Familial hemophagocytic lymphohistiocytosis

Familial hemophagocytic lymphohistiocytosis (FHL) also called primary hemophagocytic lymphohistiocytosis is a rare, genetic inborn errors of immunity causing aggressive life-threatening intense immune activation syndrome in which your body makes too many activated immune cells called macrophages (histiocytes) and lymphocytes (T cells, natural killer cells and B cells) ¹⁾, ²⁾, ³⁾, ⁴⁾, ⁵⁾, ⁶⁾. Hemophagocytic lymphohistiocytosis (HLH) is caused by an overactive and abnormal immune response resulting in an uncontrolled excessive inflammatory and ineffective immune response resulting in progressive multi-organ damage and potentially death if untreated ⁷⁾, ⁸⁾. In hemophagocytic lymphohistiocytosis (HLH), the immune system responds to a stimulus or ‘trigger’, often an infection, cancer or rheumatologic disease, but the response is ineffective and abnormal. This ineffective, abnormal response, causes a variety of signs and symptoms, which, if not treated, can potentially become life-threatening. Consequently, infants with HLH are born seemingly healthy, but upon encountering an immunological insult such as a viral infection (e.g., Epstein-Barr virus [EBV]), their lymphocytes cannot execute cytotoxic degranulation, leading to a positive feedback loop of massive inflammation without clearing the infectious trigger ⁹⁾, ¹⁰⁾, ¹¹⁾. Left untreated, familial hemophagocytic lymphohistiocytosis is fatal ¹²⁾, ¹³⁾.

Most children with familial hemophagocytic lymphohistiocytosis (FHL) are born healthy and develop the illness in the first 2 to 6 months of life ¹⁴⁾, ¹⁵⁾. Familial hemophagocytic lymphohistiocytosis (FHL) with onset in the neonatal period is rare ¹⁶⁾. Up to 20% of patients present at more than 2 years of age, and in rare cases, patients with FHL remain asymptomatic until adulthood ¹⁷⁾, ¹⁸⁾, ¹⁹⁾, ²⁰⁾.

Familial hemophagocytic lymphohistiocytosis (FHL) was first described in 1952 by Farquhar and Claireaux, who identified the disease in two fatal cases involving siblings, with the major findings being a marked increase of histiocytes in the bone marrow at the expense of blood-forming cells ²¹⁾. Familial hemophagocytic lymphohistiocytosis (FHL) was shown to be more prevalent in consanguineous marriages (marriage between two blood-related individuals who are second cousins or closer), suggesting an autosomal recessive pattern of inheritance ²²⁾, ²³⁾, ²⁴⁾, ²⁵⁾, ²⁶⁾, ²⁷⁾, ²⁸⁾, ²⁹⁾.

Familial hemophagocytic lymphohistiocytosis (FHL) is caused by mutations to the PRF1, UNC13D, STX11, STXBP2, and other genes that are associated with the exocytosis of lytic granules from cytotoxic cells ³⁰⁾, ³¹⁾. Familial hemophagocytic lymphohistiocytosis is classified into six different types based on genetic linkage analysis and chromosomal localization; five specific genetic defects have been identified, which account for approximately 90% of all patients ³²⁾. Familial hemophagocytic lymphohistiocytosis type 1 is due to as yet unidentified gene defect located on chromosome 9. Familial hemophagocytic lymphohistiocytosis type 2 is caused by mutations in the perforin (PRF1) gene, familial hemophagocytic lymphohistiocytosis type 3 by mutations in the Munc-13–4 (UNC13D) gene, familial hemophagocytic lymphohistiocytosis type 4 by mutations in the syntaxin 11 (STX11) gene and familial hemophagocytic lymphohistiocytosis type 5 due to mutations in the gene encoding syntaxin binding protein 2 (STXBP-2) ³³⁾. Twenty to fifty per cent of FHL cases worldwide are caused by mutations in perforin (PRF1) gene. Perforin is a critical protein for cytotoxicity mediated by granules present in NK cells and cytotoxic T lymphocytes, and it is required to fight viral infections as well as to keep the immune system, especially histiocytes, under control. Furtjermore, at least three genetic diseases other than FHL have been associated with the hemophagocytic syndrome, including mutations in SH2D1A (X-linked lymphoproliferative disease) ³⁴⁾, ³⁵⁾, mutations in CHS1 (Chediak-Higashi disease) ³⁶⁾ and mutations in RAB27A gene (Griscelli syndrome type-2) ³⁷⁾.

The reported incidence of FHL varies among different studies, probably reflecting different prevalence within different ethnic groups and the higher index of suspicion and improved diagnosis in more recent studies. Familial hemophagocytic lymphohistiocytosis (FHL) has been reported to be 0·12 to 1 cases per 100,000 children per year in Sweden ³⁸⁾, whereas it was found to be 0·342 per 100,000 in Japan ³⁹⁾. The frequency of familial hemophagocytic lymphohistiocytosis (FHL) among hospitalized patients in Turkey is 7·5 per 10,000, which is due most probably to the high rate of consanguineous marriage in Turkish pedigrees ⁴⁰⁾.

HLH should be suspected in all patients with prolonged high-grade fever associated with abnormally large spleen (splenomegaly) and multiple organ involvement ⁴¹⁾. The clinical spectrum of hemophagocytic lymphohistiocytosis disease is wide, ranging from mild organ dysfunction to multiorgan failure requiring intensive care. Central nervous system (CNS) involvement is frequent and often severe, even though it is not included in the official diagnostic criteria ⁴²⁾.

The typical patient is an infant under 1 year of age, ill-appearing (toxic aspect), occasionally with a critical sepsis-like aspect ⁴³⁾. A younger age at onset suggests an underlying genetic basis, as seen in familial hemophagocytic lymphohistiocytosis (F-HLH) or in HLH forms arising from genetic primary immunodeficiencies; however, familial HLH can present at any age, including adulthood ⁴⁴⁾.

Progressive splenomegaly (abnormally large spleen) is typically observed in patients, and can be associated with liver involvement, neurological signs, respiratory and kidney failure ⁴⁵⁾. Skin rashes, reddening of the skin because of inflammation (erythroderma), swelling (edema) or tiny spots on the skin due to bleeding under the skin (petechiae) have been reported ⁴⁶⁾. Atypical forms, usually seen in children older than 1 year, include isolated fever of unknown origin (FUOs), isolated central nervous system (CNS) involvement ⁴⁷⁾ or isolated acute liver failure ⁴⁸⁾, ⁴⁹⁾. Enlarged lymph node (lymphadenopathy) is uncommon in patients and indicates a potential underlying lymphoma ⁵⁰⁾.

Typical laboratory findings include cytopenias (low levels of red blood cells [anemia], white blood cells [leukopenia] or platelets [thrombocytopenia]), high level of triglycerides in blood (hypertriglyceridemia), low fibrinogen in blood (hypofibrinogenemia) are suggestive of HLH in the context of general inflammation and hyperferritinemia. Liver function tests are frequently altered ⁵¹⁾. Leukocytosis (elevated white blood cell (WBC) count) is not typical of HLH except in HLH-associated with defined rheumatological conditions or macrophage activation syndrome-HLH (MAS-HLH) ⁵²⁾. Hemophagocytosis describes the pathognomonic findings where highly activated macrophages taking up different cells including lymphocytes, erythrocytes, leukocytes, and platelets in different tissues, producing excessive cytokines and an uncontrolled inflammatory reaction ⁵³⁾, ⁵⁴⁾, ⁵⁵⁾.

To date, the only curative treatment available for familial hemophagocytic lymphohistiocytosis is an allogenic hematopoietic stem cell transplantation (HSCT) ⁵⁶⁾, ⁵⁷⁾. However, inflammation must be adequately controlled before undergoing hematopoietic stem cell transplantation (HSCT), and despite recommendations for intensification of therapy in the HLH-2004 international treatment study, the reported pre-HSCT mortality was still high at 19% ⁵⁸⁾. Survival rates vary depending on the conditioning protocol ⁵⁹⁾, ⁶⁰⁾, ⁶¹⁾, ⁶²⁾, ⁶³⁾, ⁶⁴⁾, ⁶⁵⁾, ⁶⁶⁾, ⁶⁷⁾, ⁶⁸⁾. Thus far, results from clinical trial NCT01998633 suggest that reduced intensity conditioning can improve overall 1-year survival of HLH patients to 82.4%, although larger and longer term studies will be needed to follow-up on these results ⁶⁹⁾. For patients who do receive hematopoietic stem cell transplantation (HSCT), the 5-year survival rate has improved from 54% to 61% ⁷⁰⁾, ⁷¹⁾, ⁷²⁾, ⁷³⁾.

Figure 1. Familial hemophagocytic lymphohistiocytosis pathogenesis

Footnote: The function of LYST gene, probably important for correct size and function of lytic granules, is not entirely understood. Note the empty granula in perforin deficiency. Hemophagocytic lymphohistiocytosis (HLH) is thought to develop due to abnormal reciprocal activation of mononuclear phagocytes (MNPs; monocytes, macrophages, and dendritic cells) and type 1 lymphocytes (NK cells and Th1, CD8+ cytotoxic T lymphocyte, and NKT cells). T cells with normal cytotoxic granules release them to induce mononuclear phagocyte apoptosis and terminate the synapse, whereas impaired or perforin-deficient granules (gray) cannot terminate mononuclear phagocyte activation. Immune synapse prolongation and excess IL-18 (from mononuclear phagocyte and/or epithelial sources) both amplify production of lymphocyte cytokines like IFN-γ, which in turn further activates mononuclear phagocytes and promotes hemophagocytosis and release of HLH biomarkers like ferritin, CXCL9, and interleukin-18-binding protein (IL-18BP). Activated lymphocytes upregulate the IL-2 receptor, which is cleaved by proteases released by activated mononuclear phagocytes. The absence of X-linked inhibitor of apoptosis protein (XIAP) may permit pathogenic inflammasome activation and lymphocyte apoptosis, while SLAM-associated protein (SAP) deficiency impairs restimulation-induced cell death (RICD) and, like CD27 and CD70 deficiency, prevents normal killing of EBV-infected B cells

Abbreviations: CHS = Chédiak Higashi syndrome; CTL = CD8+ cytotoxic T lymphocyte; FHL = familial hemophagocytic lymphohistiocytosis; GSII = Griscelli syndrome type 2; HPSII = Hermansky-Pudlak syndrome type 2; NK = natural killer.

[Source ⁷⁴⁾ ]

Familial hemophagocytic lymphohistiocytosis types

Familial hemophagocytic lymphohistiocytosis (F-HLH) is subclassified based on the underlying genetic mutation (Table 1). Familial hemophagocytic lymphohistiocytosis is a genetically heterogeneous disorder caused by mutations in genes involved in the secretory lysosome-dependent exocytosis pathway ⁷⁵⁾. More than 60 years after the first FHL case was reported, five independent, FHL-causing loci have been identified and the underlying genetic defect has been described for four of them accounting for over 90% of familial hemophagocytic lymphohistiocytosis (Table ​1) ⁷⁶⁾, ⁷⁷⁾, ⁷⁸⁾, ⁷⁹⁾, ⁸⁰⁾, ⁸¹⁾, ⁸²⁾. They encode the proteins perforin, MUNC13-4, syntaxin-11 and MUNC18-2, all of which play a key role in lymphocyte cytotoxicity (Figure ​1) ⁸³⁾.

Around 70% of familial hemophagocytic lymphohistiocytosis cases are explained by mutations in only two genes: PRF1 and UNC13D, which cause familial hemophagocytic lymphohistiocytosis type 2 and familial hemophagocytic lymphohistiocytosis type 3 subgroups, respectively ⁸⁴⁾. In addition to the described four subgroups of familial hemophagocytic lymphohistiocytosis, in which HLH is usually the primary manifestation, a few additional genetic conditions and other immune deficiencies may cause a clinical syndrome largely overlapping that of FHL but in which additional, distinctive clinical features occur.

Table 1. Familial hemophagocytic lymphohistiocytosis genetic causes and types

HLH type/association	Gene or locus (protein)	HLH-related dysfunction	Unique clinical features
Familial HLH
Type 1 (F-HLH 1)	9q21.3-q22 (unknown)	Unknown	N/A
Type 2 (F-HLH 2)	PRF1 (perforin)	Cytolytic pore formation	None
Type 3 (F-HLH 3)	UNC13D (Unc-13 homolog D)	Cytolytic vesicle priming	Increased incidence of central nervous system (CNS) disease
Type 4 (F-HLH 4)	STX11 (Syntaxin 11)	Cytolytic vesicle fusion	Mild, recurrent HLH, colitis
Type 5 (F-HLH 5)	STXBP2 (Syntaxin binding protein 2)	Cytolytic vesicle fusion	Colitis; hypogammaglobulinemia, and sensorineural hearing loss
Primary Immunodeficiency Syndromes
Chédiak-Higashi syndrome	LYST (Lysosomal trafficking regulator)	Cytolytic vesicle trafficking	Partial oculocutaneous albinism, recurrent pyogenic infections, and bleeding tendency
Hermansky-Pudlak syndrome type 2	AP3B1 (Adaptor Related Protein Complex 3 Subunit Beta 1)	Cytolytic vesicle trafficking	Partial albinism; immunodeficiency, bleeding tendency, and platelet storage pool deficiency
Griscelli syndrome type 2	RAB27A (Rab-27A protein)	Cytolytic vesicle docking	Partial albinism and silvery grey hair
Epstein-Barr virus (EBV) triggered
X-linked lymphoproliferative disorder type 1 (XLP1)	SH2D1A (SH2 Domain Containing 1A)	Defective killing of EBV-infected B cells	Severe HLH; Hypogammaglobulinemia, and lymphoma
X-linked lymphoproliferative disorder type 2 (XLP2)	XIAP (X-linked inhibitor of apoptosis protein)	Increased inhibition of apoptosis in EBV-infected cells (gain of function)	Mild, recurrent HLH (may be triggered by non-EBV viruses)
IL-2-inducible T cell kinase deficiency	ITK (IL2 inducible T cell kinase)	Impaired proliferation of EBV-specific T cells	Hodgkin lymphoma
CD27 deficiency	CD27	Impaired proliferation of EBV-specific T cells	Combined immunodeficiency
X-linked immunodeficiency with magnesium defect (XMEM)	MAGT1 (Magnesium transporter 1)	Impaired proliferation of EBV-specific T cells	Combined immunodeficiency, chronic viral infections, and lymphoma
Inflammasome activation
Autoinflammatory disease	NLRC4 (NLR Family CARD Domain Containing 4)	Increased inflammasome activation (gain of function)	Mild and recurrent HLH

Abbreviations: CNS = central nervous system; EBV = Epstein-Barr virus.

[Source ⁸⁵⁾, ⁸⁶⁾ ]

Primary HLH	Gene	Protein	Pathophysiology	Functional testing
Familial HLH (FHL)
FHL-1	Unknown	Unknown
FHL-2	PFR1	Perforin	Lack of perforin expression in lytic granules	Perforin expression
FHL-3	UNC13D	Munc13-4	Deficiency in fusion of lytic granule with plasma membrane	Degranulation
FHL-4	STX11	Syntaxin11	Deficiency in fusion of lytic granule with plasma membrane	Degranulation
FHL-5	STXBP2	Munc18-2	Deficiency in fusion of lytic granule with plasma membrane	Degranulation
Other immunodeficiency syndromes with defect in degranulation
GS-II	RAB27A	Rab27a	Deficiency in docking of lytic granule to the plasma membrane	Degranulation hair microscopy
CHS	LYST	Lyst	Defect in maturation of vesicles into secretory cytotoxic granules	Degranulation hair microscopy
—
Other inborn errors of immunity	Gene	Protein	Pathophysiology	Functional testing
Immunodeficiency syndromes with HLH as a frequent manifestation
XLP-1	SH2D1A	SAP	Defective killing of EBV infected B-cells by CD8 and NK cells	SAP expression
XLP-2	BIRC4	XIAP	Impaired inhibition of inflammasome activity	XIAP expression L18MDP assay
TIM3 deficiency	HAVCR2	TIM3	Persistent T cell activation and increased production of inflammatory cytokines	TIM3 expression
Immunodeficiency syndromes with HLH as an occasional manifestation
Chronic granulomatous disease (CGD)	CYBB, CYBA, NCF1, NCF2, NCF4	Components of NADPH oxidase	Excessive inflammatory responses due to altered inflammasome regulation by NADPH oxidase?	Oxidative Burst
(S)CID	>50 genes	various	Lack of pathogen control	Lymphocyte phenotyping
Wiskott-Aldrich syndrome	WAS	WASP	Lack of pathogen control Impaired cytoskeleton-inflammasome interaction?	WASP expression (FACS)
CD27 deficiency	CD27	CD27	Impaired co-stimulation of T cells Lack of EBV control	CD27 expression
ITK deficiency	ITK	ITK	Impaired TCR mediated signaling Lack of EBV control	ITK expression
IFNγ receptor deficiency	IFNGR1 IFNGR2	IFN-gamma receptor	Lack of pathogen control (mycobacteria, salmonella)	STAT1 phosphorylation
ALPS	FAS (het) FASLG	FAS FASLG	Defects in Fas ligand-mediated elimination of activated lymphocytes	TCR DNT Vitamin B12, soluble FasL
Autoinflammatory diseases with HLH as a frequent manifestation
NLRC4 gain of function	NLRC4 (het)	NLRC4	Constitutive inflammasome activation IL-1β/IL-18 production	Genetic testing
CDC42 mutations	CDC42 (het)	CDC42	Impaired cytoskeleton-inflammasome interaction?	Genetic testing

[Source ⁸⁷⁾ ]

Familial hemophagocytic lymphohistiocytosis type 2

Familial hemophagocytic lymphohistiocytosis type 2 accounts for 20 to 50% of all FHL cases, depending on the cohort studied ⁸⁸⁾, ⁸⁹⁾, ⁹⁰⁾. Mutations in the perforin gene (PRF1) are responsible for familial hemophagocytic lymphohistiocytosis type 2 ⁹¹⁾, ⁹²⁾, ⁹³⁾. Perforin (PRF1) gene has three exons: exon 2 and 3 code a 555 amino-acid polypeptide. Over 120 different mutations have been identified to date: 101 missense/non-sense mutations and 21 deletion/insertion mutations ⁹⁴⁾, ⁹⁵⁾, ⁹⁶⁾, ⁹⁷⁾, ⁹⁸⁾. Since the pathogenic role of some mutations, especially some single amino-acid substitutions, is difficult to assess, their functional characterization is still required ⁹⁹⁾. In a recent comprehensive in silico analysis of 76 missense mutations in PRF1, An et al. ¹⁰⁰⁾ used a structural approach to explain the effect of a mutation on the ability of perforin to oligomerize, thereby offering an explanation for the observed defect in cytotoxicity in familial hemophagocytic lymphohistiocytosis type 2 patients.

Accumulation of a sufficient number of cases helped to establish connections between specific mutations and particular ethnic groups: c.1122G > A (p.W374X) was found to have a high incidence in Turkish patients ¹⁰¹⁾; mutation c.50delT (p.L17FsX) is very frequent in patients of African-American origin ¹⁰²⁾; while the c.1090-1091delCT (p.L364fsX) mutation has only been identified in Japanese patients ¹⁰³⁾.

Mutation c.272C > T (p.A91V) has a particularly high frequency in Southern European population, ranging from 2.5 up to 10% ¹⁰⁴⁾, ¹⁰⁵⁾. It seems to be present at a very low frequency in African-American subjects and Sub-Saharan Africans, with no reported cases of the polymorphism in Japan, supporting the concept of a Mediterranean origin of the mutation. Although its impact on the protein structure and function has been documented ¹⁰⁶⁾, ¹⁰⁷⁾, its pathogenic role in FHL has been considered controversial. It is now clear that patients with Familial hemophagocytic lymphohistiocytosis type 2 frequently show A91V mutation in combination with another pathogenic mutation; association of A91V with another missense/hypomorphic mutation frequently results in a late-onset of familial hemophagocytic lymphohistiocytosis type 2 ¹⁰⁸⁾. Interestingly, in addition to the 106 mutations described in Familial hemophagocytic lymphohistiocytosis type 2 patients, other mutations have been associated with different phenotypes.

Familial hemophagocytic lymphohistiocytosis type 3

Familial hemophagocytic lymphohistiocytosis type 3 is caused by mutations in the UNC13D gene, which encodes the 1,090 amino-acids-long protein Munc13-4. Munc13-4 is involved in the priming of secretory granules and their fusion with the plasma membrane, and its loss-of-function consequently impairs the release of perforin and granzyme into the synaptic cleft ¹⁰⁹⁾.

Familial hemophagocytic lymphohistiocytosis type 3 covers between 30 and 40% of FHL patients, based on different geographic areas and ethnic groups ¹¹⁰⁾. The clinical picture of familial hemophagocytic lymphohistiocytosis type 3 patients is undistinguishable from that of familial hemophagocytic lymphohistiocytosis type 2 patients. However, evaluation of patient cytotoxic T lymphocyte (CTL) and NK cells by flow-cytometry showed a clear difference: while familial hemophagocytic lymphohistiocytosis type 2 patients have no perforin but show no degranulation defect, cells from familial hemophagocytic lymphohistiocytosis type 3 patients have normal perforin expression but reduced to absent degranulation ¹¹¹⁾.

UNC13D consists of 32 coding exons. To date at least 112 different mutations in UNC13D have been reported as a cause of Familial hemophagocytic lymphohistiocytosis type 3: 60 missense/non-sense, 25 splicing/regulatory, 25 deletion/insertion mutations, as well as 2 complex gene rearrangements ¹¹²⁾, ¹¹³⁾, ¹¹⁴⁾, ¹¹⁵⁾. After some initial difficulties in recognizing biallelic mutations in patients with familial hemophagocytic lymphohistiocytosis type 3, it became evident that not only exonic mutations, but also variations outside exons and splice sites are a common cause of familial hemophagocytic lymphohistiocytosis type 3 ¹¹⁶⁾. Since then, additional contribution confirmed this issue. In 2011, Meeths et al. ¹¹⁷⁾ described two novel mutations frequently occurring in Northern European populations: the deep intronic mutation c.118-308G > A selectively impairs UNC13D transcription in lymphocytes, thus abolishing Munc13-4 expression while the 253-kb inversion affects the 3′-end of the transcript, also abolishing Munc13-4 expression. Another deep intronic mutation, c.118-307G > A, was recently reported in a Chinese patient and documented to impair UNC13D transcription, possibly by disrupting a transcription factor binding-site or enhancer element ¹¹⁸⁾. Two mutations have been described in specific populations: the c.1596 + 1G > C mutation is described as the most common UNC13D mutation in Japan ¹¹⁹⁾ while mutation c.754-1G > C is predominantly found in Korean patients ¹²⁰⁾. Altogether, the mutation analysis of patients with familial hemophagocytic lymphohistiocytosis type 3 turned out to be far more engaging than that of patients with familial hemophagocytic lymphohistiocytosis type 2.

Recently in a genotype–phenotype study of 84 patients with familial hemophagocytic lymphohistiocytosis type 3 from Italy, Germany, and Sweden, Sieni et al. ¹²¹⁾ described that central nervous system (CNS) involvement is more common in patients with familial hemophagocytic lymphohistiocytosis type 3 than with familial hemophagocytic lymphohistiocytosis type 2. Moreover, the combination of fever, splenomegaly, thrombocytopenia, and hyperferritinemia appears to be the most easily and frequently recognized clinical pattern in familial hemophagocytic lymphohistiocytosis type 3, and in association with a defective granule release assay may lead to clinical suspicion of familial hemophagocytic lymphohistiocytosis type 3 ¹²²⁾.

While familial hemophagocytic lymphohistiocytosis type 2 and familial hemophagocytic lymphohistiocytosis type 3 subgroups account for the majority of patients with FHL, additional genetic subgroups have been progressively identified.

Familial hemophagocytic lymphohistiocytosis type 4

Familial hemophagocytic lymphohistiocytosis type 4 is caused by mutations in the STX11 gene ¹²³⁾. The STX11 gene consists of two exons and encodes the 287 amino-acid-long SNARE protein Syntaxin 11 (Stx11).

Although original reports of familial hemophagocytic lymphohistiocytosis type 4 were restricted to families of Turkish or Kurdish origin, more recently patients of different origins have been identified with a defect in STX11. Despite the initial clustering of cases, at least 12 different STX11 mutations have been described to date: 5 missense/non-sense mutations, 5 small deletions, 1 small deletion, and 1 gross deletion ¹²⁴⁾, ¹²⁵⁾, ¹²⁶⁾. Patients with familial hemophagocytic lymphohistiocytosis type 4 seem to have a later onset and a less severe clinical presentation of the disease compared to familial hemophagocytic lymphohistiocytosis type 2 and familial hemophagocytic lymphohistiocytosis type 3 ¹²⁷⁾. Mutations in STX11 have never been associated with variant phenotypes, different from familial hemophagocytic lymphohistiocytosis type 4.

Recently, Sepulveda et al. ¹²⁸⁾ tried to elucidate the role of STX11 mutations in the pathogenesis of FHL. They generated a Stx11-deficient (Stx11−/−) murine model that faithfully reproduced the manifestations of HLH and represented a suitable model for studying familial hemophagocytic lymphohistiocytosis type 4 in vivo and the role of Stx11 in vitro. By comparing the severity of HLH in Stx11−/− mice with that observed in Rab27a−/− and Prf1−/− mice, they established a correlation between the murine mutants and the age at HLH onset in their human counterparts ¹²⁹⁾. Furthermore, in a recent report the STX11 L58P mutation revealed that both the N-terminus and Habc domain of Stx11 are required for binding to Munc18-2, implying similarity to the dynamic binary binding of neuronal syntaxin 1 to Munc18-1 ¹³⁰⁾.

Familial hemophagocytic lymphohistiocytosis type 5

Familial hemophagocytic lymphohistiocytosis type 5 is due to mutations in STXBP2 (also named MUNC18-2) ¹³¹⁾, ¹³²⁾ and has been reported to account for up to 20% of cases with FHL in the German series ¹³³⁾. Since 2010, 40 different mutations of STXBP2 have been described: 15 missense/non-sense, 10 splicing/regulatory, and 15 deletion/insertion mutations ¹³⁴⁾, ¹³⁵⁾, ¹³⁶⁾, ¹³⁷⁾, ¹³⁸⁾. Familial hemophagocytic lymphohistiocytosis type 5 does not appear to be restricted to a specific geographic region.

In contrast to what was observed for the comparison between familial hemophagocytic lymphohistiocytosis type 2 and familial hemophagocytic lymphohistiocytosis type 3, some clinical presentations of familial hemophagocytic lymphohistiocytosis type 5 seem to be different from other classical manifestations of FHL. Gastrointestinal symptoms, such as chronic diarrhea, gastro-esophageal reflux, and abdominal pain are present in a significant number of patients. Renal tubular dysfunction was also observed in one patient. This could be explained by an impaired expression and function of Munc18-2/STXBP2 protein in cells other than cytotoxic lymphocytes, including intestinal and renal epithelium ¹³⁹⁾. The defect caused by insufficient function of STXBP2 protein in the neutrophils is associated with defective mobilization of the granules. As a result, the cell is unable to kill bacteria; insufficient clearance of E. coli might be one or the main reason for the frequency of gastrointestinal symptoms in patients with familial hemophagocytic lymphohistiocytosis type 5 ¹⁴⁰⁾, ¹⁴¹⁾, ¹⁴²⁾, ¹⁴³⁾.

Since platelets contain syntaxin-binding proteins with non-redundant functional roles, platelets from familial hemophagocytic lymphohistiocytosis type 5 patients have defective secretion, with decreased Munc18-2 and Stx11 levels. These data demonstrated a key role for Munc18-2, perhaps as a limiting factor, in platelet exocytosis, suggesting that it regulates Stx11 ¹⁴⁴⁾. These data together with those of Pagel et al. ¹⁴⁵⁾ suggest that, although bleeding histories may be too variable to be a sufficient diagnostic criteria, platelet function assays may be worth investigating in patients with FHL.

X-linked lymphoproliferative disease 1 (Duncan disease)

X-linked lymphoproliferative disease 1 (XLP-1) is a rare congenital immunodeficiency caused by mutations in SH2D1A (Xq25), the gene encoding the signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) ¹⁴⁶⁾, ¹⁴⁷⁾.

In immune-competent individuals Epstein–Barr virus (EBV) causes infectious mononucleosis, a common, usually self-limited disease. In X-linked lymphoproliferative disease 1 (XLP-1), the lack (or dysfunction) of SLAM-associated protein (SAP) causes the selective inability to control infection by EBV, a γ-herpes virus that infects B-cells ¹⁴⁸⁾, ¹⁴⁹⁾, ¹⁵⁰⁾, ¹⁵¹⁾. Several immunological defects have been identified, including defective NK and CD8+ T-cell-mediated cytolytic responses against EBV-infected cells, which lead to B-cell accumulation and persistence of reactive inflammatory responses ¹⁵²⁾. In the absence of SAP, 2B4 receptor (member of SLAM family), when engaged by its ligand CD48, delivers inhibitory instead of activating signals ¹⁵³⁾. It has been recently demonstrated that in X-linked lymphoproliferative disease 1 (XLP-1) NK cells the co-engagement of 2B4 with different activating receptors inhibits NCR, CD16, and activating KIRs, characterized by ITAM-dependent signaling pathways. In contrast, the 2B4 dysfunction does not affect the activity of DNAM-1 and NKG2D triggering receptors. Thus, while CD48+ B-EBV and lymphoma B-cells devoid of NKG2D and DNAM-1 ligands were resistant to lysis, the preferential usage of these receptors allowed XLP-1 NK cells to kill lymphomas that expressed sufficient amounts of the specific ligands ¹⁵⁴⁾. Better knowledge of the underlying dysfunction could be turned into a diagnostic tool. Patients with XLP-1 may present with different phenotypes: fulminant mononucleosis, B-cell lymphoma, lymphoproliferation, and dysgammaglobulinemia ¹⁵⁵⁾, ¹⁵⁶⁾, ¹⁵⁷⁾, but also with hemophagocytic lymphohistiocytosis ¹⁵⁸⁾. Thus, differential diagnosis is relevant. To this issue, immunological screening for intra-cytoplasmic SAP expression and rapid assays to examine 2B4 receptor function, which is inhibitory instead of activating in SH2D1A mutated patients ¹⁵⁹⁾, ¹⁶⁰⁾, may be applied.

In the literature, 100 SH2D1A mutations were found: 46 missense/non-sense, 14 splicing, 2 regulatory mutations, 9 small deletions, 6 small insertions, and 23 gross deletions ¹⁶¹⁾, ¹⁶²⁾. Interestingly, this gene is characterized by a high number of deletions including the entire gene. Intronic mutations have also been described affecting SH2D1A transcription but not mRNA splicing, and leading to markedly reduced level of SAP protein ¹⁶³⁾. Therefore, the strategy of mutation analysis of SH2D1A gene must be designed to include these possible variants.

X-linked lymphoproliferative syndrome type 2

A subset of patients with an X-linked lymphoproliferative-like phenotype was recently found to have mutations in BIRC4, the gene encoding the X-linked inhibitor of apoptosis protein (XIAP) and has been linked to another subgroup, named X-linked lymphoproliferative syndrome type 2 (XLP-2) ¹⁶⁴⁾.

X-linked inhibitor of apoptosis protein is an essential ubiquitin ligase for pro-inflammatory signaling downstream of the nucleotide-binding oligomerization domain containing (NOD)-1 and -2 pattern recognition receptors. Recently, the X-linked inhibitor of apoptosis protein (XIAP) baculovirus IAP repeat (BIR2) domain was recognized as a hotspot for missense mutations in X-linked lymphoproliferative syndrome type 2 (XLP-2). XLP-2-BIR2 mutations severely impair NOD-1/2-dependent immune signaling in primary cells from X-linked lymphoproliferative syndrome type 2 (XLP-2) patients and in reconstituted XIAP deficient cell lines. XLP-2-BIR2 mutations abolish the XIAP–RIPK2 interaction resulting in impaired ubiquitylation of RIPK2 and recruitment of linear ubiquitin chain assembly complex (LUBAC) to the NOD-2-complex. These new findings document that impaired immune signaling in response to NOD-1/2 stimulation is a general defect in X-linked lymphoproliferative syndrome type 2 (XLP-2) and demonstrate that the XIAP BIR2–RIPK2 interaction might be even targeted pharmacologically to modulate inflammatory signaling ¹⁶⁵⁾.

In a comparison of the clinical phenotypes associated with X-linked lymphoproliferative disease 1 (XLP-1) and X-linked lymphoproliferative syndrome type 2 (XLP-2), EBV infection was the common trigger of HLH in 92% of XLP-1 and 83% of XLP-2. HLH (XLP-1, 55%; XLP-2, 76%) and hypogammaglobulinemia (XLP-1, 67%; XLP-2, 33%) occurred in both groups, although with different proportions. Survival rates and mean ages at the first HLH episode did not differ for both groups, but HLH was more severe with lethal outcome in X-linked lymphoproliferative disease 1 (XLP-1) ¹⁶⁶⁾. Only X-linked lymphoproliferative disease 1 (XLP-1) patients developed lymphomas while X-linked lymphoproliferative syndrome type 2 (XLP-2) patients preferentially displayed chronic hemorrhagic colitis, recurrent splenomegaly often associated with cytopenia and fever ¹⁶⁷⁾.

In a similar study, Marsh et al. ¹⁶⁸⁾ reported an early disease onset during infancy for X-linked lymphoproliferative syndrome type 2 (XLP-2)-linked HLH and a high relapse rate; however, this seemed to occur even in the absence of an EBV infection. Some X-linked lymphoproliferative syndrome type 2 (XLP-2) patients develop hypo/dysgammaglobulinemia resulting from humoral immune system derangement. Intriguingly, and in contrast to X-linked lymphoproliferative disease 1 (XLP-1), XLP-2 was never associated with common variable immunodeficiency ¹⁶⁹⁾.

In a third consortium review of 25 patients ¹⁷⁰⁾, the majority initially presented with manifestations other than HLH, such as Crohn-like bowel disease (n = 6), severe infectious mononucleosis (n = 4), isolated splenomegaly (n = 3), uveitis (n = 1), periodic fever (n = 1), fistulating skin abscesses (n = 1), and severe Giardia enteritis (n = 1). Subsequent manifestations included celiac-like disease, antibody deficiency, splenomegaly, and partial HLH. Screening by flow-cytometry identified 14 of 17 patients in this cohort ¹⁷¹⁾. Given these clinical differences, XIAP deficiency must be considered in a wide range of clinical presentations. It has recently been suggested that XIAP deficiency would be better classified if defined as an X-linked subtype of FHL, rather than as a second type of X-linked lymphoproliferative disease ¹⁷²⁾.

To date, 41 mutations are known in BIRC4: 20 missense/non-sense, 2 splicing mutations, 2 regulatory mutations, 16 deletions/insertions, and 1 complex rearrangement ¹⁷³⁾, ¹⁷⁴⁾, ¹⁷⁵⁾, ¹⁷⁶⁾. The phenotypic differences may be the result of differences in the molecular basis of each disease. However, neither genotype, nor protein expression, nor results from cell death studies were clearly associated with the clinical phenotype.

Although HSCT remains the milestone for cure of FHL, some discrepancy recently emerged in the outcome of patients with X-linked lymphoproliferative syndrome type 2 (XLP-2). In an international survey of 19 patients, 7 received myeloablative (MAC) regimens, 1 received an intermediate-intensity regimen, and 11 received reduced intensity conditioning (RIC) regimens predominantly consisting of alemtuzumab, fludarabine, and melphalan. The probability of survival was very low in the MAC group, with all but one patient dying from transplantation-related toxicities (especially veno-occlusive disease and pulmonary hemorrhage); otherwise, 55% of those who received RIC survived at a median of 570 days after HSCT. The probability of surviving in the RIC was enhanced by disease inactivity at the time of HSCT. Based on these findings, MAC regimens should not be used for patients with XIAP deficiency. The reason may be connected with the loss of XIAP anti-apoptotic functions in X-linked lymphoproliferative syndrome type 2 (XLP-2) patients ¹⁷⁷⁾.

Immunodeficiencies associated with HLH and partial albinism

FHL associated with albinism is found as having abnormal lymphocyte cytotoxicity. Similarities were noted between molecular vesicle trafficking, especially pigment transport in the skin and hair. Moreover, the degranulation of platelets, mast cells, and perforin was shown to be impaired ¹⁷⁸⁾. At present three syndromes are known to cause HLH and manifest with partial albinism.

Chédiak–Higashi syndrome

Around 85% of Chédiak–Higashi syndrome patients develop HLH in the first decade of life. They show markedly defective cytotoxicity of both NK cells and CTL. The genetic defect is caused by mutations in the LYST gene (69), which encodes a 3,801 amino-acid protein. Each clinical manifestation of Chédiak–Higashi syndrome (albinism, bleeding tendency, recurrent bacterial infections, neurologic dysfunction, and HLH) (70, 71) is associated with a defect of a specific cell type and the formation of enlarged lysosomes in these cells.

The presence of giant inclusion bodies of lysosomal origin in a variety of granule-containing cells, including hematopoietic cells and melanocytes, has thus become the hallmark of the disease (72). This feature together with HLH and oculo-cutaneous albinism can address clinical suspicion toward Chédiak–Higashi syndrome and direct molecular analysis to LYST sequencing. In our revision of the available literature, a total of 56 mutations were found to be reported at the time of writing: 23 missense/non-sense, 4 splicing, 20 small deletions, 8 small insertions, and 1 gross deletion. As expected, disruptive mutations correlated with the severe form of the disease (73). The wider diffusion of mutation analysis, despite the big size of the gene, provided an increased number of reports from different geographic areas during the last few years, confirming that Chédiak–Higashi syndrome has no ethnic or geographic boundaries.

A rare neurologic disorder, named hereditary spastic paraplegia (HSP) and characterized by leg spasticity, weakness, hyperreflexia, and additional neurological symptoms, was recently reported in two adult siblings with hereditary spastic paraplegia and homozygous LYST pathogenic mutation. Large peroxidase-positive granules were observed in both patients’ granulocytes, while pigment deficiency, immune deficiency, and bleeding tendency were not observed. This example illustrates nicely how the clinical spectrum of Chédiak–Higashi syndrome may be much broader than recognized at present (74).

Griscelli syndrome type 2

Griscelli syndrome is a rare autosomal-recessive disorder that results in pigmentary dilution of the skin and hair, the presence of large clumps of pigment in hair shafts, and an accumulation of melanosomes in melanocytes and is characterized by partial oculo-cutaneous albinism and HLH ¹⁷⁹⁾. Among Griscelli syndrome subtypes, only patients with Griscelli syndrome type 2 (GS2) develop hemophagocytic lymphohistiocytosis (HLH) ¹⁸⁰⁾. Mutations causing Griscelli syndrome type 2 (GS2) were mapped to the RAB27A gene located on chromosome 15q15-21.1, which is composed of 5 coding exons and encodes for a 221 amino-acid protein that belongs to the superfamily of small Rab GTPase proteins ¹⁸¹⁾. Cytotoxic T lymphocytes (CTLs) and NK cell activity defect results from the inability of cytotoxic granules to dock to the plasma membrane whereas hypopigmentation is accounted for by a defective release of melanosomes from melanocyte dendrites ¹⁸²⁾. Change in phagosomal function and antigen cross-presentation of Rab27a-deficient dendritic cells has been reported in vitro. In the mouse model (ashen mice), the dendritic cells are unable to perform a sufficient antigen cross-presentation ¹⁸³⁾.

To date 34 RAB27A mutations are known: 15 missense/non-sense, 4 splicing, and 15 deletions/insertion. In 2010, Meeths et al. ¹⁸⁴⁾ sequenced RAB27A in patients diagnosed as HLH and found one mutated family. Since the clinical picture of the two syndromes is indistinguishable, they concluded that the diagnosis of Griscelli syndrome type 2 (GS2) may be overlooked, particularly in fair-haired patients with hemophagocytic syndromes ¹⁸⁵⁾.

Hermansky–Pudlak type 2

The term Hermansky-Pudlak syndrome encompasses nine different human autosomal-recessive genetic disorders, sharing partial oculo-cutaneous albinism and bleeding disorders (78, 79). Furthermore, patients with Hermansky–Pudlak type 2 (Hermansky-Pudlak syndrome type 2) also show an increased susceptibility to infections, resulting from both congenital neutropenia and impaired cytotoxic activity. Mutations of the gene encoding the β-3A subunit of adaptor protein-3 (AP-3) complex are the cause of Hermansky-Pudlak syndrome type 2 (80). To date, 20 mutations are known in this gene and are associated with Hermansky-Pudlak syndrome type 2: 7 missense/non-sense, 1 splicing, 11 deletions/insertion, 1 complex rearrangement, and a chromosome 5 inversion that disrupts the gene sequence (81). To date, only one Hermansky-Pudlak syndrome type 2 patient has been reported who developed HLH. However, since this patient also carried a potentially contributing heterozygous RAB27A mutation, the risk to develop HLH in Hermansky-Pudlak syndrome type 2 remains unclear (82). The pearl mouse model of Hermansky-Pudlak syndrome type 2, upon infection with lymphocytic choriomeningitis virus, developed all the key features of the disease, which yet was only transient (82). In a cohort of 22 Hermansky-Pudlak syndrome type 2 patients, only one additional patient with HLH was identified; two developed incomplete, transient HLH-like episodes, although the cytotoxicity or degranulation capacity was impaired in all 16 patients tested (83). Although future reports might clarify the genotype–phenotype correlations, the risk for HLH in Hermansky-Pudlak syndrome type 2 appears lower than in Griscelli or Chédiak–Higashi syndrome (79, 82).

Familial hemophagocytic lymphohistiocytosis causes

Familial hemophagocytic lymphohistiocytosis (F-HLH) or primary HLH is caused by changes or mutations of specific areas within a person’s genes. Genes provide instructions for creating proteins that play a critical role in many functions of the body. When a mutation of a gene occurs, the protein product may be faulty, inefficient, absent, or overproduced. Depending upon the functions of the particular protein, this can affect many organ systems of the body.

Several genes have been shown to be abnormal in different people with familial hemophagocytic lymphohistiocytosis (FHL) and researchers are actively working to discover these genes. At least four different genes (i.e., PRF1, UNC13D, STX11 and STXBP2) have been identified that result in a genetic predisposition to developing hemophagocytic lymphohistiocytosis. A genetic predisposition means a person has a gene or genes for a particular disorder, but the disorder will not develop unless other factors help to trigger the disorder. Because familial hemophagocytic lymphohistiocytosis manifests shortly after birth (usually in the first few weeks to months), and varies between siblings with identical mutations, it is thought that an infectious trigger is required for disease expression, even though an infectious trigger is usually not identified.

Interestingly, familial hemophagocytic lymphohistiocytosis may develop in utero ¹⁸⁶⁾, suggesting HLH-related mutations may be important in maintaining immune homeostasis in the absence of infection ¹⁸⁷⁾.

Primary hemophagocytic lymphohistiocytosis associated with lymphocyte cytotoxic defects:

• The genes that are associated with familial hemophagocytic lymphohistiocytosis (FHL) are ¹⁸⁸⁾, ¹⁸⁹⁾:
  • Unidentified gene defect located on chromosome 9 in Familial hemophagocytic lymphocytosis type 1,
  • Perforin (PRF1) gene in Familial hemophagocytic lymphocytosis type 2,
  • Munc-13–4 (UNC13D) in Familial hemophagocytic lymphocytosis type 3,
  • Syntaxin-11 (STX11) in Familial hemophagocytic lymphocytosis type 4,
  • Syntaxin binding protein 2 (STXBP-2) in Familial hemophagocytic lymphocytosis type 5.
  • SH2D1A gene in X-Linked Lymphoproliferative Disease 1 (XLP1)
  • RAB27A gene in Griscelli Syndrome type 2
  • LYST gene (also known as CHS1) in Chediak-Higashi syndrome
• Ethnic variations in causative mutations have also been reported, with 47% of white familial hemophagocytic lymphohistiocytosis (FHL) patients having mutations in UNC13D, 22% in STXBP2, and 20% in PRF1. In contrast, Hispanics and blacks were more likely to have PRF1 mutations (71% and 98%, respectively), versus Arabs, who had a 36% PRF1 mutation rate ¹⁹⁰⁾.

Primary hemophagocytic lymphohistiocytosis associated with abnormalities of inflammasome activation ¹⁹¹⁾:

• BIRC4 gene in XIAP deficiency
• NLR family CARD domain containing 4 protein (NLRC4) gene in familial cold autoinflammatory syndrome (FCAS)

Primary hemophagocytic lymphohistiocytosis associated with defined Mendelian disorders affecting inflammation

• SLC7A7 gene in Lysinuric protein intolerance
• Familial (apparently Mendelian) HLH of unknown origin

Identified causative mutations in familial hemophagocytic lymphohistiocytosis (F-HLH) result in impaired CD8+ T cell and NK cell cytotoxicity or altered lymphocyte activation and survival ¹⁹²⁾. Around 70% of familial hemophagocytic lymphohistiocytosis (F-HLH) is caused by the loss of function mutations in PRF1, the gene encoding perforin, a key cargo of cytolytic vesicles, or in UNC13D, encoding Munc13-4, which is critical for the release of cytolytic vesicles from T and NK cells ¹⁹³⁾. Mutations in other genes involved in cytolysis, such as STX11 and STXP2, are associated with familial hemophagocytic lymphohistiocytosis as well ¹⁹⁴⁾. Cytolytic dysfunction caused by abnormal granule exocytosis causes certain primary immunodeficiency (PID) syndromes associated with HLH, such as Chédiak-Higashi syndrome (CHS), Griscelli syndrome type 2 (GS2), and Hermansky-Pudlak syndrome type 2 (HPS2). Familial hemophagocytic lymphohistiocytosis type 1 to 5 develops within the first year of life ¹⁹⁵⁾.

Familial hemophagocytic lymphohistiocytosis inheritance pattern

Apart from the egg and sperm cells, each cell of your body normally has two working copies of each gene. One copy of each gene comes from your biological mother, and one comes from your biological father. Most of the time, familial hemophagocytic lymphohistiocytosis (F-HLH) or primary HLH is an autosomal recessive disorder. This means that for HLH to occur, both copies of the same HLH-associated genes must be mutated. In this situation, the parents are considered HLH “carriers” because they carry one normal and one altered copy of an HLH-associated gene in the cells of their bodies. Although they carry an altered gene copy, HLH carriers usually remain healthy and do not develop the signs and symptoms of HLH. However, they have a 50% chance of passing on the defective gene to their children.

Familial hemophagocytic lymphohistiocytosis autosomal recessive inheritance pattern means that a person must inherit two changed copies of the same gene (one abnormal gene from each parent) in order to have the condition. If a person inherits one abnormal gene and one normal gene, then in most cases that person will be a healthy carrier because the normal gene compensates for the abnormal gene. Being a carrier means that you do not have the condition, but carry a changed copy of the gene on one of a pair of genes.

• If both partners are carriers of the same abnormal gene, they may pass on either their normal gene or their abnormal gene to their child. This occurs randomly.
• Each child of parents who both carry the same abnormal gene therefore has a 25% (1 in 4) chance of inheriting a abnormal gene from both parents and being affected by the condition.
• This also means that there is a 75% ( 3 in 4) chance that a child will not be affected by the condition. This chance remains the same in every pregnancy and is the same for boys or girls.
• There is also a 50% (2 in 4) chance that the child will inherit just one copy of the abnormal gene from a parent. If this happens, then they will be healthy carriers like their parents.
• Lastly, there is a 25% (1 in 4) chance that the child will inherit both normal copies of the gene. In this case the child will not have the condition, and will not be a carrier.

These possible outcomes occur randomly. The chance remains the same in every pregnancy and is the same for boys and girls.

Figure 2 illustrates autosomal recessive inheritance. The example below shows what happens when both dad and mum is a carrier of the abnormal gene, there is only a 25% chance that they will both pass down the abnormal gene to the baby, thus causing a genetic condition.

Key points to remember

• A person must inherit two copies of a abnormal gene, one from each parent, in order to be affected by the condition (25% chance). If a person inherits only one abnormal gene then they will be a carrier (50% chance). These outcomes occur randomly. They remain the same in every pregnancy and are the same for boys and girls.
• A abnormal gene cannot be corrected – it is present for life.
• A abnormal gene is not something that can be caught from other people. They can still be a blood donor, for example.
• People often feel guilty about a genetic condition which runs in the family. It is important to remember that it is no-one’s fault and no-one has done anything to cause it to happen.

Figure 2. Familial hemophagocytic lymphohistiocytosis autosomal recessive inheritance pattern

Familial hemophagocytic lymphohistiocytosis pathophysiology

The pathogenesis of HLH has mostly been studied in familial hemophagocytic lymphohistiocytosis (FHL). An inherited defect in the perforin/granzyme pathway or in the fusion of cytotoxic lytic granules with the surface of natural killer (NK) cells causes, in the presence of an external trigger, an over-response by cytotoxic CD8+ T lymphocytes ¹⁹⁶⁾. Viral infections are the most common triggers ¹⁹⁷⁾.

Cytotoxic CD8+ T cells produce large amounts of interferon gamma (IFN-γ), which in turn activates macrophages ¹⁹⁸⁾. Overstimulated macrophages release large amounts of inflammatory cytokines, such as interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-12 (IL-12), interleukin-18 (IL-18) and tumor necrosis factor alpha (TNFα). There is also an increased production of interleukin-10 (IL-10), with inhibitory activity, but not sufficient to limit the phenomenon ¹⁹⁹⁾. IL-12 and IL-18 produced by macrophages in turn stimulate CD8+ T cells, amplifying the inflammatory response ²⁰⁰⁾. The resulting tissue damage causes a release of interleukin-33 (IL-33) and IL-1β, which further activates the macrophages. Activated macrophages engulf blood cells and produce large amounts of ferritin. The ‘cytokine storm’ causes all the clinical manifestations of HLH, from endothelial damage to coagulopathy and multi-organ failure ²⁰¹⁾, ²⁰²⁾, ²⁰³⁾, ²⁰⁴⁾, ²⁰⁵⁾. The central role of interferon gamma (IFN-γ) in the pathogenesis of familial hemophagocytic lymphohistiocytosis has been demonstrated in a perforin-deficient mouse model ²⁰⁶⁾.

A similar pathogenesis of HLH can also be observed in patients with primary immunodeficiencies involving granule trafficking or exocytosis, such as Hermansky-Pudlak syndrome type 2, Griscelli syndrome type 2 and Chediak-Higashi syndrome 2, all with reduced cytotoxic T lymphocytes (CTL) cytotoxicity ²⁰⁷⁾, ²⁰⁸⁾. Advances in genetic diagnosis suggest that cell killing by cytotoxic T lymphocytes (CTLs) and NK cells can be affected from mildly to severely, thus explaining the different HLH phenotypes as a continuum ²⁰⁹⁾. The known mutations in genes related to granule-mediated killing account for familial hemophagocytic lymphohistiocytosis (FHL) and primary forms in general ²¹⁰⁾. Minor alterations of the cytotoxic T lymphocytes (CTL) and NK cells activity, together with an external trigger, account for secondary HLH, severe sepsis, multi-organ failure and HLH in the context of rheumatologic diseases ²¹¹⁾.

In all cases, a cytokine storm causing a devastating inflammation is the primal agent of the multiorgan failure, regardless of the underlying defect ²¹²⁾, ²¹³⁾, ²¹⁴⁾, ²¹⁵⁾, ²¹⁶⁾, ²¹⁷⁾ and HLH should be considered a clinical syndrome of hyperinflammation with different phenotypes ²¹⁸⁾. Moreover, this finding likely explains the numerous similarities and overlaps between HLH and other systemic inflammatory syndromes, such as septic shock, cytokine release syndrome following viral infections ²¹⁹⁾ and acute liver failure ²²⁰⁾.

The case of malignancy-associated HLH includes two completely heterogeneous mechanisms. Firstly, when HLH is a manifestation of the underlying malignancy, particularly in the case of lymphomas, the overproduction of inflammatory cytokines such as INF-γ and TNF-α is likely caused by neoplastic cells ²²¹⁾, ²²²⁾. Secondly, when HLH develops as the consequence of chemotherapy, the process is more likely to be caused by the association of drug-induced cytotoxic T lymphocytes (CTL) suppression and infection ²²³⁾.

Figure 3. Familial hemophagocytic lymphohistiocytosis pathophysiology

Footnotes: (A) Impaired cytotoxicity (red) leads to uncontrolled T cell activation by antigen-presenting cell (APC). T cell secreted IFN-gamma (IFN-γ) is the key driver of macrophage activation. Cellular and cytokine targets of therapy are indicated in green. (B) Pathogenesis of HLH in the context of impaired inflammasome homeostasis (simplified). Inborn errors of immunity shown (NLRC4, XIAP) or assumed (Cytoskeletal disorders, CGD) to be involved in inflammasome homeostasis are indicated in red. Cytokine targets of therapy are indicated in green. Macrophages are the key cells involved, T cells play a less prominent role. Cell-directed therapies are rarely used. (C) HLH pathogenesis in the context of inborn errors of immunity with impaired pathogen control (simplified). Inborn errors of immunity impairing virus control (mainly EBV) and/or bacterial/fungal control are indicated in red. Subsequent immune stimulation leads to hypersecretion of variable cytokines. Macrophage activation can occur in the absence of T cells, but T cells can be involved depending on the genetic defect and the trigger. Cell-directed therapies further impair pathogen control and should only be used in exceptional cases.

[Source ²²⁴⁾ ]

Familial hemophagocytic lymphohistiocytosis signs and symptoms

The onset and severity of hemophagocytic lymphohistiocytosis can vary greatly from one person to another ²²⁵⁾. The specific symptoms that develop can also vary greatly, although HLH often causes multiorgan involvement. Generally, affected individuals develop fevers, a rash, an abnormally large liver (hepatomegaly), and an abnormally large spleen (splenomegaly). Fevers of unknown origin may be prolonged and persistent, often failing to respond to antibiotics ²²⁶⁾. Sometimes, the lymph nodes are also abnormally large (lymphadenopathy). Lymph nodes are part of your lymphatic system, a circulatory network of vessels, ducts, and nodes that filter and distribute certain protein-rich (lymph) and blood cells throughout the body. Lymph nodes are small structures, found in groups throughout the body, that help to filter or drain out harmful substances from the body.

These initial sign and symptoms are described as nonspecific. This means that these signs and symptoms are common to many other different disorders or conditions, which can make getting a correct diagnosis difficult.

Affected individuals may also have low levels circulating red blood cells (anemia) and low levels of circulating platelets (thrombocytopenia). Red blood cells deliver oxygen to the body and platelets allow the body to form clots to stop bleeding. Individuals with anemia may experience tiredness, increased need for sleep, weakness, lightheadedness, dizziness, irritability, headaches, pale skin color, difficulty breathing (dyspnea), and cardiac symptoms. Individuals with thrombocytopenia (low levels of circulating platelets) are more susceptible to excessive bruising following minimal injury and to spontaneous bleeding from the mucous membranes, especially those of the gums and nose.

Some affected individuals may develop neurological symptoms including seizures, changes in mental status and irritability, paralysis (palsy) of certain cranial nerves, and problems coordinating voluntary movements (ataxia). Affected individuals are at risk of developing posterior reversible encephalopathy syndrome, which causes a rapid onset of headaches, altered consciousness, seizures, and disturbances in vision. Neurological problems are most common with familial hemophagocytic lymphohistiocytosis.

Additional symptoms can occur depending upon the specific organ system involved in an individual. These symptoms can include significant problems breathing (lung dysfunction), severe low blood pressure (hypotension), liver inflammation (hepatitis), kidney dysfunction, yellowing of the skin and whites of the eyes (jaundice), swelling due to fluid accumulation (edema), abdominal swelling due to fluid accumulation (ascites), and a variety of skin problems including widespread, reddening of the skin because of inflammation (erythroderma), rashes, blood spots (purpura), and tiny spots on the skin due to bleeding under the skin (petechiae).

Infant with fever

While “classic” familial hemophagocytic lymphohistiocytosis may present at almost any age, it most commonly presents in infants under 1 year of age, and neonatal or in utero presentations are well described ²²⁷⁾. This “typical” patient with HLH usually presents with fever and is then noted to have cytopenias and splenomegaly. Further investigation reveals an illappearing patient, sometimes with a critical sepsis-like appearance. Marrow biopsy is often obtained to evaluate for leukemia and may (variably) reveal hemophagocytosis, while not displaying malignancy. Though infection and malignancy must be carefully considered, a triggering infection may not be identified ²²⁸⁾. While these patients are well described by current diagnostic criteria, their most striking features are typically the degree of cytopenias, hypofibrinogenemia (in the face of inflammation), and hepatosplenomegaly. This patient population is capable of sudden, severe, and unpredictable worsening, necessitating rapid diagnosis and aggressive intervention.

Older children with infection

Children over the age of 1 year with hemophagocytic lymphohistiocytosis typically have definable triggers, most commonly viral infection. Epstein-Barr virus (EBV) is the most frequently identified infection triggering HLH, followed by cytomegalovirus (CMV), though numerous other viral, bacterial, parasitic, and fungal infections have been reported ²²⁹⁾, ²³⁰⁾, ²³¹⁾. Though underlying genetic mutations may not be found in patients with EBV-HLH, disease is often responsive to standard treatments and rituximab may be a helpful adjunct, as it may decrease viral burden ²³²⁾. EBV-associated HLH is usually seen with primary infection, though some patients may present with HLH (or HLH recurrence) associated with chronic active EBV infection ²³³⁾. These patients typically have predominant EBV infection of T cells (less often NK cells), instead of B cells. Overall, clinical presentations in older patients are similar to those in younger patients, though they may vary widely in severity. While patients presenting with relatively milder acuity may only require corticosteroids or intravenous immune globulin, aggressive treatment may be appropriate, as early initiation of etoposide is associated with improved survival in severe EBV-HLH ²³⁴⁾, ²³⁵⁾, ²³⁶⁾. Ferritin values are consistently high in older patients, and sCD25 is always elevated, though often not to the degree seen in infants.

Central nervous system involvement

Patients with familial hemophagocytic lymphohistiocytosis present with central nervous system (CNS) involvement about one-third of the time with symptoms including seizures, meningismus, signs of cranial nerve involvement, ataxia, dysarthria, and encephalopathy ²³⁷⁾, ²³⁸⁾. However, patients may also rarely present with isolated central nervous system (CNS) inflammation, without complete or even any systemic signs of HLH. Though rare, this isolated disease is an important forme fruste of HLH, which often presents insidiously with patients experiencing substantial delays in diagnosis ²³⁹⁾. Cerebrospinal fluid analysis shows pleocytosis and hyperproteinemia, and brain biopsy reveals T-cell and histiocytic infiltrates. MRI findings in isolated or non-isolated CNS HLH typically include increased signal in T2-weighted images of widespread areas, with symmetric periventricular white matter hyperintensity, meningeal enhancement, diffusion restriction, and may progress to include necrosis ²⁴⁰⁾, ²⁴¹⁾, ²⁴²⁾. Age of onset of isolated CNS HLH varies greatly, but is most often seen in children over a year of age, as younger children will usually have accompanying systemic HLH ²⁴³⁾.

Familial hemophagocytic lymphohistiocytosis diagnosis

Diagnosing familial hemophagocytic lymphohistiocytosis can be challenging because many of its initial symptoms mimic other common conditions. Symptoms such as persistent fevers, respiratory issues, rash, anemia, seizures and enlarged liver, spleen or lymph nodes may be cause other conditions.

Familial hemophagocytic lymphohistiocytosis diagnosis is based upon identification of characteristic symptoms, a detailed patient history, a thorough clinical evaluation and a variety of specialized tests. Guidelines have been published that detail the criteria necessary for a diagnosis of hemophagocytic lymphohistiocytosis (see Table 1).

Familial hemophagocytic lymphohistiocytosis is diagnosed when an individual meets established HLH-2004 diagnostic criteria ²⁴⁴⁾. HLH-2004 criteria for diagnosis of HLH requires either 1 or 2.

1. The presence of a known HLH-causing genetic mutation, OR
2. Has five or more of the clinical or laboratory findings listed below:
   1. High and often prolonged periods of fever (more than 7 days);
   2. Rash, irritability and/or seizures;
   3. An abnormally large spleen (splenomegaly);
   4. Low blood cells (cytopenias): red blood cells (hemoglobin less than 9 g/dL, hemoglobin less than 10 g/dL in infants <4 week), white blood cells (neutrophils less than 1 × 10⁹/L), or platelets (platelets count less than 100 × 10⁹/L);
   5. Abnormally high levels of a type of fat called a triglyceride in the blood (hypertriglyceridemia) triglyceride ≥ 3.0 mmol/L (≥ 265 mg/dl);
   6. Abnormally low levels of fibrinogen (a specific blood clotting protein) (hypofibrinogenemia) serum fibrinogen < 150 mg/dL;
   7. Destruction of blood cells by macrophages (hemophagocytosis) in the bone marrow, spleen, or lymph nodes and/or cerebrospinal fluid with no evidence of malignancy;
   8. Low or absent natural killer (NK) cell activity;
   9. Abnormal high levels of ferritin (a protein that binds to iron) in the blood (ferritin ≥ 500mcg/L); and
   10. Elevated soluble interleukin-2 receptor alpha chain (sCD25 ≥ 2400 U/mL), a specialized protein that builds up in the blood when the immune system is stimulated.
   11. Reduced expression of proteins such as perforin, signaling lymphocytic activation molecule-associated protein (SAP), X-linked inhibitor of apoptosis protein (XIAP), and/or depressed cell functions

On occasion, HLH may be strongly considered, and HLH-directed therapy may be initiated, even though the 5 HLH-2004 diagnostic criteria are not fulfilled ²⁴⁵⁾.

If hemophagocytic activity is not proven at the time of presentation, further search for hemophagocytic activity is encouraged. If the bone marrow specimen is not conclusive, material may be obtained from other organs. Serial marrow aspirates over time may also be helpful. The following findings may provide strong supportive evidence for the diagnosis: spinal fluid pleocytosis (mononuclear cells) and/or elevated spinal fluid protein and histological picture in the liver resembling chronic persistent hepatitis (biopsy). Other abnormal clinical and laboratory findings consistent with the diagnosis are cerebromeningeal symptoms, lymph node enlargement, jaundice, edema, skin rash, hepatic enzyme abnormalities, hypoproteinemia, hyponatremia, and elevated very low-density lipoprotein (VLDL↑)/low high-density lipoprotein (HDL↓).

Figure 3. Hemophagocytic lymphohistiocytosis diagnostic algorithm

[Source ²⁴⁶⁾ ]

Table 2. Hemophagocytic lymphohistiocytosis diagnostic criteria

HLH2004 entry criteria	Comment
A. Molecular diagnosis consistent with HLH: Pathologic mutations of PRF1, UNC13D, STXBP2, Rab27a, STX11, SH2D1A, or XIAP or	In a patient with known genetic defects, treatment before full development of HLH may be appropriate, but genetic studies usually just help to define HLH recurrence risk, not the presence of an active disease state.
B. Five of the eight criteria listed below are fulfilled
1. Fever ≥38.3°C	Nearly universal in untreated HLH.
2. Splenomegaly	While splenomegaly and hepatomegaly are very common in HLH, adenopathy is not.
3. Cytopenias (affecting at least two of three lineages in the peripheral blood): Hemoglobin <9 g/dL (in infants <4 weeks: hemoglobin <10 g/dL) Platelets <100 × 10³/mL Neutrophils < 1 × 10³/mL	Cytopenias are ubiquitous in HLH. Lack of cytopenias should make one doubt a diagnosis of HLH, except in the special case of isolated, CNS-only disease.
4. Hypertriglyceridemia (>265 mg/dL) and/or hypofibrinogenemia (<150 mg/dL)	Low fibrinogen in the context of inflammation is paradoxical and one of the more distinctive features of HLH.
5. Hemophagocytosis in bone marrow or spleen or lymph nodes or liver	Not specific to HLH, or essential for the diagnosis, but helpful as a disease marker. Of note, it is often not evident early after disease onset.
6. Low or absent NK-cell activity	More modern and robust assays measuring perforin levels and its degranulation should replace this assay for reliable diagnosis of HLH. This assay is not specific for primary HLH.
7. Ferritin >500 ng/mL	Most patients have much higher levels than this threshold suggests.
8. Elevated Soluble CD25 (soluble IL-2 receptor alpha)	As HLH is a T-cell driven disease, this assay is extremely informative for diagnosis and response to therapy. See Clinical Testing and Workup for patients with suspected HLH disease for more information.

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Clinical testing and workup for patients with suspected HLH disease

The initial workup is two-pronged and aims (a) to establish the diagnosis of HLH promptly and (b) to identify mimickers of HLH disease (if present), or potentially treatable underlying HLH disease triggers.

Common laboratory studies

• Complete blood count (CBC), hepatic panel, fibrinogen, triglycerides. Essential to define typical features of HLH. Though not part of diagnostic criteria, hepatitis is extremely common in HLH.
• Ferritin. Usually (but not always) very elevated
• Search for triggering infection with cultures, viral PCRs: EBV, CMV, adenovirus

Specialized laboratory studies

• Markers of immune activation: soluble interleukin-2 receptor alpha chain (sCD25), granzyme B expression and CXCL9. Essentially always elevated in untreated HLH; very sensitive, but not specific. sCD25, and perhaps CXCL9, are also useful for monitoring response to therapy
• Interlukin-18 (IL-18) level. Elevated in HLH associated with inflammasome-opathies (XIAP, NLRC4, systemic onset juvenile arthritis)
• Measurement of proteins affected in familial HLH: perforin, SAP, XIAP. Helpful to quickly confirm a suspected familial HLH diagnosis
• Functional studies: lymphocyte degranulation (CD107a mobilization), NK cell function. May help to fulfill diagnostic criteria. Degranulation is the preferred functional assay over NK cell function ²⁴⁸⁾.
• Lymphocyte subsets, T-cell subsets, immune globulin levels. Screening studies for primary immune deficiency
• Genetic testing (multigene panel or whole exome). Essential for defining HLH recurrence risk

Imaging studies (should be routinely performed to help rule out malignancy, or unusual infections if trigger is unknown)

• Body cavity CT’s (chest/abdomen/pelvis)
• Consider 18F-fluorodeoxyglucose (FDG) positron emission tomography-computed tomography (PET-CT) if suspicion of lymphoma
• MRI of brain. Brain MRI complements lumbar puncture for central nervous system (CNS) assessment

Tissue sampling (essential to rule out malignancy, identify hemophagocytosis, and identify central nervous system (CNS) involvement)

• Bone marrow biopsy
• Lumbar puncture
• Other tissue biopsies as appropriate: liver, lymph node, masses

Physicians may order blood tests to take a complete blood cell count, which will measure the levels of red cells, white cells and platelets. Blood tests can also reveal abnormally high ferritin levels, or abnormal high levels of triglycerides. Physicians may also use blood tests to look for signs of infection in the blood and conduct tests to determine how well the blood clots (coagulation studies). Physicians may also order tests that can assess the health and function of the liver.

Sometimes, a bone marrow biopsy (the surgical removal and microscopic examination of a tissue sample) may be taken and studied for signs of hemophagocytosis, signs of infection or infectious organisms, and the accumulation of macrophages.

Molecular genetic testing can confirm a diagnosis of hemophagocytic lymphohistiocytosis in certain people. Molecular genetic testing can detect mutations in one of the four specific genes known to cause familial forms of this disorder, but is available only as a diagnostic service at specialized laboratories.

It should be emphasized that since T-cell activation is central to HLH pathogenesis, elevated soluble IL2 receptor alpha chain (sCD25) should always be observed in untreated HLH ²⁴⁹⁾, ²⁵⁰⁾, ²⁵¹⁾. If sCD25 is not elevated, then one should doubt a diagnosis of HLH ²⁵²⁾. Similarly, though not as well established because HLH appears to be largely driven by interferon gamma (IFN-γ), elevations of CXCL9 (a sensitive indicator of IFN-γ bioactivity) should be seen in untreated cases of HLH disease ²⁵³⁾, ²⁵⁴⁾, ²⁵⁵⁾. Additionally, elevated expression of granzyme B in NK cells has been shown to be similarly ubiquitous in HLH; normal expression levels would suggest that HLH is not the correct diagnosis ²⁵⁶⁾. Ferritin levels above 10,000 ng/mL appear to be relatively specific for HLH but are not very sensitive ²⁵⁷⁾. While specialized immunologic testing may facilitate diagnosis, if a diagnosis can be made without them then treatment should not be delayed pending these results. Likewise, treatment should not be delayed for assessment of central nervous system (CNS) involvement, though this should always be conducted (once a lumbar puncture may be safely performed).

Practical examples of when to not diagnose HLH disease

Certain clinical features may suggest mimics of HLH disease ²⁵⁸⁾:

• Despite the common presence of splenomegaly, prominent or abnormal lymphadenopathy is not typically seen in HLH. This finding strongly suggests lymphoma, Castleman disease, or unusual infection such as HIV, histoplasmosis or mycobacterial disease ²⁵⁹⁾. Similarly, leukocytosis is not typical of HLH (except in R-HLH/MAS) and should prompt a search for an alternative diagnosis.
• An extremely elevated sCD25 (>10- to 20-fold above normal) in a noninfantile patient suggests undiagnosed lymphoma, especially when ferritin is not similarly elevated ²⁶⁰⁾, ²⁶¹⁾.
• The early occurrence of isolated and aggressive “CNS relapse” during treatment of systemic HLH may suggest an undiagnosed malignant disorder relapsing in the CNS.
• A normal or only modestly elevated sCD25, despite extremely elevated ferritin, suggests disseminated infection in the context of primary immune deficiency, especially in an infant ²⁶²⁾.

Other diagnostic tools

Other features supporting an HLH diagnosis that are not part of the HLH-2004 diagnostic criteria include hyperbilirubinemia, hepatomegaly, transaminitis (present in the vast majority of patients with HLH), and elevated lactate dehydrogenase and d-dimer levels, with the latter usually elevated even when international normalized ratio, partial thromboplastin time, and fibrinogen are normal ²⁶³⁾. These findings may help to discriminate HLH from septic shock and conditions such as autoimmune hemolytic anemia, and they are also useful in assessing response to therapy.

H Score

The HLH-probability calculator (HScore), with graded clinical and laboratory parameters, is a Web-based online calculator developed retrospectively in adult patients that may be a helpful diagnostic tool (Table 3) ²⁶⁴⁾. The pattern of inflammatory cytokines (elevated levels of interferon-γ [IFN-γ] and IL-10, with only modestly elevated IL-6 levels) has high diagnostic accuracy for secondary hemophagocytic lymphohistiocytosis and may be a useful approach to differentiate HLH from infection and to monitor patients; however, the utility of this pattern of changes needs to be verified in children and adults outside of China ²⁶⁵⁾, ²⁶⁶⁾, ²⁶⁷⁾.

Table 3. Parameters and points in the HScore

Parameter	No. of points (criteria for scoring)
Known underlying immunosuppression*	0 (no) or 18 (yes)
Temperature (°C)	0 (<38.4), 33 (38.4–39.4), or 49 (>39.4)
Organomegaly	0 (no), 23 (hepatomegaly or splenomegaly), or 38 (hepatomegaly and splenomegaly)
No. of cytopenias†	0 (1 lineage), 24 (2 lineages), or 34 (3 lineages)
Ferritin (μg/L)	0 (<2000), 35 (2000-6000), or 50 (>6000)
Triglyceride (mmol/L)	0 (<1.5), 44 (1.5-4), or 64 (>4)
Fibrinogen (g/L)	0 (>2.5) or 30 (≤2.5)
Aspartate aminotransferase (U/L)	0 (<30) or 19 (≥30)
Hemophagocytosis on bone marrow aspirate	0 (no) or 35 (yes)

Footnote:

* HIV positive or receiving long-term immunosuppressive therapy (ie, glucocorticoids, cyclosporine A, azathioprine).
† Defined as a hemoglobin level of 9.2 g/L and/or a leukocyte count ≤5 × 10⁹/L and/or a platelet count ≤110 × 10⁹/L.

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Familial hemophagocytic lymphohistiocytosis treatment

The treatment of hemophagocytic lymphohistiocytosis is directed toward the specific symptoms that are apparent in each individual. Treatment may require the coordinated efforts of a team of specialists. Pediatricians, specialists in diagnosing and treating blood disorders (hematologists), specialists in diagnosing and treating cancer (oncologists), specialists in diagnosing and treating immune system diseases (immunologists), geneticists (for familial forms), social workers, and other healthcare professionals may need to systematically and comprehensively plan treatment. Psychosocial support for the entire family is essential as well. Genetic counseling may be of benefit for affected individuals and their families. At most institutions, the treatment of children with familial hemophagocytic lymphohistiocytosis is managed by hematologists.

The aim of hemophagocytic lymphohistiocytosis treatment is to suppress the life-threatening inflammation that leads to organ damage ²⁶⁹⁾. Specific therapeutic procedures and interventions may vary, depending upon numerous factors, such as the underlying cause; the presence or absence of certain symptoms; the overall severity of the symptoms and the disorder; an individual’s age and general health; and/or other elements. Decisions concerning the use of particular drug regimens and/or other treatments should be made by physicians and other members of the health care team in careful consultation with the patient based upon the specifics of his or her case; a thorough discussion of the potential benefits and risks, including possible side effects and long-term effects; patient preference; and other appropriate factors.

For children with familial hemophagocytic lymphohistiocytosis, the first step in treatment is generally to suppress the overactive immune system. This is often done using a combination of steroids and chemotherapy, with the goal of putting the disease into remission. The Histiocyte Society first published international treatment protocol for HLH in 1994 (HLH-94), which combined chemotherapy and immunotherapy (etoposide and cyclosporine A [CsA]) for 8 weeks with or without intrathecal methotrexate in select patients to control the inflammatory response as a bridge to hematopoietic stem cell transplantation (HSCT) ²⁷⁰⁾, ²⁷¹⁾. The HLH-94 protocol was reserved for patients with familial, relapsing, or severe/persistent HLH ²⁷²⁾. HLH-94 dramatically improved survival in familial hemophagocytic lymphohistiocytosis from an estimated 5-year rate of 22% to an estimated 3-year rate of 51% ±20 ²⁷³⁾. The HLH-94 protocol (minus HSCT) was also found to be beneficial in secondary forms of HLH ²⁷⁴⁾. For this reason, HLH-94 is often recommended by oncologists for the treatment of nonfamilial forms of HLH that are severe or relapsing ²⁷⁵⁾.

Based on experience with HLH-94, the Histiocyte Society in 2004 modified the protocol (HLH-2004) in several ways, most notably by adding early and continuous cyclosporine A (CsA) therapy ²⁷⁶⁾. During therapy with either protocol (HLH-94 or HLH-2004), patients should be monitored closely for clinical and laboratory response, and relapses should be expected as steroids are weaned (around week 6 in HLH-94) ²⁷⁷⁾. HSCT remains the only long-term curative treatment, and achieving disease remission of HLH prior to transplant is essential for good prognosis, because HSCT complications (infection, excessive bleeding, organ failure, graft rejection, and graft versus host disease) and mortality are associated with HLH disease activity ²⁷⁸⁾.

The HLH-94 induction chemotherapy is based on steroids and etoposide administration for 8 weeks with or without intrathecal methotrexate, followed by maintenance with cyclosporine A (CSA), etoposide and dexamethasone pulses while waiting for allogeneic hematopoietic stem cell transplantation (allo-HSCT) ²⁷⁹⁾, ²⁸⁰⁾. Chemotherapy might be suspended after 8 weeks of treatment once clinical remission is obtained and a clear genetic base is excluded ²⁸¹⁾. Otherwise, if a clear genetic base is proved, or in case of reactivation, hematopoietic stem cell transplantation (HSCT) is recommended ²⁸²⁾.

After initial treatment, children with familial hemophagocytic lymphohistiocytosis usually undergo an allogeneic stem cell transplant (allo-HSCT), which replaces their defective immune system with a healthy one from a donor. Donor cells may come from a sibling, a parent or an unrelated donor. A stem cell transplant offers familial hemophagocytic lymphohistiocytosis patients the best chance for a cure ²⁸³⁾, ²⁸⁴⁾.

For disease refractory to HLH-94/2004, an anti-CD52 monoclonal antibody called alemtuzumab (which depletes T and B cells) has shown effectiveness as a salvage therapy ²⁸⁵⁾, ²⁸⁶⁾. Salvage therapy should be directed by oncologists and hematologists ²⁸⁷⁾.

Figure 4. HLH-94 protocol

Footnotes: The HLH-94 protocol is based on immunochemotherapy including dexamethasone, etoposide, and cyclosporine A (CSA). After an intensive phase of 2 weeks with high doses of dexamethasone and twice weekly administration of etoposide, dexamethasone is tapered until week nine. Cyclosporine A (CSA) is used from week three onward to prevent reactivation. Intrathecal therapy with methotrexate is recommended in patients with central nervous system (CNS) involvement. Immunological testing and genetic confirmation of the underlying genetic disease is required in all patients and should provide the basis for hematopoietic stem cell transplantation (HSCT) within 8 weeks.

[Source ²⁸⁸⁾ ]

HLH-94 protocol

2018 consensus statements by the HLH Steering Committee of the Histiocyte Society recommending the use of HLH-94. The HLH-94 protocol is based on immuno-chemotherapy including dexamethasone, etoposide and cyclosporine A to achieve remission of the hyperinflammatory state and to maintain remission until hematopoietic stem cell transplantation (HSCT) can be performed ²⁸⁹⁾. Functionally, all agents target lymphocytes, macrophages and antigen presenting cells. The cytostatic agent etoposide induces cell death mainly in activated T cells ²⁹⁰⁾, but also in macrophages and dendritic cells. The use of the calcineurin inhibitor cyclosporine A leads to an inhibition of the transcription factor NFAT (nuclear factor of activated T-cells) and thus to a reduced activation and proliferation of T cells. Steroids slow down inflammation by reducing cytokine secretion, but in addition, they have a moderate cytotoxic effect on activated T cells. It is recommended to treat patients with central nervous system (CNS) involvement also with intrathecal methotrexate ²⁹¹⁾, although there is no clear evidence of benefit. The protocol has a 2-week intensive phase with dexamethasone and twice weekly administration of etoposide, followed by 6 weeks of weekly etoposide and steroid tapering. In this second phase, cyclosporine A is used to prevent reactivation ²⁹²⁾. Rapid immunological testing followed by genetic confirmation of the underlying genetic disease is required in all patients and should provide the basis for hematopoietic stem cell transplantation (HSCT) within these 8 weeks. In the international multicenter registry-based HLH-2004 study, 5-year probability of survival for children with genetically verified familial HLH treated with this protocol was 59% ²⁹³⁾. Dexamethasone/etoposide-based protocols have been successfully used in X-linked lymphoproliferative disorder or patients with TIM3 deficiency. It remains an ultimate choice also in other inborn errors of immunity, but the toxicity and immunosuppression associated with etoposide asks for more targeted therapies in these conditions.

Hematopoietic Stem Cell Transplantation (HSCT)

To prevent recurrences, allogenic stem cell transplantation (HSCT) should be carried out as soon as possible after achieving initial remission in the “primary” HLH ²⁹⁴⁾, ²⁹⁵⁾. Hematopoietic stem cell transplantation (HSCT) remains the only curative option for familial hemophagocytic lymphohistiocytosis. The timing of HSCT has to balance the risks between achieving full remission versus reactivation. Although active disease at conditioning remains a risk factor, full remission of all clinical symptoms is not required for successful HSCT. In particular, active neurological disease should prompt aggressive management including early HSCT ²⁹⁶⁾.

Allogeneic stem cell transplantation (HSCT) is also the definitive treatment of choice for HLH in the context of several other inborn errors of immunity, including X-linked lymphoproliferative disorder type 1 (XLP1) or X-linked lymphoproliferative disorder type 2 (XLP2) and patients with TIM3 deficiency ²⁹⁷⁾, ²⁹⁸⁾. Furthermore, allogenic stem cell transplantation (HSCT) has been successfully performed in a patient with a CDC42 mutation ²⁹⁹⁾. However, there has been no report of HSCT in NLRC4 deficiency, where it is unlikely to impact on IL-18 hypersecretion by intestinal epithelial cells ³⁰⁰⁾. Careful and broad genetic and functional evaluation is therefore mandatory before proceeding to HSCT based on clinical grounds in rare cases of familial or recurrent HLH without detection of a genetic cause.

In cases with suspected “primary” HLH, donor search and pretransplantation diagnostics should be carried out promptly during the initial presentation ³⁰¹⁾. Bi-allelic mutations should be ruled out in potential related donors ³⁰²⁾. In autosomal-recessive disease, heterozygous carriers are in most cases appropriate donors ³⁰³⁾. In the X-linked conditions, skewed X-inactivation should be excluded in potential female carrier donors ³⁰⁴⁾.

Antithymocyte Globulin (ATG)

Antithymocyte globulin directly targets T cells and other lymphocytes, to a minor extent also granulocytes and monocytes ³⁰⁵⁾. In a retrospective single-center analysis of 38 patients with familial hemophagocytosis, a protocol consisting of steroids, cyclosporine A and first-line ATG resulted in a higher initial remission rate compared to the HLH-94 protocol (active disease in 26% of patients versus 53%, after 2 months of therapy) but was associated with a higher percentage of relapses before HSCT (32% versus 13%) ³⁰⁶⁾. In an attempt to combine advantages of both protocols, a trial combining ATG and etoposide has been performed (NCT01104025), but the results have not yet been reported. There is no clear role for ATG beyond “primary” HLH ³⁰⁷⁾.

Alemtuzumab (Anti-CD52)

More recently, the humanized monoclonal anti-CD52 antibody alemtuzumab has been used in patients with “primary” HLH ³⁰⁸⁾. It is directed against the CD52 antigen (CAMPATH 1) which is a surface protein on mature lymphocytes and antigen-presenting cells (APCs). First promising results have been achieved when used in a bridging to transplant setting ³⁰⁹⁾. Excellent initial results have been orally reported from a trial evaluating Alemtuzumab as first-line treatment for “primary” HLH (in combination with methyl-prednisolone and cyclosporine A) (NCT02472054). The profound and long-lasting immune suppression and limits this drug to “primary” HLH, where induction of remission is rapidly followed by HSCT. The problem of viral (re-)activation is an important caveat when using alemtuzumab in X-linked lymphoproliferative disorder.

Emapalumab

Emapalumab is a recombinant human monoclonal antibody against interferon gamma (IFN-γ) ³¹⁰⁾. It has received FDA approval in November 2018 for the treatment of pediatric and adult patients with primary HLH with refractory, recurrent, or progressive disease or intolerance to HLH therapy ³¹¹⁾. An international multicenter follow-up study to further assess the efficacy and safety of emapalumab is still ongoing (NCT03312751). This trial will also provide data on its use as first-line therapy in “primary” HLH. Serum levels of CXCL9 emerge as an interesting biomarker for increased interferon gamma (IFN-γ) activity ³¹²⁾ and may be particularly helpful when considering the use of emapalumab in first-line treatment of HLH in the context of other inborn errors of immunity. Notably, a patient with CDC42 mutation who did not respond to steroids, cyclosporine A, and anakinra was successfully treated with emapalumab ³¹³⁾.

Ruxolitinib

Janus kinase inhibitors represent interesting therapeutic compounds in the context of HLH, since they not only inhibit signaling downstream of interferon gamma (IFN-γ), but also of several other pro-inflammatory cytokines. In the mouse-model of LCMV-induced “primary” HLH, the disease manifestations, including CNS involvement, were reduced upon JAK1/2 blockade by ruxolitinib ³¹⁴⁾, ³¹⁵⁾. The successful individual use of ruxolitinib reported in single cases of secondary HLH ³¹⁶⁾, ³¹⁷⁾, ³¹⁸⁾ has resulted in its prospective evaluation for this indication (NCT03795909). Preliminary results of a single-center phase 2 pilot study on the efficacy of ruxolitinib in secondary HLH demonstrate good tolerance to ruxolitinib in a small cohort of five patients ³¹⁹⁾. However, “primary” HLH is excluded in these studies. A trial investigating the benefit of JAK inhibition in first-line treatment of human “primary” HLH is in preparation.

IL-18 Binding Protein

IL-18 is released by activated macrophages and can induce interferon gamma (IFN-γ) and other pro- inflammatory cytokines ³²⁰⁾. Several reports have found elevated free IL-18 concentrations (i.e., IL-18 not bound to its binding protein IL-18BP) in the serum of patients with both “primary” and secondary HLH as well as in animal models and IL- 18 levels correlated with the presence of HLH-criteria and disease progression ³²¹⁾. In a murine model of “primary” HLH it was shown that treatment with IL-18BP can reduce severe organ damage, but does not improve survival ³²²⁾. An ongoing multicenter, double-blind, placebo-controlled, randomized withdrawal trial evaluates efficacy and safety of IL-18 Binding Protein (tadekinig alfa) in pediatric patients with NLRC4 associated hyperinflammation including HLH or X-linked inhibitor of apoptosis (XIAP) deficiency, diseases, in which IL-18 levels are particularly elevated (NCT03113760). While this treatment seems promising to attenuate the autoinflammatory manifestations of X-linked inhibitor of apoptosis (XIAP) deficiency, it remains to be seen whether it also has a role in acute EBV-induced HLH in this disease.

IL-1, IL-6, and TNF alpha blockade

The pro-inflammatory cytokines elevated in “primary” HLH also include IL-1, IL-6, and TNF alpha, which can be targeted by monoclonal antibodies and other blocking agents. They have been used successfully in the context of secondary HLH ³²³⁾, ³²⁴⁾ and related conditions associated with a “cytokine storm” such as hyperinflammation associated with CAR-T cell therapy ³²⁵⁾. Moreover, case reports have illustrated a partial effect of IL-1 blockade in patients with NLRC4 or CDC42 mutations ³²⁶⁾, ³²⁷⁾ and in patients with chronic granulomatous disease ³²⁸⁾. Anecdotal reports have reported efficacy of IL-6 blockade in manifestations of X-linked inhibitor of apoptosis (XIAP) deficiency different from acute EBV-induced HLH ³²⁹⁾. However, efficacy of IL-1, IL-6, or TNF alpha blockade in “primary” HLH has not been clearly documented, neither in first-line nor in rescue therapy ³³⁰⁾.

Control of Triggers

Infections should be diagnosed and treated aggressively in all forms of HLH. When active EBV infection is present, rituximab (anti-CD20 antibody) can help controlling the immune stimulation by eliminating EBV infected B cells ³³¹⁾, ³³²⁾. However, in some states of persistent EBV replication, EBV has been demonstrated in T or NK cells leading to resistance against rituximab treatment ³³³⁾. Cell-targeted therapy results in significant immunosuppression, such that reverse isolation, aspergillus-effective antifungal and Pneumocystis jirovecii prophylaxis should be administered ³³⁴⁾. Weekly monitoring for infection or reactivation of latent pathogens (EBV, CMV, adenovirus, aspergillus antigen) is recommended ³³⁵⁾.

Monitoring treatment response

Monitoring response to therapy and detecting early signs of reactivation is crucial in patients with “primary” HLH ³³⁶⁾, ³³⁷⁾. Fever, aminotransferase levels, ferritin, and coagulation parameters are practical, sensitive, and dynamic measures useful in the day-to-day monitoring of HLH patients ³³⁸⁾, ³³⁹⁾. Blood counts must be interpreted with the consideration of effects of myelosuppressive therapies (i.e., etoposide). Soluble IL-2 receptor (sCD25) and sCD163 appear to be useful indicators of active disease, but the restricted availability of these assays limits their utility in day-to-day management ³⁴⁰⁾. Monitoring coagulation parameters is essential, but they may not show marked abnormalities until late in the disease course ³⁴¹⁾. Other biomarkers for disease activity such as free IL-18 or CXCL9 are being explored. If more rapid turnaround times can be achieved, they might be valuable for guiding therapy in the future ³⁴²⁾.

In general, persistent or progressive disease in the face of standard initial therapy, should prompt continued investigation for an underlying infectious or malignant trigger (especially in adults). At the same time, HLH-targeted therapies should be intensified under the guidance of an experienced oncologist and/or hematologist ³⁴³⁾.

Salvage therapy in Refractory HLH

Early mortality of acute HLH remains a major concern. 25–50% of patients with acute “primary” HLH fail to achieve rapid and sustained initial remission after etoposide-based therapy. If cytopenia [in particular thrombocytopenia with platelets less 50 x 10⁹/L (less than 50,000 platelets per microliter)] and ferritin and/or sCD25 fail to respond after 2 weeks, the risk for an adverse outcome increases, justifying consideration of alternative (salvage) therapy ³⁴⁴⁾. There are no standard recommendations for the treatment of relapsing or refractory HLH. The salvage therapies published so far include alemtuzumab, anakinra, antithymocyte globulin (ATG), and regimens consisting of liposomal doxorubicin, etoposide, and dexamethasone ³⁴⁵⁾. In an observational study reporting on treatment of 22 patients with refractory HLH with alemtuzumab, 86 percent of patients showed partial response, and 77 percent were able to receive HSCT ³⁴⁶⁾. Notably, CMV and adenovirus viremia occurred in 23–32% of patients.

Emapalumab (GAMIFANT), a neutralizing antibody against interferon gamma (INF-γ), has recently been licensed as the first drug for the treatment patients with “primary” hemophagocytic lymphohistiocytosis (HLH) with refractory, recurrent or progressive disease or intolerance with conventional HLH therapy ³⁴⁷⁾. The recommended starting dose is 1 mg/kg twice per week with dexamethasone as a background treatment, but doses can be increased up to 10 mg/kg based on clinical response ³⁴⁸⁾. Due to the risk of serious infections (frequent during therapy of primary HLH patients and observed in 32% of patients in the trial) patients should receive prophylaxis for Herpes Zoster, Pneumocystis jirovecii, and fungal infections and should be monitored for tuberculosis, adenovirus, EBV and CMV.

The study included 27 patients with a mean age of 1 year (range: 0.1 to 13 years), with a “primary” HLH in 82% of patients. Patients had received various combinations of dexamethasone, etoposide, cyclosporine A, and anti-thymocyte globulin (ATG) prior to emapalumab. Full response was defined as normalization of all, while partial response was defined as normalization of ≥3 HLH parameters and HLH improvement was defined as ≥3 HLH abnormalities improved by at least 50% from baseline. Twenty patients completed the 8-week study, while seven were prematurely withdrawn. Seventy percent (19/27) of patients proceeded to HSCT. The overall response rate was 63%, the median time to response was 8 days. A complete response was achieved in 7 patients, partial response in 8 patients and HLH improvement in 2 patients ³⁴⁹⁾, ³⁵⁰⁾. Since refractory primary HLH has poor prognosis, these data are encouraging. However, the exact place of this drug in the context of existing and emerging therapies remains to be defined ³⁵¹⁾.

Familial hemophagocytic lymphohistiocytosis prognosis

The advent of HLH-94 and HLH-2004 treatment protocols markedly improved the survival of children with familial hemophagocytic lymphohistiocytosis. Nevertheless, familial hemophagocytic lymphohistiocytosis survivors may suffer long-term side effects such as cognitive and growth delay, hearing impairment, and obstructive lung disease ³⁵²⁾. Prognosis for adults with HLH remains poor. Overall mortality rates ranged between 42 and 88% ³⁵³⁾. In a series of 68 adult patients with HLH from multiple tertiary care centers, half of whom had an underlying malignancy, only 31% of patients were alive at a median follow up of 32 months ³⁵⁴⁾. Predictors of mortality in adult HLH include older age and underlying lymphoma ³⁵⁵⁾. These data illustrate a pressing need for better diagnostics and therapies for HLH, particularly in the adult population ³⁵⁶⁾.