IntroductionAcute myeloid leukemia (AML) refers to a genetically and biologically heterogeneous group of diseases characterized by an abnormal increase of myeloblasts in the bone marrow (BM) and peripheral blood (PB) circulation. Chemotherapy and hematopoietic stem cell transplantation (HSCT) are the mainstays of treatment, but these modalities have reached an impasse with an overall cure rate of only 30%-40%. 1 An attractive strategy is to target specific genetic and biochemical alterations in AML, thereby providing an alternative treatment modality that may improve patient outcome. 2,3 FLT3 (fms-like tyrosine kinase-3) is a receptor tyrosine kinase (RTK) that is highly expressed in hematopoietic stem and progenitor cells. It includes an extracellular domain (ECD), a transmembrane domain (TMD), a juxtamembrane domain (JMD), and 2 tyrosine kinase domains (TKDs) separated by a kinase insert. 4 On binding with FLT3 ligand secreted by BM stromal cells, FLT3 undergoes dimerization, phosphorylation, and TKD activation. The FLT3 gene is mutated in approximately 30% of AMLs, particularly those with normal karyotypes, t(6;9), t(15;17), or trisomy 8. 5,6 The most common mutation is an internal tandem duplication (ITD) up to a few hundred base-pairs within the JMD. Single-base mutations have also been described, most commonly resulting in a substitution of aspartic acid with tyrosine or less commonly a histidine at residue 835 in the TKD. 7,8 At a molecular level, these mutations result in constitutive activation of the FLT3 receptor and hence downstream PI3K/AKT/mTOR, Ras/Raf/MEK/ERK, and JAK/ STAT pathways. 9,10 The biologic consequences are enhanced proliferation and reduced apoptosis of the myeloblasts, which contribute to leukemogenesis. [11][12][13][14][15] Patients with FLT3-ITD respond poorly to conventional chemotherapy and have an inferior prognosis, 16,17 particularly in those with a high FLT3-ITD ϩ cell burden, 18 long ITD sequences, 19 and multiple FLT3-ITD ϩ subclones, 20 underscoring a pathogenetic role of FLT3-ITD in human AML. In mice, knock-in of a heterozygous FLT3-ITD resulted in a preleukemic model of a myeloproliferative disease, providing an in vivo demonstration of the important role of FLT3 in leukemia initiation. 21 We 22 and others 23,24 have shown that the FLT3-ITD allele can be found in leukemia initiating cells (LICs), as distinguished by their capability of regenerating leukemic progeny in transplanted immunodeficient mice. Therefore, targeting FLT3-ITD might provide a novel approach to therapeutic intervention.Several clinical trials on multi-TK inhibitors with different FLT3 specificities and in vitro efficacies have been reported, including the use of midostaurin, 25 lestautinib, 26 tandutinib, 27 sunitinib, 28 and sorafenib. 29 In most studies, clinical efficacy was restricted to FLT3-ITD ϩ AML and correlated with inactivation of FLT3 phosphorylation. 25,[30][31][32] Complete remission was rare and limited to anecdotal reports in relapsed AML after allogeneic HSCT. 33 Furthermore, after ...

Aldehyde dehydrogenase (ALDH) activity is used to define normal hematopoietic stem cell (HSC), but its link to leukemic stem cells (LSC) in acute myeloid leukemia (AML) is currently unknown. We hypothesize that ALDH activity in AML might be correlated with the presence of LSC. Fifty-eight bone marrow (BM) samples were collected from AML (n ¼ 43), acute lymphoblastic leukemia (ALL) (n ¼ 8) and normal cases (n ¼ 7). In 14 AML cases, a high SSC lo ALDH br cell population was identified (ALDH þ AML) (median: 14.89%, range: 5.65-48.01%), with the majority of the SSC lo ALDH br cells coexpressing CD34 þ . In another 29 cases, there was undetectable (n ¼ 23) or rare (p5%) (n ¼ 6) SSC lo ALDH br population (ALDH À AML). Among other clinicopathologic variables, ALDH þ AML was significantly associated with adverse cytogenetic abnormalities. CD34 þ BM cells from ALDH þ AML engrafted significantly better in NOD/ SCID mice (ALDH þ AML: injected bone 21.1179.07%; uninjected bone 1.5270.75% vs ALDH À AML: injected bone 1.7771.66% (P ¼ 0.05); uninjected bone 0.2370.23% (P ¼ 0.03)) with the engrafting cells showing molecular and cytogenetic aberrations identical to the original clones. Normal BM contained a small SSC lo ALDH br population (median: 2.92%, range: 0.92-5.79%), but none of the ALL cases showed this fraction. In conclusion, SSC lo ALDH br cells in ALDH þ AML might denote primitive LSC and confer an inferior prognosis in patients.

Key Points• The use of barcoding to track lineages in 196 human CD34 1 CB clones in serially sampled primary and secondary transplanted NSG mice is described.• Detection of early transient clones with later, more stable clones and definitive evidence of sustained self-renewal of multipotency is presented.Human cord blood (CB) offers an attractive source of cells for clinical transplants because of its rich content of cells with sustained repopulating ability in spite of an apparent deficiency of cells with rapid reconstituting ability. Nevertheless, the clonal dynamics of nonlimiting CB transplants remain poorly understood. To begin to address this question, we exposed CD341 CB cells to a library of barcoded lentiviruses and used massively parallel sequencing to quantify the clonal distributions of lymphoid and myeloid cells subsequently detected in sequential marrow aspirates obtained from 2 primary NOD/SCID-IL2Rg 2/2 mice, each transplanted with ∼10 5 of these cells, and for another 6 months in 2 secondary recipients. Of the 196 clones identified, 68 were detected at 4 weeks posttransplant and were often lymphomyeloid. The rest were detected later, after variable periods up to 13 months posttransplant, but with generally increasing stability throughout time, and they included clones in which different lineages were detected. However, definitive evidence of individual cells capable of generating T-, B-, and myeloid cells, for over a year, and self-renewal of this potential was also obtained. These findings highlight the caveats and utility of this model to analyze human hematopoietic stem cell control in vivo. (Blood. 2013;122(18):3129-3137)