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Myeloid-derived suppressor cells (MDSCs) are a compendium of immature myeloid cells that exhibit potent T-cell suppressive capacity and expand during pathological conditions such as cancer and chronic infections. Although well-characterized in cancer, the physiology of MDSCs in the infection setting remains enigmatic. Here, we integrated single-cell RNA sequencing (scRNA-seq) and functional metabolic profiling to gain deeper insights into the factors governing the generation and maintenance of MDSCs in chronic Staphylococcus aureus infection. We found that MDSCs originate not only in the bone marrow but also at extramedullary sites in S. aureus-infected mice. scRNA-seq showed that infection-driven MDSCs encompass a spectrum of myeloid precursors in different stages of differentiation, ranging from promyelocytes to mature neutrophils. Furthermore, the scRNA-seq analysis has also uncovered valuable phenotypic markers to distinguish mature myeloid cells from immature MDSCs. Metabolic profiling indicates that MDSCs exhibit high glycolytic activity and high glucose consumption rates, which are required for undergoing terminal maturation. However, rapid glucose consumption by MDSCs added to infection-induced perturbations in the glucose supplies in infected mice hinders the terminal maturation of MDSCs and promotes their accumulation in an immature stage. In a proof-of-concept in vivo experiment, we demonstrate the beneficial effect of increasing glucose availability in promoting MDSC terminal differentiation in infected mice. Our results provide valuable information of how metabolic alterations induced by infection influence reprogramming and differentiation of MDSCs.

Although the mixed lineage leukemia 5 (MLL5) gene has prognostic implications in acute promyelocyte leukemia (APL), the underlying mechanism remains to be elucidated. Here, we demonstrate the critical role exerted by MLL5 in APL regarding cell proliferation and resistance to drug-induced apoptosis, through mtROS regulation. Additionally, MLL5 overexpression increased the responsiveness of APL leukemic cells to all-trans retinoic acid (ATRA)-induced differentiation, via regulation of the epigenetic modifiers SETD7 and LSD1. In silico analysis indicated that APL blasts with MLL5high transcript levels were associated with retinoic acid binding and downstream signaling, while MLL5low blasts displayed decreased expression of epigenetic modifiers (such as KMT2C, PHF8 and ARID4A). Finally, APL xenograft transplants demonstrated improved engraftment of MLL5-expressing cells and increased myeloid differentiation over time. Concordantly, evaluation of engrafted blasts revealed increased responsiveness of MLL5-expressing cells to ATRA-induced granulocytic differentiation. Together, we describe the epigenetic changes triggered by the interaction of MLL5 and ATRA resulting in enhanced granulocytic differentiation.

Background Peripheral blood stem cell (PBSC) collection places a burden on the patient and ideally should be completed in a single procedure. Consequently, a convenient predictive factor is needed for clinical use. Methods This retrospective study included 72 patients who underwent autologous PBSC collection. A median volume of 3.9×106 CD34-positive cells/kg (range: 0.3-47.4×106 cells/kg) was collected on the first day. We defined failure as an inability to collect 2.0×106 cells/kg on the first day. Patients were classified into failure (n = 25, 34.7%) and success groups (n = 47, 65.3%), and their clinical backgrounds were analyzed. Results The success group included a significantly larger number of cases in which a differential white blood cell count of the peripheral blood on the day of PBSC collection detected promyelocytes (n = 34, 72.3% vs. failure group: n = 11, 44.0%; P＝0.008). Sixty-two patients underwent autologous PBSC transplantation with a median of 5.6×106 transplanted cells/μL (range: 1.60-47.4×106 cells/μL). Among transplanted patients, no significant differences were observed between the success and failure groups in terms of the intervals until neutrophil, platelet, and red blood cell engraftment. Conclusion The presence of promyelocytes in peripheral blood may be a useful indicator of the optimal timing for single-step PBSC collection.

The patient was a 62-year-old Caucasian man with blood smear and flow cytometry concerning for acute promyelocytic leukemia with FISH ultimately confirming PML-RARA translocation. He had a 30-year history of employment at a nuclear power plant. He presented with diffuse intravascular coagulation, hyperleukocytosis, and quickly developed acute respiratory distress syndrome. On day four of ATRA + Hydrea, a bronchoalveolar lavage was performed and was non-bloody. On microscopic fluid review, abnormal immature cells with bilobed nuclear contours were identified, similar in morphology to those seen on the diagnostic blood smear review, amidst background alveolar-type macrophages. Subsequent flow cytometric analysis showed these cells to be abnormal promyelocytes; however, they differed from the blood flow cytometry study performed prior to initiation of ATRA by showing maturational immunophenotypic changes. While the leukemic promyelocytes on bronchoalveolar lavage were morphologically immature, these immunophenotypic changes somewhat recapitulated those seen in normal granulocyte maturation and were thus suggestive of so-called differentiation syndrome. Unfortunately, the patient passed away during induction chemotherapy due to complications from diffuse intravascular coagulation and differentiation syndrome. Important pathobiological information can be gathered from fluid review and concomitant flow cytometric analysis.

Under the differentiation induction therapy with all-trans retinoic acid and arsenic trioxide, nearly 95% of typical acute promyelocyte leukemia (APL), which is characterized by the presence of PML-RARA, patients can be cured. Though its good prognosis, if left untreated, the natural survival duration of typical APL patients is only 1 month, but some exceptional cases also exist. Occasionally, we have observed the entire natural clinical course of one extremely indolent APL patient, who developed from pre-APL stage (

Acute promyelocytic leukemia (APL) is a type of acute myeloid leukemia characterized by predominating abnormal promyelocytes with a PML-RARA rearrangement or a variant thereof. BCR-ABL1 rearrangement is an oncogenic event that is usually associated with chronic myeloid leukemia but also occurs in both acute lymphoblastic and acute myeloid leukemias and in healthy individuals. However, APL with concurrent PML-RARA and BCR-ABL1 rearrangements has rarely been reported. Herein, we describe a patient with APL exhibiting a BCR-ABL1 rearrangement in a minor clone and discuss the importance of evaluating this genetic alteration in terms of pathogenesis and treatment.

The process of diagnosing AML is based on the complete blood-count analysis of the patients. As such, it involves high energy consumption, long completion times, and is rather expensive compared to conventional medical practices. One of the methods for identifying tumor cells involves the utilization of image-processing techniques based on the morphology of white blood cells (WBCs). The principal objective of this study involves the identification of AML cells—especially of the AML M1 and AML M2 types—through morphological imaging of WBCs using the Naive Bayes' Classifier. The Image-processing methods used in this study include YCbCr color space classification, image thresholding, morphological operations, chain code representation, and the use of bounding boxes. Regardless of the processing technique used, all identification procedures, performed in this study, were based on the Naive Bayes' Classifier. The test process was performed on 30 images of each of the AML M1 and M2 cell types. The use of the cell identification method proposed in this study demonstrated an accuracy of 73.33%. While the accuracy of cell type identification is 54.92%. Based on the results obtained in this study, it is inferred that the Naive Bayes' Classifier method can be employed in the process of identifying dominant AML cell types amongst AML M1 and AML M2 (myeloblast, promyelocyte, myelocyte, and metamyelocyte) based on the morphology of WBCs.

SUMMARYThere is a critical need to develop small molecule inhibitors of mucin-type O-linked glycosylation. The best known reagent currently is peracetylated benzyl-GalNAc, but it is only effective at millimolar concentrations. This manuscript demonstrates that Ac5GalNTGc, a peracetylated C-2 sulfhydryl substituted GalNAc, fulfills this unmet need. When added to cultured leukocytes, breast and prostate cells, Ac5GalNTGc increased cell surface VVA-binding by ~10-fold, indicating truncation of O-glycan biosynthesis. Cytometry, mass spectrometry and Western blot analysis of HL-60 promyelocytes demonstrate that 50-80μM Ac5GalNTGc prevented elaboration of 30-60% of the O-glycans beyond the Tn-antigen (GalNAcα1-Ser/Thr) stage. The effect of the compound on N-glycans and glycosphingolipids was small. Glycan inhibition induced by Ac5GalNTGc resulted in 50-80% reduction in leukocyte sialyl-Lewis-X expression, and L-/P-selectin mediated rolling under flow. Ac5GalNTGc was pharmacologically active in mouse. It reduced neutrophil infiltration to sites of inflammation by ~60%. Overall, Ac5GalNTGc may find diverse applications as a potent inhibitor of O-glycosylation.

A microfluidic assay for the detection of promyelocytic leukemia (PML)-retinoic acid receptor &alpha, (RAR&alpha, ) fusion protein was developed. This microfluidic-based system can be used for rapid personalized differential diagnosis of acute promyelocyte leukemia (APL) with the aim of early initiation of individualized therapy. The fusion protein PML-RAR&alpha, occurs in 95% of acute promyelocytic leukemia cases and is considered as diagnostically relevant. The fusion protein is formed as a result of translocation t(15,17) and is detected in the laboratory by fluorescence in situ hybridization (FISH) or reverse transcriptase polymerase chain reaction (RT-PCR). Diagnostic methods require many laboratory steps with specialized staff. The developed microfluidic assay includes a sandwich enzyme-linked immunosorbent assay (ELISA) system for PML-RAR&alpha, on surface of magnetic microparticles in a microfluidic chip. A rapid detection of PML-RAR&alpha, in cell lysates is achieved in less than one hour. A biotinylated PML-antibody on the surface of magnetic streptavidin coated microparticles is used as capture antibody. The bound translocation product is detected by a RAR&alpha, antibody conjugated with horseradish peroxidase and the substrate QuantaRed. The analysis is performed in microfluidic channels which involves automated liquid processing with stringent washing and short incubation times. The results of the developed assay show that cell lysates of PML-RAR&alpha, positive cells (NB-4) can be clearly distinguished from PML-RAR&alpha, negative cells (HL-60, MV4-11).

Mutations in ELANE cause severe congenital neutropenia (SCN), but how they affect neutrophil production and contribute to leukemia predisposition is unknown. Neutropenia is alleviated by CSF3 (granulocyte colony-stimulating factor) therapy in most cases, but dose requirements vary between patients. Here, we show that CD34+CD45+ hematopoietic progenitor cells (HPCs) derived from induced pluripotent stem cell lines from patients with SCN that have mutations in ELANE (n = 2) or HAX1 (n = 1) display elevated levels of reactive oxygen species (ROS) relative to normal iPSC-derived HPCs. In patients with ELANE mutations causing misfolding of the neutrophil elastase (NE) protein, HPCs contained elevated numbers of promyelocyte leukemia protein nuclear bodies, a hallmark of acute oxidative stress. This was confirmed in primary bone marrow cells from 3 additional patients with ELANE-mutant SCN. Apart from responding to elevated ROS levels, PML controlled the metabolic state of these ELANE-mutant HPCs as well as the expression of ELANE, suggestive of a feed-forward mechanism of disease development. Both PML deletion and correction of the ELANE mutation restored CSF3 responses of these ELANE-mutant HPCs. These findings suggest that PML plays a crucial role in the disease course of ELANE-SCN characterized by NE misfolding, with potential implications for CSF3 therapy.

Osteocytes are the most abundant (90% to 95%) cells in bone and have emerged as an important regulator of hematopoiesis, but their role in neutrophil development and the underlying mechanisms remain unclear. Interleukin 19 (IL-19) produced predominantly by osteocytes stimulated granulopoiesis and neutrophil formation, which stimulated IL-19 receptor (IL-20Rβ)/Stat3 signaling in neutrophil progenitors to promote their expansion and neutrophil formation. Mice with constitutive activation of mechanistic target of rapamycin complex (mTORC1) signaling in osteocytes (Dmp1-Cre) exhibited a dramatic increase in IL-19 production and promyelocyte/myelocytic expansion, whereas mTORC1 inactivation in osteocytes reduced IL-19 production and neutrophil numbers in mice. We showed that IL-19 administration stimulated neutrophil development, whereas neutralizing endogenous IL-19 or depletion of its receptor inhibited the process. Importantly, low-dose IL-19 reversed chemotherapy, irradiation, or chloramphenicol-induced neutropenia in mice more efficiently than granulocyte colony-stimulating factor. This evidence indicated that IL-19 was an essential regulator of neutrophil development and a potent cytokine for neutropenia treatment.

Leukocyte ECRG4 amplifies neutrophil recruitment and regulates CD44 expression to mediate inflammation in cutaneous injury., The complex molecular microenvironment of the wound bed regulates the duration and degree of inflammation in the wound repair process, while its dysregulation leads to impaired healing. Understanding factors controlling this response provides therapeutic targets for inflammatory disease. Esophageal cancer–related gene 4 (ECRG4) is a candidate chemokine that is highly expressed on leukocytes. We used ECRG4 knockout (KO) mice to establish that the absence of ECRG4 leads to defective neutrophil recruitment with a delay in wound healing. An in vitro human promyelocyte model identified an ECRG4-mediated suppression of the hyaluronic acid receptor, CD44, a key receptor mediating inflammation resolution. In ECRG4 KO mouse leukocytes, there was an increase in CD44 expression, consistent with a model in which ECRG4 negatively regulates CD44 levels. Therefore, we propose a previously unidentified mechanism in which ECRG4 regulates early neutrophil recruitment and subsequent CD44-mediated resolution of inflammation.

Epigenomic regulation plays a vital role in cell differentiation. The leukemic HL-60/S4 [human myeloid leukemic cell line HL-60/S4 (ATCC CRL-3306)] promyelocytic cell can be easily differentiated from its undifferentiated promyelocyte state into neutrophil- and macrophage-like cell states. In this study, we present the underlying genome and epigenome architecture of HL-60/S4 through its differentiation. We performed whole-genome bisulphite sequencing of HL-60/S4 cells and their differentiated counterparts. With the support of karyotyping, we show that HL-60/S4 maintains a stable genome throughout differentiation. Analysis of differential Cytosine-phosphate-Guanine dinucleotide methylation reveals that most methylation changes occur in the macrophage-like state. Differential methylation of promoters was associated with immune-related terms. Key immune genes, CEBPA, GFI1, MAFB and GATA1 showed differential expression and methylation. However, we observed the strongest enrichment of methylation changes in enhancers and CTCF binding sites, implying that methylation plays a major role in large-scale transcriptional reprogramming and chromatin reorganisation during differentiation. Correlation of differential expression and distal methylation with support from chromatin capture experiments allowed us to identify putative proximal and long-range enhancers for a number of immune cell differentiation genes, including CEBPA and CCNF. Integrating expression data, we present a model of HL-60/S4 differentiation in relation to the wider scope of myeloid differentiation., Summary: Epigenomics play a role in cell identity and differentiation. We present the DNA methylation landscape of leukemic cells during in vitro differentiation, adding another omics layer to better understand differentiation mechanisms.

The promyelocytic leukemia (PML)/retinoic acid receptor-alpha (RARα) onco-fusion protein that is generated from t(15;17) chromosome translocation is crucial for the leukemogenesis of acute promyelocytic leukemia (APL) and is well documented as a transcriptional repressor. To understand the relationship between PML/RARα and the oncogene in the development of APL, we investigate the regulation mechanism of PML/RARα to MYB proto-oncogene and the role of this regulation on the proliferation and differentiation of APL cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays show that MYB expression was significantly higher in PML/RARα positive cell lines. Microarray data verify that the MYB expression was significantly higher in APL patient samples than in normal promyelocyte samples. Further expression analysis from RT-qPCR and microarray data verifies that the expression of MYB is upregulated by PML/RARα. Transcriptional factor binding analysis shows that MYB is directly bound by PML/RARα and its cofactors. Luciferase assays show that PML/RARα transactivated MYB promoter activity through the RARα binding site and the coexistence of CCAAT enhancer binding protein ε. We also find that PML/RARα increases the acetylation level of the promoter region of MYB. Further evidence demonstrates that PML/RARα regulates MYB expression through long-range interaction. Functionally, PML/RARα increases the cell proliferation and blocks the differentiation through activating MYB expression. Collectively, this study uncovers a novel mechanism of PML/RARα-mediated transcriptional activation and enriches our knowledge of the onco-fusion protein-mediated transcription activation.

The classical paradigm suggests that PML/RARA fusion protein is the main driver of pathogenesis in APL. It is believed that the fusion oncogenic protein mediates this effect by potentially repressing key myeloid maturation genes involved in differentiation processes. However, the underlying mechanism is not completely understood. We recently challenged this model re-opening fundamental questions as to understand the precise contribution of the fusion protein to leukemic transformation. This knowledge on the mechanistic pathways can lead to better tailored combinatorial therapeutics. To understand the role of the PML/RARA fusion protein in leukemogenesis, we initially did a transcriptome analysis in our murine MRP8-PML/RARA APL model. Interestingly, we observed only moderate alterations in gene expression pattern of the key myeloid genes that we thought to be actively involved in differentiation processes. Of particular note, we found significant downregulation of the Irf8 in our promyelocyte compartments. IRF8 is a known regulator of hematopoiesis. The IRF8 myeloid transcription factor (TF) is expressed in several lineages of the hematopoietic tree and plays an important role in orchestrating specification and differentiation of B cells, dendritic cells and monocytes. Herein, we speculate lower levels of IRF8 could potentially impact tumorigenesis in the context of PML/RARA. In order to address this question, technically, we employed stringent staining and sorting strategy to distinctly differentiate early and late promyelocytes and looked at the expression pattern of Irf8 gene both at the transcript and protein levels. Results from qRT-PCR demonstrated 4.8 fold decrease in Irf8 expression compared to wildtype controls both in preleukemic promyelocytes and fully differentiated leukemic cells suggesting PML/RARA could be a target of IRF8 and this association could potentially be involved in the emergence and maintenance of leukemia. We next asked whether these changes are reflective at the protein levels and performed a Western blot analysis in our highly purified promyelocyte population and found a dramatic decrease in IRF8 levels in comparison to wild type controls again suggesting a possible protein-protein interaction under normal conditions that may provide an advantage for the cells from turning oncogenic. In order to study how low levels of IRF8 impact promyelocyte expansion, we generated double knock-outs of mice harboring both PML/RARA Irf8-/- mutations and compared their phenotype with mice harboring single mutations in either PML/RARA or Irf8 gene. As previously observed, young PML/RARA mice had a substantially increased number of marrow promyelocytes in comparison to wild-type mice. Fascinatingly, loss of Irf8 alone resulted in an essentially identical expansion of promyelocytes (as well as a loss of earlier myeloid progenitors in the bone marrow, not seen in PML/RARA mice) and a combination of PML/RARA expression and IRF8 loss did not result in a statistically significant further expansion of promyelocytes. These results suggest an epistatic relationship between PML/RARA and IRF8, compatible with downregulation of IRF8 by PML/RARA as being a key mechanism by which t(15;17) expands promyelocytes in the initiation of APL. Furthermore, in order to assess the impact of single/double genetic alterations on the overall and leukemia free survival we transplanted lethally irradiated mice with bone marrow cells derived from PML/RARA, Irf8-/- and PML/RARA Irf8-/-double knock outs and followed these mice over a period of one year. We observed there is no difference in their overall survival rate among the different groups of mice. However, looking specifically at the acute leukemic deaths, we observed a reduced latency in our PML/RARA Irf8-/- cohorts compared to mice carrying single mutation at PML/RARA loci. We also noticed that all the acute leukemias in the PML/RARA Irf8-/- cohort occurred prior to the first appearance of acute leukemia in the PML/RARA cohorts. Altogether, these data support a model of APL leukemogenesis in which the translocation of chromosomes 15 and 17 initiates leukemia development, in part by downregulating IRF8, and in which the resulting expansion of the promyelocyte compartment contributes to acquisition of additional cooperating events (e.g. trisomy of chromosome 8, mutation of FLT3) that complete leukemic transformation. Disclosures No relevant conflicts of interest to declare.

Acute Promyelocytic Leukemia (APL) is characterized by a block in differentiation where leukemic cells are halted at the promyelocyte stage. A characteristic balanced chromosomal translocation between chromosomes 15 and 17 t (15;17) (q24; q21) is seen in 95% of cases - the translocation results in the formation of the PML-RARA fusion protein. The introduction of retinoic acid (RA) and arsenic trioxide (ATO) has been responsible for initially remarkable cure rates. However, relapsed APL, particularly in the high-risk subset of patients, remains an important clinical problem. In addition, despite the success of ATRA & ATO, many clinicians still elect to use cytotoxic chemotherapy in the treatment of APL. Patients who become resistant to ATO have an increased risk of mortality. The probability of relapse is significantly higher in the high-risk subset of patients undergoing treatment for APL; overall approximately 10-20% of APL patients relapse regardless of their risk stratification. Furthermore, 20-25% of patients undergoing treatment will develop differentiation syndrome, a common side effect of differentiation agents. Recent evidence using in vitro models has shown that mutations in the B2 domain of the PML protein, mediate arsenic resistance. Alternative agents and approaches considering these clinical outcomes are needed to address ATO resistance as well as the relapse rate in high risk APL.

Acute Promyelocytic Leukemia (APL) is a subtype of AML with a defined clinical course and biology that is distinct from otherforms of AML. The term M3-AML was assigned to the hyper granular promyelocytic leukaemia that is characterized by blast cells withazurophilic granules, bundles of auer rods and reniform or bilobed nucleus. Clinically, APL is related to disseminate intravascularcoagulation and abnormal fibrinolysis. Cytogenetically, APL may cause translocation on the promyelocytic leukaemia (PML) gene,and chromosome 15 and with the retinoic acid receptor α (RARA) gene, on chromosome 17. The diagnosis of APL is shown by bonemarrow morphologically The majority of cells in the bone marrow are abnormal, having some similarities with promyelocytes. Themalignant cells bear numerous large granules and several auer rods. Aspirates of bone marrow are also taken for cytogenesis evaluationand for detecting the translocation. A twelve years girl was admitted to the hospital with haemorrhage from the gums during 5 dayafter extraction of a tooth. On physical examination there is no organomegaly shown. The laboratory examination found normocyticnormochromic anaemia, leucopoenia and thrombocytopenia with 15% blast cell and 5% promyelocytes with multiple auer rods. Thebone marrow aspirates showed predominant of promyelocyte cells (70%) with multiple auer rods.

Background: Chronic myeloid leukemia (CML) is one of the indolent myeloproliferative neoplasms. It is characterized by the presence of the Philadelphia chromosome, a translocation between chromosomes 9 and 22 or BCR-ABL1 gene. Objectives: The aims of this study were to evaluate characteristics of CML patients and their molecular response to tyrosine kinase inhibitors (TKI) in Erbil city in Iraq. Patients and Methods: Seventy-six patients with CML were recruited in this retrospective and prospective study from February 2014 to March 2016, at Nanakaly Hospital for Blood Diseases in Erbil city – Kurdistan region of Iraq. They were evaluated from clinical point of view and their laboratory data, and molecular responses to TKI based on polymerase chain reaction were analyzed. Results: The median age of participants was 45 years; the male: female ratio was 1:0.9. The main presenting features were abdominal fullness in 66% and splenomegaly in 95% of patients. Nearly 66% of them had low European Treatment and Outcome Study (EUTOS) score; 70% of patient had major or complete molecular responses (MMR/CMR). There was a significant difference between patients who did versus who did not achieve MMR/CMR in hemoglobin level, promyelocyte, and myelocyte percent, EUTOS, and Sokal scores (P = 0.02, 0.006, 0.03, 0.001, and 0.02, respectively). Conclusion: In the current study, CML patients were at a younger age of onset, and more high EUTOS score. The majority of patients achieved MMR with frontline Imatinib or Nilotinib and those who switched from Imatinib to Nilotinib as well.

Tyrosol is a phenolic found in extra virgin olive oil (EVOO). In a Maltese monocultivar EVOO, it was present at a concentration of 9.23 ppm. The HL-60 acute myeloid leukaemia cell line, which can be differentiated to both monocytes and neutrophils, was exposed to tyrosol at this concentration and analysed for evidence of differentiation and effects of cytotoxicity. The polyphenol induced a 1.93-fold increase in cellular oxidative activity (p-value 0.044) and enhanced surface expression of CD11b and CD14. This indicates that tyrosol induces monocytic-like differentiation. An RNA-seq analysis confirmed the upregulation of monocyte genes and the loss of neutrophil genes concomitant with the bi-potential promyelocyte precursor moving down the monocytic pathway. A cell cycle analysis showed an accumulation of cells in the Sub G0/G1 phase following tyrosol exposure for 5 days, which coincided with an increase in apoptotic and necrotic markers. This indicates differentiation followed by cell death, unlike the positive monocyte differentiation control PMA. This selective cytotoxic effect following differentiation indicates therapeutic potential against leukaemia., peer-reviewed

On rare occasions, a promonocyte may be difficult to morphologically distinguish from a promyelocyte, giving rise to the diagnosis of acute promyelocytic leukemia, particularly the microgranular variant. It is necessary to correctly diagnose these two types of leukemia, as treatment is different for each. Flow cytometry and cytogenetic/molecular studies are useful in distinguishing the two when morphology alone is equivocal. We report a case of acute monocytic leukemia in which the promonocytes morphologically masqueraded as promyelocytes.

Our previous work has demonstrated that some acute promyelocytic leukemia (APL) patients had significantly elevated circulating CD34+ cell count (≥ 10 × 10(6)/L), and these patients with higher CD34+ cell level usually presented with high-risk disease (WBC > 10,000/μL). The aim of this study was to investigate whether circulating CD34+ cell count is a prognostic marker in intermediate-low risk APL patients. In this study, 76 intermediate-low risk APL patients and 56 age-adjusted healthy volunteers were evaluated. Enumeration of CD34+ cells was investigated before the treatment. A cut-off value of 10 × 10(6)/L CD34+ cells could just distinguish APL patients with adverse prognostic factors from others and may have the power to predict shorter progression-free survival (PFS) and poor prognosis. Higher count of CD34+ cells was usually associated with nonclassical chromosomal translocation, PML/RARα gene complex fusion, APL history, chemotherapy-related APL, disease progression, second tumor, extramedullary infiltration, FLT3-ITD positive mutation, atypical morphology, BM promyelocyte CD56/CD34 positive expression, myelofibrosis, PCR-positive PML/RARa gene fusion but FISH-negative, marrow necrosis and shorter PFS. Our results suggest that the level of CD34+ cells can be further the stratification of disease risk, a higher CD34+ cell count may be indicative of inferior survival and serve as an adverse biomarker for intermediate-low risk APL.

Background.Acute promyelocytic leukemia is characterized by t(15;17). This leads to the formation of PML/RARαwhich blocks the differentiation of blasts at the stage of promyelocytes. This is reversed by all-trans-retinoic acid (ATRA), a vitamin A derivative. Acute myocardial ischemia is a rare side effect of ATRA.Case Report.We report a case of acute coronary syndrome manifesting as an adverse effect of ATRA in a lady with APL who had no other risk factors for cardiovascular disease.Conclusions.We emphasize the need for high index of suspicion for the diagnosis of this entity. In the light of this case, the rare instances of ATRA associated acute myocardial ischemia recorded in the literature and the options available for treatment of acute promyelocytic leukemia sans ATRA have been reviewed.

Summary Neutrophils are the most abundant peripheral immune cells and thus, are continually replenished by bone marrow-derived progenitors. Still, how newly identified neutrophil subsets fit into the bone marrow neutrophil lineage remains unclear. Here, we use mass cytometry to show that two recently defined human neutrophil progenitor populations contain a homogeneous progenitor subset we term “early neutrophil progenitors” (eNePs) (Lin−CD66b+CD117+CD71+). Surface marker- and RNA-expression analyses, together with in vitro colony formation and in vivo adoptive humanized mouse transfers, indicate that eNePs are the earliest human neutrophil progenitors. Furthermore, we identified CD71 as a marker associated with the earliest neutrophil developmental stages. Expression of CD71 marks proliferating neutrophils, which were expanded in the blood of melanoma patients and detectable in blood and tumors from lung cancer patients. In summary, we establish CD117+CD71+ eNeP as the inceptive human neutrophil progenitor and propose a refined model of the neutrophil developmental lineage in bone marrow.

Congenital neutropenia (CN) is a bone marrow failure syndrome which is characterized by a maturation arrest of myeloid progenitors in the bone marrow. The pathomechanism leading to the defective granulopoiesis in CN patients is not fully elucidated. We recently reported the inability of G-CSF to induce GADD45β (Growth arrest and DNA damage-inducible 45, beta) expression in myeloid progenitors of CN patients harboring ELANE or HAX1 mutations. GADD45β functions as stress sensor downstream of myeloid cytokines and is important in murine stress-induced hematopoiesis. Moreover, we could show that C/EBPα, which is severely diminished in CN cells, binds and activates the GADD45β promoter. Importantly, rescue of the GADD45β expression in ELANE-CN HSPCs by lentivirus-based transduction of GADD45B cDNA restored defective granulocytic differentiation compared to control transduced cells. Transduction of mouse HSCs with ectopic Gadd45β resulted in drastic decrease of immature and increase of mature myeloid cells over time. Colony forming unit (CFU) and CFU re-plating assay showed that Gadd45β overexpressing mouse HSCs exhibit lower proliferation capacity. To determine the role of GADD45β in vivo, we performed CRISPR/Cas9 knockout of gadd45bb in wild type and Tg(mpo:gfp) zebrafish embryos, in which GFP expression is activated by the mpo promoter. We found that gadd45bb-deficiency lead to dramatic reduction in neutrophil numbers as assessed by counting mpo+ cells. Ectopic expression of G-CSF using a heat-inducible system increased the number of mpo+ cells in gadd45bb crispants but not to a level similar to wild type zebrafish expressing G-CSF. GADD45 proteins are implicated in active DNA demethylation by recruiting the DNA repair machinery to specific sites in the genome to convert 5-methylcytosine into unmethylated cytosine. Nothing is known about active DNA demethylation downstream of G-CSF and the role of GADD45β in this process. We sought to unveil whether GADD45β mediates G-CSF triggered myeloid differentiation by specific gene demethylation. To address this, we performed RNA sequencing and DNA methylation analysis of wild type (WT) and GADD45β knockout (KO) CD34+ HSPCs treated or untreated with G-CSF for 72h. The RNA-seq analysis demonstrated a GADD45β-dependent upregulation of genes which play a role in neutrophil differentiation and function as for instance RXRA, ITGAM (CD11b), FPR1/2, MEFV and BATF upon G-CSF treatment. Strikingly, these genes were also upregulated in promyelocytes of healthy controls upon G-CSF treatment, but not in CN patient promyelocytes. Genomatix-based gene ontology analysis of significantly upregulated genes in the G-CSF-treated WT but not GADD45β KO group displayed that diseases associated with this expression signature included neutropenia, leukocyte disorders and AML. Motif enrichment analysis of GADD45β-responsive genes predicted mainly motifs for myeloid-specific transcription factors (e.g. RXRA, SPI1 and C/EBPβ) to be enriched. Finally, DNA methylation analysis revealed significant differences in methylation pattern between GADD45β WT and KO groups upon G-CSF treatment. In WT cells G-CSF caused the hypomethylation of 13.516 CpGs and in GADD45β KO cells, only 8.440 CpGs were hypomethylated. Methylation changes between WT and KO groups mainly occurred in myeloid-specific genes, e.g. ELANE, MPO, CSF3R and AZU1. We also identified myeloid differentiation-related genes of which mRNA expression was regulated by GADD45β-mediated DNA demethylation, as for instance RXRA, CXCR1, MEFV, FPR2 and SERPINA1. Importantly, diseases associated with the defective gene demethylation downstream of G-CSF were neutropenia, immune system diseases, pre-leukemia and AML, as assessed by Genomatix analysis. Taken together, we report for the first time the importance of GADD45β-dependent DNA demethylation for human and zebrafish G-CSF-triggered neutrophil differentiation. The failure of G-CSF to induce GADD45β in CN patients might be a reason for the maturation arrest of granulopoiesis. Disclosures No relevant conflicts of interest to declare.

Background: (-)-Epigallocatechin-3-gallate (EGCG) is a gallate ester obtained by the condensation of gallic acid with the (3R)-hydroxy group of (-)-epigallocatechin. This component, extracted from green tea, has multiple effects on signal transduction pathways and enzyme activities which could enhance apoptosis and suppress of cell proliferation, invasion, angiogenesis and metastasis in cancers. This study aims to evaluate the effect of EGCG in an experimental model of leukemia (PML-RARα mice). Methods: NOD.CB17-Prkdcscid/J mice (12-16 weeks old) received 2Gy irradiation followed by transplantation of leukemia cells obtained from hCG-PML-RARα transgenic mice by i.v. injection in the caudal vein. Establishment of disease was confirmed at day 12 through presence of leukocytosis (>30x103/µL), and/or anemia ( Results: Hematological analysis revealed that EGCG treatment reversed leukocytosis (54.09±57.71 vs 11.45±16.08; p=0.0371), anemia (9.60±1.50 vs 11.32±1.36; p=0.0155) and thrombocytopenia (238.5±146.43 vs 475.8±247.91; p=0.0179) and prolonged survival of PML/RARα mice (13 vs 15 days; p=0.0017). Notably, EGCG reduced leukemia immature cells (CD45+CD34+) (8.04±2.49 vs 5.13±1.58; p=0.0060) and promyelocytes (CD45+CD117+) (73.54±12.85 vs 56.26±15.93; p=0.0157) in bone marrow of mice whereas increased mature myeloid cells (CD11b+Gr-1+) (6.15±3.00 vs 14.60±7.83; p=0.0051), possibly by inducing cellular differentiation. These results were corroborated by the reduction in promyelocytes (45.97±11.72 vs 30.29±11.01; p=0.0154), and the increase in neutrophils (CD45+Gr-1+) (38.20±14.34 vs 54.88±14.25; p=0.0178) and monocytes (CD45+CD11b+) (60.22±18.87 vs 76.79±15.59; p=0.0463) detected in peripheral blood. We then evaluated the effect of EGCG on cellular differentiation by studying degradation of PML/RARα oncoprotein. EGCG increased the percentage of cells with aggregated PML bodies stain in the bone marrow of PML-RARα mice, suggestive of higher degradation of oncoprotein, parallel to a reduction in PIN1 expression in bone marrow cells. Higher intracellular levels of reactive oxygen species (ROS) were also detected in leukemia immature cells (2101±1025 vs 3544±614; p=0.0051), promyelocytes (1765±1176 vs 3090±1282; p=0.0271) and neutrophils (1830±1093 vs 3532±1157; p=0.0033) of bone marrow. These results are consistent with literature data demonstrating that the ablation of PIN1 and/or induction of ROS could trigger PML/RARα degradation. EGCG has been reported to inhibit PIN1, a peptidyl isomerase overexpressed and/or over activated in human cancers, which is described as a key target in PML/RARα. Furthermore, apoptosis was detected in spleen cells of PML-RARα mice (5.97±4.19 vs 10.42±3.54; p=0.0197) in parallel to increased expression of BAX, reduced expression of BCL-2, and reduction of spleen weight (0.5587±0.05 vs 0.3949±0.10; p=0.0085). Conclusion: Collectively, our results support further evaluation of EGCG in clinical trials for acute myeloid leukemia. Disclosures No relevant conflicts of interest to declare.

Acute promyelocytic leukemia, a form of acute myeloid leukemia, is characterized by cell differentiation arrest at the promyelocyte stage. Current therapeutic options include administration of all trans-retinoic acid (ATRA), but this treatment produces many side effects. ATRA is known to induce differentiation of leukemic cells into granulocytes, but the mechanism of this process is poorly studied. We performed comparative proteomic profiling of HL-60 promyelocytic cells at different stages of ATRA-induced differentiation to identify differentially expressed proteins by high-resolution mass spectrometry and relative quantitative analysis without isotope labels. A total of 1162 proteins identified by at least two unique peptides were analyzed, among them 46 and 172 differentially expressed proteins were identified in the nuclear and cytosol fractions, respectively. These differentially expressed proteins can represent candidate targets for combination therapy of acute promyelocytic leukemia.

Acute Myeloid Leukemia (AML) is one of cancer type that attack white blood cells in myeloid descendants. On the clinical examination of leukemia, the number of each blast cell in the laboratory is calculated. However, in some subtype of AML like M4, M5 dan M7 are affected by the same type of precursor cells. The precursor cell of them are myeloblast, monoblast and megakaryoblast, which needs more detailed analysis to distinguish. This research tries to help overcome the problem by doing cell type automatic classification from cells images. Classification is performed on cell types of precursors cells derived from bone marrow preparations. The stages that have been completed are preprocessing, segmentation, extraction and feature selection, and classification. Features used as input of classification stage are area, nucleus ratio, circularity, perimeter, mean, and standard deviation. The results showed the success rate of cell segmentation reached 87.72% of total 1710 cells. The support vector machine classification results in the best performance test data are achieved by Linear kernel. The performance was obtained by combining six features for eight cell types from the maturation of the three precursor cells. These cell types are myeloblast, promyelocyte, granulocyte, monoblast, promonocyte, monocyte, megakaryoblast and support cell with sequential accuracy of 98.67%, 98.01%, 84.05% 99.67%, 95.35%, 89.70%, 99.34% and 98.01% respectively.

The hematopoietically expressed homeobox (Hhex) transcription factor is overexpressed in human myeloid leukemias. Conditional knockout models of murine acute myeloid leukemia indicate that Hhex maintains leukemia stem cell self-renewal by enabling Polycomb-mediated epigenetic repression of the Cdkn2a tumor suppressor locus, encoding p16Ink4a and p19Arf However, whether Hhex overexpression also affects hematopoietic differentiation is unknown. To study this, we retrovirally overexpressed Hhex in hematopoietic progenitors. This enabled serial replating of myeloid progenitors, leading to the rapid establishment of interleukin-3 (IL-3)-dependent promyelocytic cell lines. Use of a Hhex-ERT2 fusion protein demonstrated that continuous nuclear Hhex is required for transformation, and structure function analysis demonstrated a requirement of the DNA-binding and N-terminal-repressive domains of Hhex for promyelocytic transformation. This included the N-terminal promyelocytic leukemia protein (Pml) interaction domain, although deletion of Pml failed to prevent Hhex-induced promyelocyte transformation, implying other critical partners. Furthermore, deletion of p16Ink4a or p19Arf did not promote promyelocyte transformation, indicating that repression of distinct Hhex target genes is required for this process. Indeed, transcriptome analysis showed that Hhex overexpression resulted in repression of several myeloid developmental genes. To test the potential for Hhex overexpression to contribute to leukemic transformation, Hhex-transformed promyelocyte lines were rendered growth factor-independent using a constitutively active IL-3 receptor common β subunit (βcV449E). The resultant cell lines resulted in a rapid promyelocytic leukemia in vivo. Thus, Hhex overexpression can contribute to myeloid leukemia via multiple mechanisms including differentiation blockade and enabling epigenetic repression of the Cdkn2a locus.

The chemotherapy-free treatment of acute promyelocytic leukemia (APL) is a modern success story. Early clinical trials in China during the 1980s suggested the potent activity of all-trans retinoic acid (ATRA) in APL, leading to differentiation of the malignant cells and complete remission. In the 1990s, arsenic trioxide (ATO) was also shown to lead to differentiation of the promyelocytes. Thus, the current treatment for newly diagnosed standard-risk APL (white blood cell count

Acute myeloid leukemia (AML) is a disorder involving hematopoietic stem cells, characterized by blockage of hematopoietic cell differentiation and an increase in clonal neoplastic proliferation. AML is associated with poor patient outcome. PBK/TOPK is a protein kinase derived from PDZ-binding kinase (PBK)/T-lymphokine-activated killer (T-LAK) cell-originated protein kinase (TOPK). Previous studies have shown that PBK/TOPK is expressed in hematologic tumors. In the present study, we aimed to investigate the role of PBK/TOPK in promyelocyte proliferation and to clarify the molecular mechanism. PBK/TOPK knockdown (KD) significantly decreased cell proliferation and viability in the NB4 and HL-60 promyelocytes. PBK/TOPK KD resulted in G2/M cell cycle arrest that attributed to a decrease in cdc2 and cyclin B expression. In addition, PBK/TOPK KD caused apoptosis, as evidenced by activation of the mitochondrial apoptotic pathway and an increase in TUNEL-positive cells. PBK/TOPK KD induced mitochondrial dysfunction and ROS generation, and inhibition of mitochondrial dysfunction and ROS production suppressed PBK/TOPK KD-induced cell cycle arrest and apoptosis. Moreover, PBK/TOPK KD decreased Nrf2 expression and ARE-binding activity. Overexpression of Nrf2 inhibited the PBK/TOPK KD-induced decrease in cdc2 and cyclin B expression and cell cycle arrest, and blocked ROS production and apoptosis. Based on literature and our results, it was demonstrated that Nrf2 may be a crucial regulator that mediates PBK/TOPK-exerted promotion of cell proliferation. PBK/TOPK stabilizes Nrf2, strictly regulates the ROS level, promotes cell cycle progression and inhibits apoptosis, contributing to the proliferation of promyelocytes. Our results provide new insights into the molecular mechanism of PBK/TOPK-mediated promyelocyte proliferation and shed light on the pathogenesis of AML.

Severe congenital neutropenia (SCN) is often associated with inherited heterozygous point mutations in ELANE, which encodes neutrophil elastase (NE). However, a lack of appropriate models to recapitulate SCN has substantially hampered the understanding of the genetic etiology and pathobiology of this disease. To this end, we generated both normal and SCN patient-derived induced pluripotent stem cells (iPSCs), and performed genome editing and differentiation protocols that recapitulate the major features of granulopoiesis. Pathogenesis of ELANE point mutations was the result of promyelocyte death and differentiation arrest, and was associated with NE mislocalization and activation of the unfolded protein response/ER stress (UPR/ER stress). Similarly, high-dose G-CSF (or downstream signaling through AKT/BCL2) rescues the dysgranulopoietic defect in SCN patient-derived iPSCs through C/EBPβ-dependent emergency granulopoiesis. In contrast, sivelestat, an NE-specific small-molecule inhibitor, corrected dysgranulopoiesis by restoring normal intracellular NE localization in primary granules; ameliorating UPR/ER stress; increasing expression of CEBPA, but not CEBPB; and promoting promyelocyte survival and differentiation. Together, these data suggest that SCN disease pathogenesis includes NE mislocalization, which in turn triggers dysfunctional survival signaling and UPR/ER stress. This paradigm has the potential to be clinically exploited to achieve therapeutic responses using lower doses of G-CSF combined with targeting to correct NE mislocalization.

A 20 year old man was referred to the emergency department by his general practitioner with a one week history of easy bruising, gum bleeding, and lethargy. He had no medical history and took no regular medications. He did not smoke or drink alcohol and had no travel history. On examination, he appeared unwell and had widespread petechiae with evidence of gum bleeding. The rest of the clinical examination, including funduscopy, was normal. His observations were: heart rate 110 beats per minute, blood pressure 100/80 mm Hg, temperature 38.5°C, respiratory rate 20 breaths/minute, and oxygen saturations 100% on room air. Blood results showed: haemoglobin 77 g/L (133-167 g/L), white blood cell count 6×109 cells/L (3.5-11×109 cells/L), neutrophils 1.1×109 cells/L (2.0-7.0×109 cells/L), platelets 45×109/L (150-410×109/L), and C reactive protein level 160 nmol/L (0.76 −28.5 nmol/L). A coagulation screen showed INR 1.9 (0.8-1.1), APTT ratio 1.07 (0.85-1.15), fibrinogen 0.8 g/L (2-4 g/L), and D-dimer 3334 ng/ml (

AML is one type of cancer of the blood and spinal cord. AML has a number of subtypes including M0 and M1. Both subtypes are distinguished by the dominant blast cell type in the WBC, the myeloblast cells, promyelocyte, and myelocyte. This makes the diagnosis process of leukemia subtype requires identification of blast cells in WBC. Automatic blast cell identification is widely developed but is constrained by the lack of data availability, and uneven distribution for each type of blast cell, resulting in problems of data imbalance. This makes the system developed has poor performance. This study aims to classify blast cell types in WBC identified AML-M0 and AML-M1. The method used is divided into two stages, first pre-processing, image segmentation and feature extraction. The second stage, perform resample, which is continued over sampling with SMOTE. The process is done until the amount of data obtained is relatively the same for each blast cell, then the process of elimination of data duplication, randomize, classification and performance measurement. The validation method used is k-fold cross-validation with k=10. Performance parameters used are sensitivity, specifyicity, accuracy, and AUC. The average performance resulting from classification of cell types in AML with Random Forest algorithm obtained 82.9% sensitivity, 92.1% specificity, 89.6% accuracy and 87.5% AUC. These results indicate a significant improvement compared to the system model without using SMOTE. The performance generated by reference to the AUC value, the proposed system model belongs to either category, so it can be used for further stages of leukemia subtype AML-M0 and AML-M1.

Acute promyelocytic leukaemia (APL) is characterized by t(15;17)(q22;q21), resulting in a PML-RARA fusion that is the master driver of APL. A few cases that can't be identified with PML-RARA by using routine methods (karyotype analysis, FISH, and RT-PCR) involve abnormal promyelocytes that are fully in accordance with APL in morphology, cytochemistry, and immunophenotype. To explore the mechanisms involved in pathogenesis and recurrence of morphologically diagnosed APL, we performed comprehensive variant analysis from structural variants (SVs) to single-nucleotide variants (SNVs) in paediatric patients mophologically diagnosed as APL by next generation sequencing. SV analysis using targeted gene sequencing(TGS) in 120 DNA samples from both diagnosis and relapse stage identified 95 samples with RARA rearrangement (including 94 with PML-RARA and one with NPM-RARA) and two samples with KMT2A rearrangement. In the cases failed to be identified with any RARA rearrangement, transcriptome sequencing was applied in the available 13 RNA samples at diagnosis. One case each with CPSF6-RARG, NPM1-CCDC28A, and TBC1D15-RAB21 and two cases with a TBL1XR1-RARB fusion were discovered, which strongly suggested their contributions to leukemogenesis as driver alternations and APL phenotype is inextricably linked to rearrangement of RARA gene. SNV analysis in 75 primary APL samples with RARA rearrangement showed recurrent alternations in FLT3(27%), WT1(13%), USP9X(9%), NRAS(7%), and ARID1A(4%), with a strong potential for involvement in pathogenesis, and WT1as the only gene recurrently mutated in 4/5 samples(3/4 cases) in relapsed APL. WT1 (17.6%), NPM1 (11.8%), NRAS (11.8%), FLT3 (11.8%), and NSD1 (11.8%) were identified as recurrently mutated in 17 primary samples without RARA rearrangement and WT1(44.4%), NPM1 (33.3%), TP53 (33.3%), and RARA (33.3%)as recurrently mutated in 9 relapsed samples. The retrospective study showed the survival rates of APL without RARA rearrangement(8y-EFS=38.7%±17.8%) were much worse than APL with RARA rearrangement(8y-EFS=90.9%±3.9%), which is similar to AML (non-M3) patients(8y-EFS=57.7%±3.0%). Thus,NGS is necessary in cases failed to be identified with RARA rearrangement by karyotype analysis, FISH, and RT-PCR, which can afford accurate diagnosis and treatment guidance. APL phenotype may arise by abnormities of other members of the nuclear receptor superfamily involved in retinoid signaling(RARB or RARG) or even by mechanisms distinct from the formation of aberrant retinoid receptors. Differences in genetic alternations and survival indicated that APL that cannot be identified as having a RARA rearrangement are more reasonably classified as a subclass of AML other than APL, and an AML treatment regimen or individualized treatment should be considered according to the genetic abnormalities. Figue1. (A)Mutational profile between APL with and without RARA rearrangement. (B)Comparison of estimated probability of EFS and OS among APL with and without RARA rearrangement and AML(non-M3) group . “Typical APL” means APL with RARA rearrangement and “Atypical APL” means APL without RARA rearrangement. Disclosures No relevant conflicts of interest to declare.

[Figure][1] A 70-year-old man with a history of treated immunoglobulin G (IgG)–κ plasma cell myeloma was referred to our institution for continuation of care. A preliminary differential cell count demonstrated 10% promyelocytes and 2% plasma cells. The second review of Wright-stained bone

Acute myeloid leukemia (AML) with t(8;16)(p11;q13) is a distinct clinical and morphological entity with poor prognosis, which is characterized by a high frequency of extramedullary involvement, most commonly leukemia cutis; association with therapy related AML; frequent coagulopathy and morphologic features overlapping acute promyelocytic leukemia(APL). Herein, we present a case of 47 year-old post-menopausal woman developing secondary AML with t(8;16)(p11;q13) after 1 year of completion of therapy for breast carcinoma. Blasts were granulated with few showing clefted nucleus resembling promyelocytes and immnuophenotyping showed high side scatter with MPO positivity and CD 34 and HLA-DR negativity. In view of promyelocyte like morphology and immunophenotyping of blasts, possibility of APL was considered but, reverse transcription polymerase chain reaction (RT-PCR) for PML-RARα fusion transcript came out to be negative. Conventional cytogenetics showed t(8;16)(p11;q13). So, we should keep possibility of t(8;16) (p11;q13) in therapy related acute myeloid leukemia in patient showing clinical and morphological features of acute promyelocytic leukemia.