Lucien, Fabrice

e14551 Background: Extracellular vesicles (EVs) are microscopic particles released by all cell types with a distinct fingerprint reflecting the cell of origin. In patients with prostate cancer, PSMA+EVs have been associated with clinical outcomes. In this study, we investigated tumor-derived EVs (tdEVs) using the tumor-specific CD326 marker as a novel biomarker for pancreas ductal adenocarcinoma (PDAC). Methods: The study included patients with localized PDAC treated on a prospective phase 2 clinical trial evaluating a 15-fraction chemoradiation (CRT) regimen. Blood samples were obtained after induction chemotherapy (C) (prior to CRT) and in follow-up. 20 healthy controls were included. High-resolution flow cytometry enumerated levels of fluorescent antibody labelled tumor-specific CD326 (EpCAM)-positive EVs. All samples were de-identified and randomly coded. The technologist responsible for EV analysis was blinded to patient information and clinical data. Each sample was run in triplicate. Flow cytometry data was analyzed with custom-built software to automatically determine and apply gates and generate reports with scatter plots and EV concentrations for each combination of markers and data summaries. Results: tdEV data were analyzed from 70 patients with pre-CRT samples. Patients had mean age 66 years (9.0 standard deviation), with 60% ECOG 0, 43% borderline resectable and 48% locally advanced. Patients had a median 8 cycles of C prior to CRT, and 70% proceeded to surgery after CRT. 2-year overall survival (OS) was 54%. Patient samples showed elevated total CD326 EVs compared to the healthy controls (p = 0.0005). Furthermore, patient CD326 EVs revealed two distinct subpopulations: small (120-1,000nm) and large (1,000-3,000nm) size CD326 EVs. No large CD326 subpopulation was found in healthy donor samples. Higher pre-CRT levels of total CD326 and large CD326 EVs were associated with decreased progression-free survival (PFS) (hazard ratio (HR) = 2.38, p = 0.013 and HR = 2.017, p = 0.021) on multivariable models with surgical resection. Resection was associated with increased PFS (p < 0.0001), and the interaction terms did not meet significance (p = 0.083 and p = 0.21). No differences in pre-CRT total or large CD326 EVs were identified between patients with or without surgical resection (p = 0.72 and p = 0.83). CA19-9 levels were not predictive of clinical outcomes in this trial and showed no association with EV levels. Conclusions: We present an association between clinical outcomes and tdEVs in PDAC, highlighting their potential as a novel, independent biomarker. If validated, tdEVs may offer an opportunity to personalize care and improve risk stratification.

Abstract Objectives We aimed to assess the expression of biomarkers of response to antibody-drug conjugates (TROP2 and nectin-4) and immune microenvironment (NKG7, PD-L1, and B7-H3) in penile squamous cell carcinoma (pSCC). Methods Our archive was queried for patients who had a penectomy for pSCC between 2000 and 2022. Primary tumors were immunostained for B7-H3 and NKG7, while metastatic specimens were immunostained for TROP2 and nectin-4. Expression of PD-L1, TROP2, and nectin-4 in primary tumors was previously characterized. H-scores (0-300) were used to quantify expression. Associations between biomarkers, tumor-infiltrating lymphocytes (TILs), and clinicopathologic and outcome parameters were evaluated. Results For both TROP2 and nectin-4, H-scores within the lymph node metastases were higher compared to those within the primary tumors (mean, 264.5 vs 244.8, P = .0003; mean, 170.6 vs 146.7, P = .05, respectively; 33 paired specimens). For B7-H3 (n = 107), 32.7% of the primary tumors had an H-score of more than 0. In 34.8% of the cases, NKG7 expression was observed in 25% to 50% of the TILs. A significant association was noted between TIL density, B7-H3, NKG7, and PD-L1 expression. Conclusions Therapeutic strategies targeting TROP2 and nectin-4 hold promise for patients with advanced pSCC. The potential of PD-L1, B7-H3, and NKG7 for predicting response to immunomodulatory treatment warrants further research.

Abstract Addressing the limited responsiveness to immune checkpoint blockade (ICB) in human cancers remains a critical challenge. Efforts over the past two decades have primarily focused on enhancing CD8 T cell function and preventing their exhaustion. However, less attention has been given to understanding the molecular mechanisms that contribute to the immunosuppressive tumor microenvironment (TME), which impedes CD8 T cell infiltration and fosters ICB resistance. The TME in ICB-resistant tumors is characterized by Transforming Growth Factor beta (TGF-β) cytokine signaling, leading to a fibrotic extracellular matrix (ECM) and the accumulation of immunosuppressive fibroblasts and myeloid cells. These features hinder CD8 T cell penetration, thereby promoting immune escape. The immune checkpoint molecule B7-H3, overexpressed in ICB-resistant cancers, has been linked to impaired CD8 T cell infiltration and poor prognosis. However, the molecular mechanisms governing B7-H3 function remain poorly understood. We investigated the relationship between B7-H3 gene expression and ICB response using transcriptomic data from two randomized phase 3 trials (IMvigor010 and IMvigor210). Bulk RNAseq data, tissue staining, and spectral flow cytometry were used to characterize the TME composition of B7-H3 high and low tumors. Multi-omic profiling of tumor cell lines with manipulated B7-H3 expression was performed to identify downstream targets of B7-H3. Syngeneic mouse tumor models were used to assess the impact of B7-H3 on tumor growth and antitumor immunity. High B7-H3 expression was associated with shorter overall survival in both IMvigor010 (HR=0.48, 95% CI: 0.33-0.70, p&lt;0.0001) and IMvigor210 trials (HR=0.68, 95% CI: 0.51-0.92, p=0.01). In ICB-refractory cancers, B7-H3 expression correlated with high levels of fibroblasts, collagen deposits, and M2 macrophages. Gene set enrichment analysis revealed a positive association between B7-H3 and TGF-β signaling. Genetic deletion of B7-H3 in tumor cells reduced the surface expression of MMP14, a metalloprotease that activates TGF-β. Pan-cancer analysis identified MMP14 as the gene most correlated with B7-H3 (Pearson r=0.69, p&lt;0.0001). TGF-β activation was reduced by 70% in B7-H3 knockout cells compared to wild-type. Loss of B7-H3 led to MMP14 accumulation in lysosomes, resulting in its degradation. We generated a B7-H3 inhibitor, 5D10, which promotes MMP14 degradation and inhibits TGF-β activation. In humanized B7-H3 mouse models, 5D10 significantly reduced TGF-β activation, enhancing CD8 T cell infiltration and impairing tumor growth. Our research identifies B7-H3 as a driver of immune exclusion and ICB resistance. We describe a novel, receptor-independent mechanism where B7-H3 promotes TGF-β activation via stabilization of MMP14. Targeting B7-H3 represents a strategy to modulate the TME, transforming immune-excluded tumors into immune-inflamed states that may respond better to ICB. Citation Format: Fabrice Lucien, Roxane Lavoie, Jack Korleski, Yohan Kim, Bharath Wootla, Liguo Wang, Ava Farrell, Kelly Harper, Martine Charbonneau, Claire Dubois, Igor Frank, Sounak Gupta, John Cheville, Jacob Orme, Stephen Boorjian, Parash Shah, Eugene Kwon, Sean Park, Haidong Dong. The immune checkpoint molecule B7-H3 is a driver of tumor immune exclusion by mediating TGF-β activation [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B034.

ABSTRACTBackgroundCommercial biomarkers and multiparametric MRI (mpMRI) have been utilized to triage men with elevated prostate specific antigen (PSA) and determine patients most likely to harbor clinically‐significant prostate cancer (csPCa). We studied combinations of mpMRI, PSA‐based and novel extracellular vesicle (EV)‐based biomarkers to determine the optimal pre‐biopsy testing to predict csPCa at biopsy.MethodsMen presenting with elevated PSA (≥ 2 ng/mL) were prospectively enrolled and all men underwent clinically indicated mpMRI and blinded study blood draws to determine PSA, prostate health index (PHI) scoring, and EV serum levels. MRI‐fusion transperineal prostate biopsy was performed in all patients. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated, and receiver operator characteristic (ROC) curves were constructed. Bootstrapping analysis was performed to provide more accurate assessment of predictive abilities.ResultsUltimately, 175 consecutive men were prospectively enrolled. Median age in the study population was 65 years. Combinations of biomarkers and MRI demonstrated better predictive ability for csPCa on biopsy than individual modalities. Predictive ability was greatest for PHI density (PHID)‐mpMRI with an AUC of 0.86 (95% CI: 0.80–0.92) while combinations of PSA density ‐mpMRI (AUC: 0.81; 95% CI: 0.73–0.89) and STEAP1‐mpMRI (AUC: 0.77; 95% CI: 0.70–0.86) demonstrated similar ability to predict csPCa. The study is limited by small, predominantly white patient cohort and requires external validation.ConclusionsInclusion of prostate density with biomarkers increases prognostic ability for detecting csPCa. EV density can refine prediction of csPCa and in combination with PHID‐mpMRI leads to superior specificity, thereby decreasing unnecessary biopsies.

Abstract Purpose: Two randomized clinical trials (STOMP and ORIOLE) demonstrated that stereotactic ablative radiotherapy (SABR) can prolong androgen-deprivation therapy–free survival or progression-free survival (PFS) in patients with metachronous oligometastatic castration-sensitive prostate cancer (omCSPC). Although most patients with omCSPC have a more modest delay in progression, a small subset achieves a durable response following SABR. We investigated the prognostic and predictive value of circulating prostate-specific membrane antigen-positive (PSMA+) extracellular vesicles (EV) and PSA in a biomarker correlative study using blood samples from three independent patient cohorts. Experimental Design: Plasma samples from 46 patients with omCSPC on the ORIOLE trial and 127 patients with omCSPC on the STOMP trial protocol treated with SABR were included in the study. Pre-SABR PSMA+EV levels (EV/mL) were measured by nanoscale flow cytometry. Kaplan–Meier curves and logistic regression models were used to determine the association of PSMA+EV and PSA levels with clinical outcomes. Results: In the pooled cohorts, the median biochemical PFS were 26.1 and 15.0 months (P = 0.005), and the median radiographic PFS were 36.0 and 25.0 months (P = 0.003) for PSMA+EV-low and -high groups, respectively. The combination of pre-SABR low levels of both PSMA+EV and PSA was associated with a lower risk of radiographic progression (HR, 0.34, 95% confidence interval, 0.18–0.58; P = 0.0002). In the ORIOLE cohort, which included both an SABR arm and an observation arm, low PSMA+EV was predictive of benefit from SABR (P = 0.012). Conclusions: PSMA+EV is a novel prognostic and predictive biomarker of radiographically occult tumor burden in omCSPC. PSMA+EV may inform clinical decisions about identifying patients who will achieve a durable benefit from consolidative SABR alone.

Abstract Background. Extracellular vesicles (EV) are emerging as new biomarkers for cancer diagnostics and therapeutic monitoring1. For example, in patients that receive PD1/PD-L1 immune checkpoint inhibitors (a key pillar of cancer immunotherapy), circulating EV-derived PD-L1 is gaining attention as a non-invasive biomarker for therapeutic efficacy. Standard methodologies to measure EV-derived PD-L1 requires ultracentrifugation to first separate EVs and then quantify those that express PD-L1 using nano flow cytometry. These are tedious processes that require trained technologists in centralized facilities, which is a key barrier to frequently monitoring EV-based biomarkers. Yet, frequent monitoring is crucial to quickly identify and stop an ineffective therapy while more fruitful options are still available. Method. To bridge the gap, we develop a proof-of-concept digital microfluidic (DMF) device that can separate EVs directly from biofluids and quantify PD-L1+ EVs automatedly. Briefly, DMF device supports automatedly droplet handling, including droplet transport, splitting and mixing, based on electrowetting principles2. Immunomagnetic beads were used to capture EVs from biofluids, and EVs were then detected by electrochemical sensors inserted into the device. These tips of the electrochemical sensors were modified with gold nanoparticle self-assembled layers to enhance target binding, and CD9 antibodies were immobilized on the tips to bind to EVs. We then used anti-PD-L1 as secondary antibodies for target detection. Differential Pulse Voltammetry (DPV)3 were performed to obtain the results. Results. We used the DMF device to extract 109- 1011 #/mL EVs secreted by a human breast cancer cell line (MDA-MB-231). Briefly, 10 mL of cell culture media was loaded into the DMF device, and the sample was mixed and incubated with a 10 mL droplet of immunomagnetic beads to capture EVs. After 5 min, EVs were eluted in 20 mL of elution buffer, and the beads were discarded. We estimated the number of EVs using Nanoparticle Tracking Analysis; the number of EVs extracted on-chip was comparable with those that were extracted using standard in-tube method. We also verified the purity of the EVs extracted on-chip using Western Blot. Our results showed that the electrochemical sensors can detect as low as 104 #/mL PD-L1+ EVs, with a linear range of 104 – 107 #/mL (R2=0.9701). The entire process can be completed within 1 hour. Conclusion. We integrated automated EV extraction and their surface marker detection in a single DMF device. Our next step is to extract and detect EV-based biomarkers from plasma samples. This platform holds promise for the regular monitoring of EV-based biomarkers that are indicators of therapeutic responses in patients that receive cancer immunotherapies. References1. F. Lucien, D. Gustafson, M. Lenassi et al, Journal of extracellular vesicles 12 (12), 12385 (2023). 2. Y. Zhang and Y. Liu, Sensors & Diagnostics (2022). 3. Y. Zhang, J. Liu, T.-W. Lo et al, Biosensors and Bioelectronics: X, 100490 (2024). Citation Format: Ting-Wen Lo, Yuqian Zhang, Jing Liu, Yohan Kim, Haidong Dong, Fabrice Lucien, Yuguang Liu. Rapid extraction and detection of extracellular vesicle-derived PD-L1 in a microfluidic platform [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A030.

Abstract Introduction: Immune checkpoint inhibitors (ICI) are an essential systemic therapy for advanced melanoma. However, most melanomas develop resistance to ICI. Tumor-derived soluble PD-L1 (sPD-L1) and other soluble immunosuppressive factors drive checkpoint inhibitor resistance and correlate with inferior survival. We previously showed that therapeutic plasma exchange (TPE) removes sPD-L1 from circulation. Thus, we hypothesized that TPE-mediated removal of sPD-L1 and other immunosuppressive factors could overcome immunotherapy resistance in refractory melanoma. Methods: In this clinical trial (NCT04581382), we prospectively enrolled eighteen (18) patients with widely metastatic melanoma with progression despite anti-PD-1 ICI and elevated sPD-L1 by ELISA (≥1.7ng/mL). Each patient received radiotherapy to between one and three metastatic lesions (at least two unirradiated lesions) followed by three TPE sessions on consecutive days and re-challenge with checkpoint inhibitor. The primary safety and efficacy endpoints of the study were adverse events (AEs) and sPD-L1 reduction by TPE, respectively. Secondary endpoints included RECIST-based response in unirradiated lesions and overall survival. Correlative studies included kinetics of sPD-L1 and soluble immonsuppressive factors and dynamics of peripheral immune cell phenotypes. Results: Mean age was 62 (SD 13) and seven of eighteen (39%) were female. Mean baseline sPD-L1 was 26.31 ng/mL (40.01). The treatment was well-tolerated with one (6%) patient with grade 3 and 4 adverse events from a central line infection after TPE. Levels of sPD-L1 were significantly reduced by TPE (mean 80.2% reduction, p<0.0001). Two patients (11.1%) experienced complete response (CR), one (5.6%) partial response, three (16.7%) stable disease, and 12 (66.7%) progressive disease. In one case, immunotherapy was discontinued after two years due to no detectable lesions. Changes in tumor-reactive (TTR) GZMB+/CX3CR1+/CD11ahigh and other peripheral immune cell populations predicted overall survival in this cohort. In addition to sPD-L1, other soluble mediators of ICI resistance were also reduced by TPE and predicted overall survival in this cohort. Summary/Conclusion: sPD-L1 and other soluble immunoregulatory signaling molecules are important mediators of ICI resistance. SBRT and TPE can resensitize ICI-refractory melanoma by removing these factors. Patients with persistently elevated or rapid rebound of sPD-L1 following TPE experienced poor response and overall survival. Serial monitoring of sPD-L1 may predict response to ICI and multiple courses of TPE may be necessary. Our findings may apply in other ICI-resistant cancers with elevated sPD-L1. ClinicalTrials.gov registration: NCT04581382, ReCIPE-M1 (Rescuing Cancer Immunotherapy with Plasma Exchange in Melanoma 1).

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AbstractBackgroundNeoadjuvant platinum‐based chemotherapy offers a modest survival advantage in muscle‐invasive bladder cancer (MIBC) for patients with pathologic response. B7‐H3 (CD276), an immune checkpoint overexpressed in various cancers, including urothelial‐cell carcinoma (UCC), has been associated with chemoresistance and poor oncologic outcomes. We aimed to explore if B7H3 expression on bladder biopsy samples was a predictive biomarker for pathologic response to neoadjuvant platinum‐based chemotherapy.MethodsThis was a retrospective cohort study among MIBC patients receiving neoadjuvant platinum‐based chemotherapy followed by radical cystectomy. All patients underwent routine preoperative biopsy of their tumour. Immunohistochemistry was used to evaluate B7‐H3 expression from pre‐operative specimens. The primary outcome of interest was pathologic complete response (pCR). Statistical analysis included Mann–Whitney U test, Fisher's exact test, Kaplan–Meier method, and Cox regression for survival analysis.ResultsAmong 87 patients analysed, high B7‐H3 expression was found in 44.8% (n = 39) of patients. The median follow‐up periods were similar between the high and low B7‐H3 groups (high expression; 4.29 years [SD 3.04], low expression 3.94 years [SD 3.04], p = 0.60). Only 20.5% of patients with high B7‐H3 expression achieved pCR, compared to 41.7% in the low expression group (p = 0.04). Cox regression showed no significant differences in recurrence‐free or cancer‐specific survival between the high and low B7‐H3 expression groups (p > 0.05).ConclusionHigh B7‐H3 expression is associated with a reduced likelihood of achieving pCR in MIBC patients undergoing neoadjuvant chemotherapy. This suggests B7‐H3's potential as a predictive biomarker for chemotherapy response. Further research is needed to explore the role of B7‐H3 on platinum‐based chemotherapy response in urothelial cancer.