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A precise understanding of the role of miR-223 in human hematopoiesis and in the pathogenesis of acute myeloid leukemia (AML) is still lacking. By measuring miR-223 expression in blasts from 115 AML patients, we found significantly higher miR-223 levels in patients with favorable prognosis, whereas patients with low miR-223 expression levels were associated with worse outcome. Furthermore, miR-223 was hierarchically expressed in AML subpopulations, with lower expression in leukemic stem cell-containing fractions. Genetic depletion of miR-223 decreased the leukemia initiating cell (LIC) frequency in a myelomonocytic AML mouse model, but it was not mandatory for rapid-onset AML. To relate these observations to physiologic myeloid differentiation, we knocked down or ectopically expressed miR-223 in cord-blood CD34⁺ cells using lentiviral vectors. Although miR-223 knockdown delayed myeloerythroid precursor differentiation in vitro, it increased myeloid progenitors in vivo following serial xenotransplantation. Ectopic miR-223 expression increased erythropoiesis, T lymphopoiesis, and early B lymphopoiesis in vivo. These findings broaden the role of miR-223 as a regulator of the expansion/differentiation equilibrium in hematopoietic stem and progenitor cells where its impact is dose- and differentiation-stage-dependent. This also explains the complex yet minor role of miR-223 in AML, a heterogeneous disease with variable degree of myeloid differentiation., (Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Identification of the molecular mechanisms that can promote human hematopoietic stem cell amplification is a major goal in experimental and clinical hematology. Recent data indicate that a variety of regulatory molecules active in early development may also play a role in the maintenance of hematopoietic stem cells with repopulating activity. One important class of early developmental genes determining hematopoietic development are homeobox transcription factors. Here, we report that retrovirally mediated expression of the homeobox gene HOXB4 rapidly triggers an increase in the number of human hematopoietic cord blood cells with stem cell and progenitor cell properties detected both by in vitro and in vivo assays. This growth enhancement extended across primitive myeloid-erythroid and B-lymphoid progenitors but did not lead to alterations in the balance of lymphomyeloid reconstitution in vivo, suggesting that HOXB4 does not affect control of end-cell output. These findings reveal HOXB4 as a novel, positive regulator of the primitive growth activity of human hematopoietic progenitor cells and underline the relevance of early developmental factors for stem cell fate decisions.

Objective: To study the clonality of cytogenetically normal progenitors detected in the peripheral blood (PB) of acute myeloid leukemia (AML) patients., Methods: Five female patients with cytogenetically abnormal, newly-diagnosed AML who were heterozygous for informative alleles of the androgen receptor (AR) gene were studied using the human androgen receptor allele (HUMARA) assay., Results: PB mononuclear cells and bone marrow (BM) fibroblasts from these patients were monoclonal and polyclonal, respectively. Both cytogenetically normal and abnormal colony-forming cells (CFC) were detected from 3 AML samples and the HUMARA assay determined that most of these CFC were part of the leukemic clone. The fourth sample generated colonies that were 100% normal by cytogenetics and polyclonal by HUMARA. In contrast, 5-week-old long-term culture (LTC)-derived colonies were 100% cytogenetically normal by FISH and polyclonal by HUMARA in 4 of the 5 samples. The fifth sample, which showed a small number of karyotypically abnormal LTC-derived colonies, nevertheless showed amplification of the "leukemia-associated" AR allele in 46/50 LTC-derived colonies as well as all 40 directly clonogenic cells tested., Conclusions: Thus in 4 of 5 AML samples tested, both cytogenetics and the HUMARA assay indicate that a substantial number of normal, polyclonal hematopoietic progenitors often persist in AML PB at diagnosis in spite of the predominance of malignant blasts and the severe cytopenias of normal mature blood cells that are typically seen clinically.

Human hematopoietic stem cells reside in the CD34+CD38−CD90+ population in cord blood and bone marrow. However, this cell fraction is heterogeneous, and the phenotype of the rare primitive stem cells remains poorly defined. We here report that primitive cord blood CD34+CD38−CD90+ stem cells, with the ability to reconstitute NOD/SCID-IL2Rγcnull (NSG) mice long-term, at 24 weeks after transplantation, can be prospectively isolated at an increased purity by using integrin α2 receptor as an additional stem cell marker. Using a limiting dilution transplantation assay, we found a highly significant enrichment of multilineage reconstituting stem cells in the CD34+CD38−CD90+ cell fraction expressing the integrin α2 receptor, with a frequency of 1/29 cells, as compared to a frequency of 1/157 in the corresponding integrin α2− cells. In line with this, long-term reconstituting stem cells within the cord blood CD34+CD38− cell population were significantly enriched in the integrin α2+ fraction, while stem cells and progenitors reconstituting short-term, at 8-12 weeks, were heterogeneous in integrin α2 expression. Global gene expression profiling revealed that the lineage-marker negative (Lin−) CD34+CD38−CD90+CD45RA− integrin α2+ cell population was molecularly distinct from the integrin α2− cell population and the more mature Lin−CD34+CD38−CD90−CD45RA− cell population. Our findings identify integrin α2 as a novel stem cell marker, which improves prospective isolation of the primitive human hematopoietic stem cells within the CD34+CD38−CD90+ cell population for experimental and therapeutic stem cell applications. S TEM C ELLS 2013;31:360-371 [ABSTRACT FROM AUTHOR]

High dose chemoradiotherapy and haematopoietic stem cell transplantation (SCT) is used as primary therapy for patients diagnosed with Burkitt lymphoma (BL). Forty-three adults presented with sporadic BL in British Columbia between 1987 and 2003. Twenty patients had bone marrow involvement. Sixteen patients did not proceed to SCT because of chemorefractory disease ( n = 9) or other reasons ( n = 7). Twenty-seven patients proceeded to SCT and had a 3-year event-free survival of 51%. In conclusion, approximately 50% of patients with chemosensitive BL who undergo SCT can be cured; however, a significant number of patients will not proceed to SCT because of early resistance or recurrence. [ABSTRACT FROM AUTHOR]

Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only known curative therapy for chronic myeloid leukemia (CML); however, it is rarely utilized given the excellent long-term results with tyrosine kinase inhibitor (TKI) treatment. The purpose of this study is to examine HSCT outcomes for patients with CML who failed TKI therapy or presented in advanced phase and to identify predictors of survival, relapse, and nonrelapse mortality (NRM). Fifty-one patients with CML underwent HSCT for advanced disease at diagnosis (n = 15), TKI resistance as defined by the European LeukemiaNet guidelines (n = 30), TKI intolerance (n = 2), or physician preference (n = 4). At a median follow-up of 71.9 months, the 8-year overall survival (OS), event-free survival (EFS), relapse, and NRM were 68%, 46%, 41%, and 23%, respectively. In univariate analysis, predictors of OS included first chronic phase (CP1) disease status at HSCT ( P = .0005), European Society for Blood and Marrow Transplantation score 1 to 4 ( P = .04), and complete molecular response (CMR) to HSCT ( P < .0001). Donor (female) to patient (male) gender combination ( P = .02) and CMR to HSCT ( P < .0001) predicted lower relapse. In multivariate analysis, CMR to HSCT remained an independent predictor of OS (odds ratio [OR], 43), EFS (OR, 56) and relapse (OR, 29). This report indicates that the outlook is excellent for those patients who remain in CP1 at the time of HSCT and achieve a CMR after HSCT. However, only approximately 50% of those in advanced phase at HSCT are long-term survivors. This highlights the ongoing need to try to identify patients earlier, before disease progression, who are destined to fail this treatment to optimize transplantation outcomes. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the possibility of improved selective killing of acute myeloid leukemia (AML) cells with CPX-351 (a liposomal formulation of cytarabine and daunorubicin). CPX-351 and the same molar ratio of free drugs were compared for cytotoxicity against colony-forming cells (CFCs) and subpopulations of cells enriched for primitive progenitors from AML patients and normal granulocyte colony-stimulating factor−mobilized peripheral blood (PB) and bone marrow (BM) donors. Materials and Methods: AML blasts (n = 13) and normal PB and BM cells (n = 7) were incubated for 24 hours in various concentrations of CPX-351 or free drugs before plating in CFC assay or staining with anti-CD34 and anti-CD38 antibodies, Annexin-V, and propidium iodide followed by fluorescence-activated cell sorting analysis. High performance liquid chromatography was used to measure intracellular daunorubicin accumulation. Results: AML blasts and progenitors from patients who achieved complete remission were more sensitive to both CPX-351 and free drugs than the same cells from patients with chemotherapy refractory leukemia. However, AML CFCs and CD34+CD38− AML blasts (enriched for candidate leukemia stem cells) from the same patient showed similar sensitivity to the liposomal or free drug formulations. In contrast, CFCs and CD34+CD38− cells from normal PB and BM were fivefold more sensitive to the free drugs than to CPX-351. Consistent with these observations, preferential intracellular accumulation of CPX-351 in AML over normal cells was observed, while there was little difference in drug uptake between AML and normal cells with the free drug cocktail. Conclusions: CPX-351, as compared to free cytarabine:daunorubicin, shows enhanced selective in vitro cytotoxicity for AML rather than normal progenitors. [Copyright &y& Elsevier]

Tyrosine kinase inhibitors (TKI) have been used to treat relapse of chronic myelogenous leukemia (CML) after allogeneic stem cell transplant (HSCT), with responses seen predominantly in chronic phase (CP) patients. This study aimed to analyze the response to TKI therapy and overall survival for patients relapsing predominantly in advanced phase. We retrospectively reviewed 22 patients treated with imatinib (n=20) and/or dasatinib (n=6) for relapsed CML after HSCT; 8 patients were in CP, and 14 patients had advanced disease. Seven patients also received donor lymphocyte infusions. Hematologic, cytogenetic, and molecular responses were analyzed. Nineteen patients (86%) achieved complete hematologic response (CHR), 17 patients (77%) achieved complete cytogenetic response (CCR), and 14 patients (64%) achieved complete molecular response (CMR). In advanced phase patients, 11 (79%) achieved CHR, 10 (71%) CCR, and 8 (57%) achieved CMR. Grade 3 or 4 cytopenias occurred in 10 cases. With median follow-up of 31.5 months from relapse, 14 (64%) patients remain alive, 13 in CMR. In multivariate analysis, the achievement of CMR was significantly correlated with OS with an odds ratio of 20.5 (95% confidence interval 2.3-182) P =.007. TKI therapy is capable of inducing durable molecular responses for CML relapsing after HSCT, both in chronic and advanced phases. The achievement of CMR appears to be crucial in providing long-term disease control for these patients. [Copyright &y& Elsevier]

Retroviral transduction of hematopoietic stem cells (HSCs) offers an attractive strategy for treating malignancies that home to the marrow. This approach should therefore be of interest for evaluating the therapeutic activity of anti-angiogenic agents on hematopoietic malignancies whose growth has been associated with enhanced angiogenesis. A variety of studies have indicated endostatin to be a potent anti-angiogenic agent both in vitro and in vivo, and a human malignancy that might be sensitive to endostatin is human B-lineage acute lymphoblastic leukemia (B-ALL). The demonstrated ability of human B-ALL cells to engraft the marrow of immunodeficient mice suggested the potential of this system for testing an endostatin delivery strategy using co-transplanted non-obese diabetic–scid/scid (NOD/SCID) HSCs engineered to express endostatin. Here we show that, in spite of their mutant scid gene, NOD/SCID HSCs can be transduced with an endostatin-encoding retrovirus at efficiencies that result in a several-fold increase in endostatin serum levels in transplanted recipients. However, this did not alter the regrowth of co-transplanted human B-ALL blasts. These findings validate this gene transfer approach for investigating effects of novel therapeutics on primary human malignant cells that engraft NOD/SCID mice and question the utility of native endostatin for controlling human B-ALL in vivo. [Copyright &y& Elsevier]

Background FLT3-ITD mutations occur in about 25% of patients (pts) with acute myeloid leukemia (AML) and are associated with poor outcomes. Pts with relapsed/refractory (R/R) FLT3-ITD AML have worse prognosis and high unmet medical need. Quizartinib (Q) is a potent and selective FLT3i with promising activity and a manageable safety profile. QuANTUM-R was a global, phase 3, randomized trial of Q vs chemotherapy (SC) in pts with R/R FLT3-ITD AML (NCT02039726). Methods Pts with R/R FLT3-ITD AML w/wo hematopoietic stem cell transplant (HSCT) were randomized to receive Q or a preselected investigator choice SC: low-dose cytarabine; mitoxantrone, etoposide, and intermediate-dose cytarabine (MEC); or fludarabine, cytarabine, and G-CSF with idarubicin (FLAG-IDA). Prior midostaurin was allowed. Pts receiving HSCT after Q could resume Q after HSCT. Primary and secondary endpoints were overall survival (OS) and event-free survival (EFS), respectively. Exploratory endpoints included response rate, time to and duration of response, and transplant rate. Results 367 pts were randomized; 245 to Q and 122 to SC. Median follow-up was 23.5 mo. OS hazard ratio (HR) of Q relative to SC was 0.76 (95% CI, 0.58-0.98; P=.0177). Median OS was 6.2 (95% CI, 5.3-7.2) vs 4.7 (95% CI, 4.0-5.5) mo in Q and SC arms, respectively. EFS HR was 0.90 (95% CI, 0.70-1.16; P=.1071); median EFS was 1.4 (95% CI, 0.0-1.9) vs 0.9 (95% CI, 0.4-1.3) mo, respectively. Sensitivity analyses and OS subgroup analyses supported Q vs SC. Composite complete response (CRc) was 48% and 27% in Q and SC arms, respectively. Transplant rate was 32% (Q) and 12% (SC). Median time to first CRc was 4.9 wk for Q. The most common grade ≥ 3 TEAEs in both arms were infections and those associated with cytopenia. Conclusion OS benefit was observed with single-agent Q vs SC in pts with R/R FLT3-ITD AML with a favorable Q safety profile, providing evidence of meaningful clinical benefit in pts with limited treatment options. [ABSTRACT FROM AUTHOR]