Hemophagocytic Lymphohistiocytosis: Clinical Presentations and Diagnosis

Grand Rounds Review

Hemophagocytic Lymphohistiocytosis: Clinical Presentations and Diagnosis Kimberly A. Risma, MD, PhDa, and Rebecca A. Marsh, MDb Cincinnati, Ohio Hemophagocytic lymphohistiocytosis (HLH) is an overwhelming clinical syndrome associated with extreme immune activation. Familial HLH is caused by autosomalrecessive inheritance of gene mutations that cripple lymphocyte cytotoxicity. X-linked lymphoproliferative diseases and mutations in Nod-like receptor caspase activation and recruitment domain containing protein 4 (NLRC4) also feature HLH as a predominant manifestation. In addition, “secondary” HLH may occur in immunocompromized patients or in individuals with previously intact immune responses in the context of strong immunologic triggers such as EBV infection, malignancy, rheumatologic disease, and drug hypersensitivity. Regardless of the etiology, HLH is often fatal unless recognized and treated aggressively. Research over the last 20 years has led to many advances in diagnosis and treatment. Rapid testing strategies designed to quickly screen for immune activation and cytotoxic lymphocyte dysfunction are now clinically available and genetic panels/testing algorithms may accelerate a genetic diagnosis. Immunosuppressive treatment protocols have been refined, and experience is gaining with alternative and salvage approaches. However, these advances improve the outcome of patients only when the diagnosis of HLH is made. Ongoing education is needed to ensure medical providers can appropriately recognize and diagnose HLH. This Grand Rounds Review will summarize the clinical and diagnostic features of HLH and highlight known genetic causes. Ó 2018 American Academy of Allergy, Asthma & Immunology (J Allergy Clin Immunol Pract 2019;-:---) Key words: Hemophagocytic lymphohistiocytosis Lymphocyte cytotoxicity; Perforin; Inflammasome

(HLH);

INTRODUCTION Hemophagocytic lymphohistiocytosis (HLH) presents as a systemic disorder of immune activation typified by infiltration of a

Division of Allergy/Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio b Division of Bone Marrow Transplantation and Immune Deficiency, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio Conflicts of interest: The authors declare that they have no relevant conflicts of interest. Received for publication September 11, 2018; revised November 19, 2018; accepted for publication November 22, 2018. Available online -Corresponding author: Rebecca A. Marsh, MD, Division of Bone Marrow Transplantation and Immune Deficiency, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH 45229. E-mail: Rebecca.Marsh@ cchmc.org. 2213-2198 Ó 2018 American Academy of Allergy, Asthma & Immunology https://doi.org/10.1016/j.jaip.2018.11.050

the bone marrow, liver, skin, lymph nodes, spleen, and central nervous system with activated lymphocytes and macrophages. These activated cells cause hematologic cytopenias, hepatic dysfunction, coagulopathy, lymphadenopathy, splenomegaly, rash, hectic fevers, and central nervous system (CNS) symptoms ranging from seizures and/or focal deficits to encephalopathy. HLH is life-threatening due to rapid progression to multisystem organ failure if the diagnosis is not considered and immunosuppression confidently initiated. On recognition of the clinical syndrome, treatment is begun without delay while searching for primary genetic or secondary etiologies. Hematopoietic cell transplant (HCT) is currently the only curative therapy for primary HLH. Research over the last 2 decades has led to (1) enhanced diagnostic testing designed to rapidly screen for immune activation and cytotoxic lymphocyte dysfunction, (2) accelerated identification of genetic causes of primary HLH, and (3) refined immunosuppressive and allogeneic HCT protocols. However, these approaches improve the outcome of patients only when the diagnosis of HLH is made expediently. Ongoing education is needed to ensure medical providers are knowledgeable about the typical and atypical presentations of HLH. We present here a case that assists in recognition and then summarize the clinical and diagnostic features of HLH and known genetic causes.

CLINICAL CASE: INFANT WITH FAILURE TO THRIVE, FEVER, PANCYTOPENIA, AND LIVER DYSFUNCTION A 3-month-old girl was brought to the emergency department by her parents due to rapid breathing, poor feeding, and fever. The infant had already been hospitalized twice on the general pediatric and then gastroenterology service with failure to thrive and diarrhea, responding minimally to elemental formula given by nasogastric feeds for presumed “allergic colitis.” Her family history revealed that 2 siblings had died in early infancy of an unknown disorder that caused them to develop jaundice. At presentation she was alert, thin, pale, and febrile (38.8
C) and exhibited tachypnea and mild retractions. She had a mildly enlarged liver. She had no rash, lymphadenopathy, or focal neurologic deficits. She was admitted for concerns of viral infection. Routine laboratory evaluations revealed that she had pancytopenia (hemoglobin, 9 g/dL; platelets, 60,000/mL; absolute neutrophil count, 500 cells/mL); peripheral smear was notable for 20% atypical lymphocytes; lactate dehyrdrogenase (LDH) was elevated at 577 U/L, and liver function was abnormal with transaminitis (alanine aminotransferase [ALT], 515 U/L; aspartate aminotransferase [AST], 431 U/L) and mildly elevated INR (1.4). She was screened for viral and bacterial infections and treated with antibiotics on admission. 1

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Abbreviations used CNS- central nervous system CTL- cytotoxic T lymphocyte HCT- hematopoietic cell transplant HLH- hemophagocytic lymphohistiocytosis MAS- macrophage activation syndrome NK- natural killer NLRC4- Nod-like receptor caspase activation and recruitment domain containing protein 4 NR- normal range PID- primary immunodeficiency sIL2R- soluble IL2 receptor sJIA- systemic juvenile idiopathic arthritis SLAM- signaling lymphocyte associated molecule XIAP- X-linked inhibitor of apoptosis

Because of persistent and worsening fever, pancytopenia, transaminitis, and coagulopathy, HLH was suspected and a workup was rapidly initiated using the established clinical and laboratory criteria (Table I). Ferritin was mildly elevated at 630 ng/mL (normal range [NR], 5-204 ng/mL), fibrinogen was low at 154 mg/dL (NR, 189-458 mg/dL), and triglycerides were normal. Abdominal ultrasound revealed mild ascites and hepatosplenomegaly. The patient showed evidence of immune activation with increased soluble IL2 receptor (sIL2R) of 27,466 U/mL (NR, 334-3026 U/mL) and expanded granzyme B expression (75%; NR, 0%-61%) in her CD8þ T-cell compartment (Figure 1, A). Expression of perforin was normal and granzyme B increased in her natural killer (NK) cells (mean channel fluorescence, 1279; NR, 152-825) (Figure 1, B), ruling out perforin deficiency. She had low NK-cell degranulation (3%; NR, 11%-35%) (Figure 1, C). On hospital day 3, a bone marrow aspirate and biopsy were performed and revealed hemophagocytosis. Lumbar puncture showed mild leukocytosis (9 white blood cells). Infectious workup was negative for EBV, cytomegalovirus (CMV), adenovirus, hepatitides, enteroviruses, respiratory viruses, or bacterial infection. Her hemoglobin and fibrinogen plummeted, and she was transfused and given cryoprecipitate. As she met 7 of 8 clinical criteria for HLH (Table I), induction therapy was begun on day 4 of hospitalization, starting with 10 mg/m2 of dexamethasone.2-4 Etoposide was initiated 4 days later with good response.5 One month after admission, HLH gene panel results revealed the primary cause—biallelic mutations in STXBP2—and she was referred for allogeneic HCT.

GENETICS OF PRIMARY HLH The first genetic cause of familial or primary HLH, PRF1 mutations, was discovered by Stepp et al6 in 1999. PRF1 encodes perforin, a pore-forming protein stored in the cytotoxic lymphocyte secretory granules. On target cell recognition, NK cells and cytotoxic T lymphocytes (CTLs) degranulate and kill target cells via polarized delivery of perforin to the immune synapse (Figure 2). Target cell death proceeds within minutes of perforin pore formation.7 Following the identification of PRF1 mutations, other genetic causes have been identified that impair NK-cell and CTL degranulation (Table II): mutations in UNC13D, STXBP2, STX11, RAB27A, LYST, and AP3B1 all prevent perforin from

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TABLE I. Diagnostic criteria commonly used to aid the diagnosis of HLH* 1. Fever ‡38.5
C 2. Splenomegaly 3. Cytopenias (affecting at least 2 of the below): Hemoglobin < 9 g/dL (in infants < 4 wk: hemoglobin < 10 g/dL) Platelets < 100 3 109/L Neutrophils < 1 3 109/L 4. Hypertriglyceridemia and/or hypofibrinogenemia (<150 mg/dL) 5. Hemophagocytosis in bone marrow, spleen, lymph nodes, liver, or other tissue 6. Low or absent NK-cell activity 7. Ferritin > 500 ng/mL 8. Elevated sIL2R: >2400 U/mL or elevated based on the laboratorydefined normal range *A diagnosis is consistent with HLH if 5 of 8 of the below criteria are met, or if the patient has a molecular diagnosis of genetic HLH. Patient features from clinical case are in bolded type.1

ever reaching the immune synapse.8-14 Patients with mutations in RAB27A (Griscelli syndrome type 2), LYST (ChediakHigashi syndrome), and AP3B1 (Hermansky-Pudlak syndrome type 2) may have pigment abnormalities due to abnormal melanocyte granules. Impaired delivery of perforin and cytotoxic granule contents by any of these genetic defects is thought to prevent rapid killing of antigen-presenting and/or damaged target cells and to initiate runaway immune activation, normally controlled by NK-cell/CTL function. It is interesting to note that the infant in the case presented initially with failure to thrive and diarrhea without metabolic or inflammatory cause. Although this is not a typical feature of fulminant HLH, biallelic STXBP2 mutations have been identified in patients with early-onset failure to thrive and diarrhea.15 The relationship of STXPB2 mutations to enteropathy may be related to the expression of syntaxin binding protein 2 (Munc 18-2) in the intestinal epithelium rather than due to a defect in immune cells.16,17 Because the initial description of HLH involved disorders of NK-cell function against tumor target cells, it was somewhat surprising to find that other classes of genetic mutations did not exhibit impaired NK-cell killing against tumor target cells (Table II). Patients with mutations in SH2D1A (X-linked lymphoproliferative disease type 1) develop HLH on exposure to EBV, and may also present with lymphoma, hypogammaglobulinemia, aplastic anemia, and/or vasculitis.18-20 SH2D1A encodes signaling lymphocyte associated molecule (SLAM)-associated protein, a protein that is critical to the signaling of SLAM receptors in T cells and NK cells. The development of HLH may relate to several defects caused by SLAM-associated protein deficiency, including defective 2B4 receptoremediated killing and defective T-cell restimulation-induced cell death. Patients may also develop HLH due to hyperactivation of inflammasomes: intracellular protein complexes that detect pathogenic stimuli. The end point of inflammasome activation is production of proinflammatory IL-1 and/or IL-18. Patients with XIAP or NLRC4 mutations may present with symptoms of HLH and markedly elevated levels of IL-18,21-23 whereas NK-cell cytotoxicity is intact (Table II). Patients who have mutations in XIAP encoding X-linked inhibitor of apoptosis (XIAP) have X-linked lymphoproliferative

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FIGURE 1. Results of clinical perforin flow cytometry and CD107 degranulation from patient. Perforin (Prf) and granzyme B (Grz B) are measured by flow cytometry in both CD8 Tcells and NK cells. A, CD8 Tcells express granzyme B only after activation and differentiation into CTL. The infant with HLH has an expanded population (75%) of CTL expressing granzyme B (green shading), a marker of T-cell activation. B, Evaluation of perforin in NK cells is a rapid screening test for perforin deficiency. Granzyme B expression is also measured in NK cells as a (þ) control. Isotype () controls are shaded in gray. C, The patient has normal detection of perforin. Evaluation of degranulation of NK cells. NK cells only express granule-associated CD107a on their cell surface after activation by a target cell. The infant with HLH has impaired degranulation-CD107a detection was minimal (1.9%) after exposure of her NK cells to tumor target cells.

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and gastrointestinal epithelial cells.22,23 The clinical phenotype in its most severe form may present like XIAP deficiency with early-onset colitis and HLH symptomatology. The overlap between HLH associated with XIAP deficiency and NLRC4 gainof-function mutations certainly suggests that the immunopathology may be even more complicated than initially understood.

FIGURE 2. NK-cell and CTL granule-mediated cytotoxicity pathway. Following immune synapse formation between NK/ CTL and target cells, secretory granules (SGs) are polarized toward the target cell and their protein machinery (Munc 13-4, Syntaxin 11, Munc 18-2, Rab27a, LYST, and AP3) assembled to deliver perforin and granzymes to the target cell in a process called degranulation. Perforin and granzymes are death effectors when appropriately delivered to the immune synapse. Degranulation is dependent on SG formation (AP3, LYST); SG trafficking to the immune synapse (Rab27a); and SG docking, priming, and fusion (Munc 13-4, syntaxin 11, and Munc 18-2) with the plasma membrane. In the process of degranulation, perforin forms a multimeric pore on the target cell membrane and a membrane-bound protein appears on the NK/CTL cell surface that used to be “hidden” within SGs, CD107a. Intracellular, monomeric perforin, and extracellular CD107a are readily measurable in NK cells and CTL by flow cytometry as shown by red- and yellow-labeled antibodies in the figure, aiding in the diagnosis of primary HLH. Genetic testing is recommended when perforin or CD107a detection is abnormal. Modified from Marsh and Haddad.2

disease type 224 and may present with fulminant HLH as the herald illness or may exhibit atypical HLH-like episodes. In addition, patients with XIAP mutations may be diagnosed with inflammatory bowel disease, and/or humoral immunodeficiency with rare inflammatory complications of the eyes, liver, or lungs before ever presenting with HLH.24-29 The relationship of how XIAP deficiency leads to dysregulated IL-18 production due to presumed inflammasome dysregulation is still being defined. In contrast, patients with monoallelic, activating mutations in NLRC4 have constitutively active inflammasomes in hematologic

SECONDARY HLH HLH has been observed in the context of infections with or without primary immunodeficiency (PID). Treatable infections that should be screened for beyond typical bacterial and fungal cultures include systemic viruses (EBV, HIV, and other herpes viruses being predominant), tick-born bacteria, mycobacterial and histoplasmosis infections, and in geographically susceptible patients, Leshmania.30 The finding of HLH triggered by infections should also raise the possibility of PID with a defect in T-cell and/ or phagocyte function.31,32 PID associated with HLH include defects in MAGT1, ITK, CD27, IKBKG, or GATA 2.33-41 Patients with Wiskott-Aldrich syndrome, DiGeorge syndrome, chronic granulomatous disease, or signal transducer and activator of transcription 1 gain-of-function mutations have also been reported with HLH.31,32,42,43 Recent diagnostic approaches using wholeexome sequencing have yielded additional genetic mutations associated with HLH such as additional defects in inflammasome components43 and sporadic metabolic disorders.44 There is a growing recognition of malignancy-associated HLH,45-47 identified retrospectively in pediatric and adult patients with malignancies. HLH may present during chemotherapy when there is typically substantial lymphopenia and infectious trigger, or HLH may present at the time of diagnosis. Finally, patients with rheumatologic disorders such as lupus in adults and systemic juvenile idiopathic arthritis (sJIA) in children may present with macrophage activation syndrome (MAS).48 MAS is an inflammatory disorder with substantial overlap with HLH; for example, patients present with fever, cytopenias, splenomegaly, hepatic dysfunction, disseminated intravascular coagulation, hemophagocytosis, and CNS symptoms. Some patients with sJIA/ MAS have monoallelic mutations in the same genes that cause primary HLH (reviewed in Bracaglia et al49), and studies have documented the presence of impaired NK-cell function in a fraction of patients with MAS.50 However, in MAS, the patient is typically older, biallelic mutations in HLH genes are absent, and a clinical history of rheumatic disease is frequently present. Hypercytokinemia is a unifying feature of MAS and primary HLH, and a recent study demonstrated baseline elevations in IL-18, an inflammasome-dependent cytokine, in patients with sJIA,51 with MAS levels exceeding what is typically measured in primary HLH. Patients with sJIA/MAS flares52 and patients with HLH53 also have elevated IFN-g levels in the blood. Distinguishing features between MAS and primary HLH include higher levels of platelets and fibrinogen in MAS. MAKING THE DIAGNOSIS OF HLH Although the typical presentation of HLH in a young infant includes a classic constellation of hectic fevers of unknown origin, cytopenias, splenomegaly, and hemophagocytosis in the bone marrow or other tissues, these signs/symptoms are not specific for HLH. In addition, not all features may be present at presentation, leading to an alternate diagnosis. For example, if fever, cytopenias, and bleeding related to coagulopathy are initial

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TABLE II. Most frequent genetic causes of HLH with rapid screening tests Diseases

Gene

Diseases of abnormal cytotoxicity FHL2* PRF1

FHL3*

UNC13D

FHL4* STX11 FHL5* STXBP2 Griscelli syndrome RAB27A Chediak-Higashi syndrome LYST Diseases of abnormal SLAM family receptor XLP1 SH2D1A

Diseases of abnormal inflammasome XLP2 XIAP

NLRC4 Mutation

NLRC4

Protein

Inheritance

Perforin

AR

Munc13-4

AR

Syntaxin 11 Munc18-2 Rab27a LYST signaling SLAM-associated protein (SAP)

AR AR AR AR

Special clinical features

Missense mutations may have atypical presentations

Rapid screening test

Flow cytometry—intracellular perforin expression in NK/CTL Flow cytometry— Degranulation assay—Surface CD107a on NK cells or CTL after trigger of degranulation

Early-onset diarrhea; low IgG Albinism variably present

May present with lymphoma, CVID, EBV drives HLH

Flow cytometry—Intracellular SAP expression in T and NK cells

XIAP

May present with IBD, CVID; Females skewed ionization may also be affected

NLR Family, CARD domain-containing protein 4

May present with IBD

Flow cytometry—Intracellular XIAP expression in leukocytes, NOD2 signaling in monocytes in response to muramyl peptide, serum IL-18 Serum IL-18

CVID, Common variable immunodeficiency; FHL, familial hemophagocytic lymphohistiocytosis [FHL1 remains genetically undefined]; IBD, inflammatory bowel disease; XLP1, X-linked lymphoproliferative disease type 1; XLP2, X-linked lymphoproliferative disease type 2.

features, then sepsis is typically considered before HLH. Pancytopenia and fever are also sentinel features in leukemia. Inadvertent treatment of leukemia or lymphoma with dexamethasone may cause temporary remission but delay definitive diagnosis and treatment. When rashes and fever are predominant, juvenile idiopathic arthritis, other autoinflammatory fever disorders, or rickettsial infections may be contemplated. When the primary complaint is acute liver failure without other features of typical HLH, then congenital metabolic disorders may be considered. Infants presenting with primary CNS symptoms and fever may be mistaken for having herpes simplex virus (HSV) encephalitis. Fever, pancytopenia, transaminitis, gut inflammation, and rash may be noted in patients with Omenn syndrome related to severe combined immunodeficiency. In drug hypersensitivity reactions such as Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS), many features of HLH may be present, but the disease is readily treated with discontinuation of the drug and administration of systemic steroids. It is obvious from the variety of presentations that primary care physicians, intensivists, and subspecialists (rheumatology, gastroenterology, allergy/immunology, neurology, genetics, and hematology/oncology) all need to recognize the early signs of HLH and pursue appropriate diagnostic evaluations. A general approach to the evaluation of a patient with suspected HLH is presented in Figure 3. Making the diagnosis of HLH is aided by the use of the clinical and laboratory criteria that were established by the

Histiocyte Society for use in the HLH-2004 clinical trial.1 However, many patients with HLH may not meet strict 5 of 8 criteria early during disease as noted above. In the case described, the early death of 2 siblings was an ominous sign and likely accelerated the diagnosis of primary HLH. In the absence of this history, her respiratory symptoms may have been attributed to elevated fever, cytopenias related to viral infection, and her minimally elevated ferritin and INR (if ordered) would not have supported a rapid diagnosis and treatment of HLH. Strict adherence to the HLH criteria may also prevent the diagnosis of HLH. For example, some clinicians may feel hesitant to diagnose HLH in the absence of hemophagocytosis despite the fact that it may not be present early in disease. In addition, clinicians may overinterpret the finding of hemophagocytosis as a specific marker of HLH, which it is not.54-57 Historical laboratory tests of immune activation included altered triglycerides, fibrinogen, and ferritin. The first biomarker of T-cell activation to be assessed in HLH was sIL2R,58 a marker of T-cell proliferation and activation.59 SIL2R is also not specific for HLH and is not only a marker of T-cell activation in inflammatory disorders but also a measure of lymphocyte proliferation associated with malignancy.60,61 Elevated sCD163 may be added as a marker of macrophage activation in patients with sJIA.62 Finally, newer adjunctive testing may include measurement of levels of CXCL9, which indicate IFN-g pathway activity52,63 or IL-18, indicative of inflammasome activity.51

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FIGURE 3. Suggested diagnostic strategy for the syndrome of HLH. ALT, Alanine aminotransferase; AST, aminotransferase; CBC, complete blood cell count; CNS, central nervous system; CMV, cytomegalovirus; CT, computed tomography; EBV, Epstein-Barr virus; DRESS, Drug Reaction with Eosinophilia and Systemic Symptoms; HSV, herpes simplex virus; LDL, lactate dehydrogenase; MRI, magnetic resonance imaging; PET-CT, positron emission tomography-computed tomography; sILR2, soluble IL-2 receptor.

Flow cytometric measurement of T-cell activation may also be used as an indication of HLH. For example, Ammann et al64 recently described upregulation of HLA-DR. Some centers use this marker, which is easy to measure by flow cytometry, in place of sIL2R. Other flow cytometry measurements that may aid in diagnosis included increased expression of granzyme B in NK and CD8 T cells.65 Expanded granzyme B in CTL was noted in the patient described above (Figure 1). Granzyme B expression in CD8 T cells occurs only after T-cell activation and differentiation into CTL and is therefore a marker of recent MHC Iedriven immune activation. Elimination of dendritic

cells bearing foreign antigen on MHC I is critically dependent on perforin-dependent cytotoxicity in murine models of HLH.66 NK-cell functional testing has also evolved. Absent lytic killing of tumor target cells by a patient’s NK cells was initially described as the criterion standard diagnostic test. However, because of limitations related to shipping, this test is not widely available for screening patients and its diagnostic accuracy appears to be somewhat limited compared with those of newer assays of degranulation coupled with evaluation of perforin expression.67

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DIAGNOSING GENETIC HLH A definitive diagnosis of genetic HLH can take time because genetic testing generally still takes several weeks. Several clinical laboratories now offer panel-oriented genetic testing options that can make testing simpler for physicians; that is, a panel of HLHassociated genetic diseases can be ordered instead of ordering individual genes sequentially or in tandem. Rapid screening diagnostics are available to screen for genetic defects associated with HLH, and results are usually available within a few days. Flow cytometric assays can quickly screen for deficiencies of perforin, SLAM-associated protein, and XIAP. Evaluation of NK-cell or CTL degranulation quickly screens for abnormalities caused by mutations in genes that are critical for degranulation such as UNC13D, STX11, STXBP2, RAB27A, and LYST. A general approach to the use of diagnostic screening and genetic testing for the most common genetic causes of HLH is presented in Table II. TREATMENT OF HLH In much of North America and Europe, the mainstays of primary HLH treatment have been dexamethasone and etoposide. Clinical treatment is often modeled on HLH-1994 and HLH-2004 study protocols used for clinical trials by the Histiocyte Society.3,4 HLH-2004 incorporated the early use of cyclosporine; however, no clear benefit was shown, and cyclosporine can be associated with serious complications including posterior reversible encephalopathy syndrome and renal toxicity.4 Based on trial experience, approximately half the patients can be expected to achieve a complete response at 2 months, and another third of patients can be expected to achieve a partial response.3 Patients with CNS HLH should generally receive intrathecal steroids and methotrexate.68 There are several small reports of treatment of HLH with alternative therapeutics directed against IL-1beta, IL-6, and TNF, but no large-scale studies have been conducted. Several clinical trials are currently being conducted for HLH including a “Hybrid Immunotherapy” study of steroids, etoposide, and ATG69 and separate trials evaluating the use of an antieIFN-g mAb,70,71 alemtuzumab,72 tocilizumab,73 and ruxolitinib.74 Recombinant human IL-18 binding protein is also being studied specifically in patients with XIAP deficiency and NLRC4 mutations.75 The evidence for several salvage approaches has recently been reviewed.76 Treatment for secondary HLH includes both immunosuppressive therapy and treatment of the underlying disorder, such as infection and malignancy (Figure 3). Treatment of MAS flares in patients with sJIA typically includes high-dose, systemic steroids, and agents such as cyclosporine, anakinra, or other steroidsparing agents are often used.48,77 Because of the risk of adverse effects from etoposide, a consensus statement was recently published for consideration of its use in all forms of HLH.5 Patients with HLH may be critically ill and require intensive care support and monitoring for complications of treatments such as infections and toxicities. Consideration should also be given to providing antimicrobial prophylaxis to patients when indicated such as antiePneumocystis jirovecii prophylaxis, general antifungal prophylaxis, and antiviral prophylaxis depending on age and previous exposures. Immunoglobulin replacement is also a general consideration. Infusions of blood products are usually needed early in the course of HLH.

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Allogeneic HCT is the definitive cure for patients with most genetic forms of HLH. Patients with HLH have an increased risk of toxicities, and myeloablative regimens are associated with high rates of hepatic veno-occlusive disease, pulmonary hemorrhage, and early death in these patients. Survival following myeloablative conditioning regimens has generally ranged from approximately 40% to 70%.3,27,78-85 More recent reducedintensity approaches are generally associated with greater than 70% survival but can be associated with increased rates of mixed chimerism and secondary graft failure.27,86-90 Transplant outcomes are still significantly lower for patients with HLH compared with patients with other primary immune deficiencies. Recent data from the Center for International Blood and Marrow Transplant Research revealed that 3-year overall survival was 71% among 413 patients with HLH treated at Center for International Blood and Marrow Transplant Research centers between 2010 and 2016, whereas survival for the previously mentioned PIDs was 80% or greater.91 Patients with XIAP deficiency appear to fare particularly poorly with allogeneic HCT, though reduced-intensity regimens can result in good outcomes.92,93 Given that XIAP deficiency is highly variable, not all patients should be considered for allogeneic HCT.

CONCLUSIONS Many advances have been made in the last 20 years in the HLH field. With proper recognition of the clinical syndrome of HLH, physicians should be able to rapidly instigate diagnostic evaluations. It is important to have a good understanding of the unique caveats and pitfalls of making the diagnosis of HLH, and advanced testing strategies can help physicians rapidly progress through diagnostic algorithms and begin treatment in appropriate patients. Ongoing education and forthcoming advances will continue to improve outcomes for patients. REFERENCES 1. Henter JI, Horne A, Arico M, Egeler RM, Filipovich AH, Imashuku S, et al. HLH-2004: diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 2007;48:124-31. 2. Marsh RA, Haddad E. How I treat primary haemophagocytic lymphohistiocytosis. Br J Haematol 2018;182:185-99. 3. Henter JI, Samuelsson-Horne A, Arico M, Egeler RM, Elinder G, Filipovich AH, et al. Treatment of hemophagocytic lymphohistiocytosis with HLH-94 immunochemotherapy and bone marrow transplantation. Blood 2002; 100:2367-73. 4. Bergsten E, Horne A, Arico M, Astigarraga I, Egeler RM, Filipovich AH, et al. Confirmed efficacy of etoposide and dexamethasone in HLH treatment: long term results of the cooperative HLH-2004 study. Blood 2017;130:2728-38. 5. Ehl S, Astigarraga I, von Bahr Greenwood T, Hines M, Horne A, Ishii E, et al. Recommendations for the use of etoposide-based therapy and bone marrow transplantation for the treatment of HLH: consensus statements by the HLH Steering Committee of the Histiocyte Society. J Allergy Clin Immunol Pract 2018;6:1508-17. 6. Stepp SE, Dufourcq-Lagelouse R, Le Deist F, Bhawan S, Certain S, Mathew PA, et al. Perforin gene defects in familial hemophagocytic lymphohistiocytosis. Science 1999;286:1957-9. 7. Lopez JA, Susanto O, Jenkins MR, Lukoyanova N, Sutton VR, Law RH, et al. Perforin forms transient pores on the target cell plasma membrane to facilitate rapid access of granzymes during killer cell attack. Blood 2013;121:2659-68. 8. Feldmann J, Callebaut I, Raposo G, Certain S, Bacq D, Dumont C, et al. Munc13-4 is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (FHL3). Cell 2003;115:461-73. 9. zur Stadt U, Schmidt S, Kasper B, Beutel K, Diler AS, Henter JI, et al. Linkage of familial hemophagocytic lymphohistiocytosis (FHL) type-4 to chromosome 6q24 and identification of mutations in syntaxin 11. Hum Mol Genet 2005;14: 827-34.

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