Characterization of the endotheliopathy, innate-immune activation and hemostatic imbalance underlying CAR-T cell toxicities: laboratory tools for an early and differential diagnosis

Abstract

Background: Chimeric antigen receptor (CAR)-T cell-based immunotherapy constitutes a revolutionary advance for treatment of relapsed/refractory hematological malignancies. Nevertheless, cytokine release and immune effector cell-associated neurotoxicity syndromes are life-threatening toxicities in which the endothelium could be a pathophysiological substrate. Furthermore, differential diagnosis from sepsis, highly incident in these patients, is challenging. Suitable laboratory tools could be determinant for their appropriate management.

Methods: Sixty-two patients treated with CAR-T cell immunotherapy for hematological malignancies (n=46 with CD19-positive diseases, n=16 with multiple myeloma) were included. Plasma samples were obtained: before CAR-T cell infusion (baseline); after 24-48 hours; at suspicion of any toxicity onset and 24-48 hours after immunomodulatory treatment. Biomarkers of endothelial dysfunction (soluble vascular cell adhesion molecule 1 (sVCAM-1), soluble TNF receptor 1 (sTNFRI), thrombomodulin (TM), soluble suppression of tumorigenesis-2 factor (ST2), angiopoietin-2 (Ang-2)), innate immunity activation (neutrophil extracellular traps (NETs), soluble C5b-9 (sC5b-9)) and hemostasis/fibrinolysis (von Willebrand Factor antigen (VWF:Ag), ADAMTS-13 (A13), α2-antiplasmin (α2-AP), plasminogen activator inhibitor-1 antigen (PAI-1 Ag)) were measured and compared with those in cohorts of patients with sepsis and healthy donors.

Results: Patients who developed CAR-T cell toxicities presented increased levels of sVCAM-1, sTNFRI and ST2 at the clinical onset versus postinfusion values. Twenty-four hours after infusion, ST2 levels were good predictors of any CAR-T cell toxicity, and combination of ST2, Ang-2 and NETs differentiated patients requiring intensive care unit admission from those with milder clinical presentations. Association of Ang-2, NETs, sC5b-9, VWF:Ag and PAI-1 Ag showed excellent discrimination between severe CAR-T cell toxicities and sepsis.

Conclusions: This study provides relevant contributions to the current knowledge of the CAR-T cell toxicities pathophysiology. Markers of endotheliopathy, innate immunity activation and hemostatic imbalance appear as potential laboratory tools for their prediction, severity and differential diagnosis.

Keywords: antineoplastic protocols; cytotoxicity, immunologic; hematologic neoplasms; immunity, cellular; immunotherapy.

Figure 1

Prediction of toxicity. Predictive model of soluble suppression of tumorigenesis-2 factor (ST2) at sample collected 24–48 hours after infusion (24-INF) point for the outcome ‘presence of any chimeric antigen receptor (CAR)-T cell-related toxicity’ meant as clinical detection of cytokine release syndrome (CRS) and/or immune effector cell-associated neurotoxicity (ICANS) of any severity grade. Area under the curve (AUC) 0.7 (95% CI 0.54 to 0.81, p=0.020).

Figure 2

Prediction of severity. Receiver operating characteristic (ROC) curve after the application of a regression model for the predictive value of the composed variable created from soluble suppression of tumorigenesis-2 factor (ST2), angiopoietin-2 (Ang-2) and neutrophil extracellular traps (NETs) levels at sample collected 24–48 hours after infusion (24-INF) point for the outcome ‘intensive care unit (ICU) admission’. Area under the curve (AUC) 0.8 (95% CI 0.67 to 0.93, p<0.001). The values of the AUC and 95% CI, and the proposed cutoffs for each biomarker are shown below (graph not shown): Ang-2: AUC 0.7 (95% CI 0.501 to 0.835, p=0.043). Levels of Ang-2 >1877 pg/mL have a sensitivity of 70% and a specificity of 57% for the prediction of ‘ICU admission’. ST2: AUC 0.8 (95% CI 0.626 to 0.93, p=0.001). Levels of ST2 >38.7 ng/mL have a sensitivity of 82% and a specificity of 70% for the prediction of ‘ICU admission’. NETs: AUC 0.7 (95% CI 0.559 to 0876, p=0.009). Levels of NETs >7.5 µg/mL have a sensitivity of 70% and a specificity of 88% for the prediction of ‘ICU admission’.

Figure 3

Differential diagnosis: toxicity versus sepsis. (A) Receiver operating characteristic (ROC) curve for the diagnostic value of angiopoietin-2 (Ang-2), neutrophil extracellular traps (NETs), soluble C5b9 (sC5b-9), von Willebrand Factor antigen (VWF:Ag) and plasminogen activator inhibitor-1 (PAI-1) at Tox-onset point, for the outcome ‘sepsis’. The values of the area under the curve (AUC) and 95% CI, and the proposed cutoffs for each variable are shown below: Ang-2: AUC 0.861 (95% CI 0.744 to 0.977). Levels of Ang-2 >4823 pg/mL have a sensitivity of 80% and a specificity of 74% for the diagnosis of sepsis over chimeric antigen receptor (CAR)-T toxicity. NETs: AUC 0.887 (95% CI 0.76 to 1); p<0.001. Levels of NETs >16.5 µg/mL have a sensitivity of 80% and specificity of 84% for the diagnosis of sepsis over CAR-T toxicity. sC5b-9: AUC 0.795 (95% CI 0.64 to 0.943); p=0.002. Levels of sC5b-9 >1109 ng/mL have a sensitivity of 75% and specificity of 73% for the diagnosis of sepsis over CAR-T toxicity. VWF:Ag: AUC 0.868 (95% CI 0.757 to 0.898); p<0.001. Levels of VWF:Ag >345% have a sensitivity of 75% and specificity of 84% for the diagnosis of sepsis over CAR-T toxicity. PAI-1 Ag: AUC 0.853 (95% CI 0.73 to 0.975); p<0.001. Cut-off value of PAI-1 Ag >73.6 ng/mL have a sensitivity 70% and specificity of 90% for the diagnosis of sepsis over CAR-T toxicity. (B) Predictive model for the outcome ‘sepsis’ with the composed variable created from Ang-2, NETs, sC5b-9, VWF:Ag and PAI-1 Ag at the onset of the toxicity in CAR-T cell-patients (Tox-onset point) or at the onset of sepsis. AUC 0.992 (95% CI 0.934 to 1); p<0.001.