Miscellaneous Complications of Chimeric Antigen Receptor T-Cell Therapy

Hemophagocytic Lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is a rare and potentially life-threatening complication of autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy, reported in 1% to 3.5% of patients with relapsed or refractory (r/r) large B-cell lymphoma (LBCL). In the ZUMA-1 trial, only one of the 108 patients treated with axicabtagene ciloleucel (axi-cel) developed HLH, resulting in patient’s death, while no cases of HLH were reported in the 269 patients who received lisocabtagene maraleucel (liso-cel) in the TRANSCEND study or the 111 patients who received tisagenlecleucel (tisa-cel) in the JULIET study. Additional information about the incidence of HLH derives from real-world data. In a real-world study of axi-cel, only 1 of the 275 patients who received axi-cel for LBCL developed HLH, leading to death. In another study by Sandler et al., the rate of HLH was found to be 3.5%. However, the study reported significant heterogeneity in diagnostic criteria for HLH, with most centers using HLH-2004 criteria (43%), followed by the H-score (15%).

HLH can occur as a primary or a secondary disorder. Primary or familial HLH occurs mostly in the pediatric setting and is caused by inherited mutations in genes involved in the elimination of activated macrophages by natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). The common genetic defects in familial HLH include PRF1/perforin, UNC13D/Munc13-4, STX11/Syntaxin 11, and STXBP2/Munc18-2. The defects in perforin-dependent cytotoxicity result in subsequent macrophage persistence, in the presence of appropriate triggers, leading to overproduction of myeloid-derived cytokines and increased T-cell activation, ultimately culminating in a hyperinflammatory syndrome ( Fig. 37.1 ).

HLH can also be secondary to macrophage activation because of infections, malignancies, or autoimmune diseases, in the absence of genetic predisposition. While secondary HLH (sHLH), also called macrophage activation syndrome ( MAS ), can occur in any patient, individuals with heterozygous mutations in primary HLH-related genes are at a higher risk. In addition, high tumor burden and marked T-cell expansion can increase the risk of developing HLH/MAS in patients treated with CAR T-cell therapy. The exact mechanism of HLH caused by CAR T-cell therapy remains unknown. CAR T-cell and related non-CAR T-cell proliferation is typically associated with significant cytokine release, and HLH potentially represents an extreme of the spectrum of cytokine release syndrome (CRS). The types of cytokines involved in both these events are similar and include Th1 and myeloid-driven inflammatory molecules such as interleukin (IL)-18, IL-8, IP10, MCP1, MIG, and MIP1β. This partially explains the similarity in the clinical findings and the laboratory abnormalities observed in both CRS and HLH.

The diagnosis of HLH is currently based on clinical and laboratory criteria. The original diagnostic criteria for HLH were defined and revised by the Histiocyte Society in 2004 and are known as HLH-2004 ( Table 37.1 ). According to the latter, HLH can be diagnosed if a patient has either a molecular diagnosis of HLH, based on a mutation in one of the genes outlined earlier; or five out of the eight following criteria: fever, splenomegaly, cytopenia (affecting two of three lineages in the peripheral blood), hypertriglyceridemia or hypofibrinogenemia, hyperferritinemia, elevated soluble CD25, hemophagocytosis in bone marrow (BM), spleen, lymph nodes, or low/absent NK-cell cytotoxicity. As the HLH-2004 criteria were developed primarily for familial HLH and pediatric patients, new diagnostic criteria were subsequently proposed for secondary HLH/MAS, namely the H-score (see Table 37.1 ). The H-score was developed by assessing 10 variables known to be associated with HLH. On multivariate analysis of 209 patients, 9 of the 10 variables were found to remain associated with the diagnosis of HLH. These included underlying immunosuppression, fever, organomegaly, number of cytopenias, hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia, elevated aspartate aminotransferase, and BM evidence of hemophagocytosis. These were assigned scores varying from 18 for immunosuppression to 64 for hypertriglyceridemia, with an H-score of 90 being associated with < 1% probability and a score of 250 being associated with > 99% probability of HLH.

However, because of the similarity of biologic mechanisms and clinical-laboratory presentation of CRS and HLH, the HLH-2004 and the H-score cannot be homogeneously applied to the diagnosis of HLH after CAR T-cell therapy, and their applicability in this setting remains unclear. To this regard, new criteria for the diagnosis of sHLH caused by CAR T-cell therapy have been proposed by Neelapu et al., with the goal to help better differentiate it from CRS (see Table 37.1 ). These novel diagnostic criteria include the concurrent presence of peak ferritin levels > 10,000 ng/mL during the CRS phase and development of any two of the following organ toxicities: grade ≥ 3 liver toxicity, grade ≥ renal toxicity, grade ≥ 3 respiratory toxicity, BM, and/or tissue hemophagocytosis. Prospective validation of these criteria is needed before supporting its consistent use across patients treated with CAR T-cell therapy.

Effective guidelines for the management of sHLH/MAS in patients treated with CAR T-cell therapy are missing. Most patients developing HLH in this setting are typically managed according to CRS guidelines, which includes the use of IL-6–directed therapy (tocilizumab) and corticosteroids, based on efficacy observed in cases of HLH secondary to bispecific antibodies, such as blinatumumab. However, in severe cases, resistant both to corticosteroids and tocilizumab, other therapies should be considered.

One of these includes etoposide, a standard treatment option for sHLH/MAS arising from causes other than CAR T-cell therapy. In the setting of sHLH/MAS caused by CAR T-cell therapy, etoposide should only be used if no improvement is seen after at least 48 hours of treatment with tocilizumab and corticosteroids and may be repeated if no improvement is observed after 4 to 7 days from the first dose.

Other agents that have been used for the management of refractory HLH include cyclosporine, methotrexate, and intrathecal cytarabine, typically used for patients with HLH-related neurotoxicity. Anakinra has been used for refractory sHLH/MAS in small studies, both in the pediatric and adult population, with variable efficacy. Emapalumab, a monoclonal antibody targeting interferon-gamma, has been approved for the treatment of refractory primary HLH, based on a study in 34 pediatric patients, which resulted in a 65% overall response rate. Its safety and efficacy for sHLH is being currently investigated in an ongoing clinical trial (NCT03985423), and its use in this setting remains experimental.

While these agents have been used mainly in non-CAR-T settings, increasing, though anecdotal, evidence of efficacy is also being reported in patients treated with CAR T-cell therapy.

Given the rarity of this condition and the lack of definitive guidelines for its diagnosis and management, prospective studies aimed at shedding further light on the biologic mechanisms of post-CART HLH and identifying novel targets for its treatment are desperately needed.

Graft-Versus-Host Disease

Graft-versus-host disease (GVHD) has been rarely reported with autologous CAR T-cell therapy, but it represents a major concern with the use of experimental allogeneic products. Although no cases of GVHD were reported in the ZUMA-1, TRANSCEND, and JULIET studies, a recent study that focused on late adverse events of CAR T-cell therapy reported GVHD in 20% (3/15) of patients who had a history of allogeneic hematopoietic cell transplant (allo-HCT). This included 86 patients with r/r acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL), and chronic lymphocytic leukemia (CLL), treated with second-generation autologous CAR T-cell therapy. No GVHD was reported in patients who had not previously received an allo-HCT. Acute GVHD was reported in 6.6% (1/15) of patients who had no previous history of GVHD, 1.9 months after CAR T-cells infusion, while chronic GVHD was described in 13% (2/15) of patients, either with or without previous GVHD, 2.8 months and 3.2 months after CAR T-cells infusion. Of interest, the median time from allo-HCT to CAR T-cell therapy in this study was 37 months (range, 3.2–143.6 months), and no active GVHD was observed at the time of CAR T-cell infusion. All cases required treatment: acute GVHD resolved after receiving prednisone 1 mg/kg, while the two cases of chronic GVHD required additional immunosuppression. In another study, acute GVHD was reported in 67% (10/15) of B-ALL patients who received autologous anti-CD19 CAR T-cell therapy after allo-HCT, including grade I–II in six cases, and grade III–IV in four, but no GVHD-related mortality.

The biologic mechanism of GVHD with the use of autologous CAR T-cell products remains largely unknown and is thought to be mainly driven by previous and recent exposure to allogeneic stem cells. To this regard, the T-cell receptor (TCR) on donor-derived αβ T-cells, once immunosuppression is discontinued or tapered to allow CAR T-cell infusion, can react with the host antigen-presenting cells, leading to release of FAS ligand, granzyme, perforin, and serine protease, resulting in cell death and organ damage. However, despite the potentially higher risk of GVHD with experimental allogeneic CAR T-cell products, the rates of GVHD reported in most studies are relatively low. To this regard, recent mouse models have shown progressive loss of effector function, proliferative potential, and clonal deletion for allogeneic reactive T-cells in response to excessive and dual stimulation from TCR and CAR signaling. Significantly higher expressions of PD-1, LAG3, and TIM3 as well as higher levels of phosphorylated PKCa, pERK1/2, pS6, pSTAT1, pSTAT3, and pSTAT5 were observed in this model, suggesting an enhanced T-cell stimulation followed by exhaustion caused by dual CAR and TCR stimulation ( Fig. 37.2 ). On the other hand, nonreactive allogeneic CAR T-cells were able to retain their antitumoral activity in the same model without producing GVHD because of isolated CAR stimulation.

Several steps have been taken to reduce the risk of GVHD with experimental allogeneic CAR T-cell products. As GVHD is mainly driven by activation of the αβ TCR, CAR T-cells with disrupted αβ TCR have been developed through gene editing using zinc-finger nucleases, transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) system. Through the use of TALEN, CAR T-cells with disrupted αβ TCR and CD52 domains have demonstrated tumor response but no GVHD in mouse models. In this model, alemtuzumab was used for the destruction of TCR+CD52+ CAR T-cells, thereby mediating lymphodepletion and promoting engraftment of the TCR/CD52-deficient CD19 CAR T-cells.

Two multicenter phase 1 trials, which enrolled pediatric (7) or adult (14) patients with r/r B-ALL, investigated the safety and efficacy of UCART19, an allogeneic product in which the αβ TCR and CD52 regions were disrupted through TALEN, showed grade 1 acute GVHD in only 10% (1 child and 1 adult) of patients. CRISPR/Cas-9 has since been widely used for genome editing at various targets, including CAR T-cells, to enhance their potency and uniform expression. Eyquem et al. designed anti-CD19 CAR+ TCR cells through the use of CRISPR/Cas9 directed to the T-cell receptor α constant (TRAC) locus, resulting in 95% CAR+ TCR cells, with enhanced antitumor effect in mouse models. A recent phase 1 study using universal CD19/CD22-targeting CAR T-cells (CTA101) in six patients with r/r ALL, which had CRISPR/Cas9-disrupted TRAC region and CD52 gene, demonstrated no cases of GVHD or any other genome editing-associated adverse events. A final method to decrease the risk of GVHD is the use of non–αβ-T cells, which include NK cells (CAR-NK cells) and γδ CAR T-cells. In a recent study of 11 patients with r/r CD19 positive hematologic malignancies treated with allogeneic CAR-NK cells, no cases of GVHD were reported, despite human leukocyte antigen mismatch. Also γδ CAR T-cells targeting CD19 have shown effectiveness in vitro and in vivo, and their safety is being currently investigated in an ongoing phase 1 study (NCT0473547). However, the use of NK-CAR and γδ CAR T-cells is limited by interindividual disparity of response against the same target, potential for in vivo tumorigenicity, risk of alloimmune responses, and lack of transduction efficacy/activity. Also, γδ CAR T-cells require a long expansion time, leading to T-cell exhaustion. To overcome these limitations, alternative sources have been used for these cells, including same donor, umbilical cord blood, placenta-derived stem cells, and induced pluripotent stem cells. Studies are ongoing to assess the safety and efficacy of these novel products, and their use is to be considered experimental at this point.

The treatment of GVHD caused by CAR T-cell currently follows standard treatment protocols for GVHD. For acute GVHD, the initial therapy is typically represented by corticosteroids, with an initial dose of 2 mg/kg/day of methylprednisolone (or equivalent) followed by a gradual taper, based on clinical improvement. Other immunosuppressive agents can be added to corticosteroids in case of corticosteroid resistance or dependency (i.e., inability to taper), or used as a second-line therapy, including etanercept, infliximab, methotrexate, and mycophenolate mofetil. However, the use of corticosteroids and/or other immunosuppressive agents may hamper CAR T-cell activity, and should be performed with caution. A deeper understanding of the biologic mechanisms of GVHD with autologous and allogeneic CAR T-cells may help in developing more targeted therapeutic strategies and contribute to better preserve CAR T-cell antitumoral activity.

Cytopenia

Cytopenia, including neutropenia, anemia, and thrombocytopenia, is commonly observed with the use of CAR T-cell therapy, with early onset in the majority of patients, and persistence beyond day 30 in about one-third of individuals. Neutropenia, anemia, and thrombocytopenia of any grade during the first 30 days after CAR T-cells infusion have been reported, respectively, in 84%, 66%, and 58% of patients who received axi-cel in the ZUMA-1 trial; 60%, 48%, and 13% of patients who received liso-cel in the TRANSCEND study; and 20%, 48%, and 31% of patients who received tisa-cel in the JULIET study. Severe cytopenia can occur as early as the day of CAR T-cell infusion, and persist up to 2 years afterward.

It remains unclear whether early cytopenia is merely caused by lymphodepleting chemotherapy or acute inflammation-related BM suppression. The lymphodepleting chemotherapy regimens typically used for CAR T-cell therapy consist of fludarabine and cyclophosphamide, a regimen typically used for CLL treatment, and associated with early and prolonged cytopenia in that setting. However, of interest, in a retrospective study including 83 patients with LBCL, B-ALL and multiple myeloma treated with different CAR T-cell products, early cytopenia was more common in patients who developed CRS, immune effector cell-associated neurologic syndrome (ICANS), and those with elevated C-reactive protein and ferritin, suggesting this may be caused by CAR T-cell activity. In particular, patients with grade 4 CRS developed more rapid and severe cytopenia, with a longer time to recovery as compared to patients with lower grade of CRS. These associations were not observed at 3 months, suggesting different biologic mechanisms for persistent cytopenia in these patients.

In a post hoc analysis of the ZUMA-1 and ZUMA-9 trials including 31 patients, 15 of whom developed persistent severe cytopenia, the latter was associated with the number of previous systemic therapies, low absolute lymphocyte count, and poor performance status, suggesting a potential role for CAR T-cell engraftment in this phenomenon, in addition to marrow reserve. In another retrospective study, 8 out 21 patients who received commercial axi-cel for LBCL were diagnosed with persistent severe cytopenia, and the latter was associated with baseline thrombocytopenia and with the onset of CRS, again suggesting that lymphodepleting chemotherapy may not be the only predisposing factor.

While lack of marrow reserve could be responsible for persistent cytopenia, as suggested by its higher incidence among previous recipients of autologous or allo-HCT, novel biologic mechanisms have been recently unveiled. Serum levels of stromal-derived factor-1 (SDF-1)/CXC-ligand 12 were measured in 16 patients who received experimental anti-CD-19 CAR T-cell therapy for B-ALL and lymphoma and found to correlate with absolute neutrophil count levels 21 days after CAR T-cell therapy. Delayed B-cell recovery following CAR T-cell therapy, in fact, may lead to low SDF-1 levels, resulting in low egress of neutrophils, similarly to what was observed during CD20+ B-cell recovery after rituximab use, the latter considered to be the mechanism of rituximab-induced late onset netropenia.

No formal guidelines are currently available for the management of early-onset and/or persistent cytopenia in patients treated with CAR T-cell therapy ( Table 37.2 ). Granulocyte colony-stimulating factors (G-CSF) is typically used for severe neutropenia in this setting. Concern has been raised regarding its possible association with increased CRS rates, likely caused by an immunostimulatory effect and subsequent increase in IL-6 levels, mostly observed with the use of granulocyte macrophage CSF. Of note, G-CSF has instead been safely used as a prophylactic strategy after either axi-cel or tisa-cel for patients with r/r LBCL, and no increase in toxicity rates was observed. Anemia and thrombocytopenia are typically treated according to transfusional guidelines, but in case of excessive or prolonged transfusional dependence, therapy-related myelodysplastic syndrome needs to be ruled out. The latter was diagnosed in 4 out of 15 patients with persistent cytopenia after axi-cel, after a median of 13.5 months (range, 4–26 months) from CAR T-cell therapy infusion. It remains unclear whether erythropoietic and/or thrombopoietic stimulating factors could be effective in this setting. Erythropoietin and thrombopoietin have shown a favorable response and safety profile in patients with anemia and thrombocytopenia after HCT, but no data are available for CAR T-cell therapy. In rare instances, treatment strategies typically reserved for aplastic anemia, such as antithymocyte globulin and cyclosporine, or autologous and allo-HCT, have been successfully used for its management, whereas preclinical data supporting the use of dasatinib to suppress CAR T-cell function and associated toxicities are being increasingly reported ( Table 37.3 ).