Prognostic markers in core-binding factor AML and improved survival with multiple consolidation cycles of intermediate-/high-dose cytarabine.

OBJECTIVES: Core-binding factor acute myeloid leukaemia (CBF AML) defined by t(8;21)(q22;q22) or inv(16)(p13q22)/t(16;16)(p13;q22) has a favourable prognosis; however, 30%-40% of patients still relapse after chemotherapy. We sought to evaluate the risk factors for relapse in a de novo CBF AML cohort. PATIENTS/MATERIALS/METHODS: A retrospective review of patients from four Australian tertiary centres from 2001 to 2012, comprising 40 t(8;21) and 30 inv(16) AMLs. RESULTS: Multivariate analysis identified age (P = .032) and white cell count (WCC)>40 (P = .025) as significant predictors for inferior OS and relapse, respectively. Relapse risk was higher in the inv(16) group vs the t(8;21) group (57% vs 18%, HR 4.31, 95% CI: 1.78-10.42, P = .001). Induction therapy had no bearing on OS or relapse-free survival (RFS); however, consolidation treatment with >3 cycles of intermediate-/high-dose cytarabine improved OS (P = .035) and RFS (P = .063). Five patients demonstrated post-treatment stable q PCR positivity without relapse. CONCLUSIONS: >3 consolidation cycles of intermediate-/high-dose cytarabine improves patient outcomes Age and inv(16) CBF AML subtype are predictors of inferior OS and RFS, respectively. Stable low-level MRD by qPCR does not predict relapse Similar OS in the inv(16) cohort compared to the t(8;21) cohort, despite a higher relapse rate, confirms salvageability of relapsed disease.

The dynamics of hepcidin-ferroportin internalization and consequences of a novel ferroportin disease mutation.

The hepcidin-ferroportin axis underlies the pathophysiology of many iron-associated disorders and is a key target for the development of therapeutics for treating iron-associated disorders. The aims of this study were to investigate the dynamics of hepcidin-mediated ferroportin internalization and the consequences of a novel disease-causing mutation on ferroportin function. Specific reagents for ferroportin are limited; we developed and characterized antibodies against the largest extracellular loop of ferroportin and developed a novel cell-based assay for studying hepcidin-ferroportin function. We show that hepcidin-mediated ferroportin internalization is a rapid process and could be induced using low concentrations of hepcidin. Targeted next-generation sequencing utilizing an iron metabolism gene panel developed in our group identified a novel ferroportin p.D84E variant in a patient with iron overload. Wild-type and mutant ferroportin constructs were generated, transfected into HEK293 cells and analysed using an all-in-one flow-cytometry-based assay to study the effects on hepcidin-mediated internalization and iron transport. Consistent with the classical phenotype of ferroportin disease, the p.D84E mutation results in an inability to transport iron and hepcidin insensitivity. These results validate a recently proposed 3D-structural model of ferroportin and highlight the significance of this variant in the structure and function of ferroportin. Our novel ferroportin antibody and assay will be valuable tools for investigating the regulation of hepcidin/ferroportin function and the development of novel approaches for the therapeutic modulation of iron homeostasis.

Myelodysplastic Syndrome with t(1;7) Associated with Marked Dysmegakarypoiesis & Severe Thrombocytopenia: A Case Report and Review of the Literature.

We present the case of a 70-year-old woman who had a bone marrow examination performed to investigate marked thrombocytopenia in the context of a recent history of metastatic glucagonoma. Surprisingly this identified marked dysmegakaryopoiesis and fulfilled diagnostic criteria for refractory cytopenia with multilineage dysplasia, with a relatively uncommon associated cytogenetic lesion t(1;7). We present the case and review the literature of this cytogenetic lesion.

Audit of safety and quality of the use of enoxaparin for anticoagulation in continuous renal replacement therapy.

OBJECTIVE: To evaluate the safety and efficacy of enoxaparin for anticoagulation during continuous renal replacement therapy (CRRT). DESIGN: Six-month prospective audit on filter life, anti-Xa activity and bleeding complications among patients undergoing continuous venovenous haemodiafiltration with 1.5mg/kg enoxaparin per 24 hours. The audit was conducted between June and December 2009. RESULTS: Thirteen patients were included. The median overall filter survival time was 22 hours (range, 2-176 hours). Two patients experienced minor bleeding events, but there were no major bleeding events. Systemic activity of enoxaparin was demonstrated, with a significant rise in median anti-Xa activity between assays before and during filtration (0.00 [range, 0.00-0.13] v 0.31 [range, 0.07-1.26] anti-Xa U/mL; P = 0.03). CONCLUSIONS: Enoxaparin at 1.5mg/kg/24 h appears to be effective for circuit anticoagulation in CRRT and provides significant systemic anticoagulation. Further research is required to evaluate its safety, particularly in the absence of routine anti-Xa monitoring.

Resolution of platelet function defects with imatinib therapy in a patient with chronic myeloid leukaemia in chronic phase.

Platelet function defects are frequently found in patients with chronic myeloid leukaemia. Major clinical bleeding, however, is a rare and infrequently reported complication. Platelet function abnormalities have also not been previously correlated with molecular monitoring of BCR-ABL in chronic myeloid leukaemia. We report a case of a patient with major clinical bleeding as a presenting feature of chronic myeloid leukaemia. The patient developed compartment syndrome of the thigh secondary to a haematoma developing after minor trauma. Fasciotomy was complicated by severe bleeding requiring massive transfusion. Haemostasis was only obtained after activated recombinant factor VII was administered. Laboratory investigations revealed a platelet function defect with reduced platelet aggregation to collagen, epinephrine and arachidonic acid. As imatinib therapy commenced, molecular response was associated with near-normalization of platelet function, which subsequently became significantly abnormal with molecular relapse. Electron microscopy demonstrated normal platelet ultrastructure. We conclude that dysregulated Abelson kinase plays a pathogenic role in platelet function defects associated with chronic myeloid leukaemia, and discuss the management of clinically significant bleeding in patients with platelet function defects associated with myeloproliferative disorders.

Abbreviated dose rituximab for immune-mediated hematological disorders.

As rituximab, a monoclonal antibody targeting CD20, depletes B cells, there is considerable interest in its use in immune-mediated disorders. The majority of studies employ a four-dose regimen based on practice in lymphoma therapy. We describe our experience with one or two doses of 375 mg/m(2) of rituximab in 12 patients with relapsed and refractory immune-mediated hematological disorders. Eleven patients (92%) achieved a complete remission that has been sustained in 7 for a median of 14.5 (10.5-22) months. Four patients (36%) relapsed after responses of 1-18.5 months. This preliminary experience suggests that one or two doses of rituximab may have response and relapse rates comparable to multidose regimens while potentially providing considerable cost savings. Further prospective studies are planned.

Genetic heterogeneity of granulocytes for the JAK2 V617F mutation in essential thrombocythaemia: implications for mutation detection in peripheral blood.

AIMS: The molecular pathogenesis of essential thrombocythaemia (ET) is heterogeneous. We aimed to determine the relative sensitivity of four separate molecular assays used to detect the presence of the JAK2 V617F mutation in peripheral blood from patients with essential thrombocythaemia and related myeloproliferative disorders. METHODS: Purified granulocytes from 60 patients were analysed for the presence of the JAK2 V617F mutation by direct sequencing, denaturing high-performance liquid chromatography (DHPLC), allele-specific polymerase chain reaction (PCR) and allele-specific enrichment. Clinical data were collected for all patients and correlated with assay results. RESULTS: Direct sequencing and DHPLC were relatively insensitive assays for mutation detection, together identifying only 53% of the JAK2 V617F positive cases of ET. Allele-specific PCR and allele-specific enrichment were significantly more sensitive assays and were able to identify additional ET patients that were positive for the JAK2 V617F mutation in only a minority of circulating granulocytes. Enrichment for the mutation was demonstrated in blood platelets from two of these patients. CONCLUSIONS: The observed biological difference in circulating granulocyte involvement by the JAK2 V617F clone necessitates a sensitive molecular assay for the diagnostic investigation of thrombocytosis.

Recombinant activated factor VII: treating postoperative hemorrhage in cardiac surgery.

BACKGROUND: The purpose of this study is to review the effect of recombinant activated factor VII (rFVIIa) as rescue therapy in continuing severe postoperative hemorrhage, despite conventional measures in a series of cardiac patients at our institution. METHODS: A series of all patients who received rFVIIa as rescue therapy for uncontrollable postoperative hemorrhage after cardiac surgery over a 2-year period was analyzed. We assessed and compared the use of blood products, coagulation indicators (international normalized ratio [INR], activated partial thromboplastin [APTT], and fibrinogen), and platelet levels immediately before and after the rFVIIa was given. RESULTS: Twelve patients received rFVIIa. Eight patients (75%) had thoracic aortic surgery. Bleeding stopped in all cases. Prior to the administration of rFVIIa, mean blood product usage was the following: fresh frozen plasma (FFP) 18.7 units (range, 10-40); packed cells 7.7U (range, 0-18); cryoprecipitate 19.5U (range, 8-32); and platelets 22.5U (range, 10-40). The mean coagulation results immediately prior to rFVIIa were the following: INR 2.0 (range, 1.3-8.5); APTT 60 seconds (range, 30-220); fibrinogen 3.2 gm/L (range, 1.6-6.4), and platelet count was 174,000 (range, 78,000-257,000). After rFVIIa administration the mean blood product usage was the following: FFP 0U (range, 0-2); red cells 0U (range, 0-1); cryoprecipitate 0 (range, 0); and platelets 0 (range, 0); p less than 0.0005. The mean INR was 0.9 (range, 0.7-1.5), p less than 0.001; mean APTT was 42 seconds (range, 30-87), mean fibrinogen was 3.1 (range, 1.7-4.5), and the mean platelet count was 170,000 (range, 93,000-289,000); p values not significant. There were no thrombotic complications, no cardiac ischemic events, and no deaths. CONCLUSIONS: Our results support the use of rFVIIa as rescue therapy in severe, uncontrollable, nonsurgical, postoperative hemorrhage after cardiac surgery as efficacious and safe.

A highly conserved novel family of mammalian developmental transcription factors related to Drosophila grainyhead.

The Drosophila transcription factor Grainyhead regulates several key developmental processes. Three mammalian genes, CP2, LBP-1a and LBP-9 have been previously identified as homologues of grainyhead. We now report the cloning of two new mammalian genes (Mammalian grainyhead (MGR) and Brother-of-MGR (BOM)) and one new Drosophila gene (dCP2) that rewrite the phylogeny of this family. We demonstrate that MGR and BOM are more closely related to grh, whereas CP2, LBP-1a and LBP-9 are descendants of the dCP2 gene. MGR shares the greatest sequence homology with grh, is expressed in tissue-restricted patterns more comparable to grh and binds to and transactivates the promoter of the human Engrailed-1 gene, the mammalian homologue of the key grainyhead target gene, engrailed. This sequence and functional conservation indicates that the new mammalian members of this family play important developmental roles.

Binding of the RING polycomb proteins to specific target genes in complex with the grainyhead-like family of developmental transcription factors.

The Polycomb group (PcG) of proteins represses homeotic gene expression through the assembly of multiprotein complexes on key regulatory elements. The mechanisms mediating complex assembly have remained enigmatic since most PcG proteins fail to bind DNA. We now demonstrate that the human PcG protein dinG interacts with CP2, a mammalian member of the grainyhead-like family of transcription factors, in vitro and in vivo. The functional consequence of this interaction is repression of CP2-dependent transcription. The CP2-dinG interaction is conserved in evolution with the Drosophila factor grainyhead binding to dring, the fly homologue of dinG. Electrophoretic mobility shift assays demonstrate that the grh-dring complex forms on regulatory elements of genes whose expression is repressed by grh but not on elements where grh plays an activator role. These observations reveal a novel mechanism by which PcG proteins may be anchored to specific regulatory elements in developmental genes.