Patient-reported outcomes and neurotoxicity markers in patients treated with bispecific LV20.19 CAR T cell therapy.

Background: With the rising number of chimeric antigen receptor (CAR) T cell treated patients, it is increasingly important to understand the treatment's impact on patient-reported outcomes (PROs) and, ideally, identify biomarkers of central nervous system (CNS) adverse effects.
Methods: The purpose of this exploratory study was to assess short-term PROs and serum kynurenine metabolites for associated neurotoxicity among patients treated in an anti-CD20, anti-CD19 (LV20.19) CAR T cell phase I clinical trial (NCT03019055). Fifteen CAR T treated patients from the parent trial provided serum samples and self-report surveys 15 days before and 14, 28, and 90 days after treatment.
Results: Blood kynurenine concentrations increased over time in patients with evidence of neurotoxicity (p = 0.004) and were increased in self-reported depression (r = 0.52, p = 0.002). Depression improved after CAR T infusion (p = 0.035). Elevated 3-hydroxyanthranilic acid (3HAA) concentrations prior to cell infusion were also predictive of neurotoxicity onset (p = 0.031), suggesting it is a biomarker of neurotoxicity following CAR T cell therapy. Conclusions: Elevated levels of kynurenine pathway metabolites among CAR T cell recipients are associated with depressed mood and neurotoxicity. Findings from this exploratory study are preliminary and warrant validation in a larger cohort.

Introduction

The use of chimeric antigen receptor (CAR) T cell therapy is rising rapidly in recent years, increasing from only a few US patients receiving it in 2017, to over 1000 patients in 2019[1]. These numbers are expected to increase near exponentially, replacing other therapeutic options for the treatment of blood cancers, including hematopoietic stem cell transplant (HCT)[2].

Some patients receiving CAR T cell therapies experience central nervous system (CNS) neurotoxicity that presents in the form of delirium, confusion, tremor, seizures, and in rare cases life-threatening cerebral edema[3]. Although the pathophysiology of these neurological effects is not clear, inflammatory cytokines are thought to be significant contributors[3]. While CAR T neurotoxicity itself has not yet been established as a precursor to subsequent changes in mood and other related symptoms, neuroinflammation, in general, is associated with such changes in other populations[4], with the kynurenine pathway as a critical link between inflammation, mood disorders and related symptomatology, and alteration of brain signaling pathways[5-9].

There is evolving evidence that CAR T in general can have more subtle effects on patient-reported outcomes (PROs) as a result of CNS effects. For example, Maziarz et al. demonstrated that patients achieving complete or partial response had sustained PRO improvement at 12 and 18 months[10], while Ruark et al. found that patients receiving CAR T cell therapy are at risk for longer-term PRO impairments, including the development of depression, anxiety, fatigue, sleep disturbances, and pain[11]. Given that these effects can also be associated with neuroinflammation, it has been suggested that CAR T use results in a CNS inflammatory state, which can lead to both reduced quality of life (QOL) and neurotoxicity[3,12].

There are several potential mechanisms by which CAR T mediated therapeutic and adverse effects could influence and/or be affected by PRO measures. First, CAR T treatments are associated with immune activation, inflammation, and cytokine signaling[3,12], all of which are known to affect cognitive and emotional functioning[13], including among cancer patients[14]. Second, CAR T therapy is associated with significant CNS toxicity. Not only do negative emotional and cognitive responses influence immunity and T cell function among cancer patients generally[15], but a recent review also highlighted that CRS and neurotoxicity delayed QOL improvement among CAR T recipients specifically[16]. Finally, these adverse psychosocial responses are predictive of compromised immune reconstitution[17] and adverse outcomes following HCT[18-21]. Therefore, it is critical to understand the interaction between behavioral and biological processes in the setting of CAR T cell therapy and identify predictive characteristics and biomarkers of neurotoxicity that will allow for risk stratification and potentiate early intervention.

The kynurenine pathway is activated by inflammatory cytokines released in the brain and leads to the production of kynurenine from tryptophan (TRP) as a result of upregulation of the enzyme indoleamine 2,3-dioxygenase (IDO) by astrocytes and microglia (brain-resident macrophages). Kynurenine is further metabolized to the neurotoxic metabolites 3-hydroxykynurenine (3HK), 3-hydroxyanthranilic acid (3HAA), and quinolinic acid (QA) and to the neuroprotective metabolite, kynurenic acid (KA) (Fig. 1). The ratio of serum KA/3HK has been used previously as an index of the role of this inflammatory pathway on brain function[22], with lower ratios noted in patients with mood disorders as compared to healthy individuals[23]. Santomasso et al.[24] observed significantly elevated levels of QA in the cerebral spinal fluid (CSF) of CAR T recipients during neurotoxicity compared to pretreatment; however, they did not evaluate other kynurenine pathway metabolites or examine their presence in circulation.

Fig. 1

Kynurenine metabolic pathway.

3-HK 3-Hydroxykynurenine, 3-HAA 3-Hydroxyanthranilic acid, QA Quinolinic acid, NMDA-R N-methyl-D-aspartate receptor, nAChR Nicotinic acetylcholine receptor.

Despite substantial interest in understanding the impact of CAR T cell therapy on PROs and identifying predictive biomarkers[25-28], there are limited data available currently. Biologic indicators such as ferritin, platelet numbers, lactate dehydrogenase (LDH)[29], and fibrinogen[30] have been associated with CAR T neurotoxicity with single targeting CD19 CAR products. Elevated cytokines from cerebral spinal fluid were also associated with neurotoxicity in a cohort of CAR T patients[24]. However, it is unknown whether disruption in the kynurenine pathway—a link between inflammation and the brain – is associated with PROs or predictive of CAR T neurotoxicity.

Here, we describe the PRO trajectory in the early treatment period of CAR T therapy among patients treated in phase 1, first-in-human, bispecific, lentiviral, anti-CD20, anti-CD19 (LV20.19) CAR T cell clinical trial[31]. Further, we provide preliminary data that circulating concentrations of kynurenine pathway components are associated with both neurotoxicity and PROs. Blood kynurenine concentrations increased over time in patients with evidence of neurotoxicity and were increased in self-reported depression, with depression improving after CAR T infusion. Elevated baseline 3HAA concentrations prior to cell infusion were also predictive of neurotoxicity onset. These exploratory findings suggest that blood kynurenine pathway metabolites could be used as biomarkers of and potentially targeted to prevent neurotoxicity following CAR T cell therapy.

Methods

Study population

The current study population is derived from a parent study evaluating 22 patients treated with LV20.19 CAR T cells in Phase I/Ib clinical trial (NCT03019055). See Shah et al. for full study details and for original data please contact the corresponding author[31]. No deaths were attributed to LV20.19 CAR T cell therapy. In this study we conducted a PRO and neurotoxicity biomarker sub-study focused on 15 patients with relapsed, refractory B-cell non-Hodgkin’s lymphoma or chronic lymphocytic leukemia/small lymphocytic lymphoma all treated at the selected dose from the phase 1 trial (2.5 × 106 cells/kg) for homogeneity. Patients were recruited for this sub-study following Institutional Review Board (IRB) approval of a PRO and biomarker amendment (starting with patient 7 of the parent study; IRB PRO00037171). No blinding was used in the open-label study. The sub-study cohort included patients who provided PRO data and survived through a day 28 assessment. Administration of CAR T cells occurred through the Medical College of Wisconsin (MCW) HCT Program under an FDA IND 17518. All participants provided written informed consent and all procedures were approved in advance by the MCW IRB.

Patient-reported outcomes (PROs)

Study subjects completed a series of self-report surveys at the following timepoints: baseline/apheresis (Day −15 with respect to Day 0 being day of CAR T cell infusion), Day +14 (D14), D28, and D90 post-infusion. To provide a dimensional assessment of depressive symptoms, the 20-item General Depression subscale from the Inventory of Depression and Anxiety Symptoms (IDAS) was used[32]. Questionnaires administered at timepoints listed above included the Inventory of Depression and Anxiety Symptoms (IDAS)[32], Fatigue Symptom Inventory (FSI; fatigue)[33], Pittsburgh Sleep Quality Index (PSQI; sleep)[34], and Brief Pain Inventory (BPI; pain)[35]. IDAS scoring ranges from 20 to 100 for depression and anxiety, with a mean depression subscale score for a community-dwelling adult of 44.99 (SD = 14.75)[36]. Anxiety was assessed as a sum of two IDAS subscales scores—panic disorder (healthy population mean = 12.58, SD = 5.26) and traumatic intrusions (healthy population mean = 7.60, SD = 4.20)[36]. FSI was evaluated based on fatigue intensity (FSII) and interference (FSIF) (both scales ranged 0–10 with >3 being clinically significant; FSII) and the number of days within the past week patients felt fatigued (0–7; FSID)[37]. Sleep scores >5 are considered disturbed sleep as adjusted for cancer populations[34,38]. Brief Pain Index scores were subdivided and assessed based on pain intensity (0–40; BPII) and pain interference in daily activities (0–10; BPIF)[39]. Any patients endorsing thoughts of suicidality or self-harm per the IDAS were contacted by Dr. Knight and offered appropriate follow-up care.

Neurotoxicity

Study participants were evaluated for neurotoxicity using the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) v5[40] and CRS was graded using the Lee et al. CRS grading system[41]. Neurotoxicity was retrospectively regraded utilizing the updated American Society for Transplantation and Cellular Therapy (ASTCT) system[42] with no change compared to the CTCAE (see Supplemental Appendix Table 3 in Shah et al.)[31].

Measurement of tryptophan and metabolites

Blood was obtained from subjects at D−15 (considered baseline values), D14, D28, and D90 and serum were harvested and frozen at −80 °C until assay. Serum samples were thawed and 200 µL was transferred to 1 mL of acetonitrile containing 100 ng each of deuterated tryptophan ([2H5]‐TRP), kynurenic acid ([2H5]‐KA), quinolinic acid ([2H3]‐QA), and 3-hydroxyanthralinic acid ([2H2]‐3HAA). Samples were vortexed and then placed into an ultrasonication bath for 2 min. Protein precipitation was facilitated by transferring samples to −20 °C freezer for thirty minutes, followed by centrifugation at 14,000 rcf for 10 min at 4 °C. The resulting supernatants were dried down at room temperature under a gentle nitrogen stream. After resuspension in 100 µL acetonitrile, samples were redried, resuspended in 100 µL acidified mobile phase, transferred to autosampler vials, and stored at 4 °C until analysis.

The concentrations of TRP and metabolites (KA, QA, 3HAA, kynurenine, and 3HK) were quantified in 5 μl of the mobile phase extract using stable isotope‐dilution liquid chromatography/mass spectrometry of the daughter ions (LC‐MS‐MS). Reversed-phase high-performance liquid chromatography (HPLC) using a Kromasil C18 column (150 × 2.1 mm, 5 µm particle size) and a mobile phase of 0.1% formic acid, 2 mM Ammonium acetate in ddH2O (phase A) and acetonitrile (phase B) was used to separate the metabolites. Analytes were separated at a flow rate of 0.2 mL/min with a step gradient of 0% B for 0–1.5 min, 75% B from 3.5–5 min, 85% B from 8–9 min, and 0% B at 11–14 min. Mass spectrometric detection was performed using a tandem quadrupole mass spectrometer (Agilent 6460) equipped with an electrospray ionization (ESI) source. Detection was performed in the positive mode. The quantification was performed using the multiple reaction monitoring modes with the following m/z transitions monitored: 205.1/146 for TRP, 209.1/94 for kynurenine, 225.1/110.1 for 3HK, 190/144 for KA, 168/78.1 for QA, 154/80.1 for 3HAA, 210.1/150.1 for [2H5]‐TRP, 195.1/149.1 for [2H5]‐KA, 171/81.1 for [2H3]‐QA, and 156/82 for [2H2]‐3HAA. The assay utilized was designed to analyze all kynurenine metabolites in a single serum sample and was therefore not optimized to measure QA in particular; subsequently, there were only values for 6 participants at baseline and 4 at D90.

Standard curves were generated for all analytes in a range of 10–5000 pg/µL, and internal standards [2H5]‐TRP, [2H5]‐KA, [2H3]‐QA, and [2H2]‐3HAA (each at 1000 pg/µL). Concentrations of the analytes in the samples were determined from standard curves of the area ratios (standard/analyte) versus the concentration ratios (standard/analyte); the corresponding deuterated compounds were used as the standard for the unlabeled analytes; [2H5]‐TRP was used as the standard for kynurenine and 3HK in addition to TRP.

Serum cytokine assessment

Peripheral blood serum samples were collected from each subject at several timepoints during the first 28 days following LV20.19 CAR cell infusion. Samples were immediately frozen at −80 °C and then shipped on dry ice to Eve Technologies (Calgary, Alberta Canada), where they were analyzed using a 65-plex human cytokine and chemokine panel (HD65). Each sample was analyzed in duplicate, and the average values determined. Peak levels of 10 cytokines in the panel were found to be significantly elevated in the serum of patients who experienced neurotoxicity[31].

Statistical analysis

Descriptive analyses

Categorical variables were summarized using counts and percentages, while continuous variables with median and range or interquartile range, as indicated. Between-group comparisons were performed using the chi-square test and Wilcoxon’s rank-sum test, respectively. Student’s t-tests were used to evaluate baseline differences in PROs between patients who completed visit 4 on D90 vs those that did not.

Primary analysis

A mixed-effects longitudinal model was fitted for all eligible patients to evaluate the impact of time on all PRO variables over 4 encounters through 90 days of post-study intervention. The measurement time was a fixed categorical predictor, and a subject-specific random intercept was used to account for within-patient dependence. This approach allows for an unbalanced design in which not every subject is measured at every timepoint. A mixed-effects logistic regression model was used to evaluate the changes in sleep quality over time.

Secondary analyses

Neurotoxicity was evaluated as a categorical variable with levels 0–1 and 3–4, as no subject in our study had grade 2 toxicity. Kynurenine metabolite values were background corrected. Zero values were considered to be below the limit of detection and replaced by half of the smallest non-negative value. The measurements were log-transformed to stabilize the variances and allow modeling of the expected multiplicative effects. The effect of time and neurotoxicity grade were evaluated separately for each metabolite using mixed-effects linear models with a random subject intercept, and fixed effects of time, neurotoxicity grade, and their interaction. Spearman’s rank correlation was used to evaluate the association between changes in kynurenine and concurrent changes in depression scores, and to compare kynurenine metabolites at baseline/D−15, D14, and D28 with peak concentrations of inflammatory cytokines by D28.

Table 1

Baseline patient demographics.

Baseline characteristics	Overall, N = 15a	NTX Grade 0–1, N = 12a	NTX Grade 3–4, N = 3a	p-value
Age	61 (38–72)	60 (38–69)	66 (47–72)	0.61
Male sex	14 (93%)	12 (100%)	2 (67%)	0.20
Level of education				0.76
High school	2 (13%)	2 (17%)	0 (0%)
Trade school	4 (27%)	4 (33%)	0 (0%)
Some college	3 (20%)	2 (17%)	1 (33%)
College graduate	3 (20%)	2 (17%)	1 (33%)
Post graduate degree	3 (20%)	2 (17%)	1 (33%)
Income				0.94
$10,001–25,000	2 (13%)	1 (8.3%)	1 (33%)
$25,001–40,000	3 (20%)	2 (17%)	1 (33%)
$40,001–55,000	3 (20%)	2 (17%)	1 (33%)
$55,001–70,000	3 (20%)	3 (25%)	0 (0%)
$85,001–100,000	2 (13%)	2 (17%)	0 (0%)
>$100,000	2 (13%)	2 (17%)	0 (0%)
Histology				>0.99
CLL	2 (13%)	2 (17%)	0 (0%)
DLBCL	9 (60%)	7 (58%)	2 (67%)
FL	1 (6.7%)	1 (8.3%)	0 (0%)
MCL	3 (20%)	2 (17%)	1 (33%)
Baseline LDH	203 (121–2,074)	196 (121–490)	1283 (590–2074)	0.004
Lines of prior therapy	4 (3–6)	4 (2–7)	4 (3–11)	0.55
Prior allogeneic HCT	1 (6.7%)	1 (8.3%)	0 (0%)	>0.99
Prior autologous HCT	5 (6.7%)	5 (42%)	0 (0%)	0.51
Clinical response on day 28				0.23
CR	12 (80%)	10 (83%)	2 (67%)
PD	1 (6.7%)	0 (0%)	1 (33%)
PR	2 (13%)	2 (17%)	0 (0%)
CRS (Yes)	12 (80%)	9 (75%)	3 (100%)	>0.99
Day to CRS	2.5 (0.0–10.0)	6.0 (1.0–10.0)	1.0 (0.0–3.0)	0.16
Max Grade CRS				0.32
0	3 (20%)	3 (25%)	0 (0%)
1	7 (47%)	6 (50%)	1 (33%)
2	4 (27%)	3 (25%)	1 (33%)
4	1 (6.7%)	0 (0%)	1 (33%)
NTX (Yes/no)	5 (33%)	2 (17%)	3 (100%)
Max NTX Grade
0	10 (67%)	10 (83%)	0 (0%)
1	2 (13%)	2 (17%)	0 (0%)
3	2 (13%)	0 (0%)	2 (67%)
4	1 (6.7%)	0 (0%)	1 (33%)
Days to NTX	6.0 (0.0–9.0)	7.5 (6.0–9.0)	1.0 (0.0–6.0)

CLL chronic lymphocytic leukemia, CR complete response, CRS cytokine release syndrome, DLBCL diffuse large B-cell lymphoma, FL follicular lymphoma, HCT hematopoietic cell transplantation, LDH lactate dehydrogenase, MCL mantle cell lymphoma, NTX neurotoxicity, PD progressive disease, PR partial response.

aN (%), median (range).

Table 2

Patient-reported outcome (PRO) means over timea.

PROs	Baseline, N = 15		Day 14, N = 13		Day 28, N = 14		Day 90, N = 13		P-value
	Mean	IQR	Mean	IQR	Mean	IQR	Mean	IQR
Anxiety	16	14, 18	15	12, 22	20	13, 21	13	12, 18	0.95
Depression	37	30, 42	39	26, 42	37	30, 43	31	26, 35	0.035
BPIF	1.3	0.4, 2.1	0.0	0.0, 1.7	0.29	0.0, 4.3	0.57	0, 1.0	0.067
BPII	2.0	1.0, 2.9	0.8	0.1, 3.1	1.8	0.5, 2.8	2.0	0.0, 3.3	0.67
FSID	7.0	5.5, 9.0	9.0	3.0, 13.5	8.0	5.0, 12.0	7.0	4.5, 11.5	0.88
FSIF	1.57	0.9, 2.9	1.1	0.2, 3.6	1.4	0.0, 4.0	1.1	0.1, 1.6	0.17
FSII	3.5	2.5, 4.1	3.9	2.0, 4.8	3.3	2.5, 4.3	3.2	2.1, 4.4	0.42
PSQI	5.0	2.5, 6.5	3.0	3.0, 5.0	6.0	3.2, 7.0	4.0	2.0, 6.0	0.92

BPIF Brief Pain Inventory Fatigue, BPII Brief Pain Inventory Intensity, FSID Fatigue Symptom Inventory Duration, FSIF Fatigue Symptom Inventory Interference, FSII Fatigue Symptom Inventory Intensity, PSQI Pittsburgh Sleep Quality Index

aMixed effects model with random ID effect utilized to derive p-values.

Table 3

Patient sleep quality scores.

Timepoint	Sleep quality	Number of patients N = 15 (%)
Baseline (N = 15)	Good sleep	9 (60%)
	Poor sleep	6 (40%)
Day 14 (N = 9)	Good sleep	4 (44%)
	Poor sleep	5 (56%)
Day 28 (N = 12)	Good sleep	7 (58%)
	Poor sleep	5 (42%)
Day 90 (N = 10)	Good sleep	5 (50%)
	Poor sleep	5 (50%)

Table 4

Spearman ranked correlation between kynurenine fold-change from previous timepoint and change in depression from previous timepoint.

Time change	Estimate	P-value
Baseline to 14	0.62	0.05
14 to 28	0.48	0.227
28 to 90	0.34	0.304
Pooled	0.52	0.002