indices were monitored as errors were introduced sequentially into individual catheters, simulating potential errors throughout treatment. Whenever DVH indices were outside institution thresholds: prostate V100% <95%, urethra D0.1cc >118% and rectum Dmax >80%, the plan was adapted using remaining catheters (i.e., simulating previous catheters as previously delivered). The final DVH indices were recorded. RESULTS: Prostate coverage (V100% >95%) could be maintained for source position errors up to 6 mm through online plan adaptation. The source position error at which the urethra D0.1cc and rectum Dmax was able to return to clinically acceptable levels using online adaptation varied between 6 mm to 1 mm, depending on the direction of the source position error and patient anatomy. After introduction of catheter swap errors to patient plans, prostate V100% was recoverable using online adaptation to near original plan characteristics. Urethra D0.1cc and rectum Dmax showed less recoverability. CONCLUSION: Online adaptive HDRPBT maintains the prostate V100% to clinically acceptable values for majority of directional shifts. However, the current online adaptive method may not correct for source position errors near organs at risk.

What is the effect of a low literacy talking book on patient knowledge, anxiety and communication before radiation therapy starts? A pilot study.

INTRODUCTION: Radiation therapy is a common cancer treatment, requiring timely information to help patients prepare for treatment. We pilot tested a low literacy, psycho-educational talking book (written booklet, with accompanying audio recording) to examine (i) the effect of the tool on knowledge, anxiety and communication; (ii) acceptability, and (iii) how it was used in appointments. METHODS: A pre-post design was employed. Patients scheduled to receive radiation therapy for any cancer were recruited from two hospitals in Sydney, Australia. Participants were sent the talking book before treatment planning and completed baseline and follow-up surveys, before and after the intervention. RESULTS: Forty participants were recruited, and 39 completed all study assessments. Overall, knowledge increased after receiving the talking book by 3.8 points from 13.9 to 17.7/20 (95% confidence interval (CI) 2.7, 4.8, P < 0.001). Anxiety and concerns were significantly lower after receiving the talking book (P = 0.015 and P = 0.004, respectively). Nearly half of participants (s = 17, 48%) reported using the book during appointments. Most reported finding it easier to communicate (n = 31, 89%) and to ask more questions (n = 21, 62%). CONCLUSION: The talking book shows promise in improving knowledge, reducing anxiety and enhancing communication. Strategies to support the implementation of the talking book are required. Further studies to translate the book into different languages are also planned.

HDR prostate brachytherapy plan robustness and its effect on in-vivo source tracking error thresholds: A multi-institutional study.

PURPOSE: The purpose of this study was to examine the effect of departmental planning techniques on appropriate in-vivo source tracking error thresholds for high dose rate (HDR) prostate brachytherapy (BT) treatments, and to determine if a single in-vivo source tracking error threshold would be appropriate for the same patient anatomy. METHODS: The prostate, rectum, and urethra were contoured on a single patient transrectal ultrasound (TRUS) dataset. Anonymized DICOM files were disseminated to 16 departments who created an HDR prostate BT treatment plan on the dataset with a prescription dose of 15 Gy in a single fraction. Departments were asked to follow their own local treatment planning guidelines. Source positioning errors were then simulated in the 16 treatment plans and the effect on dose-volume histogram (DVH) indices calculated. Change in DVH indices were used to determine appropriate in-vivo source tracking error thresholds. Plans were considered to require intervention if the following DVH conditions occurred: prostate V100% < 90%, urethra D0.1cc > 118%, and rectumtt Dmax > 80%. RESULTS: There was wide variation in appropriate in-vivo source tracking error thresholds among the 16 participating departments, ranging from 1 to 6 mm. Appropriate in-vivo source tracking error thresholds were also found to depend on the direction of the source positioning error and the endpoint. A robustness parameter was derived, and found to correlate with the sensitivity of plans to source positioning errors. CONCLUSIONS: A single HDR prostate BT in-vivo source tracking error threshold cannot be applied across multiple departments, even for the same patient anatomy. The burden on in-vivo source tracking devices may be eased through improving HDR prostate BT plan robustness during the plan optimisation phase.

Use of deformable image registration techniques to estimate dose to organs at risk following prostate external beam radiation therapy and high-dose-rate brachytherapy.

PURPOSE: The purpose of this investigation was to examine differences in estimates of accumulated rectal dose when using deformable image registration (DIR) compared with rigid image registration (RIR) methods, and parameter addition methods for combined transrectal ultrasound (TRUS)-based high-dose-rate brachytherapy (HDR-BT) and external beam radiation therapy (EBRT) treatments of prostate cancer. MATERIAL AND METHODS: In this retrospective study, data from 10 patients who had previously received HDR-BT in one 15 Gy fraction, followed by 46 Gy EBRT in twenty-three fractions were used. To estimate total combined dose to the rectum, dose accumulation using both DIR and RIR methods were compared with parameter addition methods, which assume the same region of rectal anatomy receives the maximum dose from both treatment modalities. For both rigid and deformable image registration techniques, the quality of image registration was evaluated through metrics, including mean distance to agreement and dice similarity coefficient of prostate contours. Total D1cc and D2cc for the rectum was calculated and compared using each method. RESULTS: The parameter addition methods predicted the highest accumulated dose to the rectum. On average, the predicted D2cc dose was higher than that calculated by the DIR method by 6.59 Gy EQD2 (range, -3.03 to 13.68 Gy EQD2) for partial parameter addition (PPA), and 4.88 Gy EQD2 (range, -3.41 to 11.97 Gy EQD2) for the full parameter addition (FPA) methods. Similarly, RIR predicted higher average doses compared with DIR, with a difference of 3.46 Gy EQD2 (range, -5.50 to 7.90 Gy EQD2). The results showed a significant difference between DIR and parameter addition methods for dose estimation. CONCLUSIONS: This retrospective study demonstrates significant differences in accumulated rectal dose prediction using different image registration methods. Each method has limitations in its application, and when used with real-time HDR-BT dose planning, awareness of these limitations is essential.

Consistency of small-field dosimetry, on and off axis, in beam-matched linacs used for stereotactic radiosurgery.

PURPOSE: Stereotactic radiosurgery (SRS) can be delivered with a standard linear accelerator (linac). At institutions having more than one linac, beam matching is common practice. In the literature, there are indications that machine central axis (CAX) matching for broad fields does not guarantee matching of small fields with side ≤2 cm. There is no indication on how matching for broad fields on axis translates to matching small fields off axis. These are of interest to multitarget single-isocenter (MTSI) SRS planning and the present work addresses that gap in the literature. METHODS: We used 6 MV flattening filter free (FFF) beams from four Elekta VersaHD® linacs equipped with an Agility™ multileaf collimator (MLC). The linacs were strictly matched for broad fields on CAX. We compared output factors (OPFs) and effective field size, measured concurrently using a novel 2D solid-state dosimeter "Duo" with a spatial resolution of 0.2 mm, in square and rectangular static fields with sides from 0.5 to 2 cm, either on axis or away from it by 5 to 15 cm. RESULTS: Among the four linacs, OPF for fields ≥1 × 1 cm2 ranged 1.3% on CAX, whereas off axis a maximum range of 1.9% was observed at 15 cm. A larger variability in OPF was noted for the 0.5 × 0.5 cm2 field, with a range of 5.9% on CAX, which improved to a maximum of 2.3% moving off axis. Two linacs showed greater consistency with a range of 1.4% on CAX and 2.2% at 15 cm off axis. Between linacs, the effective field size varied by <0.04 cm in most cases, both on and off axis. Tighter matching was observed for linacs with a similar focal spot position. CONCLUSIONS: Verification of small-field consistency for matched linacs used for SRS is an important task for dosimetric validation. A significant benefit of concurrent measurement of field size and OPF allowed for a comprehensive assessment using a novel diode array. Our study showed the four linacs, strictly matched for broad fields on CAX, were still matched down to a field size of 1 x 1 cm2 on and off axis.

Quality Assessment and Comparison of Plasma-Derived Extracellular Vesicles Separated by Three Commercial Kits for Prostate Cancer Diagnosis.

INTRODUCTION: Current standard biomarkers in clinic are not specific enough for prostate cancer (PCa) diagnosis. Extracellular vesicles (EVs) are nano-scale vesicles released by most mammalian cells. EVs are promising biomarker source for PCa liquid biopsy due to its minimal invasive approach, rich information and improved accuracy compared to the clinical standard prostate-specific antigen (PSA). However, current EV separation methods cannot separate pure EVs and the quality characteristics from these methods remain largely unknown. In this study, we evaluated the quality characteristics of human plasma-derived EVs by comparing three clinical suitable separation kits. METHODS: We combined EV separation by commercial kits with magnetic beads capture and flow cytometry analysis, and compared three kits including ExoQuick Ultra based on precipitation and qEV35 and qEV70 based on size exclusion chromatography (SEC). RESULTS: Our results indicated that two SEC kits provided higher EV purity and lower protein contamination compared to ExoQuick Ultra precipitation and that qEV35 demonstrated a higher EV yield but lower EV purity compared to qEV70. Particle number correlated very well particularly with CD9/81/63 positive EVs for all three kits, which confirms that particle number can be used as the estimate for EV amount. At last, we found that several EV metrics including total EVs and PSA-specific EVs could not differentiate PCa patients from health controls. CONCLUSION: We provided a systematic workflow for the comparison of three separation kits as well as a general analysis process in clinical laboratories for EV-based cancer diagnosis. Better EV-associated cancer biomarkers need to be explored in the future study with a larger cohort.

A Clinician's Guide to Cancer-Derived Exosomes: Immune Interactions and Therapeutic Implications.

Understanding of the role of immunity in the regulation of cancer growth continues to rapidly increase. This is fuelled by the impressive results yielded in recent years by immune checkpoint inhibitors, which block regulatory pathways to increase immune-mediated cancer destruction. Exosomes are cell-secreted membranous nanoscale vesicles that play important roles in regulating physiological and pathophysiological processes. Cancer-derived exosomes (CDEXs) and their biologically-active cargos have been proven to have varied effects in malignant progression, including the promotion of angiogenesis, metastasis, and favorable microenvironment modification. More recently, there is an increasing appreciation of their role in immune evasion. In addition to CDEXs, there are immune-derived exosomes that facilitate communication between immune cells in the non-malignant setting. Investigation of cancer-mediated mechanisms behind interruption or modification of these normal exosomal pathways may provide further understanding of how malignant immune evasion is accomplished. Accumulating evidence indicates that immune-active CDEXs also have the potential to impact clinical oncological management. Whilst immune checkpoint inhibitors have well-established pharmacologically-targeted pathways involving the immune system, other widely used treatments such as radiation and cytotoxic chemotherapies do not. Thus, investigating exosomes in immunotherapy is important for the development of next-generation combination therapies. In this article, we review the ways in which CDEXs impact individual immune cell types and how this contributes to the development of immune evasion. We discuss the relevance of lymphocytes and myeloid-lineage cells in the control of malignancy. In addition, we highlight the ways that CDEXs and their immune effects can impact current cancer therapies and the resulting clinical implications.

Towards real time in-vivo rectal dosimetry during trans-rectal ultrasound based high dose rate prostate brachytherapy using MOSkin dosimeters.

PURPOSE: To compare the dose measured by MOSkin dosimeters coupled to a trans-rectal ultrasound (TRUS) probe to the dose predicted by the brachytherapy treatment planning system (BTPS) during high dose rate (HDR) prostate brachytherapy (pBT), and to examine the feasibility of performing real-time catheter-by-catheter analysis of in-vivo rectal dosimetry during TRUS based HDR pBT. METHOD: Four MOSkin dosimeters were coupled to a TRUS probe during 20 TRUS-based HDR pBT treatment fractions. The measured MOSkin doses were retrospectively compared to those predicted by the BTPS for the total treatment fraction, as well as on a per catheter basis. RESULTS: The average relative percentage difference between MOSkin measured and BTPS predicted doses for a total treatment fraction was 0.3% ± 11.6% (k = 1), with a maximum of 23.2% and a minimum of -29.0%. The average relative percentage difference per catheter was +2.5% ± 16.9% (k = 1). The majority (64%) of per catheter MOSkin measured doses agreed with the treatment planning system within the calculated uncertainty budget of 12.3%. CONCLUSION: The results of the study agreed well with previously published data, despite differences in clinical workflows. To improve the redundancy to potential dosimeter errors, a minimum of 4 MOSkin dosimeters should be used when performing real-time in-vivo rectal dosimetry for HDR pBT, and error thresholds should be based off the total combined uncertainty estimate of measurement. 'Real time' error thresholds can be more confidently applied in the future through enhanced integration between IVD systems with both the imaging device and the BTPS/afterloader.

Extracellular vesicles: the next generation of biomarkers for liquid biopsy-based prostate cancer diagnosis.

Prostate cancer (PCa) is a leading cause of cancer death for males in western countries. The current gold standard for PCa diagnosis - template needle biopsies - often does not convey a true representation of the molecular profile given sampling error and complex tumour heterogeneity. Presently available biomarker blood tests have limited accuracy. There is a growing demand for novel diagnostic approaches to reduce both the number of men with an abnormal PSA/ DRE who undergo invasive biopsy and the number of cores collected per biopsy. 'Liquid biopsy' is a minimally invasive biofluid-based approach that has the potential to provide information and improve the accuracy of diagnosis for patients' treatment selection, prognostic counselling and development of risk-adjusted follow-up protocols. Extracellular vesicles (EVs) are lipid bilayer-delimited particles released by tumour cells which may provide a real-time snapshot of the entire tumour in a non-invasive way. EVs can regulate physiological processes and mediate systemic dissemination of various types of cancers. Emerging evidence suggests that EVs have crucial roles in PCa development and metastasis. Most importantly, EVs are directly derived from their parent cells with their information. EVs contain components including proteins, mRNAs, DNA fragments, non-coding RNAs and lipids, and play a critical role in intercellular communication. Therefore, EVs hold promise for the discovery of liquid biopsy-based biomarkers for PCa diagnosis. Here, we review the current approaches for EV isolation and analysis, summarise the recent advances in EV protein biomarkers in PCa and focus on liquid biopsy-based EV biomarkers in PCa diagnosis for personalised medicine.