Glial injury in neurotoxicity after pediatric CD19-directed chimeric antigen receptor T cell therapy

Abstract

Objective: To test whether systemic cytokine release is associated with central nervous system inflammatory responses and glial injury in immune effector cell-associated neurotoxicity syndrome (ICANS) after chimeric antigen receptor (CAR)-T cell therapy in children and young adults.

Methods: We performed a prospective cohort study of clinical manifestations as well as imaging, pathology, CSF, and blood biomarkers on 43 subjects ages 1 to 25 who received CD19-directed CAR/T cells for acute lymphoblastic leukemia (ALL).

Results: Neurotoxicity occurred in 19 of 43 (44%) subjects. Nine subjects (21%) had CTCAE grade 3 or 4 neurological symptoms, with no neurotoxicity-related deaths. Reversible delirium, headache, decreased level of consciousness, tremor, and seizures were most commonly observed. Cornell Assessment of Pediatric Delirium (CAPD) scores ≥9 had 94% sensitivity and 33% specificity for grade ≥3 neurotoxicity, and 91% sensitivity and 72% specificity for grade ≥2 neurotoxicity. Neurotoxicity correlated with severity of cytokine release syndrome, abnormal past brain magnetic resonance imaging (MRI), and higher peak CAR-T cell numbers in blood, but not cerebrospinal fluid (CSF). CSF levels of S100 calcium-binding protein B and glial fibrillary acidic protein increased during neurotoxicity, indicating astrocyte injury. There were concomitant increases in CSF white blood cells, protein, interferon-γ (IFNγ), interleukin (IL)-6, IL-10, and granzyme B (GzB), with concurrent elevation of serum IFNγ IL-10, GzB, granulocyte macrophage colony-stimulating factor, macrophage inflammatory protein 1 alpha, and tumor necrosis factor alpha, but not IL-6. We did not find direct evidence of endothelial activation.

Interpretation: Our data are most consistent with ICANS as a syndrome of systemic inflammation, which affects the brain through compromise of the neurovascular unit and astrocyte injury. ANN NEUROL 2019.

FIGURE 1:

Kinetics and treatment of CAR-T cell treatment-related neurotoxicity (ICANS). (A) Swimmer plot showing kinetics and treatment of neurotoxicity for each of the 19 subjects who had neurotoxicity. Each lane represents 1 subject, and the colors indicate the overall neurotoxicity CTCAE grade on a given day. The daily CTCAE grade is indicated by the color code on the right. Stars indicate days when subjects received steroids, wedges indicate tocilizumab, and X indicates anakinra administration. (B) Neurotoxicity risk peaks on day 8. The y-axis indicates the mean CTCAE neurotoxicity grade averaged across all subjects in the study on any given day. (C) Early tocilizumab treatment is not associated with a change in neurotoxicity risk relative to CRS severity. Each subject who received tocilizumab is represented by one dot. NT > CRS: grade 3/4 neurotoxicity (NT) and mild CRS; NT = CRS: grade 3/4 NT and severe CRS, or grade 1/2 NT and mild CRS; CRS > NT: grade 1/2 NT and severe CRS, or no NT and mild CRS; CR≫NT: no NT and severe CRS. (D) CAPD scores for 3 individual patients over the course of ICU admission after CAR-T cell infusion. The y-axis indicates the highest CAPD score for any individual day, and the color of the dots shows the CTCAE neurotoxicity grade for that day. CAPD scores on or above the dashed line at CAPD = 9 are consistent with delirium. (E) Correlation of CAPD scores and CTCAE grades. Each dot represents the overall CTCAE score and highest CAPD score on any given patient day for the entire cohort. The dashed line at CAPD = 9 shows the delirium cutoff. Long bars indicate the median, and short bars the interquartile range. CAPD = Cornell Assessment of Pediatric Delirium; CAR = chimeric antigen receptor; CRS = cytokine-release syndrome; CTCAE = Common Terminology Criteria for Adverse Events; ICU = intensive care unit.

FIGURE 2:

Brain MRI patterns and evidence of chronic injury on brain histopathology in CAR-T cell neurotoxicity. (A) Pattern 1: acute patchy supratentorial white matter hyperintensities (arrowheads) are seen on this fluid-attenuated inversion recovery (FLAIR) image. The white matter lesions were not present on baseline imaging for this patient. (B) Additional subcortical white matter FLAIR hyperintensities (arrowhead). (C) Pattern 3: FLAIR hyperintensities in the brain stem and (D) bilateral thalami (arrowheads), with additional white matter lesions (arrow). (E,F) Pattern 3: acute edema in area of past injury. (E) FLAIR image obtained before CAR-T cell treatment shows cerebellar microhemorrhages (arrow) related to past cerebellar leukemic infiltrate and radiation therapy. (F) Acute edema of the cerebellar white matter (arrowheads) occurred during CAR-T cell treatment, as seen on this FLAIR image, as well as possible new microhemorrhages (arrow). (G,H) Pattern 4: cortical injury. (G) Diffusion restriction of the occipital cortex, right > left (arrowheads), is seen on this diffusion weighted image. (H) Ten months later, the same subject showed right occipital hypometabolism (arrowhead) on FDG-PET. (I–L) Brain histopathology from autopsy 3 years after initial CAR-T cell infusion, same subject as (G) and (H). (I) Right occipital cortex shows prominent gliosis of Chaslin (a pattern classically associated with chronic seizures), seen as a darker band of staining in the subpial region (double headed arrow), glial fibrillary acidic protein (GFAP) stain at 20× magnification. (J) Higher-power view of reactive astrocytes, GFAP stain, 60×. (K) Hemosiderin-laden macrophages in a perivascular space in the basal ganglia, hematoxylin and eosin stain, 40×. (L) Basal ganglia white matter with multiple round, darkly staining corpora amylacea suggestive of neuronal degeneration. Periodic acid Schiff stain at 20×; inset at 40×. CAR = chimeric antigen receptor; FDG-PET = fluorodeoxyglucose positron emission tomography; MRI = magnetic resonance imaging.

FIGURE 3:

Inflammatory infiltrate and elevated GFAP and S100b levels in CSF during neurotoxicity. (A) CSF protein and (B) white blood cell (WBC) counts. (C) CSF GFAP and (D) S100b levels. In all graphs, each point represents data from an individual subject. Empty circles, no neurotoxicity; filled circles, grade 1 to 4 neurotoxicity. The number of individual subject samples in each group is given below the plots. Bars show the median (long bar) and interquartile range (short bars). “Pre”: sample obtained before CAR-T cell infusion; “Acute”: sample obtained while symptomatic with CRS and/or neurotoxicity (days 6–16); “D21”: day 21 after treatment. *p < 0.05; **p < 0.01; ***p < 0.001, Kruskal-Wallis with uncorrected Dunn’s posttest. CAR = chimeric antigen receptor; CRS = cytokine-release syndrome; CSF = cerebrospinal fluid; GFAP = glial fibrillary acidic protein.

FIGURE 4:

Enrichment of CD4⁺ CAR-T cells in the CSF. (A) Peak and day 21 CAR-T cell counts in the blood. (B) CSF CAR-T cell counts during the acute phase and on day 21. (C) The y-axis indicates the percentage of CD3⁺ T cells that were CAR-T cells in blood and CSF samples. (D) Percentage of CAR-T cells that were CD4⁺ in blood and CSF. In all graphs, each point represents data from an individual subject. Empty circles, no neurotoxicity; filled circles, grade 1 to 4 neurotoxicity. The number of individual subject samples in each group is given below the plots. Bars show the median (long bar) and interquartile range (short bars). “EGFRt⁺”: CAR-T cells labeled with Erbitux; “Acute”: sample obtained while symptomatic with CRS and/or neurotoxicity (days 6–16); “Peak”: highest CAR-T cell density in each subject’s course of treatment; “D21”: day 21 after treatment; “No NT”: no neurotoxicity; “Grade 1-4 NT”: grade 1 to 4 neurotoxicity. The number of CAR-T cells detected in the acute CSF of patients without neurotoxicity was too low to quantify expression patterns; therefore, no data are shown for this group in (C) and (D). n.s., not significant at 95% confidence level. *p < 0.05; **p < 0.01; ***p < 0.001, Kruskal-Wallis with uncorrected Dunn’s posttest. CAR = chimeric antigen receptor; CRS = cytokine-release syndrome; CSF = cerebrospinal fluid.

FIGURE 5:

Markers of CNS inflammation are elevated during acute CAR-T neurotoxicity. (A) Cytokine levels in CSF samples from subjects with neurotoxicity. (B) Serum cytokine levels on day 7 after CAR-T cell infusion, stratified by CRS grade. (C) Paired same-day CSF and serum cytokine levels. Empty circles, no neurotoxicity; filled circles, grade 1 to 4 neurotoxicity. The number of individual subject samples in each group is given below the plots. Bars show the median (long bar) and interquartile range (short bars). “Pre”: before CAR-T cell infusion; “Acute”: sample obtained while symptomatic with CRS and/or neurotoxicity (days 6–16); “D21”: on day 21 after treatment. “Gz B”: granzyme B. n.s., not significant at 95% confidence level. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, Kruskal-Wallis with uncorrected Dunn’s posttest for (A) and (B), paired Wilcoxon signed-rank test for (C). CAR = chimeric antigen receptor; CNS = central nervous system; CRS = cytokine-release syndrome; CSF = cerebrospinal fluid; IFN = interferon; IL = interleukin.