Title: A scopic review on the diagnostic dilemmas and newer treatment
modalities in Hemophagocytic Lymphohistiocytosis
Abstract:
Hemophagocytic lymphohistiocytosis is a rare and fatal systemic illness
arising secondary to an immune dysregulation .Primary HLH is due to
genetic defects and secondary HLH is caused due to unchecked macrophage
recruitment following an acquired trigger. It is often diagnosed late in
view of its rarity and similarities of presentation to sepsis and SIRS.
A compelete curative solution to this problem is hematopoietic stem cell
transplant, though secondary cases are often seen to have sustained
remission with immune-chemotherapy COVID 19 has also been postulated to
cause a HLH like scenario with blunted NK cell number and function.
Recent advances in this field comprising of various immunosuppressant
based regimens, myeloablative therapies preceding stem cell transplant
and improved techniques of stem cell transplant have improved the
outcomes. Here we try to present the pathogenesis, etiology, diagnostic
criteria and the dilemmas, various treatment strategies, prognostic
markers and the most recent researches regarding this rare disease.
Introduction :
Hemophagocytic lymphohistiocytosis (HLH) is an immune dysregulation
syndrome which is caused due to defect of the Natural killer (NK) cells.
There are 2 variants – primary (familial) and secondary. Primary HLH is
caused due to mutations of certain genes which leads to improper
activation and functioning of NK cells leading to an uncontrolled
cytokine storm. Secondary HLH is secondary to infections (e.g.: EBV,
Dengue), rheumatological issues and malignancy.
History:
HLH was first noted way back in 1939, when Scott and Robb Smith
construed this condition to be an atypical presentation of Hodgkin’s
lymphoma. However, post mortem histopathology showed histiocytic
infiltration in the bone marrow with phagocytosed erythrocytes. Hence
they named it histiocytic medullary reticulosis
(HMR).1 It was later in 1959 that similar findings
were identified in pediatric population by Farquhar and Claireaux. Two
siblings presented with the same during infancy and rapidly progressed
into multi organ failure leading to death in both.2
Pathophysiology:
In a normal body NK cells and cytotoxic T cells (CTLs) identify the
Antigen presenting cells (APC - dendritic cells and histiocytes) and
then release perforin and granzyme in a well-regulated fashion through a
newly formed synapse called immunological synapse
(IS).3 Perforins and granzymes are normally stored in
granules in lysosomes of NK cells and CTLs.4 Limited
amounts of required Inflammatory mediators are also transported across
the IS towards the APCs. Perforins form pores on the APCs and later
granzymes help in apoptosis.4 Together all these
processes orchestrate in a well regulated manner to first destroy the
antigen and then set stage for apoptosis.3 Following
apoptosis, the dying cells secrete enzymes which stimulate each other in
a cascade fashion to destroy the IS, hence controlling the release of
inflammatory mediators.5 Any hindrance in this pathway
gives a longer life to the IS causing more uncontrolled and unnecessary
release of inflammatory mediators – cytokines, tumour necrosis factor
(TNF) and interferons. This inflammatory cocktail stimulates macrophages
and turns on the secretion of IL-6.5 The unchecked
Macrophages hence activated, phagocytose the other blood cells causing
cytopeneias.6 Also the antigens presented by the
antigen presenting cells, are not effectively killed causing persistent
activation of these cells and leading to further recruitment of NK cells
and immunologic synapses, thus leading to further hypercytokinemia
(cytokine storm).7 The macrophages and histiocytes
with the phagocytosed cells, infiltrate various organs, impeding their
function like liver, spleen, and lymph nodes leading to
hepatosplenomegaly and lymphadenopathy.
Etiology:
Familial HLH:
The incidence of familial (primary) HLH was seen to be 1.2 in 1000000
children per year, according to a study conducted in
2009.8 An Indian meta – analysis review showed that
the incidence of primary HLH in India is 1 in 50000.9They are inherited in an autosomal recessive pattern. There are 5 main
gene loci identified currently, which could cause familial HLH – a
genetic loci on chromosome 9, PFR1, UNC13D, STX11, STXBP2. (Table 1)
Most common mutations noted are those of PRF1 and UNC13D causing FHL2
and FHL3 respectively. Together they account for 70% of all the
familial HLH cases.10 Age of presentation is mostly
during early infancy .11
Other genetic diseases – Griscelli syndrome, Chediak Higashi,
Hermansky-Pudlak and X linked lymphoproliferative disease (type 1 and 2
) could also pre-dispose to HLH.12,13,14 (Table 2)
Secondary HLH:
This is caused due to conditions which initiates NK cells or cytotoxic T
lymphocytes induced inflammation. NK cells constitute the early
defenders against intracellular pathogens such as viruses. Hence viruses
are the most common infectious agents leading to HLH.8In a meta-analysis by Srinivas Rajagopala and Navneet singh it was found
that viral infections were the most common cause of Secondary HLH. EBV
and dengue were the most common viruses to cause HLH in children. In the
current period, it is worth mentioning that many of the deaths occurring
due to COVID 19 are also due to severe HLH like cytokine
storm.9,15 COVID 19 causes blunting of NK cell number
and functions leading to improper clearance of the APCs and unchecked
elevation in tissue damaging inflammatory mediators.10Leishmaniasis, Ricketsia, Malaria, Histoplasmosis, Enteric fever, and
Tuberculosis were also responsible agents in adults. The other causes
were connective tissue disorders such as stills disease, juvenile
idiopathic arthritis (JIA) and SLE.9 Malignancies
especially those affecting T cells – like anaplastic T cell lymphoma
and acute lymphoblastic lymphoma were seen to cause HLH in a study by
Lehmberg et al in 2015.16 Few B cell lineage
neoplasias like hodgkins lymphoma were also seen. Post bone marrow
transplant, a HLH like scenario could arise.
Diagnosis:
Diagnosis of this condition is based on symptoms, signs, and markers of
inflammation. Prolonged high grade fever is present in 90-100% of the
children, and hence HLH is one of the differential diagnosis of Pyrexia
of unknown origin.17 Splenomegaly is also seen in more
than 80% of children according to all the studies
reviewed11,17,18 and lymphadenopathy is seen in
approximately 50% of cases.19 The child at
presentation appears very sick with features mimicking sepsis and septic
shock ( tachycardia, tachypnea, hyperpyrexia and occasionally
hypotension ). Pancytopenia or bicytopenia is always present because of
significant phagocytosis, and the differential count always show
lymphocyte predominance. Neutropenia is occasionally noted. CRPH which
is a Marker of inflammation is seen to be very high. Activated
histiocytes bind with factor 10, hence activating the common pathway of
coagulation and consuming fibrinogen leading to
hypofibrinogenemia.20 ESR is hence low, since
erythrocyte sedimentation is dependent on fibrinogen. Ferritin is
elevated because ferroportin mediated iron efflux increases due to
increase in growth differentiation factor 15 during the
hyperinflammation in HLH. Inflammation also causes upregulation of
hemoxygenase – which breaks heme to iron. Other markers of inflammation
which increase are IL-6, IL-18, IFN-gamma, TNF-alpha.
Hypertriglyceridemia occurs due to inhibition of lipo-protien lipase by
TNF alpha.21
Diagnostic criteria for HLH was formulated in 2004 and confirms the
diagnosis if any of the gene abnormalities can be detected ( molecular
diagnosis ) or any 5 of the 8 below mentioned criteria are fulfilled:
- Fever > 38.5 degree centigrade
- Splenomegaly
- Cytopenia (at least 2 cell lines):
a) Hb<9; b) Absolute neutrophil count <1000 c)
Platelet<100000/microL
4) Hypertriglyceridemia >265 mg/dl
5) Hemophagocytosis in bone marrow, spleen, lymph node
6) Low or absent NK cell activity
7) Ferritin > 500ng/ml
8) Elevated soluble CD25 ( IL-2 receptor – on T cell
)20
These diagnostic criteria was formulated according to prevalence of
these features in 369 patients in HLH study of 2004. More than 70% of
children in this study showed all these features.22
Ferritin levels for diagnosis of HLH is a very debated criteria. Though
500ng/ml is considered significant, various authors have opined about
increasing the threshold for confirmation of the diagnosis. Ferritin
levels of 10000 ng/ml was considered significant by Allen et al and 6000
ng/ml by Belfike et al.23,24 CD107a was seen to be
upregulated in all activated NK cells and hence was considered to be a
marker of NK cell activity.
As mentioned earlier, infiltration of macrophages and histiocytes in
various organs cause multi organ failure.
Liver involvement is seen in most of the children with HLH. Hence HLH is
considered to be a differential diagnosis of Acute Liver Failure (ALF)
during the first year of life. In a study on 251 children with HLH in
Korea, 86% of the children were seen to have hepatomegaly, with 63%
having transaminitis and 35% showing icterus. Around 27% of them had
prolonged APTT and hypofibrinogenemia was seen in 62% of the
children.19 Histology of affected liver shows
lymphohistiocytic infiltration in the portal system, along with bile
duct injury. However lobular histology is preserved. Kupffer cell
hyperplasia is also noted along with sinusoidal congestion with
histiocytes, along with hemosiderosis. However liver biopsy is usually
avoided due to the catastrophic bleeds which may
ensue.25
Symptoms and signs of lung infiltration are very difficult to be
differentiated from other infections.12
CNS infiltration is seen in mostly the familial variant, and its
symptoms are varied ranging from neck rigidity due to meningeal
involvement to irritability due to encephalitis. Three stages were
described by Henter and Nennesmo in 1997, where stage 1 showed only
meningeal infiltration in the mildest form and stage 3 showed diffuse
infiltration of parenchyma, meninges, along with multifocal tissue
necrosis.26 Features of raised ICP – bulged
fontanelle, vomiting, blurring of vision are also seen. Convulsions also
are occasionally seen when parenchyma is infiltrated. Cranial nerves 6
and 7 are occasionally involved.12
It must be noted that the diagnosis of HLH is mostly made with
progression of symptoms, not responding to the usual therapies, and
hence the commoner possibilities must always be considered first. SIRS,
sepsis induced multi-organ dysfunction (MODS), and macrophage activation
syndrome (MAS) secondary to JIA share the same clinical and laboratory
features as that of HLH. This dilemma leads to over diagnosis of
HLH.27 Arico et al hence made an algorithm with three
tests – perforin expression ( by flow cytometry ), lymphocyte 2B4
receptor function (antibody dependent cellular cytotoxicity assay ) and
NK cell activity.28 In centers which do not have such
facilities- supportive therapies for sepsis and MODS should be initiated
and chemotherapy for HLH should be reserved for children who do not
respond to them.27 However delay in treatment of HLH
is also a determinant of mortality.
Treatment:
The most important goal in treatment is to arrest the stormy
hyperinflamatory response within the body and to promote apoptosis of
the antigen presenting cell to prevent further inflammatory cells’
recruitment. Etoposide is a chemotherapeutic drug which does both the
above functions. Once this is achieved- the familial, persistent or
relapse disease is treated with hemopoeitic stem cell transplant
(HSCT).22
To achieve appropriate immune suppression HLH 94 consensus had come up
with dxamethasone and etoposide regimen for 8 weeks and cyclosporine was
added from the 9th week. Intrathecal methotrexate was given to children
with CNS involvement. However HLH 2004 study suggested addition of
cyclosporine in the begining itself along with dexamethasone and
etoposide. CNS involvement was treated with intrathecal methotrexate and
hydrocortisone as per 2004 guidelines. There were marginal improvement
in the survival with HLH 2004 as compared to HLH 94 guidelines – 62%
with the former and 54% with the latter.22 In v/o of
significant complications of cyclosporine (renal toxicity and posterior
reversible encephalopathy syndrome) many experts do not start early
cyclosporine therapy, though some clinicians opine that it helps in
maintaining remission for longer time.29 Early
initiation of steroids after ruling out malignancy must be considered
even before all investigation reports are obtained.29Treatment should be tailored according to the disease severity and liver
and renal functions. Children who show a very rapid response should be
weaned off faster. Dexamethasone which is started at
10mg/m2 could also be tapered down as per severity
either weekly or once in two weeks.29 At the same time
any flare in the disease warrants further intensification of both
steroid dose and etoposide frequency.
Anti thymocyte globulin (ATG), which causes T cell lymphopenia, has also
been used along with steroids instead of etoposide and has shown to have
good efficacy with lesser degree of cytopenia. A study done by Mahlaoui
et al from showed this regimen to have complete remission in 73% and
partial remission in 24% of the 38 children
enrolled.30
Intravenous immunoglobulins could be used in the treatment of HLH as a
primary agent or an adjunct. In a multi centre study conducted by
Larroche C et al it was noticed that 17 children (9 primary and 8
secondary) showed remission to 2 IV-IG doses of 1.6g/kg per dose. They
also mention about the importance of early delivery of the same, and
report inadequate results with late administration.31Also few Indian studies Divya nandhakumar et al and Sarala Rajajee et al
showed good response to IVIG with steroid regimens in secondary HLH –
17 children in the former and 19 children in the latter studies achieved
remission. These studies have used IV IG in secondary HLH
only.32,33 However few studies such as Imashuku et al
suggest that IV IG based primary regimens are not adequate in treating
HLH.34
EBV induced HLH has shown good response to addition of Rituximab (anti
CD 20 antibody) and Anakinra (IL1 recptor antagonist) was found to be
beneficial as an early add on in Systemic onset juvenile idiopathic
arthritis induced HLH.35,36 HLH like presentation
following bone marrow transplant can be treated by adalimumab – a TNF
alpha inhibitor.37 (Table 3)
Response to therapy could be monitored using ferritin levels or soluble
IL2 receptor concentration. A drop of 15% of the former within first 48
hours and any decrease in the latter is considered to be a positive
response.29 Decrease in triglyceride level also
suggest improvement.21
Refractory cases (nil response after 8 weeks standard therapy) could be
treated with Alemtuzumab (anti CD 52 antibody) along with steroids,
etoposide and cyclosporine as shown in Marsh et al. 1 mg/kg of
alemtuzumab was given as an add on to 14 children and 25% of them
showed partial remission.38 Doxorubicin (liposomal),
Etoposide and Methylprednisolone (DEP) is another regimen used as a
salvage therapy especially in adult refractory
cases.39
Hematopoietic stem cell transplant (HSCT):
HSCT for this disease was first carried out in 1995, and advances from
then in this field have led to better outcomes. The stem cells are
usually harvested from peripheral blood (78%) or bone marrow (22%) of
the donor.40 Umblical cord stem cells have also been
used for transplant and have shown good results with better survival in
infants with primary HLH.41 However this cannot be
done in bigger children and adults, since umblical cord may not have
sufficient stem cells. Finding a fully matched donor for HSCT could be
difficult, and hence partially matched family member’s stem cells are
used in few centers – haploidentical HSCT. The chances of the donor
also having the abnormal gene causing HLH is high in such cases and if
so the recipient would go into relapse.38
Following HSCT, it takes 10-20 days for engraftment to occur. If all
hematopoietic cells post-transplant are of donor origin, then the
recipient is called a complete chimera and shows complete chimerism.
20-30% stable chimerism is required for a successful transplant, and to
keep the child in sustained remission.38 A donor
chimerism of 20% means that following the transplant, 20% of the cells
in the marrow and the circulating blood of the recipient would be that
of the donor origin. Increasing level of recipient cells could lead to
graft rejection, and the opposite may result in the complication of
graft vs host disease (GVHD). During mixed chimerism, whether an
increased recipient origin cells indicates graft failure or a graft,
recipient peaceful coexistence is difficult to
assert.42 Hence clinical monitoring of remission, and
early identification of GVHD is prime following the procedure.
Myelo-ablative conditioning regimens are nowadays being used to kill the
recipient marrow cells, so that there is enough room for the donor cells
in the recipient marrow. The advent of this therapy, also known as
conditioning, preceding transplant has improved the outcomes in HLH
quite significantly. Initially Busulphan and cyclophosphamide were used.
However adverse effects such as veno-occlusive disorders ensued leading
to adverse outcomes.38 Better outcomes were noticed
with reduced intensity conditioning regimens like melphalan and
fludarabine, or treosulfan and thiotepa, though at the expense of higher
rates of mixed chimerism. The results were further enhanced with
addition of serotherapy with alemtuzumab which is a monoclonal antibody
of CD52 ( present on all mature lymphocytes, but absent in the stem
cells ).43 Alemtuzumab could be given at the proximal,
distal or intermediate phase of the regimen. Data from various studies
favor both proximal and intermediate phase administration. Mixed
chimerism is seen in such cases but with adequate tolerance, because the
levels of alemtuzumab is seen to be high enough till around 60
days.44 Also Alemtuzumab mediated depletion of T cells
and APCs bring down the inflammation prior to HSCT – improving the
chances of survival. However it is to be noted that low donor chimerism
lesser than 20% warrants post HSCT cell therapy, which could be either
donor lymphocyte infusion (DLI) or a second HSCT.
GVHD is a dreaded complication of HSCT. In acute GVHD, occurring within
100 days, fever, rash, hepatitis with hyperbilirubinemia, vomiting,
diarrhoea, abdominal pain and weight loss are the presenting complaints.
Chronic GVHD affects the skin predominantly with lichenoid rash and
sclerotic changes. Hepatobiliary system is also affected and significant
immunodeficiency occur frequently. Cyclosporine and methylprednisolone
are tailored into the HSCT regimen, starting 2-3 days prior to
transplant. Cyclosporine is usually continued for upto 100 days
post-transplant. Methotrexate and tacrolimus could also be used.
Post-transplant, another cytokine storm can occur which looks like the
original HLH itself. This condition is seen to have good response to
adalimumbab.37
Carl et al from United states of America, studied 36 children with HLH
underwent stem cell transplant, following reduced intensity conditioning
with melphalan and fludarabine, with intermediate alemtuzumab. The 1
year overall survival was 82.4% and 18 months overall survival of 68%
was noted.45 Another study by Katharina et al on 60
children who underwent HSCT for HLH showed similar promising results
with 5 year overall survival at 75%, though 30% of them required
secondary post HSCT cell therapy (DLI or second HSCT). Both melphalan
and treosulfan based regimens were used for conditioning with
serotherapy given in the proximal limb of therapy with alemtuzumab and
ATG. All the regimens were found to be equally
efficacious.46
Outcomes :
Children with this condition do not survive more than 2 months without
treatment.11 Younger age, CNS involvement, severe
elevation in transaminase, severe cholestasis, and coagulation
abnormalities are considered to be indicators of poor outcomes according
to a study by Koh et al.18 Outcomes are also negative
if the treatment is delayed, and hence at least steroids should be
commenced immediately after ruling out all other possibilities such as
malignancy. In some children repeated, multiple blood and plasma
exchange have shown positive response, and have been used to buy some
time till diagnosis is confirmed.47 Hematopoietic stem
cell transplant is the final curative treatment available, and in
familial cases, this must be performed as soon as possible during
remission. Hence HLA typing and searching an appropriate donor is
commenced as soon as the disease is confirmed in such cases. Having
active disease during transplant, using haploidentical stem cells and a
poor match in HLA typing were seen to have high rates of failure
following HSCT.38,46
Recent updates and research:
Pre B cell colony enhancing factor (PBEF) - a marker of severe
inflammation, has been shown to be a very good marker for diagnosis and
disease activity. This has shown a better specificity because with
remission it touches the baseline levels, as compared to IL2 receptor
levels which stays mildly elevated for some time.48
Hybrid immunotherapy is a new treatment modality which is being tried in
few centers where steroid, etoposide and ATG are being used. Other
regimens with the primary agent being alemtuzumab and tocilizumab (IL-6
receptor inhibitor) are being studied in France and Philadelphia
respectively. However there are no published results from these studies.
Cytokines cause the inflammatory storm through Janus kinase (JAK1,2) and
signal transducer and activator of transcription (STAT) associated
receptors. Ruxolitinib causes inhibition of JAK1,2 receptors with
amelioration of inflammation in murine models.49 This
agent as primary agent is being tried in Michigan. Anti IFN-gamma
antibodies – Emapalumab is also being tried. A study on 13 children
treated with Emapalumab showed improvement of parameters in 9 children
and 7 of them went for HSCT.50
Hematopoietic stem cell gene therapy with selected cells of high level
perforin expression led to better survival in animal model studies.
Perforin gene transfer using lenti virus as vector into progenitor cells
of perforin deficient mice was performed and significant reduction in
cytokine secretion in mice, with more than 30% engraftment, was
noted.51
In another study Micro RNA -126 (enhancer of perforin expression) was
placed in the perforin DNA loci of the stem cells, and was transplanted
into perforin deficient mice. These mice showed a dose dependent
increase in survival and better clearance of
pathogen.52 Such gene therapy if successful in humans
could improve the outcome remarkably.
Extracorporeal adsorption technique has shown success in adults. It was
used in 2 adults with HLH secondary to herpes infection, and was found
to be effective in bringing the cytokines’ level down. An adsorption
cartridge made up of polystyrene divinylbenzene copolymer beads is used,
which binds to hydrophobic compounds with a molecular weight of 10-55
kDa.53
Conclusion:
HLH is a fatal storm of inflammation in the body, which mimics other
severe illnesses such as SIRS and sepsis. Differentiating between them
clinically is a challenge. Treatment of both sepsis and HLH are also
very different with former being treated with broad spectrum antibiotics
and latter with immunosuppressants. The recent diagnostic guidelines
have led to an over diagnosis of HLH leading to flare up of infections
and missing malignancies at an earlier stage due to the
immunosuppressants used for the same. At the same time delaying the
treatment of HLH also causes mortality. This dilemma warrants immediate
but prudent treatment with exercise of cautious monitoring.
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Table 1: Genes involved in primary HLH