Efficacy of T cell assays for the diagnosis of primary defects in cytotoxic lymphocyte exocytosis.

Primary hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder associated with autosomal recessive variants in genes required for perforin-mediated lymphocyte cytotoxicity. A rapid diagnosis is crucial for successful treatment. Although defective cytotoxic T lymphocyte (CTL) function causes pathogenesis, quantification of natural killer (NK) cell exocytosis triggered by K562 target cells currently represents a standard diagnostic procedure for primary HLH. We have prospectively evaluated different lymphocyte exocytosis assays in 213 patients referred for evaluation for suspected HLH and related hyperinflammatory syndromes. A total of 138 patients received a molecular diagnosis consistent with primary HLH. Compared to routine K562 cell-based assays, assessment of Fc receptor-triggered NK-cell and T cell receptor-triggered CTL exocytosis displayed higher sensitivity and improved specificity for the diagnosis of primary HLH, with these assays combined providing a sensitivity of 100% and specificity of 98.3%. By comparison, NK-cell exocytosis following K562 target cell stimulation displayed a higher inter-individual variability, in part explained by differences in NK-cell differentiation or large functional reductions following shipment. We thus recommend combined analysis of T cell receptor-triggered CTL and Fc receptor-triggered NK-cell exocytosis for the diagnosis of patients with suspected familial HLH or atypical manifestations of congenital defects in lymphocyte exocytosis.

Malignancy-associated hemophagocytic lymphohistiocytosis in Sweden: incidence, clinical characteristics, and survival.

ABSTRACT: We evaluated malignancy-associated hemophagocytic lymphohistiocytosis (mal-HLH) in Sweden regarding population-based incidence, clinical features, and survival. From 1997 to 2018, we identified 307 adults (≥18 years old) and 9 children (209 males, 107 females; P < .001) with both an HLH-related diagnosis and malignant disease, corresponding to 0.19 per 100 000 adults annually (0.15/100 000 for the entire population), increasing from 0.026 (1997-2007) to 0.34 (2008-2018) (P < .001). In the latest 7-year period (2012-2018), the annual incidence was 0.45 per 100 000 adults (n = 246). This incidence varied between the 6 health care regions in Sweden, from 0.18 to 0.71 (Region Stockholm) per 100 000 adults annually (P < .001), likely due to variable awareness. Mal-HLH was reported in 0.6% of all hematological malignancies, with the highest proportion (2.5%) in young males. Among the 316 patients, the 1-month probability of survival, likely representing the HLH episode, increased significantly from 52% (95% confidence interval [CI], 40-63) (1997-2007) to 71% (95% CI, 65-76) (2008-2018), whereas 2-year survival remained poor (25%; 95% CI, 20-30). Altogether, 52% were lymphomas, 29% leukemias, 8% other hematological malignancies, and 11% solid tumors. Males were more affected than females by mal-HLH, also taking the over-representation of males with hematological malignancies into account (P = .0012). Validation by medical-file reviews revealed 13% over-reporting of HLH. We conclude that the annual mal-HLH incidence has increased 10-fold and was at least 0.71 per 100 000 adults from 2012 to 2018, that is, 0.62 per 100 000 adults considering 13% estimated HLH over-reporting, and that early survival improved significantly, likely due to increased awareness and more HLH-directed therapy.

Rubella vaccine-induced granulomas are a novel phenotype with incomplete penetrance of genetic defects in cytotoxicity.

BACKGROUND: Rubella virus-induced granulomas have been described in patients with various inborn errors of immunity. Most defects impair T-cell immunity, suggesting a critical role of T cells in rubella elimination. However, the molecular mechanism of virus control remains elusive. OBJECTIVE: This study sought to understand the defective effector mechanism allowing rubella vaccine virus persistence in granulomas. METHODS: Starting from an index case with Griscelli syndrome type 2 and rubella skin granulomas, this study combined an international survey with a literature search to identify patients with cytotoxicity defects and granuloma. The investigators performed rubella virus immunohistochemistry and PCR and T-cell migration assays. RESULTS: This study identified 21 patients with various genetically confirmed cytotoxicity defects, who presented with skin and visceral granulomas. Rubella virus was demonstrated in all 12 accessible biopsies. Granuloma onset was typically before 2 years of age and lesions persisted from months to years. Granulomas were particularly frequent in MUNC13-4 and RAB27A deficiency, where 50% of patients at risk were affected. Although these proteins have also been implicated in lymphocyte migration, 3-dimensional migration assays revealed no evidence of impaired migration of patient T cells. Notably, patients showed no evidence of reduced control of concomitantly given measles, mumps, or varicella live-attenuated vaccine or severe infections with other viruses. CONCLUSIONS: This study identified lymphocyte cytotoxicity as a key effector mechanism for control of rubella vaccine virus, without evidence for its need in control of live measles, mumps, or varicella vaccines. Rubella vaccine-induced granulomas are a novel phenotype with incomplete penetrance of genetic disorders of cytotoxicity.

Genetics and pathophysiology of haemophagocytic lymphohistiocytosis.

Haemophagocytic lymphohistiocytosis (HLH) represents a life-threatening hyperinflammatory syndrome. Familial studies have established autosomal and X-linked recessive causes of HLH, highlighting a pivotal role for lymphocyte cytotoxicity in the control of certain virus infections and immunoregulation. Recently, a more complex etiological framework has emerged, linking HLH predisposition to variants in genes required for metabolism or immunity to intracellular pathogens. We review genetic predisposition to HLH and discuss how molecular insights have provided fundamental knowledge of the immune system as well as detailed pathophysiological understanding of hyperinflammatory diseases, highlighting new treatment strategies.

Efficacy of Moderately Dosed Etoposide in Macrophage Activation Syndrome-Hemophagocytic Lymphohistiocytosis.

OBJECTIVE: Macrophage activation syndrome (MAS) constitutes 1 subtype of the hyperinflammatory syndrome hemophagocytic lymphohistiocytosis (HLH), and the term MAS-HLH was recently proposed for HLH with underlying autoimmune/autoinflammatory conditions. The mortality of MAS-HLH has been estimated at 5-10%. Here we report our experiences with moderately dosed etoposide in severe MAS-HLH; the objective was to effectively reduce severe hyperinflammatory activity with limited side effects. METHODS: In addition to conventional antiinflammatory treatment, moderately dosed etoposide was administered to 7 children affected by rapidly progressing MAS-HLH with central nervous system (n = 5) and/or pulmonary (n = 5) involvement. Three had underlying systemic juvenile idiopathic arthritis (sJIA), 2 had atypical sJIA (no arthritis at diagnosis), and 2 had systemic lupus erythematosus. We performed lymphocyte cytotoxicity analyses in all 7 and genetic analyses in 6. RESULTS: All children promptly responded to moderately dosed etoposide (50-100 mg/m2 once weekly), added to conventional MAS-HLH treatment that was considered insufficient. The mean accumulated etoposide dose was 671 mg/m2 (range 300-1050 mg/m2) as compared to 1500 mg/m2 recommended in the first 8 weeks of the HLH-94/HLH-2004 protocols. One child developed neutropenic fever and another neutropenic sepsis (neutrophils 0.3 × 109/L at therapy onset). Five of 7 children had low percentages (< 5%) of circulating natural killer (NK) cells prior to or in association with diagnosis; NK cell activity was pathologically low in 2 of 5 children studied. Disease-causing variants in HLH-associated genes were not found. All children were alive at latest follow-up (2-9 yrs after onset); neurological symptoms had normalized in 4 of 5 affected children. CONCLUSION: Moderately dosed etoposide may be beneficial in severe and/or refractory MAS-HLH.

Chondrodysplasia and growth failure in children after early hematopoietic stem cell transplantation for non-oncologic disorders.

Bone dysplasias (osteochondrodysplasias) are a large group of conditions associated with short stature, skeletal disproportion, and radiographic abnormalities of skeletal elements. Nearly all are genetic in origin. We report a series of seven children with similar findings of chondrodysplasia and growth failure following early hematopoietic stem cell transplantation (HSCT) for pediatric non-oncologic disease: hemophagocytic lymphohistiocytosis or HLH (five children, three with biallelic HLH-associated variants [in PRF1 and UNC13D] and one with HLH secondary to visceral Leishmaniasis), one child with severe combined immunodeficiency and one with Omenn syndrome (both children had biallelic RAG1 pathogenic variants). All children had normal growth and no sign of chondrodysplasia at birth and prior to their primary disease. After HSCT, all children developed growth failure, with standard deviation scores for height at or below -3. Radiographically, all children had changes in the spine, metaphyses and epiphyses, compatible with a spondyloepimetaphyseal dysplasia. Genomic sequencing failed to detect pathogenic variants in genes associated with osteochondrodysplasias. We propose that such chondrodysplasia with growth failure is a novel, rare, but clinically important complication following early HSCT for non-oncologic pediatric diseases. The pathogenesis is unknown but could possibly involve loss or perturbation of the cartilage-bone stem cell population.

Diagnostic challenges for a novel SH2D1A mutation associated with X-linked lymphoproliferative disease.

Mutations in SH2D1A, encoding the intracellular adaptor signaling lymphocyte activation molecule associated protein (SAP), are associated with X-linked lymphoproliferative disease type 1 (XLP1). We identified a novel hemizygous SH2D1A c.49G > A (p.E17K) variant in a 21-year-old patient with fatal Epstein-Barr virus infection-associated hemophagocytic lymphohistiocytosis. Cellular and biochemical assays revealed normal expression of the SAP variant protein, yet binding to phosphorylated CD244 receptor was reduced by >95%. Three healthy brothers carried the SH2D1A c.49G > A variant. Thus, data suggest that this variant represents a pathogenic mutation, but with variable expressivity. Importantly, our results highlight challenges in the clinical interpretation of SH2D1A variants and caution in using functional flow cytometry assays for the diagnosis of XLP1.

Long-term outcome of LRBA deficiency in 76 patients after various treatment modalities as evaluated by the immune deficiency and dysregulation activity (IDDA) score.

BACKGROUND: Recent findings strongly support hematopoietic stem cell transplantation (HSCT) in patients with severe presentation of LPS-responsive beige-like anchor protein (LRBA) deficiency, but long-term follow-up and survival data beyond previous patient reports or meta-reviews are scarce for those patients who do not receive a transplant. OBJECTIVE: This international retrospective study was conducted to elucidate the longitudinal clinical course of patients with LRBA deficiency who do and do not receive a transplant. METHOD: We assessed disease burden and treatment responses with a specially developed immune deficiency and dysregulation activity score, reflecting the sum and severity of organ involvement and infections, days of hospitalization, supportive care requirements, and performance indices. RESULTS: Of 76 patients with LRBA deficiency from 29 centers (median follow-up, 10 years; range, 1-52), 24 underwent HSCT from 2005 to 2019. The overall survival rate after HSCT (median follow-up, 20 months) was 70.8% (17 of 24 patients); all deaths were due to nonspecific, early, transplant-related mortality. Currently, 82.7% of patients who did not receive a transplant (43 of 52; age range, 3-69 years) are alive. Of 17 HSCT survivors, 7 are in complete remission and 5 are in good partial remission without treatment (together, 12 of 17 [70.6%]). In contrast, only 5 of 43 patients who did not receive a transplant (11.6%) are without immunosuppression. Immune deficiency and dysregulation activity scores were significantly lower in patients who survived HSCT than in those receiving conventional treatment (P = .005) or in patients who received abatacept or sirolimus as compared with other therapies, and in patients with residual LRBA expression. Higher disease burden, longer duration before HSCT, and lung involvement were associated with poor outcome. CONCLUSION: The lifelong disease activity, implying a need for immunosuppression and risk of malignancy, must be weighed against the risks of HSCT.

Publisher Correction: Human DEF6 deficiency underlies an immunodeficiency syndrome with systemic autoimmunity and aberrant CTLA-4 homeostasis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Human DEF6 deficiency underlies an immunodeficiency syndrome with systemic autoimmunity and aberrant CTLA-4 homeostasis.

Immune responses need to be controlled tightly to prevent autoimmune diseases, yet underlying molecular mechanisms remain partially understood. Here, we identify biallelic mutations in three patients from two unrelated families in differentially expressed in FDCP6 homolog (DEF6) as the molecular cause of an inborn error of immunity with systemic autoimmunity. Patient T cells exhibit impaired regulation of CTLA-4 surface trafficking associated with reduced functional CTLA-4 availability, which is replicated in DEF6-knockout Jurkat cells. Mechanistically, we identify the small GTPase RAB11 as an interactor of the guanine nucleotide exchange factor DEF6, and find disrupted binding of mutant DEF6 to RAB11 as well as reduced RAB11+CTLA-4+ vesicles in DEF6-mutated cells. One of the patients has been treated with CTLA-4-Ig and achieved sustained remission. Collectively, we uncover DEF6 as player in immune homeostasis ensuring availability of the checkpoint protein CTLA-4 at T-cell surface, identifying a potential target for autoimmune and/or cancer therapy.

Haploinsufficiency of UNC13D increases the risk of lymphoma.

BACKGROUND: Experimental models have demonstrated that immune surveillance by cytotoxic lymphocytes can protect from spontaneous neoplasms and cancer. In humans, defective lymphocyte cytotoxicity is associated with the development of hemophagocytic lymphohistiocytosis, a hyperinflammatory syndrome. However, to the best of the authors' knowledge, the degree to which human lymphocyte cytotoxicity protects from cancer remains unclear. In the current study, the authors examined the risk of lymphoma attributable to haploinsufficiency in a gene required for lymphocyte cytotoxicity. METHODS: The authors exploited a founder effect of an UNC13D inversion, which abolishes Munc13-4 expression and causes hemophagocytic lymphohistiocytosis in an autosomal recessive manner. Within 2 epidemiological screening programs in northern Sweden, an area demonstrating a founder effect of this specific UNC13D mutation, all individuals with a diagnosis of lymphoma (487 patients) and matched controls (1844 controls) were assessed using polymerase chain reaction for carrier status. RESULTS: Among 487 individuals with lymphoma, 15 (3.1%) were heterozygous carriers of the UNC13D inversion, compared with 18 controls (1.0%) (odds ratio, 3.0; P = .002). It is interesting to note that a higher risk of lymphoma was attributed to female carriers (odds ratio, 3.7; P = .004). CONCLUSIONS: Establishing a high regional prevalence of the UNC13D inversion, the authors have reported an overrepresentation of this mutation in individuals with lymphoma. Therefore, the results of the current study indicate that haploinsufficiency of a gene required for lymphocyte cytotoxicity can predispose patients to lymphoma, suggesting the importance of cytotoxic lymphocyte-mediated surveillance of cancer. Furthermore, the results of the current study suggest that female carriers are more susceptible to lymphoma.

Differences in Granule Morphology yet Equally Impaired Exocytosis among Cytotoxic T Cells and NK Cells from Chediak-Higashi Syndrome Patients.

Chediak-Higashi syndrome (CHS) is caused by autosomal recessive mutations in LYST, resulting in enlarged lysosomal compartments in multiple cell types. CHS patients display oculocutaneous albinism and may develop life-threatening hemophagocytic lymphohistiocytosis (HLH). While NK cell-mediated cytotoxicity has been reported to be uniformly defective, variable defects in T cell-mediated cytotoxicity has been observed. The latter has been linked to the degree of HLH susceptibility. Since the discrepancies in NK cell- and T cell-mediated cellular cytotoxicity might result from differences in regulation of cytotoxic granule release, we here evaluated perforin-containing secretory lysosome size and number in freshly isolated lymphocytes from CHS patients and furthermore compared their exocytic capacities. Whereas NK cells from CHS patients generally contained a single, gigantic perforin-containing granule, cytotoxic T cells predominantly contained several smaller granules. Nonetheless, in a cohort of 21 CHS patients, cytotoxic T cell and NK cell granule exocytosis were similarly impaired upon activating receptor stimulation. Mechanistically, polarization of cytotoxic granules was defective in cytotoxic lymphocytes from CHS patients, with EEA1, a marker of early endosomes, mislocalizing to lysosomal structures. The results leads to the conclusion that lysosome enlargement corresponds to loss of distinct organelle identity in the endocytic pathway, which on a subcellular level more adversely affects NK cells than T cells. Hence, vesicular size or numbers do not per se dictate the impairment of lysosomal exocytosis in the two cell types studied.

Successful Hematopoietic Stem Cell Transplantation in a Patient with LPS-Responsive Beige-Like Anchor (LRBA) Gene Mutation.

PURPOSE: Autosomal recessive mutations in LRBA, encoding for LPS-responsive beige-like anchor protein, were described in patients with a common variable immunodeficiency (CVID)-like disease characterized by hypogammaglobulinemia, autoimmune cytopenias, and enteropathy. Here, we detail the clinical, immunological, and genetic features of a patient with severe autoimmune manifestations. METHODS: Whole exome sequencing was performed to establish a molecular diagnosis. Evaluation of lymphocyte subsets was performed for immunological characterization. Medical files were reviewed to collect clinical and immunological data. RESULTS: A 7-year-old boy, born to consanguineous parents, presented with autoimmune hemolytic anemia, hepatosplenomegaly, autoimmune thyroiditis, and severe autoimmune gastrointestinal manifestations. Immunological investigations revealed low immunoglobulin levels and low numbers of B and NK cells. Treatment included immunoglobulin replacement and immunosuppressive therapy. Seven years after disease onset, the patient developed severe neurological symptoms resembling acute disseminated encephalomyelitis, prompting allogeneic hematopoietic stem cell transplantation (HSCT) with the HLA-identical mother as donor. Whole exome sequencing of the patient uncovered a homozygous 1 bp deletion in LRBA (c.7162delA:p.T2388Pfs*7). Importantly, during 2 years of follow-up post-HSCT, marked clinical improvement and recovery of immune function was observed. CONCLUSIONS: Our data suggest a beneficial effect of HSCT in patients with LRBA deficiency.

Targeted high-throughput sequencing for genetic diagnostics of hemophagocytic lymphohistiocytosis.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a rapid-onset, potentially fatal hyperinflammatory syndrome. A prompt molecular diagnosis is crucial for appropriate clinical management. Here, we validated and prospectively evaluated a targeted high-throughput sequencing approach for HLH diagnostics. METHODS: A high-throughput sequencing strategy of 12 genes linked to HLH was validated in 13 patients with previously identified HLH-associated mutations and prospectively evaluated in 58 HLH patients. Moreover, 2504 healthy individuals from the 1000 Genomes project were analyzed in silico for variants in the same genes. RESULTS: Analyses revealed a mutation detection sensitivity of 97.3%, an average coverage per gene of 98.0%, and adequate coverage over 98.6% of sites previously reported as mutated in these genes. In the prospective cohort, we achieved a diagnosis in 22 out of 58 patients (38%). Genetically undiagnosed HLH patients had a later age at onset and manifested higher frequencies of known secondary HLH triggers. Rare, putatively pathogenic monoallelic variants were identified in nine patients. However, such monoallelic variants were not enriched compared with healthy individuals. CONCLUSIONS: We have established a comprehensive high-throughput platform for genetic screening of patients with HLH. Almost all cases with reduced natural killer cell function received a diagnosis, but the majority of the prospective cases remain genetically unexplained, highlighting genetic heterogeneity and environmental impact within HLH. Moreover, in silico analyses of the genetic variation affecting HLH-related genes in the general population suggest caution with respect to interpreting causality between monoallelic mutations and HLH. A complete understanding of the genetic susceptibility to HLH thus requires further in-depth investigations, including genome sequencing and detailed immunological characterization.

Cancer risk in relatives of patients with a primary disorder of lymphocyte cytotoxicity: a retrospective cohort study.

BACKGROUND: Mutations in genes for perforin-dependent lymphocyte cytotoxicity are associated with haemophagocytic lymphohistiocytosis, a rare disease of severe hyperinflammation that typically becomes evident in early childhood. It has been suggested that individuals with hypomorphic biallelic mutations in genes associated with haemophagocytic lymphohistiocytosis are at increased risk of developing haematological malignancies. We aimed to assess whether relatives of patients with primary haemophagocytic lymphohistiocytosis (ie, heterozygous carriers of these mutated genes) were more likely to develop cancer. METHODS: In this retrospective cohort study, we used a multigeneration registry to identify relatives (parents and grandparents) of 79 Swedish children (<15 years) with primary haemophagocytic lymphohistiocytosis diagnosed between 1971 and 2011. For each relative, we randomly selected eight matched individuals from the Swedish total population registry, stratified for sex, birth year, and birth region. Relatives and matched controls were cross-linked with the Swedish Cancer Registry to establish cancer incidence rate. We then calculated the incidence rate ratio between first-degree and second-degree relatives and the matched controls. Additionally, we assessed natural-killer-cell-mediated cytotoxicity in a subgroup of first-degree relatives using standard 4 h (51)Cr assay and flow cytometry quantification of the upregulation of surface CD107a. FINDINGS: We identified 346 first-degree and second-degree relatives from 67 families (67 mothers, 66 fathers, 106 grandmothers, and 107 grandfathers) and 2768 matched controls. Median follow-up was 49 years, range 0-54 years. By death or last follow-up (Dec 31, 2012), first-degree relatives had a significantly increased incidence rate of malignancies than did controls (incidence rate per 1000 person-years 2.78 [95% CI 1.42-4.15] vs 1.56 [1.16-1.95]; incidence rate ratio 1.79 [95% CI 1.06-3.03]; p=0.030). Mothers had a particularly increased risk (incidence rate per 1000 person-years 4.43 [95% CI 1.99-6.87] vs 1.60 [1.08-2.11]; incidence rate ratio 2.78 [95% CI 1.48-5.21]; p=0.0014), whereas no difference was found between fathers and controls (1.24 [0.00-2.51] vs 1.52 [0.89-2.15]; 0.82 [0.29-2.29]; p=0.70) or between grandparents and controls (7.24 [5.44-9.04] vs 6.36 [5.70-7.03]; 1.14 [0.88-1.48]; p=0.33). Functional analysis of heterozygous carriers of mutations associated with haemophagocytic lymphohistiocytosis could not show significantly reduced lymphocyte cytotoxicity. INTERPRETATION: Heterozygous mutations in genes associated with haemophagocytic lymphohistiocytosis might be a new risk factor for cancer. The increased risk of cancer might imply haploinsufficiency of cytotoxic lymphocyte-mediated immunosurveillance of cancer in carriers of these mutations. Our findings might support intensified screening for malignancies in relatives of patients with haemophagocytic lymphohistiocytosis. FUNDING: Swedish Children's Cancer Foundation, Swedish Research Council, Histiocytosis Association, Swedish Cancer Society, Swedish Cancer and Allergy Foundation, Mary Béve Foundation, Karolinska Institutet Research Foundation, Stockholm County Council (ALF-project).

Spectrum of Atypical Clinical Presentations in Patients with Biallelic PRF1 Missense Mutations.

BACKGROUND: Perforin, encoded by PRF1, is a pore-forming protein crucial for lymphocyte cytotoxicity. Biallelic PRF1 nonsense mutations invariably result in early-onset hemophagocytic lymphohistiocytosis (HLH), termed familial HLH type 2 (FHL2). In contrast, biallelic PRF1 missense mutations may give rise to later-onset disease and more variable manifestations. PROCEDURE: We retrospectively searched our database for patients from families with siblings carrying biallelic PRF1 missense mutations where at least one sibling did not develop HLH, and for patients with biallelic PRF1 missense mutations and an atypical presentation of disease. We reviewed their clinical, genetic, and immunological characteristics. RESULTS: In all, we identified 10 such patients, including three sibling pairs with discordant manifestations. Interestingly, in two families, siblings of late-onset HLH patients developed Hodgkin lymphoma but no HLH. In a third family, one sibling presented with recurrent HLH episodes, whereas the other remains healthy. Of note, the affected sibling also suffered from systemic lupus erythematosus. Additional unrelated patients with biallelic PRF1 missense mutations were affected by neurological disease without classical signs of HLH, gastrointestinal inflammation as initial presentation of disease, as well as a hematological malignancy. Compared to early-onset FHL2 patients, the patients with an atypical presentation displayed a partial recovery of NK cell cytotoxicity upon IL-2 stimulation in vitro. CONCLUSIONS: Our findings substantiate and expand the spectrum of clinical presentations of perforin deficiency, linking PRF1 missense mutations to lymphoma susceptibility and highlighting clinical variability within families. PRF1 mutations should, therefore, be considered as a cause of several diseases disparate to HLH.

Incidence and clinical presentation of primary hemophagocytic lymphohistiocytosis in Sweden.

BACKGROUND: Primary hemophagocytic lymphohistiocytosis (HLH) represents a group of inherited hyperinflammatory immunodeficiencies, including familial HLH (FHL), Griscelli syndrome type 2 (GS2), and X-linked lymphoproliferative syndrome (XLP). We previously reported an annual incidence of suspected primary HLH in Sweden 1971-1986 of 0.12 per 100,000 children. Here, we determined if the incidence had increased with concomitant awareness. PROCEDURE: Children <15 years old presenting with HLH 1987-2006 in Sweden were identified through the national mortality registry as well as by nation-wide inquiries to all pediatric centers. HLH was diagnosed according to the HLH-2004 diagnostic guidelines (in case of missing data of at least three of the eight diagnostic criteria, fulfillment of four was sufficient for inclusion). We defined primary HLH as patients presenting with HLH requiring transplantation or dying of disease. RESULTS: Remarkably, the minimal annual incidence rate of primary HLH remained 0.12 per 100,000 children, equating to 1.8 per 100,000 live births. Notably, an increased overall survival was observed in 1997-2006, relative to the period 1987-1996. During the subsequent 5-year period, 2007-2011, the incidence of genetically and/or functionally verified primary HLH was 0.15 per 100,000 children per year, suggesting that new assays may aid the identification of patients with primary HLH. CONCLUSION: The annual incidence of primary HLH in Sweden is 0.12-0.15 per 100,000 children per year. Pediatr Blood Cancer 2015;62:346-352. © 2014 Wiley Periodicals, Inc.