Pre-existing clonal hematopoiesis in CAR-T recipients is not associated with increased immunotoxicity and inflammatory serum signatures

Abstract Introduction: Clonal hematopoiesis of indeterminate potential (CHiP) represents a myeloid precursor lesion described by the presence of clonal hematopoiesis without signs of neoplasia and cytopenia. Previous studies have highlighted its high frequency in lymphoma and multiple myeloma (MM) patients treated with chimeric antigen receptor T-cell (CAR-T) therapy – with a reported CHiP prevalence of 33-56% before treatment initiation. CHiP has been extensively studied in the context of chemotherapy and hematopoietic cell transplantation (HCT). However, its immunologic impact and potential influence on CAR-T-associated toxicities, inflammation, and treatment outcomes remain poorly defined. Methods: This observational study aimed (i) to investigate the impact of pre-existing CHiP clones on CAR-T-associated toxicity and treatment outcomes, and (ii) to explore correlations between CHiP mutations and the inflammatory serum proteome that drives immune-related toxicities. Outcome data and toxicity were analyzed from 65 large B-cell lymphoma, 13 mantle-cell lymphoma, 19 MM, and 2 follicular lymphoma patients treated with CD19- or BCMA CAR-T constructs. Peripheral blood mononuclear cells (PBMCs) and serum samples were collected before and after CAR-T infusion. Targeted next-generation sequencing for CHiP mutations was performed on 99 PBMC samples. Proteomic profiling was conducted using the 92-plex proximity extension assay on the Olink® platform (Immuno-Oncology Panel); serum samples were collected from 80 patients on days 0, 4, 14, 28, 90, 180, 360 post-infusion, censoring at disease progression. Results: The median age of the patient cohort was 64 years (range: 19-85) and the median follow up was 23.3 months (mo). Patients had received a median of three prior treatment lines (IQR=2-4), including 36.4% who had undergone autologous HCT. Sequencing of the total cohort identified CHiP-related mutations in 37 patients (37.4%, VAF≥1%) prior to CAR-T cell infusion. The most frequently mutated gene was DNMT3A (n=18), followed by TP53 and ASXL1 (n=7), PPM1D (n=5), TET2 and SF3B1 (n=2), and NRAS and BRAF (n=1). Among CHiP patients, 28 had a single mutation, 8 had two mutations, and 1 patient had three mutations. The incidence of grade ≥3 CRS and ICANS was comparable between CHiP and non-CHiP patients (CRS: 14% vs 15%, p0.9; ICANS: 14% vs 13%, p0.9). Early and late grade ≥3 ICAHT occurred in 22% vs 23% (p0.9) and 24% vs 37% (p=0.27) of CHiP vs non-CHiP patients. Neutrophil recovery phenotypes were similar between groups, with an aplastic phenotype observed in 19% of CHiP and 18% of non-CHiP patients (p0.9). Progression-free survival (PFS) and overall survival (OS) did not differ significantly between the two groups (median PFS: 6.4 vs 5.0 mo, p=0.58; median OS: 18.1 vs 21.9 mo, p0.9). Longitudinal inflammatory profile of CHiP (n=30) and non-CHiP (n=50) patients was performed by Olink®. Importantly, the CHiP cohort was not characterized by a distinct immune dysregulation signature compared to the non-CHiP cohort. Accordingly, not a single serum protein differed significantly across the patient cohorts (corrected p0.05). On subgroup analysis, patients with a TP53 CHiP mutation (n=7) were characterized at baseline by lower platelets (Plt) and hemoglobin (Hb) counts compared to all other patients independent of CHiP status (median Plt: 124 vs 178.5 e9/L, p=0.03; median Hb: 8.6 vs 10.3 g/dL, p=0.01). A baseline inflammatory signature was detected in the TP53 CHiP patients characterized by higher levels of C-reactive protein (CRP) and ferritin compared to the non-TP53 CHiP group (median CRP: 2.3 vs 0.6 mg/L, p=0.04; median ferritin: 1450 vs 331 mg/L, p=0.001). Interestingly, the Olink® analysis confirmed an inflammatory profile showing upregulation of IL-6, IL-18, PGF, Gal-9, MIC-A/B and HO-1 between days 0 and 28. Notably, one patient in the TP53 CHiP group subsequently developed a therapy related myeloid neoplasm. Conclusions: In this exploratory analysis of 99 CAR-T recipients, CHiP status was not associated with toxicity, outcomes, or systemic inflammatory profiles. These findings challenge the clinical relevance of CHiP as a biomarker for toxicity in the CAR-T setting. Notably, TP53 CHiP mutations were linked to lower platelet and hemoglobin levels and a distinct inflammatory signature, warranting further investigation. Ongoing studies aim to characterize CHiP clonal dynamics and their potential link to secondary malignancies post-CAR-T.