1. Hemophagocytic Syndromes
Brant Ward, MD, PhD
Allergy & Immunology

2. Goals 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

3. Hemophagocytosis
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

4. Hemophagocytosis
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)

5. Diagnosis of 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 in children
Ferritin >10,000 ng/ml is >90% specific for HLH in children
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

6. Pathophysiology of HLH
Basic science alert!

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

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

9. Primary HLH FHL can be divided into 5 subtypes:
FHL1 – caused by unknown defect on chromosome 9
FHL2 – caused by deficiency of Perforin
FHL3 – caused by deficiency of Munc 13-4
FHL4 – caused by deficiency of Syntaxin 11
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

10. FHL
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

11. FHL1
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

12. FHL2 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):

13. Perforin
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

14. MACPF Structure & Function
Kondos, et al. Tissue Antigens Nov;76(5):

15. FHL3 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):

16. Munc 13-4 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

17. FHL4 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):

18. Syntaxin 11
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

19. FHL5 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):

20. Munc 18-2 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

21. Chediak-Higashi Syndrome
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):

22. LYST 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

23. Griscelli Syndrome Type 2
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

24. Rab27a
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

25. XLP
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

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

27. Models of Pathogenesis
Virus-infected
Cells
CTL
APC
IFN-

28. Models of Pathogenesis
Virus-infected
Cells
CTL
APC
CTL
IFN-

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

30. Role of Interferon 
Jordan, et al. Blood Aug 1;104(3):

31. Role of CTLs in HLH
Jordan, et al. Blood Aug 1;104(3):

32. Most Applicable to Non-Pediatricians
Causes of RHL
Most Applicable to Non-Pediatricians

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

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

35. Role of Macrophages in HLH
Schaer, et al. Eur J Haematol Nov;77(5):432-6.

36. Macrophage Activation Syndrome
First description of the disorder may have been as early as 1970s
‘Macrophage activation syndrome’ (MAS) first used in 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

37. MAS and Autoimmunity
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

38. Autoimmune Diseases Associated with MAS
MAS and Autoimmunity
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

39. Diagnosis of MAS
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

40. ≥ 3 cm below costal margin
Diagnosis of MAS
Laboratory Criteria
Value
Thrombocytopenia
≤ 262 x 106/l
Elevation in AST
> 59 U/L
Leukocytosis
≤ 4.0 x 106/l
Hypofibrinogenemia
≤ 250 mg/dL
Diagnosis requires:
>2 Laboratory criteria
>2 Lab + Clinical criteria
Clinical Criteria
Manifestation
CNS dysfunction
Irritability
Headache
Lethargy
Disorientation
Seizures
Coma
Hemorrhages
Ecchymoses
Purpura
Mucosal bleeding
Hepatomegaly
≥ 3 cm below costal margin
Addition of ferritin >500 ng/ml may better discriminate MAS vs systemic infection.
Adapted from: Davi, et al. Arthritis Rheumatol Oct;66(10):

41. Diagnosis of MAS MAS/HLH (MH) Score Criteria Points Age 1.6 y 37
Splenomegaly 12
PMN 1.4x109/ml
Plts 78x109/ml 11
Fibrinogen 131 mg/dL 15
Hgb 8.3 g/dL
Optimal cutoff value (60)
Adapted from: Minoia, et al. J Pediatr Oct;189:72-78.e3.

42. Infection and HLH
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)

43. Infections Associated with HLH
Infection and HLH
Infections Associated with HLH
Epstein-Barr virus
Escherichia coli
Cytomegalovirus
Salmonella sp.
Varicella virus
Enterococcus sp.
HHV6
Mycoplasma sp.
Parvovirus B19
Tick-born bacteria
Hepatitis A
Tuberculosis
HIV
Visceral leishmaniasis
Adenovirus
Plasmodium sp.
Influenza
Toxoplasma sp.
Coxsackievirus
Pneumocystis jiroveci
Torovirus
Candida sp.

44. EBV and HLH
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

45. Medication-Induced HLH
Medications Associated with HLH
Aspirin
Morniflumate
NSAIDs
Methotrexate
Sulfasalazine
Infliximab
Etanercept
Penicillamine
Anakinra
Vancomycin
Gold salts
Parenteral lipids
Autologous stem cell transplantation

46. HScore for Diagnosing RHL
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):

47. sIL-2R for Diagnosing RHL
Hayden, et al. Blood Adv Dec 6;1(26):

48. sIL-2R for Diagnosing RHL
Optimal: 2515 U/ml
Sensitivity: 100%
Specificity: 72.5%
Exclude HLH: <2400 U/ml
Rule-in HLH: >10,000 U/ml
Specificity: 93%
Higher mean sIL-2R in malignancy vs infectious or MAS
sIL-2R >10,000 does not correlate with prognosis
Hayden, et al. Blood Adv Dec 6;1(26):

49. FHL/RHL Overlap
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 & RHL patients
Current recommendation is to perform genetic analyses on ALL patients suspected or confirmed to have HLH
15%
Cetica, et al. J Allergy Clin Immunol Jan;137(1): e4.
Kaufman, et al. Arthritis Rheumatol Dec;66(12):

50. Treatment of HLH
Finally!

51. Treatment of HLH
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

52. Stratified Treatment Guidelines

53. HLH 2004 Protocol Systemic Therapy Dexamethasone Etoposide
Cyclosporine
Week 1
10 mg/m2 daily
150 mg/m2 IV biw
3 mg/kg bid
Week 2
To Trough 200 g/L
Week 3
5 mg/m2 daily
150 mg/m2 IV qwk
Week 4
Week 5
2.5 mg/m2 daily
Week 6
Week 7
1.25 mg/m2 daily
Week 8
Taper and d/c

54. Anti-IFN (NI-0501) for FHL
Abstract: Jordan, et al. Blood :LBA-3

55. RHL/MAS Treatment
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
Other Proposed Treatments
IL-1 blockade (anakinra, etc.)
IL-6 blockade (tocilizumab)
JAK inhibitors (ruxolitinib)
B cell depletion (rituximab)
IFN blockade (NI-0501)
Secondary Therapy
Intravenous immunoglobulin (1-3 g/kg)
Cyclosporine A, etoposide

56. Anakinra for RHL
SOFA (sequential organ failure assessment) score >11 predicts 95% mortality
Wohlfarth, et al. J Intensive Care Med Jan 1:

57. Tocilizumab for RHL
Faquer, et al. Hematol Oncol Mar;34(1):55-7.

58. Personalized Treatment for RHL
Survival in 14/19 (73%)
Kumar, et al. J Clin Immunol Oct;37(7):

59. Salvage Therapy in Adults
Only prospective study for treatment of adults with HLH
Enrolled patients that did not achieve at least partial response on HLH-94 protocol (± RTX)
Unknown
Genetic
(100 mg/m2)
(25 mg/m2)
Wang, et al. Blood Nov 5;126(19):

60. Proposed Treatments for Autoimmune-Associated HLH
MAS Treatment Options
Proposed Treatments for Autoimmune-Associated HLH
Cyclosporine A
Plasmaphoresis
Etanercept
Abatacept
Anakinra
Antithymocyte globulin
Intravenous immunoglobulin
Corticosteroids
Etoposide
Naproxen
Splenectomy

61. Treatment Notes
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

62. Don’t worry, just one more slide…
Takeaways
Don’t worry, just one more slide…

63. Summary
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