ALBERT

Description: Key Points Synergistic effects were observed in the granule mediated lymphocyte cytotoxicity. Digenic pathogenesis contributed to the development of hemophagocytic lymphohistiocytosis.

Description: Platelets are vital for hemostasis because they release their granule contents in response to vascular damage. Platelet exocytosis is mediated by soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs), whose interactions are governed by regulators, eg, Sec/Munc18 proteins. These proteins chaperone syntaxin t-SNAREs and are required for exocytosis. Platelets contain 3 Munc18 isoforms: Munc18a, Munc18b, and Munc18c. We report that Munc18b is the major isoform and is required for platelet secretion. Familial hemophagocytic lymphohistiocytosis type 5 (FHL5) is caused by defects in the Munc18b/STXBP2 gene. We confirm a previous report showing that platelets from FHL5 patients have defective secretion. Serotonin, ADP/ATP, and platelet factor 4 release was profoundly affected in the 2 biallelic patients and partially in a heterozygous patient. Release of lysosomal contents was only affected in the biallelic platelets. Platelets from the FHL5 biallelic patients showed decreased Munc18b and syntaxin-11 levels were significantly reduced; other syntaxins were unaffected. Munc18b formed complexes with syntaxin-11, SNAP-23, and vesicle-associated membrane protein-8 in human platelets. Other potential secretion regulators, Munc13-4 and Rab27, were also found associated. These data demonstrate a key role for Munc18b, perhaps as a limiting factor, in platelet exocytosis and suggest that it regulates syntaxin-11.

Description: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder of immune regulation leading to widespread lymphocytic and hemophagocytic infiltration of vital organs. Apparent cure has only been achieved with allogeneic bone marrow transplantation (BMT). This report describes 20 consecutive patients, who underwent either matched sibling donor (n = 4) or unrelated donor (URD; n = 16) BMT. Age at the time of BMT was 0.4 to 5.3 years (median, 0.8 years). Central nervous system disease was present at diagnosis in 13 patients. At BMT, 14 patients were in a clinical remission, whereas 6 patients had active HLH. All patients were engrafted after cytoreduction with busulfan, cyclophosphamide, and etoposide. The probability of grade II-III acute graft-versus-host disease (GVHD) for all patients was 57% (95% confidence limit [CL], 0.28, 0.86), and 73% (95% CL, 0.44, 1.0) in URD patients. The overall probability of survival at 3 years was 45% (95% CL, 0.23, 0.67) and 44% (95% CL, 0.19, 0.68) when URD BMT was evaluated separately. Favorable BMT outcome was associated with clinical remission status at the time of BMT. The preparative regimen was well tolerated, and in the 9 surviving patients it provided durable engraftment and was effective at eradicating the underlying disease.

Description: Abstract 2557 Monosomy 7 is a common chromosomal abnormality in adults with myelodysplasia (MDS) and AML, seen most frequently in older persons. It is believed to be one of a series of stochastically acquired genetic anomalies that accumulate over many years to complete the process of leukemogenesis. MDS in general and monosomy 7 in particular are rarely seen in children, although cases in very young children are reported. Monosomy 7 arising in very young children likely has a different etiology and may contribute differently to leukemogenesis, as the young age of the children does not allow sufficient time for stochastic acquisition of mutation in the same way as in elderly persons. A proportion of young children with monosomy 7 have an associated DNA instability disorder such as Fanconi anemia (FA) or Dyskeratosis Congenita, and this genetic background likely plays a key role in the evolution of malignancy. In a few reported cases, the occurrence of monosomy 7 is familial, and in a smaller number of cases yet, the clone may resolve spontaneously or wax and wane, as the associated pancytopenia waxes and wanes. We report 5 young children with monosomy 7 in whom we have explored the origins of monosomy 7. In case 1, who presented with MDS and monosomy 7 at 2 years of age, a stored cord blood was available for analysis. The sample was flow sorted and FISH for monosomy 7 was performed on different populations. The monosomy 7 clone was present in approximately 10% of the cord blood cells overall, and the same clone was found in the most primitive population, i.e. CD34+CD38- cells, suggesting in utero origin of this abnormal clone in a very early precursor or stem cell population (Table 1). Similar analyses done on BM or PB samples of 4 additional young patients with monosomy 7, with age range of 4 months to 42 months (3 ½ years)(median 22.5 months), also showed the abnormal clone to be present in CD34+ cells, supporting our hypothesis that monosomy 7 arises in early precursors or stem cells (Table 2, below). In fact, we were able to confirm the presence of monosomy 7 in the CD34+CD38- population in one of these additional cases for which sufficient material was available. Interestingly, in one patient (#4) where we had sequential samplings nine months apart, total BM showed a decrease in the percentage of cells with monosomy 7, from 2.6% to 1.1%. However, in sorted CD34+ cells, we detected a nearly 10 fold increase in cells with monosomy 7 (Table 2). Clinical significance of this finding remains to be determined In summary, our preliminary studies provide proof of concept that in children, the abnormal monosomy 7 clone is present in the hematopoietic stem cell compartment, and can arise in utero in the absence of any known genetic susceptibility syndrome. Table 1. Patient 1 (UCB) FACS Analysis: % of Cord Blood Cells % Monosomy 7 by FISH CD3 (T cells) 23.1% 6/66 (9.1%) CD 19 (B cells) 9.1% 1/44 (0.02%) CD 14 (monocytes) 4% 1/8 (12.5%) CD 16 (granulocytes) 21.5% 0/20 (0%) CD34 1% 70% CD34+CD38- (primitive progenitor) NA 124/136 (91.2%) CD34+38+19-7- (myeloid progenitor) NA 8/13 (61.5%) Table 2 Patient # (BM or PB) % of total sample % Monosomy 7 by FISH % of total sample % Monosomy 7 by FISH % of total sample % Monosomy 7 by FISH % of total sample % Monosomy 7 by FISH % of total sample % Monosomy 7 by FISH % of total sample % Monosomy 7 by FISH CD3 (T cells) CD 19 (B cells) CD 14 (monocytes) CD 16 (granulocytes) CD34+38+ or CD34+ ( progenitor) CD34+CD38- (primitive progenitor) 2 BM 27.0 0.4 1.9 10.9 5.9 46.4 11.3 16.9 1.2 77 (34+) NA NA 3 BM 40.3 2.5 6.6 1.7 2.4 94 15.9 92.4 0.24 78.3 (34+) .01 NA 4-1 BM 37.4 0 13.3 0 3.7 5.9 15.0 3.8 3.9 1.3 1.7 2.8 4-2 BM 13.0 2 9.5 4 0.4 0 35.6 0.4 0.2 12.2 (34+) NA NA 5 PB 21.8 1.5 3.4 NA 22.4 100 9.1 100 NA NA NA NA Disclosures: No relevant conflicts of interest to declare.

Description: X-linked lymphoproliferative disease (XLP1), described in the mid-1970s and molecularly defined in 1998, and XLP2, reported in 2006, are prematurely lethal genetic immunodeficiencies that share susceptibility to overwhelming inflammatory responses to certain infectious triggers. Signaling lymphocytic activation molecule-associated protein (SAP; encoded by SH2D1A) is mutated in XLP1, and X-linked inhibitor of apoptosis (XIAP; encoded by BIRC4) is mutated in XLP2. XLP1 is a disease with multiple and variable clinical consequences, including fatal hemophagocytic lymphohistiocytosis (HLH) triggered predominantly by Epstein-Barr virus, lymphomas, antibody deficiency, and rarer consequences of immune dysregulation. To date, XLP2 has been found to cause HLH with and without exposure to Epstein-Barr virus, and HLH is commonly recurrent in these patients. For both forms of XLP, the only curative therapy at present is allogeneic hematopoietic cell transplantation. Beyond their common X-linked locus and their requirement for normal immune responses to certain viral infections, SAP and XIAP demonstrate no obvious structural or functional similarity, are not coordinately regulated with respect to their expression, and do not appear to directly interact. In this review, we describe the genetic, clinical, and immunopathologic features of these 2 disorders and discuss current diagnostic and therapeutic strategies.

Description: Abstract SCI-9 Hemophagocytic lymphohistiocytosis (HLH) is a group of immunodeficiencies characterized by clinical signs and symptoms of extreme inflammation. HLH is now more widely recognized and no longer viewed as a disorder of young children only, as more adults are being diagnosed and treated. HLH is defined by a unique pattern of clinical findings. In addition to fevers, cytopenias, hepatitis, and splenomegaly, markedly increased levels of inflammatory markers in the blood (ferritin and sCD163 reflecting activation of antigen presenting cells; sIL2Ra and neopterin reflecting activation of T cells) constitute the collection of diagnostic criteria. Activation of inflammatory cells within the central nervous system (CNS) is found in approximately 50 percent of children at diagnosis and requires targeted therapy. In many cases immune defects affecting cytotoxicity of T cells and natural killer cells underlie the susceptibility to HLH. Autosomal recessive disorders include perforin deficiency (the major cytotoxin of the immune system), or defects in proteins involved in degranulation and exocytosis of perforin and granzyme B (MUNC 13–4, MUNC18-2, STX11, Rab27a). The latter proteins are involved in degranulation generally within the hematopoietic system, thus impacting the function of neutrophils and platelets as well. A rare defect of granulogenesis, Chediak Higashi syndrome, is also associated with a high incidence of HLH. Two forms of X-linked lymphoproliferative syndrome (XLP1 – SAP deficiency, and XLP2 – XIAP deficiency), as well as the rare autosomal recessive disorder ITK (IL-2 inducible T cell kinase) deficiency, are characterized by a high incidence of Epstein-Barr virus-driven HLH and lymphoproliferation. A common pathogenic mechanism underlying these consequences has not yet been elucidated. Effective initial treatment for HLH consists of cytotoxic and anti-inflammatory agents. The most widely used over the past 20 years has been a combination of CNS-penetrating steroid (Decadron) and etoposide. Another approach has been to use anti-thymocyte globulin (ATG) as induction therapy. Both treatments have resulted in approximately 60 percent responses during the first month of therapy. Supportive care with broad-spectrum antimicrobials is a critical adjunct. More recently, a new induction protocol—hybrid immunotherapy for HLH, combining the features of early ATG followed by etoposide, with steroids—has been opened in the United States (http://clinicaltrials.gov/ct2/show/NCT01104025) and Europe. However, HLH persists or reactivates in nearly half of patients as immune suppression is reduced. While a common approach to reactivation is to reintensify previous therapy, no clear guidelines have been developed for this complication. The use of Campath, a humanized monoclonal anti-CD52 antibody, as salvage therapy prior to hematopoietic cell transplantation (HCT) is being tested, as both activated T cells and activated monocyte/macrophages (histiocytes) are targeted through CD52. Historically, the three-year survival after HCT in patients treated with HLH-94 was 60 percent. More recently, use of Campath-based reduced intensity conditioning protocols have led to improved results after HCT. Campath has the advantage of reducing graft-versus-host disease if properly timed prior to HCT. In a recent contemporaneous series of HCT from unrelated adult donors, three-year posttransplant survival improved from 43 percent to 92 percent with no early transplant-related mortality. Disclosures: No relevant conflicts of interest to declare.

Description: Mutations in the perforin gene have been described in some patients with hemophagocytic lymphohistiocytosis (HLH), but the role of perforin defects in the pathogenesis of HLH remains unclear. Four-color flow cytometric analysis was used to establish normal patterns of perforin expression for control subjects of all ages, and patterns of perforin staining in cytotoxic lymphocytes (natural killer [NK] cells, CD8+ T cells, CD56+ T cells) from patients with HLH and their family members were studied. Eleven unrelated HLH patients and 19 family members were analyzed prospectively. Four of the 7 patients with primary HLH showed lack of intracellular perforin in all cytotoxic cell types. All 4 patients showed mutations in the perforin gene. Their parents, obligate carriers of perforin mutations, had abnormal perforin-staining patterns. Analysis of cytotoxic cells from the other 3 patients with primary HLH and remaining family members had normal percentages of perforin-positive cytotoxic cells. On the other hand, the 4 patients with Epstein-Barr virus–associated HLH typically had depressed numbers of NK cells but markedly increased proportions of CD8+ T cells with perforin expression. Four-color flow cytometry provides diagnostic information that, in conjunction with evidence of reduced NK function, may speed the identification of life-threatening HLH in some families and direct further genetic studies of the syndrome.

Description: X-linked inhibitor of apoptosis (XIAP) deficiency, caused by BIRC4 mutations, is described to cause X-linked lymphoproliferative disease (XLP) phenotypes. However, compared with XLP caused by SLAM-Associated Protein deficiency (SH2D1A mutation), XIAP deficiency was originally observed to be associated with a high incidence of hemophagocytic lymphohistiocytosis (HLH) and a lack of lymphoma, suggesting that classification of XIAP deficiency as a cause of XLP may not be entirely accurate. To further characterize XIAP deficiency, we reviewed our experience with 10 patients from 8 unrelated families with BIRC4 mutations. Nine of 10 patients developed HLH by 8 years of age. Most patients presented in infancy, and recurrent HLH was common. There were no cases of lymphoma. Lymphocyte defects thought to contribute to HLH development in SLAM-Associated Protein deficiency were not observed in XIAP deficiency. We conclude that XIAP deficiency is a unique primary immunodeficiency that is more appropriately classified as X-linked familial hemophagocytic lymphohistiocytosis.

Description: Key Points Alemtuzumab levels impact acute GVHD, mixed chimerism, and lymphocyte recovery after alemtuzumab, fludarabine, and melphalan RIC HCT. An in vivo lytic threshold appears to lie near 0.1 to 0.16 μg/mL; targeted dose trials are warranted to optimize outcomes.

Description: Recent experience suggests that reduced-intensity conditioning (RIC) regimens can improve the outcomes of patients with hemophagocytic lymphohistiocytosis (HLH) undergoing allogeneic hematopoietic cell transplantation (HCT). However, studies directly comparing RIC to myeloablative conditioning (MAC) regimens are lacking. Forty patients with HLH underwent allogeneic HCT between 2003-2009 at Cincinnati Children's Hospital. Fourteen patients received MAC consisting of busulfan, cyclophosphamide, and antithymocyte globulin plus or minus etoposide. Twenty-six patients received RIC consisting of fludarabine, melphalan, and alemtuzumab. All patients engrafted. Acute graft-versus-host disease grades II to III occurred in 14% of MAC patients and 8% of RIC patients (P = .3171). Posttransplantation mixed donor/recipient chimerism developed in 18% of MAC patients and 65% of RIC patients (P = .0110). The majority of patients with mixed chimerism received intervention with reduction of immune suppression plus or minus donor lymphocyte infusion or stem cell boost, which stabilized or increased donor contribution to hematopoiesis and prevented relapse of HLH in all but 1 patient. Grade II to III graft-versus-host disease occurred in 5 of 14 RIC patients after donor lymphocyte infusion. The overall estimated 3-year survival after HCT was 43% (confidence interval = ± 26%) for MAC patients and 92% (confidence interval = ± 11%) for RIC patients (P = .0001). We conclude that RIC significantly improves the outcome of patients with HLH undergoing allogeneic HCT.