Immature Precursors Research Articles

Newly diagnosed adult AML and MPAL patients frequently show clonal residual hematopoiesis

Adult acute myeloid leukemia (AML) is a highly heterogeneous stem cell malignancy characterized by the clonal expansion of immature myeloid precursors. AML may emerge de novo, following other hematopoietic malignancies or after cytotoxic therapy for other disorders. Here, we investigated the clonal vs reactive nature of residual maturing bone marrow cells in 59 newly diagnosed adult AML and mixed phenotype acute leukemia (MPAL) patients as assessed by interphase fluorescence in situ hybridization analysis of AML and myelodysplastic syndrome-associated cytogenetic alterations and/or the pattern of chromosome X inactivation, in females. In addition, we investigated the potential association between the degree of molecular/genetic involvement of hematopoiesis and coexistence of altered immunophenotypes by flow cytometry. Our results indicate that residual maturing neutrophils, monocytes and nucleated red cell precursors from the great majority of newly diagnosed AML and MPAL cases show a clonal pattern of involvement of residual maturing hematopoietic cells, in association with a greater number of altered immunophenotypes. These findings are consistent with the replacement of normal/reactive hematopoiesis by clonal myelopoiesis and/or erythropoiesis in most newly diagnosed AML and MPAL cases, supporting the notion that in most adults presenting with de novo AML, accumulation of blast cells could occur over a pre-existing clonal hematopoiesis.

Human umbilical cord Wharton's jelly-derived oligodendrocyte precursor-like cells for axon and myelin sheath regeneration.

Human umbilical mesenchymal stem cells from Wharton's jelly of the umbilical cord were induced to differentiate into oligodendrocyte precursor-like cells in vitro. Oligodendrocyte precursor cells were transplanted into contused rat spinal cords. Immunofluorescence double staining indicated that transplanted cells survived in injured spinal cord, and differentiated into mature and immature oligodendrocyte precursor cells. Biotinylated dextran amine tracing results showed that cell transplantation promoted a higher density of the corticospinal tract in the central and caudal parts of the injured spinal cord. Luxol fast blue and toluidine blue staining showed that the volume of residual myelin was significantly increased at 1 and 2 mm rostral and caudal to the lesion epicenter after cell transplantation. Furthermore, immunofluorescence staining verified that the newly regenerated myelin sheath was derived from the central nervous system. Basso, Beattie and Bresnahan testing showed an evident behavioral recovery. These results suggest that human umbilical mesenchymal stem cell-derived oligodendrocyte precursor cells promote the regeneration of spinal axons and myelin sheaths.

A critical role for immature muscle precursors in myositis

The innate and adaptive immune responses contribute to the development of inflammatory myopathies; the innate immune system does so through activation of the type I interferon and Toll-like receptor pathways. Dendritic cells have a pivotal role in the development of both adaptive and innate immune responses. Equipped with a range of pattern-recognition receptors, dendritic cells link innate and adaptive immunity. This Perspectives article discusses novel concepts in myositis, focusing on immature muscle precursors. Of interest, the immature muscle precursors involved in regeneration are associated with upregulation of HLA class I antigens and myositis-associated autoantigens, as well as activation of the Toll-like receptor pathway and production of type I interferon, and could have a critical contribution to the pathogenesis of myositis. These regenerating immature muscle cells might also be a target of the immune response in myositis, thereby explaining why muscle regeneration is not effective in the context of such inflammation.

Clustered immature myeloid precursors in intertrabecular region during remission evolve from leukemia stem cell near endosteum and contribute to disease relapse in acute myeloid leukemia

Most acute myeloid leukemia (AML) cannot be cured because leukemia stem cells (LSC) will contribute to eventual relapse. However, how LSC initiate relapse is not yet fully understood. We performed a retrospective study on bone marrow sections from AML patients during complete remission (CR), demonstrating that single and double immature myeloid precursors were located near endosteum and clustered precursors (⩾3cells/group) in intertrabecular region. Based on our observations, we hypothesize that after retrieval of myelotoxic regimen, LSC harboring near endosteum divide and differentiate into progeny cells which proliferate and then form colony clusters. Meanwhile, these clusters may migrate to intertrabecular region under the actions of cell migration factors. Without any interventions, clustered immature myeloid precursors may proliferate and hemotologic relapse is then unavoidable.

SPARC Regulates Microgliosis and Functional Recovery following Cortical Ischemia

Secreted protein acidic rich in cysteine (SPARC) is a matricellular protein that modulates the activity of growth factors, cytokines, and extracellular matrix to play multiple roles in tissue development and repair, such as cellular adhesion, migration, and proliferation. Throughout the CNS, SPARC is highly localized in mature ramified microglia, but its role in microglia--in development or during response to disease or injury--is not understood. In the postnatal brain, immature amoeboid myeloid precursors only induce SPARC expression after they cease proliferation and migration, and transform into mature, ramified resting microglia. SPARC null/CX3CR1-GFP reporter mice reveal that SPARC regulates the distribution and branching of mature microglia, with significant differences between cortical gray and white matter in both controls and SPARC nulls. Following ischemic and excitotoxic lesion, reactive, hypertrophic microglia rapidly downregulate and release SPARC at the lesion, concomitant with reactive, hypertrophic perilesion astrocytes upregulating SPARC. After photothrombotic stroke in the forelimb sensorimotor cortex, SPARC nulls demonstrate enhanced microgliosis in and around the lesion site, which accompanies significantly enhanced functional recovery by 32 d after lesion. Microglia from SPARC nulls also intrinsically proliferate at a greater rate in vitro--an enhanced effect that can be rescued by the addition of exogenous SPARC. SPARC is thus a novel regulator of microglial proliferation and structure, and, in addition to regulating glioma progression, may play an important role in differently regulating the gray and white matter microglial responses to CNS lesion--and modulating behavioral recovery--after injury.

Prognostic significance of Bcl-2 and p53 protein expressions and Ki67 proliferative index in diffuse large B-cell lymphoma

To the Editor Here we present a case of multiple granulocytic sarcomas with monocytic differentiation involving the skin and retroperitoneal area. A 19-year-old man presented with the complaints of generalized arthralgia and fixed non-tender nodular lesions (Figure 1). Physical examination revealed generalized immobile, painless violaceous erythematous nodules on his back, bilateral arms and legs, and joints. The largest nodule was 3x2 cm, located on his right posterolateral upper leg. Abnormal laboratory values included hemoglobin of 84 g/L, hematocrit of 27.2%, erythroid sedimentation rate of 58 mm/h, and lactate dehydrogenase of 613 IU/L. Blood smear revealed hypochromia and microcytosis, and bone marrow was normocellular without blasts. However, monocyte and macrophage marker CD163 was positive in 10%-15% of the bone marrow cells. The pathological examination of the nodular lesions revealed medium-sized, irregular-shaped blastic cells with frequent mitotic activity, which infiltrated the dermis. The tumor tissue contained tingible body macrophages with nuclear debris. In immunohistochemical study, these cells were positive for CD163, lysozyme, CD4, and CD43, while CD117, MPO, CD68, langerin, CD1a, S–100, CD35, CD21, clusterin, fascin, factor 13a, CD123, TCL–1, TdT, CD3, CD20, and CD30 were all negative. The Ki–67 proliferation index of the neoplastic cells was 80%. In situ hybridization study with the EBER probe for Epstein-Barr virus was negative. Figure 1 Multiple violaceous skin nodules. Abdominal and thorax computerized tomography (CT) was performed, which revealed a right retroperitoneal mass with blurred margins. A CT-guided biopsy was performed. The biopsy showed pleomorphic cell infiltration with large mononuclear and binuclear cells with clear nucleoli, which were CD163-positive, CD117-negative, and focal MPO-positive. These atypical pleomorphic, monocytic-histiocytic cells did not have blastic morphology. A high dose of cytosine-arabinoside and idarubicin was started. The nodular lesions disappeared 20 days after starting chemotherapy. Arthralgia, dyspnea, and respiratory distress developed consequently. Right pleural effusion was demonstrated in the chest X-ray. The cytological examination of the pleural fluid revealed blastic infiltration. Since the retroperitoneal mass persisted in follow-up abdominal CT, a new chemotherapy regimen containing fludarabine, cytosine arabinoside, and idarubicin was started. However, pleural effusion persisted and required Pleurocan evacuation. Methotrexate (Mtx) was given intrapleurally accompanied with systemic high-dose Mtx. Tumor lysis syndrome developed 28 days after this regimen and required intubation; the patient died due to cardiac arrest 3 days later. Granulocytic sarcoma (GS) is the tumoral infiltration of extramedullary sites with immature myeloid precursor cells; it is associated with 3%-8% of acute myeloid leukemia (AML) cases and rarely with chronic myeloproliferative disorders [1]. Monocytic differentiation of GS is seen mostly in AML M4 and M5 subtypes according to the French-American-British (FAB) classification [2]. Any extramedullary anatomic site may be involved with GS and it might rarely precede but usually occurs simultaneously with acute leukemia. CD163 positivity is controversial in GS. While Lau et al. claimed that CD163 is a highly specific marker for cells of monocyte/macrophage lineage, they did not demonstrate CD163 positivity in GS in their series [2]. Backe et al., however, demonstrated CD163 positivity in myeloid leukemias with monocytic differentiation [3], while Benet et al. demonstrated CD163 positivity in 52% of their cases of leukemia cutis [4]. The prevalence of skin involvement in myeloid malignancies is approximately 3%, and it is usually associated with cases with monocytic differentiation. It is difficult to identify the origin of the tumor when it is not associated with bone marrow infiltration, namely “aleukemic leukemia cutis” [4]. In a series of 173 patients with leukemia cutis, 7.5% of the patients with leukemic skin infiltration had no underlying myeloid neoplasm [4). Skin is one of the most common sites of involvement in GS, with approximately one-quarter of patients with GS having skin involvement [4]. The presented patient had arthralgia and multiple granulocytic nodules on various parts of the body and joints without bone morrow involvement. The morphologic appearance of the skin lesions was typical for leukemia cutis in its color and nodular form, as is immunohistochemistry with monocytic differentiation. Nevertheless, most of the aleukemic GS of skin is known to be associated with infiltration with cells of monocytic differentiation [5]. In conclusion, myeloid leukemias might present with granulocytic sarcomas, particularly in skin without bone marrow involvement. Multiple skin lesions of nodular pattern with violaceous color might suggest an underlying myeloid neoplasm. The positivity of monocytic markers such as CD68 and CD163 is typical for leukemia with a predilection to the skin. Patients with aleukemic granulocytic sarcoma might have a very aggressive course, with evolution to tumor lysis syndrome. Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

Myeloid sarcoma of the cheek and the maxillary sinus regions

Myeloid sarcoma (MS) is a rare, extramedullary malignant tumor composed of immature myeloid precursor cells and myeloblast. Most MSs occur in the subperiosteal region of the bone, with the skull, sternum, ribs, and proximal portions of the long bones being the common sites of involvement. It is thought that the MS tumor originates in the bone marrow, and traverses the Haversian canals to reach the subperiosteum. Various reports have also described the involvement of the liver, spleen, brain, heart, pharynx, uterus, vagina, skin, kidney, and other soft tissues in the formation of the tumor.

CEBPA-CEBPG axis as a novel promising therapeutic target in acute myeloid leukemia

Disruption of normal differentiation is an important link in tumorigenesis, but understanding the molecular mechanisms underlying this process is still limited. Acute myeloid leukemia (AML) is characterized by an accumulation of immature myeloid precursor cells in the bone marrow, which has been extensively used as a model for studying genetic/epigenetic regulation of differentiation and cancer progression. Intensive studies using cultured AML cells and animal models have identified well-known aberrant transcription factors, such as CEBPA, RARα, RUNX1, PU.1, GATA2, etc1. Consequently, pharmacological restoration of their function represents a promising therapeutic strategy. Noteworthily, all-trans retinoic acid was shown to be capable of inducing the terminal differentiation of immature leukemic promyelocytes, and firstly used clinically in 19882. Thus, identifying new molecular mechanisms that control myeloid differentiation is fundamental in developing novel anti-leukemic therapies. In a recent issue of Journal of Clinical Investigation, Meritxell and colleagues have systematically elucidated the role of CEBPA-CEBPG pathway in myeloid differentiation3. The authors found in the gene expression profile analysis that CEBPG mRNA was significantly upregulated in a small subset of AML patients with CEBPA hypermethylation/silencing (8/526). They further demonstrated in a CEBPA conditional knockout mice model that ablation of CEBPA led to upregulation of CEBPG in early stem/progenitor cells. CEBPA could bind to CEBPG proximal promoter and downregulate CEBPG expression by affecting E2F1 transcriptional activity. Furthermore, they showed that CEBPG was downregulated during neutrophilic differentiation in a 32D/G-CSF–R murine cell line model, whereas overexpression of CEBPG blocked granulocytic differentiation. Conversely, inhibition of CEBPG by specific shRNA in vitro or in vivo restored granulocytic differentiation in a CEBPA-knockout early hematopoietic stem/progenitor cells. Intriguingly, the authors found that the DNA demethylating agent 5-aza-2'-deoxycitidine (decitabine) restored the CEBPA-CEBPG balance and induced myeloid differentiation in CEBPA-silenced primary AML blasts. Thus, the authors concluded that targeting the CEBPA-CEBPG pathway represented a novel therapeutic approach in a particular subtype of AML. CEBPA is important for regulating of myeloid development, directing granulocyte and monocyte differentiation. CEBPA function can be disrupted either through mutation or transcriptional repression. Given the essential role of the CEBPA transcription factor in hematopoiesis, it is crucial to elucidate its mechanisms and the full spectrum of target genes that mediate its activity. This paper demonstrates for the first time that a new CEBPA-CEBPG axis can block myeloid differentiation in subsets of AML, thus revealing novel mechanistic implications for the pathogenesis of AML. DNA methylation plays a critical role in regulation gene expression during development. However, the mechanisms of how DNA methylation contributes to malignancy development, and how hypomethylation agents exert their effects, are poor understood. This study not only reveals a novel mechanism of AML differentiation failure, but also suggests the necessity of investigating the induction of epigenetic differentiation by DNA hypomethylating agents. DNA hypomethylating agents, including azacitidine and decitabine, have been recently approved for treatment of patients with high-risk myelodysplastic syndrome (MDS)4,5. Of particular interest, azacitidine and decitabine, have been used alone or in combination with conventional chemotherapy drugs to treat AML6,7,8. As inhibitors of DNA methylation, the anti-leukemic activity of these agents is believed to reactivate silenced tumor suppressor genes by epigenetic event, but a link between clinical outcomes and the specific gene targets of decitabine is not yet clear. This study provided intriguing data showing the CEBPA-CEBPG axis as a target of decitabine, thus deepened our understanding of the mechanism of the drug's action. What' more, the identification of biomarkers of decitabine action would help to select distinct subgroups of AML patients, who may benefit from the drug. At this stage, it will be of great interest to explore the relationship between aberrated CEBPA-CEBPG axis and response to decitabine in future clinical trials. Recently, cancer genome sequencing studies have revealed recurrent mutation of epigenetic modifying genes, such as DNMT3A, TET2, IDH1/2, and ASXL1 in the pathogenesis of AML9. Moreover, aberration of epigenetic regulator genes, such as IDH1/2, EZH2, and CREBBP were also found in solid tumors. Increased evidence indicates that carcinogenesis is a multistep process driven by interplay between genomic and epigenomic alterations10. The direct gene therapy for certain mutatant genes would be especially challenging. Since epigenetic modifications are reversible, they are interesting targets for specific pharmacological interventions. It will be of interest to target downstream and potentially reversible epigenetic consequences with more specific epigenetic agents, such as targeted DNMT inhibitors or specific methylating agents. We are advancing towards an era of personalized medicine in AML, and it is hoped that AML treatment will be greatly improved by integrative pharmacological interventions at both genetic and epigenetic levels in the future, which will provide a useful model for other types of human cancers.

Ku Haploinsuffiency Causes a Lymphoproliferative Disorder of Immature T-cell Precursors due to Ikaros Malfunction

Ikaros (IK) malfunction has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL), the most common form of childhood cancer. Therefore, a stringent regulation of IK activity is very important. Here we provide unique genetic and biochemical evidence that the Ku protein components Ku70 and Ku80 act as positive regulators of IK function via formation of IK-Ku70 and IK-Ku80 heterodimers with augmented sequence-specific DNA binding activity. siRNA-mediated depletion of Ku70 or Ku80 reduced the sequence-specific DNA binding activity of IK in EMSA as well as the RT-PCR measured IK target gene expression levels in human cells. The interaction of Ku components with IK likely contributes to the anti-leukemic effects of IK as a tumor suppressor, because Ku70 as well as Ku80 haploinsuffiency in mice caused development of a lymphoproliferative disorder (LPD) involving CD2+CD4+CD8+CD1+IL7R+ thymic T-cell precursors with functional IK deficiency.

Testicular myeloid sarcoma: case report

Myeloid sarcomas are extramedullary solid tumors composed of immature granulocytic precursor cells. In association with acute myeloid leukemia and other myeloproliferative disorders, they may arise concurrently with compromised bone marrow related to acute myeloid leukemia, as a relapsed presentation, or occur as the first manifestation. The testicles are considered to be an uncommon site for myeloid sarcomas. No therapeutic strategy has been defined as best but may include chemotherapy, radiotherapy and/or hematopoietic stem cell transplantation. This study reports the evolution of a patient with testicular myeloid sarcoma as the first manifestation of acute myeloid leukemia. The patient initially refused medical treatment and died five months after the clinical condition started.

Tethering bi-functional protein onto mineralized polymer scaffolds to regulate mesenchymal stem cell behaviors for bone regeneration.

Modifying three-dimensional scaffolds with bioactive extracellular matrix (ECM) molecules enhances their potential use in tissue engineering, by providing natural biochemical/physical cues for cell recognition. Here, we engineered the surface of poly(caprolactone) (PCL) scaffolds, first with bone mineral hydroxyapatite (HA), and then with fibronectin-osteocalcin (FN-OCN) bi-functional protein by means of affinity binding between OCN and HA. While FN is expected to enhance initial adhesion of immature precursor cells, OCN is considered to regulate osteogenic differentiation. Quartz crystal microbalance dissipation analysis revealed FN-OCN protein had a more stable and stronger adherence to the HA-mineralized surface than to the native PCL-surface. Initial adhesion and the spreading of rat mesenchymal stem cells were significantly enhanced on the FN-OCN tethered scaffold. Expression of bone-associated genes (osteopontin, bone sialoprotein II and OCN) was significantly higher on the FN-OCN tethered scaffold. Moreover, those proteins were more abundantly found when cultured on the scaffolds with FN-OCN than those without, as confirmed by immunofluorescence cell labeling and fluorescence activated cell sorting analysis. All taken, the tethering of FN-OCN to a HA-mineralized surface is an effective strategy to provide biopolymer scaffolds improved bi-functional capacity for bone tissue engineering, in terms of initial cell adhesion and osteogenic differentiation.

Dual effects of increased glycogen synthase kinase-3β activity on adult neurogenesis

Adult neurogenesis, the generation of new neurons during the adulthood, is a process controlled by several kinases and phosphatases among which GSK3β exerts important functions. This protein is particularly abundant in the central nervous system, and its activity deregulation is believed to play a key role in chronic disorders such as Alzheimer's disease. Previously, we reported that in vivo overexpression of GSK3β (Tet/GSK3β mice) causes alterations in adult neurogenesis, leading to a depletion of the neurogenic niches. Here, we have further characterized those alterations, finding a delay in the switching-off of doublecortin marker as well as changes in the survival and death rates of immature precursors and a decrease in the total number of mature neurons. Besides, we have highlighted the importance of the inflammatory environment, identifying eotaxin as a possible modulator of the detrimental effects on adult neurogenesis. Taking advantage of the conditional system, we have also explored whether these negative consequences of increasing GSK3 activity are susceptible to revert after doxycycline treatment. We show that transgene shutdown in symptomatic mice reverts microgliosis, abnormal eotaxin levels as well as the aforementioned alterations concerning immature neurons. Unexpectedly, the decrease in the number of mature neurons and neuronal precursor cells of the subgranular zone of Tet/GSK3β mice could not be reverted. Thus, alterations in adult neurogenesis and likely in neurodegenerative disorders can be restored in part, although neurogenic niche depletion represents a non-reversible damage persisting during lifetime with a remarkable impact in adult mature neurons.

Endothelial Cells Are Essential to Sense Lipopolysaccharide in a MYD88-Dependent Manner and to Subsequently Induce Emergency Myelopoiesis

AbstractAbstract 641Severe systemic infections evoke a number of characteristic clinical signs such as fever, neutrophilia and the appearance of immature myeloid precursors in the circulation (left-shift). This reflects a well-regulated hematopoietic response program to enhance myeloid cell output during times of increased hematopoietic demand, a condition which is referred to as ’emergency myelopoiesis’. Important molecular components of the emergency myelopoiesis cascade, such as cytokines and transcription factors involved, have been elucidated. However, the initial steps of emergency myelopoiesis involving pathogen recognition and translation into accelerated bone marrow (BM) myelopoiesis have only been inferred from findings on Toll-like receptor (TLR) expression on immature hematopoietic stem and progenitor cells (HSPCs) as well as on mature hematopoietic cells (e.g. macrophages). Accordingly, it has been assumed that both immature as well as mature hematopoietic cells are involved in sensing infection and inducing emergency myelopoiesis directly and indirectly, respectively. Surprisingly, by generating reciprocal BM chimeric animals mice with TLR4−/− hematopoiesis on a wild-type (WT) nonhematopoietic background (TLR4−/−→WT mice) and WT hematopoiesis on a TLR4−/− nonhematopoietic background (WT→TLR4−/−mice), we demonstrated that LPS-Induced emergency myelopoiesis depends on TLR4-expressing nonhematopoietic cells (Boettcher et al., J Immunol. 2012 Jun 15;188(12):5824–8.). However, the precise identity and localization of the nonhematopoietic cell type crucial for sensing gramnegative infection-derived lipopolysaccharide (LPS) has remained elusive to date.We now have addressed this fundamental question using BM transplantation experiments and Cre-loxP recombination technology. BM chimeric mice with a myeloid differentiation primary response gene 88 (Myd88)-deficiency in the hematopoietic lineage (MYD88−/−→WT mice) showed a normal LPS response indistinguishable to control (WT→WT) mice, while knocked out Myd88 within the nonhematopoietic compartment (WT→MYD88−/− mice) led to a non-responsiveness towards LPS similar to controls (Myd88−/−→Myd88−/− mice). These results are in line with our earlier data, thus confirming the critical role of the TLR4/MYD88 pathway in nonhematopoietic cells for the induction of emergency myelopoiesis. In order to specifically delete TLR-MyYD88-downstream signaling in various nonhematopoietic cells including BM Nestin+ mesenchymal stem cells (MSCs) and their progeny, perivascular cells, endothelial cells, and hepatocytes, we generated Nes-Cre;Myd88fl/fl, Pdgfrb-Cre;Myd88fl/fl, Tek-Cre;Myd88fl/fl, and Alb-Cre;Myd88fl/fl mice, respectively. We observed a normal increase in the frequency of BM CD11b+Gr-1low immature myeloid precursors accompanied by a decrease of BM CD11b+Gr-1high mature myeloid cells upon LPS stimulation characteristic for efficient emergency myelopoiesis in Nes-Cre;Myd88fl/fl, Pdgfrb-Cre;Myd88fl/fl, and Alb-Cre;Myd88fl/fl mice as compared to control mice. Furthermore, we measured highly-elevated plasma G-CSF levels in these mouse strains upon LPS injection. Hence, intact TLR signaling in mesenchymal stromal cells incl. Nestin+ MSCs, perivascular cells as well as hepatocytes is dispensable for induction of emergency myelopoiesis. Strikingly, Tek-Cre;Myd88fl/fl mice were completely non-responsive towards LPS stimulation as assessed by the above-mentioned parameters. Our results thus demonstrate a fundamental and unanticipated role of the endothelium for sensing of systemically spread pathogens and subsequent stimulation of BM emergency myelopoiesis. Disclosures:No relevant conflicts of interest to declare.

Genetic Loss of Tmprss6 Increases Effective Erythropoiesis in a Mouse Model of β-Thalassemia

Abstract 482β-thalassemia is a disorder of ineffective erythropoiesis in which oxidative damage caused by unpaired α-globin chains leads to erythroid apoptosis, increased proliferation of erythroid precursors, and impaired erythroid differentiation. Patients develop systemic iron overload that is caused by red blood cell transfusions and by insufficient inhibition of gastrointestinal iron absorption by the iron regulatory hormone hepcidin. Previously we reported that homozygous genetic loss of Tmprss6, a hepatic transmembrane serine protease that inhibits hepcidin expression by the liver, led to hepcidin elevation and systemic iron deficiency in Hbbth3/+ mice, a model of β-thalassemia intermedia. Interestingly, we also found that while maintaining similar hemoglobin levels, Hbbth3/+mice with homozygous loss of Tmprss6 showed a significant reduction in splenomegaly and marked improvement in peripheral red blood cell (RBC) morphology.Here, we investigated the effects of genetic loss of Tmprss6 on erythropoiesis in Hbbth3/+ mice. In mice of different Tmprss6-Hbb genotypes, we used flow cytometry to quantify the proportion of total bone marrow cells of the erythroid lineage by measuring expression of TER119, an antigen expressed from the pro-erythroblast through the mature erythrocyte stage. Additionally, within the TER119+ population, we quantified the different erythroblast subpopulations by analyzing the intensity of forward scatter and CD44 expression. Compared to wild type (Tmprss6+/+Hbb+/+) controls, Hbbth3/+ mice with 2 wild-type Tmprss6 alleles (Tmprss6+/+Hbbth3/+) showed a significant increase in the proportion of total erythroid cells in the bone marrow, significant increases in the proportion of immature erythroid precursors (basophilic and polychromatic erythroblasts) within the erythroid population, and a significant decrease in the proportion of mature RBCs, resulting in a marrow profile consistent with ineffective erythropoiesis. In Hbbth3/+ mice with homozygous Tmprss6 disruption (Tmprss6−/−Hbbth3/+), the proportion of immature erythroid precursors (basophilic and polychromatic erythroblasts) within the erythroid population remained significantly elevated; however, the proportion of total erythroid cells in the bone marrow was no longer increased. Compared to Tmprss6+/+Hbbth3/+ mice, Tmprss6−/−Hbbth3/+ mice showed a significant increase in the proportion of mature RBCs; this was accompanied by a reduction in reactive oxygen species (ROS) production (as assessed by the indicator CM-H2DCFDA) and apoptotic cells (as assessed by annexin V binding) within both the orthochromatic erythroblast/reticulocyte and mature red cell subpopulations. Additionally, compared to Tmprss6+/+Hbbth3/+ mice, Tmprss6−/−Hbbth3/+ mice showed a marked reduction in α-globin precipitates in membrane fractions prepared from peripheral RBCs. Interestingly, when normalized to α-globin mRNA expression, bone marrow mRNA encoding α-hemoglobin stabilizing protein (AHSP), an α-globin chaperone, was significantly higher in Tmprss6−/−Hbbth3/+ mice compared to Tmprss6+/+Hbbth3/+ mice, compatible with the known stabilization of AHSP mRNA under low iron conditions. Together, these findings suggest a model in which genetic loss of Tmprss6 in Hbbth3/+ mice leads to a systemically iron-deficient state in which reduced iron availability to erythroid precursors leads to stabilization of free α-globin chains, a reduction in both ROS formation and erythroid apoptosis, and ultimately more effective erythropoiesis. In the context of previous findings, these results indicate that hepcidin-elevating strategies based on pharmacological inhibition of Tmprss6 might alter the clinical phenotype of β-thalassemia not only by reducing systemic iron loading but also by altering erythroid maturation. Disclosures:Rivella:Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding.

Langerhans Cell Histiocytosis: Back to Histiocytosis X

Abstract SCI-8Langerhans cell histiocytosis (LCH) is a disorder characterized by inflammatory lesions that include pathologic CD207+ dendritic cells. LCH has pleotropic clinical presentations ranging from single lesions cured by curettage to potentially fatal multisystem disease. The first descriptions of LCH, including Hand-Schüller-Christian disease and Letterer-Siwe disease, were based on anatomic location and extent of the lesions. Despite clinical heterogeneity, LCH lesions are generally indistinguishable by histology, which led to the notion that the spectrum of clinical manifestations represents a single disorder, histiocytosis X. The designation “Langerhans cell histiocytosis” was subsequently proposed with discovery of cytoplasmic Birbeck granules in the pathologic infiltrating dendritic cells in histiocytosis X lesions, a feature shared by epidermal Langerhans cells. The etiology of LCH remains elusive, and debate of LCH as an inflammatory versus malignant disorder remains unresolved. However, recent discoveries question the model of LCH arising from transformed or pathologically activated epidermal Langerhans cells. We found cell-specific gene expression signature in CD207+ dendritic cells within LCH lesions to be more consistent with immature myeloid dendritic cell precursors than epidermal Langerhans cells. Furthermore, recent mouse studies demonstrate that CD207+ is more promiscuous than previously appreciated. Langerin (CD207) expression can be induced in many dendritic cell lineages, supporting the plausibility of a spectrum of candidates for an LCH cell of origin, including circulating dendritic cell precursors. Finally, recurrent activating BRAF mutations in LCH lesions suggest a role for a hyperactive RAS pathway in LCH pathogenesis, and possibly in normal dendritic cell development. This presentation will discuss the historical background and recent advances in LCH biology, along with a proposal to reframe “histiocytosis X” as a myeloid neoplasia caused by aberrant maturation and migration of myeloid dendritic cell precursors. Disclosures:No relevant conflicts of interest to declare.

Prognostic Impact of the Levels of Expression of Cell Surface Proteins Commonly Expressed by Blasts and Hematopoietic Precursor Cells in De Novo AML Patients: A Report From the Spanish Cetlam Study Group

AbstractAbstract 1452▪▪This icon denotes a clinically relevant abstract Introduction:Acute myeloid leukemia (AML) is a heterogeneous group of neoplastic disorders characterized by the presence of acquired genetic alterations in hematopoietic progenitor and stem cells. The prognostic impact of immunophenotypic markers has long been controversial. Despite this, only a very limited number of studies have investigated the prognostic impact of the levels of cell surface membrane (Sm) proteins commonly expressed on AML blasts. In this study we analyzed the prognostic impact of the levels of expression of several markers associated with AML blasts and normal hematopoietic precursors, as asessed by Multiparameter Flow Cytometry (MFC) in de novo AML patients (pts). Methods:Overall, 122 adult de novo AML pts diagnosed between 12/2003 and 08/2011 were studied, after excluding acute promyelocytic leukemia. All cases were diagnosed and classified according to the WHO criteria and they were immunophenotyped using an extensive 4-color panel of monoclonal antibodies by MFC, as previously described. Patients were treated according to the multicenter CETLAM AML-03 trial. Blast cells were gated after excluding all other cell populations, as well-delineated clusters of SSC low/int/CD45dimevents (“blast gate”) using the merge function of the Infinicyt software (Cytognos SL, Salamanca, Spain). For each case, the reactivity for the following markers was evaluated in terms of mean fluorescence intensity (MFI; arbitrary units) in bone marrow samples: CD123, CD117, CD34 and CD7. Normal residual bone marrow lymphocytes were used as reference for internal quality control purposes. Cytogenetic and molecular risk stratifications were based on the European LeukemiaNet (ELN) recommendations and the molecular lesions were investigated using well established protocols. Overall survival (OS), disease-free survival (DFS) and relapse rate (RR) were measured by the Kaplan–Meier method and curves were compared with the log-rank test. Multivariate analysis was performed using the Cox regression model. P-values ≤0.05 were considered to be statistically significant. Results:Median AML patient age was of 54 years (range: 20–70 y) with a M/F ratio of 63/59. Twelve pts (10%) had good cytogenetic/molecular risk, 83 (68%) intermediate, 17 (14%) high risk and 10 (8%) pts were unknown. Fifteen cases (12%) had NPM1mut, 33 (27%) showed FLT3-ITDmut, 5 (4%) pts carried CEBPAmut, 41 (33.6%) pts with no mutations and 28 (23%) pts were unknown. The median follow-up of pts alive was 19 months (range 0–97 months) and the 5-year OS of all pts was 42±5%. Univariate analysis of prognostic factors showed an association between higher CD45 (MFI >232; p=.01), CD117 (MFI>259; p=.008), CD34 (MFI >242; p=.007) and CD7 (MFI> 19; p=.03) expression and both a shorter OS and DFS. In addition, high CD123 expression (MFI >248; p=.04) was associated with a shorter DFS. In multivariate analysis only a high CD45 (p=.03) and a high CD34 (p=.03) expression were identified as independent predictors for a shorter OS. In turn, higher levels of CD123 (p=.03) and of CD34 (p=.02) were independent predictors for DFS and relapse, even when age, gender and WBC were taken in account. Conclusion:In this study, we show that higher levels of expression of immunophenotypic markers associate with immature myeloid precursors (CD45, CD117, CD34 and CD7) as assessed by MFC, have a significantly adverse prognostic impact in AML, both as regards OS and DFS. Accordingly, higher levels of CD45 and CD34 were independent predictors for both a shorter OS whereas, greater CD123 and CD34 values were associated with an increased risk of relapse and a shorter DFS, supporting the adverse prognostic impact of a more immature blast cell phenotype in de novo AML. Disclosures:No relevant conflicts of interest to declare.

Sotatercept, an Activin Receptor IIa Ligand Trap, Acts Through Bone Marrow Accessory Cells to Promote Late-Stage Erythropoiesis and a Rapid Induction of Red Blood Cell Number and Hemoglobin

Abstract 372The regulation of erythropoiesis requires stem cell factor and erythropoietin (EPO) for the proliferation and survival of erythroid progenitor and early precursor cells. While recombinant EPO is widely used for treating various types of anemia, it often lacks efficacy in cases of anemia due to ineffective erythropoiesis in which immature erythroid precursors undergo apoptosis. Thus, there is an need for new therapies to treat the later stages of erythropoiesis.Members of the transforming growth factor beta (TGFβ) superfamily have been studied as potential regulators of erythropoiesis, iron regulation and globin expression. Sotatercept (ACE-011), a recombinant fusion protein consisting of the extracellular domain of the human activin receptor IIA (ActRIIA) linked to the human immunoglobulin G1 (IgG1) Fc domain, is a ligand trap which binds a number of TGFβ superfamily ligands including activin A, activin B, growth differentiation factor-11 (GDF-11) and bone morphogenetic protein-10 (BMP-10). Administration of sotatercept led to substantial increases in red cell number and hemoglobin in human subjects, but the mechanism is not fully understood. We utilized both mouse in vivo and human in vitro models to investigate the mechanism of sotatercept in promoting erythropoiesis.In order to compare the effects of RAP-011 (the murine version of sotatercept) to EPO on red blood cell (RBC) parameters, C57/Bl mice were dosed with RAP-011, EPO or control vehicle. RAP-011-treated mice had a rapid and statistically significant increase in hematocrit, hemoglobin, and RBC number in less than 72-hours. As rapidly as 24 hours after treatment, RAP-011 induced a significant increase in RNA-negative, enucleated cells in the bone marrow (BM). RAP-011 also rapidly increased BM BFU-e and CFU-e erythroid progenitors, while EPO was more effective on spleen-derived progenitors. These data suggest that RAP-011 acts primarily on both bone marrow progenitor cells and late erythroblasts to promote erythropoiesis.In order to investigate the cellular mechanism by which RAP-011 increases red blood cell parameters, we conducted a series of in vitro experiments and found no evidence to support direct effects of RAP-011 on human CD34+ cells assessed in colony formation assays and in erythroid differentiation in liquid culture. As both clinical and pharmacological findings point to a clear role for RAP-011 in stimulating RBC parameters, we hypothesized that RAP-011 effects may be mediated by accessory cells in the BM microenvironment. Human CD36+ cells, which are highly enriched for erythroid progenitors, were co-cultured with long-term BM cultures and erythroid differentiation was assessed following 6 days of culture in EPO (2U/mL)-supplemented media. At day 6 the output of these cultures was predominantly characterized as EryA (∼basophilic erythroblast) but with the addition of RAP-011 (50μM), a significant fraction of CD36+ cells matured into EryB/C cells (polychromatic/orthochromatic erythroblasts), suggesting that factors produced by BM accessory cells mediate RAP-011 erythropoietic effects and that, in contrast to EPO, RAP-011 may play a role in the latter stages of erythroblast maturation. To identify cytokines that may mediate RAP-011 effects, CD36+ cells were treated with several activin receptor IIA ligands. GDF-11 treatment significantly decreased proliferation of GPA+ cells during the differentiation process and RAP-011 effectively reversed this effect, but had no consequence on untreated cells. These data suggest that GDF-11 may mediate the erythroipoietic stimulatory effects of RAP-011.In summary, RAP-011 induced a rapid increase in RBC parameters in mice (reflected in the number of enucleated cells found in the bone marrow), likely mediated by BM accessory cells. Our data also suggest that effects of sotatercept may be mediated at least partly by GDF-11, acting as a potential negative regulator of the terminal stages of erythropoiesis. The ability of sotatercept to reverse this inhibition would lead to a rapid release of terminal erythroid cells into the circulation. These data support the rationale to develop sotatercept for the treatment of anemia and ineffective erythropoiesis, especially in patients who may not respond to EPO. Disclosures:Carrancio:Celgene Corporation: Employment. Markovics:Celgene Corporation: Employment. Wong:Celgene Corporation: Employment. Leisten:Celgene Corporation: Employment. Groza:Celgene Corporation: Employment. Raymon:Celgene Corporation: Employment. Heise:Celgene Corporation: Employment. Chopra:Celgene Corp: Employment, Equity Ownership. Daniel:Celgene Corporation: Employment. Sung:Celgene Corporation: Employment.

MiRNA-130a Regulates C/EBP-ε During Granulopoiesis.

AbstractAbstract 2137Granulopoiesis is a tightly regulated process. A strictly regulated temporal expression of different transcription factors is crucial for proper neutrophil development. Dysregulation of transcription factors may result in impaired antimicrobial function of the neutrophils and may cause leukemia.microRNAs (miRNAs) are small noncoding RNAs, that post-transcriptionally regulate protein expression. Dysregulation of miRNA expression has been shown to contribute to the development of neoplastic myeloid diseases such as myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) and point to an important role of miRNAs in fine tuning of protein regulation in granulopoiesis.We found that miR-130a is highly expressed in immature proliferating granulocytic precursors, and has a low expression in more mature cells in granulopoiesis. Using microRNA target-prediction software we found the transcription factor C/EBP-ε to be a putative target for miR-130a. C/EBP-ε is essential for expression of the genes encoding the proteins stored in the specific granules of the neutrophil. C/EBP-ε also play an important role for cessation of cell division at the myelocyte stage and the further progress of differentiation.The biological effect of lacking C/EBP-ε can be observed in the C/EBP-ε knock-out mouse, with accumulation of immature granulocytic precursors in the bone marrow and a severely reduced number of terminally differentiated granulocytes in both bone marrow and peripheral blood. The anti-microbial defense is impaired in the C/EBP-ε knock-out mice which are significantly more susceptible to infection than wild-type mice.We demonstrate that the mRNA for C/EBP-epsilon is present already in the late promyelocytes while the protein does not appear until the myelocyte stage. This indicates a posttranscriptional regulation of C/EBP-epsilon expression e.g. by microRNAs.In-silico analysis has shown that the 3′-UTR of the C/EBP-epsilon mRNA contains one binding site for miR-130a. We have verified this by analysis of luciferase reporter constructs carrying the 3′-UTR of C/EBP-ε with the miR-130a binding-site. The repressive effect of miR-130a was abolished by point mutations in the miRNA–binding site.Next we analyzed a murine granulocytic cell line (MPRO), which stably over expressed miR-130a, and found a reduction of the protein level of C/EBP-ε. We also found a significant decrease in the expression of different genes encoding proteins stored in specific granules such as lactoferrin and hCAP18. Interestingly, we also found elevated expression of MPO mRNA in these cells indicating a more immature phenotype of the cells.Finally, we transiently transfected myelocytes and metamyelocytes from human bone marrow with pre-miR-130a and demonstrated that this has a significant effect on C/EBP-ε protein levels also in human cells. The reverse result was obtained when transfecting the cells with an anti-miR-130a oligonucleotide.These results indicate that miR-130a plays an important regulatory role in granulopoiesis and if dysregulated, could lead to a compromised immune function and developmental progression through its regulation of C/EBP-ε.Grant acknowledgment: The Danish Cancer Society, Lundbeck foundation, Brøchner Mortensen foundation, Medical Research Council. Disclosures:No relevant conflicts of interest to declare.

The Use of Pegylated G-CSF in Chronic Neutropaenia of Childhood. A Single Centre Experience

AbstractAbstract ▪3268▪This icon denotes a clinically relevant abstractChronic Neutropenia of Childhood (CNC) is defined as an absolute neutrophil count that is persistently below 0.5×109/L and leaves patients at risk of severe, systemic bacterial and fungal infections. The standard treatment to raise the neutrophil count is daily injections of recombinant G-CSF to maintain the neutrophil count above 1.0 × 109/L and the child infection free.A long acting Pegylated G-CSF (PEG G-CSF) is available but is unlicensed for use in chronic neutropenia and in children. Concern has been expressed regarding long term exposure to PEG G-CSF and the potential for neutralising antibodies; there is a lack of clinical evidence and experience in its use for CNC. Potential advantages exist in its use including a more stable and sustained neutrophil count and fewer subcutaneous injections; the latter an important consideration in a paediatric population.We report a single centre experience of seven consecutive cases using PEG G-CSF in CNC. Five boys and two girls age range five months to six years had a total of thirty four presentations in Accident and Emergency for bacterial infection; eleven episodes required hospital admission and intravenous antibiotics. All had a history of persistent neutropenia ranging from one to thirty two months. Prophylactic oral anti-infectives; ciprofloxacin and itraconazole were prescribed and once bone marrow examination had excluded evidence for other causes of persistent neutropenia standard G-CSF was commenced.All seven patients were responsive to G-CSF and so following two – four weeks of treatment a switch to PEG G-CSF was made. Neutrophil response kinetics was monitored to tailor individual dose and frequency of injections.In all cases there was an increased longevity of neutrophil response over time. Treatment with PEG G-CSF was required initially every seven days, but the neutrophil count was ultimately sustained by one injection every three to four weeks. We hypothesise that this is due to gradual recruitment of immature precursors and stem cells towards the neutrophil lineage; whereas the initial neutrophil responses are the result of more mature committed precursors.Once on PEG G-CSF there was a marked reduction in serious bacterial infections with a total of only six urgent medical assessments sought. This resulted in one admission each for three of the children. Four children had no further hospital admissions.The neutrophil count responded and was maintained in all seven cases. Prophylactic anti-infectives were discontinued once a stable neutrophil count was achieved. Six patients continued the PEG G-CSF successfully whilst one discontinued its use due to maternal concerns of an associated behaviour change and sleep disturbance, not an expected side effect of PEG G-CSF.These cases demonstrate the effective, safe use of PEG G-CSF in chronic neutropenia of childhood, with a more sustained neutrophil count and thus reduced risk of infection. There was no evidence of a reduced response to PEG C-CSF suggesting no development of neutralising antibodies.The decreased frequency of injections has led to an improved quality of life for these children and their families, with fewer hospital visits for treatment, blood sampling and review. Disclosures:Off Label Use: PEGylated G-CSF used in chronic neutropaenia of childhood.

Failure of axonal transport induces a spatially coincident increase in astrocyte BDNF prior to synapse loss in a central target

Failure of anterograde transport to distal targets in the brain is a common feature of neurodegenerative diseases. We have demonstrated in rodent models of glaucoma, the most common optic neuropathy, early loss of anterograde transport along the retinal ganglion cell (RGC) projection to the superior colliculus (SC) is retinotopic and followed by a period of persistence of RGC axon terminals and synapses through unknown molecular pathways. Here we use the DBA/2J mouse model of hereditary glaucoma and an acute rat model to demonstrate that retinotopically focal transport deficits in the SC are accompanied by a spatially coincident increase in brain-derived neurotrophic factor (BDNF), especially in hypertrophic astrocytes. These neurochemical changes occur prior to loss of RGC synapses in the DBA/2J SC. In contrast to BDNF protein, levels of Bdnf mRNA decreased with transport failure, even as mRNA encoding synaptic structures remained unchanged. In situ hybridization signal for Bdnf mRNA was the strongest in SC neurons, and labeling for the immature precursor pro-BDNF was very limited. Subcellular fractionation of SC indicated that membrane-bound BDNF decreased with age in the DBA/2J, while BDNF released from vesicles remained high. These results suggest that in response to diminished axonal function, activated astrocytes in the brain may sequester mature BDNF released from target neurons to counter stressors that otherwise would challenge survival of projection synapses.