Emapalumab for adult and pediatric patients with hemophagocytic lymphohistiocytosis
Chiara Garonzi, Matteo Chinello and Simone Cesaro
Pediatric Hematology Oncology, Department of Mother and Child, Azienda Ospedaliera Universitaria Integrata Verona, Verona, Italy

Introduction: Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening hyperinflammatory syndrome. Standard treatment is based on immunosuppressive, cytotoxic drugs and hematopoietic stem cell transplantation (HSCT) in primary HLH. Interferon-gamma (IFN-γ) plays a key pathogenic role. Emapalumab, a monoclonal antibody directed against IFN-γ, is the first target therapy approved for primary HLH with refractory, recurrent or progressive disease or intolerance to conventional therapy. Areas covered: We reviewed the pharmacological characteristics, safety, efficacy and clinical uses of emapalumab. We summarized the results of current standard treatment based on chemo-immuno suppressive protocols and outlined the alternative options available.
Expert opinion: Emapalumab is an effective treatment for HLH with a good safety profile. Its efficacy was demonstrated in a phase II/III study on primary HLH pediatric patients with refractory, relapsing HLH or intolerance to first-line treatment. The use of emapalumab allowed most patients to proceed to HSCT, with a high estimated probability of survival 12 months after transplantation. The outcomes in patients who underwent transplantation compare favorably with those reported previously with either myeloablative or reduced-intensity conditioning regimens. The potential role of emapalumab in the treatment of secondary HLH and as a prevention of graft failure after HSCT deserves to be further assessed.
ARTICLE HISTORY Received 31 December 2020 Accepted 8 March 2021
KEYWORDS Emapalumab; hemophagocytic lymphohistiocytosis;
chemotherapy; interferon-γ; monoclonal antibodies

Hemophagocytic lymphohistiocytosis (HLH) is a rare life- threatening hyperinflammatory syndrome characterized by a severe immune dysregulation which clinically manifests with multiple organ involvement, such as fever, hepatosple- nomegaly, cytopenia, hypertriglyceridemia, hypofibrinogen- emia, and hyperferritinemia. HLH is classically divided into primary and secondary forms. Primary HLH comprises several inherited genetic disorders that cause HLH syndrome and most of the gene mutations implicated encode components of the secretory granule-dependent death pathway, the prin- cipal mechanism of cytotoxic T lymphocytes (CTLs) and nat- ural killer (NK) cell cytotoxicity [1–3]. Secondary HLH includes acquired forms associated with infections, malignancy or rheu- matic conditions (i.e. macrophage activation syndrome (MAS- HLH) [1]. Of note, this dichotomy is debated due to the finding that some genetic defects may contribute to the development of secondary forms [4]. For this reason, a new terminology of HLH-disease and HLH-disease mimic has been proposed, recently [5].
HLH epidemiology is difficult to establish, probably because the condition is frequently underdiagnosed. Worldwide, few epidemiologic data are available, especially for secondary HLH cases [6]. The incidence of primary HLH in Sweden is estimated to be 1 in 50.000 live-born children, with the highest incidence in early life (<3 months). The sex ratio is close to 1:1 [7]. An

estimate of the prevalence of HLH in people under the age of 18 in Texas is 1.07 cases per 100,000 [8].
Without treatment, HLH can be rapidly fatal, with a median survival between 2 and 3 months [7]. In 1983, long-term survival was <5% [9]. Considering the first 122 patients recorded on Histiocyte Society’s FHL Registry, set in 1989, the estimated 5-year survival was 21% for all patients, being 66% after allogeneic hematopoietic stem-cell transplantation (HSCT) and only 10.1% after chemotherapy alone [10].
The first international treatment study on HLH was HLH-94, which consisted of an initial therapy with immunosuppressive and cytotoxic agents for 8 weeks, followed by a continuation therapy for patients with familial, persistent or relapsing dis- ease, until an acceptable donor was available for HSCT. In detail, the initial therapy included etoposide (VP-16) and daily dexamethasone. For patients with progressive neurologic symptoms and/or persisting abnormal cerebrospinal fluid find- ings, intrathecal (IT) therapy with methotrexate (MTX) was recommended. The continuation therapy involved pulses of dexamethasone in combination with VP-16 and cyclosporine A (CSA). HSCT was recommended as soon as a suitable donor was available. The protocol improved the survival of HLH patients, showing an estimated 5-year probability of survival (pSUR) of 54% ± 6%. Pre-HSCT mortality was 29%. In patients who underwent HSCT, 5-year cumulative survival was 66% ± 8%. Considering patients with ‘familial’ disease (i.e. patients

CONTACT Simone Cesaro [email protected] Pediatric Hematology Oncology Department of Mother and Child, Azienda Ospedaliera Universitaria Integrata Verona, Verona, Italy.
© 2021 Informa UK Limited, trading as Taylor & Francis Group

(JAK-STAT), CD52, tumor necrosis factor alfa (TNF-α), IL-1, and

Article highlights
● Patients affected by HLH have augmented circulating levels of IFN-γ, an inflammatory cytokine that showed a key role in the pathogenesis of HLH.
● Emapalumab is a fully human IgG1 monoclonal antibody directed against IFN-γ that binds to both free and receptor-bound IFN-γ, neutralizing its biological activity.
● The efficacy and safety of emapalumab in patients with primary HLH were demonstrated in a phase II/III study, mostly led in patients with refractory, relapsing HLH or intolerance to first-line treatment. Emapalumab controlled the disease in almost two-third of the patients, without significant toxicity and infectious complications.
● Emapalumab was approved in November 2018 in the United States by the Food and Drug Administration for the treatment of pediatric and adult patients with primary HLH and refractory, recurrent or progressive disease or intolerance to conventional HLH therapy.
● The recommended starting dose of emapalumab is 1 mg/kg by intravenous infusion twice a week with concomitant dexamethasone (5-10 mg/m2/day). Emapalumab dosage can be increased according to clinical and laboratory criteria, up to 10 mg/kg.

with an affected sibling), the 5-year survival was 50% ± 13%. None of them survived without HSCT [11,12].
Subsequently, HLH-2004 was presented as a new treat- ment protocol. The study was based on HLH-94 and included updated diagnostic and therapeutic guidelines. HLH-2004 diagnostic criteria are still currently used. For the diagnosis of HLH at least five out of the eight diagnostic criteria need to be fulfilled (fever, splenomegaly, bi- or tri- lineage cytopenias in peripheral blood, hypertriglyceridemia and/or hypofibrinogenemia, hemophagocytosis in bone marrow, spleen or lymph nodes, low or absent NK-cell activ- ity, hyperferritinemia and high levels of soluble interleukin (IL)-2 receptor), and/or molecular diagnosis in FHL-causative genes. Major differences in treatment were the intensifica- tion of the initial therapy by administrating CSA upfront and the addition of prednisolone to IT MTX in the cases where IT therapy was recommended. Among the 369 patients enrolled, HLH-2004 showed an estimated 5-year pSUR of 61% for the entire cohort (confidence interval (CI) 56%- 67%) and 59% (CI 52%-67%) for patients with family his- tory/genetically verified FHL. Overall, 20% of the patients died without HSCT. Five-year pSUR after HSCT was 66% (CI 59%-73%). Compared to the previous HLH-94 protocol, HLH- 2004 did not improve the outcome significantly. For this reason, HLH-94 remains the standard of care [13,14].
An alternative immunotherapeutic approach for HLH was developed in France. It combined antithymocyte globulin (ATG), methylprednisolone, IT MTX and corticosteroids during the early phase. CSA was administered during the mainte- nance phase, until HSCT was performed. The study, which involved 38 patients, with 45 courses of ATG administered, resulted in a complete response in 73% of the patients, partial response in 24%, and no response in 2% [15].
Biologic treatments open up new prospects on HLH therapy. Since the hyperactive immune response and hypercytokinemia play a key role in HLH pathogenesis, several targets have been proposed in the last years, including interferon gamma (IFN-γ), Janus kinase-signal transducer, and activator of transcription
IL-6 [16].
This review focuses on the pharmacological characteristics, safety, efficacy and clinical uses of emapalumab, a monoclonal antibody directed against IFN-γ, the first targeted therapy approved for the treatment of primary HLH patients with refractory, recurrent or progressive disease or intolerance to conventional therapy.

2.1.Role of IFN-γ in HLH pathogenesis
It has long been known that patients affected by HLH have augmented circulating inflammatory cytokines, including IFN- γ. Indeed, several studies showed that IFN-γ is increased in the peripheral blood of both primary and secondary HLH affected patients, assuming a pathogenic role of this cytokine [17–21]. In detail, IFN-γ level exhibited a correlation with the clinical condition, since it was elevated in active HLH but below the detection level in patient in remission of HLH, in healthy controls and during infections [17].
By studying murine models of HLH, the key role of IFN-γ in the pathogenesis was confirmed. One of the most used animal models of primary HLH was based on perforin-deficient (Prf -/-) mice. These mice were seen developing HLH clinical, laboratory and histopathologic features after infection with lymphocytic choriomeningitic virus (LCMV). The neutralization of IFN-γ pre- vented the development of multiple HLH manifestations and allowed most LCMV-infected Prf -/- mice to survive, highlighting the crucial role in the development of the disease [22]. The repeated stimulation of Toll-like receptor 9 (TLR9) using CpG DNA (a potent stimulator of TLR9) led to clinical manifestations that were consistent with a MAS-like syndrome and served as a model for secondary HLH. Even in this model, the develop- ment of MAS was found to be IFN-γ-dependent [23]. Several other murine models of HLH validated these findings [24,25], although recent studies are evidencing possible differences, e.g. when using alternative triggers or in different secondary HLH models or some IFN-γ-independent manifestations [26–29]. Among the latter, a recent study showed a dichotomist patho- genic mechanism of HLH, in which IFN-γ has a key role in the hematological manifestations of HLH, with a strict dependence on its production, while immunologic features are driven by the excessive consumption of IL-2 [28,30].
These preclinical data laid the basis for IFN-γ inhibition as a treatment for HLH.

Emapalumab is a fully human IgG1 monoclonal antibody directed against IFN-γ, as a noncompetitive inhibitor. In detail, it binds to both free and receptor-bound IFN-γ, preventing receptor dimerization and signaling transduction, and neutra- lizes its activity [31,32]. Emapalumab lowered the plasma con- centrations of CXCL9, an IFN-γ induced chemokine [33].

After administering emapalumab at 1 mg/kg dose, the median steady state peak and the median steady state serum trough level were, respectively, 44 μg/mL and 25 μg/mL, which were 2.9 and 4.3 times higher than following the first dose. The average steady state concentration exhibited a slightly greater than proportional increase at a dose range of emapalumab of 1–3 mg/kg, and a less than proportional increase at doses of 3, 6 and 10 mg/kg.
Emapalumab showed target-mediated clearance, which was dependent on the production of IFN-γ. At moderate IFN- γ production, steady state was achieved by the seventh ema- palumab infusion whereas at high IFN-γ production it was reached earlier due to a shorter half-life. In healthy subjects, emapalumab clearance was about 0.007 L/h and the elimina- tion half-life about 22 days, whereas in HLH patients the elimination half-life ranged between 2.5 and 18.9 days and the total clearance was significantly influenced by IFN-γ production.
In a subject with a body weight of 70 kg, the central and the peripheral volumes of distribution were, respectively, 4.2 and 5.6 L.
Emapalumab is expected to be degraded into small pep- tides and amino acids, like other protein therapeutics, although the actual metabolic pathway has not yet been identified.
Body weight-based dosing of emapalumab was supported by the significant influence of body weight on the pharmaco- kinetics. Age, sex, race, renal or hepatic impairment did not show significant clinical impact on the pharmacokinetics [33].

2.4.Drug interactions
No direct drug interaction has been demonstrated up to now, even if a possible effect of emapalumab on cytochrome P450 substrates has been reported. In fact, since increased levels of cytokines (including IFN-γ) may suppress the formation of CYP450 enzymes, by neutralizing IFN-γ CYP450 activities may be normalized. Moreover, the use of emapalumab may increase the metabolism of drugs that are CYP450 substrates and reduce their efficacy. Therefore, it is important to monitor for reduced efficacy and eventually adjust the dosage of CYP450 substrate drugs when concomitant emapalumab is started or discontinued [33].

2.5.Phase II/III study
The efficacy and safety of emapalumab in patients affected by primary HLH were examined in an open-label, single group, phase II/III study. Overall, 34 pediatric patients (18 years old or younger) with active disease were included in the trial and received emapalumab. The study involved both previously untreated (n = 7) and previously treated patients (n = 27). The patients who had received previous treatment had wor- sened or reactivated the disease, had an unsatisfactory response to conventional therapy, or had to withdraw it because of adverse effects. The median age of study patients was 1.0 year (range, 0.1 to 13.0). Most patients had a genetic

mutation consistent with primary HLH (79%). Patients with active mycobacteria, histoplasma capsulatum, shigella, campy- lobacter, leishmania or salmonella infections were excluded, since those infections are potentially associated with IFN-γ neutralization. Patients with secondary HLH were excluded from the study as well. Emapalumab was administered at a starting dose of 1 mg/kg dose by intravenous infusion every 3 days in association with dexamethasone at 5–10 mg/
m2/day. IT therapy could be combined, when indicated. Following doses of emapalumab could be increased up to 10 mg/kg, according to clinical and laboratory criteria. The planned treatment period lasted 8 weeks, although it could be shortened to a minimum of 4 weeks or extended, depend- ing on the availability of HSCT. In addition, 28 patients entered a long-term follow-up [32].

Overall response (at 8 weeks or at the end of treatment), which included complete or partial response or improvement of HLH in the previously treated patients, was the primary end point for efficacy. In all patients who received emapalumab the overall response was 65% (95% CI 46–80) that was con- sidered complete in 21%, partial in 32% whereas 12% improved. Among previously treated patients overall response was 63% (95% CI 42–81), the corresponding complete, partial, improved responses were 26%, 30%, and 7%, respectively. In both groups, the median time to response was 8 days. Interestingly, central nervous system involvement, present in 12 patients, normalized in 6 and improved in 4, while it could not be assessed in 2 patients, due to the worsening of HLH. Secondary end points included the number of patients pro- ceeding to HSCT and overall survival. Considering all patients treated with emapalumab, 65% underwent HSCT, 71% were alive at last observation with an estimated 12-month pSUR of 69.3% (95% CI 50.3–82.2). In the previously treated patients, 70% underwent HSCT, 74% were alive at last observation, and the estimated 12-month pSUR was 73.4% (95% CI 52.2–86.4). The estimated 12-month pSUR after transplantation was 90.2% (95% CI 66.2–97.5) in all patients treated with emapalumab and 89.5% (95% CI 64.1–97.3) in previously treated patients. Worth mentioning, two study patients did not receive HSCT because of the non-availability of compatible donor and, until the end of the study, HLH was resolved and they survived. Note that during the emapalumab treatment CXCL9 serum levels were monitored. It was seen that clinical HLH improve- ment corresponded to CXCL9 levels decline and that there was an association between the probability of response and a decrease of CXCL9 levels at 8 weeks. Since CXCL9 is exclu- sively induced by IFN-γ, this observation confirms the impor- tance of neutralizing IFN-γ in HLH treatment [32].

2.7.Adverse events
Ten patients who received emapalumab died (eight before and two after HSCT); however, study investigators did not consider those deaths related to emapalumab. Before or after conditioning periods, every patient experienced at least one adverse event. To note, 35% of the patients had already

had a concomitant infection or positive microbiological tests at the beginning of the study. The most frequent adverse reactions reported during emapalumab treatment, before con- ditioning, were infections that were seen in 56% of the patients who received emapalumab and 32% of them were severe. One patient died from septic shock and one patient discontinued the therapy because of disseminated histoplas- mosis. Other common adverse events (≥20%) were hyperten- sion (35%), infusion reactions (27%), and pyrexia (24%). Importantly, investigators reported the case of disseminated histoplasmosis and one case of necrotizing fasciitis as related to emapalumab [32].

2.8.Clinical indications and usage
Emapalumab was approved in November 2018 in the United States by the Food and Drug Administration for the treatment of pediatric and adult patients with primary HLH with refrac- tory, recurrent or progressive disease or intolerance to con- ventional HLH therapy. The recommended starting dose is 1 mg/kg by intravenous infusion (over 1 h) twice a week with concomitant dexamethasone (5–10 mg/m2/day). Following emapalumab dosage, increases should be deter- mined upon clinical and laboratory criteria, up to 10 mg/kg. Importantly, before starting the treatment with emapalumab, it is recommended to perform tests for detecting latent tuber- culosis (TB) infections and evaluate risk factors for TB, in order to start appropriate prophylaxis in patients at risk or with positive tests. Prophylaxis for Herpes Zoster virus (HZV), Pneumocystis jirovecii and fungal infections is also recom- mended. The treatment with emapalumab must not be given in patients with mycobacteria, HZV, and Histoplasma Capsulatum infections, as long as the beginning of appropriate treatment [33,34].

2.9.Ongoing clinical trials
Pediatric patients with primary HLH who have already received emapalumab in a previous clinical trial could enter an international, multicenter, long-term, follow-up study (NCT02069899), which is still underway. This study monitors the long-term efficacy, safety, tolerability, and pharmacoki- netics of emapalumab. In addition, an open-label, single arm, phase III study (NCT03312751) on primary HLH patients (up to 18 years) allows a broadening access to emapalumab, focusing on long-term outcome and quality of life. Moreover, it is still ongoing a pilot, open-label, single arm, phase II study in pediatric patients with systemic juvenile idiopathic arthritis developing MAS with an inadequate response to high-dose glucocorticoid treatment receiving emapalumab twice weekly (NCT03311854). Data from six patients enrolled in this study were reported, showing a complete response in all patients. Emapalumab was well tolerated and none of the patients discontinued the treatment. Interestingly, the study reported that both IFN-γ was rapidly neutralized and T-cells were deac- tivated, as showed respectively by the reduction of CXCL-9 and soluble IL-2 R levels [35]. Finally, the efficacy, safety and pharmacokinetics of emapalumab in adult patients with HLH

are currently being assessed in an open-label, single arm, phase II/III clinical trial (NCT03985423).

2.10.Other clinical uses of emapalumab
The use of emapalumab was described in several case reports or case-series in patients with HLH or other disease with immunological dysfunction, as reported below [36–42]. A recent study reported a group of patients with therapy refractory HLH and concomitant complement-mediated thrombotic microangiopathy (TMA). Among patients who were treated with the complement inhibitor eculizumab (n = 10), six received emapalumab and four HLH conventional therapy (corticosteroids with or without VP-16). Interestingly, all patients who received emapalumab in addition to eculizu- mab survived, showing a complete resolution of TMA. One of them did not proceed to HSCT because of the lack of donor, while the other five patients successfully underwent HSCT. Among patients who did not receive emapalumab, none of them underwent HSCT and two of them died due to active TMA. This study highlighted a possible co-activation of IFN-γ and complement pathways in the development of TMA in patients with inflammatory disorders [36].
Several case reports described other uses of emapalumab. Emapalumab compassionate use was reported in a 20-months patient with treatment refractory HLH, multiple viral (including EBV), bacterial and fungal infections and multiple organ fail- ure. Emapalumab was started in addition to proper antimicro- bial therapies. Dexamethasone was stopped because of persistent fungemia. Emapalumab therapy led to clinical and laboratory resolution of patient’s manifestations. Interestingly, despite the IFN-γ inhibition, the patient succeeded in recover- ing from the multiple infections [37]. A 4-year-old child with severe combined immunodeficiency due to adenosine deami- nase deficiency, experienced two graft failure after gene- therapy and HLA-haploidentical HSCT, associated with concur- rent multiple infections (including disseminated TB, due to the bacille Calmette-Guérin vaccine strain reactivation), and sec- ondary HLH. The compassionate use of emapalumab led to keep HLH under control and perform the third haplo-HSCT, preventing graft failure (GF). Interestingly, during IFN-γ inhibi- tion, infections properly treated with the antimicrobial drugs improved and TB did not reactivate [38]. GF pathophysiology was analyzed in 15 patients who experienced HSCT-related GF who were enrolled in a prospective study. In detail, serum levels of several cytokines/chemokines were investigated, highlighting that patients developing GF had significantly higher IFN-γ and CXCL9 levels than controls. Supporting this evidence, the inhibition of IFN-γ with the use of emapalumab in primary HLH pediatric patients who were re-transplanted after experiencing GF, allowed a successful donor engraftment in 2 out of 3 patients [39].
Another case report described a one-month-old patient affected by primary HLH, who received dexamethasone and CSA, followed by emapalumab, who successfully underwent HLA-matched unrelated donor HSCT at 6 months of life. To note, in the pre-engraftment period, the patient experienced Pseudomonas aeruginosa sepsis and lobar pneumonia and, at 17 months, developed severe chronic graft versus host

disease-related polymyositis [40]. The effective use of emapa- lumab in a 22-year-old female patient with adult-onset Still’s disease complicated with MAS was also reported. The patient did not respond to corticosteroids and anakinra, while she significantly improved after emapalumab treatment. Of note are the elevated IFN-γ levels prior to IFN-γ blockade treat- ment [41].
NOCARH syndrome (neonatal onset, cytopenia, autoinflam- mation, rash, HLH) is a newly identified hematological/autoin- flammatory condition, in which cytopenia combined with dyshematopoiesis and HLH manifestations is secondary to CDC42 mutation. The use of emapalumab in one patient affected by this condition, allowed the patient to survive and successfully undergo HSCT [42].

3.Other treatments for HLH
JAK-STAT pathway is involved in the signal transduction acti- vated by the binding of many HLH-associated cytokines, such as IFN-γ, IL-2 and IL-6, to specific receptors. The hypothesis that JAK-STAT inhibition would be effective in HLH was con- firmed in murine models of both primary and secondary HLH. In particular, JAK1/2 inhibitor, ruxolitinib, significantly reduced the manifestations of HLH and improved survival, limiting CD8 + T-cell expansion and pro-inflammatory cytokine pro- duction [43]. Preliminary data on the efficacy of ruxolitinib were shown in an open-label, single-center, pilot study led in five adult patients with secondary HLH. In detail, during the follow-up period of the study (median of 490 days) no patients died, with a 2-months overall survival of 100% (57–100%). All five patients showed a response (complete or partial). The drug exhibited a good tolerance, with a single serious adverse event (grade 4 febrile neutropenia) and a treatment disconti- nuation in a patient due to grade 2 extremity pain [44]. A prospective, pilot study demonstrated the efficacy and safety of the use of ruxolitinib as a front-line therapy on 12 pediatric patients affected by secondary HLH. In particular, at the end of the treatment (28 days) the overall response rate was 83.3% (66.7% complete, 8.3% partial, 8.3% HLH improve- ment), with a clinical and laboratory resolution of manifesta- tions. No serious adverse effects were reported. To note, patients who had a complete response maintained the condi- tion for more than 6 months, except for one patient who relapsed without responding to the subsequent conventional therapy and died. Among patients who did not respond or discontinued the treatment, all of them had a good response to the following HLH-1994 regimen [45]. According to a retrospective study on nine children with recurrent or refrac- tory HLH, ruxolitinib is considered a tolerable salvage therapy. In detail, within 48 hours from the administration of this drug all patients’ body temperature lowered to normal values, even if 72 hours later, fever recurred in six patients. Among these six patients, one died because of the persistence of HLH, while the other five patients received salvage chemotherapy and three of them underwent HSCT and survived. During 1 week of ruxolitinib administration, three patients had partial remis- sion and got complete remission until the last follow-up. To note, the poorer response to ruxolitinib was seen among EBV-

HLH patients [46]. Importantly, concerns about lymphoma progression were reported in two patients during ruxolitinib therapy for refractory lymphoma-associated-HLH. Thus, cau- tion was advised in the use of this drug in lymphoma- associated-HLH, until more studies are available [47]. Few data are available in patients primary HLH, even if favorable results were reported in a patient with refractory familial HLH, using ruxolitinib as a bridge to HSCT [48]. The efficacy and safety of ruxolitinib in patients with treatment-naïve or relapsed/refractory HLH are under assessment in four ongoing clinical trials [30].
Alemtuzumab is a monoclonal antibody, which targets CD52 antigen, expressed on B, T and NK lymphocytes, monocytes, macrophages and dendritic cells. It may be an effective salvage therapy for patients with refractory HLH. Of the 22 patients treated with this drug, 14 had a partial response and five had clinical or laboratory HLH improve- ment. In addition, 17 out of 22 patients survived to undergo HSCT [49].
Anakinra is a recombinant human IL-1 receptor antagonist, which blocks IL-1 biological activity. The blockade of IL-1 has shown favorable results in the treatment of many diseases, including systemic juvenile idiopathic arthritis (JIA) and JIA- related MAS [50]. Anakinra has also been reported to be a successful treatment in 12 cases of MAS associated with non–systemic JIA rheumatic disorders [51]. A retrospective case series showed its use in eight critically ill patients with supposed secondary HLH, and seven of them survived [52]. Another retrospective study showed data from 44 pediatric patients with secondary HLH/MAS treated with anakinra. Overall mortality rate was 27%. Among systemic JIA patients, no deaths were reported, but mortality rate was 100% (n = 3) among patients with an underlying hematologic malignancy. In addition, mortality was reduced when anakinra was started early in the disease course. Thus, anakinra seems to be effec- tive in pediatric patients with nonmalignant associated HLH/
MAS, and in particular in those with an underlying rheumatic disease [53].
Tocilizumab is a monoclonal antibody that targets the IL-6 receptor. Its use has been studied in the cytokine release syndrome after chimeric antigen receptor (CART) T-cell ther- apy, which resulted in clinical improvement [54]. Since cyto- kine release syndrome mimics in some ways HLH/MAS, tocilizumab was used to treat severe secondary HLH patients. A recent study reviewed the outcomes of nine critically ill patients with HLH secondary to autoimmune diseases (n = 4) and bacterial or viral infections (n = 3) or idiopathic HLH (n = 2), who received tocilizumab. Remission was seen in 8/9 patients, while one patient had refractory HLH. During hospi- talization, one patient relapsed and overall four patients died. Thus, tocilizumab may be an alternative in critically ill patients with HLH, but further studies are needed [55].
Available data regarding the efficacy and the limits of target therapy in HLH are summarized in Table 1.

HLH therapy is undergoing a deep change in the last years, of which target therapies play a key role. In the HLH hyperactive

Table 1. Table of comparison between new biologics in HLH therapy. 5. Expert opinion

Drug (target) Emapalumab

Ruxolitinib (JAK-STAT)

Alemtuzumab (CD-52)

Anakinra (IL- 1Ra)

Tocilizumab (IL-6 R)
Effectiveness Murine models of
both primary and secondary HLH; approved by the FDA for primary HLH with refractory, recurrent or progressive disease or intolerance to conventional therapy. Preliminary data on sJIA developing MAS.
Murine models of both primary and secondary HLH; preliminary data on primary and secondary HLH; potential salvage therapy in children with recurrent or refractory HLH.

Therapy for patients with refractory HLH.

Preliminary data on pediatric patients with nonmalignant associated HLH/

Preliminary data on critically ill HLH patients.
Limitations Ongoing clinical
trials on sJIA developing MAS and on adult patients.

Ongoing clinical trials in treatment-naïve patients or with relapsed/
refractory HLH; concern about lymphoma progression in in a patient with refractory lymphoma- associated-HLH receiving ruxolitinib.
Few data available, more studies are needed in HLH therapy.
High mortality among HLH patients with an underlying hematologic malignancy; lack of data on primary HLH; more studies are needed to validate available findings.
Few data available; more studies are needed in HLH therapy.
References [22,23,32,35]
Clinical trials: NCT02069899, NCT03312751, NCT03311854, NCT03985423.




HLH has represented for decades a severe clinical syndrome characterized by an uncertain pathogenesis, limited possibili- ties of interventions, and high mortality. The demonstration that the clinical manifestations of the HLH were the conse- quence of a deep dysregulation of immune system disease brought to the introduction of a chemo-immunosuppressive based on dexamethasone and etoposide to obtain the control of the disease, and of allogeneic stem cell transplantation as definitive curative therapy for the primary HLH where the defective mechanism of cytoxicity is genetically determined. Animal and clinical studies have demonstrated that IFN-γ is the cytokine that play a key role in maintaining the status of abnormal hyperinflammation in patients with HLH so that blocking this mediator the process of cell hyperactivation (lymphocytes, macrophages) can be switched off. Emapalumab is a humanized monoclonal antibody directed against circulating or tissue-associated IFN-γ that administered intravenously, was able to control HLH disease in almost two- third of the patients, without significant toxicity and infectious complications. The clinical efficacy of emapalumab has been assessed mainly in patient’s refractory, relapsing, or intolerant to conventional treatment that represent a poor prognosis category with a major incidence of transplant-related compli- cations and graft failure. The possibility to rescue these patients with a safe and effective biological target therapy is a major step forward that allows to bring the patient to the transplant with a better control of the underlying disease, mostly free from active infections, and without the burden of the hematological toxicity of chemotherapy or of metabolic sequelae of prolonged exposure to high-dose of steroids. The importance of this point is underlined by the favorable survi- val results of allogeneic transplantation in patients who had been treated with emapalumab, compared to the past [56], including either myeloablative or reduced-intensity condition- ing regimens [57,58]. In the phase II study, the clinical and safety data on 7 ‘naive’ patients treated with emapalumab as first-line were superimposable to that observed in the refrac- tory/relapsing/intolerant cohort, but these promising results need to be confirmed in a larger cohort. Considering the rarity of primary HLH, the possibility to test emapalumab versus the standard of care for the first-line therapy (dexamethasone and

IFN-γ: interferon-gamma; FDA: Food and Drug Administration; JAK-STAT: Janus kinase-signal transducer and activator of transcription; IL-1Ra: interleukin-1 receptor antagonist; IL-6 R: interleukin-6 receptor; HLH: hemophagocytic lymphhistiocytosis; sJIA: systemic juvenile idiopatic arthritis; MAS: macrophage activation syndrome.

immune response, IFN-γ has proved to be an important target to neutralize. In fact, the IFN-γ neutralization improved HLH manifes- tations and survival in both murine models and human clinical trials. Emapalumab, an IFN-γ inhibitor, is the first target therapy approved for the treatment of primary HLH patients with refrac- tory, recurrent or progressive disease or intolerance to conven- tional therapy, showing efficacy and low toxicity. Further studies are needed to widen our knowledge on its use in HLH patients and find out other possible clinical applications of this drug.
etoposide) in a formal phase III study is not feasible in an acceptable window of time so other types of assessment need to be explored, for example, a prospective study where the experimental arm is matched with a historical control. We believe this could be an important aim for the future because this biological drug has the potentiality to conjugate efficacy and velocity in controlling HLH without worsening the patient clinical conditions for the minor impact on innate and adap- tive immunity compared to polyclonal rabbit antilymphocyte serum, anti-CD52 monoclonal antibody (alemtuzumab), etopo- side, and high-dose of steroids. Finally, the demonstration of the key role of IFN-γ in secondary HLH and the mechanism of rejection and graft failure open the way for a broader applica- tion of this drug outside of primary HLH. On the basis of these considerations, we believe that the treatment of HLH is going to change in the few next years because emapalumab

represents a valid option for the cases needing a rescue treat- ment to bridge the patient to the transplant.

Author contributions
SC and CG thought the review; SC, MC, and CG performed the literature search; CG and SC wrote the manuscript. All authors read and approved the manuscript.

This paper was not funded

Declaration of interest
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Papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers.
1.Emile JF, Abla O, Fraitag S, et al. Revised classification of histiocy- toses and neoplasms of the macrophage-dendritic cell lineages. Blood. 2016 Jun 2 127;(22)2672–2681.
2.Marsh RA, Haddad E. How i treat primary haemophagocytic lymphohistiocytosis. Br J Haematol. 2018 Jul;182(2):185–199.
3.Voskoboinik I, Dunstone MA, Baran K, et al. Perforin: structure, function, and role in human immunopathology. Immunol Rev. 2010 May;235(1):35–54. .
4.Miao Y, Zhu HY, Qiao C, et al. Pathogenic gene mutations or variants identified by targeted gene sequencing in adults with hemophagocytic lymphohistiocytosis. Front Immunol. 2019 Mar;7 (10):395. .
5.Jordan MB, Allen CE, Greenberg J, et al. Challenges in the diagnosis of hemophagocytic lymphohistiocytosis: recommendations from the North American Consortium for Histiocytosis (NACHO). Pediatr Blood Cancer. 2019 Nov;66(11):e27929. .
6.George MR. Hemophagocytic lymphohistiocytosis: review of etiol- ogies and management. J Blood Med. 2014 Jun;12(5):69–86.
7.Henter JI, Elinder G, Soder O, et al. Incidence in Sweden and clinical features of familial hemophagocytic lymphohistiocytosis. Acta Paediatr Scand. 1991 Apr;80(4):428–435.
8.Niece JA, Rogers ZR, Ahmad N, et al. Hemophagocytic lymphohis- tiocytosis in Texas: observations on ethnicity and race. Pediatr Blood Cancer. 2010 Mar;54(3):424–428. .
9.Janka GE. Familial hemophagocytic lymphohistiocytosis. Eur J Pediatr. 1983 Jun-Jul;140(3):221–230.
10.Aricò M, Janka G, Fischer A, et al. Hemophagocytic lymphohistio- cytosis. report of 122 children from the International Registry. FHL Study Group of the Histiocyte Society. Leukemia. 1996 Feb;10 (2):197–203.

11.Trottestam H, Horne AC, Aricò M, et al. Chemoimmunotherapy for hemophagocytic lymphohistiocytosis: long-term results of the HLH-94 treatment protocol. Blood. 2011 Oct 27 118;(17) 4577–4584.
12.Henter JI, Arico M, Egeler RM, et al. HLH-94: a treatment protocol for hemophagocytic lymphohistiocytosis. HLH study Group of the Histiocyte Society. Med Pediatr Oncol. 1997 May 28;(5)342–347.
13.Bergsten E, Horne AC, Aricó M, et al. Confirmed efficacy of etopo- side and dexamethasone in HLH treatment: long-Term results of the cooperative HLH-2004 study. Blood. 2017 Dec 21 130;(25) 2728–2738.
14.Henter JI, Horne AC, Aricó M, et al. HLH-2004: diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer. 2007 Feb 48;(2)124–131.
15.Mahlaoui N, Ouachée-Chardin M, De Saint Basile G, et al. Immunotherapy of familial hemophagocytic lymphohistiocytosis with antithymocyte globulins: a single-center retrospective report of 38 patients. Pediatrics. 2007 Sep 120;(3)e622–8.
16.McClain KL. Treatment of hemophagocytic lymphohistiocytosis in the era of new biologics. Pediatr Blood Cancer. 2020 Oct;67(10): e28631.
17.Henter JI, Elinder G, Soder O, et al. Hypercytokinemia in familial hemo- phagocytic lymphohistiocytosis. Blood. 1991;78(11):2918–2922. .
18.Put K, Avau A, Brisse E, et al. Cytokines in systemic juvenile idiopathic arthritis and haemophagocytic lymphohistiocytosis: tipping the bal- ance between interleukin-18 and interferon-γ. Rheumatology (Oxford). 2015 Aug 54;(8)1507–1517.
19.Tang Y, Xu X, Song H, et al. Early diagnostic and prognostic significance of a specific Th1/Th2 cytokine pattern in children with haemophagocytic syndrome. Br J Haematol. 2008 Oct 143;(1) 84–91.
20.Xu XJ, Tang YM, Song H, et al. Diagnostic accuracy of a specific cytokine pattern in hemophagocytic lymphohistiocytosis in children. J Pediatr. 2012 Jun;160(6):984–990. e1.
21.Bracaglia C, De Graaf K, Pires Marafon D, et al. Elevated circulating levels of interferon-γ and interferon-γ-induced chemokines charac- terize patients with macrophage activation syndrome complicating systemic juvenile idiopathic arthritis. Ann Rheum Dis. 2017;76 (1):166–172. .
22.Jordan MB, Hildeman D, Kappler J, et al. An animal model of hemophagocytic lymphohistiocytosis (HLH): CD8+ T cells and inter- feron gamma are essential for the disorder. Blood. 2004 Aug 1; 104 (3):735–743.
23.Behrens EM, Canna SW, Slade K, et al. Repeated TLR9 stimulation results in macrophage activation syndrome-like disease in mice. J Clin Invest. 2011 Jun 121;(6)2264–2277.
24.Pachlopnik Schmid J, Ho CH, Chrétien F, et al. Neutralization of IFNgamma defeats haemophagocytosis in LCMV-infected perforin- and Rab27a-deficient mice. EMBO Mol Med. 2009 May 1;(2) 112–124.
25.Kögl T, Müller J, Jessen B, et al. Hemophagocytic lymphohistiocy- tosis in syntaxin-11-deficient mice: t-cell exhaustion limits fatal disease. Blood. 2013 Jan 24 121;(4)604–613.
26.Gather R, Aichele P, Goos N, et al. Trigger-dependent differences determine therapeutic outcome in murine primary hemophagocy- tic lymphohistiocytosis. Eur J Immunol. 2020 Nov 2 50;(11) 1770–1782.
27.Brisse E, Imbrechts M, Put K, et al. Mouse cytomegalovirus infection in balb/c mice resembles virus-associated secondary hemophago- cytic lymphohistiocytosis and shows a pathogenesis distinct from primary hemophagocytic lymphohistiocytosis. J Immunol. 2016 Apr 1 196;(7)3124–3134.
28.Humblet-Baron S, Franckaert D, Dooley J, et al. IFN-γ and CD25 drive distinct pathologic features during hemophagocytic lympho- histiocytosis. J Allergy Clin Immunol. 2019 [Jun 1];143(6):2215– 2226.e7. .
29.Burn TN, Weaver L, Rood JE, et al. Genetic deficiency of interferon-γ reveals interferon-γ–independent manifestations of murine hemo- phagocytic lymphohistiocytosis. Arthritis Rheumatol. 2020 Feb;72 (2):335–347. .

30.Merli P, Quintarelli C, Strocchio L, et al. The role of interferon-gamma and its signaling pathway in pediatric hematological disorders. Pediatr Blood Cancer. 2021 Apr;68(4):e28900.
31.Hatterer E, Richard F, Malinge P, et al. P156 Investigating the novel mechanism of action for NI-0501, a human interferon gamma monoclonal antibody. Cytokine. 2012 Sep 1 59;(3)570.
32.Locatelli F, Jordan MB, Allen C, et al. Emapalumab in children with primary hemophagocytic lymphohistiocytosis. N Engl J Med. 2020 May 7;382(19):1811–1822. .
•• Phase II/III trial in pediatric patients with primary HLH.
33.Novimmune SA. GAMIFANTTM (emapalumab-Izsg): prescribing information. 2018.[Internet]. cited 2020 Nov 11]. Available from. : https://www.accessdata.fda.gov/drugsatfda_docs/label/2018/
● Prescribing information for emapalumab
34.Al-Salama ZT. Emapalumab: first global approval. Drugs. 2019 Jan;79(1):99–103.
36.Gloude NJ, Dandoy CE, Davies SM, et al. Thinking beyond HLH: clinical features of patients with concurrent presentation of hemo- phagocytic lymphohistiocytosis and thrombotic microangiopathy. J Clin Immunol. 2020 Jul;40(5):699–707. .
37.Lounder DT, Bin Q, De Min C, et al. Treatment of refractory hemo- phagocytic lymphohistiocytosis with emapalumab despite severe concurrent infections. Blood Adv. 2019 Jan 8; 3(1):47–50.
38.Tucci F, Gallo V, Barzaghi F, et al. Treatment with emapalumab in an ADA-SCID patient with refractory hemophagocytic lympho histiocytosis-related graft failure and disseminated BCGitis. Haematologica. 2020 Aug;13:2020.255620. haematol.
39.Merli P, Caruana I, De Vito R, et al. Role of interferon-γ in immune- mediated graft failure after allogeneic hematopoietic stem cell transplantation. Haematologica. 2019 Nov;104(11):2314–2323. .
40.Chinello M, Balter R, De Bortoli M, et al. Chronic graft-versus- host-disease-related polymyositis: a 17-months-old child with a rare and late complication of haematopoietic stem cell transplantation. Mediterr J Hematol Infect Dis. 2020 Jan 1 12; (1)e2020002.
41.Gabr JB, Liu E, Mian S, et al. Successful treatment of secondary macrophage activation syndrome with emapalumab in a patient with newly diagnosed adult-onset still’s disease: case report and review of the literature. Ann Transl Med. 2020 Jul;8(14):887. .
42.Lam MT, Coppola S, Krumbach OHF, et al. A novel disorder invol- ving dyshematopoiesis, inflammation, and HLH due to aberrant CDC42 function. J Exp Med. 2019 Dec 2 216;(12)2778–2799.
43.Das R, Guan P, Sprague L, et al. Janus kinase inhibition lessens inflammation and ameliorates disease in murine models of hemo- phagocytic lymphohistiocytosis. Blood. 2016 Mar 31 127;(13) 1666–1675.
44.Ahmed A, Merrill SA, Alsawah F, et al. Ruxolitinib in adult patients with secondary haemophagocytic lymphohistiocytosis: an

open-label, single-centre, pilot trial. Lancet Haematol. 2019 Dec 1 6;(12)e630–7.
45.Zhang Q, Wei A, Ma HH, et al. A pilot study of ruxolitinib as a front-line therapy for 12 children with secondary hemophagocytic lymphohistiocytosis. Haematologica. 2020 Jul; 30: haematol 2020.253781
46.Wei A, Ma H, Li Z, et al. Short-term effectiveness of ruxolitinib in the treatment of recurrent or refractory hemophagocytic lymphohistio- cytosis in children. Int J Hematol. 2020 Oct;112(4):568–576. .
47.Trantham T, Auten J, Muluneh B, et al. Ruxolitinib for the treatment of lymphoma-associated hemophagocytic lymphohistiocytosis: a cautionary tale. J Oncol Pharm Pract. 2020 Jun;26(4):1005–1008.
48.Ramanan KM, Uppuluri R, Ravichandran N, et al. Successful remis- sion induction in refractory familial hemophagocytic lymphohistio- cytosis with ruxolitinib as a bridge to hematopoietic stem cell transplantation. Pediatr Blood Cancer. 2020 Mar;67(3):e28071. .
49.Marsh RA, Allen CE, McClain KL, et al. Salvage therapy of refractory hemophagocytic lymphohistiocytosis with alemtuzumab. Pediatr Blood Cancer. 2013 Jan;60(1):101–109. .
50.Ravelli A, Grom AA, Behrens EM, et al. Macrophage activation syndrome as part of systemic juvenile idiopathic arthritis: diagnosis, genetics, pathophysiology and treatment. Genes Immun. 2012 Jun;13(4):289–298.
51.Miettunen PM, Narendran A, Jayanthan A, et al. Successful treat- ment of severe paediatric rheumatic disease-associated macro- phage activation syndrome with interleukin-1 inhibition following conventional immunosuppressive therapy: case series with 12 patients. Rheumatology (Oxford). 2011 Feb 50;(2) 417–419.
52.Rajasekaran S, Kruse K, Kovey K, et al. Therapeutic role of anakinra, an interleukin-1 receptor antagonist, in the management of sec- ondary hemophagocytic lymphohistiocytosis/sepsis/multiple organ dysfunction/macrophage activating syndrome in critically ill children. Pediatr Crit Care Med. 2014 Jun 15;(5)401–408.
53.Eloseily EM, Weiser P, Crayne CB, et al. Benefit of anakinra in treating pediatric secondary hemophagocytic lymphohistiocytosis. Arthritis Rheumatol. 2020 Feb;72(2):326–334. .
54.Fitzgerald JC, Weiss SL, Maude SL, et al. Cytokine release syndrome after chimeric antigen receptor T cell therapy for acute lympho- blastic leukemia. Crit Care Med. 2017 Feb;45(2):e124–e131. .
55.Dufranc E, Del Bello A, Belliere J, et al. TAIDI (Toulouse Acquired Immune Deficiency and Infection) study group. IL6-R blocking with tocilizumab in critically ill patients with hemophagocytic syndrome. Crit Care. 2020 Apr 22 24;(1)166.
56.Bergsten E, Horne A, Hed Myrberg I, et al. Stem cell transplanta- tion for children with hemophagocytic lymphohistiocytosis: results from the HLH-2004 study. Blood Adv. 2020 Aug 11 4; (15)3754–3766.
57.Allen CE, Marsh R, Dawson P, et al. Reduced-intensity conditioning for hematopoietic cell transplant for HLH and primary immune deficiencies. Blood. 2018 Sep 27 132;(13)1438–1451.
58.Messina C, Zecca M, Fagioli F, et al. Outcomes of children with hemophagocytic lymphohistiocytosis given allogeneic hemato- poietic stem cell transplantation in italy. Biol Blood Marrow Transplant. 2018 Jun;24(6):1223–1231. .