Progression To Acute Myelogenous Leukemia Research Articles

LSP1 promotes the progression of acute myelogenous leukemia by regulating KSR/ERK signaling pathway and cell migration

ABSTRACT We aimed to investigate the role and mechanism of LSP1 in the progression of acute myelogenous leukemia. In this study, we established shLSP1 cell line to analyze the function of LSP1 in AML. We observed high expression of LSP1 in AML patients, whereas it showed no expression in normal adults. Furthermore, we found that LSP1 expression was associated with disease prognosis. Our results indicate that LSP1 plays a crucial role in mediating proliferation and survival of leukemia cells through the KSR/ERK signaling pathway. Additionally, LSP1 promotes cell chemotaxis and homing by enhancing cell adhesion and migration. We also discovered that LSP1 confers chemotactic ability to leukemia cells in vivo. Finally, our study identified 12 genes related to LSP1 in AML, which indicated poor survival outcome in AML patients and were enriched in Ras and cell adhesion signaling pathways. Our results revealed that the overexpression of LSP1 is related to the activation of the KSR/ERK signaling pathway, as well as cell adhesion and migration in AML patients. Reducing LSP1 expression impair AML progression, suggesting that LSP1 may serve as a potential drug therapy target for more effective treatment of AML.

The RNA m6A Reader YTHDF1 Is Required for Acute Myeloid Leukemia Progression.

The m6A reader YTHDF1 is required for progression of acute myelogenous leukemia and can be targeted with the FDA-approved drug tegaserod to suppress leukemia growth.

Acceleration of AML Progression By Cigarette Smoke Exposure or Condensate Exposure and Associated DNA Methylation Alterations

Subsets of acute myelogenous leukemia (AML) are characterized by molecular alterations with prognostic significance, however, little is known about how modifiable behaviors, such as cigarette smoking, intersect with genetic factors. Mutations rendering the Fms-like tyrosine kinase 3 (FLT3) constitutively active, such as internal tandem duplication (ITD), are associated with refractory disease and therapy resistance. Inhibition of the FLT3 kinase shows some benefit in this population, as highlighted by the FDA approvals of midostaurin and gilteritinib, but overall outcomes remain poor. Cigarette smoke exposure (CSE) also marks a population of patients with poor prognosis. Current and former smokers who develop AML are known to have worse survival as compared to never smokers (Alfayez M., et al. ASCO 2019), but the impact of FLT3 mutation and subsequent associated treatment response has not been studied. Also, the underlying mechanism of how history of cigarette smoking influences leukemia biology and response to therapy is poorly understood. In order to model a history of smoking in AML patients, NOD-SCID mice (n=25) were exposed to CSE using a smoking robot for 2 hours, 5 days/week, for 2 weeks. Mice were then inoculated via tail-vein injection with luciferase tagged human FLT3-ITD cells and leukemia burden was monitored through noninvasive imaging. CSE continued through the duration of the experiment, post engraftment. Control "non-smoking" mice (n=15) were only injected with leukemia cells. Within one week post leukemic introduction, a significant increase in leukemic burden as measured by bioluminescence was apparent in mice exposed to CSE versus control mice (P-value<0.0001). To model the impact of smoking cessation upon diagnosis of AML in patients, experiments were modified to halt CSE once leukemic engraftment was detectable by non-invasive imaging. Smoking cessation versus continuous smoke exposure yielded reduced relative leukemic burden. Mice with continuous smoke exposure had higher rates of leukemia compared to mice who ceased smoking (n=10) one week prior (P-value =0.0064). These rapid changes in leukemic burden suggest that CSE may prime the microenvironment to promote leukemia progression or directly affect leukemia cells. To address the latter possibility, human AML cells were exposed to cigarette smoke condensate (CSC), which contains the chemicals present in cigarettes, for two weeks before introducing the cells into mice. A significant increase in leukemic burden was observed in mice injected cells exposed to CSC compared to mice injected with unexposed leukemia cells (P-value <0.001), highlighting a direct role for the chemicals in cigarettes on in vivo leukemia proliferative factors. Smokers are known to carry altered global DNA methylation signatures that persist decades after quitting. To measure DNA methylation changes in the in vivo models described above, we examined spleens of non-smoking and smoke exposed mice by reduced representative bisulfite sequencing (RRBS). Sequences were mapped to either the human or mouse genome, (enabling identification of leukemia specific versus microenvironment specific alterations) and were compared in the smoking and non-smoking mice. Over 200 genes exhibited significant DNA methylation alterations in their promoter regions. Genes involved in RNA polymerase activity and chromatin remodeling were highly represented amongst those with altered DNA methylation. The clinical significance of our observations was confirmed in a cohort of 58 treatment naïve FLT3-ITD AML patients at MD Anderson receiving intensive induction therapy: 41 never smokers and 17 ever smokers. Smokers had significantly reduced survival as compared to the never smokers (median overall survival of 18 vs 23 months, P-value 0.0092). Collectively our findings indicate that short-term CSE is sufficient to alter DNA methylation patterns and accelerate the early progression of FLT3-ITD AML in vivo. Smoking cessation upon diagnosis may slow leukemic growth relative to smoking throughout AML therapy prompting the consideration of behavioral interventions for smokers with AML. Improved understanding of the mechanism of leukemic progression and drug resistance from CSE is expected to lead to improved treatment paradigms designed for patients with a history of cigarette smoking. Disclosures Konopleva: Genentech: Honoraria, Research Funding; Kisoji: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Ascentage: Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding. Jabbour:Cyclacel LTD: Research Funding; Pfizer: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; BMS: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding. DiNardo:agios: Consultancy, Honoraria; abbvie: Consultancy, Honoraria; jazz: Honoraria; medimmune: Honoraria; notable labs: Membership on an entity's Board of Directors or advisory committees; daiichi sankyo: Honoraria; syros: Honoraria; celgene: Consultancy, Honoraria.

Transcription factor Oct1 protects against hematopoietic stress and promotes acute myeloid leukemia

A better understanding of the development and progression of acute myelogenous leukemia (AML) is necessary to improve patient outcome. Here we define roles for the transcription factor Oct1/Pou2f1 in AML and normal hematopoiesis. Inappropriate reactivation of the CDX2 gene is widely observed in leukemia patients and in leukemia mouse models. We show that Oct1 associates with the CDX2 promoter in both normal and AML primary patient samples, but recruits the histone demethylase Jmjd1a/Kdm3a to remove the repressive H3K9me2 mark only in malignant specimens. The CpG DNA immediately adjacent to the Oct1 binding site within the CDX2 promoter exhibits variable DNA methylation in healthy control blood and bone marrow samples, but complete demethylation in AML samples. In MLL-AF9-driven mouse models, partial loss of Oct1 protects from myeloid leukemia. Complete Oct1 loss completely suppresses leukemia but results in lethality from bone marrow failure. Loss of Oct1 in normal hematopoietic transplants results in superficially normal long-term reconstitution; however, animals become acutely sensitive to 5-fluorouracil, indicating that Oct1 is dispensable for normal hematopoiesis but protects blood progenitor cells against external chemotoxic stress. These findings elucidate a novel and important role for Oct1 in AML.

CD82 supports survival of childhood acute myeloid leukemia cells via activation of Wnt/β-catenin signaling pathway.

Stem cell marker CD82 plays a vital role in the oncogenesis and progression of acute myelogenous leukemia (AML), especially in sharing properties of leukemia stem cells (LSCs). The Wnt/β-catenin pathway is required for the development of LSCs in AML. The present study aimed to validate whether CD82 supports the survival of LSCs in pediatric AML via activation of Wnt/β-catenin signaling pathway. CD82 expression and its correlation with molecules downstream of Wnt/β-catenin pathway in samples from pediatric AML patients were analyzed. Forced or downregulated expression of CD82 in AML cells was evaluated for the effects of CD82 on cell proliferation, cycle regulation, apoptosis, and adriamycin chemoresistance and to validate the underlying mechanism. Aberrant expression of CD82 in pediatric AML patients was found. CD82 messenger RNA expression correlated positively with downstream molecules of Wnt/β-catenin pathway in AML children. Knockdown of CD82 induced apoptosis, suppressed growth, and decreased adriamycin chemoresistance in AML cells. CD82 accelerated β-catenin nuclear location and then stimulated the expression of downstream molecules of Wnt/β-catenin pathway. CD82 regulates the proliferation and chemotherapy resistance of AML cells via activation of the Wnt/β-catenin pathway, which suggest that the CD82 may be a potential therapeutic target in AML children.

Targeting the Second Bromodomain (BDII) of BET Family with Abbv-744 Results in Robust Anti-Leukemia Activity in AML Models with Excellent In Vivo Tolerability

The BET family proteins consist of BRD2, BRD3, BRD4 and BRDT, with each of these proteins containing two highly conserved bromodomains (BDI and BDII). First generation BET inhibitors, including ABBV-075, are pan-BET inhibitors that bind with nearly equimolar affinity to BDI and BDII. Pan-BET inhibitors generally block transcription and inhibit the proliferation of a large spectrum of tumor types, but exhibit a narrow therapeutic index. ABBV-744 is a highly selective inhibitor for the BDII of BET family proteins, exhibiting greater than 300-fold more potent binding affinity to the BDII bromodomain of BRD4 relative to BDI (Warren Kati AACR 2018; Xiaoyu Lin AACR 2018). In this study, we evaluated the anti-leukemia activities of ABBV-744 in acute myelogenous leukemia (AML) cell lines and primary samples.In AML cell lines, ABBV-744 induced G1 cell cycle arrest and apoptosis with caspase-3 activation and PAPR cleavage through regulation of the key proteins that are involved in cell cycle and apoptosis pathways (e.g. (BCL-2, BCL-XL, MCL-1, c-Myc). Mechanistically, ABBV-744 displaced BRD4 from the regulatory region of BCL2, RUNX1, BCL2L1 and IL8 genes by ChiP-Seq. MLL-rearranged cells were most sensitive to BDII inhibition, and ABBV-744 displaced both, BRD4 and MLL from the regulatory regions of BCL2 and c-Myc genes in MV4;11 (MLL-AF4) and Nomo-1 (MLL-AF9) cells. We examined anti-leukemia activity of ABBV-744 in 12 primary AML samples with diverse genetic alterations. ABBV-744 inhibited cell growth in 8/12 primary samples (20.2%-77.9%) and induced apoptosis (26.7 ± 9.6% in ABBV-744 group vs 14.1± 3.9% in DMSO group, p=0.007, Fig 1A). We next performed the whole genome transcriptome analysis of pre-treatment AML cells by RNA-sequencing (RNA-seq). The samples which were sensitive to ABBV-744 tend to have higher pro-apoptosis (TXNIP, DR5) and lower anti-apoptosis (BCL2, EXPO) than resistant samples.For efficacy assessment in vivo, we established a patient-derived xenograft (PDX) from an AML patient with FLT3-ITD, DNMT3A, IDH1 and NPM1 mutations in NSG mice. Upon engraftment, mice were randomized to receive vehicle or single agent ABBV-744 at a well-below MTD dose of 9.4 mg/kg for 21 days. The median survival time of mice treated with ABBV-744 (Median survival: 76 days) was significantly higher compared to untreated mice (Median survival: 67.5 days, p=0.007, Fig 1B). Next, the anti-leukemia efficacy of ABBV-744 was tested in 7 additional AML PDX models. NSG mice were injected with 7 AML PDX (one mouse per cohort design). After engraftment was confirmed by retro-orbital bleeding, mice were treated with vehicle or ABBV-744. Circulating tumor burden was measured by flow cytometry in peripheral blood samples from mice collected on indicated days of therapy. In five of the 7 models, ABBV-744 treatment delayed AML progression compared to untreated mice (survival days: 135 vs 105, 205 vs 153, 62 vs 46, 118 vs 77, 97 vs 86). No significant impact on mice' weight was noted (Fig 1C), and no clinical signs of toxicity recorded over the course of therapy.In summary, blockade of BET family bromodomain BDII promotes apoptotic cell death and suppresses proliferation in the majority of primary AML cells. Importantly, ABBV-744 therapy reduced AML tumor burden and extended survival in AML PDX models at a well-below MTD dose. Together, these results provide proof of concept that highly selective inhibitors of the second bromodomain of BET family may maintain robust anti-leukemia efficacy in AML while exhibiting improved tolerability relative to pan-BET inhibitors. [Display omitted] DisclosuresUziel:AbbVie Inc.: Employment. Shen:AbbVie Inc: Employment. Konopleva:Stemline Therapeutics: Research Funding. Lin:AbbVie Inc: Employment. Lu:AbbVie Inc: Employment. Zhang:AbbVie Inc: Employment.

Extracellular Vesicles (EVs) Shape the Leukemic Microenvironment

BackgroundExtracellular Vesicles (EVs) compose a naturally occurring, heterogeneous group of membrane-bound, nano-sized particles shed by all cells. Depending on cellular type, physiological state, and mode of secretion some harbor potent regenerative properties while others have the propensity to induce disease. Human bone marrow mesenchymal stem cell (MSC)-derived EVs harbor regenerative potential. Our own studies have shown MSC-EVs are able to mitigate radiation damage to bone marrow, and to reverse the malignant phenotype in prostate and colorectal cancer. On the contrary, EVs isolated from neoplastic cells induce a neoplastic phenotype in non-cancerous cells. Leukemic EVs also potentiate the phenotypic change of healthy MSCs into cancer associated fibroblasts. The role of EVs within the leukemic microenvironment may provide insight for therapeutic advances. We hypothesize that EVs derived from normal MSCs inhibit the growth of nascent acute myelogenous leukemia (AML), and that the predominant EV population changes during leukemia progression. Neural network machine learning will allow us to capture these changes in order to build predictive models.MethodsKasumi AML cells lines were seeded with various concentrations of MSC-derived EVs. Vesicles were isolated using an established differential centrifugation technique, and co-cultured with Kasumi. To study cellular proliferation we employed a fluorescence-based method for quantifying viable cells (CyQuant). We also investigated the modes of death (apoptosis vs necrosis) EVs may induce on AML via a three die fluorescent system. Fluorescence intensities were normalized to control wells containing non-EV treated cells alone.Our neural network achieved 90.16% classification accuracy with cell culture data, and was tasked to classify the similarity of patient samples to the AML-derived EVs.ResultsProliferation of AML cells after one day of co-culture with 2.6E8 &1.3E10 MSC-EVs respectively was inhibited in a dose dependent manner: with 2.6E8 EVs leading to ~ 15% reduction in growth, and 1.3E10 EVs leading to ~60% reduction when normalized to non-EV treated controls.3 days of co-culture with similar doses resulted in ~40% and ~80% reduction in proliferation when normalized to control.At day 6 of co-culture growth was inhibited by ~80% at both EV concentrations.At multiple time points hMSC-EV treated AML cells showed a significantly higher proportion of apoptosis. Cellular necrosis was negligible.There was no statistically significant change in proliferation of MSC exposed to MSC-derived EVs when compared to non-EV treated controls.There was also no statistically significant change in proliferation of AML cells exposed to AML-derived EVs.Samples of AML, Chronic Myelomonocytic Leukemia, and Multiple Myeloma entered into our machine alogrithm were calculated to be 100%, 100%, and 66%, respectively, similar to the AML-derived EVs.Summary/ConclusionMSC-derived EVs inhibits the proliferation of the AML cell line in vitro via an apoptotic mechanism. This effect is seen as early as one day of co-culture and persists out to three, and six days, implicating an miRNA-mediated mechanism that has been discussed in previous works. We have also shown that when cells are exposed to their own EV there is no change or (in the case of leukemia) a statistically insignificant increase in proliferation. We feel this is perhaps a model of how a normal marrow works to suppress early cancer. As leukemia develops the cross-talk between AML and its microenvironment, via direct EV mediated effects, alters the MSCs to promote a survival signal favoring AML growth. This restructuring is EV mediated. Future work involves the capacity of AML-derived EVs to alter the phenotype of normal marrow towards a pro-leuekmic phenotype. This also includes the use of AML mouse models to further investigate the therapeutic potential MSC-derived EVs may have as single or adjunct therapy; as well as to study potential cellular mediators that may be involved in EV-direceted AML progression. Lastly we endeavor to employ machine learning networks to characterize and predict the dynamic restructuring the EV milieu undergoes as leukemia progresses. Capturing these alteration will allow the creation of reliable predictive models that will have direct inferences on clinical diagnosis and prognosis. DisclosuresNo relevant conflicts of interest to declare.

CD98-Mediated Adhesive Signaling Enables the Establishment and Propagation of Acute Myelogenous Leukemia

Acute myelogenous leukemia (AML) is an aggressive disease associated with drug resistance and relapse. To improve therapeutic strategies, it is critical to better understand the mechanisms that underlie AML progression. Here we show that the integrin binding glycoprotein CD98 plays a central role in AML. CD98 promotes AML propagation and lethality by driving engagement of leukemia cells with their microenvironment and maintaining leukemic stem cells. Further, delivery of a humanized anti-CD98 antibody blocks growth of patient-derived AML, highlighting the importance of this pathway in human disease. These findings indicate that microenvironmental interactions are key regulators of AML and that disrupting these signals with targeted inhibitors such as CD98 antibodies may be a valuable therapeutic approach for adults and children with this disease.

Myelodysplastic syndromes with 5q deletion: pathophysiology and role of lenalidomide

Myelodysplastic syndrome (MDS) is a hematopoietic stem cell disorder primarily affecting CD34+ cells, characterized by ineffective hematopoiesis, often transforming into acute myelogenous leukemia (AML). A subset of patients has 5q deletion (del(5q)) as the culprit pathogenetic trigger. Del(5q) affects critical regions 5q31 and 5q33, leading to gene haplodeficiency with subsequent RPS14 haplodeficiency and P53 activation. Subsequent to P53 activation, erythroid cell apoptosis and ineffective erythropoiesis occur. Other pathogenetic elements include protein phosphatase 2a and CDC25C haplodeficiency and decreased miR-145 and miR-146a expression. Lenalidomide is an immunomodulatory agent that selectively suppresses the del(5q) clone. While the mechanism is not fully understood, it is associated with diverse molecular changes including stabilization of MDM2 with subsequent enhanced P53 degradation. Lenalidomide showed success in low- and intermediate-1-risk MDS as reported in the 002, 003, and 004 trials. However, in higher-risk MDS, the results of lenalidomide monotherapy were modest, mandating the use of combination therapy. The role and priority of lenalidomide varies between different guidelines, and accordingly, future efforts are necessary to reach a unified therapeutic algorithm. TP53 mutations are important predictors of AML progression and possible resistance to lenalidomide. It is recommended to identify TP53 mutation early in the disease since it may change the decision regarding choice of therapy. Challenges with lenalidomide therapy remain the long-term effects and timing of its discontinuation.

WTAP is a novel oncogenic protein in acute myeloid leukemia

WTAP is a novel oncogenic protein in acute myeloid leukemia

Sequential Acquisition of AML1-ETO and c-Kit Mutation Leads to Formation of Human t(8;21) Acute Myelogenous Leukemia Stem Cells,

Abstract 3584The 8;21 translocation, one of the most general chromosomal abnormalities in acute myelogenous leukemia (AML), encodes the AML1-ETO chimeric fusion gene. Because AML1-ETO can inhibit the CBF complex to transactivate myeloid-lineage genes in a dominant negative fashion, the high level expression of this gene plays a critical role in inhibiting differentiation of target cells, which leads to progression of AML. We, however, have reported that patients maintaining a long-term remission retain AML1-ETO expression at a very low level that can be detected by nested RT-PCR. The AML1-ETO transcripts in these patients were derived from a small fraction of t(8;21)+ hematopoietic stem cells (HSCs) capable of multilineage differentiation (PNAS 2000). In fact, previous data shown that AML1/ETO knock-in or AML1/ETO transgenic mice did not develop AML. These data suggest that acquisition of the AML1-ETO fusion is not sufficient to develop t(8;21) AML. Since t(8;21) AML cells frequently possess constitutive active mutation of c-Kit, we hypothesized that the c-Kit mutation may work as a second oncogenic hit in t(8;21)+ HSCs to transform into AML. To test the hypothesis, we extensively analyzed the existence of c-Kit mutation within AML1-ETO+ HSCs from patients maintaining remission for a long-term. CD34+CD38− HSCs were purified from the bone marrow of patients in long-term remission, and were cultured in vitro to form colonies. These HSC-derived colonies were picked up, and tested for the presence ofAML1-ETO and c-Kit mutation. Five t(8;21) AML patients with c-Kit mutation were enrolled in this study. All of 1020 blastic colonies at diagnosis were positive for both AML1-ETO and c-Kit mutation. In 7187 colonies formed in the culture of remission marrow, almost 1% (89 colonies) of these colonies expressed AML1-ETO. Surprisingly, none of these colonies possessed c-Kit mutation, indicating that AML1-ETO+ clones in remission are not identical to these in t(8;21) AML. Accordingly, it is highly likely that HSCs first acquire AML1-ETO, and a fraction of these cells additionally mutated c-Kit, resulting in transformation into AML stem cells. This is the first clear-cut evidence that human HSCs transform into AML via multi-step oncogenesis in vivo. Disclosures:No relevant conflicts of interest to declare.

A cost-effectiveness analysis of using azacitidine vs. decitabine in treating patients with myelodysplastic syndromes

Objective:Azacitidine and decitabine are used to treat patients with myelodysplastic syndromes (MDS) in the United States (US). This study sought to assess their relative cost-effectiveness.Design and methods:The authors developed a cost-effectiveness Markov model (1-month cycles) tracking hypothetical cohorts of MDS patients treated with azacitidine or decitabine over 2 years. The model used a US payer perspective and 2009 costs. Health states modeled included MDS with Transfusion Dependence, MDS with Transfusion Independence, Progression to Acute Myelogenous Leukemia (AML), and Death. Incremental cost-effectiveness outcomes included cost per quality-adjusted life year (QALY), cost per life year (LY), cost per patient-month of transfusion independence, and cost per case of AML progression avoided. One-way sensitivity analyses were performed on key model parameters.Results:Compared to decitabine, azacitidine was associated with better survival (1.512 LYs vs 1.292), more QALYs gained (1.041 vs 0.870), more patient-months with transfusion independence (8.328 vs 6.224), and a greater proportion of patients avoiding progression to AML (50.9% vs 28.5%). Total per-patient costs over 2 years for azacitidine were lower than for decitabine ($150,322 vs $166, 212).Limitations:To inform and update the model over time, it will be important that randomized or observational clinical studies be conducted to directly compare azacitidine and decitabine, provide new information on how these medicines are used, and on their relative clinical effectiveness.Conclusion:Results demonstrate that azacitidine provides greater clinical benefit and costs less than decitabine across all key outcomes. These results accentuate the positive role of azacitidine in providing cost-effective care for MDS.

294 Prognostic significance of beta 2 microglobulin in survival and transformation to acute myelogenous leukemia in patients with myelodysplastic syndrome

s of the 11th International Symposium on Myelodysplastic Syndromes (MDS) 18 May 2011 21 May 2011 S116 Posters / Leukemia Research 35 (2011) S27–S142 294 Prognostic significance of beta 2 microglobulin in survival and transformation to acute myelogenous leukemia in patients with myelodysplastic syndrome A. Galanopoulos, A. Aggelidis, D. Gogos, A. Megalakaki, I. Kotsianidis, M. Dimou, P. Panagiotidis, N. Zoumbos, N.I. Anagnostopoulos, M. Voulgarelis, H. Papadaki, A. Symeonidis. Clinical Hematology, G. Gennimatas General Hospital, Athens, Clinical Hematology, Metaxas Hospital, Piraeus, Clinical Hematology, Dimokritio University of Thraki, Alexandroupolis, Clinical Hematology, Laikon General Hospital/National and Kapodistrian University of Athens, Athens, Clinical Hematology, University of Patras, Medical School, Patras, Clinical Hematology, University of Crete School of Medicine, Heraklion, Greece Background: Beta 2 microglobulin (b2M) is a 12 kDa light chain protein that binds to the a chain of class I MHC (MHC1) molecule. Serum b2M levels are known to reflect renal function and membrane turnover, which is associated with tumor mass and growth rate. Elevated serum levels of b2M are reported to predict poor survival in several hematologic malignancies. Purpose: To investigate the prognostic relevance of b2M with regard to survival and the risk of transformation into acute myelogenous leukemia (AML) in patients with myelodysplastic syndrome (MDS). Material and Methods: We evaluated serum b2M levels in 407 MDS patients from the Hellenic MDS Study Group. From these 152 patients were excluded from further analysis for various reasons such as serum creatinine levels greater than 1.5mg/dl, treatment with intensive therapy and blood stem cell transplantation or insufficient data. B2M and karyotype were recorded along with other hematologic, biochemical and clinical covariates. Serum b2M levels were quantified by a radioimmunoassay. The log rank test was used to compare overall survival and AML transformation. Multivariate analysis was conducted utilizing the Cox proportional hazards regression model with a stepwise backward selection. Results: Thirty patients had refractory anemia (RA), 64 refractroy anemia with excess of blasts-1(RAEB-1), 34 refractory anemia-2 (RAEB-2), 24 refractory anemia with ringed sideroblasts (RARS), 69 refractory cytopenia with multilineage dysplasia (RCMD), 13 refractory cytopenia with multilineage dysplasia with ringed sideroblasts (RCMD-RS) and 11 5q− syndrome. The median value for b2M was 2.67(±1.53). The univariate analysis revealed that patients with b2M below 3mg/dl had a higher overall survival compared to patients with b2M above 3mg/dl (log-rank=7.42; p = 0.006). Other factors that exerted a statistically significant effect on overall survival were the medullary blasts (log-rank =13.5; p 15%. MDS with del(5q) was identified in 135 (6.6%) of the cases, RARS-T in 76 patients (3.7%). There were 122 (6%) cases with MDS unclassifiable, including 95 (4.7%) with 1% peripheral blasts, 26 (1.3%) with pancytopenia, and only one patient without signs of dysplasia despite chromosomal abnormalities. Survival data (months) and AML progression rates (%) at 2 and 5 years are shown in the tables. Types with unilineage Dysplasia WHO 2008 Hb g/dl PLT/ nl ANC/ ml Abnormal karyotype in % Complex karyotype in % IPSS low/ int 1/int 2 median survival in month AML progression in % after 2 years AML progression in % after 5 years RCUDbi 10.3 116 2000 50 19 30/57/13 64 9 14 RA 8.5 248 3900 40 14 91/9/0 48 2 2 RT 11.7 56 2650 57 0 80/20/0 not reached 0 11 RN 11.9 137 1300 8 0 92/8/0 141 10 10 RARS 9.5 267 3150 28 6 73/24/2 63 4 4 MDS-U-PAN 8.5 44 750 33 0 0/100/0 30 0 18 Types with multilineage Dysplasia WHO 2008 HB g/dl PLT/ nl ANC/ ml Abnormal karyotype in % Complex karyotype in % IPSS low/ int 1/ int 2 median survival in month AML progression in % after 2 years AML progression in % after 5 years RCMD 9.3 134 2030 49 19 40/51/9 36 11 17 RCMD-RS 8.8 185 2200 55 24 45/41/14 31 10 17 RARS-T 9.1 584 4750 41 18 67/33/0 55 3 8 MDS del (5q) 9.3 255 2300 100 0 73/27/0 91 4 14 MDS-U-PB 9.4 118 2300 47 20 41/52/7 35 10 13 Conclusions: 1. While RCUD carries a relatively good prognosis, pancytopenic patients (MDS-U-Pan) have a shorter survival, similar to RCMD. The separation of MDS-U-Pan therefore seems to be is justified. 2. RCUD with bicytopenia accounts for a considerable fraction (52.3%) of RCUD. The prognosis of RCUDbi is intermediate between RCUD with unilineage cytopenia and MDS-U-Pan. 3. The prognostic impact of blast cells in the peripheral blood is similar to that of pancytopenia. 4. Survival rates are not substantially different for RCMD and RCMDRS. 5. RARS-T and MDS with del5q usually present with multilineage dysplasia. From a prognostic point of view, they are similar to RCUD, but their specific pathogenetic features justify their existence as distinct subtypes in the WHO classification.

Ocular masquerade syndrome as a herald of progression of acute myelogenous leukemia

Dear Editor, A 77-year-old Indian female presented with a 5-day history of left eye redness, pain, and intermittent blurring of vision. She had been diagnosed with acute myeloid leukemia (AML) 6 months ago and decided for conservative management, with dependence on monthly packed cell transfusions. Presenting Snellen acuity was 20/50 on the right and 20/40 on the left. Anterior segment examination revealed conjunctival injection with anterior chamber inflammation of 3+ cells, 2+ retrolental cells, and vitritis 1+ cells with normal fundus and intraocular pressure (IOP). Right eye was unremarkable. Flow cytometry of aqueous revealed 12% myeloblasts. No background monocytes or neutrophils were detected, and cultures were negative. Full blood count (FBC) revealed raised total white cells 22.2×10/L, comprising of 63% blasts (13.99×10/L), myelocytes (0.22×10/L), thrombocytopenia 9×10/L (170–420), and anemia 7.6 g/dL. Erythrocyte sedimentation rate was >140 mm/h. She was treated conservatively with topical prednisolone acetate 1%. Subsequent review showed no treatment response. IOP remained normal. She was admitted for palliation of severe bone pain and received multiple packed cell transfusions, but succumbed to pneumonia 30 days later.

Comparative Cost-Effectiveness of Azacitidine Versus Decitabine for the Treatment of Myelodysplastic Syndromes (MDS).

Abstract 1377Poster Board I-399 Introduction:National Comprehensive Cancer Network guidelines recommend the use of azacitidine and decitabine to treat patients with myelodysplastic syndromes (MDS). In a Phase III clinical trial (AZA-001), MDS patients treated with azacitidine experienced a statistically significant increase in overall survival compared to patients receiving conventional care (24.5 vs. 15.0 months, p<0.0001) (Fenaux 2009). In addition, fewer azacitidine-treated patients required blood transfusions, and use of azacitidine delayed progression to acute myelogenous leukemia (AML). While the clinical benefits of azacitidine have been demonstrated, no study has evaluated its cost-effectiveness relative to decitabine. Methods:We developed a dynamic decision-analytic model (Markov process with 1-month cycles) to evaluate the cost-effectiveness of azacitidine versus decitabine for the treatment of MDS patients. Model parameters were derived from published literature, product labels, clinical trial data, and drug pricing and medical services cost databases. While patient populations in the clinical trials referenced for this model differed (i.e., approximately 1/3 of patients in the decitabine clinical trial were classified as lower-risk MDS patients, whereas approximately 90% of the azacitidine trial patients were higher-risk) (Kantarjian 2006, Fenaux 2009), data from the two trials were the highest quality available. The model tracks hypothetical cohorts of 1,000 MDS patients treated with azacitidine or decitabine over time periods of 1 and 3 years. Patients either remain in or transition among 4 health states in the model: (1) MDS with transfusion dependence; (2) MDS with transfusion independence; (3) progression to AML; or (4) dead. Clinical and economic consequences of adverse events were not included in the model. Clinical outputs generated by the model include: (1) number of patients progressing to AML; (2) number of patient months with transfusion independence; and (3) quality-adjusted life months/years (QALMs/QALYs). The primary economic output was treatment cost (including drug acquisition, administration and monitoring, blood transfusions, and iron chelation therapy). Cost-effectiveness of azacitidine versus decitabine was measured incrementally as: (1) cost per QALY gained; (2) cost per additional patient month of transfusion independence; (3) cost per additional case of AML progression avoided. The model used a US third-party payer perspective. Costs are stated as 2009 US dollars. One-way sensitivity analyses were performed on key model parameters to assess robustness of results. Results:The total number of QALYs (per 1,000 patients) for azacitidine-treated patients exceeded those for decitabine-treated patients in both the 1-year scenario (579 vs. 526) and the 3-year scenario (1,446 vs. 1,175). The total number of patient months with transfusion independence was higher for azacitidine vs. decitabine in both the 1-year scenario (4,340 vs. 3,521) and the 3-year scenario (11,836 vs. 8,553). A greater number of azacitidine-treated patients avoided progression to AML compared to decitabine-treated patients in both the 1-year scenario (664 vs. 462) and the 3-year scenario (372 vs. 231). Total per-patient costs for azacitidine-treated patients were lower than for decitabine-treated patients in both the 1-year scenario ($29,921 vs. $43,208), and the 3-year scenario ($101,052 vs. $106,806). In the 1-year scenario, costs for an azacitidine-treated patient were $13,287 less than those for a decitabine-treated patient, and azacitidine conferred a benefit of 0.0526 additional QALYs. In the 3-year scenario, costs for an azacitidine-treated patient were $5,754 less than those for a decitabine-treated patient, and azacitidine conferred a benefit of 0.2707 additional QALYs. Thus, incremental cost-effectiveness ratios (ICERs) for both the 1-year and 3-year scenarios demonstrate that azacitidine consistently had lower cost and provided greater health benefit compared to decitabine across all outcomes of interest. Conclusion:Model results suggest that despite a higher-risk profile in the underlying data for azacitidine, treatment of MDS patients with azacitidine leads to cost savings—it costs less and confers greater clinical benefit than decitabine. These results accentuate azacitidine as a major asset in providing cost-effective care for MDS patients. Disclosures:Miller: Celgene Corporation: Consultancy. Fenaux: Celgene, Ortho Biotech, Roche, Novartis, Cephalon, Epicept, Amgen, Merck: Honoraria, Research Funding. Beach: Celgene Corporation: Employment. Gidwani: Celgene Corporation: Consultancy. Khan: Celgene Corporation: Employment.

Aberrant CpG Island Methylation in AML Is Associated with Disease Progression and Relapse.

The role of DNA methylation in relapse and progression of acute myelogenous leukemia (AML) is incompletely understood. We studied DNA methylation of 9 promoter-associated CpG islands of genes frequently hypermethylated in leukemic cell lines. These were NOR1, NPM2, HIN1, SLC26A4, CDH13, PGRA, PGRB, OLIG2 and the tumor suppressor gene p15INK4b. We examined bone marrow and/or peripheral blood cells collected at the time of diagnosis and at the first relapse from 32 patients (13 females, 19 males) with AML. The median age was 58 years (20–68), the median survival was 18 months (8–80), the median blast count was 64% (20–98), and 10 patients had additional solid tumors and/or lymphatic/hematologic malignancies. Bisulfite treatment of DNA, followed by PCR and pyrosequencing were used to quantitatively measure levels of cytosine methylation in promoter-associated CpG islands. We analyzed methylation data for individual genes and for a methylation index derived after Z-score (z = [value –mean]/standard deviation) transformation to equalize absolute differences between individual genes and we used paired t-tests for statistical analysis. Abnormal hypermethylation (≥10%) in bone marrow and peripheral blood cells at diagnosis was detected in all 9 investigated genes, with a range of 7/29 (24%) for HIN1 to 24/32 (75%) for CDH13. On average, an increase in methylation between diagnosis and relapse was detected in all genes, and was significant for CDH13 (mean 10%, p=0.0006), SLC26A4 (mean 7%, p=0.0012), HIN1 (mean 8%, p=0.0037), NPM2 (mean 7%, p=0.0073), p15INK4b (mean 13%, p=0.0081), NOR1 (mean 4%, p=0.0124), PGRB (mean 6%, p=0.0144), PGRA (mean 9%, p=0.0275), and OLIG2 (mean 3%, p=0.0732). When analyzed by change in methylation status: negative (methylation below 10%) turning positive (methylation ≥ 10%) and vice versa, of 238 analyses, 39 (16%) showed a negative to positive switch, 15 (6%) showed a positive to negative switch, and the remaining 184 (77%) were either positive or negative unchanged. Finally, when analyzed in individual patients, an increase in methylation was noted in 29 of 32 patients (91%). The median increase in methylation index between diagnosis and relapse calculated as a delta-Z-score was 30% (range from −10% to 147%), and was highly significant (p&lt;0.0001). In summary, abnormal hypermethylation in bone marrow and/or peripheral blood cells from AML patients was detected in all investigated genes at diagnosis. Methylation levels further increased at relapse of the disease in 29 of 32 patients in 1 to 8 of 9 investigated genes. Based on quantitative analyses, we propose that methylation of CDH13, PGRB, PGRA and OLIG2 CpG islands are early markers for AML, while hypermethylation of HIN1, NPM2 and p15INK4b CpG islands is associated with disease progression and predominantly appears at relapse. Thus, aberrant hypermethylation is clearly associated with disease progression and relapse in AML, and likely mediates drug resistance in this setting. Increase of Methylation Index in Relapse Increase of Methylation Index in Relapse

Functional CXCR4-Expressing Microparticles and SDF-1 Correlate with Circulating Acute Myelogenous Leukemia Cells

Stromal cell-derived factor-1 (SDF-1/CXCL12) and its receptor CXCR4 are implicated in the pathogenesis and prognosis of acute myelogenous leukemia (AML). Cellular microparticles, submicron vesicles shed from the plasma membrane of various cells, are also associated with human pathology. In the present study, we investigated the putative relationships between the SDF-1/CXCR4 axis and microparticles in AML. We detected CXCR4-expressing microparticles (CXCR4(+) microparticles) in the peripheral blood and bone marrow plasma samples of normal donors and newly diagnosed adult AML patients. In samples from AML patients, levels of CXCR4(+) microparticles and total SDF-1 were elevated compared with normal individuals. The majority of CXCR4(+) microparticles in AML patients were CD45(+), whereas in normal individuals, they were mostly CD41(+). Importantly, we found a strong correlation between the levels of CXCR4(+) microparticle and WBC count in the peripheral blood and bone marrow plasma obtained from the AML patients. Of interest, levels of functional, noncleaved SDF-1 were reduced in these patients compared with normal individuals and also strongly correlated with the WBC count. Furthermore, our data indicate NH(2)-terminal truncation of the CXCR4 molecule in the microparticles of AML patients. However, such microparticles were capable of transferring the CXCR4 molecule to AML-derived HL-60 cells, enhancing their migration to SDF-1 in vitro and increasing their homing to the bone marrow of irradiated NOD/SCID/beta2m(null) mice. The CXCR4 antagonist AMD3100 reduced these effects. Our findings suggest that functional CXCR4(+) microparticles and SDF-1 are involved in the progression of AML. We propose that their levels are potentially valuable as an additional diagnostic AML variable.

Heterogeneous Patterns of CEBPα Mutation Status in the Progression of Myelodysplastic Syndrome and Chronic Myelomonocytic Leukemia to Acute Myelogenous Leukemia

We aimed to assess the role of CEBPalpha mutations in the progression of myelodysplastic syndrome (MDS) to acute myelogenous leukemia (AML) and their cooperating mutations. Mutational analysis of CEBPalpha with direct sequencing for each PCR product was done on matched bone marrow samples obtained from 50 adult patients with MDS at diagnosis and at AML transformation. Cloning analysis was used to determine the allelic distribution. CEBPalpha mutations were identified in four patients at diagnosis of MDS, including one with refractory anemia with excess blasts and three with chronic myelomonocytic leukemia. At AML transformation, three patients retained the identical mutant clones as their initial diagnosis, three acquired the mutations, and one lost CEBPalpha mutation when she gained FLT3/ITD mutation. Together, seven patients had CEBPalpha mutations throughout the disease course; four patients had NH(2)-terminal mutations resulting in a frameshift and truncation of the protein, three of them had two different mutations either on the same alleles or on different alleles, two had missense mutations, and one had a deletion in the basic region leucine zipper domain. Except for one with coexistence of N-ras mutation, no sample harbored cooperating mutations with FLT3 or N-ras genes. CEBPalpha mutations had no influence on the time to AML progression or overall survival. Our results show that CEBPalpha mutations play a role in a subset of patients with MDS, especially in chronic myelomonocytic leukemia. The mutation status was heterogeneous, exhibiting identical clone, clonal change, or clonal evolution during the progression to AML.

The Molecular Pathology of Acute Myeloid Leukemia

The pathogenesis of acute myelogenous leukemia (AML) involves an array of molecular alterations that disrupt almost every facet of cell transformation. These processes include the regulation of cell proliferation, differentiation, self-renewal, survival, cell cycle checkpoint control, DNA repair and chromatin stability, and cell dissemination. Normal regulatory networks are disrupted or usurped by these leukemogenic insults, and the understanding of these alterations is guiding the design of new therapeutic strategies. This overview describes some of the critical molecular alterations and implicates the rogue leukemogenic proteins in the onset and progression of AML.

Establishment of a cell line with AML1-MTG8, TP53, and TP73 abnormalities from acute myelogenous leukemia.

Gene alterations accumulate during the progression of acute myelogenous leukemia (AML) to a malignant clone. Here, a new myeloid cell line, designated YSK-21, with the balanced t(8;21)(q22;q22) and the unbalanced der(1)t(1;17)(p36;q21), was established. YSK-21 grows well in a medium containing recombinant human granulocyte colony-stimulating factor (rhG-CSF), granulocyte-macrophage colony-stimulating factor (rhGM-CSF), or interleukin-3 (rhIL-3). Molecular analysis using the reverse transcriptase-polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) revealed that t(8;21)(q22;q22) resulted in an AML1-MTG8 fusion transcript. FISH and spectral karyotyping (SKY) in conjunction with G-banding analysis revealed a der(1)t(1;17)(p36;q21) chromosomal translocation, which appeared in the clone developed from the original leukemic cells. Molecular analysis of the TP73 gene on 1p36 and the TP53 gene revealed a deletion of one-allele in TP73 with partial demethylation of another allele in the initial clone of YSK, and a point mutation consisting of an A-->T substitution in codon 288 of the TP53 gene in the developed clone of YSK-21. YSK-21 cells, expressing aberrant AML1-MTG8, TP53, and TP73 protein molecules, may be useful for elucidating the pathophysiology of these aberrant proteins and for studying the der(1)t(1;17)(p36;q21) chromosomal translocation.