 Brant Ward, MD, PhD Allergy & Immunology

 Recognize the diagnostic criteria for HLH  Become familiar with the genetic and mechanistic causes of HLH  Describe the differences between genetic and acquired forms of HLH  Formulate an effective treatment plan

 Pathologic finding of phagocytosis of red blood cells, leukocytes, and thrombocytes by macrophages  Thought to occur after over-activation of macrophages due to dysregulated immune responses

 In 1939, Scott & Robb-Smith described patients with “atypical Hodgkin’s disease” – proliferation of histiocytes affecting all lympho-reticular tissues  Farquhar & Claireaux described a familial syndrome with similar features in 1952  Hallmark clinical features include fever, splenomegaly, and cytopenias; hepatitis, altered mental status, and neurological involvement seen as well  Syndromes characterized by hemophagocytosis are termed ‘hemophagocytic lymphohistiocytsis’ (HLH)

 Diagnostic criteria for HLH were proposed by the Histiocyte Society in 1991  Five of the eight following criteria must be present to make the diagnosis:  Alternatively, identification of one of the known genetic defects associated with the disease  Ferritin is the most sensitive at discerning HLH from other disorders o Ferritin >10,000 ng/ml is >90% specific for HLH in children o Ferritin >50,000 ng/ml is less specific in adults, but still very sensitive Fever Cytopenias in 2 of 3 lineages Splenomegaly Hypertriglyceridemia and/or hypofibrinogenemia Hemophagocytosis Low or absent NK cell activity Hyperferritinemia Elevated plasma levels of soluble CD25

HLH Perforin deficiency Munc 13-4 deficiency Syntaxin 11 deficiency Munc 18-2 deficiency Unknown gene mutations Immune deficiencies Malignancy Autoimmune diseases Viral infections Bacterial infections Fungal infections Helminthic infections Medications

 Genetic HLH o Disorders characterized by elevated risk for HLH o Includes Familial Hemophagocytic Lymphohistiocytosis (FHL) as well as certain immunodeficiencies o Caused by defects in the cell-mediated cytotoxicity pathways  Acquired HLH o A.k.a., Reactive Hemophagocytic Lymphohistiocytosis (RHL) o Varied group of disorders that result in hemophagocytic symptoms o Caused by dysregulated immune responses leading to lymphocyte and macrophage activation

 FHL can be divided into 5 subtypes: o FHL1 – caused by unknown defect on chromosome 9 o FHL2 – caused by deficiency of Perforin o FHL3 – caused by deficiency of Munc 13-4 o FHL4 – caused by deficiency of Syntaxin 11 o FHL5 – caused by deficiency of Munc 18-2  Chediak-Higashi & Griscelli II syndromes are characterized by partial albinism and immune deficiency  XLP is characterized by massive lymphoproliferation and immune deficiency

 Sometimes referred to as Farquhar’s disease after its describer (1952)  Autosomal recessive inheritance with estimated incidence of 1:50,000 live births (male > female)  Symptoms are usually evident by 1 year (70-80% of case) and can even present at birth or in utero  Some forms can present in later childhood or even as adults  Overwhelming HLH is the primary symptom, and deficient NK cell-mediated cytotoxicity is characteristic

 Identified from four consanginous families of Pakistani descent using homozygosity mapping  First defined susceptibility locus for FHL, located at 9q  This locus contains hundreds of candidate genes, though none have been identified as the culprit

 Perforin (PRF1) was the first identified gene causing FHL  >70 different mutations have been identified  Trp374 stop has high incidence in Turkish families  L364 frame-shift is found in Japanese families  L17 frameshift found in families of African origin Stepp, et al. Science Dec 3;286(5446):

 Protein found in lytic granules of NK cells and cytotoxic T lymphocytes  Contains MACPF domain that shares a high degree of homology with complement proteins C6-9  Oligomerizes within the membrane of target cell, forming a channel in the membrane  Perforin alone is sufficient to lyse target cells at high (i.e., non-physiologic) concentrations  Perforin channels allow entry of granzymes from the immune synapse into the target cell cytoplasm

Kondos, et al. Tissue Antigens Nov;76(5):

 Due to mutations in Munc 13-4  Identified from 7 affected families (6 consanguinous)  Munc 13-4 defiency accounts for 30-35% of cases  Together with perforin gene mutations, cause up to 70% of FHL cases Feldmann, et al. Cell Nov 14;115(4):

 Member of the Munc 13-UNC 13 family of proteins  Many expressed at the neurological synapse, acting as priming factors for synaptic vesicle secretion  Deficiency causes impaired release of cytotoxic granules from cells, but no affect on interferon-  secretion  Munc 13-4 is required for priming of lytic granules that are docked at the plasma membrane  Goblet cells in lung epithelium express high levels of Munc 13-4, but deficiency causes no observable lung pathology

 Mutations in syntaxin 11 characterize FHL4  Identified in a large consanguineous Kurdish family  All identified mutations in are null mutations  Syntaxin mutations account for ~20% of FHL cases in Turkish and Kurdish populations zur Stadt, et al. Hum Mol Genet Mar 15;14(6):

 Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) family member  Phylogenetically related to the target membrane SNARE (t- SNARE) proteins syntaxin 1-4  Selective pairing of t-SNARE, v-SNARE, and adaptor proteins form a stable parallel four helical bundle  Deficiencies cause defects in NK cell, but not CTL, cytotoxic activity, which is partially rescued by IL-2  FHL4 phenotype does not differ from that of FHL 2 or 3

 Results from deficiency of Munc 18-2  Identified in patients of African, Arabian, Turkish, and European descent  Phenotype appears to correlate with genotype based on age of onset and severity of disease Cote, et al. J Clin Invest Dec;119(12):

 Also called syntaxin-binding-protein-2 (STXBP2)  Member of SM family of fusion accessory proteins — complimentary role with SNAREs in membrane fusion  Syntaxin 11 expression is impaired in Munc 18-2 deficient cells, suggesting a requirement for Munc18-2  From data on Munc 18-1, Munc 18-2/syntaxin 11 complex regulates docking and initiation of SNARE complex formation

 Pigmentary dilution, HLH, defective NK, T cell, & neutrophil function  HLH typically occurs later than in FHL (2-10 years)  Light complexion, silvery hair, and characteristic peripheral nerve disease  Infections are common, due to inability to kill organisms after phagocytosis Reddy, et al. Int J Trichology Jul;3(2):

 Caused by mutation in CHSI/LYST, a ubiquitously-expressed protein  Function of LYST has been inferred from studies of other BEACH family proteins  May regulate sorting of endosomal proteins into lysosomes or regulate fusion or fission events of lysosomes  Striking feature is the occurrence of giant intra-cytoplasmic lysosomal structures in all granulated cells, with lack of degranulation upon stimulus

 Pigmentary dilution, HLH, and pyogenic infections  Onset of HLH is later than in FHL (median age 3 years)  Patients have silvery hair and light skin  NK cells and CTLs show impaired degranulation

 Caused by mutations in the gene encoding Rab27a, a ubiquitously expressed small GTPase  Enriched on endosomal structures that fuse with cytotoxic granules before release of their contents  Deficiency renders cytotoxic granules unable to reach the immune synapse to dock with the plasma membrane  Interacts with Munc 13-4 to coordinate the final step of the exocytic process, between docking and priming of the granule

 X-linked lymphoproliferative disorder—characterized by hypogammaglobulinemia or lymphoproliferation  Caused by mutations in SLAM-associated protein (SAP) or X- linked inhibitor of apoptosis (XIAP)  Epstein-Barr virus infection results in fulminant and fatal mononucleosis  HLH is almost always associated with EBV infection  SAP deficiency results in a partial cytotoxic defect; no observable cytotoxic defect in XIAP deficiency

Vesicle Maturation (LYST) Vesicle Docking (Rab27a) Vesicle Priming (Munc 13-4) Vesicle Fusion (Syntaxin 11/Munc 18-2) Effector Function (Perforin)

APC Virus-infected Cells CTL IFN- 

APC Virus-infected Cells CTL IFN-  CTL

 Perforin deficient mice with HLH have normal total amount of dendritic cells, but… Terrell & Jordan. Blood Jun 27;121(26):

Jordan, et al. Blood Aug 1;104(3):

 The cytokines production and immune activation triggered by these cells is thought to cause the observed symptoms o Fever is induced by overproduction of IL-1 o Pancytopenia is a consequence of TNF  and IFN  o Activated macrophages actively secrete ferritin o Macrophages also secrete plasminogen activator, leading to consumption of plasma fibrinogen o Activated lymphocytes secrete soluble CD25 and infiltrate the liver and central nervous system o Proliferation of macrophages expressing CD163 in marrow and lymphoid tissue leads to hemophagocytosis

Spectrum of Cytokine-Induced Disease Normal response to infxn SIRS Severe sepsis Macrophage activation syndrome Acquired HLH Genetic HLH

Schaer, et al. Eur J Haematol Nov;77(5):432-6.

 First description of the disorder may have been as early as 1970s  ‘Macrophage activation syndrome’ (MAS) first used in 1992 by Albert, et al.  MAS occurs in both children and adults with autoimmune syndromes  Characterized by cytopenias, organ dysfunction, coagulopathy, and inappropriate activation of macrophages in a proinflammatory milieu

 MAS can be associated with a wide variety of autoimmune diseases  Strongest associations is with systemic juvenile idiopathic arthritis (sJIA), with estimated clinically apparent MAS in 7- 13% of subjects  Subclinical bone marrow evidence of MAS in >50% of sJIA patients  However, MAS also occurs with SLE and adult-onset Still’s disease, along with multiple other diseases

Autoimmune Diseases Associated with MAS Adult-onset Still’s disease Ankylosing spondylitis Dermatomyositis Enthesitis-related arthritis Inflammatory bowel disease Kawasaki disease Polyarticular JIA Sarcoidosis Systemic JIA Systemic lupus erythematosus Unidentified autoimmune disease

 sJIA and other autoimmune conditions are associated with fevers, anemia, hepatosplenomegaly, lymphadenopathy, and elevated serum ferritin  Ravelli, et al., defined a set of criteria for the diagnosis of MAS in patients with sJIA  A number of characteristic findings on routine studies were also identified  Subsequent investigation demonstrated that the criteria do not always apply to MAS in other autoimmune conditions

Laboratory CriteriaValue Thrombocytopenia ≤ 262 x 10 6 /  l Elevation in AST> 59 U/L Leukocytosis ≤ 4.0 x 10 6 /  l Hypofibrinogenemia≤ 250 mg/dL Clinical CriteriaManifestation CNS dysfunction Irritability Headache Lethargy Disorientation Seizures Coma Hemorrhages Ecchymoses Purpura Mucosal bleeding Hepatomegaly≥ 3 cm below costal margin Diagnosis requires: >2 Laboratory criteria >2 Lab + Clinical criteria Addition of ferritin >500 ng/ml may better discriminate MAS vs systemic infection. Adapted from: Davi, et al. Arthritis Rheumatol Oct;66(10):

 In 1979, Risdall, et al., described 19 patients with evidence of HLH and viral infection after transplantation  Later, it was shown that most patients had no evidence of immune system dysfunction before developing RHL  ‘Virus associated hemophagocytic syndrome’ was used to denote any case of HLH without a genetic cause  Eventually, bacteria, fungi, and even protozoa were shown to trigger RHL, leading to the term ‘infection associated hemophagocytic syndrome’ (IAHS)

Infections Associated with HLH Epstein-Barr virusEscherichia coli CytomegalovirusSalmonella sp. Varicella virusEnterococcus sp. HHV6Mycoplasma sp. Parvovirus B19Tick-born bacteria Hepatitis ATuberculosis HIVVisceral leishmaniasis AdenovirusPlasmodium sp. InfluenzaToxoplasma sp. CoxsackievirusPneumocystis jiroveci TorovirusCandida sp.

 EBV is the most common infectious trigger of RHL, accounting for 74% of viral triggers in one study  EBV carries the worst prognosis among viral triggers, with 73% mortality in one case series (before HLH ’04)  Incidence is highest in east Asians countries, possibly due to more-virulent endemic strain  Rarely detected in B-cell lymphoma-associated RHL; present in 80% with T/NK cell lymphoma-triggered RHL  Mortality was found to be 14x higher in EBV-associated RHL patients who did not receive etoposide

Medications Associated with HLH AspirinMorniflumate NSAIDsMethotrexate SulfasalazineInfliximab EtanerceptPenicillamine AnakinraVancomycin Gold saltsParenteral lipids Autologous stem cell transplantation

Included Parameters Known underlying immunosuppression Temperature Organomegaly No. of cytopenias Ferritin Triglycerides Fibrinogen AST Hemophagocytosis on BM biopsy Freely available at: Fardet, et al. Arthritis Rheumatol Sep;66(9):

 Adult-onset HLH has been associated with homozygous and heterzygous mutations in multiple FHL genes  Striking number variants of FHL-associated genes have been identified in MAS patients  Current recommendation is to perform genetic analyses on ALL patients suspected or confirmed to have HLH Kaufman, et al. Arthritis Rheumatol Dec;66(12):

 Finally!

 Immediate aim is to suppress over-whelming inflammation and immune activation; many different agents have been tried  In 1991, the Histiocyte Society developed the HLH 94 treatment protocol, improving survival in pediatric populations from ~25% to 51-55%  In 2002, Henter, et al., showed an overall survival rate of 80% in patients that underwent HSCT  Histiocyte Society updated the treatment protocol in 2004, including new treatments such as HSCT

Systemic TherapyDexamethasoneEtoposideCyclosporine Week 110 mg/m 2 daily150 mg/m 2 IV biw3 mg/kg bid Week 210 mg/m 2 daily150 mg/m 2 IV biw To Trough 200  g/L Week 35 mg/m 2 daily150 mg/m 2 IV qwk To Trough 200  g/L Week 45 mg/m 2 daily150 mg/m 2 IV qwk To Trough 200  g/L Week 52.5 mg/m 2 daily150 mg/m 2 IV qwk To Trough 200  g/L Week 62.5 mg/m 2 daily150 mg/m 2 IV qwk To Trough 200  g/L Week mg/m 2 daily150 mg/m 2 IV qwk To Trough 200  g/L Week 8Taper and d/c150 mg/m 2 IV qwk To Trough 200  g/L

 Applicability of HLH 04 protocol to RHL syndromes (e.g., MAS) and to adult populations is not been established  Mutliple groups support a graded-approach, with corticosteroids alone as initial treatment Initial Therapy High-dose corticosteroids (prednisolone 30 mg/kg x3 days) Elimination of suspected triggers, infection control Aggressive supportive measures Secondary Therapy Intravenous immunoglobulin (1-3 g/kg) Cyclosporine A, etoposide

Proposed Treatments for Autoimmune-Associated HLH Cyclosporine APlasmaphoresis EtanerceptAbatacept AnakinraAntithymocyte globulin Intravenous immunoglobulinCorticosteroids EtoposideNaproxen Splenectomy

 Several series suggest outcomes are poor in RHL if infection control measures are used alone  RHL triggered by leishmaniasis may be treated solely with amphotericin  Etoposide is crucial for EBV-associated RHL — inhibits activated T cells, plus EBV NA in infected cells  Multiple groups agree that HLH 2004 should be initiated for relapses of RHL, despite etiology  HSCT has best overall outcome among all single treatment modalities across all patient populations

 HLH is a clinical syndrome of overwhelming immune activation and cytokine production  Cytokine storm in HLH occurs due to failure to clear antigen presenting cells and/or activated T cells  Genetic variants may predispose patients to HLH at any age  Treatment is aimed at controlling the inflammatory cytokine cascade, and may require BMT in severe cases