Endothelial Activation and Blood-Brain Barrier Disruption in Neurotoxicity after Adoptive Immunotherapy with CD19 CAR-T Cells

Abstract

Lymphodepletion chemotherapy followed by infusion of CD19-targeted chimeric antigen receptor-modified T (CAR-T) cells can be complicated by neurologic adverse events (AE) in patients with refractory B-cell malignancies. In 133 adults treated with CD19 CAR-T cells, we found that acute lymphoblastic leukemia, high CD19+ cells in bone marrow, high CAR-T cell dose, cytokine release syndrome, and preexisting neurologic comorbidities were associated with increased risk of neurologic AEs. Patients with severe neurotoxicity demonstrated evidence of endothelial activation, including disseminated intravascular coagulation, capillary leak, and increased blood-brain barrier (BBB) permeability. The permeable BBB failed to protect the cerebrospinal fluid from high concentrations of systemic cytokines, including IFNγ, which induced brain vascular pericyte stress and their secretion of endothelium-activating cytokines. Endothelial activation and multifocal vascular disruption were found in the brain of a patient with fatal neurotoxicity. Biomarkers of endothelial activation were higher before treatment in patients who subsequently developed grade ≥4 neurotoxicity.Significance: We provide a detailed clinical, radiologic, and pathologic characterization of neurotoxicity after CD19 CAR-T cells, and identify risk factors for neurotoxicity. We show endothelial dysfunction and increased BBB permeability in neurotoxicity and find that patients with evidence of endothelial activation before lymphodepletion may be at increased risk of neurotoxicity. Cancer Discov; 7(12); 1404-19. ©2017 AACR.See related commentary by Mackall and Miklos, p. 1371This article is highlighted in the In This Issue feature, p. 1355.

Conflict of interest statement

Conflict of interest disclosure:

CJT, DGM and SRR receive research funding from Juno Therapeutics and hold patents and/or provisional patent applications. SRR is a co-founder of Juno Therapeutics, and holds patents. DL is an employee of and has equity in Juno Therapeutics. CY received research funding from Gilead for an unrelated project. FHCRC receives research funding from Juno Therapeutics.

©2017 American Association for Cancer Research.

Figures

Figure 1. Frequency, kinetics, and treatment of neurotoxicity

A: The numbers of patients with each overall neurotoxicity grade are shown for the entire cohort and each disease. The diameters of each pie chart indicate the relative size of each subgroup. B: The swimmer plot (bottom) shows the kinetics of the severity of neurotoxicity in each patient who developed neurotoxicity through 28 days after CAR-T cell infusion (n=53). Each row represents one patient and the colors indicate the highest grade of neurotoxicity recorded on each day. Six patients died within 28 days of CAR-T cell infusion (neurotoxicity, n=3; disease progression, n=1; CRS, n=2). The graph (top) shows the mean of the highest grade of neurotoxicity occurring in all patients on each day after CAR T cell infusion. C: Numbers of patients with each grade of neurotoxicity and CRS. D: Cumulative incidences of fever, any grade of neurotoxicity, and the peak grade of neurotoxicity are shown for patients with grade 1–2 and grade ≥3 neurotoxicity.

Figure 2. Brain magnetic resonance imaging (MRI) findings in patients with severe neurotoxicity after CD19 CAR-T cell immunotherapy

A–B: Symmetric edema of deep structures in a patient with grade 5 neurotoxicity. FLAIR hyperintensities were seen in the bilateral thalami (A, arrowheads) and the pons (B, arrowheads), consistent with vasogenic edema. Punctate hemorrhages in the most affected areas are seen as T2 dark lesions (B, arrow). C: Global edema with blurring of the gray-white junction (stars) and slit-like ventricles (arrowhead) on FLAIR imaging in a patient with grade 5 neurotoxicity. D: Diffuse leptomeningeal enhancement (arrowheads) in a patient with grade 5 neurotoxicity. E–F: White matter FLAIR hyperintensities (E, arrowheads) that in some cases were contrast enhancing (F, arrowheads; T1 + gadolinium) in a patient with grade 3 neurotoxicity without focal neurologic deficits on exam. G–I: Cytotoxic edema of the cortical ribbon is seen on diffusion weighted imaging (G, arrowheads) and concomitant cortical swelling on FLAIR (H, arrowheads). In the same patient, injury progressed to irreversible cortical laminar necrosis indicated by T1 hyperintensities within the cortical ribbon 10 days later (I, arrowheads).

Figure 3. Severe neurotoxicity is associated with vascular dysfunction

A: Absolute counts of CD4+/EGFRt+ and CD8+/EGFRt+ CAR-T cells in blood, and the percentages of CD4+/EGFRt+ cells within CD4+ T cells and of CD8+/EGFRt+ cells within CD8+ T cells in the indicated time windows after CAR-T cell infusion are shown in patients without neurotoxicity (grey) or with grade 1–2 (orange) or ≥3 (red) neurotoxicity. B: Minimum (min) or maximum (max) values of vital signs, serum protein and albumin concentration, and body weight are shown within the indicated time periods. Pre-chemo, before lymphodepletion chemotherapy. Pre-infusion, before CAR-T cell infusion. SBP, systolic blood pressure. DBP, diastolic blood pressure. HR, heart rate. RR, respiratory rate. C: Minimum (min) or maximum (max) values of coagulation parameters are shown within the indicated time periods. PT, prothrombin time. APTT, activated partial thromboplastin time. D: Maximum serum CRP, ferritin, IFN-γ, IL-6, and TNF-α concentrations within indicated time periods are shown, according to severity of neurotoxicity. Within each time window in all figures, the y-axis shows the mean +/− standard error of the mean (SEM) of the values for all patients. *0.001<p<0.005, **0.0001<p<0.001, ***p<0.0001 for the indicated time points for the comparison of grade 0 vs 1–2 vs 3–5 neurotoxicity. P values for TNF-α at 0–36 hours and 2–5 days after CAR-T cell infusion were 0.038 and 0.022, respectively.

Figure 4. Endothelial activation in neurotoxicity associated with CD19 CAR-T cell immunotherapy

A: Ang-1 (left) and Ang-2 (center) concentrations and the Ang-2:Ang-1 ratio (right) in serum collected approximately 7 days after CAR-T cell infusion from a subset of patients with grade 0–3 (n=52) or ≥4 (n=7) neurotoxicity. The median (bar) and interquartile range are shown. Each point represents data from one patient. B: VWF concentration in serum from patients with grade 0–3 (n=45) or grade ≥4 (n=7) neurotoxicity. Serum was collected approximately one week after CAR-T cell infusion. Data represent the fold change from the VWF concentration in normal reference plasma (CRYOcheck, Precision Biologic, Dartmouth, NS, Canada; VWF 12.2 μg/mL). C: Ang-2:Ang-1 ratios in serum collected before lymphodepletion chemotherapy from patients who subsequently developed grade 0–3 (n=49) or ≥4 (n=6) neurotoxicity. The median and interquartile range are shown. D: VWF string unit formation in HUVECs incubated with serum collected from day 3–5 from patients who received CD19 CAR-T cells and developed grade ≥4 neurotoxicity (n=4) or from healthy donors (n=4). E: VWF string unit formation in HUVECs incubated with serum collected from patients with grade ≥4 (n=3) or grade 0–3 (n=6) neurotoxicity between day 7 and 14 after CAR-T cell infusion. The mean value of 2 samples collected on days 7 and 10 was used for one patient without neurotoxicity. F: HMW and LMW VWF multimers in serum from patients with grade ≥4 (n=5) compared to grade 0–3 (n=6) neurotoxicity.

Figure 5. Increased permeability of the BBB during neurotoxicity

CSF was collected from patients before CAR-T cell infusion (Pre), during acute neurotoxicity (Acute), and after recovery from acute neurotoxicity or ≥21 days after CAR-T cell infusion in those without neurotoxicity (Recovery). A: Protein concentration and WBC counts in CSF in patients who did (red) or did not (grey) develop neurotoxicity. Each point represents data from a single patient. Box and whisker plots show the interquartile range. B: Paired CSF and blood samples collected on the same day from individual patients with neurotoxicity, showing CD4+ and CD8+ CAR-T cells as a percentage of total CD4+ and CD8+ cells, respectively. Each line represents data from a single patient. NS, not significant. C: CD4+ and CD8+ CAR-T cells as percentages of total CD4+ and CD8+ cells, respectively, in CSF. Each point represents data from a single patient. Box and whisker plots show the interquartile range. D: Concentrations of cytokines in paired serum and CSF samples obtained from patients who developed neurotoxicity. Box and whisker plots show the median (bar) and interquartile range (box). Each point represents data from one patient. *p<0.05, **p<0.01, ***p<0.001. Paired tests were used to compare serum and CSF cytokines at a single timepoint. Unpaired tests were used for comparisons between Pre and Acute timepoints. E: IL-6 and VEGF concentrations in supernatant from pericytes cultured with medium alone, IFN-γ or TNF-α. Data are representative of 6 experiments and are expressed as the fold change (mean +/− SEM) compared to culture in medium alone. F: PDGFRβ and activated caspase-3 expression by human brain vascular pericytes incubated with IFN-γ. Data are expressed as the fold change (mean +/− SEM) compared to culture in medium alone.

Figure 6. Endothelial activation and vascular disruption in CAR-T cell neurotoxicity

A: Hematoxylin and eosin staining of medulla showing red blood cell extravasation into the surrounding parenchyma and Virchow-Robin space in the setting of minimal arteriolar wall disruption. B: Hematoxylin and eosin staining showing fibrinoid vessel wall necrosis and vascular occlusion. C: Perivascular CD8+ T cell infiltration. D: IHC for VWF showing VWF binding to capillaries. E: IHC for CD61 demonstrates intravascular microthrombi. F: IHC for CD31 showing reduplicated and disrupted endothelium. Size bars (100μm) are shown.