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Introduction

Chimeric antigen receptor (CAR) T cell therapy serves an important role in cancer treatment (1) and CAR T cells have been approved for treating hematological malignancy (2). CAR T cells may cause lethal toxicity through several mechanisms (3–5) and cytokine release syndrome (CRS) is the most common toxic effect (6). Its symptoms often mimic those of influenza, including fever, headache, malaise, myalgia, rigor and anorexia (1). However, some severe cases progress to life-threatening symptoms, such as hypotension, hypoxia, pulmonary edema, pleural effusion and tachycardia (7). CAR T cell-associated hemophagocytic lymphohistiocytosis (CARHLH), which carries a high risk of lethality, shares similar clinical manifestations with CRS, such as fever, multiorgan dysfunction, hyperferritinemia and hypercytokinemia (8,9).

A retrospective analysis by the European Society for Blood and Marrow Transplantation (EBMT) included 201 patients receiving CAR-T therapy from 114 centers across 24 countries, with a CARHLH incidence rate of 3.48% between 2016 and 2018 (10). A retrospective study of 59 patients with relapsed/refractory B cell acute lymphoblastic leukemia/lymphoblastic lymphoma treated with CD22 CAR T cells reported that 52 patients (88.1%) developed CRS, of whom 21 (40.4%) developed CARHLH (all following CRS onset), including six of 12 patients with severe (grade3/4) CRS (11). Another study on 27 patients with relapsed and/or refractory CD19-positive acute lymphoblastic leukemia treated with CD19 CAR T cells reported that 11 (40%) had CRS and four (14.8%) had CRS and CARHLH (12); all patients with CARHLH either concurrently or previously experienced CRS. Typically, CRS resolves or is fully resolved prior to the onset of CARHLH (11,13). The progression of CRS and CARHLH is connected, making it challenging to distinguish between these (11,13).

However, previous studies have indicated that the pathogenesis, treatment and prognosis of these diseases differ (11,13,14). Patients suspected of having CARHLH should be diagnosed as soon as possible and promptly receive effective treatment to control disease progression and improve outcomes (1,5,10,11,15–18). Therefore, early identification and prompt treatment are essential for improving patient prognosis.

HLH

HLH is a severe inflammatory syndrome caused by abnormal immune regulation (19). A study of patients with primary HLH (pHLH) revealed that the outcome is usually rapidly fatal, with a median survival <2 months without timely treatment (20). HLH is characterized by uncontrolled activation of T cells and macrophages (21). The cardinal features include hyperferritinemia, fever, hepatosplenomegaly, cytopenia and hemophagocytic phenomena in the spleen, liver, bone marrow and lymph nodes (17,22,23). HLH is classified as primary or secondary, based on its etiology. pHLH arises from immune deficiencies associated with mutations in genes responsible for the cytotoxic functions of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). The onset of pHLH commonly occurs in infants aged <1 year (21). Secondary HLH (sHLH) is more common in adults with underlying conditions including infection, autoimmune disease, autoinflammatory responses and cancer (22,24). Infection is the most frequent trigger of sHLH (25).

Genetic or acquired immunoregulatory defects imbalance the regulatory pathways responsible for the natural termination of immune or inflammatory responses, leading to sustained and excessive activation of T and antigen-presenting cells. Abnormal NK cell function is also observed in patients with HLH (19). NK cells regulate the initial responses of antigen-presenting cells to pathogens and attenuate signals communicated by antigen-driven T cells. NK cells also serve a role in the contraction of immune responses by culling activated T cells and histiocytes during the later stages of antigen-driven activation (26). The abnormal function of CTLs and NK cells may lead to continuous activation and proliferation of macrophages triggered by antigens. Activated macrophages persistently proliferate and release cytokines that promote the recruitment and expansion of lymphocytes and other inflammatory cells. This can lead to organ damage and systemic manifestations of hypercytokinemia. In addition, activated macrophages non-selectively phagocytose hematopoietic components, resulting in observable hemophagocytosis in the bone marrow, lymph nodes and spleen (27).

HLH is a rare condition: The incidence of HLH (including pHLH and sHLH) in individuals aged <18 years in the United States is 1/100,000 (28). In Sweden, the incidence of familial HLH (fHLH) in children is 1.2/1,000,000 (20), whereas that of sHLH in children is 0.19/100,000 (29). HLH is a life-threatening disease. A Swedish study of 32 children with fHLH, with a median survival of only 2–3 months (20). However, mortality rates differ between subtypes, with malignancy-associated HLH having the worst prognosis (17,18). The 2-month survival rate for malignancy-associated HLH was 52% overall from 1997 to 2018 in Sweden (29).

CAR T cell therapy

CAR T cell therapy is based on the isolation of T cells from peripheral blood, which are genetically engineered to express CARs. These cells are expanded in vitro and then re-infused into patients with cancer. CAR T cells undergo non-specific expansion in vivo and simulate immune responses against cancer cells (30). This modification enables the modified T cells to recognize and attack tumor cells independently of major histocompatibility complex (MHC) engagement (2,31–33). CAR T cell therapy targets tumor cells independently of antigen processing and presentation, making them resistant to tumor escape mechanisms associated with MHC loss. Furthermore, this approach can effectively recognize antigens such as glycolipids, proteins with abnormal glycosylation and conformational epitopes (34,35). CAR T cell therapy has notable benefits in the treatment of various malignancies (33,36–38). The response rates of patients with relapsed or refractory B non-Hodgkin's lymphoma is 52–82% (38,39). In patients with relapsed or refractory B cell ALL, the rate of complete remission is 90%, with a 2-year overall survival rate of 73% and an event-free survival rate of 49.5% (40). The objective and complete remission rates in patients with relapsed or refractory multiple myeloma are 85 and 45%, respectively. The median progression-free survival is 11.8 months (41).

However, CAR T cells may damage normal tissue by cross-reacting with antigens that are not expressed in tumor cells (3,4). CAR-targeted tumor-associated antigens may be expressed in normal tissue, leading to CAR T cell-mediated damage to normal tissue cells (5). CAR T cells can cause complications such as CRS and neurotoxicity. Additionally, both the malignancies and the chemotherapy that patients may receive before CAR T cell infusion can impair immune function, thereby increasing the risk of infection (42). CAR T cell therapy has the potential to trigger life-threatening toxicity, such as CARHLH (5).

CRS

CRS is the most frequent adverse effect associated with CAR T cell therapy. A meta-analysis of 2,592 patients from 84 studies who received CAR T cell therapy reported that the all-grade CRS rate is 77%, while the grade ≥3 CRS rate is 29% (43). This is a systemic inflammatory response triggered by the release of pro-inflammatory cytokines from activated CAR T, ruptured cancer and other immune cells. The interplay between CAR T and cancer cells triggers the activation of immune cells, such as macrophages and endothelial cells, which amplifies the production of cytokines (such as IL-6, IFN-γ, IL-8 and IL-10), leading to a cytokine storm (44).

Furthermore, cytokines, perforin and granzyme from CAR T cells induce pyroptosis in tumor cells. Pyroptosis is a form of programmed cell death distinct from apoptosis, marked by cell swelling, lysis and the release of cellular contents and proinflammatory factors (Fig. 1) (45). The majority of CRS cases develop within the first 14 days of infusion and persist for 7 or 8 days (7,39,46,47). In most cases, CRS resolves naturally without intervention, and patients typically require only symptomatic care (1,5,7,15,16,48,49). The initial symptoms include fever, headache, malaise, myalgias, and rigors (1). However, some severe cases progress to life-threatening symptoms, such as hypotension, hypoxia, pulmonary edema, pleural effusion and tachycardia (7). These symptoms may lead to multiorgan failure and require intensive care management (1,7). A meta-analysis by Lei et al (43) reported a CRS mortality rate <1%.

Figure 1. Pathogenesis of cytokine release syndrome.

CARHLH

CARHLH is a severe toxicity associated with CAR T cell therapy (9). CARHLH typically manifests with symptoms such as fever, hepatosplenomegaly, peripheral blood trilineage or bilineage cytopenia, elevated cytokine levels and increased soluble CD25. CARHLH can be life-threatening and is associated with multiorgan dysfunction, infection and coagulopathy (9). A case report described a patient with multiple myeloma who achieved a stringent complete response in the primary disease evaluation before CAR T cell infusion (50). However, after the infusion of CAR T cells, the patient developed CARHLH and succumbed to multiple infections caused by bacteria, fungi and viruses (50). The cause of sHLH varies, with numerous underlying factors, and its pathophysiological mechanisms are incompletely understood. The pathogenesis of CARHLH, a subtype of sHLH associated with immunotherapy, has not been fully elucidated. Lichtenstein et al (11) observed an elevated T:NK cell ratio and a decreased percentage of peripheral blood monocytes in patients with CARHLH. Patients who develop CARHLH exhibit relative NK cell lymphopenia before infusion, which becomes more severe during peak CAR expansion in patients with CARHLH (11).

NK cells inhibit excessive activation and expansion of CD8+ T cells (51). Deficiency of NK cells may lead to the over-expansion of CAR T cells and delayed T cell contraction (51). CAR T and ruptured tumor cells release proinflammatory cytokines in HLH. Previous studies have demonstrated that tumor cell pyroptosis results in the release of IL-18 and other cytokines (52,53). Excessive IL-18 can initiate signaling cascades that activate nuclear factor κB or STAT3, MAPK and JNK pathways, ultimately leading to the robust production and dissemination of IFN-γ (52,53). This activates macrophages to release more cytokines, thereby creating a positive feedback loop of inflammation, and generating the clinical manifestations associated with CARHLH. Moreover, excessive IL-6 production during the disease may affect the synthesis of perforin and granzyme, leading to impaired NK cell activity and perpetuating immune imbalance (Fig. 2) (49,54,55).

Figure 2. Potential pathogenesis of CARHLH. CARHLH, chimeric antigen receptor T cell-associated hemophagocytic lymphohistiocytosis; NK, natural killer.

To the best of our knowledge, there is a lack of large studies investigating CARHLH (19). CARHLH is rapidly becoming a fatal condition. In a clinical study (56), 100 patients with relapsed or refractory large B cell lymphoma received CAR T cell therapy. CARHLH occurred in five cases, of which two were treated with anakinra (a recombinant human IL-1 receptor antagonist), etoposide and corticosteroids but succumbed to CARHLH on days 15 and 71 of treatment (56). A multicenter clinical trial (38) of 101 patients treated with CD19 CAR T cell therapy observed that 94 patients (93%) developed CRS, and one patient developed CRS progressed and succumbed to CARHLH. By contrast, the CRS manifestations in the other patients resolved after treatment, with one patient succumbing to cardiac arrest (38). A retrospective review (57) of 105 patients with relapsed or refractory large B cell lymphoma who received CAR T cell therapy reported that six patients (5.7%) developed CARHLH. The progression-free and overall survival rates of patients who developed CARHLH were markedly worse than those of patients without HLH (57). Hines et al (12) conducted a cohort study of 27 patients with relapsed or refractory CD19 acute lymphoblastic leukemia. Following CAR T cell therapy, four patients (14.8%) developed CARHLH, one succumbed to CARHLH and three succumbed to leukemia, with a median survival of 44.5 days. The aforementioned study revealed that patients who developed CARHLH experienced worse outcomes, a greater incidence of non-response to CAR T cell therapy and markedly decreased overall survival (12).

Although CARHLH and CRS have similar clinical manifestations, there are differences in their pathogenesis. Patients with CARHLH have more severe and persistent clinical manifestations; therefore, early recognition and rational treatment are key (11).

Diagnosis and differentiation of CRS and CARHLH

Diagnosis of CRS is primarily based on clinical symptoms. The American Society for Transplantation and Cellular Therapy (ASTCT) defines CRS following immunotherapy as ‘a supraphysiologic response following any immune therapy that results in the activation or engagement of endogenous or infused T cells and/or other immune effector cells. Symptoms can be progressive, must include fever at the onset, and may include hypotension, capillary leak (hypoxia) and end organ dysfunction’ (58). CRS should be suspected if ≥1 of the following four manifestations occurs within 3 weeks of CAR T cell infusion: Fever (temperature ≥38°C), hypotension (systolic blood pressure <90 mmHg), hypoxia (arterial oxygen saturation <90%) or organ toxicity (48). Laboratory tests are key for identifying other diseases, such as sepsis and tumor lysis syndrome, because the clinical manifestations of CRS are non-specific. If CRS is suspected, a thorough evaluation of the patient should be conducted, and treatment should be initiated accordingly (1). The ASTCT consensus provides grading criteria for CRS based on clinical manifestation (Table I) (58).

Table I. American Society for Transplantation and Cellular Therapy consensus grading for CRS.

Table I.

American Society for Transplantation and Cellular Therapy consensus grading for CRS.

Grade	CRS
1	Fevera, with or without constitutional symptoms
2	Fevera, with hypotension not requiring vasopressors and/or hypoxia requiring the use of oxygen delivered by
	low-flow nasal cannulab or blow-by
3	Fevera, with hypotension requiring one type of vasopressor with or without vasopressin and/or hypoxia
	requiring high-flow nasal cannulab, face, non-rebreather or Venturi mask
4	Fevera, with hypotension requiring multiple vasopressorsc and/or hypoxia requiring positive pressured
5	Death due to CRS in which another cause is not the principle factor leading to this outcome

a Temperature ≥38°C not attributable to any other cause;

b oxygen delivered at ≤6 l/min. Low flow also includes blow-by oxygen delivery. High-flow nasal cannula is defined as oxygen delivered at >6 l/min;

c excluding vasopressin;

d continuous positive airway pressure, bilevel positive airway pressure, intubation, mechanical ventilation. CRS, cytokine release syndrome.

To the best of our knowledge, there are currently no standardized diagnostic criteria for CARHLH, although both the guidelines released by the ASTCT in 2023 and the previous HLH diagnostic criteria are used in conjunction with the clinical manifestations of patients to diagnose CARHLH (50,59,60). The diagnosis should be accompanied by close clinical observation and repeated laboratory tests. The HLH-2004 criteria, H-score, Takagi criteria and MD Anderson criteria are used to help diagnose CARHLH (Table II) (9,10,48,61–63). The 2023 ASTCT expert recommendation on CARHLH/IEC-HS use the term ‘immune effector cell-associated HLH-like syndrome (IEC-HS)’ to describe CARHLH and describe the definition of IEC-HS. The guideline recommends a diagnosis of CARHLH based on clinical manifestations, with a considerable elevation in serum ferritin levels being the primary criterion for diagnosis (Table II) (9). ASTCT proposed a grading schema for this condition (Table III). The MD Anderson criteria (10) are the diagnostic standards for CARHLH. However, their clinical application has not yet become widespread (10). The HLH-2004 criteria are currently the accepted diagnostic protocol for HLH and are used to diagnose CARHLH (10,61). The H-score identifies patients with HLH by assigning points based on clinical manifestation (62), whereas the Takagi criteria diagnose HLH based on a combination of pathological and clinical characteristics (63). The HLH-2004 protocol, H-score and Takagi criteria were not developed specifically for CARHLH, and therefore have limitations in diagnosing CARHLH. A study (14) of 20 adult and 16 pediatric HLH patients from Belgium. The study found that the H-score demonstrated better sensitivity and specificity than the HLH-2004 protocol in the early diagnosis of the disease (14). For diagnosing HLH in adults, the H-score is more effective than the traditional HLH-2004 criteria. However, the sensitivity and specificity of the H-score decrease in the diagnosis of deteriorating diseases (14).

Table II. Criteria commonly used to diagnose CARHLH.

Table II.

Criteria commonly used to diagnose CARHLH.

Criteria	Components	(Refs.)
HLH-2004 (for familial HLH)	Molecular diagnosis consistent with HLH or diagnosis criteria for HLH fulfilled by five of: i) Fever; ii) splenomegaly; iii) cytopenia (affecting ≥2/3 lineages in the peripheral blood); hemoglobin <90 g/l, platelets <100×109/l, neutrophils <1×109/l; iv) hypertriglyceridemia and/or hypofibrinogenemia; v) hemophagocytosis in bone marrow or spleen or lymph nodes; vi) low or absent NK cell activity; vii) ferritin ≥500 µg/l and viii) soluble CD25 (soluble IL-2 receptor) ≥2,400 U/ml	(61)
H-score (for all sHLH/MAS)	Known underlying immunosuppression, fever, organomegaly, mono-, bi- or tri- lineage cytopenia, ferritin, triglyceride, fibrinogen, aspartate aminotransferase, hemophagocytosis on bone marrow aspirate	(62)
Takagi criteria (for sHLH/MAS post-HSCT)	A total of two major, or one major and all four minor criteria. Major criteria: Engraftment delay, primary or secondary failure or histopathological evidence of hemophagocytosis. Minor criteria: Fever, hepatosplenomegaly, elevated ferritin or lactate dehydrogenase	(63)
MD Anderson (for CAR T cell only)	Ferritin >10,000 µg/l and two of: i) Grade >3 increase in serum transaminases or bilirubin; ii) grade >3 oliguria or increase in serum creatinine; iii) grade >3 pulmonary oedema or iv) histological evidence of hemophagocytosis in bone marrow or organs	(10)
2023 ASTCT expert consensus recommendations (definition of IEC-HS)	Development of a pathological and biochemical hyperinflammatory syndrome independent from CRS and ICANS that manifests with features of macrophage activation/HLH, is attributable to IEC therapy and is associated with progression or new onset of cytopenias, hyperferritinemia, coagulopathy with hypofibrinogenemia and/or transaminitis	(9)
2023 ASTCT expert consensus recommendations (criteria for identifying IEC-HS)a	Clinical/laboratory manifestation. Common manifestationsb: Elevated ferritin [>2 × ULN or baseline (at time of infusion)] and/or rapidly rising (per clinical assessment; required); onset with resolving/resolved CRS or worsening inflammatory response following initial improvement with CRS-directed therapyc; hepatic transaminase elevationd [>5 × ULN (if baseline was normal) or >5 × baseline if baseline was abnormal]; hypofibrinogenemia (<150 mg/dl or <LLN)e; hemophagocytosis in bone marrow or other tissuee; cytopenias (new onset, worsening or refractoryf). Other manifestations: Lactate dehydrogenase elevation (>ULN); other coagulation abnormality (for example, elevated PT/PTT); direct hyperbiliru-binemia; new-onset splenomegaly; fever (newg or persistente); neurotoxicity; pulmonary manifestations (for example, hypoxia, pulmonary infiltrate or pulmonary edema); renal insufficiency (new onset); hypertriglyceridemia (fasting level, >265 mg/dle)	(9)

a Diagnosis made only when not attributable to alternative etiologies, including CRS, infection and/or disease progression;

b findings manifest as a temporally clustered presentation, typically emerging within a narrow observational window (e.g., ≤72 h post-onset);

c although the majority of cases of IEC-HS have been seen with antecedent CRS, this may not always be the case;

d consistent with grade 3 hepatic transaminase elevation according to Common Terminology for Adverse Events version 5.0 (94);

e according to HLH-2004;

f generally ≥1 lineage will be a grade 4 cytopenia (platelets, neutrophils or hemoglobin);

g distinguished from CRS onset or recrudescence. CARHLH, Chimeric Antigen Receptor T Cell Immunotherapy-Associated Hemophagocytic Lymphohistiocytosis; NK, Natural Killer; sHLH/MAS, Secondary Hemophagocytic Lymphohistiocytosis/Macrophage Activation Syndrome; HSCT, Hemopoietic Stem Cell Transplantation; ASTCT, The American Society for Transplantation and Cellular Therapy; ICANS, Immune Effector Cell-Associated Neurotoxicity Syndrome; IEC, Immune Effector Cell; IEC-HS, Immune Effector Cell-Associated HLH-Like Syndrome; ULN, upper Limit of Normal; LLN, Lower Limit of Normal; PT/PTT, Prothrombin Time/Partial Thromboplastin Time.

Table III. ASTCT consensus grading for IEC-HS.

Table III.

ASTCT consensus grading for IEC-HS.

Grade	Descriptiona
1	Asymptomatic or mild symptoms; requires observation and/or clinical and diagnostic evaluation. Intervention not indicated
2	Mild to moderate symptoms, with intervention indicated (for example, immunosuppressive agents directed at IEC-HS, transfusion for asymptomatic hypofibrinogenemia)
3	Severe or medically significant but not immediately life- threatening (for example, coagulopathy with bleeding requiring transfusion support, or hospitalization required for new-onset acute kidney injury, hypotension or respiratory distress)
4	Life-threatening consequences: Urgent intervention indicated (for example, life-threatening bleeding or hypotension, respiratory distress requiring intubation, dialysis indicated for acute kidney injury)
5	Death

a Not attributable to other causes; defined by development of pathological and biochemical features of macrophage activation/HLH that is attributable to IEC therapy and associated with progression or new onset of cytopenia, hyperferritinemia, coagulopathy with hypofibrinogenemia and hepatic transaminitis (>5 ULN). While HLH-like manifestations are frequently observed in patients with severe CRS (as defined by ASTCT), IEC-HS is often delayed in onset and manifests as CRS is resolved/resolving. HLH, hemophagocytic lymphohistiocytosis; ASTCT, American Society for Transplantation and Cellular Therapy; CRS, cytokine release syndrome; ULN, upper Limit of Normal; IEC-HS, Immune Effector Cell-Associated HLH-Like Syndrome.

Early recognition of CARHLH in patients with CRS and the timely initiation of effective treatment are critical for patient prognosis. The ASTCT expert recommendations state that patients with HLH-like toxicities experience either a preceding or concurrent CRS and suggest the use of markedly elevated ferritin levels as the primary criterion for diagnosing CARHLH (9). Ferritin is the most frequently used laboratory test to distinguish CRS and CARHLH; however, the threshold value is not yet standardized (9,64). Elevated ferritin levels may indicate CARHLH; however, low ferritin levels have a negative predictive value (10). Zu et al (44) retrospectively studied 99 patients treated with B cell maturation antigen (BCMA). Serum parameter monitoring revealed elevated ferritin, aspartate aminotransferase and lactate dehydrogenase (LDH) levels in patients with CARHLH and CRS, as well as a marked peak difference between the two groups; elevated parameters in patients with CARHLH persisted for an extended duration (44). Similar patterns were observed during monitoring of cytokines. Compared with patients with CRS, the levels of IFN-γ, granzyme B, IL-1a, IL-1b, IL-10, IL-12, IL-33 and chemokine are markedly increased in patients with CARHLH (11,44).

Numerous studies (11,44) have attempted to explore the predictors of CARHLH. Lichtenstein et al (11) developed a predictive model of CD22 CAR T cells using IFN-γ and baseline myeloid T:NK cell ratios on day 2 post-CRS to predict the probability that patients with CRS develop CARHLH in the future. Zu et al (44) also developed a CARHLH prediction model for BCMA therapy that used fibrinogen levels on day 3 post-CRS and LDH levels on day 3 post-CRS to predict CARHLH in patients with CRS. Further clinical studies are required to clarify the predictive effects of these models, which are still in the research stage and have not been extensively applied in clinical practice.

Treatment of CRS and CARHLH

According to MD Anderson Cancer Center, patients should be hospitalized and closely monitored for ≥7 days following CAR T cell infusion (48). The treatment protocol should be formulated based on the overall clinical condition of the patient (15). Management of ASTCT grade 1 CRS primarily involves supportive measures including rehydration, antipyretics, oxygenation and elevation of blood pressure. The option of anti-cytokine treatment may be evaluated in patients with moderate-to-severe CRS (15,48,58). IL-6 is a key factor in the pathogenesis of CRS, and its expression is associated with the severity of CRS following CAR T cell therapy. Furthermore, anti-IL-6 therapy does not affect the expansion or efficacy of CAR T cells (38,48,65,66). Consequently, anti-IL-6 is the preferred treatment option for patients with severe CRS (48,66). Numerous case reports demonstrate the efficacy of tocilizumab (an IL-6 monoclonal antibody) in managing CAR T cell-associated CRS (50,67,68). The Society for Immunotherapy of Cancer (SITC) and American Society of Clinical Oncology (ASCO) (69,70) recommend the use of anti-cytokine therapy and corticosteroids in CRS (Table IV). Compared with SITC, ASCO is more proactive in the use of anti-cytokine therapy and corticosteroids (69,70).

Table IV. Management of CRS.

Table IV.

Management of CRS.

Guideline	Recommendations	(Refs.)
SITC (2020)	For elderly patients or patients with extensive comorbidities, tocilizumab should be considered earlier in the course of CRS. For adults who develop ASTCT grade 2 CRS, tocilizumab may be considered. For pediatric patients, tocilizumab should be administered at ASTCT grade 3 CRS. For pediatric patients who develop prolonged ASTCT grade 2 CRS or intolerance to fever, tocilizumab may be administered. In both adults and children, if CRS does not improve after one dose of tocilizumab, steroids should be administered with a second dose of tocilizumab. If CRS does not improve following two doses of tocilizumab (and steroids), third-line agents, including anakinra, siltuximab and highdose methylprednisolone, should be considered. If CRS does not improve after tocilizumab and steroids, infection should be considered again in the differential diagnosis and managed appropriately. If steroids are used in the management of CRS, a rapid taper should be used once symptoms begin to improve	(69)
ASCO (2021)	CRS is primarily managed with tocilizumab in lower-grade CRS and corticosteroids in refractory, prolonged or higher-grade CRS. Alternate IL-6 antagonist therapies including siltuximab and clazakizumab may be used for CRS refractory to tocilizumab. Anakinra (an IL-1 receptor antagonist) mitigates CRS in certain CAR T cell therapy recipients with high-grade CRS	(70)

[i] SITC, Society for Immunotherapy of Cancer; ASCO, American Society of Clinical Oncology; ASTCT, American Society for Transplantation and Cellular Therapy; CRS, cytokine release syndrome; CAR, chimeric antigen receptor.

The current first-line treatment regimen for HLH consists of etoposide, cyclosporine A and dexamethasone, as well as intrathecal methotrexate and dexamethasone and is indicated for most types of HLH. Due to the toxic side effects of chemotherapy, maximum supportive treatment should be provided during the treatment period. Bone marrow transplantation is the sole curative option for treating pHLH, malignancy-associated HLH and Epstein-Barr virus-related HLH (19). Disease remission prior to transplantation is key for patient prognosis (71). Biological agent-targeted therapies are a promising treatment option: In a clinical study of 34 patients with pHLH (27 treated and seven primary), 8-week treatment with emapalumab (an IFN-γ monoclonal antibody) had effective rates of 63 and 65%, respectively (72). Ge et al (73) included 21 patients with pHLH (12 treated and nine primary) using a ruxolitinib (a JAK inhibitor)-based regimen, with a 1-year overall survival rate of 90.5%; all patients achieved objective remission within the first 8 weeks, with 19 (90.5%) achieving complete remission.

The treatment of CARHLH has not yet been standardized. The ASTCT CRS guidelines published in 2019 (58) suggest that there is no need to distinguish CARHLH from CRS. However, there is an increasing consensus that the early identification of CARHLH and prompt treatment are essential for improving patient outcomes (58,69,70). A case report described two patients with CARHLH: A patient who received timely diagnosis and treatment experienced improvement in CARHLH after therapy. By contrast, a patient who did not receive prompt medical attention, quickly succumbed from multi-organ failure (68). The MD Anderson Cancer Center recommends patients with suspected CARHLH initially be considered as having CRS and should be treated with corticosteroids and anti-IL-6 therapy (48).

A retrospective study by the EBMT reported that the most frequently used combination regimen for the treatment of CARHLH is corticosteroids combined with chemotherapy, followed by corticosteroids combined with chemotherapy and monoclonal antibodies or anti-cytokine drugs and other treatment (10). A retrospective clinical study analyzed the clinical data of patients with hematological malignancy who received CD22 CAR T cell therapy and reported that during the active phase of CARHLH, HLH-related cytokine levels are persistently elevated, including IFN-γ, IL-1 and IL-6(11). These cytokines are key contributors to disease progression (11). Rejeski (74) proposed that patients with CARHLH be treated with anakinra in combination with high-dose corticosteroids. Refractory patients can be treated with ruxolitinib and emapalumab therapy (74). Porter et al (64) reported that three patients with CD19 CARHLH demonstrated gradual improvement in laboratory manifestations after treatment with anakinra and/or ruxolitinib. Repeat PET-CT scans in two patients revealed complete remission, but relapse or progression of the disease was observed (64). A clinical study analyzed data from 58 patients who received CD22+ CAR T cell therapy: CRS was observed in 50 patients and HLH-like toxicity was identified in 19 patients (38%). Notably, the toxic manifestations improved in all patients following administration of anakinra or the addition of corticosteroids (13). In one patient, all abnormal laboratory parameters returned to normal following 1 month of treatment with anakinra (13).

SITC guidelines suggest that tocilizumab treatment should be promptly initiated in patients with suspectedCARHLH. Anakinra and corticosteroids may be considered for tocilizumab-refractory HLH/MAS-like toxicity (69). Corticosteroids have side effects, including hypertension, metabolic derangement, fractures and increased risk of infection (75). According to the ASCO guidelines (70), corticosteroids and IL-6 antagonist therapy should be used to manage CARHLH with grade ≥3 organ toxicity. Anakinra is administered to patients with refractory CARHLH (74). Tocilizumab demonstrates limited therapeutic effectiveness in adult patients with sHLH and may elevate the risk of infection-related complications when compared with conventional treatment regimens (76). ASTCT expert recommendations for CARHLH suggest that anakinra and/or corticosteroids are first-line agents (Table V). Anakinra is an IL-1 receptor antagonist; clinical trials of anakinra for sHLH have demonstrated benefit without compromising the efficacy of CAR T cells (77–79). Common toxicities associated with anakinra include infection, headaches and fever. For patients needing further intervention to manage CARHLH, particularly patients with worsening inflammatory markers or signs of progressive end-organ damage, second-line treatments such as ruxolitinib may be considered as a therapeutic option (80). However, ruxolitinib may increase the risk of infection and exacerbate cytopenia and there is still a lack of research and clinical experience (80). Etoposide and emapalumab may serve as alternative treatment options for patients with life-threatening refractory CARHLH (9). Etoposide is the preferred T cell-depleting agent owing to the large amount of data available in both pHLH and sHLH (81–84). The side effects of etoposide include dose-limiting myelosuppression, particularly neutropenia, which may increase the risk of infection (85). Although IFN-γ serves a key role in the development of CARHLH, research on anti-IFN therapy for the treatment of CARHLH is limited (11,86,87). Antagonizing IFN-γ may not affect the efficacy of CAR T cells (86). The application of emapalumab for treating adults with CARHLH is largely speculative. Although emapalumab has been approved by the US Food and Drug Administration for the treatment of pHLH and yields good results, there is debate regarding its efficacy and safety in the treatment of CARHLH (88,89). Currently, targeted therapy is the primary treatment approach for CARHLH. The pathophysiological mechanisms underlying CARHLH are under investigation, and future research may uncover novel mechanisms, facilitating development of targeted therapies based on new molecular targets (19,90). Owing to limited literature on the management of CARHLH and a lack of prospective clinical trials for CARHLH, there is no standardized and unified treatment for CARHLH. However, current guidelines and expert consensus suggest that anti-cytokine therapy serves an important role in the treatment of CARHLH.

Table V. Management of CARHLH.

Table V.

Management of CARHLH.

Guideline	Description	(Refs.)
SITC (2020)	CRS and HLH/MAS substantially overlap. However, lateonset, tocilizumabrefractory HLH/MASlikesymptoms may represent a distinct and separate pathology than conventional CRS. Delayed coagulopathy may be a hallmark of delayed onset HLH/MASlike toxicity, typically hypofibrinogenemia disproportionately worse than changes in PT/PTT, which requires close followup and replacement with cryoprecipitate. Etoposide should only be administered to patients experiencing lateonset, tocilizumabrefractory HLH/MASlike symptoms following CAR T cell therapy as a last resort. For treatment of late-onset, HLH/MASlike pathology, which may be tocilzumabrefractory, thirdline CRS agents such as anakinra and steroids may be considered	(69)
ASCO (2021)	Corticosteroids and IL-6 antagonist therapy are used to treat HLH in CAR T cell therapy recipients with grade ≥3 organ toxicity. Etoposide may be used; however, data in CAR T cell therapy recipients are lacking. Anakinra is used for refractory HLH in CAR T cell therapy recipients; however, clinical efficacy is unclear	(70)
ASTCT expert opinion (2023)	Therapy generally should be initiated with corticosteroids with or without anakinra before the development of life-threatening complications and, if possible, a time interval to evaluate for efficacy (for example, 48 h) should elapse before use of additional agents to avoid cumulative toxicity of multiple immunosuppressive agents. The best approach to tapering and withdrawal of therapy is unclear, particularly in patients with protracted IEC-HS. When clinical and laboratory parameters have stabilized, gradually tapering therapy is encouraged while monitoring for recrudescence of symptoms.	(9)
	First-line therapy can start with anakinra with or without corticosteroids (or vice versa). Use of one or both agents and/or low vs. higher dose is dependent on the clinical presentation of the patient and how rapidly the inflammatory response is worsening. For patients with grade 2 IEC-HS, starting with the lower end of the range for dual therapy or higher dosing for single-agent intervention could be considered. For second-line therapy, in patients with clearly worsening inflammatory parameters who develop more severe manifestations of IEC-HS, recommendations are for dual-agent therapy at the higher end of the range, with consideration for additional agents (ruxolitinib) if the patient is rapidly worsening. For third-line therapy, typically, corticosteroids/anakinra would continue as other therapies are added to achieve control of the inflammatory response. Choice of therapy beyond ruxolitinib can be informed by patient considerations (for example, etoposide for CAR T cell-associated lymphocytosis or emapalumab for highly elevated IFN-γ). An ongoing assessment for alternative etiology should continue throughout the IEC-HS manifestations and treatment course. As clinical and laboratory parameters stabilize, gradually tapering immunosuppression is encouraged while monitoring for recrudescence of symptoms

[i] CARHLH, chimeric antigen Receptor T-cell Associated Hemophagocytic Lymphohistiocytosis; SITC, Society for Immunotherapy of Cancer; CRS, Cytokine Release Syndrome; MAS, Macrophage Activation Syndrome; PT/PTT, Prothrombin Time/Partial Thromboplastin Time; CAR, Chimeric Antigen Receptor; ASCO, American Society of Clinical Oncology; ASTCT, American Society for Transplantation and Cellular Therapy; IEC-HS, Immune Effector Cell-Associated HLH-Like Syndrome.

Conclusion

CARHLH and CRS exhibit distinct pathogenic mechanisms, although their clinical manifestations are overlapping, with all patients with CARHLH presenting concurrent or prior CRS (8,9,11). The pathogenesis of CARHLH is still being explored. Excessive CAR T cell expansion, NK cell decrease and delayed T cell contraction are the potential mechanisms of action (11,51–53). There are no standardized diagnostic or treatment criteria for CARHLH. ASCO guidelines recommend tocilizumab (an IL-6 monoclonal antibody) for CRS treatment (58). The timing of targeted therapy is determined by the CRS grade (58). During the diagnostic workup for CARHLH, it is key to differentiate whether sHLH is driven by CAR T therapy (59). Regular monitoring of CAR T cell expansion dynamics can aid this distinction (59). Patients at a high risk of HLH can be identified early using indicators such as ferritin and HLH-associated cytokine levels (9). Patients with suspected CARHLH should receive CARHLH treatment as soon as possible (9). Currently, there is no unified therapeutic regimen for CARHLH. The 2023 ASTCT expert consensus recommends anakinra and corticosteroids as first-line agents, while ruxolitinib, etoposide and emapalumab may serve as alternative therapies for refractory cases (58). Corticosteroids, anti-IL-1 and anti-IL-6 therapies are the most frequently used, followed by etoposide (12,50,59,67,68,91–93). Given the abnormal CAR T cell proliferation during CARHLH progression, T cell-targeted therapy could be considered in rapidly progressing or life-threatening scenarios when first- and second-line treatments fail. However, research and clinical experience remain scarce, necessitating further investigation.

In conclusion, the specific pathogenesis of CARHLH is unclear, and the diagnostic and treatment criteria need to be improved and standardized. CARHLH is a rare disease, and the mechanisms of its occurrence and development are not fully understood. Larger multicenter studies need to be conducted or insights should be extrapolated from other sHLH cases to develop diagnosis, treatment and prediction models and systematic understanding of CARHLH.

Acknowledgements

Not applicable.

Funding

The present study was supported by the National Natural Science Foundation of China (grant no. 82170122).

Availability of data and materials

Not applicable.

Authors' contributions

JH, CF, LH and YW wrote the manuscript. CF, LH and YW revised the manuscript. All authors have read and approved the final manuscript. Data authentication is not applicable.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References