Hemophagocytic Syndromes

Brant Ward, MD, PhDAllergy & Immunology

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

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

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)

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 disorderso 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

Causes of HLH

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

Subtypes of HLH 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

Primary HLH FHL can be divided into 5 subtypes:

o FHL1 – caused by unknown defect on chromosome 9

o FHL2 – caused by deficiency of Perforino FHL3 – caused by deficiency of Munc 13-4o FHL4 – caused by deficiency of Syntaxin 11o 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

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

FHL1 Identified from four consanginous families of

Pakistani descent using homozygosity mapping

First defined susceptibility locus for FHL, located at 9q21.3-22

This locus contains hundreds of candidate genes, though none have been identified as the culprit

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. 1999 Dec 3;286(5446):1957-9.

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

MACPF Structure & Function

Kondos, et al. Tissue Antigens. 2010 Nov;76(5):341-51.

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. 2003 Nov 14;115(4):461-73.

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

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. 2005 Mar 15;14(6):827-34.

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

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. 2009 Dec;119(12):3765-73.

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

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. 2011 Jul;3(2):107-11.

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

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

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

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

Granule Formation and Function

Vesicle Maturation

(LYST)

Vesicle Docking(Rab27a)

Vesicle Priming(Munc 13-4)

Vesicle Fusion(Syntaxin 11/Munc

18-2)

Effector Function(Perforin)

Models of Pathogenesis

APC

Virus-infected Cells

CTL

IFN-

Models of Pathogenesis

APC

Virus-infected Cells

CTL

IFN-

CTL

Impaired DC Deletion Perforin deficient mice with HLH have normal

total amount of dendritic cells, but…

Terrell & Jordan. Blood. 2013 Jun 27;121(26):5184-91.

Role of Interferon

Jordan, et al. Blood. 2004 Aug 1;104(3):735-43.

Role of CTLs in HLH

Jordan, et al. Blood. 2004 Aug 1;104(3):735-43.

Cytokine Storm The cytokines production and immune activation

triggered by these cells is thought to cause the observed symptomso Fever is induced by overproduction of IL-1o Pancytopenia is a consequence of TNF and IFNo Activated macrophages actively secrete ferritino Macrophages also secrete plasminogen activator,

leading to consumption of plasma fibrinogeno Activated lymphocytes secrete soluble CD25 and

infiltrate the liver and central nervous systemo Proliferation of macrophages expressing CD163 in

marrow and lymphoid tissue leads to hemophagocytosis

Cytokine Storm

Spectrum of Cytokine-Induced Disease

Normal respons

e to infxn

SIRS

Severe

sepsis

Macrophage

activation syndrome

Acquired HLH

Genetic HLH

Role of Macrophages in HLH

Schaer, et al. Eur J Haematol. 2006 Nov;77(5):432-6.

Macrophage Activation Syndrome

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 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

MAS and AutoimmunityAutoimmune 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

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

Diagnosis of MASLaboratory Criteria Value

Thrombocytopenia ≤ 262 x 106/l

Elevation in AST > 59 U/L

Leukocytosis ≤ 4.0 x 106/l

Hypofibrinogenemia ≤ 250 mg/dL

Clinical Criteria Manifestation

CNS dysfunction

IrritabilityHeadacheLethargy

DisorientationSeizures

Coma

HemorrhagesEcchymoses

PurpuraMucosal 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. 2014 Oct;66(10):2871-80.

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)

Infection and HLH

Infections Associated with HLHEpstein-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.

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

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

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:http://saintantoine.aphp.fr/score/

Fardet, et al. Arthritis Rheumatol. 2014 Sep;66(9):2613-20.

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 patients

Current recommendation is to perform genetic analyses on ALL patients suspected or confirmed to have HLH

Kaufman, et al. Arthritis Rheumatol. 2014 Dec;66(12):3486-95.

Treatment of HLHFinally!

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

Stratified Treatment Guidelines

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 10 mg/m2 daily 150 mg/m2 IV biw

To Trough 200 g/L

Week 3 5 mg/m2 daily 150 mg/m2 IV qwk

To Trough 200 g/L

Week 4 5 mg/m2 daily 150 mg/m2 IV qwk

To Trough 200 g/L

Week 5 2.5 mg/m2 daily 150 mg/m2 IV qwk

To Trough 200 g/L

Week 6 2.5 mg/m2 daily 150 mg/m2 IV qwk

To Trough 200 g/L

Week 7 1.25 mg/m2 daily 150 mg/m2 IV qwk

To Trough 200 g/L

Week 8 Taper and d/c 150 mg/m2 IV qwk

To Trough 200 g/L

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

Secondary Therapy

Intravenous immunoglobulin (1-3 g/kg)

Cyclosporine A, etoposide

MAS Treatment Options

Proposed Treatments for Autoimmune-Associated HLH

Cyclosporine A Plasmaphoresis

Etanercept Abatacept

Anakinra Antithymocyte globulin

Intravenous immunoglobulin Corticosteroids

Etoposide Naproxen

Splenectomy

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

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