Myeloid Hyperplasia Research Articles

Enhanced stability of tristetraprolin mRNA protects mice against immune-mediated inflammatory pathologies

Tristetraprolin (TTP) is an inducible, tandem zinc-finger mRNA binding protein that binds to adenylate-uridylate-rich elements (AREs) in the 3'-untranslated regions (3'UTRs) of specific mRNAs, such as that encoding TNF, and increases their rates of deadenylation and turnover. Stabilization of Tnf mRNA and other cytokine transcripts in TTP-deficient mice results in the development of a profound, chronic inflammatory syndrome characterized by polyarticular arthritis, dermatitis, myeloid hyperplasia, and autoimmunity. To address the hypothesis that increasing endogenous levels of TTP in an intact animal might be beneficial in the treatment of inflammatory diseases, we generated a mouse model (TTPΔARE) in which a 136-base instability motif in the 3'UTR of TTP mRNA was deleted in the endogenous genetic locus. These mice appeared normal, but cultured fibroblasts and macrophages derived from them exhibited increased stability of the otherwise highly labile TTP mRNA. This resulted in increased TTP protein expression in LPS-stimulated macrophages and increased levels of TTP protein in mouse tissues. TTPΔARE mice were protected from collagen antibody-induced arthritis, exhibited significantly reduced inflammation in imiquimod-induced dermatitis, and were resistant to induction of experimental autoimmune encephalomyelitis, presumably by dampening the excessive production of proinflammatory mediators in all cases. These data suggest that increased systemic levels of TTP, secondary to increased stability of its mRNA throughout the body, can be protective against inflammatory disease in certain models and might be viewed as an attractive therapeutic target for the treatment of human inflammatory diseases.

Increased Serum Alkaline Phosphatase in a FeLV-Positive Cat

Background: Feline Leukemia Virus (FeLV) is an oncogenic virus that usually invades bone marrow causing non-regenerative anemia and thrombocytopenia. In FeLV positive cats, the myeloid leukemia is characterized by intense proliferation of blast cells in a bone marrow with myeloid precursor predominance and several malignancies degree. The leukocyte alkaline phosphatase is found secondary granules of mature neutrophils of humans, horses and cows but not in dogs and cats,which demonstrate the enzyme activity in myeloblast and promyeloblasts cells. In this case is describing a FeLV-positive case with leukemic disorder accompanied by extremely high levels of serum alkaline phosphatase.Case: A 8-year-old castrated male Domestic Short Hair cat with a 3-month history of convulsions, pica, vomits, lethargy, hypodipsia and anorexia was examined and showed mild dehydration, severe jaundice and hyperpnoea. Laboratory results evidenced severe normocytic hypochromic non-regenerative anemia, moderated leukocytosis with severe degenerativeneutrophil left shift, moderated lymphocytosis and severe thrombocytopenia. Serum biochemical profle showed mild hyperalbuminemia, moderated increment in alanine transaminase and severe increment in alkaline phosphatese. The cat waspositive for ELISA point of care FeLV antigen and negative for FIV antibodies. Urinalysis revealed moderated bilirubinuria and proteinuria. The bone marrow cytology showed high cellularity, increased of M:E ratio, rare megakaryocyte lineage, erythroid hypoplasia, myeloid hyperplasia with blast excess and dysgranulopoiesis.Discussion: FeLV is a virus that usually causes cytopenias and in this case this was well evidenced through a severe non regenerative anemia and thrombocytopenia. Generally, anemia is a non-neoplastic complication of FeLV mainly related to suppressive effects hematopoietic and stromal cells of the bone marrow. Nevertheless, in this case, the anemia also maybe attributable to the observed acute myeloid leukemia. Neutrophilia is an uncommon fnding in FeLV-infected cats, even more with the severe degenerative left shift as in the present case. This fnding could be associated to a myeloid hyperplasia. Some biochemical alterations, as hyperalbuminemia and increment in values of serum ALT could be explained by clinical observations like dehydration and some degree of hepatic enlargement, respectively. However, the excessive value forserum alkaline phosphatase activity is an unpublished fnding in cats. In humans high levels of ALP has been associated with neutrophilia and it is quite likely that a great amount of degenerative neutrophils may contribute for high leakage of ALP than in other circumstances of neutrophilia. This is the frst report of a FeLV-positive cat exhibiting extremely high serum values of the enzyme ALP, possibly attributable to acute myeloid leukemia.Keywords: neutrophilia, cytopenia, non-regenerative anemia, acute leukemia.

Myeloproliferative Neoplasm or Reactive Process? A Rare Case of Acute Myeloid Leukemia and Transient Posttreatment Megakaryocytic Hyperplasia with JAK-2 Mutation.

Myeloproliferative neoplasms (MPNs) are hematopoietic malignancies characterized by unchecked proliferation of differentiated myeloid cells. The most common BCR-ABL1-negative MPNs are polycythemia vera, essential thrombocythemia, and primary myelofibrosis. The discovery of JAK2 V617F mutation has improved our understanding of the molecular basis of MPN. The high frequency of JAK2 mutation in MPN makes JAK2 mutation testing an essential diagnostic tool and potential therapeutic target for MPN. Here, we present a rare case of a 34-year-old patient who was initially diagnosed with acute myeloid leukemia (AML) with mutated NPM1. After chemotherapy treatment followed by granulocyte colony stimulating factor administration, the patient achieved complete remission of AML. However, the bone marrow showed hypercellularity with granulocytic hyperplasia, markedly increased atypical megakaryocytes (50.2/HPF) with focal clustering, and reticulin fibrosis (3/4). JAK2 V617F mutation was also detected. Considering the possibility of AML transformed from a previous undiagnosed MPN, patient underwent peripheral blood allogenic stem cell transplant. This case illustrates the diagnostic challenges of firmly establishing a diagnosis between similar, but distinct, disease entities and an accurate clinicopathological differentiation is crucial.

SHIP represses lung inflammation and inhibits mammary tumor metastasis in BALB/c mice.

SH2-containing-inositol-5′-phosphatase (SHIP) is a negative regulator of the phosphatidylinositol-3-kinase pathway in hematopoietic cells and limits the development of leukemias and lymphomas. The potential role of SHIP in solid tumor development and metastasis remains unknown. While SHIP restricts the aberrant development of myeloid cells in C57BL/6 mice, there are conflicting reports regarding the effect of SHIP deletion in BALB/c mice with important consequences for determining the influence of SHIP in different model tumor systems. We generated SHIP−/− BALB/c mice and challenged them with syngeneic non-metastatic 67NR or metastatic 4T1 mammary tumors. We demonstrate that SHIP restricts the development, alternative-activation, and immunosuppressive function of myeloid cells in tumor-free and tumor-bearing BALB/c mice. Tumor-free SHIP−/− BALB/c mice exhibited pulmonary inflammation, myeloid hyperplasia, and M2-polarized macrophages and this phenotype was greatly exacerbated by 4T1, but not 67NR, tumors. 4T1-bearing SHIP−/− mice rapidly lost weight and died from necrohemorrhagic inflammatory pulmonary disease, characterized by massive infiltration of pulmonary macrophages and myeloid-derived suppressor cells that were more M2-polarized and immunosuppressive than wild-type cells. Importantly, while SHIP loss did not affect primary tumor growth, 4T1-bearing SHIP−/− mice had 7.5-fold more metastatic tumor cells in their lungs than wild-type mice, consistent with the influence of immunosuppressive myeloid cells on metastatic growth. Our findings identify the hematopoietic cell-restricted protein SHIP as an intriguing target to influence the development of solid tumor metastases, and support development of SHIP agonists to prevent the accumulation of immunosuppressive myeloid cells and tumor metastases in the lungs to improve treatment of metastatic breast cancer.

Long Non-Coding RNA Induces De Novo Myelodysplastic Syndrome through Epigenetic Regulation

Long non-coding RNAs (lncRNAs) form the largest part of the mammalian non-coding transcriptome and control gene expression at various levels including chromatin modification, transcriptional and post-transcriptional processing. LncRNAs are implicated in initiation and progression of several cancers.Cancer-associated genomic regions are regions showing high frequency of cancer related abnormalities, such as loss of heterozygosity or amplifications. One such widely studied CAGR is the 8q24.21 genomic region. One SNP of particular importance present at this locus is rs6983267, with the G allele of the SNP conferring increased risk of colorectal, prostate, breast and bladder cancers. CCAT2 is a lncRNA that spans this highly conserved region. CCAT2 has been shown to play an important role in inducing chromosomal instability and supporting cell proliferation and cell cycle arrest. Despite advances in diagnosis of MDS patients, the underlying mechanisms that lead to spontaneous induction of MDS remains poorly understood. Here we attempted to elucidate the role of CCAT2 and its specific alleles (G/T) in regulation of cellular processes that drive spontaneous tumorigenesis using a genetically engineered mouse model. We generated transgenic mice for each CCAT2 allele using random integration approach in C57Bl6/N background, expressing CCAT2 in all tissues of mice.In this study, we identified that CCAT2 plays an important role in regulation of normal hematopoiesis. Constitutive in vivo overexpression of each CCAT2 transcript in the mice resulted in spontaneous induction of widespread pancytopenias. CCAT2(G/T) BM biopsies displayed severe myeloid or erythroid hyperplasia, and dysplastic megakaryocytic proliferation, along with enhanced proliferation and excessive apoptosis. Interestingly, we identified two distinct phenotypes in CCAT2(G/T) mice with equal prevalence of MDS or mixed MDS/MPN. This suggests that CCAT2 overexpression might affect regulation of hematopoietic stem cells, disturbing their self-renewal or maturation capacity, and subsequently resulting in BM failure. Percentage of HSPCs was significantly reduced in BM of MDS mice, with increased presence of immature erythroid blasts and granulocyte-macrophage progenitors suggesting a block in differentiation. HSPCs of CCAT2(G/T) mice also showed increased frequency of cytogenetic aberrations, including breaks and chromosomal fusions. However, these mice don't develop sAML, suggesting CCAT2 is critical in initiation of MDS. We further identified significantly higher CCAT2 expression in the MDS patients as compared to healthy volunteers. Patients with sAML had significantly lower expression of CCAT2 as compared to patients with only MDS.To determine the mechanism by which CCAT2 induces genomic instability and myelodysplasia, we screened for several genes that have been previously reported to induce myelodysplasia as potential targets of CCAT2. Interestingly, EZH2 was downregulated in the BMCs of CCAT2(G/T) mice compared to WT littermates. EZH2 downregulation was observed in both MDS only and MDS/MPD mice. In CCAT2(G/T) mice, EZH2 and H3K27Me3 reduction was observed in hematopoietic stem and progenitor cells (HSPCs) as well as lineage positive bulk cells,suggesting that CCAT2 might induce alteration in EZH2 levels in the HSC compartment. Interestingly, we identified miR-26a and miR-26b, that were already reported to target EZH2, were significantly overexpressed only in BM of CCAT2-G mice. These data suggests that CCAT2-G regulates EZH2 expression primarily through regulation of target miRNAs. On the other hand, we identified EZH2 to interact preferentially to CCAT2-T compared to WT or CCAT2-G transcript. These data confirmed that EZH2 preferentially binds to the CCAT2 in an allele-specific manner.In conclusion, deciphering the role of CCAT2 in spontaneously induced myelodysplasia and cytopenias will help us further characterize the poorly understood MDS/MPN phenotype. CCAT2 mice can serve as a robust model for studying initiation of de novo MDS/MPN that does not progress to secondary AML, and as a pre-clinical model for evaluation of new therapies for MDS. It has high translational potential as CCAT2 can be developed into a diagnostic and prognostic marker, as well as a novel intervention target for MDS therapy. DisclosuresNo relevant conflicts of interest to declare.

Acute Promyelocytic Leukemia: An Unsual Presentation

IntroductionFive to eight percent of acute promyelocytic leukemia (APL) occurs in mid-life with disseminated intravascular coagulation (DIC) being a classic presentation. Laboratory investigation usually reveals high white cell count (WBC), promyelocytes and early myeloid series with abundant auer rods, with the exception of the microgranular variant. The bone marrow in APL is usually hypercellular with positive myeloperoxidase stains and the characteristic CD33, CD13, CD15, and CD117 positivity on immunohistochemistry and flow cytometry. The presence of a reciprocal translocation t(15;17)(q22;q12) on chromosome 17 causing a fusion of the promyelocytic - and retinoic acid receptor-alpha genes (PML-RARA) is the hallmark of APL, which is found in 95% of cases. The diagnosis of APL becomes challenging when it presents with uncharacteristic features, causing a delay in early commencement of treatment. A wide index of suspicion arises from being aware of unusual presentations.Case presentationWe therefore present a 52 year old diabetic male who while been treated for hyperosmolar non ketotic hyperglycemia (HONK) was found with persistent isolated leucopenia with severe neutropenia (WBC: 0.7, absolute neutropenia: 100) and weight loss. This was initially reckoned as an adverse reaction to medication. He was treated with granulocyte colony stimulating factor (GCSF) and he responded with an unsustained increase in WBC and declining platelet counts. His medical history was insignificant for malignancy, splenomegaly, adenopathy nor sepsis. His medications comprised of antihypertensives, antidiabetics and the statins. Bone marrow findings included patchy cellularity, marked myeloid hyperplasia, few cells with auer rods, and myeloid maturation (1% blasts, 26% promyelocytes, and 22% myelocytes). The maturation and other listed bone marrow findings were initially attributed to the effect of the GCSF, but were also highly suspicious for APL. The diagnosis of APL was not given until a molecular analysis was positive for PML-RARA fusion protein. Following a diagnosis of APL with translocation, induction chemotherapy was commenced with all-trans retinoic acid (ATRA) and arsenic trioxide, during which the patient achieved molecular remission with no evidence of residual leukemia in the bone marrow (0% blasts, 1% promyelocytes, and 6% myelocytes) and a negative flow cytometric analysis. The consolidation phase is ongoing and the patient has consistently maintained remission and normal complete blood count (WBC: 1.1, absolute neutrophil count: 300).DiscussionThis case highlights the importance of having a high index of suspicion for APL. In such clinical settings, an early bone marrow examination is imperative and can be the first pointer to the diagnosis of APL. The prior use of GCSF created a distraction by adding an increased myelocyte population to the patient's differential. Fortunately, molecular testing for PML-RARA detected the fusion protein and treatment was then initiated. Any increased promyelocyte population, regardless of the remaining differential, should always prompt a discussion regarding the high value of ruling out APL by molecular testing. DisclosuresNo relevant conflicts of interest to declare.

Functional analysis of TET2 using a knockdown mouse model

In myeloid malignancies, mutations have occurred in epigenetic regulator genes, including Ten-Eleven-Translocation 2 (TET2). TET2 is an enzyme that catalyzes the conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5-hmC) which is a key intermediate for oxidative DNA demethylation. We analyzed the in vivo phenotype of TET2 failure using Ayu17-449 (TET2(trap/trap)) mice created by the gene-trap method in which TET2 mRNA levels were decreased to about 20% of the level in wild-type (WT) mice. In TET2(trap/trap) mice the levels of 5-hmC in genomic DNA from bone marrow (BM) cells were decreased in comparison to WT mice. TET2(trap/trap) mice were born at an expected Mendelian frequency but died at a high rate by postnatal day 3, indicating TET2 to be essential for survival. In analysis of the hematopoietic system, transplantation of TET2(trap/trap), but not WT fetal liver cells, led to mild myeloid hyperplasia and splenomegaly in WT recipient mice, but no onsets of lethal hematological malignancies were observed during a follow-up period of 12 months. TET2 knockdown led to an increased serial replating capacity of BM cells in vitro and increased hematopoietic stem cell (HSC) self-renewal in vivo in competitive repopulation and serial transplantation assays. These data indicate that TET2 has a critical role in survival and HSC homeostasis.

MiR-155 regulative network in FLT3 mutated acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) represents a heterogeneous disorder with recurrent chromosomal alterations and molecular abnormalities. Among AML with normal karyotype (NK-AML) FLT3 activating mutation, internal tandem duplication (FLT3-ITD), is present in about 30% of patients, conferring unfavorable outcome. Our previous data demonstrated specific up-regulation of miR-155 in FLT3-ITD+ AML. miR-155 is known to be directly implicated in normal hematopoiesis and in some pathologies such as myeloid hyperplasia and acute lymphoblastic leukemia. Methods and resultsTo investigate about the potential influence of miR-155 de-regulation in FLT3-mutated AML we generated a transcription factors regulatory network and combined this with data from multiple sources that predict miR-155 interactions. From these analyses, we derived a sub-network, called “miR-155 module” that describes functional relationship among miR-155 and transcription factors in FLT3-mutated AML. We found that “miR-155 module” is characterized by the presence of six transcription factors as central hubs: four miR-155 regulators (JUN, RUNX1, FOSb, JUNB) and two targets of miR-155 (SPI1, CEBPB) all known to be “master” genes of myelopoiesis. We found, in FLT3-mutated AML, a significant down-regulation of miR-155 target genes CEBPB and SPI1 and up-regulation of miR-155 regulator genes JUN and RUNX1. We also showed that PKC412-related FLT3 inhibition, in MV4-11 cell line, causes down-regulation of miR-155 and increased level of mRNA and protein of miR-155 target SPI1. We showed in experiments of miR-155 mimic in K562 cell line, a high increase of miR-155 and an inverse correlation with the mRNA levels of its targets SPI1 and CEBPB. Moreover silencing of miR-155 in primary AMLs causes mRNA up-regulation of its target SPI1 and CEBPB. ConclusionOur results suggest that activating mutation of FLT3 in AML can lead, through the induction of JUN, to an increased expression of miR-155, which then causes down-regulation of SPI1 and CEBPB and consequently may causes block of myeloid differentiation.

Natural course and biology of CML.

Chronic myeloid leukaemia (CML) is a myeloproliferative disorder arising in the haemopoietic stem cell (HSC) compartment. This disease is characterised by a reciprocal t(9;22) chromosomal translocation, resulting in the formation of the Philadelphia (Ph) chromosome containing the BCR-ABL1 gene. As such, diagnosis and monitoring of disease involves detection of BCR-ABL1. It is the BCR-ABL1 protein, in particular its constitutively active tyrosine kinase activity, that forges the pathogenesis of CML. This aberrant kinase signalling activates downstream targets that reprogram the cell to cause uncontrolled proliferation and results in myeloid hyperplasia and 'indolent' symptoms of chronic phase (CP) CML. Without successful intervention, the disease will progress into blast crisis (BC), resembling an acute leukaemia. This advanced disease stage takes on an aggressive phenotype and is almost always fatal. The cell biology of CML is also centred on BCR-ABL1. The presence of BCR-ABL1 can explain virtually all the cellular features of the leukaemia (enhanced cell growth, inhibition of apoptosis, altered cell adhesion, growth factor independence, impaired genomic surveillance and differentiation). This article provides an overview of the clinical and cell biology of CML, and highlights key findings and unanswered questions essential for understanding this disease.

Spontaneous Soft Tissue Haematomas- A Rare Presentation of Chronic Myeloid Leukemic (CML)

Spontaneous soft tissue haematomas are rarely found in haematological malignancies. Chronic myeloid leukemia (CML) is a myeloproliferative disorder which rarely present with thrombo-haemorrhagic phenomenon. It is a malignant clonal disorder of pleuripotent stem cells that results in increase in myeloid, erythroid and platelets cells in peripheral blood and marked myeloid hyperplasia in bone marrow. It is characterized by the presence of Philadelphia chromosome (Ph) with BCR - ABL 1 fusion gene. This gene is responsible for the formation of 210 KDa chimeric proteins with enhanced tyrosine kinase activity which leads to the abnormal bone marrow cell proliferation and to the clinical and morphologic manifestations of leukemia. Cutaneous and mucous membrane bleeding is common in CML whereas bleeding in deep soft tissue is rarely found because of qualitative and quantitative platelet abnormalities. Here, we report a case of CML (BCR-ABL rearrangement positive) who presented with large haematoma in the anterior as well as posterior compartment of left thigh and treated successfully with hydroxyurea and imatinib.

HnRNP K: A Tumor Suppressor or Oncogene?

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA and DNA binding protein that regulates critical pathways controlling differentiation and proliferation programs. While alterations in hnRNP K expression are associated with neoplastic malignancies, we currently do not understand how changes in hnRNP K expression contribute to tumor phenotypes in vivo. Previous biochemical and cell line studies demonstrate that hnRNP K transcriptionally regulates p53-dependent activities, suggesting it functions as a potential tumor suppressor. However, hnRNP K has also been shown to positively regulate c-Myc expression, indicating it may behave as an oncogene.The HNRNP K gene maps to a region of chromosome 9 (9q21.32), which is lost in a subset of patients with acute myeloid leukemia (AML). RNA expression analyses of patient samples with AML that harbor 9q21.32 deletions revealed a significant reduction in HNRNP K expression compared to wild type control samples, supporting the notion that hnRNP K acts as a tumor suppressor (Figure 1A). However, patients with AML who do not harbor a 9q21.32 deletion displayed a significant increase in hnRNP K expression (Figure 1A). Thus, to examine the association between altered hnRNP K expression and disease status in patients with AML, we performed reverse phase protein array (RPPA) analysis on CD34+ bone marrow cells from 415 de novo AML patient as well as healthy donor controls. Interestingly, we observed a significant correlation between elevated hnRNP K levels and poor outcomes, which supports the idea that hnRNP K has oncogenic potential (Figure 1A). Together, these observations indicate that any change in hnRNP K expression may contribute to the etiology of AML and supports the idea that hnRNP K may potentially act as either a haploinsufficient tumor suppressor or oncogene in AML.To directly interrogate these possibilities in vivo, we generated mouse models that either harbor a deletion of one hnRNP K allele (hnRNP K+/-) or overexpressed hnRNP K (hnRNP KTg) in the hematological compartment. Western blot analyses demonstrated that hnRNP K haploinsufficiency results in a significant reduction in hnRNP K expression while tissue-specific activation of hnRNP K resulted in overexpression of hnRNP K.Similar to our observation in AML patients, either hnRNP K haploinsufficiency or overexpression resulted in similar phenotypes in vitro and in vivo. Lin-CD117+ hematopoietic stem cells (HSCs) from hnRNP K+/- and hnRNP KTg mice had significant increases in differentiation and proliferation potential as determined by colony formation assays. In these experiments, we observed a significant increase in the number of total colonies and number of cells per colony in both hnRNP K+/- and hnRNP KTg HSCs as compared to wild type HSCs (Figure 1B). In vivo analyses of the hnRNP K+/- and hnRNP KTg mice revealed a significant increase in myeloid hyperplasia in the peripheral blood and bone marrow, increased tumor formation, genomic instability, and decreased survival compared to wild type mice (Figure 1C). Interestingly, both increased and decreased hnRNP K expression resulted in alterations in similar pathways that regulate differentiation and proliferations potential (e.g.; p53 and c-Myc pathways and alterations in C/EBP expression).Together, these clinical and animal model studies illustrate that either over-expression or under-expression of hnRNP K lead to strikingly similar phenotypes that directly impact the etiology of AML. Furthermore, these data not only implicate that hnRNP K behaves as both a tumor suppressor and oncogene, but also suggest that it functions as a master toggle that dictates the proliferation and differentiation potential of HSCs. We are currently using Whole Transcriptome Shotgun Sequencing (RNA-Seq) and ChIP-Seq to evaluate the mechanisms by which increased and decreased hnRNP K expression impact hematologic malignancies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

LAMTOR2 Regulates Stem Cell Maintenance and Acts in Lineage Choice By Modulating Cytokine Receptor-Mediated Signaling

BackgroundWe have previously reported a novel primary immunodeficiency syndrome caused by a mutation in the 3’ untranslated region of the gene p14 (also known as late endosomal/lysosomal adaptor, MAPK and MTOR activator 2; LAMTOR2) resulting in impaired endosomal biogenesis (Bohn et al, Nature Medicine 2007). The p14-deficient patients are characterized by congenital neutropenia associated with growth failure, partial albinism, as well as B cell and cytotoxic T cell deficiency. Here, we studied the role of the endosomal adaptor protein p14 in hematopoieses using a Cre/loxP-based conditional knockout mouse model.MethodsWe have generated a conditional Mx1Cre-p14flox/flox (p14-/-) mouse model, in which postnatal deletion of p14 can be induced by intraperitoneal injection of polyinosinic-polycytidylic acid (pIpC, 5 x 250 µg, every other day). The hematopoietic system of p14-/- mice was analyzed by tissue sections, flow cytometry, biochemical assays, and electron microscopy studies. Hematopoietic stem cells were analyzed by competitive transplantation studies and in vitro colony-forming unit assays.Resultsp14-/- mice die 3 - 6 weeks after treatment with pIpC. Disruption of p14 was associated with anemia, thrombocytopenia, and moderate leukocytosis in the peripheral blood. Pathological examination of p14-/- mice revealed a splenomegaly and myeloproliferative infiltrations in the bone marrow and spleen. Flow cytometric analysis of p14-deficient nucleated bone marrow cells showed a massive expansion of immature myelomonocytic cells (Gr-1lowCD11b+, pre-M), whereas the granulocytic (Gr-1highCD11b+), B cell (CD19+B220+), and erythroid (TER-119+) lineages were severely reduced. Similarly, FACS analysis revealed a remarkable accumulation of the pre-M population in the spleen, whereas the erythroid lineage was increased at various developmental stages as determined by expression of TER-119 and CD71, indicating enhanced extramedullary hematopoiesis. Immunophenotypic characterization of the hematopoietic stem cell (HSC) compartment showed a significant depletion of long-term HSCs (lin-Sca-1+c-kit+CD150+CD48-) accompanied by an increase in the proportion of short-term HSCs (lin-Sca-1+c-kit+CD150+CD48+) and multipotent progenitors (lin-Sca-1+c-kit+CD150-CD48+). BrdU incorporation assays documented enhanced cycling of lin-Sca-1+c-kit+ cells (LSK) 4 days after pIpC injection. Transcriptome analysis detected a strong enrichment of myeloid-specific and a loss of “stemness” gene sets within the p14-deficient LSK gene signature. Bone marrow transplantation studies showed that the p14-deficient bone marrow cells are less competent in repopulating lethally irradiated mice and that the phenotype of myeloid hyperplasia in p14-/- mice is transplantable and caused by cell-intrinsic effects. As a potential pathomechanism for the myeloproliferative phenotype in p14-/- mice we documented an altered cytokine receptor-mediated signaling in p14-deficient hematopoietic progenitor cells (HPC). Whereas p14-deficient HPC exhibited an increased response to GM-CSF and IL-3 with respect to ERK1/2 phosphorylation and colony formation, G-CSF signaling was substantially impaired corroborating with the decreased frequency of granulocytic Gr-1highCD11b+ cells in p14-/-mice.ConclusionOur data suggest that conditional deletion of p14 in mice causes a myeloproliferative disorder associated with increased sensitivity of HPC to GM-CSF receptor-mediated signaling. DisclosuresNo relevant conflicts of interest to declare.

Neutropenia during Hematopoietic Stem Cell Mobilization with G-CSF

A 36-year-old female was referred to our hematology department for HSC donation by leukapheresis of peripheral blood after mobilization. On the first day she was informed of adverse events of HSC harvesting and then signed an agreement. She denied of having any severe or chronic disorders before. No abnormal signs are found in physical and laboratory examination. Laboratory data showed normal blood routine test (WBC 3.77X10E9/L, LYM 0.61X10E9/L NEU 1.68X10E9/L, HGB 128 g/L and PLT 174X10E9/L), negative test for HBV, HCV, HIV, Toxoplasmosis and Treponemapallidum. Quantitation of both cytomegalovirus and Epstein-Barr virus DNA were <500 copies/mL. She was administrated with 300u G-CSF (FILGRASTIM, Kirin-Amgen) by subcutaneous injection at 16:00, then once a day in the morning. Norethisterone tablets were given 5000ug TID daily to delay the coming menstruation, which had been used routinely for many years safely for this purpose in our hospital, altogether total dose of Norethisterone tablets 30000ug in 48 hours.On the third day of G-CSF administration, blood routine test amazingly showed a sharply declined Neutrophilic Granulocyte count (NEU) of 0.54X10E9/L, reduced lymphocyte count (LYM) of 0.77X10E9/L and normal Monocyte count (MON) of 0.56X10E9/L, normal Hematoglobin (HGB) of 127 g/L and normal platelet count (PLT) of 150X10E9/L. She was diagnosed with Neutropenia. During re-inquiring for her medical history, she admitted that she was diagnosed with Adult Onset Still’s Disease (AOSD) when she was 18 y.o. which relapsed at 24. She was given aspirin and achieved remission of the symptoms. She denied any history of allergy. The donor did not complain about any symptoms and her body temperature was 36°C. Norethisterone tablets administration was stopped. At 16:00 of the third day, additional 300u G-CSF was administrated for Neutropenia. At 17:21, blood sample showed NEU 0.50X10E9/L, Agranulocytosis indicated, and an evaluated MON of 0.77X10E9/L; normal coagulation function, evaluated ESR of 31mm/h, slightly reduced complement 3 of 0.87 g/L (normal: 0.9 to 1.8 g/L) and evaluated total complement of 50.1 U/mL (normal: 23.0 to 46.0 U/mL) was showed; on the next day afternoon, strong positive (3+) test for anti-RO52 and negative test for ANA, anti-DS-DNA, anti-Jo-1, ANCA, anti-SS-A or anti-SS-B in autoimmune antibody was reported. Abdominal ultrasonography reported normal size of her liver and spleen.On the fourth day, 300u G-CSF was administrated the fifth time at 10:00. After that, blood sample was collected and showed WBC of 2.22X10E9/L, LYM of 0.86X10E9/L, NEU of 0.52X10E9/L, MON of 0.83X10E9/L, HGB of 133 g/L and PLT of 153X10E9/L. Bone marrow aspiration showed myeloid hyperplasia (-), hypoplasia and abnormal maturation in granule cell, left shift with toxic granulation. CD34+ cell ratio reported 1.4% (marrow) and 0.2% (peripheral blood), which revealed HSC mobilization was failed.On the fifth day, G-CSF was not administrated. Blood sample collected at 12:00, 26 hours after the last G-CSF administration, revealed WBC of 3.94X10E9/L, LYMX1.31 10E9/L, NEU 1.08 X10E9/L, MON 1.48X10E9/L, HGB of 125 g/L and PLT of 151X10E9/L. The donor seemed recovering from Neutropenia.At 12:00 on the sixth day, bone marrow was collected for transplantation. Blood routine test showed WBC of 4.03X10E9/L, LYM 0.84X10E9/L, NEU 2.38X10E9/L, MON 0.78X10E9/L, HGB of 94 g/L and PLT of 129X10E9/L.We described a HSC donor with AOSD history who developed Neutropenia subsequent to subcutaneous injection of G-SCF and recovered in 2 days after the last administration. Neutropenia caused by Norethisterone tablets was not common and we didn’t find any report that combined medication of norethisterone tablets and G-CSF would cause neutropenia. The donor didn’t complain any symptoms and we couldn’t find any typical signs in the process, which indicated Neutropenia might be unrelated with infection or allergy.It may be the first case of this situation, we have not yet confirmed the cause of her Neutropenia. We preserved samples of blood and marrow aspiration under the donor’s consent and we will have further research and follow-up with this case. DisclosuresLiu:National Natural Science Foundation of China (81270647, 81300445, 81200388): Research Funding; National High Technology Research and Development Program of China (863 Program) (2011AA020105): Research Funding; National Public Health Grand Research Foundation (201202017): Research Funding; Natural Science Foundation of Guangdong Province (S2012010009299): Research Funding; the project of health collaborative innovation of Guangzhou city (201400000003-4, 201400000003-1): Research Funding; the Technology Plan of Guangdong Province of China (2012B031800403): Research Funding; the project of the Zhujiang Science & Technology Star of Guangzhou city (2013027): Research Funding.

Myeloproliferative Neoplasm or Reactive Process? A Rare Case of Acute Myeloid Leukemia and Transient Post-Treatment Megakaryocytic Hyperplasia with JAK-2 Mutation

Myeloproliferative neoplasms (MPNs) are hematopoietic malignancies characterized by unchecked proliferation of differentiated myeloid cells. The most common BCR-ABL1-negative MPNs are polycythemia vera, essential thrombocythemia, and primary myelofibrosis. The discovery of JAK2 V617F mutation has improved our understanding of the molecular basis of MPN. The high frequency of JAK2 mutation in MPN makes JAK2 mutation testing an essential diagnostic tool and potential therapeutic target for MPN. Here, we present a rare case of a 34-year-old patient who was initially diagnosed with acute myeloid leukemia (AML) with mutated NPM1. After chemotherapy treatment followed by granulocyte colony stimulating factor administration, the patient achieved complete remission of AML. However, the bonemarrow showed hypercellularity with granulocytic hyperplasia, markedly increased atypical megakaryocytes (50.2/HPF) with focal clustering, and reticulin fibrosis (3/4). JAK2 V617F mutation was also detected. Considering the possibility of AML transformed from a previous undiagnosedMPN, patient underwent peripheral blood allogenic stem cell transplant. This case illustrates the diagnostic challenges of firmly establishing a diagnosis between similar, but distinct, disease entities and an accurate clinicopathological differentiation is crucial.

Gfi-1 regulates the erythroid transcription factor network through Id2 repression in murine hematopoietic progenitor cells

AbstractGrowth factor independence 1 (Gfi-1) is a part of the transcriptional network that regulates the development of adult hematopoietic stem and progenitor cells. Gfi-1-null (Gfi-1−/−) mice have reduced numbers of hematopoietic stem cells (HSCs), impaired radioprotective function of hematopoietic progenitor cells (HPCs), and myeloid and erythroid hyperplasia. We found that the development of HPCs and erythropoiesis, but not HSC function, was rescued by reducing the expression of inhibitor of DNA-binding protein 2 (Id2) in Gfi-1−/− mice. Analysis of Gfi-1−/−;Id2+/− mice revealed that short-term HSCs, common myeloid progenitors (CMPs), erythroid burst-forming units, colony-forming units in spleen, and more differentiated red cells were partially restored by reducing Id2 levels in Gfi-1−/− mice. Moreover, short-term reconstituting cells, and, to a greater extent, CMP and megakaryocyte-erythroid progenitor development, and red blood cell production (anemia) were rescued in mice transplanted with Gfi-1−/−;Id2+/− bone marrow cells (BMCs) in comparison with Gfi-1−/− BMCs. Reduction of Id2 expression in Gfi-1−/− mice increased the expression of Gata1, Eklf, and EpoR, which are required for proper erythropoiesis. Reducing the levels of other Id family members (Id1 and Id3) in Gfi-1−/− mice did not rescue impaired HPC function or erythropoiesis. These data provide new evidence that Gfi-1 is linked to the erythroid gene regulatory network by repressing Id2 expression.

Mycobacterium leprae in bone marrow.

The case of a 54-year-old man, who was referred to evaluate a consumptive syndrome with anemia and fever of unknown origin, is reported. A physical examination revealed madarosis, nodular lesions on the ears, nasal soft tissue collapse, subcutaneous nodules on arms and generalized lymphadenopathy. Laboratory tests showed anemia (Hb 8.8 g/dL, mean corpuscular volume 82.4 fL, mean corpuscular hemoglobin 24.5pg), normal reticulocyte count (0.5%), thrombocytosis (482×109/L), normal serum ferritin (268ng/mL) and elevated C-reactive protein (124mg/L). Serologies (viral hepatitis and human immunodeficiency virus) were non-reagent. Lepromatous leprosy was confirmed by staining for acidfast bacilli using samples from the ear and subcutaneous nodules and bone marrow smears. Bone marrow was hypercellular with myeloid hyperplasia (myeloid–erythroid ratio 7:1) but without dysplasia. Mycobacterium leprae was detected lying free and in foamy histiocytes named Virchow cells (Figures 1 and 2).1,2 A multidrug therapeutic regimen (clofazimine, dapsone, rifampicin) was established with progressive improvement.

Deviation from Normal Values of Leukocyte and Erythroblast Parameters in Complete Blood Count is a Messenger for Platelet Abnormalities.

Automated blood cell counters have undergone a formidable technological evolution owing to the introduction of new physical principles for cellular analysis and the progressive evolution of software [1,2]. The results have been an improvement in analytical efficiency and an increase in information provided with new parameters. A 61-year-old male patient had the diagnosis of diffuse large B-cell lymphoma 6 years ago, and after chemotherapy, he was still in remission. He was hospitalized for high fever, fatigue, acute renal failure, and bibasilar crepitant rales. Complete blood count measured with a Beckman Coulter LH 780 hematology analyzer revealed an uncorrected leukocyte count (UWBC) of 63.5x109/L, leukocyte count (WBC) of 22.1x109/L, erythroblast count (NRBC) of 21.4x109/L, and platelet count of 197x109/L (Figure 1). Upon peripheral blood smear examination, we detected 5% neutrophils, 22% band forms, 61% metamyelocytes, 5% myelocytes, 1% promyelocytes, 2% myeloblasts, 2% lymphocytes, and 2% eosinophils. We also detected rare erythroblasts and large platelets with profuse platelet clumps (Figures 2 ). Routine biochemical analysis revealed high fasting glucose, blood urea nitrogen, creatinine, serum glutamic oxaloacetic transaminase, alkaline phosphatase, direct and indirect bilirubin, albumin, and lactate dehydrogenase. The erythrocyte sedimentation rate was 100 mm/h, and serum ferritin was 2944 ng/mL. High-resolution computed tomography of the thorax revealed bilateral diffuse infiltrations, nodular opacities, right pleural effusion, and mediastinal lymphadenopathies. Clarithromycin and imipenem/cilastatin were administered for a probable diagnosis of pneumonia. Bone marrow examination revealed myeloid hyperplasia but nothing else significant. No endobronchial mass was detected in bronchoscopy, but mucopurulent secretion was present in the right upper and lower lobes. Biopsy reports showed non-neoplastic bronchial mucosa epithelium. Sputum, blood, and urine cultures; sputum mycobacterial examination; and serum galactomannan antigen were all negative. After the general condition, fever, acute renal failure, signs, and symptoms were relieved, the patient was discharged. Informed consent was obtained. Figure 1 Complete blood count. Figure 2 Peripheral smear. Large platelets with profuse platelet clumps are noteworthy. When we subtracted the WBC and NRBC from the UWBC (=20.0x109/L), a significanT-cell group was composed of big platelets. It is probable that this ratio was higher than calculated. Rare erythroblasts in the peripheral blood smear with high NRBC values support the idea of large platelets as cellular origin. In fact, the peripheral blood smear revealed large, profuse platelet clumps, contradictory to the platelet count. We conclude that complete blood counts should be examined carefully; despite the essential role of automation in the modern hematology laboratory, microscopic control of pathologic samples (i. e. peripheral blood smear) remains indispensable, so much so that in certain cases, it alone is diagnostic.

Epidermal overexpression of transgenic ΔNp63 promotes type 2 immune and myeloid inflammatory responses and hyperplasia via NF-κB activation

ΔNp63 is known to be critical in skin development and cancer; however, how it triggers proliferation and inflammation in vivo remains to be elucidated. Here, we find that induced ΔNp63 expression in skin of transgenic mice (TG) results in a hyperproliferative epidermis coupled with inflammatory infiltrates. In situ, infiltrating cells include CD45(+) leukocytes, CD19(+) B lymphocytes, CD3(+) T lymphocytes, CD4(+) T helper, CD25(+)/Foxp3(+) Treg, Ly6B(+) neutrophils, S-100(+) dendritic cells, and macrophages bearing CD11b(+), F4/80(+), CD68(+), and CD206(+) M2 type markers. Transcriptional profiling of TG skin revealed increased gene expression involved in inflammation and immune responses, including Th2/M2 cytokines and chemokines. These genes were co-regulated by ΔNp63 and NF-κB RelA or cRel, and enhanced by TNF-α. Elevated cRel, RelA, and IKKs were observed in TG mouse skin and human squamous carcinomas with ΔNp63 overexpression. Thus, our findings unveil a missing link connecting overexpressed ΔNp63 with aberrant NF-κB activation, pro-inflammatory and type 2 cytokines and chemokines, and host infiltrates during skin inflammation and hyperplasia. Our findings provide a missing link between ΔNp63 overexpression and NF-κB-mediated inflammation, of potential relevance to the pathogenesis of squamous carcinoma.

Impaired Erythropoiesis Of Gfi-1 Null Hematopoietic Progenitor Cells Is Rescued By Reducing Id2 Levels

The survival, self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPC) are tightly regulated by extrinsic signals, and intrinsically by transcription factors and their regulatory networks. The molecular and cellular mechanisms, which regulate the complex process of hematopoiesis, depend upon the correct expression of transcription factors and their regulators. One such family of regulators is the inhibitor of DNA binding/differentiation (Id), which is helix-loop-helix proteins that function by acting as dominant negative regulators of transcription factors such as E proteins, ETS, Pax, and retinoblastoma proteins. Expression of Id2, one of the Id family proteins, is regulated by growth factor independence-1 (Gfi-1) encoding a transcriptional repressor. Gfi-1 is required for the development of multiple cell lineages including HSPC and ultimately differentiated blood cells. Although genes have been identified to mediate hematopoietic defects observed in Gfi-1 knockout (Gfi-1 KO) mice including the maturational and developmental defects in granulocyte (CSF-1, RasGRP1, and PU.1) and B cell (PU.1 or Id2), and myeloid hyperplasia (Id2 or HoxA9), Gfi-1-target genes that mediate the defects in radioprotection, maintenance of HSC, and erythroid hyperplasia in Gfi-1 KO mice are unknown.Since Id2 expression is elevated in HSPC of Gfi-1 KO mice and Id2 promotes cell proliferation, we hypothesized that lowering Id2 expression could rescue the HSPC defects in the Gfi-1 KO mice. By transplanting Gfi-1 KO mouse bone marrow cells (BMC) into lethally-irradiated recipient mice, we observed that short-term reconstituting cell (STRC) activity in Gfi-1 KO BMC is rescued by transplanting Gfi-1 KO; Id2 Het (heterozygosity at the Id2 locus) BMC, while the long-term reconstitution defect of HSC was not. Interestingly, lineage- Sca-1- c-Kithi HPC, which enriched for megakaryocyte-erythroid progenitor (MEP) as one of the STRC, were fully restored in mice transplanted with Gfi-1 KO; Id2 Het BMC, in contrast to lack of the HPC in Gfi-1 KO BM-transplanted mice. The restoration of donor c-Kithi HPC was directly correlated with increased red blood cell (RBC) levels in recipient mice, which was produced after donor BM engraftment. Furthermore, we identified that reduced Id2 levels restore erythroid cell development by rescuing short-term hematopoietic stem cell, common myeloid progenitor and MEP in the Gfi-1 KO mice. In addition, burst forming unit-erythroid (BFU-E) colony assay showed that hemoglobinized BFU-E development was restored in Gfi-1 KO BM and spleen by lowering Id2 levels. Unlike Id2 reduction, reducing other Id family (Id1 or Id3) levels in Gfi-1 KO mice does not rescue the impaired development of erythroid and other hematopoietic lineages including myeloid, T and B cells.Abnormal expansion of CD71+ Ter119-/low erythroid progenitor cells was rescued in Gfi-1 KO; Id2 Het BMC compared to those in Gfi-1 KO mice. Thus, we hypothesized that erythroid development was blocked at the early stage of erythropoiesis due to the ectopic expression of Id2 in Gfi-1 KO mice. Using Id2 promoter-driven YFP reporter mice, we found that Id2 is highly expressed in the CD71+ Ter119-/low erythroid progenitors, and decreases as the cells mature to pro-erythroblasts and erythroblasts, suggesting that repression of Id2 expression is required for proper erythroid differentiation in the later stages. The dramatic changes of Id2 expression during erythroid development support our findings that the overexpression of Id2 in the absence of Gfi-1-mediated transcriptional repression causes impaired erythropoiesis at the early stage. To identify the molecular mechanisms that could account for how reduced Id2 levels rescue erythropoiesis in Gfi-1 KO mice, we compared the expression of genes and proteins in Gfi-1 KO; Id2 Het and Gfi-1 KO BMC. Using microarray, qRT-PCR and western blot, we discovered that reduction of Id2 expression in Gfi-1 KO BMC results in increased expression of Gata1, EKlf, and EpoR genes, which are required for erythropoiesis. However, the expression levels of cell cycle regulators were not altered by lowering Id2 expression in Gfi-1 KO mice. These data suggest a novel molecular mechanism in which Gfi-1 modulates erythropoiesis by repressing the expression of Id2 that reduce the levels of Id2 protein, binding to E2A and inhibiting the formation of E2A/Scl transcription enhancer complex. Disclosures:No relevant conflicts of interest to declare.

Evaluation Of The Effects Of Long-Term Treatment With Eltrombopag On Bone Marrow In Patients With Chronic Immune Thrombocytopenia (ITP) — Data From The EXTEND Study

▪ IntroductionEltrombopag, a thrombopoietin receptor agonist (TPO-RA), increases platelet counts in patients with cITP. TPO-RAs have been associated with varying degrees of bone marrow (BM) reticulin increases. Patients in the EXTEND study, an ongoing, open-label extension study of eltrombopag in patients with heavily pretreated cITP who received eltrombopag or placebo in a previous eltrombopag study, underwent annual BM biopsies while on study. MethodsCentral morphology review was done by 2 hematopathologists blinded to clinical information who assessed specimens for cellularity, megakaryocyte, erythroid, and myeloid quantity and appearance; trabecular bone changes; and reticulin and collagen deposition (European Consensus scale). ResultsIn EXTEND, 302 patients with cITP were treated with eltrombopag for ≤6.5 years as of February 2013. Of these, 232 BM biopsy specimens collected from 117 patients from pretreatment and up to 5.5 y on treatment were available at the time of this analysis, and were centrally reviewed (Table). Of these, 5 patients provided a biopsy sample within <10 mo on treatment, 48 patients at 1 y, 68 at 2 y, 51 at 3 y, 30 at 4 y, and 11 at 5 y on treatment.Median age (range) of the 117 patients was 50 (20-78) y; 69 (59%) patients were female; 85% were Caucasian/European, 9% were East Asian, 2% were South East Asian, and 4% were Arabic/North African. Median time since ITP diagnosis was 58.6 (8.5-421) mo.Two hundred and nine on-treatment biopsies, collected from 115 patients, were graded for reticulin using the European Consensus scale. Most (63-80%) of the on-treatment biopsies, collected between 1 y and 5 y, were MF-0 or MF-1 (17-33%) (Figure). Reticulin fibers were increased (MF ≥2) in 5 specimens from 2/115 (1.7%) patients. All 5 specimens were positive for collagen, and 3 of the 5 specimens in the 2 patients were judged as hypercellular. Of the 2 patients, 1 patient (East Asian) had reticulin documented after 2 y (970 days) of treatment, and was preemptively withdrawn from the study. Per protocol, a post-treatment biopsy was performed; a BM specimen taken 7 mo following withdrawal was graded as MF-0 for reticulin and negative for collagen. The second patient (Caucasian) had MF-2 at 1 y, which increased to MF-3 at 2 y and contained megakaryocytes with myeloproliferative neoplasm-like features, and then returned to MF-2 at the 3 y and 4 y assessments; however, these biopsies at 3 y and 4 y showed no signs of myeloproliferative neoplasm-like features and had reverted to normal. The patient remains on treatment at the time of this analysis. Neither patient experienced any adverse event or abnormality in hematologic parameters potentially related to impaired BM function.Cellularity was normal in the majority of patients at each assessment timepoint. Most (82-99%) patients demonstrated megakaryocytic hyperplasia. Megakaryocyte morphology was normal in all patients in all years except for 1 patient in year 1. Seven patients (6%) had megakaryocytes reminiscent of those seen with polycythemia vera or essential thrombocythemia. Erythroid hyperplasia was observed in 13-27% and myeloid hyperplasia in 6-13% of patients. Trabecular bone thinning was found in 39-70% of patients over the 5-year observation period. No pathologic pattern of changes was identified among the on-treatment specimens. DiscussionReticulin fibrosis was considered a potential risk for cITP patients receiving TPO-RAs. In the ongoing EXTEND study, at the time of this analysis, reticulin was either absent or mildly increased in 113/115 (98%) patients treated with eltrombopag up to 5.5 years at the time of their biopsy. No patient with reticulin or collagen increases showed clinical signs or symptoms indicative of BM dysfunction, and the findings were reversible upon discontinuation of treatment. No pattern of increasing reticulin deposition with longer treatment periods was observed based on BM biopsy data available to date. These data suggest that for most cITP patients, treatment with eltrombopag is not associated with the development of BM reticulin or collagen fibrosis; however, the potential association of TPO-RAs and increased BM fibrosis in cITP still needs to be better understood.▪ [Display omitted] Disclosures:Brynes:GlaxoSmithKline: Research Funding. Bakshi:GlaxoSmithKline: Employment, Equity Ownership. Bailey:GlaxoSmithKline: Employment, Equity Ownership. Chan:GlaxoSmithKline: Employment.