Lucien, Fabrice

337 Background: Prostate cancer remains the most common solid organ malignancy among males in the United States. Commercial biomarkers and multiparametric MRI have been utilized to better triage men with elevated PSA and determine patients most likely to have clinically significant prostate cancer (csPCa) upon prostate biopsy. We studied the combination of MRI with PSA-based and novel extracellular vesicle (EV)-based biomarkers to determine the optimal pre-biopsy testing to reduce the number of unnecessary biopsies. Methods: Men presenting to our clinic with elevated PSA (≥ 2 ng/ml) were prospectively enrolled from October 2019 to March 2022. All men underwent multiparametric MRI and blood draws to determine prostate health index (PHI) scoring, EV density, and PSA density. MRI-fusion biopsy was performed with both systematic and targeted prostate sampling, with clinicians blinded to prior blood testing results. Blood concentrations of prostate EV (ProsEVs) were measured by nanoscale flow cytometry using antibodies against PSMA and STEAP1. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated, and receiver operator characteristic (ROC) curves were constructed separately for all patients, those without prior diagnosis of PCa, and biopsy-naïve patients. Bootstrapping analysis was performed to provide more accurate assessment of the predictive ability of all combinations. Results: Ultimately, 175 men enrolled and underwent MRI, blood testing, and prostate biopsy. Among the 175 patients included, 101 (58%) had never undergone biopsy, 38 (22%) had a previous negative biopsy, and 36 (21%) were on active surveillance (AS) for GS6 disease. AUC of mpMRI (PI-RADS≥3) to predict clinically significant PCa was 0.66 with a sensitivity, specificity, NPV and PPV of 95.2%, 37.6%, 76.0% and 79.26%. MRI combined with STEAP1-EV density had an AUC of 0.77 with a sensitivity, specificity, NPV and PPV of 90.1%, 46.7%, 64.4% and 80.9%. A multivariable model including MRI, STEAP1-EV density, PSA density and PHI density outperformed other combinations with an AUC, sensitivity, specificity, NPV and PPV of 0.90, 89.2%, 68.7%, 72.8% and 88.1%. Conclusions: Combination of mpMRI with ProsEV and PSA derivatives demonstrated better predictive ability for csPCa on biopsy compared to imaging or serum testing alone. Inclusion of prostate volume with biomarkers further increased predictive ability which could lead to a decrease in number of unnecessary biopsies for men with benign conditions and indolent cancer.

334 Background: Prostate cancer (PCa) screening has relied on prostate-specific antigen (PSA) levels to screen patients for biopsy. However, the limited specificity of serum PSA leads to high rates of negative biopsies and detection of clinically insignificant cancers. Tumor-derived extracellular vesicles, microscopic particles released by tumor cells and ubiquitously found in all bodily fluids, have emerged as novel biomarkers for a diverse set of malignancies. This study explored the utility of urine prostate-specific extracellular vesicles (ProsEVs) identified by unique surface protein markers with novel flow cytometry-based techniques as biomarkers for PCa screening. Methods: This prospective study recruited at a tertiary care center between Oct 2019 and Mar 2022. Patients with PSA≥2 ng/ml underwent prebiopsy mpMRI followed by transperineal prostate biopsy (PI-RADS 1-2) or combined MRI-guided biopsy (PI-RADS ≥ 3). Patients with metastatic PCa or prior systemic therapy were excluded. Urine samples were collected at MRI using Colli-Pee. ProsEVs were labeled using monoclonal antibodies for PSMA and STEAP1 and ProsEV concentrations were determined using nanoscale flow cytometry. EV densities were calculated by normalizing EV counts to prostate volume. Multivariable logistic regression models were constructed and model performance was compared used ROC curve analysis. Results: This study had a total of 179 patients prospectively recruited, 95 were diagnosed with Gleason score (GS) ≥ 7 prostate cancer (csPCa), while the remaining had GS <7 or benign disease. Analysis of urinary ProsEV densities revealed a significant dose-response relationship between benign, GS <7 PCa and GS ≥ 7 PCa groups and ProsEV densities (p=0.01 for PSMA, and <0.001 for STEAP1). The log sum of the PSMA and STEAP1 EV densities exhibited the strongest correlation with other ProsEV variables and was selected for model development. ROC curve analysis showed that the final model including Log Sum ProsEVs, PSA, and PI-RADS outperformed one including PSA and PI-RADS (0.733 vs 0.651, p=0.02). Specificity for detecting csPCa at a sensitivity of 90% was 39.5% in the final model compared to 37% for PSA and PI-RADS (Table). Conclusions: This study highlights the potential of urine ProsEVs as novel biomarkers for improving screening specificity for csPCa detection. Future works should explore analysis of intra-ProsEV content to further improve specificity and advance liquid biopsy techniques. [Table: see text]

191 Background: Two randomized clinical trials have demonstrated that SABR can prolong time to progression in oligometastatic castration-sensitive prostate cancer (omCSPC) patients. However, there is a need for predictive tools to identify patients who will benefit from SABR alone or with systemic therapy. We investigated the prognostic value of PSMA-positive extracellular vesicles (PSMA+EVs) in a blinded observational study using blood samples from three independent patient cohorts. Methods: We obtained plasma samples from 157 omCSPC patients treated with SABR from the ORIOLE cohort (N=30, JHU) and STOMP-like cohorts (N=80, UGhent; N=47, Iridium Net). Baseline PSMA+EV levels (EV/ml) were measured by nanoscale flow cytometry. Primary clinical endpoints were biochemical progression (bPFS) and radiographic progression (rPFS). Optimal cutoffs for PSA (ng/ml) and PSMA+EV (EVs/ml) were defined as the point with the most significant log-rank test split. Kaplan-Meier curves and univariate Cox regression models were used to determine the association of PSMA+EV levels with clinical outcomes. Results: Twenty percent of patients (N=157 total) were diagnosed with bone scan, 79% with PET imaging, and 1% with other modalities. Ninety-four percent of patients presented with ≤3 metastases on imaging. In the pooled STOMP-like cohorts, median bPFS was 27.9 and 18.0 months for PSMA+EV low and high groups, respectively (p=0.039). Median rPFS was 36.0 versus 27.0 months (p=0.029). In the ORIOLE cohort, median bPFS was 24.3 and 5.9 months in PSMA+EV low and high, respectively (p=0.021). Median rPFS was 36.0 versus 11.1 months (p=0.019). High baseline PSMA+EV levels were associated with higher risk of both biochemical and radiographic progression (Table). Combination of baseline PSA low and PSMA+EV low outperformed either biomarker alone and better predicted risk of disease progression after SABR. Conclusions: While prospective biomarker-guided trials are warranted, a PSMA+EV blood test can aid patient selection by identifying omCSPC patients who may have durable responses to SABR without ADT. [Table: see text]

Background: Approximately 30% of renal cell carcinoma (RCC) cases present with de novo metastatic disease, while 20% to 30% of those with localized disease will develop metastases following surgical resection. Various drug classes have been investigated to treat RCC, including cytokine-based therapies, small molecule Vascular Endothelial Growth Factor (VEGF) tyrosine kinase inhibitors (TKIs) and antibody-based therapies. Up to 58% of patients fail to respond to primary immune checkpoint inhibitor (ICI) therapy, and nearly all initial responders experience disease progression due to the development of secondary resistance. Consequently, novel treatment options are being investigated. Objective: Review the rapidly evolving ADC therapeutic landscape in metastatic RCC including recent trials, emerging ADCs targets, and future directions for ADCs in the treatment of advanced RCC. Methods: Literature review using the MEDLINE database on important trials and presentations from the American Society of Clinical Oncology (ASCO), and the European Society for Medical Oncology (ESMO) conferences. Key words used included “renal cell carcinoma,” “RCC,” “metastatic RCC,” “advanced RCC,” “antibody-based therapies,” “immunotherapy,” “clinical trials,” and “emerging drugs.” Specifically for review of ADCs in RCC, the following search string was used with additional review of bibliographies from retrieved papers: “((antibody drug conjugate) OR (antibody-dependent cellular cytotoxicity) OR (chimeric antigen receptor)) AND ((kidney cancer) OR (renal cell carcinoma))”. Results: Several promising targets including MMP14, EGFR, MCT4, CA9, MET, CDH13, B7-H3, and PSMA were identified with relevant preclinical and clinical studies reviewed. Conclusions: While ADCs therapeutics have not shown benefit to date for renal cell carcinoma, there are ample promising candidates and targets for future research.

Introduction: Platelets are heavily implicated in angiogenesis; however, the underlying mechanism remains relatively uncharacterized. The goal of this study was to evaluate the angiogenic potency of a platelet-derived regenerative exosome product (PEP) in an effort to develop a novel treatment for peripheral arterial disease. Here, we evaluated the mechanistic basis of PEP in angiogenesis and it’s use In Vivo in a rodent hind limb ischemia model. Methods/Results: Under Current Good Manufacturing Practices, conditioned medium from apheresis platelet cultures was purified to acquire the exosomes. PEP, enriched in exosome markers CD63, CD9 and Flotillin, induced human umbilical vein endothelial cell (HUVEC) proliferation, migration, and vascular tube formation In Vitro. Proteomic evaluation of PEP highlighted receptor tyrosine kinase (RTK) pathways and western blot validation demonstrated induction of phospho-MAPK and phospho-AKT following PEP treatment of HUVECs. Systematic evaluation of RTK receptor involvement was performed using small molecule inhibitors and receptor knock-down in HUVECs, ruling out RTK activation as a mode of action. However, small molecule inhibition of PI3 kinase demonstrated elimination of PEP-induced AKT phosphorylation and reduction in PEP-induced angiogenesis In Vitro measured by HUVEC proliferation and migration. In Vivo, evaluation of angiogenesis was performed in a rodent model of hindlimb ischemia treated with PEP embedded in fibrin glue (PEP/TISSEEL). Blood flow was evaluated by SPY angiography on day 0 pre- and post-operation, day 21, and day 28. Animals treated with PEP/TISSEEL showed a significant increase in blood flow compared to sham and TISSEEL treated animals (percent perfusion at 28 days: sham 40.9%, TISSEEL 44.3%, PEP/TISSEEL 100.2%), with significant increase in vascular area on histology (at 28 days vascular area (μm 2 )/muscle area(μm 2 ): healthy control 12.71, sham 4.66, TISSEEL 4.76, PEP/TISSEEL 15.54). Conclusions: Exosome driven activation of PI3 kinase can induce angiogenesis both In Vitro and In Vivo. PEP provides a novel, clinical-grade, off-the-shelf therapy to promote angiogenesis for the treatment of peripheral arterial disease.

Abstract Blood is the most commonly used body fluid for extracellular vesicle (EV) research. The composition of a blood sample and its derivatives (i.e., plasma and serum) are not only donor‐dependent but also influenced by collection and preparation protocols. Since there are hundreds of pre‐analytical protocols and over forty variables, the development of standard operating procedures for EV research is very challenging. To improve the reproducibility of blood EV research, the International Society for Extracellular Vesicles (ISEV) Blood EV Task Force proposes standardized reporting of (i) the applied blood collection and preparation protocol and (ii) the quality of the prepared plasma and serum samples. Gathering detailed information will provide insight into the performance of the protocols and more effectively identify potential confounders in the prepared plasma and serum samples. To collect this information, the ISEV Blood EV Task Force created the Minimal Information for Blood EV research (MIBlood‐EV), a tool to record and report information about pre‐analytical protocols used for plasma and serum preparation as well as assays used to assess the quality of these preparations. This tool does not require modifications of established local pre‐analytical protocols and can be easily implemented to enhance existing databases thereby enabling evidence‐based optimization of pre‐analytical protocols through meta‐analysis. Taken together, insight into the quality of prepared plasma and serum samples will (i) improve the quality of biobanks for EV research, (ii) guide the exchange of plasma and serum samples between biobanks and laboratories, (iii) facilitate inter‐laboratory comparative EV studies, and (iv) improve the peer review process.

Abstract Polycaprolactone fumarate (PCLF) is a cross‐linkable PCL derivative extensively considered for tissue engineering applications. Although injection molding has been widely used to develop PCLF scaffolds, platforms developed using such technique lack precise control on architecture, design, and porosity required to ensure adequate cellular and tissue responses. In particular, the scaffolds should provide a suitable surface for cell attachment and proliferation, and facilitate cell–cell communication and nutrient flow. 3D printing technologies have led to new architype for biomaterial development with micro‐architecture mimicking native tissue. Here, we developed a method for 3D printing of PCLF structures using the extrusion printing technique. The crosslinking property of PCLF enabled the unique post‐processing of 3D printed scaffolds resulting in highly porous and flexible PCLF scaffolds with compressive properties imitating natural features of cancellous bone. Generated scaffolds supported excellent attachment and proliferation of mesenchymal stem cells (MSC). The high porosity of PCLF scaffolds facilitated vascularized membrane formation demonstrable with the stringency of the ex ovo chicken chorioallantoic membrane (CAM) implantation. Furthermore, upon implantation to rat calvarium defects, PCLF scaffolds enabled an exceptional new bone formation with a bone mineral density of newly formed bone mirroring native bone tissue. These studies suggest that the 3D‐printed highly porous PCLF scaffolds may serve as a suitable biomaterial platform to significantly expand the utility of the PCLF biomaterial for bone tissue engineering applications.

Patients with advanced cancers who either do not experience initial response to or progress while on immune checkpoint inhibitors (ICIs) receive salvage radiotherapy to reduce tumor burden and tumor-related symptoms. Occasionally, some patients experience substantial global tumor regression with a rebound of cytotoxic CD8 + T cells. We have termed the rebound of cytotoxic CD8 + T cells in response to salvage therapy as T cell resilience and examined the underlying mechanisms of resilience. Resilient T cells are enriched for CX3CR1 + CD8 + T cells with low mitochondrial membrane potential, accumulate less reactive oxygen species (ROS), and express more malic enzyme 1 (ME1). ME1 overexpression enhanced the cytotoxicity and expansion of effector CD8 + T cells partially via the type I interferon pathway. ME1 also increased mitochondrial respiration while maintaining the redox state balance. ME1 increased the cytotoxicity of peripheral lymphocytes from patients with advanced cancers. Thus, preserved resilient T cells in patients rebound after salvage therapy and ME1 enhances their resiliency.