Distinguishing Early Infections from CRS with Routine and Exploratory Serum Proteomics and the HT10 Score Following CD19 CAR-T for Relapsed/Refractory B-NHL

Background: Early fever after CAR-T therapy can reflect both an infection or CRS. Distinguishing both etiologies in these commonly neutropenic patients represents an unresolved clinical challenge. Accordingly, the vast majority of patients receive empiric broad-spectrum antibiotics, which can promote antibiotic resistance and negatively influence the gut microbiome, which has recently been linked to inferior CAR-T outcomes (Smith et al, Nature Medicine 2022). Here, we aimed to characterize infection- vs. CRS-specific differences in routine and exploratory serum proteomics, and to develop a clinical tool that reliably discriminates between both.

Methods: Early fever events (day 0-30) were retrospectively characterized as CRS vs. infection in 62 patients treated with standard-of-care CD19 CAR-T for r/r B-NHL (23 Axi-cel, 30 Tisa-cel, 9 Brexu-cel). Severe infections were defined as requiring IV anti-infectives and/or hospitalization in the presence of microbiologic evidence or distinct clinical signs of infection; CRS was graded according to ASTCT criteria. Cumulative incidence curves were calculated as time-to-event-analysis for CRS 1°, ≥2° and severe infections (day of event = index event).

Serum inflammatory markers (CRP, interleukin-6 [IL-6], procalcitonin [PCT]) were measured daily from CAR infusion until discharge and on subsequent outpatient visits. Inflammatory markers were tested using binary logistic regression for severe infection vs. CRS^onlyindex events. Discriminatory thresholds were determined from receiver operating characteristic (ROC) curves by optimizing the Youden J statistic. Longitudinal plasma proteomics was performed in 52 patients across four time points (day 0, 4, 14, 28) using a 92-protein multiplex proximity extension assay (Olink Proteomics). We applied a linear mixed model (LMM) accounting for both infection vs. CRS status and time effects.

Results: The cumulative 30-day incidence of CRS 1°, CRS ≥2° and severe infections was 47%, 43%, and 25%, respectively. Five patients had neither CRS nor an infection, and were subsequently excluded from further analysis. Tocilizumab was applied in the majority of CRS patients (1°: 90%, ≥2°: 100%). We did not find a significant correlation between CRS grade and severe infections (G² = 0.13, p = n.s.). Median onset of CRS and infection was 3 and 8 days, respectively. Most events (54%) occurred in the setting of coincident severe neutropenia (ANC <500/µl).

While event-day CRP was comparable between infection and CRS (median 1.8 vs. 3.4 mg/dL, n.s.), we noted increased serum IL-6 (median 2243 vs. 64 pg/mL, p=0.03) and particularly high PCT levels (median 1.6 vs. 0.3 µg/L, p<0.0001) in the infection cohort. For PCT, an optimal discriminatory threshold of 1.5 µg/L was established (AUC_ROC 0.78). In previous work, we demonstrated that a CAR-HEMATOTOX (HT) score ≥2 identifies patients with a high infection risk (Rejeski et al, JITC 2022). Indeed, the infection cohort displayed higher baseline HT scores (median 5 vs. 2, p=0.002). By incorporating day-of-fever PCT levels with the HT score, the discriminatory capacity for severe infection events was improved. On ROC analysis, the 'HT10 score' (addition of 10 points to the patient-specific HT score in case event-day PCT ≥1.5 µg/L) exhibited superior discrimination for severe infections (AUC 0.92, p<0.0001, sens. 80%, spec. 91%, Fig. 1A).

Additionally, we identified protein-level differences between the infection (n=13) vs. CRS^only (n=39) cohorts from the Olink dataset. On day 0, we noted marked upregulation of the angiogenetic factor TNFRSF12A in the infection cohort (q=4.8E-05). Notably, we observed progressive endothelial dysfunction and immune dysregulation in the patients developing severe infections (day 28, Fig. 1B). This included an increased ANG2/1 ratio and dysregulated CD40/CD40L axis. Consistent with a response to microbial exposure, the innate immune regulators CXC3L1, CCL20, and CXCL13 were upregulated in the infection group.

Conclusions: The high discriminatory capacity of the HT10 score for infections highlights the advantage of dynamic risk-assessment and emphasizes the utility of procalcitonin in routine inflammatory panels. If validated prospectively, the score will enable more patient-individual decisions on antibiotic use. Finally, the identified Olink candidate markers may further refine the discriminatory capacity of the HT10 score.

Rejeski:Kite/Gilead: Other: Travel Support, Research Funding; Novartis: Honoraria. Blumenberg:Gilead: Consultancy, Honoraria, Research Funding; Celgene: Research Funding; Novartis: Honoraria, Research Funding; Janssen: Research Funding. Schmidt:Janssen: Other: Travel Support; Bayer Healthcare: Research Funding; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel Support; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel Support; Kite Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Support, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Support. Dreyling:Abbvie, Bayer, BMS/Celgene, Gilead/Kite, Janssen, Roche: Research Funding; Amgen, Astra Zeneca, Gilead/Kite, Janssen, Lilly, Novartis, Roche: Honoraria; Astra Zeneca, Beigene, BMS/Celgene, Gilead/Kite, Janssen, Lilly/Loxo, Novartis, Roche: Consultancy. Subklewe:Gilead: Consultancy, Research Funding, Speakers Bureau; Morphosys: Research Funding; Takeda: Other: Travel support; Janssen: Consultancy, Speakers Bureau; Roche: Consultancy, Research Funding; Miltenyi Biotech: Research Funding; Seagen: Research Funding; Pfizer: Consultancy; Seattle Genetics: Research Funding; Bristol-Myers Squibb: Research Funding; Novartis: Consultancy, Speakers Bureau; Celgene/BMS: Consultancy, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau. Buecklein:Amgen: Honoraria; Pfizer: Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Miltenyi Biotech: Research Funding; Gilead: Honoraria.