Abstract 4231: Factors predictive of CAR T cell associated hemophagocytic lymphohistiocytosis (HLH)
Authors: Lichtenstein Daniel A. , Steinberg Seth M. , Highfill Steven L. , Yates Bonnie , Jin Ping , Jin Jianjian , Panch Sandhya , Shalabi Haneen , Stroncek David F. , Fry Terry J. , Shah Nirali N. 
Published: September 2020

Abstract
Introduction: HLH is a T cell mediated inflammatory syndrome associated with immune activation and NK cell dysfunction. In the context of CAR T cells, HLH occurs in a subset of those with cytokine release syndrome (CRS), yet predisposing factors are not yet well established. Utilizing our ongoing phase I study of CD22/4-1BB CAR T cells in children and young adults with CD22+ B cell malignancies (NCT02315612), we evaluated patient and product characteristics to elucidate factors associated with HLH.
Methods: We retrospectively reviewed multiple variables to identify correlates of HLH in those infused. Definitions for CRS and HLH are in Table 1. Multiple univariate analyses were performed to identify risk factors for HLH; variables with significance were incorporated into a multiple logistic regression model to predict those at risk of HLH-like CRS.
Table 1.Patient and Product Characteristics Amongst those with CRS, Distinguished by HLH versus non HLH-like toxicitiesNo HLH^HLH ^pBaseline DemographicsHLH manifestations amongst those with CRS, n, (%)31 (59.6%)21 (40.4%)Age (years), median (range)17 (4 - 30)13 (4 - 30)nsTime to CRS onset, day post infusion, median (range)8 (3-13)7.5 (3-13)nsTime to HLH onset, day post infusion, median (range)N/A14 (7-26)N/A# patients with prior HSCT, n (%)23/31 (74.2%)13/21 (61.9%)ns# patients with prior CAR, n (%)20/31 (64.5%)11/21 (52.4)nsPatient Factors; median (IQR)Baseline# PB12 (7.1-23.6)7.4 (4.4-14.6)0.04NK%Baseline# PB75 (58.5 - 88.5)83.9 (75.3-92.1)0.05CD3%Baseline# PB2.3 (1.2 - 4.3)6.1 (2.6 - 10.8)0.008CD8/NK ratioBaseline# BM3.6 (1.6 - 6.1)8.4 (4.9 - 15.7)0.0009CD3/NK ratioBaseline# Disease burden, % marrow blasts50 (18.9 - 90)71.4 (40.8 - 88.5)nsBaseline# Soluble IL2Ra (pg/mL)728 (460 - 1001)1176 (556.3 - 1550)0.07Product Factors; median (IQR)TCS of apheresis product, n, (%)13/31 (41.9%)18/21 (85.7%)0.002Product culture day 7, CD3%99.6 (99.3-99.8)99.8 (99.7 - 99.8)0.02Product culture day 7, NK%.1 (.01 - .23)0 (0 -.10)0.002Final product, CD3%99.7 (99.6 - 99.9)99.9 (99.8 - 99.9)0.01Final product, NK%.03 (0 - .12)0 (0 - .04)0.1Max Grade CRSGrade 121/31 (67.7%)5/21 (23.8%)0.004Grade 27/31 (22.3%)14/21 (66.7%)Grade 3&43/31 (9.6%)2/21 (9.5%)CAR T-cell Expansion (PB); median (IQR)Day 14, %CD871.6 (60.3 - 79.4)85.9 (61.1-92.9)0.02Day 14, %CD419.8 (11.9 - 27.2)7.8 (4.5 - 32.7)0.06Day 14, %NK4.0 (1.9 - 5.4)1.7 (.7 - 5.2)nsDay 14, CD8:NK ratio16.7 (10.5 - 41.1)42.1 (12.3 - 151.1).05Day 14, CD4:NK5.3 (1.8 - 8.1)5.5 (1.7 - 15)nsDay 28, %CD843.6 (37.8 - 55.7)69.8 (43.6 - 81.7)0.02Day 28, %CD435.8 (26 - 47.2)22.3 (10.7 - 44.9)0.07Day 28, %NK9 (4.5 - 15.5)1.9 (.7 - 11.7)0.02Day 28, CD8:NK ratio4.5 (2.6 - 12.8)21.5 (2.9 - 109.2)0.03Day 28, CD4:NK3.9 (1.8 - 7.6)7.7 (3.9 - 19.6)0.05Inflammatory markers and select cytokine profiling**; median (IQR)Ferritin (ng/mL)Baseline#1979 (992.5 - 3802)1942 (1397 - 4192)nsDay 51433 (888.5 - 2491)3621 (1587 - 5024)0.02Day 95920 (1097 - 18321)14063 (5367 - 61503)0.02IL18 (pg/mL)Day 0347.1 (251.8 - 544.4)645 (331.7 -863.3)0.02Day 5450 (232.5 - 589)578.6 (426.5 - 887.4)0.05Day 9835 (586.8 - 1035)894.4 (843.5 - 2971)nsDay 131494 (843.3 - 1970)2058 (1386 - 5001)0.04IL-1B (pg/mL)Day 0.29 (.28 - .33).28 (.28 - .29)nsDay 5.29 (.28 - .33).28 (.28 - .29)nsDay 9.33 (.28. - .82).53 (.28 - 3.36)nsDay 13.29 (.28 - .645).89 (.28 - 1.528)0.03IL-6** (pg/mL)Day 02.9 (1.4 - 8.9)4.1 (2.1 - 8.5)nsDay 53.3 (1.7 - 14.9)6.2 (2.7 - 9.3)nsDay 920.5 (5.0- 59.1)43.0 (15.5 - 983)0.04Day 139.025 (4.118 - 22.54)127.2 (20.92 - 597.4)0.0008IFNy (pg/mL)Day 011.1 (3.8 - 17.4)8.5 (3.2 - 15.4)nsDay 518.01 (5.0 - 46.9)13.6 (4.0 - 25.5)nsDay 9169.5 (93.3 - 665.4)485.2 (188.2 - 1887).06Day 1381.07 (21.1 - 132.8)287.3 (123 - 829.7)0.001Predictive model1.9882 x (TCS, 1=Yes/0=No) + 0.8013 x (max grade CRS) + 0.2368 x (bone marrow T/NK ratio); values &gt; 4.47 were predictive for HLH.(Model based on 59/60 infused patients, with full data available for 55 subjects. 1 subject excluded due to rapidly progressive disease requiring steroids prior to CAR expansion)*CRS (cytokine release syndrome) was defined by Lee et al. (Blood 2014).^HLH was defined by the following criteria, modified from Neelapu et al.: peak ferritin &gt;100,000 µg/L with at least two of the following criteria: a) hepatic transaminases or bilirubin &gt; grade 3, b) creatinine &gt; grade 3, c) pulmonary edema &gt; grade 3 and/or d) evidence of hemophagocytosis on bone marrow aspirate/biopsy.#Baseline: Timepoint prior to apheresis.**IL-6R antagonist (Tocilizumab) administration confounds results.Abbreviations: PB: peripheral blood; BM: bone marrow; TCS: T-cell selection of the apheresis product using CD4/CD8 beads; IQR: interquartile range; N/A: not applicable; ns: not significant.P values determined using Fisher's exact and Mann Whitney U tests for categorical and continuous variables, respectively, except Max Grade CRS which utilized a Chi-Squared test.
Results: 52 of 60 (86.7%) treated subjects developed CRS; 21 (40.4%) of whom subsequently developed HLH. The following factors were associated with HLH: lower baseline blood and marrow NK% and higher CD3% cells; CD4/CD8 T cell selection (TCS) of the apheresis product (which increased CD3 and reduced NK cell populations); and higher IL18. Following infusion, distinct cytokine profiles and prolonged predominance of CD8 T cells distinguished those with HLH from those without it. Multiple logistic regression identified a) use of TCS, b) higher grade CRS and c) high baseline bone marrow T/NK ratio as most predictive of HLH; leading to a model with 75% sensitivity and 80% specificity.
Conclusion: Our results provide novel insights into factors associated with CAR T cell related HLH and implicate preexisting T and NK cell populations, baseline cytokines, apheresis selection methodologies and CAR T cell expansion parameters in the pathophysiology. With a goal of earlier intervention to prevent HLH, the predictive model will be prospectively evaluated. Biologic correlative studies and assessment of generalizability in alternate CAR T cell constructs are planned.
Citation Format: Daniel A. Lichtenstein, Seth M. Steinberg, Steven L. Highfill, Bonnie Yates, Ping Jin, Jianjian Jin, Sandhya Panch, Haneen Shalabi, David F. Stroncek, Terry J. Fry, Nirali N. Shah. Factors predictive of CAR T cell associated hemophagocytic lymphohistiocytosis (HLH) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4231.