Age has been suggested as a risk factor for the development of ICANS (Table 2). However, evidence is conflicting and strongly biased by agent examined: The original ZUMA-1 clinical trial which lead to approval for axi-cel for DLBCL reported modest increased risk of neurologic events in patients ≥65 years old (n = 27, median age 69) vs. <65 years of age (n = 81, median age 55). Grade 3+ neurologic toxicities included delirium (11 vs. 0%), agitation (11 vs. 2%), and encephalopathy (30 vs. 22%), but interestingly not aphasia (0 vs. 10%) (50). Rubin et al. prospectively examined a cohort of 204 patients receiving axi-cel (median age 60 years) and found 58% developed neurotoxicity, with 43% developing grade 3+ neurotoxicity (39). Univariate regression revealed an association between age and ICANS (odds ratio (OR) 1.05). Once axi-cel was FDA approved for use, Jacobson et al. examined outcomes in a large international cohort of 1,297 patients who received the standard of care axi-cel, of which 739 (57%) would have been illegible for the original ZUMA-1 clinical trial (16) due to age or other ineglibility criteria. In the clinical practice, 55% of patients developed ICANS, with 24% developing grade 3+ ICANS. Multivariate analysis revealed a significant increased risk for patients aged 65 and older of developing any grade ICANS and grade 3+ ICANS with an odds ratio (OR) of 1.77 (95% CI, 1.39 to 2.26) and 1.38 (95% CI, 1.04–1.82) respectively. However, this relationship between older age and ICANS in patients treated with axi-cel has not been universally observed. Faramand and colleagues prospectively examined 75 patients (median age 63) treated with axi-cel and found no association with age. Similarly, Nastoupil and colleagues retrospectively analyzed results from the US Lymphoma CAR-T consortium totally 298 patients (median age 60, of whom, 275 were treated with axi-cel) and found no association with age and severe (Grade 3) neurotoxicity. No association with age has likewise been reported in smaller retrospective cohorts as well (40, 43, 48, 51). Like axi-cel, the investigational agent 19–28 z uses a CD28 co-stimulatory domain. Park et al. initially reported an association with age and ICANS in 51 adult patients (30), although this relationship was not observed in a second adult cohort of 51 patients (32). Overall, conflicting evidence exists for axi-cel and similar CD19-directed agents.

A relationship between age and ICANS was not observed for tisa-cel, either in the original trials (3) or later real-world cohorts (42, 43). Recently younger, not older age was reported associated with ICANS in a cohort of 15 patients (median age 59) treated with tisa-cel (44) (Table 2). Given the comparatively low incidence of ICANS for tisa-cel compared to axi-cel, statistical power is a limiting factor in drawing conclusions on the risk factors for ICANS development with tisa-cel. When examining an investigational CD19-directed CAR-T that also contains a 4-1BB costimulatory domain like tisa-cel, Gust et al. (28) also did not observe a significant relationship between neurotoxicity and age, but interestingly a trend for increased ICANS in younger, not older adult patients. Overall, age is an unclear risk factor with differences predominately related to agent. Larger studies are needed to understand the contributing factor of age to ICANS.

Functional status, like age, is a risk factor reported predominately in studies that are heavily biased toward CD19-directed agents. It is measured using Eastern Cooperative Oncology Group performance status (ECOG PS), an established ordinal scale capturing a patient's level of medical independence and activity. An ECOG PS of 0 reflects a fully independent patient, while ECOG PS of 2 reflects an ambulatory patient who can carry out self-care but unable to work and up no more than 50% of waking hours. Faramand et al. (38) prospective study of patients treated with axi-cell did not observe an association between ECOG score and ICANS. In contrast, several large retrospective studies have demonstrated a positive association: Riedell and colleagues performed a retrospective multi-center study on 240 patients undergoing cellular therapy for a B cell lymphoma. They also observed ECOG PS ≥2 was associated with an increased risk of developing ICANS (p = 0.05, OR 5.05), even when controlling for treatment type i.e., axi-cel use. Jacobson and colleagues real-world cohort of 1,297 patients treated with axi-cel also observed a significant OR for baseline ECOG PS ≥2 and both any grade ICANS (OR 2.63; 95% CI, 1.40 to 4.93) and grade 3+ ICANS (3.23; 95% CI, 1.81 to 5.74) (16). Similarly, Iacaboni et al. examined 75 patients (median age 60) treated exclusively with tisa-cel in a retrospective Spanish cohort and found an association between ICANS and ECOG FS ≥1 (vs. 0) (42). Wudhikarn et al. only observed an increased likelihood of developing ICANS in patients with ECOG ≥2, but only in patients under the age of 60 (40). Gauthier and colleagues did not observe an association in their smaller retrospectively study (43). Taken together, ECOG PS remains a possible but weak factor mainly observed in larger cohorts with limited data beyond CD19-directed agents.

Pre-infusion tumor burden has been reported as a risk factor for the development of neurotoxicity across several (17, 24, 28, 30, 32, 34) but not all studies (26, 36, 43, 44). Notably the methods used to measure tumor burden can vary widely from bone marrow assessments, to imaging-derived (including using positron emission tomography (PET), discussed below), to secondarily inferred from blood LDH levels (Table 3). As a result, tumor burden has been defined in different studies as a categorical variable (“high” vs. “low burden” based on marrow blasts or by the presence of secondary sites of disease), ordinal variable, and continuous variable highlighting the varied approaches used. To date, the association between tumor and risk for ICANS has been best characterized in adults with B cell lymphomas (17, 24, 28, 30, 32, 52) but not children (36). Studies reporting no association often limited by power (43, 44). Taken together, pre-infusion tumor burden is a possible risk factor, though standardization of measurement modality and larger studies comparing across agents is warranted.

PET-derived assessment of total metabolic tumor volume (TMTV) has been proposed as a standardized means to quantify total active tumor volume and address some of the variability in tumor burden assessment highlighted above. TMTV is calculated from pre-treatment (i.e., baseline at recurrence) PET scans of patients with B cell lymphoma using a 41% maximum standardized uptake value threshold (53, 54). Unfortunately use of TMTV has had mixed to success to date (52, 54). Dean et al. retrospectively examined baseline PET scans of 96 patients treated with axi-cel and found no relationship between TMTV and ICANS (54), in line with earlier reports (55). Likewise, Iacoboni et al. retrospectively examined a cohort of 35 patients treated with an admixture of agents (breakdown not reported) also did not observe an association with ICANS risk (52). Nor did we see a relationship between TMTV and ICANS risk in our recent retrospective study of 30 patients (46). TMTV as a surrogate for tumor burden in B cell lymphomas remains of unclear utility at this time.

Until recently, only axi-cel's multiple indications permitted comparison of ICANS incidence between different histological subtypes while controlling for agent. The original ZUMA-1 [diffused large B cell lymphoma (DLBCL)], ZUMA-5 [follicular lymphoma (FL)], and most recently ZUMA-7 (2nd line for large B cell) clinical trials all reported comparable frequency of ICANS of approximately 60% (Table 1). However, the frequency of grade 3+ ICANS was approximately half for FL (ZUMA-5) vs. DLBCL (ZUMA-1). Rubin and colleagues prospective study of 204 patients categorically clustered by patient's histological subtype: aggressive (DLBL, primary mediastinal B-cell lymphoma) or indolent (follicular lymphoma, marginal zone lymphoma) histologic subtype (39). They found a univariate association between neurotoxicity and an aggressive histological subtype (p < 0.001), and included it in their multivariate prediction model. When comparing between different aggressive subtypes of lymphoma (DLBL, primary mediastinal B-cell lymphoma, transformed FL, germinal center B cell (GCB)-like, non-GCB, double/triple hit, or double expressor), no association was observed retrospectively in the US Lymphoma CAR-T consortium (17). With the recent approval of tisa-cel for follicular lymphoma, similar future comparisons will be possible [Table 1, (5)]. Taken together, histologic subtype is a possible factor, though confounding factors such as volume and extent of tumor burden, CAR-T expansion/peak, and selected agent make clear associations challenging to infer.

The presence of microvascular and macrovascular insults, fluid attenuated inversion recovery (FLAIR) white matter lesions, and posterior reversible (leuko)encephalopathy syndrome (PRES) observed on imaging (56, 57) suggest an underlying vascular pathology may be at play in ICANS that leads to secondary central nervous system (CNS) injury after CRS and/or CAR-T expansion. Consistently reported endothelial risk factor for ICANS in adults are the agonist/antagonist pair of soluble vascular growth factors known as angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) supports this observation. Both Ang-1 and Ang-2 act on the tyrosine kinase receptor, Tie-2. Ang-1 stabilizes endothelium on binding to Tie-2, including closing inter-endothelial gaps (58). In contrast, high levels of Ang-2 inhibits Tie-2 activity resulting in increased endothelial permeability [(59); for review Saharinen et al. (60)]. Three studies which have examined levels of Ang-1, Ang-2, and the ratio of Ang-2/Ang-1 in axi-cel (38) and the investigational CD19-directed agents JCAR014 (28) and 19-28z (32) with generally congruent findings: Faramand et al. found baseline (pre-lymphodepletion) serum levels of Ang-2 (p = 0.0190) and Ang-2/Ang-1 ratio (p = 0.0056) were elevated in patients who developed grade 3+ neurotoxicity (F57). Among post-infusion factors, an elevated peak Ang-2/Ang-1 ratio (p = 0.0016) and depressed nadir Ang-1 level (p = 0.0298) were also associated with grade 3+ neurotoxicity (38). Gust and colleagues found a trend to increased baseline serum Ang-2/Ang-1 ratio (p = 0.09) and significant post-infusion peak Ang-2 (p = 0.0003), and Ang-2/Ang-1 ratio (p = 0.0014) elevations in patients with grade 4+ neurotoxicity, although no difference in nadir post-infusion Ang-1 was observed (p = 0.13) (28). Finally, Santomasso and colleagues also observed significant post-infusion day 7 Ang-2/Ang-1 ratio elevation (p = 0.0310), but not either day 7 Ang-2 elevation (p = 0.07) or day 7 Ang-1 depression (p = 0.053) in patients with grade 3+ neurotoxicity (32).

While the pre-treatment Ang-2/Ang-1 ratio may be a promising predictive biomarker for higher grade ICANS, there remain several caveats warranting examination. First, similar findings were not observed in pediatric populations receiving CD19-directed CAR-T agents, suggesting possible differences in pediatric vs. adult susceptibility factors (36). Second, other more non-specific plasma markers such as drops in platelet count and/or fibrinogen have had more varied success (Table 2). Third, data is limited for non-CD19 CAR-T cell agents. Finally, while suggestive of a link for vascular injury and subsequent ICANS risk, the mechanism remains unclear including the relationship of ICANS to blood brain barrier breakdown and systemic cytokine, myeloid, and/or CAR-T Infiltration into the CNS. Overall, Ang-1, Ang-2, and the ratio of Ang-2/Ang-1 remain a promising, if under-explored, risk factor for ICANS.

History of neurologic comorbidities or injury is another commonly reported risk factor for neurotoxicity (Table 4). Neurologic comorbidities may reflect prior CNS injury (e.g., seizures), peripheral nervous system (PNS) injury (e.g., neuropathy), CNS involvement of cancer (e.g., CNS lymphoma), or neurotoxic treatment exposure (e.g., CNS radiation, intrathecal methotrexate). Unfortunately, the definition of neurologic comorbidities varied significantly between studies. Gust and colleagues used an inclusive definition and found univariate (p = 0.006) and multivariate association (p = 0.002) when further controlling for tumor burden, increased CAR-T expansion, lymphodepletion regimen, and infused CAR-T dose in adults (28). A small recent adult cohort likewise clustered different sources of injury together and reported a possible univariate association (44). Gofshteyn and colleagues separately examined history of brain radiation, seizures, AED use, and pre-existing deficits in a pediatric cohort. Only pre-existing deficits (p = 0.01) was associated with encephalopathy, seizure, and aphasia, all key hallmarks of ICANS (31). Another small pediatric cohort observed 3 of 5 patients with history of pre-existing neurologic deficits developed ICANS (33). However, an association is not always observed in pediatric cohorts; Gust et al. did not observe a relationship when clustering different neurologic comorbidities except an abnormal MRI prior to treatment in a pediatric cohort (36). Overall, categorically-defined neurologic injury remains a possible risk factor, particularly in adults, though the underlying localization of injury remains unclear.

More recently neurofilament light chain (NfL) has been used to provide quantitative, rather than categorical, assessment of neural injury (Table 4). NfL is a structural component of long axons whose levels in cerebral spinal fluide (CSF) and blood increase after a neurological insult. Baseline elevations in NfL have been reported in two adult cohorts (45, 46), with elevations predating lymphodepletion. Levels further remain elevated throughout the course of treatment for up to 30 days after infusion (46). A similar relationship is not observed in a pediatric cohort, where global elevations were observed across all patients independent of ICANS risk (49). The disagreement between the adult (45, 46) and pediatric studies (49) may reflect different assay sensitivities or previous treatment dosing/regimens. Furthermore, like vascular injury markers listed above, the mechanistic link in ICANS between neuroaxonal injury, vascular injury, blood brain barrier breakdown, and CNS cytokine, myeloid, and/or CAR-T cross infiltration in ICANS remains unclear. While promising, further study is needed to account for possible confounding factors including neuropathy and systemic vascular injury unrelated to the blood brain barrier.

Cyclophosphamide and fludarabine containing regimens are commonly used for lymphodepletion prior to CAR-T cell infusion. The dose intensity of cyclophosphamide and addition of fludarabine are associated with improved CAR-T kinetics, expansion, and response in both pediatric and adult patients treated with CD19-directed agents (24–26, 35). Early studies demonstrated an association between the lymphodepletion regimen and ICANS in both univariate and multi-variate modeling (24, 25, 28). Notably all three of these studies all examined the same experimental agent, JCAR014. Unfortunately this relationship is not universally seen across CD19-directed agents and was not observed for investigational agents, including SCRI-CAR19v1 (26, 36) or 19–28 z (30, 35). Indeed a recent retrospective study of 152 patients treated with tisa-cel also revealed no association between low (termed “suboptimal” <13.8 mgxh/L) fludarabine exposure and ICANS (61). This variability may stem from a secondary relationship with neurotoxicity, where better lymphodepletion results in improved expansion and peak CAR-T levels, which in turn is associated with neurotoxicity. Fludarabine exposure is also known to cause a rare but separate neurotoxicity from ICANS, as was observed in the ZUMA-3 trial (10). Therefore, in select cases, misdiagnosis of neurotoxicity related to fludarabine may be another source of variability that confounds the clinical assessment for ICANS. Rates of ICANS in bendamustine and other fludarabine-free lymphodepletion regimes warrant further investigation. Overall, standardization of lymphodepletion regimens and monitoring/consideration for Flu-associated neurotoxicity remains key for risk modulation.

Higher doses of infused CAR-T cells has been associated with increased risk for CRS and neurotoxicity, particularly in patients with high tumor burden [Tisa-cel: (42, 44); JCAR014: (24, 25, 28)]. This association was not observed for other CD19-directed agents such as the investigational 19–28 z (30, 35) or SCRI-CAR19v1 (26, 36). Overall CAR-T dose remains an unclear risk factor, particularly given the standardization of target dosing per patient weight.

In contrast to dose, the degree of expansion remains a consistent risk for the development of ICANS across multiple studies and agents (Table 2). Following infusion, CAR-T cells redistribute and proliferate rapidly, with the early expansion coupled to cytokine production and CRS risk. The duration of expansion varies, with CAR-T targeting leukemia and multiple myeloma associated with a slightly longer duration than those targeting lymphoma [for detailed comparison of kinetics, see Liu et al. (62)]. The association between expansion and ICANS risk was first observed in the original trials leading to approval of axi-cel (6, 7, 10), brexu-cel (9), liso-cel (A69), and several investigational CAR-T products including JCAR014 (24, 25, 28), 19–28 z (30, 32), SCRI-CAR19v1 (36). Of note 41 who examined 19–28 z in a pediatric cohort did not observe a relationship between neurotoxicity and expansion (p = 0.14), though this may reflect a small sample size (n = 25). Overall, rate of expansion and maximum CAR-T expansion capability remains a key risk factor for the development of neurotoxicity.

Post-treatment peak IL-6 elevations remain tightly coupled with the development of ICANS across multiple prospective and retrospective cohorts (Table 2). This includes the seminal trials leading the to the approval of axi-cel (6, 7, 10) and brexu-cel (9), but interestingly not tisa-cel (45). Elevations in IL-6 are a known hallmark of CRS. Given the high degree of association between CRS and ICANS, further correlation between IL-6 and ICANS is unsurprising though it remains unclear which level of contribution they provide to ICANS independently and specifically.

Along with IL-6, the IL-1 signaling pathway is considered a key mediator in CRS. Preclinical models (intraperitoneal Raji tumor cells injected SCID-beige subsequently treated with human 1928 z CAR T cells) demonstrated a tight coupling between the IL-1 signaling cascade and severe CRS through macrophage/monocyte activity (64). Of the two major agonist IL-1 ligands IL-1α and IL-1β, IL-1β is the predominately macrophage and monocyte activator with secondary effects on non-immune cells. Currently, the role of IL-1 and associated monocyte activation beyond CRS in neurotoxicity remains under active investigation. In preclinical studies, SGM3 mice treated with human CD19 CAR T cells demonstrated seizures and paralysis may be abrogated through IL-1 but not IL-6 blockade (65). This has led to active clinical investigation of IL-1 receptor antagonism using anakinra to intervene on the development of CRS and ICANS.

Along with IL-6, IL-15, which promotes T and NK cell proliferation and activation, has been observed to be consistently elevated in individuals who develop ICANS (6, 7, 25, 28–33). The myeloid proliferation and activation cytokine GM-CSF is likewise a commonly observed blood marker whose peak levels after treatment are associated with ICANS in several retrospective and prospective studies (6, 7, 9, 30, 32, 33).

Both IL-10 and IFN-γ are immune-modulators, with IL-10 traditionally thought of as an immunosuppressive regulator that decreases cytokine production and antigen presentation by antigen-presenting cells (66–68). However, high levels of IL-10 have been associated with IFN-γ-mediated CD8(+) T cell cytotoxicity (68, 69). Given known marked elevations in acute ICANS of IFN-γ, this second pathway paradoxical coupling to IFN-γ elevations is possible mechanism warranting further study.

A subset of studies have examined baseline blood markers in 19–28 z (32) and axi-cel (7, 10, 38, 46). Santomasso et al. found baseline elevations in IL-6, IL-10, GM-CSF, and G-CSF, but not ferritin, in patients treated with 19–28 z who went on to develop ICANS. Faramand et al. (38) also observed baseline elevations in IL-6 in patients treated with Axi-cel who went on to develop ICANS. In contrast to Santomasso et al., baseline ferritin elevations were observed in this study and in more recent studies (7, 38, 46). Overall, baseline IL-6 and ferritin elevations are a possible risk factor, though data is limited beyond a subset of CD19-directed agents.