SUMMARY
1. Familial Hemophagocytic Lymphohistiocytosis (FHL) refers to a group of diseases which are characterized by a genetic predisposition to develop life-threatening episodes of hemophagocytic lymphohistiocytosis (HLH). Up to 25% of HLH cases may be familial in nature.
2. Cell-mediated contact cytotoxicity by NK cells and CD8 T cells is a key immune effector mechanism against viruses and intracellular bacteria. This cytotoxicity is mediated by the release of cytotoxic granules such as perforin and granzymes. The current known forms of FHL are caused by a deficiency of cytotoxic granules or impaired transport and release of granules.
3. Defects in cell cytotoxicity result in impaired pathogen elimination, leading to persistent antigen stimulation and proliferation of activated lymphocytes which infiltrate tissues and cause end-organ damage. Excessive cytokine stimulation by activated CTL and NK cells leads to a state of hyperinflammation.
4. IFN-gamma in particular has been identified as a key cytokine involved in disease pathogenesis. IFN gamma is toxic to hematopoietic cells and can cause the cytopenia seen in HLH. In addition, IFN gamma can increase activation of macrophages which results in phagocytosis of blood cells in infiltrated organs such as the bone marrow, liver and spleen.
5. In the majority of patients with FHL (85%), disease manifestation occurs within the first year of life. Patients present with a febrile illness, cytopenia and hepatosplenomegaly. Neurologic symptoms (irritability, hypertonia, hypotonia, seizures, or coma) may manifest at the onset of disease but typically develop later as the disease progresses.
6. Currently, four gene mutations have been linked to FHL:
FHL 1 - gene not yet reported
-Linkage analysis in four consanguineous Pakistani families with FHL identified a locus on chromosome
9q21.3-22 but a causative gene has not been identified.
FHL 2 - Perforin Deficiency (PRF1)
-Perforin is a pore-forming protein that can insert into the lipid bilayer of target cell membranes causing
cell death by osmotic lysis. It also allows for entry of apoptosis-inducing granzymes into the target cell.
Patients with PRF1 mutations are at higher risk of early onset HLH (before 6 months). Perforin
deficiency accounts for 30-35% of FHL cases.
FHL 3 - MUNC 13-4 (UNC13D)
-Munc 13-4 is involved in the priming of lytic granules docked at the plasma membrane. It likely
regulates the interaction between the vesicle (v)-SNARE and target (t)-SNARE which is required for
fusion of the vesicle with the plasma membrane. Munc 13-4 deficiency results in defective release of
cytotoxic granules despite normal polarization of granules at the immunologic synapse. Munc 13-4
deficiency accounts for 30-35% of FHL cases.
FHL 4 - Syntaxin 11 (STX11) -
-Syntaxin 11 is a member of the t-SNARE family and appears to be important for vesicle membrane
fusion and exocytosis - NK cells from syntaxin-deficient patients fail to degranulate when encountering
target cells. Syntaxin 11 deficiency accounts for 20% of FHL cases.
FHL 5 - Munc 18-2 (STXBP2)
-The syntaxin-binding protein 2 (STXBP2) gene encodes for Munc 18-2, which appears to partner with
syntaxin 11 to mediate release of cytotoxic granules. One-third of FHL-5 patients also appear to have
colitis, bleeding disorder, and hypogammaglobulinemia.
7. Clinically it is quite difficult to distinguish between primary FHL and secondary forms of HLH secondary to infections, hematopoietic malignancies or autoimmune disease. A family history of FHL or a history of consanguinity may suggest a familial form of disease.
8. In conjunction with an HLH evaluation, a careful microbiological evaluation to identify infections that could trigger HLH is recommended. This includes an evaluation for EBV, CMV, HIV, adenovirus, enterovirus, parvovirus, or HHV-6, fungal, bacterial, and parasitic infections. However, the presence of an infectious source does not discriminate between primary FHL and secondary HLH.
9. Diagnostic guidelines for the diagnosis of HLH have been established - five of the following eight criteria need to be fulfilled:
-Fever (from inflammatory cytokine release)
-Splenomegaly (from infiltration by activated lymphocytes)
-Cytopenia ≥ 2 lineages (from IFN-gamma and hemophagocytosis)
-Hypertriglyceridemia ± Hypofibrinogenemia (TNF-alpha inhibits lipoprotein lipase, which results in high triglyceride levels)
-Hemophagocytosis in the bone marrow, spleen, or lymph node
-Impaired NK cell function
-Ferritin > 500 g/L
-Soluble IL-2 receptor >2,400 u/ml
10. Absent intracellular staining for perforin on NK cells suggests a diagnosis of FHL2 (perforin deficiency). However, FHL2 patients with residual perforin expression have been reported.
11. Genetic analysis can establish a definitive diagnosis for FHL. Gene sequencing for FHL2 (PRF1), FHL3 (UNC13D), FHL4 (STX11), and FHL5 (STXBP2) is commercially available.
12. The differential diagnosis for patients should include other inherited defects of lymphocyte cell cytotoxicity (XLP, Chediak-Higashi, Hermansky-Pudlak 2, Griscelli, p14 deficiency) which can also present with life-threatening hemophagocytosis in response to viral infections.
13. The current treatment approach for patients involves initiation of an appropriate chemotherapeutic HLH protocol followed by HSCT. Current HLH protocols use treatment with etoposide and dexamethasone. Cyclosporine may also be added, but the benefit of this therapy has not been proven.
14. Most cases of FHL are lethal within the first year of life. As a result, HSCT is indicated for all patients with FHL. The success of transplant is determined by the donor type (matched related donors are optimal) and the extent of HLH control prior to HSCT.
OVERVIEW
Familial Hemophagocytic Lymphohistiocytosis (FHL) refers to a group of diseases which are characterized by a genetic predisposition to develop life-threatening episodes of hemophagocytic lymphohistiocytosis (HLH). Up to 25% of HLH cases may be familial in nature.
In the majority of patients with FHL (85%), disease manifestation occurs within the first year of life. Patients present with a febrile illness, cytopenia and hepatosplenomegaly. Neurologic symptoms (irritability, hypertonia, hypotonia, seizures, or coma) may manifest at the onset of disease but typically develop later as the disease progresses. The clinical features of a Swedish cohort of FHL patients are summarized below:
-Fever (91%)
-Hepatomegaly (90%)
-Splenomegaly (84%)
-Neurologic Symptoms (47%)
-Rash (43%)
-Lymphadenopathy (42%)
Currently, four gene mutations have been linked to FHL:nna, and nose start to develop around 3 to 5 years of age. Thus, the onset of ataxia precedes the development of telangiectasia by a few years.
FHL 1 - gene not yet reported
-Linkage analysis in four consanguineous Pakistani families with FHL identified a locus on chromosome
9q21.3-22 but a causative gene has not been identified.
FHL 2 - Perforin Deficiency (PRF1)
-Perforin is a pore-forming protein that can insert into the lipid bilayer of target cell membranes causing
cell death by osmotic lysis. It also allows for entry of apoptosis-inducing granzymes into the target cell.
Patients with PRF1 mutations are at higher risk of early onset HLH (before 6 months). Perforin
deficiency accounts for 30-35% of FHL cases.
FHL 3 - MUNC 13-4 (UNC13D)
-Munc 13-4 is involved in the priming of lytic granules docked at the plasma membrane. It likely
regulates the interaction between the vesicle (v)-SNARE and target (t)-SNARE which is required for
fusion of the vesicle with the plasma membrane. Munc 13-4 deficiency results in defective release of
cytotoxic granules despite normal polarization of granules at the immunologic synapse. Munc 13-4
deficiency accounts for 30-35% of FHL cases.
FHL 4 - Syntaxin 11 (STX11) -
-Syntaxin 11 is a member of the t-SNARE family and appears to be important for vesicle membrane
fusion and exocytosis - NK cells from syntaxin-deficient patients fail to degranulate when encountering
target cells. Syntaxin 11 deficiency accounts for 20% of FHL cases.
FHL 5 - Munc 18-2 (STXBP2)
-The syntaxin-binding protein 2 (STXBP2) gene encodes for Munc 18-2, which appears to partner with
syntaxin 11 to mediate release of cytotoxic granules. One-third of FHL-5 patients also appear to have
colitis, bleeding disorder, and hypogammaglobulinemia.
Clinically it is quite difficult to distinguish between primary FHL and secondary forms of HLH secondary to infections, hematopoietic malignancies or autoimmune disease. A family history of FHL or a history of consanguinity may suggest a familial form of disease.
In conjunction with an HLH evaluation, a careful microbiological evaluation to identify infections that could trigger HLH is recommended. This includes an evaluation for EBV, CMV, HIV, adenovirus, enterovirus, parvovirus, or HHV-6, fungal, bacterial, and parasitic infections. However, the presence of an infectious source does not discriminate between primary FHL and secondary HLH.
PATHOGENESIS
Cell-mediated contact cytotoxicity by NK cells and CD8 T cells is a key immune effector mechanism against viruses and intracellular bacteria. This cytotoxicity is mediated by the release of cytotoxic granules such as perforin and granzymes. The current known forms of FHL are caused by a deficiency of cytotoxic granules or impaired transport and release of granules.
Defects in cell cytotoxicity result in impaired pathogen elimination, leading to persistent antigen stimulation and proliferation of activated lymphocytes which infiltrate tissues and cause end-organ damage. Excessive cytokine stimulation by activated CTL and NK cells leads to a state of hyperinflammation.
IFN-gamma in particular has been identified as a key cytokine involved in disease pathogenesis. IFN gamma is toxic to hematopoietic cells and can cause the cytopenia seen in HLH. In addition, IFN gamma can increase activation of macrophages which results in phagocytosis of blood cells in infiltrated organs such as the bone marrow, liver and spleen.
EVALUATION
Diagnostic guidelines for the diagnosis of HLH have been established - five of the following eight criteria need to be fulfilled:
-Fever (from inflammatory cytokine release)
-Splenomegaly (from infiltration by activated lymphocytes)
-Cytopenia of ≥ 2 lineages (from IFN-gamma and hemophagocytosis)
-Hypertriglyceridemia ± Hypofibrinogenemia (TNF-alpha inhibits lipoprotein lipase, which results in high triglyceride levels)
-Hemophagocytosis in the bone marrow, spleen, or lymph node
-Impaired NK cell function
-Ferritin > 500 g/L
-Soluble IL-2 receptor >2,400 u/ml
Step 1a: HLH Laboratory Studies
-CBC with Differential
-Liver Function Tests
-PT, PTT
-Triglyceride and Fibrinogen levels
-Ferritin Levels
-Bone Marrow or Lymph Node biopsy
-NK cell functional Assay
-Soluble IL-2 Receptor
-Cytopenia in at least two cell lines is a diagnostic criteria for HLH (Hb < 9 mg/l, Platelets <100,000/ul, Neutrophils <1,000/ul)
-Liver dysfunction including markedly elevated liver transaminases and hyperbilirubinemia are common in HLH
-Coagulation abnormalities occur due to profound liver dysfunction
-Hypertriglyceridemia and hypofibrinogenemia may be present
-Elevated Ferritin > 500 ng/ml may be present
-Tissue demonstration of hemophagocytosis may require multiple biopsy attempts (in 20% of HLH cases, demonstration of hemophagocytosis on a first bone marrow attempt is not possible)
-Reduced NK cell lytic activity may be present
-Soluble IL-2 Receptor (sCD25) levels may be elevated.
Step 1b: Infections can be a key trigger for HLH events a careful evaluation for infectious sources should be performed in parallel with the above HLH evaluation. This should include blood/CSF/bone marrow cultures for bacteria, fungi and viruses. Serologic or nucleic acid testing for EBV, CMV, HIV, enterovirus, parvovirus B19, and HHV-6, should be considered. A review article for infectious triggers of HLH is available in the resource section.
Step 2: Immune Evaluation
-Quantitative immunoglobulins (IgG, IgM, IgA)
-Antibody titers to vaccine antigens
-Flow cytometry for B cell, T cell, and NK cell enumeration
-Low immunoglobulin levels are not a typical feature of FHL but it has been described in some patients with FHL5 (STXBP2). In addition, hypogammaglobulinemia and decreased vaccine responses can be a feature of XLP, which has significant clinical overlap with FHL. Lymphocyte subset analysis may reveal elevated levels of activated CD8 T cells, resulting in an inversion of the CD4:CD8 ratio.
Step 3: Additional Immunologic screening tests for FHL
-Perforin/Granzyme intracellular flow cytometry
-Decreased perforin may suggest FHL-2 (perforin deficiency). However, it should be noted that some patients with FHL-2 can still have residual perforin expression.
Step 4: Genetic confirmation
-FHL 2 Perforin Deficiency (PRF1)
-FHL 3 MUNC 13-4 (UNC13D)
-FHL 4 Syntaxin 11 (STX11)
-FHL 5 Munc 18-2 (STXBP2)
-Sequencing of the above genes can provide genetic confirmation for FHL (testing is available commercially through Cincinnati Childrens Hospital).
MANAGEMENT
The current treatment approach for patients involves initiation of an appropriate chemotherapeutic HLH protocol followed by HSCT. Current HLH protocols use treatment with etoposide and dexamethasone. Cyclosporine may also be added, but the benefit of this therapy has not been proven.
Most cases of FHL are lethal within the first year of life. As a result, HSCT is indicated for all patients with FHL. The success of transplant is determined by the donor type (matched related donors are optimal) and the extent of HLH control prior to HSCT.
RESOURCES
Diagnostic Resources
1. CINCINNATI CHILDRENS - NK function assay
2. CINCINNATI CHILDRENS - Perforin + Granzyme B flow cytometry
3. CINCINNATI CHILDRENS - Soluble IL-2 receptor levels
4. CINCINNATI CHILDRENS - PRF1, MUNC 13-4, STX11, STXBP2 gene sequencing
Literature Resources
Infectious triggers for HLH in children
2. Schmid 2010
Inherited defects of lymphocyte cytotoxic activity (review)
3. Jordan 2008
HSCT for HLH (review)