HEMOPHAGOCYTIC

LYMPHOHISTIOCYTOSIS
Presentor: Dr.Sivagamasundari
Moderator: Dr.A.S.Nigam
 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 (including MAS)

 Formulate an effective treatment plan

 INTRODUCTION
• HLH is a non-malignant, aggressive, life-threatening
syndrome characterized by excessive immune activation.
• HLH typically affects infants from birth to 18 months of age
but can be seen in children and adults.
• Although the distinction is blurred, HLH has been historically
divided into primary and secondary types.
• Rapid recognition and initiation of treatment is necessary for
survival.
Immune System Review
 Normal Function

NK cell/CTL
Target cell

Target Cell Activated NK cell/CTL



Apoptosis of Target Cell

Activate Macrophages for

phagocytosis
The normal elimination of activated macrophages occurs via NK
cells and CTLs that lyse target cells through perforin-dependent
cytotoxicity.
1 Create a pore in the macrophage membrane.
2 Deliver cytolytic granules into the macrophage.
3 Proteases, such as Granzyme B, initiate cell death
(apoptosis) by activating caspases
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)
 NK cell/CTL
Target Cell

Activated NK
Target Cell
Cell/CTL

↑ INF-g

Activate Macrophages
Apoptosis

↑↑ CYTOKINES

Persistence of target cells

↑ NK/CTL Proliferation
 HEMOPHAGOCYTOSIS
• The pathologic finding of activated, histologically benign
macrophages engulfing host blood cells.
• Can be seen in biopsies of lymph nodes, spleen, liver, bone
marrow, and sometimes CNS.
 HEMOPHAGOCYTOSIS contd….

• Hemophagocytosis alone is not pathognomonic, nor required,

for diagnosis of HLH.
• In 1996, of 122 children with HLH from the Histiocyte
Society’s International Registry, only 75% had evidence of
hemophagocytosis at diagnosis.
• Reactive process thought to occur after over-activation of
macrophages due to dysregulated immune responses
 HLH

• In 1939, Scott & Robb-Smith described patients with “atypical

Hodgkin’s disease” – proliferation of histiocytes affecting all
lympho-reticular tissues as “histiocytic medullary reticulosis
“(HMR)

• 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)
 HLH Pathophysiology

• Excessive inflammation occurs due to overactive but impaired

immune function.
• There is a lack of normal feedback to activated macrophages
which leads to excessive activity.
• Most patients exhibit impaired cytotoxic function of NK cells
and CTLs.
• NK cells and/or CTLs fail to eliminate activated macrophages.
• Tissue damage is caused by excessive cytokine production
otherwise known as a cytokine storm.
 Models of Pathogenesis

Virus-infected
Cells

CTL APC

IFN-
 Virus-infected
Cells

CTL APC
CTL

IFN-
 CYTOKINE STORM
• A pro-inflammatory state is created which is ultimately
responsible for multi-organ failure and high mortality without
treatment or with delayed treatment.
 CYTOKINE STORM

• The cytokines production and immune activation triggered by

these cells is thought to cause the following 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
 SPECTRUM OF CYTOKINE-INDUCED DISEASE

Genetic Macrophage
HLH activation SIRS
syndrome
Acquired Normal
Severe response
HLH
sepsis to infxn
 TYPES

• HLH classified as either primary or acquired

• Primary HLH attributed to underlying genetic defect that is
inherited in an autosomal recessive pattern
• In primary HLH, 70-80% of cases seen before the age of one
year
• Most cases of acquired HLH attributed to infection, but it’s
important to note that primary HLH is often precipitated by
infections
 Causes of HLH

Perforin Helminthic
deficiency infections

Munc 13-4
Fungal infections
deficiency

Syntaxin 11 Bacterial
deficiency infections

Munc 18-2
deficiency
HLH Viral infections

Unknown gene
Medications
mutations

Immune Autoimmune
deficiencies Malignancy diseases
 Familial HLH

• Primary HLH is caused by inborn defects in the primary

cytotoxicity effector pathway in lymphocytes and NK cells.
• Autosomal Recessive
• Incidence 1:50,000
• Median survival is <2 months if left untreated.

• Onset is typically during infancy or early childhood.

• May be triggered by infection.
 Primary HLH contd….

• Non-syndromic forms consist of 5 mutations, FHL 1-5.

• Associated with Immune Deficiencies syndromes – Chediak-
Higashi Syndrome, Griscelli syndrome, x-linked proliferative
syndrome
 Primary HLH contd…..

• 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
 Primary HLH contd….

IMMUNODEFICIENCY SYNDROMES
• Chediak Higashi syndrome
– Mutations in CHS1/LYST
• Griscelli syndrome type II

– Mutations in RAB27A
• Hermansky-Pudlak syndrome type II
– Mutations in AP3B1
• 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 causative gene
 FHL2

• Perforin (PRF1) was the first identified gene causing FHL

• Loci- 10q22

• >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

 Perforin

• Protein found in lytic granules of NK cells and

cytotoxic T lymphocytes
• Plays an important role in apoptosis and immune
modulation
• 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
 FHL3

• Due to mutations in Munc 13-4

• Loci 17q25

• Munc 13-4 defiency accounts for 30-35% of cases

• Together with perforin gene mutations, cause up to 70% of

FHL cases
 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 contd…..

• 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(STX11)

• Loci-6q24

• All identified mutations in are null mutations

• Syntaxin mutations account for ~20% of FHL cases in Turkish

populations
 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
 FHL5

• Results from deficiency of Munc 18-2

• Loci -19p13

• 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
 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
 LYST

• Caused by mutation in CHSI/LYST, a ubiquitously-expressed

protein

• Loci -1q42.1-42.2

• 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 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

• Loci-15q21

• Enriched on endosomal structures that fuse with cytotoxic

granules before release of their contents
 Rab27a contd….

• 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)

• Loci Xq25

• gene –SH2D1A and XIAP

 XLP contd…

• High susceptibility to severe EBV infection results in

fulminant and fatal mononucleosis ,Lymproliferative disorder
and hypogammaglobulinemia

• SAP deficiency results in a partial cytotoxic defect

 Secondary HLH
• sHLH develops as a consequence of a strong immunologic
response to a stimulus:
– Infection
– Rheumatologic illness such as JIA or SLE
– Malignancy, either at presentation or during treatment

– Immunodeficiency
 Secondary HLH contd….

• There is no inherited immune defect found in sHLH

• More prevalent than FHL but incidence is poorly defined
• Median age of onset is higher than FHL
 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 due to virus infection without a
genetic cause
 Infection and HLH

• 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 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 sHLH

• 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
 EBV and HLH

• 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
 DRUG-Induced HLH
Medications Associated with HLH
Aspirin Morniflumate

NSAIDs Methotrexate

Sulfasalazine Infliximab

Etanercept Penicillamine

Anakinra Vancomycin

Gold salts Parenteral lipids

Autologous stem cell transplantation

 Malignancy

• HLH has been reported with lymphomas or leukemia of the T

or NK cell lineage.
• Many patients have a simultaneous bacterial, viral, or fungal
infection that may trigger HLH.
• May occur during treatment for a known cancer in clinical
remission or as a manifestation of an aggressive uncontrolled
cancer.
– Iatrogenic immune dysregulation
2004 -Diagnostic guidelines for HLH
• Molecular diagnosis consistent with HLH.
• Or 5 of the 8 criteria listed below:
– Clinical criteria
1.Fever
2.Splenomegaly
– Laboratory criteria
3.Cytopenias: (affecting ≥2 of 3 lineages)
• Hb <9 g/dl(in infants <4 wks Hb <10 g/dl)
• platelets <100 x 109/L
• neutrophils <1x109/L
 4. Hypertriglyceridemia and/or hypofibrinogenemia
• Fasting triglycerides ≥3.0 mmol/l (i.e ≥265 mg /dl)
• Fibrinogen ≤1.5 g/dl
5.Hyperferritinemia: >500 µg/L
6.Elevated soluble CD25 (sIL-2R): ≥ 2400 U/ml
7.Low/Absent natural killer function
– Histopathologic criteria
8.Hemophagocytosis in bone marrow, spleen or lymph
node with no evidence of malignancy
 Comments

1.Absence of hemophagocytosis does not exclude a diagnosis of

HLH.
2.The following may provide strong supportive evidence for the
diagnosis:
• spinal fluid pleocytosis (mononuclear cells) and/or elevated
spinal fluid protein
• histological evidence resembling chronic persistent hepatitis by
liver biopsy.
3.Other abnormal clinical and laboratory findings consistent with
the diagnosis: Jaundice, hepatic enzyme abnormalities,
coagulopathy, lymph node enlargement, edema, skin rash,
hypoproteinemia, hyponatremia, VLDL increased, HDL
decreased.
 Clinical Presentation

• The presentation of HLH is variable and it is due to

1.Hyperactivation of CD8+ T lymphocytes and macrophages
2.Proliferation ,ectopic migration and infiltration of these cells
into variable organs
3.Hypercytokinemia with persistenly elevated levels of
multiple proinflammatory cytokines ,resulting in progressive
organ dysfunction that leads to death
• Typical findings include
 persistent fever
 splenomegaly with or without hepatomegaly
 cytopenias that affect at least two cell lines
• Less common initial findings include
 lymphadenopathy
 rash
 jaundice
 edema
• Clinical features may not be initially or simultaneously present,
however, patients are usually acutely ill at presentation.
Bone Marrow Abnormalities & Cytopenias

• Anemia and thrombocytopenia occur in >80% of patients at

presentation.
• The cellularity of bone marrow may be normocellular,
hypocellular, or hypercellular.
– Presence of hemophagocytosis ranges from 25-100%.

– IHC stain for CD163 may be helpful to identify

• The diagnosis of HLH should never be made solely based on the
presence or absence of hemophagocytosis.
 Liver Disease

• Most patients have evidence of hepatitis on presentation.

• Clinical findings include jaundice, hepatomegaly, and bleeding

due to direct result of organ infiltration by lymphocytes and
macrophages
• Autopsy findings of 22 out of 27 patients diagnosed with HLH
found infiltration of lymphocytes into the portal tracts similar
to what is seen in chronic persistent hepatitis.
• Lab findings include hypertriglyceridemia, elevated
transaminases, hyperbilirubinemia, and hyperferritonemia.
– A serum ferritin >10,000 mg/L is highly sensitive(90%)
and specific (96%) for HLH
• Impaired synthetic function and clotting factor consumption
leads to hypofibrinogenemia as well as prolongation of PT
and aPTT.
 Neurologic Symptoms

• More than 1/3 of patients will present with neurologic

symptoms.
– Seizures, meningismus, decreased level of consciousness,
cranial nerve palsy, psychomotor retardation, ataxia,
irritability, hypotonia, or peripheral neuropathy
• CSF is abnormal in more than half of patients.
– Hyperprotienemia, pleocytosis, and hemophagocytosis
• Variable MRI findings that correlate with symptoms.
– Discrete lesions, leptomeingeal enhancement, or global
edema.
• Retinal hemorrhages, swelling of the optic nerve, and
infiltration of the choroid has been seen in infants.
 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 ( Rhematology asso HLH)
 Macrophage activation
syndrome

• Definition:
It is a syndrome caused by the excessive activation and
uncontrolled proliferation of T-cells and well-differentiated
macrophages. This activation leads to widespread hemo-
phagocytosis and cytokine overproduction, a highly stimulated
but ineffective inflammatory-immune response, which can be
fatal.
• MAS has been reported in patients with almost
 Any rheumatic diseases

• Most strongly associated with Systemic Juvenile Idiopathic

Arthritis (SJIA)
• 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
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
 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 in 2016
• 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
2011-DIAGNOSTIC CRITERIA
Any 2 or more of clinical
(or) 2 or more laboratory criteria
A. Clinical criteria:
• CNS dysfunction: irritability, disorientation, lethargy,
headache, seizures, coma
• Hemorrhages: purpura, easy bruising, mucosal bleeding
• Hepatomegaly (≥3 cm below the costal margin)
B. Laboratory criteria
• Leucopenia (TLC < 4 x 109/L)
• Thrombocytopenia (Platelet count <262 x 109/L)
• Deranged LFT ( AST > 59 U/L)
• Hypofibrinogenemia (Fibrinogen ≤ 2.5 g/L)
• 2016 criteria by ravelli et al
•Platelet count and fibrinogen level included in the criteria are
within normal range of routine laboratory assessments
•The children with active systemic JIA often have increased
platelet counts (e.g. >6-8 lacks) as well as elevated fibrinogen
levels(e.g.>500-600mg/dl ) as part of underlying inflammatory
process
•Thus a paradoxically normal platelet count or fibrinogen level
in the setting otherwise prominent systemic inflammation may
raise the suspicion of MAS
 Treatment of HLH

• Immediate aim is to suppress over-whelming inflammation and

immune activation

• Many different agents have been tried

• In 1994, 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
• To control T-cell proliferation and activation, based on the
HLH-94 protocol, an 8-week course of dexamethasone and
etoposide therapy is used. After 8 wks cyclosporine started.

• Patients with proven persistent CNS disease after 2 wks of

systemic therapy should be treated with weekly Intrathecal
MTX and hydrocortisone until CSF abnormalities and

symptoms resolve.
• HSCT was recommended for all children with a suitable
allogenic donor.
• Remission rate was 71% and 5 year post-HCT survival
probability was 54%±6%.

• The HLH-2004 protocol was developed in an effort to improve

remission rates.
– Intensification of initial therapy by adding cyclosporine
and intrathecal steroids.
 Treat the Underlying Disease

• EBV
– Rituximab
• Rheumatologic disorder
– Immunosuppression, IVIG

• Malignancy
– Treat malignancy first vs. HLH
 Salvage Therapy

• If patients do not display at least a partial response within 2-3

weeks of therapy, salvage therapy should be considered
• Alemtuzumab
– Monoclonal antibody to CD52
– Will suppress T cells and histiocytes

– Induced partial response in 64% of patients

– In HLH-2004 among refractory patients salvage therapy
may be useful as a bridge to transplant
Other salvage therapies
-antithrombin III
-infliximab ( anti-TNF Ab)

-daclizumab (anti-CD25 Ab)

-plasmapheresis to remove cytokines
 Follow Up

• Relapse, if it occurs, generally occurs within the first year.

• Close monthly follow up is necessary to evaluate for recurrent

disease.
– Signs of recurrent disease: fever, hepatosplenomegaly,
neurological abnormalities.
– Lab tests that indicate recurrence: cytopenia and elevated
ferritin, serum transaminases, and sIL-2R.
 Prognosis

• Invariably fatal if not treated.

• The median survival rate- 2-6 months without treatment
• Success or failure of an allogeneic BMT is the most important
long-term prognostic factor
Stratified Treatment Guidelines
 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
• Cytokine blockers in MAS
- IL-1 blockers: anakinra and canakinumab
- IL-6 blocker: tocilizumab
 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
• Next activity on 12-12-2018
• Journal club by Dr.Nayan jyoti