BACKGROUND: Predicting immune effector cell–associated neurotoxicity syndrome (ICANS) in patients infused with CAR T cells is still a conundrum. This complication, thought to be consequent to CAR T cell activation, arises a few days after infusion, when circulating CAR T cells are scarce and specific CAR T cell–derived biomarkers are lacking. METHODS: CAR ⁺ extracellular vesicle (CAR ⁺ EV) release was assessed in human CD19.CAR T cells cocultured with CD19 ⁺ target cells. A prospective cohort of 100 patients with B cell lymphoma infused with approved CD19.CAR T cell products was assessed for plasma CAR ⁺ EVs as biomarkers of in vivo CD19.CAR T cell activation. Human induced pluripotent stem cell–derived (iPSC-derived) neural cells were used as a model for CAR ⁺ EV-induced neurotoxicity. RESULTS: In vitro release of CAR ⁺ EVs occurs within 1 hour after target engagement. Plasma CAR ⁺ EVs are detectable 1 hour after infusion. A concentration greater than 132.8 CAR ⁺ EVs/μL at hour +1 or greater than 224.5 CAR ⁺ EVs/μL at day +1 predicted ICANS in advance of 4 days, with a sensitivity and a specificity outperforming other ICANS predictors. ENO2 ⁺ nanoparticles were released by iPSC-derived neural cells upon CAR ⁺ EV exposure and were increased in plasma of patients with ICANS. CONCLUSION: Plasma CAR ⁺ EVs are an immediate signal of CD19.CAR T cell activation, are suitable predictors of neurotoxicity, and may be involved in ICANS pathogenesis. TRIAL REGISTRATION: NCT04892433, NCT05807789. FUNDING: Life Science Hub–Advanced Therapies (financed by Health Ministry as part of the National Plan for Complementary Investments to the National Recovery and Resilience Plan [NRRP]: E.3 Innovative health ecosystem for APC fees and immunomonitoring).

Background: Cutaneous melanoma (CM) is one of the most lethal tumors among skin cancers and its incidence is rising worldwide. Recent data support the role of microRNAs (miRNAs) in melanoma carcinogenesis and their potential use as disease biomarkers. Methods: We quantified the expression of miR-146a-5p and miR-21-5p in 170 formalin-fixed paraffin embedded (FFPE) samples of CM, namely 116 superficial spreading melanoma (SSM), 26 nodular melanoma (NM), and 28 lentigo maligna melanoma (LMM). We correlated miRNA expression with specific histopathologic features including Breslow thickness (BT), histological subtype, ulceration and regression status, and mitotic index. Results: miR-146a-5p and miR-21-5p were significantly higher in NM compared to SSM and LMM. The positive correlation between miR-146a-5p and miR-21-5p expression and BT was confirmed for both miRNAs in SSM. Considering the ulceration status, we assessed that individual miR-21-5p expression was significantly higher in ulcerated CMs. The increased combined expression of the two miRNAs was strongly associated with ulceration (p = 0.0093) and higher mitotic rate (≥1/mm²) (p = 0.0005). We demonstrated that the combination of two-miRNA expression and prognostic features (BT and ulceration) can better differentiate cutaneous melanoma prognostic groups, considering overall survival and time-to-relapse clinical outcomes. Specifically, miRNA expression can further stratify prognostic groups among patients with BT ≥ 0.8 mm but without ulceration. Our findings provide further insights into the characterization of CM with specific prognostic features. The graphical abstract was created with BioRender.com.