Citations

Alcohol-associated liver disease (ALD) is a growing global health concern, with alcohol-associated hepatitis (AH) leading to the highest morbidity and mortality. Available therapies are limited and often inadequate. Platelets contribute in a variety of ways to liver disease pathogenesis, but their role in AH remains largely unexplored. In this study, we addressed the hypothesis that platelets contribute to pathological inflammation in AH. Using patient samples and a multiomics approach, we found that platelets undergo proinflammatory transcriptomic and proteomic changes in AH, with 2 alarmins, S100A8 and S100A9, being among the top upregulated genes/proteins. Additionally, the abundance of platelet-derived microparticles containing S100A8 and S100A9 in AH patient plasma was increased and correlated with disease severity (assessed by model for end-stage liver disease sodium [MELD-Na]) and endotheliopathy (assessed by ICAM1, CXCL8, and vWF). We mechanistically linked S100A9 with endotheliopathy via crosstalk between primary human liver sinusoidal endothelial cells and primary human monocytes. We also demonstrated that IL-6 upregulates S100A9 in megakaryocytic cells in a JAK/STAT-dependent manner, modeling changes occurring in the bone marrow in patients with AH. Our studies establish proinflammatory platelets as important contributors to AH pathology. Moreover, antiplatelet agents — or, more specifically, S100A9 targeted drugs — are potential therapeutic strategies in AH.

5068 Background: 177 Lu-PSMA-617 (LuPSMA) is a groundbreaking therapy for mPC, but biomarkers are urgently needed to improve patient selection. Plasma-derived EVs are emerging blood-based markers of tumor burden. Here, we analyzed levels of EVs of 123 patients (pts) from 4 cohorts: a pre- and during LuPSMA treatment for a contemporary prospective Mayo Clinic cohort, pre-treatment plasma levels of EVs for a historical Tulane cohort, localized prostate cancer (LPC) after radical prostatectomy (RP), and male healthy donors (HD). Methods: Blood samples were collected from 51 pts pre-cycles (pre-C) 1-3 of LuPSMA at Mayo Clinic from 12/2024-12/2025 and 26 pts pre-C1 at Tulane Cancer Center between 12/2018-1/2025. Small and large EVs were incubated with fluorescently conjugated antibodies (PSMA, STEAP1, CEACAM5, and TROP2) and enumerated using nanoscale flow cytometry. EVs from blood samples of 10 HD and 36 LPC post-RP from 6/2020-8/2024 at Mayo Clinic as controls. AUC-ROC and logistic regression analyses were performed to identify marker signatures associated with PSA50 response. Results: Baseline demographics are shown in the table. Median levels of PSMA, STEAP1, CEACAM5, and TROP2 EV/milliliter (mL) were all significantly higher for pts with mPC (pooled Tulane and Mayo cohorts) compared to LPC post-RP (p<0.001) and HD (p<0.001). In the Mayo cohort, 10 of 51 pts (20%) had undetectable PSA pre-C1. There were no significant differences in median pre-C1 plasma EV levels for these 4 different types of EVs comparing pts with undetectable vs detectable PSA. For 41 Mayo pts with detectable PSA pre-C1, 20 achieved PSA50 response at time of data cutoff (49%), while 7 pts (17%) had persistent PSA rise on LuPSMA. For pts who achieved PSA50, there was a significant decline in both CEACAM5 and TROP2 EVs/mL pre-C1 compared to pre-C3 (19 x 10 6 vs 5.8 x 10 6 , p=0.003 and 16 x 10 6 vs 5.5 x 10 6 , p<0.001, respectively), while no significant changes occurred for the 7 pts with no PSA response. A composite score of pre-C1 CEACAM5 and TROP2 EVs (large- and small-sized EVs) demonstrated the strongest association with PSA50 response (OR=2.96, 95% CI: 1.27-6.44, p=0.038), achieving AUC of 0.73. PSMA and STEAP1 EVs did not show any significant changes from pre-C1 to pre-C3. Conclusions: Plasma-derived EVs are a promising biomarker to identify patients who will benefit from LuPSMA. Longer follow-up and enrolling a larger cohort are underway. Clinical trial information: NCT06200103 . Median values for pre-LuPSMA (Mayo and Tulane), post-RP for LPC, and HD. Mayo mPC (n=51) Tulane mPC (n=26) LPC (n=36) HD (n=10) Age (range) 73 (57-87) 71 (54-83) 66 (56-77) 55 (50-57) Prior lines (range) 1 (1-3) 4 (1-11) - - Gleason score 9 8 7 - PSA (range) 1.0 (0-182) 75 (0.8-503) - - PSMA EV/mL 3.7 x 10 6 14 x 10 6 2.0 x 10 6 1.4 x 10 6 STEAP1 EV/mL 30 x 10 6 45 x 10 6 12 x 10 6 12 x 10 6 CEACAM5 EV/mL 17 x 10 6 5.1 x 10 6 3.7 x 10 6 3.9 x 10 6 TROP2 EV/mL 13 x 10 6 5.2 x 10 6 4.5 x 10 6 3.6 x 10 6

ABSTRACT Early-life nutrition profoundly influences long-term metabolic health, and breast milk not only provides nutrients but also conveys maternal signals shaping infant metabolic development. While postpartum exercise by lactating women benefits maternal health, its impact on milk-borne signaling remains largely undefined. Small extracellular vesicles (sEVs) in breast milk are key mediators of maternal–infant communication because of their selectively packaged bioactive cargo and resistance to infant digestive enzymes and acids, enabling delivery of their cargo to peripheral tissues. Here, we show that a single session of moderate-intensity postpartum aerobic exercise robustly increases human breast milk sEV concentration, which persists for multiple post-exercise milk collections. Exercise enriches breast milk with sEVs containing regulatory metabolic cargo (proteins, miRNAs, and metabolites), which translates into enhanced mitochondrial capacity in neonatal-stage cells. These findings implicate sEVs as an exercise-responsive signaling compartment in breast milk capable of connecting postpartum maternal physical activity to beneficial infant metabolic programming. Highlights Acute moderate-intensity exercise increases human breast milk sEV concentration The exercise-mediated sEV increase lasts for multiple subsequent milk expressions Exercise coordinates a multi-omic enrichment of sEVs in breast milk Exercised breast milk sEVs enhance mitochondrial respiration in UC-MSCs

Abstract Introduction: Urine cell-free DNA (cfDNA) is a promising ultra-noninvasive analyte for genitourinary cancers (GU) cancers. In this study, we investigated urine cfDNA fragmentomic features and evaluated their ability to predict the tumor tissue of origin. Methods: A total of 204 GU cancer patients and 34 healthy adults were enrolled in this prospective study. Preoperative urine was collected from 89 bladder cancer (BC) patients (64% muscle-invasive) undergoing cystectomy, 65 renal cell carcinoma (RCC) patients undergoing nephrectomy and 50 metastatic prostate cancer (mPC) patients. Urine cfDNA was isolated and sequenced at 5x genome-wide coverage on a NovaSeq S4 flow cell. To identify malignant tissue of origin, we analyzed somatic copy number alterations (CNAs), genome-wide fragment length ratios, and 4-mer end motifs. CNAs and tumor fraction (TFx) were quantified using ichorCNA in 1-Mb windows, while chromosome arm-level fragment count z-scores and fragment length ratios were calculated in 5-Mb bins across the genome. Relative frequencies of all 256 possible 4-mer end motifs were calculated for each sample. Individual logistic regression models were developed based on each fragmentomic feature using leave-one-out cross validation (LOOCV). Outputs from these models were combined into multi-feature XGBoost models. Cohort-specific end-motif enrichments were identified using Kruskal-Wallis tests with FDR correction. Motifs were then hierarchically clustered based on their cohort-wise median frequencies to define groups of motifs with shared fragmentation patterns. Results: Across urine cfDNA in the GU cohort, BC showed the highest mean TFx at 10.0%, followed by mPC at 3.6% and RCC at 2.7%. A machine learning model based on 4-mer end motifs achieved a mean ROC AUC of 0.933 with classification accuracies of 63% for healthy, 87% for BC, 72% for mPC, and 83% for RCC. This model outperformed models based on ichorCNA (AUC = 0.767), chromosome arm fragment-count z-scores (AUC = 0.827), and fragment length ratios (AUC = 0.674). Combining end motifs with other fragmentomic features did not improve prediction accuracy. Multi-feature models incorporating end motifs with ichorCNA, chromosome arm-level z-scores, or fragment-length ratios yielded mean AUCs between 0.908-0.924, lower than the end-motif only model. Kruskal-Wallis tests identified 179 of 256 end motifs (79%) as significantly different across healthy, BC, mPC, and RCC. Hierarchical clustering of cohort-level median motifs revealed distinct end motif patterns: Motifs beginning with CT were enriched in RCC; AAAA was strongly enriched in BC; and motifs beginning with CA, AC, and AG were enriched in mPC. Conclusion: Urine cfDNA 4-mer end motifs capture GU cancer-specific biological patterns that enable accurate tissue-of-origin prediction, underscoring their promise as a diagnostic tool. Citation Format: Jessica Linford, Pradeep S. Chauhan, Irfan Alahi, Yohan Kim, Arpit Panda, Nathan Colon, Ryan Mueller, Faridi Qaium, Eric H. Kim, Melissa A. Reimers, Zachary L. Smith, Woodson W. Smelser, Fabrice Lucien-Matteoni, Aadel A. Chaudhuri. Urine cfDNA 4-mer end-motif signatures outperform other fragmentomic features for tissue-of-origin classification in genitourinary cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2590.

Abstract Introduction: Detectable circulating tumor DNA (ctDNA) is associated with inferior outcomes in Ewing sarcoma (ES); however, ctDNA clears in nearly all patients within 2 of the 6 chemotherapy cycles before radiotherapy (RT) or surgery. Given a 3-year EFS of 37% on AEWS 1221 (metastatic) and 5-year EFS of 78% on AEWS 1031 (localized), current ctDNA testing fails to predict most relapses. Extracellular vesicles (EVs) are microscopic particles studied as a biomarker in multiple cancer types. We hope to improve the sensitivity of ctDNA in ES and provide the first application of EVs in ES. Methods: cfDNA was isolated from 1mL plasma, quantified by Qubit, and sequenced as NEBNext Enzymatic Methyl-seq v2 libraries. We performed a systematic unsupervised clustering analysis of 27 ES plasma cfDNA profiles using 4 differentially methylated regions (DMR) subsets (63 strong DMRs, top 30, top 15, and 10 promoter-proximal loci), 3 clustering algorithms (k-means, Ward hierarchical, PAM), and k=2-5 clusters, testing associations with tumor burden and progression. EV analysis involved antibody detection against ES surface markers, including LINGO1, ENPP1, CDH11, and CD99. Concentrations of EVs were determined by standardized and calibrated high-resolution flow cytometry then analyzed with custom-built software to automatically determine and apply gates. Samples were de-identified, randomly coded, and run in triplicates. The senior research technologist responsible for EV analysis was blinded to patient information and clinical data. Results: EV analysis included 36 pre-RT samples from 16 metastatic and 20 localized ES patients from a prospectively maintained biobank, with 27 pre-RT samples in the cfDNA analysis and 19 healthy donor (HD) samples. 35 of 36 patients had chemotherapy before sample collection. cfDNA concentrations were markedly elevated in ES patients compared with HD (24.4 vs 4.6 ng/mL, p&lt;0.0001), and 74% of ES samples exceeded the HD 95th-percentile detectability threshold (7.0 ng/mL). Across clustering configurations, ES cfDNA profiles consistently exhibited reproducible methylation substructure. One configuration (PAM clustering, k = 5, 63 strong DMRs) showed a modest but permutation-supported association with primary tumor size (nominal p ≈ 0.04; empirical p ≈ 0.03-0.04). ES samples had increased EV concentration compared to HD (p&lt;0.001), with AUC 0.988 for distinguishing between ES and HD ENPP1 (p&lt;0.0001). The highest ENPP1 EV values were in patients with large primary tumors (&gt;8 cm vs. ≤ 8cm) and metastatic disease (p=0.066). Conclusions: cfDNA- and EV-based liquid biopsy provide encouraging signals for high ES detectability pre-RT. We also show the first study of EVs as a novel biomarker in ES, which may provide complementary information to ctDNA. Multimodal liquid biopsy in ES may identify candidate patients for systemic or local therapy escalation and facilitate personalized care. Citation Format: Roman O. Kowalchuk, Lilli J. Greiner, Yohan Kim, Rubia Noori, Jeffrey Szymanski, William S. Harmsen, Pradeep S. Chauhan, Adam C. Amundson, Andrea M. McMahon, Peter J. Schoettler, Wendy A. Allen-Rhoades, Stephanie F. Polites, Peter S. Rose, Kelly M. Bailey, Linda M. McAllister-Lucas, Anita Mahajan, Fabrice Lucien-Matteoni, Nadia N. Laack, Aadel A. Chaudhuri. Multimodal liquid biopsy of Ewing sarcoma reveals high sensitivity prior to radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1149.

TPS283 Background: Emerging evidence suggests that metastasis-directed therapy (MDT) with stereotactic body radiotherapy (SBRT) can delay systemic therapy in oligometastatic castration-sensitive prostate cancer (omCSPC). However, the optimal timing of androgen deprivation therapy (ADT) and androgen receptor pathway inhibitor (ARPI) initiation in this setting remains undefined. While deferred ADT following MDT may preserve quality of life, early short-course ADT/ARPI may eradicate micrometastatic disease and extend progression-free survival. Extracellular vesicles (EVs) carrying PSMA-positive prostate cancer (PC) signals and circulating tumor DNA (ctDNA) may serve as real-time biomarkers of MRD and treatment response. Methods: DIVINE [NCT06378866] is an open-label, phase II, randomized trial enrolling men with omCSPC defined by 1–5 metastases (≥1 extrapelvic) on conventional imaging (CT/MRI and bone scan) and testosterone >100 ng/dL. PSMA PET may be obtained for correlative purposes but does not determine eligibility. Participants (N = 220) are randomized 1:1 to: Arm A (Early systemic): SBRT plus 6 months of ADT + ARPI. Arm B (Deferred systemic): SBRT alone with observation, reserving systemic therapy until radiographic progression not amenable to further SBRT (mrPD). SBRT is delivered per institutional standard; repeat SBRT is allowed until lesions become non-addressable. The primary endpoint, modified radiographic progression-free survival (mrPFS), is the time from randomization to death or radiographic progression not amenable to further MDT, defined by PCWG3 (bone) and modified RECIST 1.1 (soft tissue) criteria on conventional imaging. Soft-tissue progression requires ≥ 20% increase in measurable disease or a new lesion; bone progression requires ≥ 2 new confirmed lesions. Secondary endpoints include OS, biochemical PFS, time to local and distant progression, and grade 3–5 adverse events during systemic therapy. Exploratory endpoints evaluate EV and ctDNA kinetics as MRD markers, correlation of PSMA-positive EV burden with outcomes, and duration of response after short-course ADT/ARPI. Correlative analyses will longitudinally quantify PSMA-positive EVs and ctDNA at baseline, on treatment, and at progression to define MRD dynamics and resistance patterns. The trial uses Pocock–Simon dynamic allocation by EV strata and metastatic burden to ensure balance across arms. The trial is open and actively enrolling across all Mayo Clinic sites; as of October 2025, 35 of the planned 220 patients have been accrued. Clinical trial information: NCT06378866 .

214 Background: 177 Lu-PSMA-617 (LuPSMA) has revolutionized care for patients (pts) with metastatic castrate-resistant prostate cancer (mCRPC), but only half achieves PSA decline ≥50%. Biomarkers are urgently needed to improve pt selection. EVs and ctDNA released by cancer cells into the blood are emerging biomarkers to estimate overall tumor burden and capture tumor heterogeneity in mCRPC. Here, we analyzed pre-treatment plasma levels of tumor EVs and ctDNA in a pilot cohort of 35 pts with mCRPC treated with LuPSMA. Methods: Blood samples were collected pre-cycle 1 of LuPSMA at Tulane Cancer Center between 12/2018-1/2025. Plasma-derived small and large EVs were incubated with fluorescently conjugated antibodies and enumerated using nanoscale flow cytometry. ctDNA fraction was estimated from plasma cfDNA methylation WGS (∼10×) with ichorCNA. We also analyzed EVs from blood samples of male healthy donors (HD) and pts with localized prostate cancer (LPC) collected 1 day to 8 months after radical prostatectomy (post-RP) from 6/2020-8/2024 at Mayo Clinic as controls. Results: Median age at collection for 10 HD was 55 yrs and for 36 pts with LPC post-RP was 65 yrs. Median PSMA+ EV level was 2.0 x 10 6 /mL for post-RP and 1.4 x 10 6 /mL for HD. For 35 pts with mCRPC, 25 pts had complete data for analysis. Median PSMA+ EV level was 1.4 x 10 7 /mL, significantly higher than post-RP (p<0.001) and HD (p<0.001). At LuPSMA start, median age was 69 yrs (range 54-83), PSA was 82 ng/mL (range 0.8-503), median time from CRPC diagnosis to start was 50 months (range 6.9-285), median time from plasma collection to treatment initiation was 0.5 months (range 0-7.3). Median number of prior lines of life-extending therapy was 4 (range 1-11), including 100% androgen receptor pathway inhibitor and 75% docetaxel. Pts received median 4 cycles of LuPSMA. Median ctDNA fraction was 14.0% (range 0-51.1). Median time to reach nadir PSA for pts with any decline was 2.6 months. 15 pts (60%) achieved PSA40 (defined ≥40% decline in PSA from start to nadir) (range -99% to -42%) compared to 10 pts (40%) who did not (range -16% to rise), which predicted better median overall survival (OS) after LuPSMA start (40.7 v 6.0 months; HR 0.23, 95% CI, 0.07-0.71; p=0.0001). Pts were classified as LuPSMA responders using PSA40. Responders had a lower ratio of large to small PSMA+ EVs (0.30 v 0.53, p=0.0042) with an area under the receiver operating characteristic curve (AUC) of 0.83 (95% CI, 0.68-0.99), and a lower median ctDNA fraction (6.1% v 26.2%, p=0.0044) with an AUC of 0.83 (95% CI, 0.68-1.00). Incorporating both PSMA+ EV and ctDNA fraction together predicted PSA40 response with an AUC of 0.93 (95% CI, 0.84-1.00, p=0.0003). Conclusions: Using pre-LuPSMA plasma samples from a heavily pre-treated historical cohort, we integrated non-invasive liquid biopsy methods to successfully predict response to LuPSMA using PSA decline. Prospective validation is underway.

ABSTRACT The concentration of cells is a key component of modern blood tests. Given the biomarker potential of extracellular vesicles (EVs) in blood, we aimed to establish reference ranges for blood cell‐derived EVs using flow cytometry. To address the orders‐of‐magnitude variability in reported EV concentrations between different flow cytometers (FCMs), we first validated a calibration methodology to enable reproducible EV concentration measurements. The methodology was evaluated in an interlaboratory comparison study and shows that calibration reduces the median absolute deviation of EV concentrations measured on 25 different FCMs from 67 % to 25 %–31 %. The calibration methodology was then used to determine reference ranges of erythrocyte‐, leukocyte‐, and platelet‐derived EVs in human blood plasma in a cohort of healthy individuals ( n = 224). This study demonstrates that calibration enables comparable concentration measurements of blood cell‐derived EVs, thereby bringing EVs one step closer to clinical applications.

Abstract Description Combination of radiotherapy (RT) and immunotherapy has gained popularity to turn up the heat on cold tumors. However, intrinsic resistance is prevalent, hence better therapeutic strategies are needed to improve the outcome of patients. We recently demonstrated in metastatic prostate cancer that circulating levels of tumor-derived extracellular vesicles (tdEVs) increased shortly after RT and was associated with poor survival. TdEVs are mediators of antitumor immunity but their role in response to RT remains unknown. Our study elucidates the impact of RT on immunomodulatory roles of tdEVs. Mouse tumors models expressing a bioluminescent EV reporter were treated with clinically used doses of RT. TdEV organotropism was measured by ex-vivo imaging at different timepoints. TdEVs were detected in blood, spleen, lymph nodes and immune cells. OT-1 transgenic mice and in vitro co-culture assays showed a reduction in CD8 T-cell cytotoxic activity in presence of tdEVs from irradiated cells. To identify key players of tdEV-mediated immunosuppression in response to RT, proteomic analysis revealed the immune checkpoint molecule B7-H3 was overexpressed in tdEVs after irradiation. Genetic deletion of B7-H3 inhibited the suppressive activity of tdEVs from irradiated cells resulting in enhanced T-cell cytotoxicity. This study describes tdEVs as a mediator of systemic immunosuppression in response to radiotherapy and B7-H3 as a potential therapeutic target to boost antitumor immunity. Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)