1. Managing HLH: Recent Improvements and Persistent Challenges

2. Introduction
syndrome of pathologic immune activation characterized by clinical signs and symptoms of extreme inflammation.
It was first recognized as a familial immune dysregulatory disorder of childhood, called familial hemophagocytic reticulosis” in 1952.
Later, HLH was described as both a familial disorder and as a sporadic one, in association with infections, malignancies, or rheumatologic disorders.

3. Hemophagocytic Lymphohistiocytosis Introduction
Group of life-threatening immunodeficiencies characterized by excessive inflammation
Due primarily to genetic defects which compromise cytotoxic lymphocytes
Leading to prolonged and uncontrolled activation of T cells and macrophages (histiocytes)
Resulting in toxic hypercytokinemia.

4. Hemophagocytic Lymphohistiocytosis Introduction
HLH today, continued
True incidence unknown
High risk of mortality if not rapidly treated
Multiple genetic causes are recognized
Autosomal recessive and X-linked
Estimated 15-20% of familial cases without known genetic defect

5. Diagnosing HLH
Diagnosing HLH is the first critical step toward successful therapy but is challenging because of
the rare occurrence,
variable presentation, and
nonspecific findings of this disorder.

7. Diagnosing HLH
these diagnostic criteria do not reflect all of the typical clinical or laboratory features.
For instance,
patients with HLH almost always have evidence of liver inflammation,
which may range from very mild elevations of transaminases to
fulminant liver failure.
Thus, unexplained liver failure with concurrent cytopenias and elevated inflammatory indices should suggest HLH,
whereas a diagnosis of HLH with normal liver indices should be considered unusual.
neurologic findings are not part of current diagnostic criteria, even though they are relatively common (30%) and a distinctive clinical feature in many patients with HLH.

8. Diagnosing HLH the diagnosis of HLH is often delayed.
the rarity of HLH,
the complexity of diagnostic criteria, and
concern regarding the specificity of current diagnostic criteria.
more prompt and accurate diagnoses
better understanding of the clinical patterns of HLH and
how they appear to relate to the underlying pathophysiology would lead to.

9. Hemophagocytic Lymphohistiocytosis Immunologic Abnormalities
T cell activation
High soluble IL2R
High gamma interferon and other cytokines
Macrophage/Histiocyte activation
High Ferritin
High CD163
High Neopterin

10. Hemophagocytosis is the hallmark of “histiocyte” activation

11. CD163 stain of BM biopsy/HLH

12. Hemophagocytic Lymphohistiocytosis Immunologic Abnormalities
Absent or profoundly decreased NK cytotoxicity has been closely associated with HLH for more than 30 years
Putative roles of NK cells in immune homeostasis
Control of viral infection
Modulation of antigen presentation (APCs)
Contraction of activated T cells
Triggered apoptosis of APCs (histiocytes)

13. Normal Immune Homeostasis
De Saint Basile, Nature Rev Imm, 2010

14. Immune Homeostasis Gone Awry
#1 Can’t Kill Target

15. Hemophagocytic Lymphohistiocytosis Pathogenesis of Illness
The systemic hypercytokinemia which results from inadequate immunologic control underlies organ dysfunction: IL1β, TNFα, IL6, IL8, gIFN, IL18, IL10
Fevers
Hyperlipidemia
Endothelial inflammation/coagulopathy
Hepatitis, triaditis
CNS vasculitis and demyelination (> 50% of cases)
Inflammatory lung disease/ARDS
Marrow hyperplasia or hypoplasia, hemophagocytosis

18. Hemophagocytic Lymphohistiocytosis Immunologic Abnormalities
Gene microarray studies of newly-diagnosed and relapsing patients with HLH reveal profound and broad downregulation of genes involved with key pathways of innate, B cell, and cytotoxic immunity.
- TLRs 4,5,7,8,10; CD1a
- BLNK, BTK, CD19
PRF1, UNC13D, GrA, SAP

19. Hemophagocytic Lymphohistiocytosis Hematologic Dysfunction
Neutrophil degranulation is abnormal
– IBD in some genetic forms of HLH
Platelet degranulation is abnormal
Bruising/bleeding in some genetic forms

20. XLP1 (SH2D1A defect) Clinical consequences SAP Interacts with SLAMs
EBV associated HLH – 70%
Hypogammaglobulinemia – 25%
Lymphoma – 15%
Vasculitis – 4 %
SAP Interacts with SLAMs
Displaces inhibitory phosphatases
Potentiating signaling and cytotoxicity

21. XLP2 =XIAP (BIRC4 defect)
Clinical consequences
EBV associated HLH – 70%
Indolent or severe
Chronic splenomegaly
Recurrent HLH
Liver failure
Colitis- 15%
Hypogammaglobulibemia..

23. If cellular perforin protein levels are low, we do PRF mutation analysis.
●If CD107alpha mobilization is low, we do UNC13D, STX11, STXBP2, and RAB27A mutation analysis.
●If SAP expression is low, we do SH2D1A mutation analysis.
●If XIAP expression is low, we do BIRC4 mutation analysis.

24. We typically perform the following immunologic testing:
●Soluble IL-2 receptor alpha (sCD25 or sIL-2R)
●Tests of NK cell function/degranulation (eg, by flow cytometry for surface expression of CD107alpha, also called LAMP-1 [lysosomal-associated membrane protein 1])
●Flow cytometry for cell surface expression of perforin and granzyme B proteins
●Flow cytometry for cell surface expression of SAP and XIAP proteins in males
●Soluble levels of the hemoglobin-haptoglobin scavenger receptor (sCD163)
●Immunoglobulin levels (eg, IgG, IgA, IgM)
●Lymphocyte subsets (underlying immune deficiency diseases are sometimes found)

25. Mutations at FHL loci — Several of the gene mutations in HLH map to familial hemophagocytic lymphohistiocytosis (FHL) loci. (See 'Terminology' above.)
●PRF1/Perforin – FHL2 results from mutations in the PRF1 gene, which encodes perforin. Perforin is delivered in cytolytic granules and forms pores in the membrane of target cells. Mutations in other genes that affect perforin expression have also been reported
●UNC13D/Munc13-4 – FHL3 results from mutations in the UNC13D gene, which encodes Munc13-4 Proteins of the Unc (uncoordinated) family regulate cytolytic granule maturation.
●STX11/Syntaxin 11 – FHL4 results from mutations in the STX11 gene, which encodes Syntaxin 11. Syntaxins control granule exocytosis. Several Syntaxin mutations were reported in a group of Kurdish families with HLH [
●STXBP2/Munc18-2 – FHL5 results from mutations in the STXBP2 gene, which encodes Munc18-2 (also called Syntaxin binding protein 2) This protein binds to Syntaxin 11 and promotes the release of cytotoxic granules.

27. Immunologic and genetic workup of HLH
Rapid immunologic testing
(which may be performed in 1-3 days)
may support a diagnosis of HLH and rovide etiologic data,
whereas gene sequencing (typically requiring 3-8 weeks)
may define the underlying genetic cause

30. Pitfalls in NK Cell Testing
Classic Cytotoxic Assay
Dependent on number of NK cells in PBMC sample
Decreased if steroids or other immunosuppressants are used
Not reliable under 2 months of age
Degranulation Assay
Can suggest defect in the pathway, but not perforin deficiency
Pantoxilux*
Visualizes the entire NK cytotoxic process

32. Variations in the HLH pattern

33. Primary HLH clear familial inheritance or genetic causes
infants or younger children,
To have fixed defects of cytotoxic function
clear risk of HLH recurrence
not likely to survive long-term without HSCT.
Although HLH in these patients can be associated with infections (such as CMV or EBV) or vaccination, the immunologic trigger is often not apparent.

34. Secondary HLH older children (or adults)
without a family history or known genetic cause
typically have concurrent infections/medical conditions that appear to trigger their HLH, such as
EBV infection,
malignancy, or
rheumatologic disorders.
risk of recurrence in cases of secondary HLH is poorly defined.
Recurrence of HLH, in the absence of autoimmune disease or malignancy, is generally considered to be good evidence that a patient has primary HLH

35. Additional clinical features of HLH
Prolonged fever
FUO
fevers above 102° F (38.9°C) for a median of 19 days (range, 4-41 days).
In patients with
FUO, cytopenias, highly elevated ferritin (> 3000 g/dL), or sCD25 significantly above age- adjusted normal ranges,
generally prompt us to pursue a complete HLH diagnostic evaluation.

36. Additional clinical features of HLH
Liver disease and coagulopathy
Most patients have variable evidence of hepatitis at presentation.
HLH should be considered in the differential diagnosis of acute liver failure, especially if lymphocytic infiltrates are noted on biopsy.
Veno-occlusive disease may arise
Spontaneously (rate = 25% after BMT.
disseminated intravascular coagulation
95% of patients have features
high risk for acute bleeding.
platelet dysfunction
resulting from degranulation defects
Bone marrow failure

37. Additional clinical features of HLH
Bone marrow failure
Anemia and thrombocytopenia occur in > 80% of patients at the time of presentation with HLH.
The cellularity of bone marrow aspirates varies from normocelluar to hypocellular or hypercellular.
Prevalence of hemophagocytosis in association with HLH diagnosis ranges from 25%-100%.
blood transfusions, infection, autoimmune disease, and other forms of bone marrow failure or causes of red blood cell destruction
diagnosis should never be made or excluded solely on the presence or absence of hemophagocytosis.

38. Additional clinical features of HLH
variety of skin manifestations manifestations (6%-65%)
generalized maculopapular erythematous rashes,
generalized erythroderma,
edema,
panniculitis,
morbilliform erythema,
petechiae, and purpura.
Kawasaki Like Features:
erythematous rashes, conjunctivitis, red lips, and enlarged cervical lymph nodes

39. Additional clinical features of HLH
Pulmonary dysfunction
leads to urgent admission to the intensive care unit
review of the radiographic abnormalities in 25 patients,
17 had acute respiratory failure with alveolar or interstitial opacities (ARDS),
with fatal outcomes in 88% of those cases.
Worsening pulmonary function is
an ominous sign and should suggest inadequate control of HLH and/or infection.

40. Additional clinical features of HLH
Brain, ophthalmic, and neuromuscular symptoms (1/3 at presentation)
seizures,
meningismus,
decreased level of consciousness,
cranial nerve palsy,
psychomotor retardation,
ataxia,
irritability, or
hypotonia.
The cerebrospinal fluid (CSF) is abnormal in > 50% of HLH patients
pleocytosis, elevated protein, and/or hemophagocytosis.
MRI findings
discrete lesions, leptomeningeal enhancement, or global edema, and
images correlate with neurologic symptoms.
Retinal hemorrhages, swelling of the optic nerve, and infiltration of the choroid have been reported in infants with HLH.
Diffuse peripheral neuropathy with pain and weakness secondary to myelin destruction by macrophages

41. Treatment Initial considerations
Often the principle challenge for treating patients with HLH is making a timely diagnosis.
It is also critical to search for and treat underlying triggers of HLH, and institute specific antimicrobial therapy.
Rituximab is often helpful in controlling EBV infection.
Intravenous immunoglobulin is an appropriate adjunct for most viral infections. visceral leishmaniasis may closely resemble HLH

42. Treatment Initial considerations In general,
if the patient is stable and not severely ill,
treating the underlying trigger with disease-specific therapy with or without corticosteroids and close follow-up.
In most cases,
an aggressive therapeutic approach is warranted and
may reasonably be initiated before obtaining final results for all diagnostic studies.
Specifically, HLH therapy should not be withheld while awaiting results of genetic testing, as our understanding of HLH-associated gene defects remains incomplete.
With the exception of autoimmune disease and malignancy, we do not differentiate initial therapy for patients with suspected familial or reactive HLH

43. Hemophagocytic Lymphohistiocytosis Treatment
Provide definitive cure with allogeneic HCT from best available donor
- genetically determined HLH
- progressive HLH
- recurrent (reactivating) HLH

44. Hemophagocytic Lymphohistiocytosis Treatment
Induction Therapy: control inflammation and it’s sequelae
Decadron – CNS penetration
Etoposide
ATG (HIT-HLH): 19 patients treated
Possible improved survival pretransplant
Campath (anti CD52), salvage therapy

45. Emerging Therapy for HLH Reactivation Biologics to Replace Chemotherapy
Novimmune 0501
Fully humanized MoAb which neutralizes gIFN
Dosing is individualized based on PK
Has been approved in Europe (EMA)
First European site opened in October 2012
U.S. FDA approved trial in spring 2013
Is safe during BMT conditioning

46. To be continued?