The effects of monoclonal anti-CD47 on RBCs, compatibility testing, and transfusion requirements in refractory acute myeloid leukemia.

BACKGROUND: CD47 is a novel therapeutic target in the treatment of solid-organ and hematologic malignancies. CD47 is also expressed on RBCs. Here, we report our experience of the RBC effects and the impact on blood bank testing and transfusion management in a Phase 1 trial of the humanized anti-CD47 monoclonal antibody Hu5F9-G4 in relapsed or primary refractory acute myeloid leukemia (AML) (NCT02678338). STUDY DESIGN AND METHODS: Nineteen patients with relapsed or primary refractory AML treated across five UK centers were included for analysis. Patients received escalating doses of Hu5F9-G4. Serial laboratory data were collected to evaluate impact on hemoglobin (Hb), markers of hemolysis (bilirubin, lactate dehydrogenase, reticulocyte count), transfusion requirements, and blood compatibility testing. RESULTS: A decline in Hb was observed with drug administration (median Hb change, -1.0 g/dL; range, 0.4-1.6) with associated increase in transfusion requirements. Patients responded to transfusion with a median Hb increment per unit of 1.0 g/dL. RBC agglutination was seen in all cases without associated change in Hb, lactate dehydrogenase, bilirubin, or reticulocyte count. Nine of 19 (47%) patients developed a newly positive antibody screen with a pan-agglutinin identified in plasma. Invalid ABO blood grouping occurred in 4 of 12 (33%) non-group O patients due to anomalous reactivity in the reverse ABO-type results. CONCLUSIONS: Treatment with Hu5F9-G4 in patients with AML resulted in an Hb decline and increased transfusion requirements. Problems with ABO blood typing and compatibility testing were widely observed and should be expected by centers treating recipients of Hu5F9-G4.

Distinct factors determine the kinetics of disease relapse in adults transplanted for acute myeloid leukaemia.

BACKGROUND: Disease recurrence remains the major cause of death in adults with acute myeloid leukaemia (AML) treated using either intensive chemotherapy (IC) or allogenic stem cell transplantation (allo-SCT). AIMS: The timely delivery of maintenance drug or cellular therapies represent emerging strategies with the potential to reduce relapse after both treatment modalities, but whilst the determinants of overall relapse risk have been extensively characterized the factors determining the timing of disease recurrence have not been characterized. MATERIALS AND METHODS: We have therefore examined, using a series of sequential landmark analyses, relapse kinetics in a cohort of 2028 patients who received an allo-SCT for AML in CR1 and separately 570 patients treated with IC alone. RESULTS: In the first 3 months after allo-SCT, the factors associated with an increased risk of relapse included the presence of the FLT3-ITD (P < 0.001), patient age (P = 0.012), time interval from CR1 to transplant (P < 0.001) and donor type (P = 0.03). Relapse from 3 to 6 months was associated with a higher white cell count at diagnosis (P = 0.001), adverse-risk cytogenetics (P < 0.001), presence of FLT3-ITD mutation (P < 0.001) and time interval to achieve first complete remission (P = 0.013). Later relapse was associated with adverse cytogenetics, mutated NPM1, absence of chronic graft-versus-host disease (GVHD) and the use of in vivo T-cell depletion. In patients treated with IC alone, the factors associated with relapse in the first 3 months were adverse-risk cytogenetics (P < 0.001) and FLT3-ITD status (P = 0.001). The factors predicting later relapse were the time interval from diagnosis to CR1 (P = 0.22) and time interval from CR1 to IC (P = 0.012). DISCUSSION AND CONCLUSION: Taken together, these data provide novel insights into the biology of disease recurrence after both allo-SCT and IC and have the potential to inform the design of novel maintenance strategies in both clinical settings.

GATA1s induces hyperproliferation of eosinophil precursors in Down syndrome transient leukemia.

Transient leukemia (TL) is evident in 5-10% of all neonates with Down syndrome (DS) and associated with N-terminal truncating GATA1 mutations (GATA1s). Here we report that TL-cell clones generate abundant eosinophils in a substantial fraction of patients. Sorted eosinophils from patients with TL and eosinophilia carried the same GATA1s mutations as sorted TL blasts, consistent with their clonal origin. TL blasts exhibited a genetic program characteristic of eosinophils and differentiated along the eosinophil lineage in vitro. Similarly, ectopic expression of Gata1s, but not Gata1, in wild-type CD34(+)-hematopoietic stem and progenitor cells induced hyperproliferation of eosinophil promyelocytes in vitro. Although GATA1s retained the function of GATA1 to induce eosinophil genes by occupying their promoter regions, GATA1s was impaired in its ability to repress oncogenic MYC and the pro-proliferative E2F transcription network. Chromatin Immunoprecipitation Sequencing (ChIP-seq) indicated reduced GATA1s occupancy at the MYC promoter. Knockdown of MYC, or the obligate E2F-cooperation partner DP1, rescued the GATA1s-induced hyperproliferative phenotype. In agreement, terminal eosinophil maturation was blocked in Gata1(Δe2) knockin mice, exclusively expressing Gata1s, leading to accumulation of eosinophil precursors in blood and bone marrow. These data suggest a direct relationship between the N-terminal truncating mutations of GATA1 and clonal eosinophilia in DS patients.

Lenalidomide monotherapy and in combination with cytarabine, daunorubicin and etoposide for high-risk myelodysplasia and acute myeloid leukaemia with chromosome 5 abnormalities.

Patients with high risk myelodysplasia (HR-MDS) and acute myeloid leukaemia (AML) with chromosomal changes involving deletion of the long arm of chromosome 5 (del5q), especially with complex karyotype, rarely have a durable response to combination chemotherapy. In the subgroup with monosomal karyotype there are no long term survivors (Fang et al., 2011) [1]. Recent experience indicates that the incidence of del5q in AML is ~20-30%, with only 20-25% of patients achieving complete remission (CR) (Farag et al., 2006) [2]. Additionally, therapy has significant toxicity, with induction death rates ~20% even in younger patients (Juliusson et al., 2009) [3]. This lack of efficacy provides the clinical rationale for combination/sequential therapy with Lenalidomide and combination chemotherapy. Dose dependent haematological toxicity is the major safety concern with such a combination protocol. Therefore we conducted a phase 2 study, AML Len5 (ISRCTN58492795), to assess safety, tolerability and efficacy of lenalidomide monotherapy, followed by lenalidomide with intensive chemotherapy in patients with primary/relapsed/refractory high risk MDS or AML with abnormalities of chromosome 5.

Azacitidine fails to eradicate leukemic stem/progenitor cell populations in patients with acute myeloid leukemia and myelodysplasia.

Epigenetic therapies demonstrate significant clinical activity in acute myeloid leukemia (AML) and myelodysplasia (MDS) and constitute an important new class of therapeutic agents. However hematological responses are not durable and disease relapse appears inevitable. Experimentally, leukemic stem/progenitor cells (LSC) propagate disease in animal models of AML and it has been postulated that their relative chemo-resistance contributes to disease relapse. We serially measured LSC numbers in patients with high-risk AML and MDS treated with 5'-azacitidine and sodium valproate (VAL-AZA). Fifteen out of seventy-nine patients achieved a complete remission (CR) or complete remission with incomplete blood count recovery (CRi) with VAL-AZA therapy. There was no significant reduction in the size of the LSC-containing population in non-responders. While the LSC-containing population was substantially reduced in all patients achieving a CR/CRi it was never eradicated and expansion of this population antedated morphological relapse. Similar studies were performed in seven patients with newly diagnosed AML treated with induction chemotherapy. Eradication of the LSC-containing population was observed in three patients all of whom achieved a durable CR in contrast to patients with resistant disease where LSC persistence was observed. LSC quantitation provides a novel biomarker of disease response and relapse in patients with AML treated with epigenetic therapies. New drugs that target this cellular population in vivo are required.

Plasma non-enzymatic antioxidants-vitamin C, E, beta-carotenes, reduced glutathione levels and total antioxidant activity in oral sub mucous fibrosis.

BACKGROUND: Oral submucous fibrosis (OSMF) is a crippling slowly progressive disease of oral cavity that predominantly affects people habit of consuming areca nut and its commercial preparations which generates high levels of reactive oxygen species (ROS) during their metabolism. OBJECTIVE: The objective of this present study is to evaluate the role of oxidative stress in causation and progression of OSMF by measuring the levels of nonenzymatic antioxidants in OSMF patients. MATERIALS AND METHODS: For this study we selected 27 newly diagnosed OSMF patients of both sex with age group between 23 to 40 years and the same number of age and sex matched healthy individuals were selected as control group. In both the groups we measured plasma non enzymatic antioxidants like vitamin A. E, C and reduced glutathione. Total antioxidant activity was also assessed in both the groups. RESULTS AND CONCLUSIONS: We observed a very low levels of plasma non-enzymatic antioxidants (p < 0.001) and at the same time a very poor antioxidant activity (p < 0.001) in OSMF patients when compared to controls. Therefore, consumption of tobacco or areca quid creates an oxidative stress environment which might plays a major role in the causation of OSMF.

Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts.

BACKGROUND: Myelodysplastic syndromes are a diverse and common group of chronic hematologic cancers. The identification of new genetic lesions could facilitate new diagnostic and therapeutic strategies. METHODS: We used massively parallel sequencing technology to identify somatically acquired point mutations across all protein-coding exons in the genome in 9 patients with low-grade myelodysplasia. Targeted resequencing of the gene encoding RNA splicing factor 3B, subunit 1 (SF3B1), was also performed in a cohort of 2087 patients with myeloid or other cancers. RESULTS: We identified 64 point mutations in the 9 patients. Recurrent somatically acquired mutations were identified in SF3B1. Follow-up revealed SF3B1 mutations in 72 of 354 patients (20%) with myelodysplastic syndromes, with particularly high frequency among patients whose disease was characterized by ring sideroblasts (53 of 82 [65%]). The gene was also mutated in 1 to 5% of patients with a variety of other tumor types. The observed mutations were less deleterious than was expected on the basis of chance, suggesting that the mutated protein retains structural integrity with altered function. SF3B1 mutations were associated with down-regulation of key gene networks, including core mitochondrial pathways. Clinically, patients with SF3B1 mutations had fewer cytopenias and longer event-free survival than patients without SF3B1 mutations. CONCLUSIONS: Mutations in SF3B1 implicate abnormalities of messenger RNA splicing in the pathogenesis of myelodysplastic syndromes. (Funded by the Wellcome Trust and others.).

c-Myb and GATA-1 alternate dominant roles during megakaryocyte differentiation.

BACKGROUND: Transcription factors are essential for blood cell formation. Mice expressing low levels of c-Myb (c-Myb(low)) have an increased number of bone marrow megakaryocytes (MKs) and corresponding thrombocytosis. In contrast, mice engineered to express low levels of GATA-1 (GATA-1(low)) in the megakaryocytic lineage exhibit aberrant megakaryocytopoiesis with hyperproliferation of progenitors and defective terminal differentiation leading to thrombocytopenia. These seemingly opposite roles may affect platelet turnover and thus be of clinical relevance. OBJECTIVE: To determine how these two transcription factors act together to control megakaryocytopoiesis and platelet formation. METHODS: We used a combination of cellular and molecular in vitro assays to examine the ability of bone marrow cells from mice expressing low levels of both c-Myb and GATA-1 (referred to as double(low)) to produce MKs and platelets. RESULTS: Double(low) cells, or those with low GATA-1 levels in which c-Myb is conditionally deleted, lack the hyperproliferative capacity of GATA-1(low) cells, allowing the cells to proceed towards more committed MKs that are, however, impaired in their capacity to produce fully differentiated cells, as confirmed by the abundance of morphologically aberrant cells that lack the ability to form proplatelets. CONCLUSION: c-Myb and GATA-1 act in concert to achieve correct megakaryocytic differentiation. GATA-1 regulates both the proliferation of megakaryocytic progenitors and their terminal maturation. c-Myb also acts at the level of the progenitor by influencing its commitment to differentiation, but in contrast to GATA-1 it does not have any effect on the process of terminal differentiation.

Mesenchymal hamartoma of the liver.

We present a multiseptated mesenchymal hamartoma of the liver in a 10-year-old male patient, a rare benign tumor of childhood. The characteristic ultrasound and CT appearances of this unusual tumor are reviewed. A single septal calcification associated with this tumor was demonstrated, an association which has not previously been reported. The differential diagnosis for cystic liver lesions is discussed in detail.

Malignant pleural mesothelioma in a 13-year-old girl.

Pleural mesothelioma is an uncommon tumor in all age groups, but is especially rare in childhood. We describe the clinical and radiological features of malignant pleural mesothelioma in a 13-year-old girl. The chest radiograph showed nearly complete opacification and loss of volume in the left hemithorax. Computed tomography demonstrated a large pleural effusion centrally surrounded by a thick enhancing rind of soft tissue. The radiological features of childhood pleural mesothelioma in our case were similar to those described in adults with this disease.

Inositol polyphosphate 4-phosphatase type I regulates cell growth downstream of transcription factor GATA-1.

Megakaryocytes lacking transcription factor GATA-1 fail to complete maturation in vivo and hyperproliferate. To define how GATA-1 regulates megakaryocyte cell growth we searched for mRNA transcripts expressed in primary wild-type, but not GATA-1(-), megakaryocytes. One differentially expressed transcript encodes inositol polyphosphate 4-phosphatase type I (4-Ptase I). This enzyme hydrolyses phosphatidylinositol 3,4-bisphosphate and also has lesser activity against soluble analogues of this lipid, inositol 3, 4-bisphosphate and inositol 1,3,4-triphosphate. Reintroduction of 4-Ptase I into both primary GATA-1(-) and wild-type megakaryocytes significantly retards cell growth, suggesting that absence of 4-Ptase I may contribute to the hyperproliferative phenotype of GATA-1(-) megakaryocytes. Overexpression of 4-Ptase I also markedly reduces growth of NIH 3T3 fibroblasts. Taken together, these data indicate that 4-Ptase I is a regulator of cell proliferation.

Longitudinal monitoring of immune reconstitution by CDR3 size spectratyping after T-cell-depleted allogeneic bone marrow transplant and the effect of donor lymphocyte infusions on T-cell repertoire.

Delayed immune reconstitution after allogeneic bone marrow transplantation (BMT) with associated infection is a major cause of morbidity and mortality. We used third complementarity region (CDR3) size spectratyping as a tool for monitoring T-cell repertoire reconstitution in 19 patients over a median time of 40 months after T-cell-depleted allogeneic BMT for chronic myeloid leukemia (CML). Furthermore, the effect of donor lymphocyte infusions (DLI) for the treatment of relapse in 18 of the 19 patients was analyzed. All BMT recipients had irregular spectratypes in the first 3- to -6 months after transplant. These evolved to more normal patterns by 12 months after transplant and continued to improve thereafter. In approximately a third of the patients, it took 2 to 3 years for all spectratypes to normalize, whereas in the other two thirds, some abnormal spectratypes persisted even after several years. In 9 patients, there was no immediate change in the CDR3 size profiles after DLI. In 3 patients, spectratypes improved slightly after DLI, whereas in 6 patients, spectratypes became more restricted and irregular. Overall, T-cell spectratypes in BMT patients were characterized by instability over time and in patients with graft-versus-host disease (GVHD), this was even more exaggerated. Several factors, such as pre-BMT conditioning, T-cell depletion of the donor marrow, loss of thymic function in adults, exposure to infectious agents, GVHD, and immunosuppressive treatment, are likely contributors to the delay in T-cell-repertoire reconstitution. (Blood. 2000;95:3990-3995)

Differential anti-inflammatory effects of immunosuppressive drugs: cyclosporin, rapamycin and FK-506 on inducible nitric oxide synthase, nitric oxide, cyclooxygenase-2 and PGE2 production.

OBJECTIVE AND DESIGN: Cyclosporin, FK-506 and rapamycin have similar but distinct modes of interaction with cyclophilins, calcineurins and transcription factors. These immunosuppressive drugs have also been shown to inhibit cytotoxic and inflammatory responses in macrophage. Therefore, we evaluated the mechanism of action of these drugs on iNOS and COX-2 expression by macrophages, the products of which (NO and PGE2) have cytotoxic and proinflammatory activities. MATERIALS AND METHODS: The murine macrophage cell line RAW 264.7 was grown as monolayer cultures. The effects of pharmacologically relevant concentrations of cyclosporin, rapamycin and FK-506 were evaluated in the presence and absence of lipopolysaccharide (LPS) which is a known inducer of iNOS and COX-2. Subsequently the expression of iNOS and COX-2 were analyzed by Western and Northern analysis. The production of NO and PGE2 were assayed by Greiss and RIA respectively. RESULTS: Cyclosporin (1-5 microg/ml) and rapamycin (1.0-10 nM) but not FK-506 (5-10 nM) inhibited both iNOS and COX-2 expression at mRNA level which led to significant inhibition of NO and PGE2 production. CONCLUSION: These studies characterize differential mechanistic capacity of the immunophilin-binding immunosuppressive drugs (comparable to hydrocortisone) to inhibit both iNOS and COX-2 expression. Inhibition of iNOS and COX-2 mRNA accumulation by cyclosporin and rapamycin seem to be distinct. These studies also highlight potential anti-inflammatory properties of these drugs in addition to their known immunosuppressive activity.

Disseminated lymphangiomatosis presenting with massive chylothorax.

BACKGROUND: Lymphangiomatosis is a disease characterized by involvement of various body constituents and can involve the skeletal system, connective tissues, and visceral organs. MATERIALS AND METHODS: We present a case of a 9-year-old girl where this entity presented with extensive right-sided chylothorax. Conventional imaging, including skeletal scintigraphy and contrast enhanced CT of the chest and abdomen, may have underestimated the extent of the disease, as seen on follow-up T2-weighted MR images of the chest and abdomen in our case. RESULTS: MRI easily demonstrated additional bone lesions as well as multiple small splenic lesions, which were difficult to appreciate on prior CT examinations. CONCLUSION: We suggest that MRI may be helpful to assess the extent of this disease more accurately.

Different sequence requirements for expression in erythroid and megakaryocytic cells within a regulatory element upstream of the GATA-1 gene.

The lineage-restricted transcription factor GATA-1 is required for differentiation of erythroid and megakaryocytic cells. We have localized a 317 base pair cis-acting regulatory element, HS I, associated with a hematopoietic-specific DNase I hypersensitive site, which lies approx. 3.7 kilobases upstream of the murine hematopoietic-specific GATA-1 IE promoter. HS I directs high-level expression of reporter GATA-1/lacZ genes to primitive and definitive erythroid cells and megakaryocytes in transgenic mice. Comparative sequence analysis of HS I between human and mouse shows approx. 63% nucleotide identity with a more conserved core of 169 base pairs (86% identity). This core contains a GATA site separated by 10 base pairs from an E-box motif. The composite motif binds a multi-protein hematopoietic-specific transcription factor complex which includes GATA-1, SCL/tal-1, E2A, Lmo2 and Ldb-1. Point mutations of the GATA site abolishes HS I function, whereas mutation of the E-box motif still allows reporter gene expression in both lineages. Strict dependence of HS I activity on a GATA site implies that assembly of a protein complex containing a GATA-factor, presumably GATA-1 or GATA-2, is critical to activating or maintaining its function. Further dissection of the 317 base pair region demonstrates that, whereas all 317 base pairs are required for expression in megakaryocytes, only the 5' 62 base pairs are needed for erythroid-specific reporter expression. These findings demonstrate differential lineage requirements for expression within the HS I element.

Mrvi1, a common MRV integration site in BXH2 myeloid leukemias, encodes a protein with homology to a lymphoid-restricted membrane protein Jaw1.

Ecotropic MuLVs induce myeloid leukemia in BXH2 mice by insertional mutagenesis of cellular proto-oncogenes or tumor suppressor genes. Disease genes can thus be identified by viral tagging as common sites of viral integration in BXH2 leukemias. Previous studies showed that a frequent common integration site in BXH2 leukemias is the Nf1 tumor suppressor gene. Unexpectedly, about half of the viral integrations at Nf1 represented a previously undiscovered defective nonecotropic virus, termed MRV. Because other common integration sites in BXH2 leukemias encoding proto-oncogenes contain ecotropic rather than MRV viruses, it has been speculated that MRV viruses may selectively target tumor suppressor genes. To determine if this were the case, 21 MRV-positive BXH2 leukemias were screened for new MRV common integration sites. One new site, Mrvi1 was identified that was disrupted by MRV in two of the leukemias. Ecotropic virus did not disrupt Mrvi1 in 205 ecotropic virus-positive leukemias, suggesting that Mrvi1 is specifically targeted by MRV. Mrvi1 encodes a novel protein with homology to Jaw1, a lymphoid restricted type II membrane protein that localizes to the endoplasmic reticulum. MRV integration occurs at the 5' end of the gene between two differentially used promoters. Within hematopoietic cells, Mrvi1 expression is restricted to megakaryocytes and some myeloid leukemias. Like Jaw1, which is down-regulated during lymphoid differentiation, Mrv1 is downregulated during monocytic differentiation of BXH2 leukemias. Taken together, these data suggest that MRV integration at Mrvi1 induces myeloid leukemia by altering the expression of a gene important for myeloid cell growth and/or differentiation. Experiments are in progress to test whether Mrvi1 is a tumor suppressor gene.

Consequences of GATA-1 deficiency in megakaryocytes and platelets.

In the absence of the hematopoietic transcription factor GATA-1, mice develop thrombocytopenia and an increased number of megakaryocytes characterized by marked ultrastructural abnormalities. These observations establish a critical role for GATA-1 in megakaryopoiesis and raise the question as to how GATA-1 influences megakaryocyte maturation and platelet production. To begin to address this, we have performed a more detailed examination of the megakaryocytes and platelets produced in mice that lack GATA-1 in this lineage. Our analysis demonstrates that compared with their normal counterparts, GATA-1-deficient primary megakaryocytes exhibit significant hyperproliferation in liquid culture, suggesting that the megakaryocytosis seen in animals is nonreactive. Morphologically, these mutant megakaryocytes are small and show evidence of retarded nuclear and cytoplasmic development. A significant proportion of these cells do not undergo endomitosis and express markedly lower levels of mRNA of all megakaryocyte-associated genes tested, including GPIbalpha, GPIbbeta, platelet factor 4 (PF4), c-mpl, and p45 NF-E2. These results are consistent with regulation of a program of megakaryocytic differentiation by GATA-1. Bleeding times are significantly prolonged in mutant animals. GATA-1-deficient platelets show abnormal ultrastructure, reminiscent of the megakaryocytes from which they are derived, and exhibit modest but selective defects in platelet activation in response to thrombin or to the combination of adenosine diphosphate (ADP) and epinephrine. Our findings indicate that GATA-1 serves multiple functions in megakaryocyte development, influencing both cellular growth and maturation.