HLH (Hemophagocytic Lymphohistiocytosis

[dr.mukesh desai]

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Introduction:
HLH (Hemophagocytic Lymphohistiocytosis) is not an uncommon disorder &
every year we diagnose 25 cases of HLH at B J Wadia Hospital for
children, Mumbai. Hemophagocytic Lymphohistiocytosis is an interim
mechanism of disorder like DIC. It is an overreaction of the immune
system & many diseases can trigger the same events.
Aetiology of HLH:
It can be familial or acquired & both share one common feature of a
highly stimulated and ineffective immune response. Familial HLH is due
to congenital defects in various proteins involved in granule
exocytosis or due to defect in Perforin granzyme pathway. HLH is also
commonly seen in primary immunodeficiency like XLP1 (X Linked
Lymphoproliferative Disorder) due to defect in SAP and XLP2 due to
defect in XIAP gene. Genes involved in Familial HLH are shown in Table
(1) & (2).

Genetic aetiology of HLH: Table (1)
FHL Gene defective % of Familial HLH cases
FHL1 Chromosome 9 -
FHL2 Perforin 20 to 25%
FHL3 Munc 13/4 15 to 20%
FHL4 Syntaxin 11 10%
FHL5 Munc 18/2 20 to 25%
XLP1 SAP -
XLP2 XIAP -
Genetic aetiology of HLH Table (2)
Pigmentary Dilution Gene Comment
Chediak Higashi Syndrome (CHS) Lyst Accelerated phase of CHS
Griscelli Syndrome (GS) Type 2 RAB 27 alpha HLH does not occur in GS
type I & 3
Hermansky Pudlak Syndrome AP3B1 Only 2 cases in world type 2 (HP)
literature

Acquired HLH is commonly seen with infections (IAHS – Infection
associated Hemophagocytic Syndrome); Malignancies and Rheumatic
diseases where it is also known as MAS (Macrophage Activating
Syndrome). Thus a host of diverse diseases can activate HLH. The most
common infections, which trigger HLH, are EBV, CMV, HSV, HHV, Koch,
Salmonella, Malaria, Kalaazar in the Indian set up but virtually any
infection can trigger HLH.
Pathophysiology & Understanding:
The Hallmark of HLH is defective NK Cell & Cytotoxic T cell activity.
NK cells & Cytotoxic T cells can be recognized morphologically as
large lymphocytes with azurophilic granules in wright preparation.
These granules contain apoptosis inducing machinery the Perforin
protein & Granzyme B.
To understand HLH we must understand the function of NK cell &
Cytotoxic T cells. Both are designed to kill virally infected cells.
NK cells are our innate immune system cell that comprise 10 to 15% of
Peripheral Blood Lymphocytes and are like Rambo’s of the immune system
with ready to kill receptors but have to be inhibited by receptors for
MHC class I molecule, which override the kill signal. Thus MHC class I
molecule protect us from NK cell toxicity.
Cytotoxic T cells are produced as an adaptive response to any
infection. They recognize cytosolic protein antigen (e.g. Viral
proteins) which are presented on the MHC class I molecule. Cytotoxic T
cells are specific for that infection and are produced later after 4
to 5 days of infection. They are the atypical lymphocytes seen in
Infectious mononucleosis & express Cd3+ & Cd8+ on their surface.
NK cell & Cytotoxic T cells eliminate a virally infected target cell
via the Perforin Granzyme pathway. Once NK cell or Cytotoxic T cell
binds the virally infected cell they form an immunologic synapse at
the contact site. All the subsequent events occur at the immunologic
synapse.
The azurophilic granule undergoes steps of vesicle maturation,
polarization to the immunologic synapse, docking, and priming before
it fuses with the surface membrane and releases its content with in
the immunological synapse.
Perforin is a protein like Complement C5-9, it perforates the target
cell membrane forming a channel thus allowing Granzyme B to enter the
target cell and induce apoptosis by activating the apoptotic
mechanism.
Proteins important in the granule exocytosis process and the disease
caused by their deficiency are shown in fig (3), (4), (5) & (6)
It is thus easy to comprehend why such diverse defects result in a
common pathophysiologic process (HLH).
XLP (X linked Lymphoproliferative disorder) is of two types XLP1 due
to defect in SAP protein & XLP2 due to defect in XIAP (X-linked
Inhibitor of Apoptosis) gene. 60% of XLP1 patients develop EBV induced
HLH while 90% of XLP2 develop HLH. Currently it XLP2 is being
reclassified as HLH causing disease rather than causing
lymphoproliferation.

Since NK cell & Cytotoxic T cells are unable to kill target cells
following events occur. There is over stimulation of the immune
system, excess antigen presentation, Excess T cell proliferation with
infiltration of various organs like CNS, Liver, Spleen, & Lymph nodes.
This results in formation of excess Cytokines like TNF alpha, INFG,
IL1, Gm-CSF. The Antigen Presenting Cell needs to be culled by NK
cells to achieve immune homeostasis. This does not happen and
persistent antigen presentation occurs with further activation of
immune system. The cytokine IFNG activate the Macrophages that result
in Hemophagocytosis, thus giving the name to this syndrome.
In normal immune response, once the infection is eliminated, the
effector immune cells need to be culled to achieve immune homeostasis.
In HLH this does not occur resulting in massive expansion of effector
T cells, organ infiltration resulting in massive tissue necrosis and
multiorgan failure.
The cells participating in HLH are not only NK cells & Cytotoxic
CD8+ve T cells but also other cells with cytotoxic activity like CD4+
cytotoxic T cells & iNKT cells.
There is some correlation between hypercytokinemia and the diverse
clinical manifestations in HLH.
IL1 is responsible for fever.
TNF alpha is responsible for Hepatic injury, coagulopathy.
TNF alpha inhibits lipoprotein lipase resulting in
hypertriglyceridemia
IFNG causes Macrophage activation, Hemophagocytosis; Cytopenias &
muscle wasting.
Soluble IL2R alpha (sCD25) & Beta 2 Microglobulin levels in serum
suggest excess T cell activation.
Elevated Serum Ferritin is due to Macrophage activation.
Does this understanding of pathophysiology & role of cytokine in HLH
have any therapeutic implications? Probably yes; clinical trials with
Anti TNF alpha or Anti IFNG antibodies are already being done with
success in animal model.
What is the clinical presentation of HLH?
HLH should be suspected in a patient when they have following signs &
Symptoms.
Fever
Rapidly progressive cytopenias
LFT abnormalities
Organomegaly like Hepatosplenomegaly, Lymphadenopathy
Coagulopathy
BM or Organ biopsy with hemophagocytosis
Hyperferritinemia
Hypertriglyceridemia
Hypofibrinogenemia
Hyponatremia.
We very often suspect HLH in case of fever, organomegaly, icterus and
rapidly evolving cytopenias; especially in an intensive care setting.
In such cases besides looking for LFT abnormalities we specifically
ask for Coagulation profile with ‘D’ Dimer; Serum Ferritin, Serum
Triglycerides & Serum Electrolytes.
A BM is very often diagnostic in HLH.
Some times the hemophagocytes may be few but if the clinical suspicion
is strong it may be worth repeating a BM examination.
Rarely Hemophagocytosis may be seen in Lymph nodes or on liver biopsy
In 2009 Filipovich et al has revised the HLH diagnostic criteria,
which are practical and very much applicable in our set up. These
criteria are shown in Table (3):
HLH Diagnostic Criteria 2009: Table (3)
Molecular Diagnosis of HLH or XLP
At least ¾
Fever
Splenomegaly
Hepatitis
Cytopenias

At least ¼
Hemophagocytosis,
Hyperferritinemia
Increased Soluble IL2Ralpha
Absent or very decreased NK cell function.

Supportive of HLH
Hypertriglyceridemia,
Hypofibrinogenemia
Hyponatraemia


Familial HLH present generally early in life with 70 to 80% less than
1 yr. of age. However presentation later on in life is also seen. In a
recent study it was shown that 37% of adult with HLH (age>18 yrs.) had
mutations in Perforin (Prf) gene. Thus today, we can better understand
the spectrum of disease.
Prf A91V mutation is common polymorphism seen in 4 to 11%
(Heterozygous) & 1 to 4% (Homozygous) of western population. It is now
believed that it significantly decreases Perforin expression due to
protein misfolding & is an HLH inducing mutation. How common is this
polymorphism In India we do not have any data.
In our experience we see HLH at all age groups and we do have
significant no of HLH in adults.
It is important to highlight over here that in India especially at B J
Wadia Hospital for children we also see Peripheral Blood HLH that was
1st reported by Dr Zinet Currimbhoy and we do consistently see cases
of PB HLH. We recently saw 2 newborns and 1 older boy with PB HLH.
This has not been reported from abroad.
Perforin mutations are also seen in patients of Chronic Active EBV
(CAEBV) disease & in 5% of patients with NHL. We have seen an
interesting case of Hodgkin’s disease being referred from TMC who
developed HLH on treatment and his Perforin levels were Zero. Thus his
first presentation was with malignancy and then developed HLH on
treatment. Why he did not develop HLH with zero Perforin level? We do
not have answer to these questions, but it opens our eyes to the
spectrum of disease and the fact that other factors besides Perforin
mutations are important for manifestation of HLH.
In 2 patients HLH presented as chronic meningoencephalitis suggesting
that a localized organ involvement may be the only presentation of
HLH.
In a patient of ALPS (Autoimmune Lymphoproliferative Syndrome) both
mutation in FAS gene and Perforin gene was detected.
HLH is a fulminant disease but some patient can have a mild chronic
relapsing course. We had a 9 yr. old boy who had persistent
pancytopenia with persistent HLH for more than 2 yrs.
Very often HLH may be triggered by infection and it may be impossible
to do distinguish primary HLH from secondary infection associated HLH.
Hence it is very important to establish molecular diagnosis.
Once HLH is diagnosed a search for triggering infection has to be done
especially for EBV, CMV, Koch, Enteric, Malaria, Kalaazar depending
upon clinical presentation.
Thus we understand that the spectrum of HLH can be as diverse as
fulminant HLH to chronic HLH. It may have multiorgan affection or
single system presentation. In setting of CAEBV or malignancy
associated HLH and MAS also possibility of Prf mutations have to be
considered.
MAS (Macrophage Activation Syndrome occur in the setting of RA, SLE &
other collagen vascular defects & more than 100 cases are published in
literature. MAS tend to occur in the early active phase of the
disease. Leucopenia, thrombocytopenia & fibrinogen levels are not
markedly decreased, as there is preexisting inflammation that results
in neutrophilic leukocytosis, thrombocytosis & elevated plasma
fibrinogen levels. Serum ferritin levels are markedly elevated and we
have seen levels up to 200,000 ng/ml in MAS secondary to RA. Besides
clinical features typical of HLH there is often pronounced
coagulopathy & cardiac involvement that is responsible for mortality
with MAS. MTX, NSAID’s & Gold salt may be the triggering event. The
mortality is high at 10 to 15%. A few patients of MAS have been found
to have Perforin & SAP mutation.
Lymphoma associated HLH very often is triggered by EBV, which infect
the NK & NKT cells unlike the B cells in Infectious mononucleosis.
Commonly associated with peripheral T cell lymphoma.
How do we work up a patient of HLH?
Laboratory evaluation of HLH is directed with following considerations
Establish diagnosis of HLH

CBC platelet
ESR
PS (look for peripheral Blood HLH)
LFT
Creatinine
LDH
Serum Electrolytes
Serum Ferritin
Serum Triglycerides
Coagulation Profile (PT, PTT, Plasma Fibrinogen & ‘D’ Dimer.)
CSF for pleocytosis & elevated proteins (50% of cases)

Supportive evidence for HLH
PB or Bone Marrow Aspiration
Liver Biopsy
Lymph node Biopsy

Sophisticated Lab investigations

sCD25 > 2400 IU/L
NK cell activity (May be normal in 30% of cases)
Serum Beta2 Microglobulin

Aetiological work up

Anti EBV VCA IgM, PCR
Anti CMB Abs, Antigen, PCR
Appropriate Microbiological Cultures
Hair Mount studies for pigmentary dilution disorders.

Work up for familial HLH

Perforin by flow cytometry
GRA (Granule Release Assay)
Gene sequencing to identify mutations

Management of HLH:
Principle of treatment:
The immediate aim of treatment is to suppress the severe hyper
inflammation. The secondary aim is to eliminate pathogen activated
APCs (Antigen Presenting Cells) so as to remove the stimulus for
ineffective activation of T cells.
Please note in secondary HLH pathogen directed therapy is not
sufficient to control the hyper inflammation. Leishmaniasis treated
with Liposomal Amphotericin B is the only exception.
The treatment recommended is HLH 2000 protocol that is devised by
Histiocytic society. It essentially consists of 3 drugs.
Dexamethasone:
Lympholytic
Inhibit expression of cytokines
Suppresses maturation of APCs
Better CNS penetration hence preferred over prednisolone
Cyclosporine A:
Prevents T cell activation & proliferation
Etoposide (VP-16):
Activity against Monocytes & Macrophages
Inhibits EBNA synthesis I EBV infected cells
IV Gammaglobulins:
Provide cytokine & pathogen specific antibodies
Immunomodulation
Etoposide is a genotoxic drug and there are concerns regarding
development of late effects like MDS (Myelodysplastic Syndrome); but
it can be life saving especially in EBV associated HLH. Cytopenias is
common in HLH and there is this constant dilemma & tussle (between the
intensivist & Immunologist) as to give Etoposide or not as Etoposide
is a myelosuppressive drug. Do not withhold Etoposide due to
cytopenias. At least the first two doses must be given before
modifying the schedule due to presence of a very low ANC (Absolute
Neutrophil Count) < 500. Unless we control the hyper inflammation
there is no chance of cytopenias improving.
The French group does not use Etoposide and they use ATG (Anti
Thymocyte Globulin) to control HLH.
Up to 4 doses of Intra Thecal MTX (Methotrexate) are recommended I HLH
2004 protocol if the 1st 2 weeks of therapy fail to improve
neurological symptoms & if there is progressive CSF pleocytosis.
Effective systemic therapy can result in CNS control of the disease.
Once inflammation is controlled a search for a potential Bone Marrow
donor is done and if a match is available the child should be
transplanted to achieve a cure. In HLH 1994 protocol median survival
was 64% with OS (Over all Survival) of 55%.
In secondary HLH treatment is given for 8 wks. HLH reevaluation is
done & if normal treatment is stopped and child is observed
periodically for recurrence.
Once hyper inflammation is controlled there can be reactivation of HLH
or development of CNS events. Intra Thecal MTX would benefit for CNS
activation.
The HLH therapy is reinforced in case of systemic reactivation.
Indications for BMT:
FHL (Familial Hemophagocytic Lymphohistiocytosis)
Relapsing secondary HLH.
Results of BMT are:
Matched Related Donor: 71 % 16%
Matched Unrelated Donor 71 %
HLA mismatched Unrelated Donor: 54% 27%
Haploidentical Related Donor: 50 % 24%
Disease directed therapy:
Anti Leishmania therapy
Anti Lymphoma therapy.

If there is poor or no response to 4 weeks of HLH 2004 protocol
treatment, HLH is probably refractory & continuing HLH 2004 protocol
will be of no further benefit. Salvage treatment options that can be
tried in such a situation are shown in table (). There is no
established salvage regime. ATG is rarely effective if Etoposide based
regimen has been ineffective. Search for BM donor should be done & one
should attempt to transplant these patients.
ATG
Fludarabine
Alemtuzumab
Daclizumab
IVGG
Chemotherapeutic agents
Anti TNF alpha receptor blocking agents
Anti Interferon Gamma Antibodies

In patients with EBV induced HLH or XLP with fulminant EBV induced HLH
we can use Injection Rituximab (monoclonal Ab to CD 20) at doses of
375 mg / m2 weekly for 4 weeks, to knock out the B cells harboring
EBV. This strategy works very well to control EBV induced HLH & in
fulminant infectious mononucleosis.
In desperate situations Anti TNF alpha-receptor blocking agents have
been tried.
Experimental works with Anti Interferon Gamma Antibodies have shown
excellent results in mouse model of HLH. Human trials are about to
begin. We are awaiting those results.
Prognosis: The prognosis of familial HLH is guarded in India. We have
seen more than 11 cases of Perforin deficiency and have lost all of
them. Most of the mortality is due to hepatic failure, multiorgan
failure, Coagulopathy, infection and CNS events. If a successful BMT
is done than the chances of cure are more than 50%. The first patient
of HLH was transplanted at Necker hospital Paris 28 years back and is
alive and fine. We need to establish a successful transplant programme
in India to save our babies with HLH
Conclusion:
HLH is not an uncommon disease. a practicing pediatrician is likely to
see these patients in a tertiary care set up. HLH should be considered
in the differential diagnosis when we have a constellation of symptoms
like fever, organomegaly, rapidly evolving cytopenias, LFT
disturbances, and coagulopathy. One should evaluate further with
estimation of serum ferritin, Serum Triglycerides, Coagulation profile
& serum electrolytes. A Bone Marrow should be done to confirm one’s
suspicion. An aggressive approach is warranted even in secondary HLH,
as the hyper inflammation is not possible to control by infection
directed therapy. Therapy should be given for at least 8 weeks in
secondary HLH. In India peculiarly peripheral blood HLH is not
uncommon. Awareness is the clue. As more pediatricians get sensitized
we should be able to diagnose more cases of HLH and offer them prompt
treatment. HLH in adults is also not uncommon and we need to create
awareness about the same in adult physicians too. There is an urgent
need to set up BMT facilities to cure some of these patients.
The Division of Immunology at B J Wadia Hospital for children along
with NIIH runs PID (Primary Immunodeficiency) services at B J Wadia
Hospital. NIIH has established Perforin & granule release assay which
can be done in suspected cases of HLH.
We hope we are able to establish transplant facilities to cure some of
our patients.

Dr Mukesh M Desai. M.D
Prof. of Pediatric Hematology Oncology (DNB)

Hon. Hematologist Oncologist & Immunologist.
Chief, Division of Immunology; Department of PHO.

B. J. Wadia Hospital for Children.

Honorary Consultant Hematologist
Nanavati Hospital.
Sir H N Hospital.
Saifee Hospital.
Asian Heart Institute
Rooms:
Hematology & Immunology Cell.
B1 Matru Ashish,
Next to Balbharti School; SV Road,
Kandivili west, Mumbai 400067.

Cell : +91 9820037087.
Clinic : +91 22 28092927;
+91 22 28092917.

Email: mmdes...@gmail.com
URL: http://sites.google.com/site/mmdesai007/
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