Biology Of Acute Myeloid Leukemia Research Articles

The heterogeneity of pediatric MLL-rearranged acute myeloid leukemia

Translocations involving the mixed-lineage leukemia (MLL) gene, localized at 11q23, comprise 15 to 20% of all pediatric acute myeloid leukemia (AML) cases. This review summarizes current knowledge about the etiology, biology, clinical characteristics and differences in outcome in MLL-rearranged pediatric AML. Furthermore, we discuss the role of cooperating events in MLL-rearranged pediatric AML, and future therapeutic strategies to improve outcome. We conclude that MLL-rearranged pediatric AML is a heterogeneous disease, and prognosis depends on various factors, for example, translocation partner, age, WBC and additional cytogenetic aberrations. The relationship of outcome with specific translocation partners requires that they be searched for in the diagnostic work-up of AML. To achieve further improvements in outcome, unraveling the biology of MLL-rearranged pediatric AML is warranted.

Stratification of pediatric acute myeloid leukemia through cancer cell gene-expression profiling

Evaluation of: Balgobind BV, Marry M, van den Heuvel-Eibrink MM et al. Evaluation of gene expression signatures predictive for cytogenetic and molecular subtypes of pediatric acute myeloid leukemia. Haematologica 96(2), 221–230 (2010).Treatment of children’s acute lymphoblastic leukemia has been at the forefront of conventional chemotherapy development. Despite outstanding results in long-term survival of acute lymphoblastic leukemia, development of therapies for acute myeloid leukemia (AML) have lagged behind. AML in children demonstrate similar long-term survival compared with adults 18–65 years of age: 40–50% overall long-term survival. AML is a heterogeneous disease in both adults and children, but the presence of recurrent chromosome translocations and mutations in children are lower than in adult AML. In particular, patients without chromosome aberrancies have been examined for stratification through examination of gene expression. The paper from Baglobind and coauthors proposes a useful prognostication by gene-expression analysis of 75 gene pairs in 40% of patient cases, accurately discriminating mixed lineage leukemia (MLL) gene rearrangement, t(8;21)(q22;q22), inv(16)(p13q22), t(15;17)(q21;q22) and t(7;12)(q36;p13)-positive AML. Gene-expression analysis of AML has provided an important research tool for uncovering information about AML biology that can be used for the development of novel therapies.

Prognostic Significance of Histone (H4) Acetylation In Newly Diagnosed Acute Myeloid Leukemia (AML) Patients with Intermediate Risk Cytogenetics

AbstractAbstract 2736 Background:Epigenetic instability and aberrant repression of tumor suppressor genes are fundamental aspects of acute myeloid leukemia (AML) biology. Current prognostic assays for AML focus on the genetic aspects of disease biology, for example, chromosome abnormalities and recurrent mutations. We hypothesized that a simple assay for an epigenetic abnormality can address the epigenetic domain of AML, with implications for prognostication and for treatment selection with chromatin relaxing drugs. We focused on patients with intermediate risk cytogenetics (CG) and evaluated the association between histone (H4) acetylation and complete remission (CR), relapse-free survival (RFS), and overall survival (OS) in newly diagnosed AML patients with intermediate risk CG. Methods:We assessed histone H4 acetylation status in bone marrow biopsies from newly diagnosed AML patients with intermediate risk CG treated at our institution between the years 2003–2005. CG risk group was ascribed per Cancer and Leukemia Group B criteria. Association with CR, RFS and OS was assessed using logistic regression and Cox proportional hazard regression models. Variables used in the multivariate models included: age, WBC at diagnosis, gender, and history of antecedent hematologic disorder (AHD). B5-fixed bone marrow core biopsies were reviewed for areas with the highest blast concentration. Immunohistochemistry was performed for acetyl-H4 (1:200 dilution; polyclonal; Upstate Biotech, Lake Placid, NY) using automated stainers and heat-induced epitope retrieval. In each case, five hundred blasts were counted and only strong nuclear staining was classified as positive. Based on the distribution of cell counts, cases were classified as strongly positive if nuclear staining occurred in > 80% of the blasts. Results:Forty-two patients had intermediate risk CG and received cytarabine-based induction chemotherapy. The median age was 56.5 years (range 17–79) and median WBC count at diagnosis was 10.9 × 109/L (range 0.8– 227.9). Twenty two percent of patients had an AHD. Thirty three patients (78%) had normal cytogenetics. Chromosomal abnormalities +8,+21, and 11q23 abnormalities were present in 3 (7.1%), 1 (2.3%) and 4 (9.4%) patients, respectively. FLT3 status was available on only 9 patients; with 7 patients having no FLT3 mutations. Twelve patients (28.5%) received an allogeneic bone marrow transplant in first remission. The median follow-up time was 22.4 months (range 0.6–65.3). Thirty-four patients (81%) achieved CR. Using the 80% cut-off, 15 patients (35.7%) were positive for histone H4 acetylation. Histone (H4) acetylation was not associated with achievement of CR (OR=3.0, 95% CI 0.3–25.3, p=0.3). However, on multivariate analysis, a significant association was found between histone acetylation and risk of relapse (HR 0.34, 95% CI 0.11–0.99, p=0.05) and overall survival (HR=0.34, 95% CI 0.15–0.80, p=0.01). Inclusion of allogeneic transplant in the multivariate models did not change these estimates. Conclusion:Histone H4 acetylation is associated with improved RFS and OS in newly diagnosed AML patients with intermediate risk CG. These results will need to be validated in a larger population of uniformly treated patients. However, histone H4 acetylation has the potential as a simple assay to assess for the contribution of abnormal epigenetics to AML biology, with prognostic and potentially therapeutic implications, given the increasing role of epigenetically targeted therapy in myeloid malignancy management. Disclosures:No relevant conflicts of interest to declare.

Acute Myeloid Leukemia and Survival on the Intensive Care Unit (ICU) - Biology- and Treatment-Related Determinants of Outcome - An Analysis of the AML-CG

AbstractAbstract 2166 Background:An established factor for predicting the mortality of patients admitted to an ICU is the Severe Acute Physiology Score (SAPS) II. However, for patients with acute myeloid leukemia (AML) it is uncertain whether factors beyond SAPS II do influence the ICU outcome. Therefore we examined additional factors including age and factors related to AML biology and its treatment in the so far largest cohort of patients with AML worldwide regarding their ICU outcome. Methods:Retrospective analysis of 256 patients with 366 admissions to medical ICU between 2004 and 2009 in 3 large German hematologic centers. Patient age and gender, reason for ICU admission, duration of intensive care, SAPSII, need for invasive mechanical ventilation (IMV), renal replacement therapy and/or vasopressors, laboratory values at ICU admission for creatinine, bilirubin and C-reactive protein, AML karyotype, presence of FLT3-ITD and/or NPM1-mutation, FAB classification, last AML treatment, AML status and allogeneic transplant status were evaluated as potential risk factors. Correlations were analyzed using the Mann-Whitney U test. Univariate analysis was performed using the log rank test for the time until death on ICU occurred. Significant risk factors were studied in multivariate analysis (Cox regression). Results:At the time of analysis (08/2010) the median age of patients was 55.3 (range 19.7–84.9) years, and 47.5% were female. 46% of ICU admissions were due to infectious complications. A respiratory problem was present in 60% of the ICU transfers. IMV, vasopressors and renal replacement therapy were necessary in 51.3%, 42.6% and 22.7%, respectively, of the ICU courses. ICU survival was 64.8%. AML status was primary diagnosis/induction phase in 53.3%, postremission phase in 15.8% and relapse/refractory in 27.6% of ICU courses. 66.4% of admissions to ICU had conservative treatment (no transplant), 15.8% underwent allogeneic hematopoietic stem cell transplantation (allo SCT) in the same hospital stay (peritransplant status) and 14.5% had had allo SCT in a former hospital stay (posttransplant status). AML karyotype was favourable in 7.1%, intermediate in 47.8% and unfavourable in 20.2%. SAPS II was available in 208 ICU transfers. Duration of intensive care was 8.1 (mean)/3.0 (median) days with a range from 0.5–76 days. In univariate analysis risk factors predicting diminished ICU survival were high SAPS II (p=0.008), sepsis as reason for ICU admission (p=0.007), need for IMV (p<0.001), use of vasopressors (p<0.001), renal replacement therapy (p=0.002), intermediate or unfavourable AML karyotype (p=0.027), FAB classification other than AML M3 (p=0.012), postremission or relapse/refractory status of AML (p=0.029) and posttransplant status of AML (p=0.002). ICU mortality was lower in primary diagnosis/induction phase and higher in posttransplant phase of AML than predicted by the median SAPS II of these cohorts. In multivariate analysis the only significant predictor of inferior ICU survival was the extent of vasopressor treatment (hazard ratio (hr) 1.83, 95% CI 1.09–3.08; p=0.022), whereas high SAPS II was of borderline significance (hr 1.02, 95% CI 1.00– 1.03; p=0.064). Conclusions:In contrast to the broad majority of ICU patients, SAPS II is not an optimal predictor of ICU survival in patients with AML. Disease status was of high relevance with an AML status of primary diagnosis/induction phase indicating a better and posttransplant (but not peritransplant) status a worse ICU survival than predicted by SAPS II. The strongest predictor for ICU mortality was the extent of vasopressor use. In contrast age up to the 8th decade had no impact on ICU survival. These results may help to better define ICU admission and treatment policies for patients with AML. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Isolation and Functional Characterization of a Novel “Oxidative State – low” Leukemic Population with Stem Cell Properties and Potential Resistance to Chemotherapy In Acute Myeloid Leukemia.

AbstractAbstract 1580Most adults with acute myeloid leukemia (AML) are not cured with current treatments due to primary chemo-resistance or relapse. Emerging evidence suggests that the pool of leukemic blasts is heterogeneous and disease persistence is due to a subset of leukemic (-stem) cells able to evade chemotherapy and sustain tumor growth. Cell surface marker expression has proven to be a valuable tool to isolate and study leukemic stem cells (LSC) which, similarly to normal hematopoiesis, are shown to reside in the CD34+/CD38- leukemic fraction. However, recent data indicate that the phenotype of LSC varies from patient to patient and it seems likely that no single phenotypic signature exists to uniformly identify LSC. Besides immunophenotype, isolating LSC on the basis of functional properties unique for these cells may enforce our understanding of AML biology and provide the basis to develop more effective therapies. Reactive oxygen species (ROS) regulate essential cellular functions such as self-renewal, proliferation and apoptosis. In normal neurogenesis and hematopoiesis, ROSlow states correlate with self-renewal and ROShigh is associated with differentiation. In malignant tissues, although cancer cells are commonly more oxidized than their normal counterparts, some cancer stem cells are shown to contain low ROS levels, a feature associated with increased resistance to therapy (Nature. 2009;458:780-783). We hypothesized that LSCs also reside in a less oxidized state than bulk leukemic cells, a condition which promotes self-renewal and confers resistance to chemotherapy. To validate this hypothesis, we evaluated the redox state of leukemic blasts isolated from bone marrow or peripheral blood from 21 AML and 2 high-risk MDS patients. Loading of cells with the fluorescent probe DCF-DA showed that primary AML specimens have a broad range of oxidative state, with cells clearly falling into ROShigh and ROSlow populations (ROSlow=11.5±9%). Phenotypic analyses of AML specimens with respect to primitive cell surface markers indicated that the ROSlow gate represented 18 ± 17% of the phenotypically primitive CD34+/CD38- cells and was significantly more enriched in CD34-/CD38- leukemic cells in comparison to ROShigh. We isolated ROSlow and ROShigh leukemic subsets by flow cytometric sorting on the basis of their DCF fluorescence from 11 AML patients' samples and analyzed them for stem cell properties and drug sensitivity. Importantly, we used the differential redox state and not phenotypic markers to isolate distinct leukemic subpopulations. Morphological evaluation of sorted CD45/SS blast gated, DCFlow and DCFhigh cells demonstrated that both subpopulations were leukemic. Comparative analysis of the cell cycle distribution after staining with Ki67 and 7AAD indicated in most cases that ROSlow cells are quiescent, in contrast to ROShigh and total blast cells which are more actively cycling. Despite their predominant quiescent state, ROSlow leukemic cells were able both to grow as colonies in CFU assays and also to engraft in NOD SCID mice in pilot experiments, suggesting the existence of both leukemic “progenitor” and “stem” cell types within the ROSlow leukemic fraction. Based on these data, we challenged primary AML specimens with conventional chemotherapy agents (daunorubicin and AraC). Intriguingly, ROSlow cells preferentially survived exposure to either antileukemic agent in vitro. Taken together, our data identify a novel quiescent “oxidative state – low” leukemic population from patients with AML/MDS at diagnosis, which displays stem cell properties and exhibits functional differences related to drug sensitivity. The detailed molecular and functional characterization of this novel leukemic population is the subject of our ongoing studies. Disclosures:No relevant conflicts of interest to declare.

Cytoplasmic FANCA-FANCC Complex Interacts and Stabilizes the Cytoplasm-dislocalized Leukemic Nucleophosmin Protein (NPMc)

Eight of the Fanconi anemia (FA) proteins form a core complex that activates the FA pathway. Some core complex components also form subcomplexes for yet-to-be-elucidated functions. Here, we have analyzed the interaction between a cytoplasmic FA subcomplex and the leukemic nucleophosmin (NPMc). Exogenous NPMc was degraded rapidly in FA acute myeloid leukemia bone marrow cells. Knockdown of FANCA or FANCC in leukemic OCI/AML3 cells induced rapid degradation of endogenous NPMc. NPMc degradation was mediated by the ubiquitin-proteasome pathway involving the IBR-type RING-finger E3 ubiquitin ligase IBRDC2, and genetic correction of FA-A or FA-C lymphoblasts prevented NPMc ubiquitination. Moreover, cytoplasmic FANCA and FANCC formed a cytoplasmic complex and interacted with NPMc. Using a patient-derived FANCC mutant and a nuclearized FANCC, we demonstrated that the cytoplasmic FANCA-FANCC complex was essential for NPMc stability. Finally, depletion of FANCA and FANCC in NPMc-positive leukemic cells significantly increased inflammation and chemoresistance through NF-κB activation. Our findings not only reveal the molecular mechanism involving cytoplasmic retention of NPMc but also suggest cytoplasmic function of FANCA and FANCC in NPMc-related leukemogenesis.

Acute Myeloid Leukemia Diagnosis in the 21st Century

Abstract Context.—Rapid advances in understanding the molecular biology of acute myeloid leukemia are transforming the approach to diagnosis, prognostication, and treatment of these cases. Objective.—To briefly review the current state of AML classification with a particular emphasis on the role of molecular studies and their impact on the management of acute myeloid leukemia and other malignancies. Data Sources.—Current literature and experience of the authors. Conclusions.—While morphology, immunophenotyping, cytogenetics, and clinical history continue to play an important role, an increasing number of molecular tests are now required to properly classify these cases.

Acute Myeloid Leukemia Diagnosis in the 21st Century

Rapid advances in understanding the molecular biology of acute myeloid leukemia are transforming the approach to diagnosis, prognostication, and treatment of these cases. To briefly review the current state of AML classification with a particular emphasis on the role of molecular studies and their impact on the management of acute myeloid leukemia and other malignancies. Current literature and experience of the authors. While morphology, immunophenotyping, cytogenetics, and clinical history continue to play an important role, an increasing number of molecular tests are now required to properly classify these cases.

Proteomics of AML1/ETO Target Proteins: AML1–ETO Targets a C/EBP–NM23 Pathway

Abstract Introduction The rational design of targeted therapies for acute myeloid leukemia (AML) requires the discovery of novel protein pathways in the systems biology of a specific AML subtype. We have shown that in the AML subtype with translocation t(8;21), the leukemic fusion protein AML1–ETO inhibits the function of transcription factors PU.1 and C/EBPα via direct protein–protein interaction. In addition, recently using proteomics, we have also shown that the AML subtypes differ in their proteome, interactome, and post-translational modifications. Methods We, therefore, hypothesized that the systematic identification of target proteins of AML1–ETO on a global proteome-wide level will lead to novel insights into the systems biology of t(8;21) AML on a post-genomic functional level. Thus, 6 h after inducible expression of AML1–ETO, protein expression changes were identified by two-dimensional gel electrophoresis and subsequent mass spectrometry analysis. Results Twenty-eight target proteins of AML1–ETO including prohibitin, NM23, HSP27, and Annexin1 were identified by MALDI-TOF mass spectrometry. AML1–ETO upregulated the differentiation inhibitory factor NM23 protein expression after 6 h, and the NM23 mRNA expression was also elevated in t(8;21) AML patient samples in comparison with normal bone marrow. AML1–ETO inhibited the ability of C/EBP transcription factors to downregulate the NM23 promoter. These data suggest a model in which AML1–ETO inhibits the C/EBP-induced downregulation of the NM23 promoter and thereby increases the protein level of differentiation inhibitory factor NM23. Conclusions Proteomic pathway discovery can identify novel functional pathways in AML, such as the AML1–ETO–C/EBP–NM23 pathway, as the main step towards a systems biology and therapy of AML.

Low SMC1A protein expression predicts poor survival in acute myeloid leukemia

Age is a strong adverse prognostic factor in acute myeloid leukemia. Little is known about the biology of acute myeloid leukemia in elderly patients. The aim of this study was to identify genes with age-dependent changes of expression in leukemic blasts and their relevance for the patient prognosis. Gene expression profiling was carried out by mRNA microarray analysis from blasts of 67 adult acute myeloid leukemia patients of different age (range, 17-80 years). Among the genes that correlated with age, PRPF4 and SMC1A were selected for protein expression studies on a tissue array containing bone marrow histologies of 135 patients with newly diagnosed AML of different ages. A significant correlation between mRNA expression levels and patient age was shown by 131 genes. Increasing age was associated with significantly decreased mRNA levels of SMC1A. On the protein level, expression of SMC1A was low or absent in 74 out of 116 acute myeloid leukemia specimens. Importantly, patients with low protein expression levels of SMC1A experienced significantly shortened event free (2.6 months versus 10.3 months, p=0.003) and overall survival (10.4 months versus 22.6 months, p=0.015). The SMC1A protein expression level remained a significant prognostic factor for event free survival (p=0.014) with a borderline significance for overall survival (p=0.066) in a multivariate analysis. SMC1A protein expression might play a role in the determination of the prognosis and might have possible implications in therapy decision in patients with acute myeloid leukemia.

MicroRNA-181b targets MLK2 in HL-60 cells

microRNAs (miRNAs) play critical roles in many different cellular processes, including metabolism, apoptosis, differentiation and development. We showed miR-181b to be highly expressed in acute myeloid leukemia (AML). Furthermore, miR-181b contributed to proliferation of AML cells by targeting MLK2. Our results demonstrated that miR-181b plays an important role in the biology of AML and may be useful in developing therapies targeting miRNAs.

The development of a three-dimensional scaffold for ex vivo biomimicry of human acute myeloid leukaemia

Acute myeloid leukaemia (AML) is a cancer of haematopoietic cells that develops in three-dimensional (3-D) bone marrow niches in vivo. The study of AML has been hampered by lack of appropriate ex vivo models that mimic this microenvironment. We hypothesised that fabrication and optimisation of suitable biomimetic scaffolds for culturing leukaemic cells ex vivo might facilitate the study of AML in its native 3-D niche. We evaluated the growth of three leukaemia subtype-specific cell lines, K-562, HL60 and Kasumi-6, on highly porous scaffolds fabricated from biodegradable and non-biodegradable polymeric materials, such as poly (L-lactic-co-glycolic acid) (PLGA), polyurethane (PU), poly (methyl-methacrylate), poly ( d, l-lactade), poly (caprolactone), and polystyrene. Our results show that PLGA and PU supported the best seeding efficiency and leukaemic growth. Furthermore, the PLGA and PU scaffolds were coated with extracellular matrix (ECM) proteins, collagen type I (62.5 or 125 μg/ml) and fibronectin (25 or 50 μg/ml) to provide biorecognition signals. The 3 leukaemia subtype-specific lines grew best on PU scaffolds coated with 62.5 μg/ml collagen type I over 6 weeks in the absence of exogenous growth factors. In conclusion, PU-collagen scaffolds may provide a practical model to study the biology and treatment of primary AML in an ex vivo mimicry.

Osteopontin - A New Angiogenic Factor Which Correlates with Circulating Endothelial Cells in Acute Myeloid Leukemia.

Abstract 1004Poster Board I-26 Objectives:There is evidence that acute myeloid leukemia (AML) development and progression could be determined by the expression profiling of specific angiogenic proteins. Previously we have found that the number of circulating endothelial cells (CEC) and endothelial progenitors (CEP) in peripheral blood of AML patients is a good noninvasive marker of angiogenesis and correlates with tumor mass and response to treatment. Osteopontin (OPN) is hematopoietic stem cell niche component which controls tumor growth and angiogenesis. It promotes VEGF dependent tumor progression, induces endothelial cell survival and migration and modulates expression of angiopoietins. The role of OPN in AML is unknown. We evaluated the level of circulating OPN, angiopoietin-1 (ANG-1) and ANG-2 in AML patients. In addition we correlated the levels of angiogenic cytokines with CEC number, known prognostic factors and response to treatment. Methods:OPN, ANG-1 and ANG-2 were measured in plasma samples in 69 newly diagnosed AML patients with median age 53 (range 18-78 years) and 16 controls with median age 36 (range 22-73 years) using enzyme-linked immunosorbent assay (ELISA). According to median level of cytokines patients were subdivided into “low-expressers” and “high”-expressers group. CEC were evaluated by the four colour flow cytometry using a panel of previously described monoclonal antibodies. CEC deriving from angiogenic microvessels were characterized by CD36 expression. Results:The median levels of circulating OPN (162,5 pg/ml) and ANG-2 (5184 pg/ml) were significantly higher in AML patients as compared to healthy controls (70 pg/ml; p<0.0001 and 2624,3 pg/ml; p<0.001 respectively). In contrast, the level of ANG-1 in AML pts (1038 pg/ml) was lower than in control group (1710 pg/ml; p<0.002). OPN plasma level was significantly higher in AML patients with more than 50% of blasts in the bone marrow (197 pg/ml) and elevated LDH activity (170 pg/ml) compared to group with BM blasts <50% (152 pg/ml; p<0.05) and normal LDH (137,5 pg/ml; p<0.03). In analogy, we found the significant positive correlation between ANG-2, but not ANG-1, with WBC count (p=0.05), circulating blast count (CBC) (p<0.005) and LDH activity (p=0.05). There was no statistical association between the levels of OPN, ANG-1, and ANG-2, and patients' age, sex, AML subtype according FAB classification and cytogenetic risk group. Interestingly, in AML patients OPN level correlated positively with the number of CEC deriving from angiogenic microvessels (p<0.02). Moreover, we observed tendency to positive correlation between ANG-2 level and CD36+ CEC count (p=0.09). In addition, in AML we have found significant positive correlation between OPN and ANG-2 level (p<0.03). From the 49 patients receiving intensive induction chemotherapy, 28 (56%) achieved complete remission (CR). We observed that the CR rate in “low-expressers” of OPN (67%) was higher than in the “high-expressers” (33%), however a difference did not reached statistical significance. This may be due to not very large number of evaluated patients. Conclusions:The levels of circulating OPN and ANG-2, but not ANG-1, are significantly higher in AML patients than in healthy subjects and correlate with tumor burden as well as the number of “angiogenic” circulating endothelial cells. This data clearly demonstrate an important role of circulating OPN as well as ANG-2 in angiogenesis and AML biology. A better understanding of precise functions of OPN and angiopoietins may create new options for therapeutic interventions in AML. Disclosures:Robak:Celgene: Consultancy; Roche: Honoraria, Research Funding; Genmab: Research Funding; Cambridge Antibody Technology: Research Funding; GlaxoSmithKline: Honoraria.

Acute myeloid leukemia with mutated NPM1: diagnosis, prognosis and therapeutic perspectives

Nucleophosmin (NPM1) gene mutations, which cause aberrant cytoplasmic expression of nucleophosmin (NPMc+), are the most frequent genetic alteration in acute myeloid leukemia (AML), being found in about 30% cases. The present review summarizes recent advances in the biology, diagnosis, prognosis and therapy of NPM1-mutated AML. Diagnostic criteria of NPM1-mutated AML are discussed in the light of its recent inclusion in the 2008 WHO classification of myeloid neoplasms. We also outline the most recent findings on prognosis and monitoring of minimal residual disease in NPM1-mutated AML and their implications for therapeutic decisions. Moreover, new insights are presented into the molecular mechanisms underlying perturbed nucleophosmin traffic in NPM1-mutated AML, which provides the rationale for the development of targeted therapies. AML with mutated NPM1 is a leukemia entity with distinct molecular, pathological, and prognostic features.

A robust xenotransplantation model for acute myeloid leukemia

Xenotransplantation of human acute myeloid leukemia (AML) in immunocompromised animals has been critical for defining leukemic stem cells. However, existing immunodeficient strains of mice have short life spans and low levels of AML cell engraftment, hindering long-term evaluation of primary human AML biology. A recent study suggested that NOD/LtSz-scid IL2Rgammac null (NSG) mice have enhanced AML cell engraftment, but this relied on technically challenging neonatal injections. Here, we performed extensive analysis of AML engraftment in adult NSG mice using tail vein injection. Of the 35 AML samples analyzed, 66% showed bone marrow engraftment over 0.1%. Further, 37% showed high levels of engraftment (>10%), with some as high as 95%. A 2-44-fold expansion of AML cells was often seen. Secondary and tertiary recipients showed consistent engraftment, with most showing further AML cell expansion. Engraftment did not correlate with French-American-British subtype or cytogenetic abnormalities. However, samples with FLT3 mutations showed a higher probability of engraftment than FLT3 wild type. Importantly, animals developed organomegaly and a wasting illness consistent with advanced leukemia. We conclude that the NSG xenotransplantation model is a robust model for human AML cell engraftment, which will allow better characterization of AML biology and testing of new therapies.

Detection of nucleophosmin gene mutations in plasma from patients with acute myeloid leukemia: clinical significance and implications.

Roughly one-third of acute myeloid leukemia (AML) patients exhibit mutations in the nucleophosmin (NPM1) gene, and multiple studies have linked these mutations with a more favorable clinical outcome. We developed an assay for the detection of NPM1 mutations in peripheral blood plasma, and compared the results with clinical outcomes from a single institution. Analyzing plasma from previously untreated AML patients revealed NPM1 insertion mutations in 24 of 98 (24%) patients, with greater sensitivity than existing peripheral blood cell-based tests which showed positivity in only 22 of the 24 patients. Plasma testing allowed the detection of a novel 4 bp deletion in NPM1 in one patient. Analysis of clinical data corroborated previous data linking NPM1 mutations with better clinical outcome. These data underline the significance of NPM1 in the biology and clinical behavior of AML, and demonstrate the reliability and efficacy of plasma-based testing for NPM1 mutations.

A Robust Xenotransplantation Model for Acute Myeloid Leukemia

Xenotransplantation of human acute myeloid leukemia (AML) in immunocompromised animals has been critical for the definition of leukemic stem cells. However, existing immunodeficient strains such as NOD/SCID and NOD/SCID/b2mnull have short life spans, age dependent leakiness of humoral immunity and low levels of AML cell engraftment making long-term evaluation of primary human AML biology difficult. A recent study suggested that the nonobese diabetic/severe combined immondeficient/IL2Rgnull (NOG) mouse has enhanced ability to engraft AML cells but this study relied on neonatal injections that are technically challenging. We performed an extensive analysis of AML engraftment in adult NOG mice using intravenous tail vein injection. Thirty-six different AML samples were analyzed including 2 samples of acute promyelocytic leukemia (APML). We used a threshold for AML engraftment of >0.5% human CD45+33+ cells in the murine bone marrow. Based on this threshold, 22 samples (61%) showed engraftment in NOG mice. Of these samples, 14 (64%) showed high levels of engraftment (greater than 10% of murine marrow replaced with human CD45+CD33+ cells). Engraftment did not correlate with FAB subtype or cytogenetic abnormalities to a statistically significant degree, however we noted that one sample with an 11q23 translocation and several samples with Flt3 ITD mutations showed consistent high level engraftment. Several samples demonstrated engraftment as high as 95% of the murine marrow with total AML cell expansion of 2-30 fold. Evaluation of AML stem cell frequency and expansion is ongoing. Engraftment in spleen was variable and in general significantly lower than in bone marrow. For most samples, peripheral blood engraftment was barely detectable. In contrast to NOD/SCID mice, both APML samples engrafted well in the NOG mouse with high levels of peripheral blood involvement. Some samples occasionally showed engraftment of a population of cells expressing CD2 and other T cell associated markers by flow cytometry, however this observation was inconsistent even between mice injected with the same sample. All samples tested (n=5) showed consistent engraftment in secondary and tertiary recipients with most samples tested showing further expansion of total AML cells in subsequent transplants. Importantly, a number of animals developed organomegaly and a wasting illness consistent with advanced leukemic disease. Several such animals showed extramedullary leukemic infiltration into non-hematopoietic tissues. Etoposide monotherapy (40 mg/kg in divided doses) of heavily engrafted mice did not induce a significant response in terms of leukemia regression. Studies of other chemotherapeutic agents are ongoing. We conclude that the NOG xenotransplantation model is a robust model for studying human AML cell engraftment which will allow for better characterization of AML biology and testing of new therapies

The DNA Crosslinking Agent SJG-136 (SG2000, BN2629) Is Highly Potent in Acute Myeloid Leukaemia and Is Synergistic with Cytarabine

Standard treatment of acute myeloid leukemia (AML) results in a median survival of approximately 1 year. However, increased understanding of the complex biology of AML has led to the introduction of more targeted therapies, including gemtuzumab ozogamycin and a number of FLT3 inhibitors, in the expectation that they will improve outcomes in this condition. The future is likely to see combinations of new targeted drugs together with more standard therapies. The challenge will be to identify the optimal combinations for individual patients based on their specific disease markers. SJG-136 (SG2000, BN2629) is a pyrrolo[2,1-c][1,4]benzodiazepine dimer which binds into the minor grove of DNA with a preference for 5′Œ-GATC-3′Œ sequences. The crosslinks that SJG-136 forms are more persistent than those formed by nitrogen mustard agents like melphalan due to their slow rate of repair. We have previously shown that SJG-136 is very potent against primary chronic lymphocytic leukaemia (CLL) cells so we set out to investigate its effect on AML cell lines and primary AML. The AML cell lines, NB4 and HL60, had LD50 values of 5.75nM and 12.10nM respectively following exposure to SJG-136 for 48 hours. Apoptosis induction was dose-dependent, as evidenced by Annexin V/propidium iodide assay and caspase-3 activation assay. Subsequently, primary AML cells from 37 diagnostic samples were isolated and treated with SJG-136 and cytarabine yielding an average LD50 of 19.4nM (±22.8) and 5.7mM (±5.6) respectively. SJG-136 was >100 times more potent than cytarabine in samples derived from the same patients (P< 0.0001). We next investigated the potential for synergy between cytarabine and SJG-136 in the AML cell lines (cytarabine:SJG-136, 100:1). NB4 cells demonstrated a combination index (CI) for the two agents of 0.37 and HL60 cells showed a CI of 0.69 indicating that these two agents were synergistic in the two cells lines under these conditions. The primary AML samples were also tested in vitro for synergy between SJG-136 and cytarabine at the same ratio. In these samples the mean CI was 0.56 (± 0.49) with synergy being demonstrated in 29/37 patient samples. Closer examination of these results showed that the 8 patient samples with a CI greater than 1 were all particularly sensitive to cytarabine as a single agent. Therefore, the increased sensitivity of these patient samples to cytarabine may account for their apparent lack of synergy as calculated by the median effect method. These results indicate that SJG-136 has significant potential as a therapy for AML both as a single agent and in combination with cytarabine.

Common Self-Renewal Pathways Contribute to the Induction of Acute Myeloid Leukemias Associated with Different Oncogenes

Self-renewal is a prerequisite for cancer initiation and maintenance, however the origins and molecular basis of self-renewal in malignant cells remain elusive. Recently, we and others have shown that certain leukemia-associated fusion oncogenes (such as MOZ-TIF2 and MLL fusions) can alter self-renewal in, and generate acute myeloid leukemia (AML) from, committed murine myeloid progenitor cells. AML is a highly heterogeneous disease, both genetically and biologically, and is associated with many differing combinations of mutations and a variable clinical course. However, it is not currently known whether an alteration of self-renewal is a more widespread finding in AML. Moreover, it is also not known whether alterations of self-renewal are mediated by common genetic programs downstream of multiple individual mutations or through the engagement of unique programs downstream of individual mutations. This distinction is important, as the demonstration of common pathways may identify common critical molecular targets for the treatment of AML. We now demonstrate, using retroviral expression of the AML1-ETO (RUNX1-RUNX1T1) and NUP98-HOXA9 fusion proteins in committed myeloid progenitors, that the ability to alter self-renewal is a more generalized property of leukemia-associated transcription factor fusion oncogenes. Furthermore, to investigate the molecular basis for the reestablishment of self-renewal we compare expression differences between granulocyte monocyte progenitors (GMP) directly transduced with MOZ-TIF2, AML1-ETO and NUP98-HOXA9 retroviruses and GMP transduced with empty vector retrovirus, to identify common candidate genes and molecular pathways involved in self-renewal downstream of these fusion oncogenes. Thereafter, we validate the expression of these early genetic programs in functionally defined murine leukemia stem cells and bulk human leukemias and perform functional experiments to validate the effects of individual candidate genes within the programs on self-renewal and transformation. Our data demonstrateAML1-ETO and NUP98-HOXA9 alter self-renewal in and NUP98-HOXA9 generates AML from committed myeloid progenitors.Disparate leukemia-associated oncogenes (MOZ-TIF2, AML1-ETO and NUP98-HOXA9) initiate an early, common self-renewal signature of 167 genes (FDR level p<0.05).Significant elements of this self-renewal signature can be detected in established, functionally defined leukemia stem cells from a murine model of AML.Significant elements of this early signature can also be detected across a large cohort of 253 patients with differing AML subtypes, where the signature predicts for disease biology and patient survival.Single genes from the signature, including Sox4 and Bmi1 can themselves phenocopy the leukemia-associated oncogenes and alter self-renewal in committed murine progenitors. Taken together, our findings have important implications for the biology of AML and, furthermore, may suggest common targets for therapeutic intervention.

New Drugs in Acute Myeloid Leukemia

Standard treatment of acute myeloid leukemia (AML) results in a median survival of approximately 1 year. Together with increasing understanding of the biology of AML, this has led to the introduction of many new anti-AML drugs. Here we discuss the most prominent of these: clofarabine, epigenetic-acting agents, gemtuzumab ozogamycin, and FLT3 inhibitors. All are indubitably active. However, their therapeutic efficacy relative to each other and to standard therapy is unclear. The future is likely to see combinations of the drugs with each other and with more standard therapies. There also will be a shift away from inquiring which therapy is best for the average patient to inquiring which therapy is best for a given patient with a specific constellation of disease markers.