Activating KIR Haplotype Influences Clinical Outcome Following HLA-Matched Sibling Hematopoietic Stem Cell Transplantation.

Natural killer cells are thought to influence the outcome of hematopoietic stem cell transplant (HSCT), impacting on relapse, overall survival, graft versus host disease and the control of infection, in part through the complex interplay between the large and genetically diverse killer immunoglobulin-like receptor (KIR) family and their ligands. This study examined the relationship between KIR gene content and clinical outcomes including the control of opportunistic infections such as cytomegalovirus in the setting of human leucocyte antigen (HLA)-matched sibling HSCT in an Australian cohort. The presence of the KIR B haplotype which contain more activating receptors in the donor, in particular centromeric B haplotype genes (Cen-B), was associated with improved overall survival of patients with acute myeloid leukemia (AML) undergoing sibling HSCT and receiving myeloablative conditioning. Donor Cen-B haplotype was also associated with reduced acute graft versus host disease grades II-IV whereas donor telomeric-B haplotype was associated with decreased incidence of CMV reactivation. In contrast, we were not able to demonstrate a reduced rate of relapse when the donor had KIR Cen-B, however relapse with a donor Cen-A haplotype was a competing risk factor to poor overall survival. Here we show that the presence of donor activating KIR led to improved outcome for the patient, potentially through reduced relapse rates and decreased incidence of acute GvHD translating to improved overall survival. This article is protected by copyright. All rights reserved.

A novel, somatic, transforming mutation in the extracellular domain of Epidermal Growth Factor Receptor identified in myeloproliferative neoplasm.

We describe a novel ERBB1/EGFR somatic mutation (p. C329R; c.985 T > C) identified in a patient with JAK2V617F Polycythaemia Vera (PV). This substitution affects a conserved cysteine residue in EGFR domain 2 and leads to the formation of a ligand-independent covalent receptor dimer, associated with increased transforming potential. Aberrant signalling from the EGFRC329R receptor is cell type-dependent and in the TF1.8 erythroid cell line expression of this mutant suppresses EPO-induced differentiation. Clonal analysis shows that the dominant JAK2V617F-positive clone in this PV patient harbors EGFRC329R, thus this mutation may contribute to clonal expansion. Somatic mutations affecting other ERBB and related receptor tyrosine kinases are observed in myeloproliferative neoplasms (MPN), and we show elevated EGFR levels in MPN samples, consistent with previous reports. Thus activation of this group of receptors, via multiple mechanisms, may contribute to clonal growth and survival of the JAK2V617F disease clone in MPN.

Consensus guidelines for the use of empiric and diagnostic-driven antifungal treatment strategies in haematological malignancy, 2014.

Invasive fungal disease (IFD) causes significant morbidity and mortality in patients undergoing allogeneic haemopoietic stem cell transplantation or chemotherapy for haematological malignancy. Much of these adverse outcomes are due to the limited ability of traditional diagnostic tests (i.e. culture and histology) to make an early and accurate diagnosis. As persistent or recurrent fevers of unknown origin (PFUO) in neutropenic patients despite broad-spectrum antibiotics have been associated with the development of IFD, most centres have traditionally administered empiric antifungal therapy (EAFT) to patients with PFUO. However, use of an EAFT strategy has not been shown to have an overall survival benefit and is associated with excessive antifungal therapy use. As a result, the focus has shifted to developing more sensitive and specific diagnostic tests for early and more targeted antifungal treatment. These tests, including the galactomannan enzyme-linked immunosorbent assay and Aspergillus polymerase chain reaction (PCR), have enabled the development of diagnostic-driven antifungal treatment (DDAT) strategies, which have been shown to be safe and feasible, reducing antifungal usage. In addition, the development of effective antifungal prophylactic strategies has changed the landscape in terms of the incidence and types of IFD that clinicians have encountered. In this review, we examine the current role of EAFT and provide up-to-date data on the newer diagnostic tests and algorithms available for use in EAFT and DDAT strategies, within the context of patient risk and type of antifungal prophylaxis used.

Diagnostic and therapeutic approach to persistent or recurrent fevers of unknown origin in adult stem cell transplantation and haematological malignancy.

Persistent or recurrent fevers of unknown origin (PFUO) in neutropenic patients on broad-spectrum antibiotics have traditionally been treated with empirical antifungal therapy (EAFT). The lack of survival benefit seen with the use of amphotericin B deoxycholate (AmB-D) as EAFT has been attributed to its toxicities. More recently, newer, less toxic and more expensive antifungal agents such as the lipid formulations of AmB, the newer azoles (fluconazole, itraconazole and voriconazole) and caspofungin have been analysed in a number of EAFT trials. Compared with AmB-D the newer agents have superior safety but are of equivalent efficacy. This lack of survival advantage is related to the fact that the trigger for commencement of EAFT is late and non-specific. Thus, alternative approaches are required. New sensitive serological and molecular tests for the detection of Aspergillus antigens and genomic DNA have been developed and evaluated in accuracy studies. These tests have been incorporated into management strategies (i.e. pre-emptive strategies) to direct antifungal therapy. The pre-emptive approach has been shown to be safe and feasible but its impact on clinically important patient outcomes such as survival is less clear. Other advances include the introduction of effective, non-toxic mould-active antifungal prophylaxis and patient risk-group stratification. In this paper we provide new evidence-based algorithms for the diagnosis and treatment of PFUO in adult patients undergoing stem cell transplantation and chemotherapy for haematological malignancy which incorporate these newer diagnostic tests and are directed by the risk category of the patient and type of antifungal prophylaxis the patient is receiving.

A prospective multicenter trial of peripheral blood stem cell sibling allografts for acute myeloid leukemia in first complete remission using fludarabine-cyclophosphamide reduced intensity conditioning.

The role of allogeneic transplantation in patients with de novo acute myeloid leukemia in first complete remission (AML-CR1) is controversial. Aiming to preserve a graft-versus-leukemia effect, but minimize morbidity and mortality from conditioning-related toxicity and graft-versus-host disease (GVHD), we conducted a prospective multicenter study of reduced-intensity conditioning (RIC) as preparation for peripheral blood stem cell sibling allografts in patients with intermediate or poor risk AML-CR1. Conditioning consisted of fludarabine 125 mg/m(2) and cyclophosphamide 120 mg/kg. Thirty-four patients were transplanted with a median age of 45 years; 85% had intermediate risk cytogenetics. Early toxicity was minimal. The overall incidence of grade II-IV acute GVHD was low (21%), but the 3 patients (9%) who developed grade IV GVHD died. Donor T cell chimerism was rapid and generally complete, but complete myeloid chimerism was delayed. Thirteen patients (38%) relapsed, 12 within a year of transplant. The estimated disease-free survival (DFS) and overall survival at 2 years was 56% (95% confidence interval [CI] 39%-71%) and 68% (95% CI 50%-81%), respectively. The incidence of extensive chronic GVHD (cGVHD) was low (24% of surviving patients at 12 months) and most survivors had an excellent performance status. These observations justify a prospective comparison of RIC versus myeloablative conditioning allografts for AML-CR1.

Mannose-binding lectin: biology and clinical implications.

The innate host defence molecule mannose-binding lectin (MBL) has attracted great interest as a potential candidate for passive immunotherapy to prevent infection. MBL is a multimeric lectin that recognizes a wide array of pathogens independently of specific antibody, and initiates the lectin pathway of complement activation. The basic structural unit is a triple helix of MBL peptides, which aggregate into complement-fixing higher-order structures (tetramers, pentamers and hexamers). The gene encoding MBL, MBL2, contains several common polymorphisms that influence transcription and assembly of the molecule into multimers. MBL2 coding alleles associated with low blood levels are present in up to 40% of Caucasoids, with up to 8% having genotypes associated with profound reduction in circulating MBL levels. Low-producing MBL2 variants and low MBL levels are associated with increased susceptibility to and severity of a variety of infective illnesses, particularly when immunity is already compromised--for example, in infants and young children, patients with cystic fibrosis, and after chemotherapy and transplantation. These observations suggest that administration of recombinant or purified MBL may be of benefit in clinical settings where MBL deficiency is associated with a high burden of infection. This review provides a background to MBL biology and disease associations, and identifies the exciting therapeutic possibilities of MBL replacement.

Fas gene promoter polymorphisms in primary Sjögren's syndrome.

BACKGROUND: Fas mediated apoptosis may be important in the pathogenesis of primary Sjögren's syndrome (pSS). OBJECTIVE: To examine genetic variation in the promoter region of the Fas gene in pSS. METHODS: Two single nucleotide polymorphisms at positions -1377(G/A) and -670(G/A) in the Fas gene promoter were genotyped by PCR-SSP in 101 patients with pSS and 108 Caucasoid controls. RESULTS: No significant differences in allele or genotype frequencies were detected between the patients with pSS and controls. However, significant associations were observed with Ro/La autoantibody negative patients, who display milder and later onset disease. The -670A allele was more frequent in Ro/La autoantibody negative patients than in Ro/La autoantibody positive patients (p = 0.04). CONCLUSION: This study does not confirm an earlier report of an association between pSS and the Fas promoter -670G allele. However, the results suggest that genetically determined variability in Fas expression may modulate Ro/La autoantibody responses in patients with pSS.

Variation in immune response genes and chronic Q fever. Concepts: preliminary test with post-Q fever fatigue syndrome.

Acute primary Q fever is followed by various chronic sequelae. These include subacute Q fever endocarditis, granulomatous reactions in various organs or a prolonged debilitating post-infection fatigue syndrome (QFS). The causative organism, Coxiella burnetii, persists after an initial infection. The differing chronic outcomes may reflect variations within cytokine and accessory immune control genes which affect regulation of the level of persistence. As a preliminary test of the concept we have genotyped QFS patients and controls for gene variants spanning 15 genes and also examined HLA-B and DR frequencies. QFS patients exhibited a significantly increased frequency of HLA-DR-11 compared with controls and also significant differences in allelic variant frequencies within the NRAMP, and IFNgamma genes. These results indicate a possible genetic role in the expression of overt chronic Q fever. Further studies will be undertaken to increase sample sizes, to survey other forms of chronic Q fever and to examine Q fever patients who have recovered without sequelae.

The addition of allogeneic peripheral blood-derived progenitor cells to bone marrow for transplantation: results of a randomised clinical trial.

BACKGROUND: The use of cytokine-mobilised peripheral-blood-derived progenitor cells (PBPC) has resulted in a significant improvement in the safety of autologous transplantation. Collections of PBPC contain large numbers of haemopoietic progenitors and T-lymphocytes when compared with bone marrow (BM). AIMS: To assess the clinical effects and safety of filgrastim-mobilised allogeneic PBPC, in particular whether PBPC would alter the kinetics of engraftment or the incidence and severity of graft-versus-host disease (GVHD). METHODS: Twenty-seven patients undergoing allogeneic transplantation were randomised to receive BM or BM supplemented by PBPC. Patients were conditioned with busulphan 16 mg/kg and cyclophosphamide 120 mg/kg. GVHD prophylaxis was with methotrexate and cyclosporin. Ganciclovir prophylaxis was administered to all cytomegalovirus seropositive patients. No haemopoietic growth factor was used after BMT. Donors of PBPC underwent a single, seven litre apheresis after four days of filgrastim, 10 microg/kg/day by subcutaneous injection. RESULTS: Donor toxicity was mild, with the most frequently reported being bone pain in the cytokine-treated donors. The patients transplanted with BM+PBPC received an eight-fold increase in CD3+ T-lymphocytes (1.65X 10(8)kg vs 0.22 x 10(8)/kg, p=0.04) and the cytokine product contained a median of 5 x 10(6) CD34+ cells/kg. BM+PBPC patients had more rapid engraftment of neutrophils to 0.5x 10(9)/L (17 days vs 19 days, p=0.02) and platelets to 50X 10(9)/L (22 days vs 35 days, p=0.04). BM megakaryocyte numbers were significantly enhanced at days 14 and 28 after BMT (both p=0.04), however platelet transfusions were not significantly different. The incidence, organ distribution, severity and time to onset of acute and chronic GVHD were not different between the treatment groups and there was no difference in overall survival or event-free survival. CONCLUSIONS: Allogeneic PBPC from HLA-identical siblings may speed engraftment of neutrophils and platelets without detrimental effects on GVHD or survival.

Human GM-CSF receptor alpha-chain gene is highly polymorphic but not rearranged in AML.

Acute myeloid leukaemia (AML) blast cells express haemopoietic growth factor receptors. However, their presence does not predict response to the cognate ligand in vitro. This suggests that haemopoietic growth factor receptor structure or function may be abnormal in some cases of acute myeloid leukaemia. The granulocyte-macrophage colony-stimulating factor receptor alpha-chain gene (GM-CSF-R) has recently been localised to the pseudoautosomal region of the sex chromosomes. A sex chromosome is lost in 25% of cases of AML FAB subtype M2. The loss of one allele of this gene may have some aetiological significance in AML if the other allele is altered leading to abnormal receptor structure, function or number. In this initial study, we have examined DNA from leukaemic cells of 29 patients with AML, including three with FAB subtype M2 with deletion of an X or Y chromosome for evidence of gross rearrangement of this gene. We report that although the gene is highly polymorphic for a number of restriction enzymes, we have found no evidence of gross rearrangement in AML.

Human interleukin-3 mRNA accumulation is controlled at both the transcriptional and posttranscriptional level.

Interleukin-3 (IL-3) is a hematopoietic growth factor that regulates the differentiation of multilineage and committed progenitor cells and the functions of some mature blood cells. The expression of human IL-3 appears to be restricted to stimulated T lymphocytes. We have investigated the kinetics and mechanisms involved in the induction of IL-3 expression in the human T lymphocytic tumor cell line Jurkat. We show that accumulation of IL-3 mRNA is controlled at both the transcriptional and posttranscriptional level. Transcription of the IL-3 gene in these cells appears to be constitutive but no IL-3 mRNA was detected in unstimulated cells, indicating that in resting cells IL-3 mRNA is highly unstable. Treatment with phytohemagglutinin (PHA) induced a small and transient increase in the IL-3 gene transcription rate and led to the production of detectable levels of IL-3 mRNA and protein. Optimal induction of IL-3 expression required a second stimulus. Costimulation of Jurkat cells with both phorbol myristate acetate and PHA caused both a transient increase in IL-3 gene transcription, which is dependent on new protein synthesis, and also a transient increase in mRNA stability.