Lucien, Fabrice

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.

Abstract Introduction Checkpoint blockade revolutionized cancer therapy. However, only a small proportion of cancers respond to checkpoint blockade. B7-H3 is a member of the B7 superfamily, which includes both co-inhibitory and co-stimulatory receptors. B7-H3 is upregulated by many tumors which is generally associated with a worse prognosis. However, the exact mechanism by which B7-H3 suppresses the immune response remains elusive. Methods We transfected human HEK293T cells and assessed the binding to recombinant Siglecs via flow cytometry. We generated monoclonal antibody F7 against human B7-H3 and tested its blocking function using B7-H3 transfected HEK293T cells. We examined the extent of degranulation and NK cell-mediated killing on B7-H3 expressing HEK293T cells in vitro, as well as the CART19 cells mediated killing in relapse model of B7-H3 expressing CD19+ leukemia Nalm6 cells NSG mice. Results We demonstrated that sialylated B7-H3 binds to the inhibitory receptor Siglec-9 on immune cells but not to the related protein Siglec-7. A high affinity monoclonal antibody (mAb) F7 against B7-H3 blocks Siglec-9 binding. Functionally, F7 enhances NK cell killing of B7-H3 expressing target cells in vitro. F7 also dramatically enhances CART19 mediated killing of B7-H3 expressing Nalm6 cells in a relapse model in NSG mice in vivo. Conclusion We have now shown for the first time that B7-H3 binds to the inhibitory receptor Siglec-9 and generated a novel monoclonal antibody F7 that target a unique sequence in B7-H3 as compared to other monoclonal antibodies being examined for clinical efficacy. F7 can promote NK cell killing of B7-H3 expressing target cells in vitro and synergize with CART19 to enhance killing of B7-H3 expressing tumor cells in vivo. As B7-H3 is overexpressed in many human cancers, this work is critical toward understanding how B7-H3 mediates immune evasion through Siglec-9 for enhancing anti-tumor immunity. Funding Source R01CA243545-01A1 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

ABSTRACT The FDA Modernization Act 3.0 represents a pivotal shift in biomedical research by formally removing the requirement for animal testing and enabling regulatory acceptance of New Approach Methodologies (NAMs). This transition creates an urgent need for robust, human‐relevant biomarkers that can anchor NAM‐generated data within regulatory decision‐making, particularly for first‐in‐human studies. Extracellular vesicles (EVs), owing to their biological stability, accessibility and rich molecular cargo, may be well positioned to fulfil this role. EVs reflect dynamic cellular states and intercellular communication, making them mechanistically informative readouts of pharmacology, toxicity, immune modulation and disease biology across human‐derived model systems. This Perspective outlines how EV‐based biomarkers can support the qualification, standardization, and regulatory acceptance of NAMs, with particular emphasis on ex vivo platforms such as organoids, microtissue systems, organ‐on‐chip devices and perfused tissue slices. We highlight the advantages of EVs for longitudinal, non‐destructive sampling, representation of human variability, and integration with quantitative systems pharmacology and physiologically based pharmacokinetic models. Finally, we discuss the scientific, operational and regulatory challenges that must be addressed and argue that coordinated leadership from the International Society for Extracellular Vesicles (ISEV) is essential. The convergence of regulatory reform and EV science presents a timely call to action to establish EVs as foundational biomarkers in next‐generation, human‐centric drug development.

6123 Background: Pemetrexed (PTX) is safe and tolerable with responses reported in pts with R/M SGC. Given enhanced responses with PTX and pembrolizumab (PMB) for lung cancer, we hypothesized that PTX and PMB will have activity for SGC. Herein we present the efficacy results in pts with adenoid cystic carcinoma (ACC). Methods: MC200708 is a single arm phase II study of PTX and PMB in pts with R/M SGC (NCT04895735) with 2 cohorts: ACC (cohort A) and non-ACC (cohort B). Key eligibility criteria: ≥18 years, ECOG 0-1, measurable disease. Prior ICI and/or PTX was allowed. Key exclusion criteria: serious comorbidities, autoimmune disease and brain metastases. Simon’s 2-stage design was used for each cohort. Primary endpoint was overall response rate (ORR) for Cohort A but was amended to clinical benefit rate (CBR=SD+PR+CR) due to prolonged SD in several pts. Secondary endpoints: progression free survival (PFS), overall survival (OS), and toxicity. Exploratory analyses in Cohort A1 (post-amendment pts) included PSMA PET imaging and circulating PSMA extravesicles (EVs) as a biomarker. All pts received PTX 500 mg/m2 IV + PMB 200 mg IV q3 weeks until progression or treatment intolerance. Imaging was q3 cycles. Results: 20 pts (11 Cohort A + 9 Cohort A1) were enrolled from August 2021-Feb 2025. All 20 patients were eligible and received ≥ 1 cycle of treatment. Median age was 60.5 yrs, 50% male, performance status 0 (80%) or 1 (20%). 11 pts had no prior therapies (55.0%). The remaining pts had 1 (35%) or 2 (10%) prior lines of therapy. 75% of pts had no prior ICI. In Cohort A, the ORR was 0% (0 of 11). Cohort A1 met criteria for success based on CBR with 1 PR (12.5%) and 5 SD (62.5%) in the first 8 pts. For the whole cohort of 20 patients, the ORR was 5% (95% CI: 0.1-24.9) and the CBR rate was 75% (95% CI: 51-91) with 1 PR and 14 pts with SD. The duration of response for the 1 PR patient was 12.4 months. The median duration of response for the 15 pts with SD was 7.5 (2.0-29.7) months. 3 pts (15%) had PD and 2 pts (10%) went off prior to response assessment. Median cycles of treatment was 4 (2-30). In the 17 pts who discontinued, treatment was stopped in 9 (53%) for PD, 4 (23.5%) due to adverse events (AE), and 4 (23.5%) due to physician/patient preference. With a median follow-up of 14.5 months (1.5-39.2), the 1-year PFS rate is 52.1% (32.6 – 83.2%) and the OS rate is 82.1% (95% CI: 65.6 – 100%). The overall grade 3+ AE rate regardless of attribution was 80% (16/20). The most common grade 3 AEs were lymphocyte count decrease (40%), hypertension (20%), fatigue (10%), and pneumonitis (10%). 2 pts had a grade 4 AE (lymphocyte count decrease and thrombotic thrombocytopenic purpura). There were no treatment-related deaths. Analysis of correlative studies for Cohort A1 with PSMA imaging and EV biomarker ongoing. Conclusions: PTX and PMB has modest activity in pts with R/M ACC with CBR of 75% and prolonged benefit for some patients. Clinical trial information: NCT04895735 .

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

Background The optimal management of biochemical recurrence (BCR) after radical prostatectomy (RP) remains controversial, particularly in the absence of clinical or radiographic evidence of disease. Salvage radiotherapy (SRT) remains standard of care, but it is unclear whether SRT benefits patients when modern imaging with a multiparametric magnetic resonance imaging (mpMRI) and prostate‐specific membrane antigen‐positron emission tomography/computed tomography (PSMA‐PET/CT) are negative for local recurrence. Retrospective data comparing SRT to observation for such patients is fraught with selection bias, underscoring the need for a prospective randomised controlled trial. Study Design and Methods The Biochemical Recurrence Irradiation or Observation (BRIO) is a randomised study under the DIVINE trial framework (NCT06378866) enrolling men with BCR (prostate‐specific antigen [PSA] level ≥0.2, up to 1.5 ng/mL) after RP with no radiographic evidence of recurrence on mpMRI and PSMA‐PET/CT. Major inclusion criteria include age ≥18 years, biochemically recurrent prostate cancer after RP with a PSA level between 0.2 and 1.5 ng/mL, and no local or metastatic recurrence on imaging. A total of 312 patients will be enrolled. Participants are randomised 1:1 to standard‐of‐care SRT followed by surveillance (Group A), or initial observation with subsequent image‐guided therapy at radiographic progression (Group B). Based on historical analysis, a total of 30 metastasis events provides 80% power to detect a significant improvement (hazard ratio 0.5 corresponding to a 5‐year metastasis‐free proportion of 80.7% in the control group and 89.8% in the experimental group with one‐sided type I error rate of 0.15). All participants undergo baseline blood collection to measure prostate cancer‐extracellular vesicles and cell‐free DNA for biomarker development. Participants are followed in a 16‐week cycle with serial PSA measurements, repeat imaging if PSA doubles, safety assessment, and patient‐reported outcomes. Endpoints The primary endpoint is distant recurrence‐free survival at 5‐years using a 12‐month landmark to mitigate bias arising from differing assessment schedules for patients receiving optional 6 months of androgen deprivation therapy. Key secondary outcomes include time to hormone therapy, quality of life measures, adverse effects, and novel blood and urine biomarkers. Conclusion The BRIO study will clarify the clinical benefit of immediate SRT vs observation with image‐guided intervention in mpMRI and PET‐negative BCR after RP.