Characteristic perforin gene mutations of haemophagocytic lymphohistiocytosis patients in Japan

Perforin gene (PRF1) mutations appear to occur in about 30% of patients with haemophagocytic lymphohistiocytosis (HLH). We tested perforin expression and gene mutations in 14 HLH patients and six patients with Epstein-Barr virus-associated HLH (EBV-HLH) in Japan. Five of the 14 HLH patients had perforin abnormalities. The presence of PRF1 genetic abnormality correlated well with the lack of perforin expression as determined by flow cytometry. Sequencing showed that four patients had a compound heterozygous mutation while the fifth patient had a homozygous mutation. Three of the mutations we detected were novel. In contrast, none of the six EBV-HLH patients showed perforin abnormalities. Our data, combined with the PRF1 mutations in three previously reported Japanese patients, suggest that the 1090-1091delCT and 207delC mutations of the perforin gene are frequently present in Japanese HLH patients (62.5% and 37.5% respectively). Examination of the geographical origins of the ancestors in the perforin-mutant HLH patients revealed that they mostly came from the Western part of Japan, suggesting that the present-day cases may largely derive from a common ancestor.     

Late-onset cases of familial hemophagocytic lymphohistiocytosis with missense perforin gene mutations

Since the discovery of perforin gene mutations in familial hemophagocytic lymphohistiocytosis (FHL) type 2, heterogeneous features in FHL2 patients have been identified in a report of Feldmann et al. as the beginning. This study was conducted to determine the impact of characteristic gene mutations on late-onset (age > or = 7 years) hemophagocytic lymphohistiocytosis episodes. We analyzed perforin gene mutations in three late-onset cases from our registry in Japan and an additional 10 cases from the literature. Of the 13 cases with onset ages of a median of 10 (range 7-49) years, nine had homozygous and four had compound heterozygous missense mutations of the perforin gene. None had homozygous nonsense mutations. Our data suggest that nonsense perforin gene mutations yield early onset and missense mutations late onset in FHL2 cases.     

Perforin defects of primary haemophagocytic lymphohistiocytosis in Japan

The perforin gene was analysed in 15 Japanese patients with primary haemophagocytic lymphohistiocytosis (HLH). Perforin gene defects were found in two out of eight patients with familial HLH (FHL), and one out of seven without affected siblings. Four novel mutations were identified. Compound heterozygous mutations (one FHL and one sporadic HLH) and only one allele mutation (one FHL) were defined. Flow cytometry revealed no perforin expression in CD8+ or CD56+ cells from a surviving patient with a mutation. The frequency of mutation was at least 20% of FHL in Japan. Flow cytometry for intracellular perforin may be useful for the screening of FHL2.     

Diagnostic evaluation of patients with suspected haemophagocytic lymphohistiocytosis

Haemophagocytic lymphohistiocytosis (HLH) is a life‐threatening hyperinflammatory syndrome characterized by severely disturbed immune homeostasis. It can affect all age groups. Diagnostic evaluation of the patient with suspected HLH has to address three main questions: (i) does the patient have HLH? There is no simple diagnostic test, but a number of clinical and laboratory criteria define this clinical syndrome. (ii) Can a trigger be identified? A variety of infections, malignant or autoimmune diseases can contribute to the disturbed immune homeostasis with important consequences for treatment. (iii) Does the patient suffer from a genetic disease predisposing to HLH? Recent advances in the understanding of the genetic and pathophysiological basis of HLH have enabled a better and more rapid answer to this question, which is relevant for prognosis and the decision to perform haematopoietic stem cell transplantation. This review summarizes the current diagnostic approach to the patient with HLH.     

Induction of apoptosis and caspase activation in cells obtained from familial haemophagocytic lymphohistiocytosis patients

Familial haemophagocytic lymphohistiocytosis (FHL) is a rare and uniformly fatal disorder of early childhood characterized by fever, hepatosplenomegaly, cytopenia and widespread infiltration of vital organs by activated lymphocytes and macrophages. In order to test whether the massive accumulation of immune cells in these patients is associated with a perturbation of apoptosis, lymphocytes were isolated from eight patients and subjected to the chemotherapeutic agent etoposide or agonistic anti-Fas monoclonal antibodies in vitro. These stimuli elicited a normal apoptotic response in FHL patient cells when compared to healthy controls, as determined by phosphatidylserine exposure, DNA fragmentation, in vitro cleavage of the caspase-3-like substrate aspartate-glutamate-valine-aspartate-7-amino-4-methyl-coumarin (DEVD-AMC) and proteolysis of the anti-apoptotic protein Bcl-2. In addition, the degree of constitutive and Fas-triggered apoptosis in freshly isolated neutrophils was monitored in three children, with similar results. These studies indicate that immune cells derived from FHL patients are not inherently resistant to apoptosis induction. Specifically, etoposide-induced and Fas-triggered activation of intracellular caspases appears to remain intact in these individuals. However, the degree of spontaneous activation of caspase-3-like enzymes in activated lymphocytes was attenuated in three of the four patients tested prior to initiation of therapy, suggesting a possible biological deficiency in these individuals.     

Haemophagocytic lymphohistiocytosis: proposal of a diagnostic algorithm based on perforin expression

Haemophagocytic lymphohistiocytosis (HLH) is a rare, fatal disorder of early infancy. Mutations of the PRF1 gene have been identified in a subset of patients. However, the distinction between the different genetically determined and environmental subtypes of the disease remains a major issue to be solved. This may result in delayed or inappropriate application of bone marrow transplantation (BMT). We propose an algorithm that uses a combination of three rapid laboratory tests, i.e. perforin expression by peripheral lymphocytes, assessment of the behaviour of the 2B4 lymphocyte receptor and natural killer (NK) cell activity, to identify the different subgroups of HLH. In 19 patients diagnosed according to current criteria, we tested perforin expression, 2B4 receptor function and NK cell activity. PRF1 mutations were found in all seven patients showing absent perforin expression. In one male with abnormal behaviour of the 2B4 receptor, SH2D1A mutation confirmed the diagnosis of X-linked lymphoproliferative disease. Four patients with normal NK cell activity had evidence of associated infections. Of the seven with impaired NK cell activity, two had a probable genetically determined subtype of HLH and five appeared as sporadic, infection-associated cases. Improving the diagnostic approach may restrict the use of BMT, the only recognized curative treatment, to HLH patients with a documented poor prognosis while patients with milder disorders may be treated less intensively. Our flow chart could also lead to better selection of patients for specific gene analysis.     

Haematopoietic stem cell transplantation in haemophagocytic lymphohistiocytosis

Haemophagocytic lymphohistiocytosis (HLH) poses major therapeutic challenges, and the primary inherited form, familial haemophagocytic lymphohistiocytosis (FHL), is usually fatal. We evaluated, including Cox regression analysis, survival in 86 children (29 familial) that received HLH-94-therapy (etoposide, dexamethasone, ciclosporin) followed by allogeneic stem cell transplantation (SCT) between 1995 and 2000. The overall estimated 3-year-survival post-SCT was 64% [confidence interval (CI) = +/-10%] (n = 86); 71 +/- 18% in those patients with a matched related donor (MRD, n = 24), 70 +/- 16% with a matched unrelated donor (MUD, n = 33), 50 +/- 24% with a family haploidentical donor (haploidentical, n = 16), and 54 +/- 27% with a mismatched unrelated donor (MMUD, n = 13). After adjustment for potential confounding factors, estimated odds ratios (OR) for mortality were 1.93 (CI =0.61-6.19) for MUD, 3.31 (1.02-10.76) for haploidentical, and 3.01 (0.91-9.97) for MMUD, compared with MRD. In children with active disease after 2-months of therapy (n = 43) the OR was 2.75 (1.26-5.99), compared with inactive disease (n = 43). In children with active disease at SCT (n = 37), the OR was 1.80 (0.80-4.06) compared with inactive disease (n = 49), after adjustment for disease activity at 2-months. Mortality was predominantly transplant-related. Most HLH patients survived SCT using MRD or MUD, and survival with partially mismatched donors was also acceptable. Patients that responded well to initial pretransplant-induction therapy fared best, but some persisting HLH activity should not automatically preclude performing SCT.     

Transient familial haemophagocytic lymphohistiocytosis reactivation post-CD34 haematopoietic stem cell transplantation

Familial haemophagocytic lymphohistiocytosis (FHLH) is a genetic disorder caused by defective lymphocyte cytotoxicity, resulting in impaired lymphocyte homeostasis and macrophage infiltration of solid tissues and bone marrow, with extensive haemophagocytosis. It is invariably fatal unless treated by allogeneic haematopoietic stem cell transplantation (HSCT). In a retrospective analysis of 11 cases of FHLH, transplanted in one centre between January 1999 and December 2003, it was found that host T cell expansion occurred early after HSCT in a setting of a viral infection (cytomegalovirus and Epstein-Barr virus respectively) in two cases who received T cell-depleted HSCT. Transient recurrence of clinical and biological manifestations of FHLH was observed, despite evidence for donor cell engraftment. Secondary development of donor T cells led to stable mixed chimaerism and sustained remission of FHLH. Detection of host-derived T cells soon after HSCT in a patient with FHLH should thus not mistakenly be taken as a manifestation of graft rejection.     

Engraftment and dissemination of T lymphocytes from primary haemophagocytic lymphohistiocytosis in scid mice

Although primary haemophagocytic lymphohistiocytosis (HLH) is a genetic disorder of T lymphocytes, it remains unclear why T lymphocytes of primary HLH patients preferentially infiltrate the central nervous system and peripheral blood, in addition to the reticuloendothelial systems. We engrafted Herpesvirus saimiri (HVS)-immortalized T-lymphocyte lines established from primary HLH patients into severe combined immunodeficient (scid) mice and examined their capacity to infiltrate mouse organs. A diffuse infiltration of human T lymphocytes was detected in each organ of scid mice treated with 1 x 10(6) T lymphocytes from all four primary HLH patients assessed, whereas no infiltration of T lymphocytes from healthy individuals was observed in any organ. The infiltration of T lymphocytes was mainly observed in the lung, brain and peripheral blood, in association with haemophagocytosis. These cells were positive for HLA-DR, CD3 and either CD8 or CD4, but negative for CD68. Certain markers of proliferation and apoptotic activities were highly positive in these cells. There was no difference between the infiltration pattern of T lymphocytes of primary HLH patients with a perforin deficiency and those without. By Southern blot analysis, T lymphocytes infiltrating mouse organs were observed to be polyclonal. These findings suggest that our murine model implementing HVS-immortalized human T lymphocytes is suitable to clarify the pathogenesis of primary HLH.     

Evidence of clonality in a child with haemophagocytic lymphohistiocytosis

DNA from the bone marrow of a child with haemophagocytic lymphohistiocytosis was examined by polymerase chain reaction for evidence of rearrangement of the T-cell receptor delta chain gene. Evidence of clonal rearrangement was detected, supporting a role for abnormalities of T-cell function in the pathogenesis of this disorder.     

Laboratory parameters identify familial haemophagocytic lymphohistiocytosis from other forms of paediatric haemophagocytosis

Haemophagocytic lymphohistiocytosis (HLH) is a life‐threatening syndrome of immune dysregulation and is classified as primary or secondary according to the underlying aetiology. The treatment strategies recommended for these two groups differ substantially; however, it is thought to be impossible to predict the underlying causes of HLH using conventional laboratory tests. Recent studies show that serum levels of soluble interleukin‐2 receptor (sIL2R) and ferritin are useful for differentiating some forms of HLH. The present study reports that combinations of common laboratory parameters, such as the percentage of total lymphocytes within the peripheral blood leucocyte population, serum levels of lactate dehydrogenase and the sIL2R/ferritin ratio, are useful for identifying patients with familial haemophagocytic lymphohistiocytosis and for differentiating the underlying aetiology of paediatric HLH during the early course of the disease. These findings suggest that the pathogenesis of HLH differs greatly in terms of innate and adaptive immunity depending on the aetiology and may provide a new approach to unravelling the complex pathophysiology underlying this syndrome.     

L-DEP regimen salvage therapy for paediatric patients with refractory Epstein-Barr virus-associated haemophagocytic lymphohistiocytosis

Epstein–Barr virus-associated haemophagocytic lymphohistiocytosis (EBV-HLH) is one of the most common subtypes of secondary HLH. However, more than 30% of patients do not respond to traditional treatment. Here, we investigated the efficacy and safety of the L-DEP regimen as a salvage therapy for paediatric refractory EBV-HLH. We retrospectively analysed 26 paediatric patients with refractory EBV-HLH who received the L-DEP regimen at Beijing Children’s Hospital from 1 January 2016 to 31 March 2019. Five of the patients achieved complete response (CR) and 11 achieved partial response (PR), indicating an overall response rate of 61·5% (CR + PR). Ten of the 16 patients who achieved CR or PR received allogenic haematopoietic stem cell transplantation (allo-HSCT), and seven were still alive at the last follow-up on 30 April 2020. Two of the 10 patients who did not respond were alive after allo-HSCT. Major side effects included increased amylase, bone marrow suppression and coagulation disorders, though these could be controlled by supportive therapy in most cases. Thus, we conclude that the L-DEP regimen is an effective and relatively safe salvage therapy for children with refractory EBV-HLH. This regimen also provides a bridge to allo-HSCT.     

An inframe perforin gene deletion in familial hemophagocytic lymphohistiocytosis is associated with perforin expression

Familial hemophagocytic lymphohistiocytosis is an autosomal recessive disease of early childhood manifested by hypercytokinemia and organ infiltration of macrophages and activated lymphocytes, and it is characterized by a fulminant clinical course. The molecular mechanism underlying this disease appears to be a deregulation of apoptosis of activated T cells and macrophages. Approximately 20-40% of patients with familial hemophagocytic lymphohistiocytosis reported worldwide had a perforin gene mutation. We report herein a novel perforin variant in the homozygous state in an Omani boy who was diagnosed 44 days after birth. Sequence analysis of the perforin gene coding region revealed a 12-base pair deletion (codon 284-287) resulting in the deletion of four amino acids in the membrane attack complex domain of the protein. This deletion maintains the reading frame of the perforin mRNA. Both parents were heterozygotes for this molecular defect. Flow-cytometric analysis revealed intracellular perforin expression at the lower end of the normal range in the cytotoxic T cells (CD3+/CD8+) and (CD3+/CD56+) and in around 50% of the natural killer cells (CD3-/CD56+). This is an additional example of a perforin variant which is associated with a significant level of cellular perforin expression and thus confirms that drastic reduction in its expression is not a constant feature in familial hemophagocytic lymphohistiocytosis type 2.     

The breakpoints of a constitutional inversion of chromosome 9 associated with haemophagocytic lymphohistiocytosis are not linked to the disease gene

Haemophagocytic lymphohistiocytosis (HLH) is a severe disorder of early infancy consistent with an autosomal recessive inheritance. Neither the genetic locus nor the biochemical defect is known for the disease. A constitutional pericentric inversion of chromosome 9, with breakpoints in bands 9p23 and 9q31, has been reported in a case of HLH, suggesting a possible relationship between this chromosome abnormality and the disease. We investigated such an association, performing a genetic linkage analysis in a set of five consanguineous HLH families. 27 polymorphic markers on chromosome 9 were studied, excluding most of chromosome 9 as a putative site for the HLH gene.     

Risk factors affecting outcome of unrelated cord blood transplantation for children with familial haemophagocytic lymphohistiocytosis

Allogeneic haematopoietic stem cell transplantation is still the only available curative option for Familial Haemophagocytic Lymphohistiocytosis (FHLH). Most studies report outcomes after bone marrow or peripheral blood stem cell transplantation. We analysed the outcomes of 118 children with FHLH undergoing single-unit umbilical cord blood transplantation performed from 1996 to 2014. Myeloablative conditioning regimen was given to 90% of the patients, and was mostly busulfan-based (n = 81, 76%), including anti-thymocyte globulin or alemtuzumab (n = 102, 86%). The cumulative incidence of Day 60 neutrophil engraftment was 85%; and that of non-relapse mortality and acute graft-versus-host disease (GvHD) was 21% and 33% at 100 days, respectively. The 6-year cumulative incidence of chronic GvHD was 17% and the 6-year probability of overall survival was 55%. In multivariate analysis, children receiving a graft with a total nucleated cell dose greater than 9·9 × 10⁷/kg had a better overall survival (hazard ratio [HR]: 0·49, 95% CI: 0·27–0·88, P = 0·02). Degree of human leucocyte antigen (HLA) matching was associated with improved disease-free survival (5/6 vs. 6/6 HR: 2·11, 95% confidence interval [CI]: 1·01–4·4, P = 0·05 and ≤4/6 vs. 6/6, HR: 2·82, CI: 1·27–6·23, P = 0·01). Umbilical cord blood transplantation with a high cell dose and good HLA match is a suitable alternative option to haematopoietic stem cell transplantation in children with FHLH who lack a HLA-matched donor.     

Epidemiology of haemophagocytic lymphohistiocytosis at the population level in Germany

We retrospectively analysed all German inpatient cases of haemophagocytic lymphohistiocytosis (HLH) from 2014 to 2020 to describe the epidemiology, clinical course, and underlying diseases of 4065 HLH patients. The age-standardized incidence rate of HLH in Germany was 0.52/100 000 people in 2014 and steadily increased by 10% per year to 0.97/100 000 in 2020 (mean 0.70/100 000). Inpatient deaths related to HLH increased from 0.84/1 000 000 people in 2014 to 2.32/1 000 000 people in 2020, caused by rising numbers of older HLH patients. Overall, HLH is more frequent than previously expected and incidence as well as HLH-related deaths increased significantly.     

Frequency and spectrum of central nervous system involvement in 193 children with haemophagocytic lymphohistiocytosis

Haemophagocytic lymphohistiocytosis (HLH) may cause meningoencephalitis and significant neurological sequelae. We examined the relationship between neurological symptoms and cerebrospinal fluid (CSF) at diagnosis, and long-term outcome, in all children enroled in the HLH-94-study prior to July 1, 2003, for whom information on CSF at diagnosis was available (n = 193). Patients were divided into four groups: (i) normal CSF (cells/protein) and no neurological symptoms (n = 71); (ii) normal CSF but neurological symptoms (n = 21); (iii) abnormal CSF but no symptoms (n = 50); and (iv) abnormal CSF with neurological symptoms (n = 51). At diagnosis, neurological symptoms were reported in 72/193 (37%) (seizures = 23); abnormal CSF in 101/193 (52%), and either or both in 122/193 (63%). Altogether 16/107 (15%) survivors had neurological sequelae at follow-up (median 5.3 years). Multivariate hazard ratios (HR) for mortality were 0.98 [95% confidence interval (CI) = 0.42-2.31], 1.52 (0.82-2.82) and 2.05 (1.13-3.72) for groups 2-4, compared with group 1. Moreover, sequelae were more frequent in group 4 (7/21, 33%) compared to groups 1-3 (9/86, 10%) (P = 0.015). Patients with abnormal CSF at diagnosis had significantly increased mortality [HR = 1.78 (95% CI = 1.08-2.92), P = 0.023]. Thus, a substantial proportion of HLH survivors suffer neurological sequelae, and children with abnormal CSF have increased risk of mortality and neurological sequelae. Prompt treatment of HLH at onset or relapse may reduce these complications.