ALBERT

Description: Acute myeloid leukemia cells have constitutive activation of phosphatidylinositol 3(PI3) kinase and require PI3 kinase activation for survival; however, the function of the PI3 kinase pathway in the survival of leukemic cells is poorly defined. We have studied the role of one PI3 kinase substrate, mTOR (mammalian target of rapamycin), in primary leukemic cells. In initial experiments, we have defined a novel growth medium that improves survival of acute myeloid leukemia (AML) blasts in long-term suspension culture and the survival of leukemic stem cells in short-term cultures. Inhibition of mTOR using rapamycin leads to a modest decrease in cell survival after 2 days of incubation with more significant decrease in survival after 7 days of culture. However, when rapamycin is added to etoposide in 2-day cultures, there is a dramatic increase in the cytotoxicity of etoposide against AML blasts. Furthermore, etoposide consistently decreased the engraftment of AML cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) animals, and this effect was enhanced by coincubation with rapamycin, demonstrating that mTOR regulates survival of AML stem cells after etoposide treatment. These results suggest that rapamycin in combination with etoposide-based chemotherapy may be efficacious in the treatment of AML.

Description: Abstract 979 Poster Board I-1 Smoking is the leading preventable cause of death. Smokers are approximately 1.5 times more likely to develop acute myeloid leukemia (AML) than non-smokers, but little is known about the relationship between cigarette smoking and AML outcome. We studied the effect of smoking on outcome in 282 newly diagnosed AML patients treated with high-dose cytarabine and idarubicin-containing regimens at Roswell Park Cancer Institute between 9/92 and 12/08. There were 161 males and 121 females (median age 56 years, range 18-85 years). The median follow up was 12.9 (range,

Description: Abstract 1763 Poster Board I-789 Introduction The myelodysplastic syndromes (MDS) are a group of aging-associated hematopoietic disorders characterized by ineffective maturation that, in 6-33% of cases, progress to acute leukemia (AML), a disease of blocked differentiation. Since microRNAs (miRNAs) regulate cell differentiation/maturation as well as cell identity, miRNAs may play critical roles in both the development of MDS as well as transformation to AML. With the advent of new therapies and treatment modalities for MDS and AML, the availability of biomarkers capable of detecting early MDS and predicting progression to AML would have tremendous impact on the management of these patients. Methods The study utilizes twenty samples of bone marrow mononuclear cells, ten from MDS patients and ten from normal controls, isolated and stored at the University of Pennsylvania Stem Cell Core Facility between 2003 and 2007. The MDS specimens included seven (7) from patients in whom it was known that no transformation to acute leukemia occurred within five (5) years of the available specimen and three (3) from patients in whom acute leukemia was diagnosed within two (2) years of the available specimen. The patient specimens were selected to represent early stage MDS and therefore not at high risk for transformation based on morphologic analysis (circulating blasts '5%) and the 2001 WHO classification system. Total RNA was obtained from each sample and arrayed on a custom Agilent miRNA microarray at the University of Pennsylvania Microarray Facility in a dual channel experiment in which each sample was arrayed against a pool of all twenty samples. Statistical analysis was performed using Genespring v 7.31 and Partek Genomics Suite v6.3. Subsequently, thirteen miRNAs of interest and one amplification control were examined by RT-PCR using ABI commercial primers for those targets. Total RNA extracted from formalin-fixed paraffin-embedded tissue was also examined for the relative expression of these fourteen miRNAs. Results Class discovery ANCOVA algorithms were applied to the data to identify 13 miRNAs which differentiate the MDS samples from the normal controls. Real time PCR was performed upon the sample RNAs to verify the microarray results. Of the thirteen candidate miRNAs, miR-150, miR-342, and miR-103, miRNAs documented in other studies to be important in hematopoiesis, demonstrate particular promise as classifiers for MDS based upon the high degree of correlation between the microarray results and the RT-PCR studies as well as their ability to independently differentiate MDS patients from normal controls. When the three miRNAs are taken together, the microarray and RT-PCR results correlate in 83% of cases, with only 2 major discrepancies. Furthermore, comparison between the total RNA isolated from the bone marrow mononuclear cells suspensions of two patients with total RNA isolated from the corresponding formalin-fixed paraffin-embedded clot sections, across fourteen different miRNAs demonstrate extremely high levels of correlation (R2 = 0.94 and 0.85), proving the feasibility of future studies utilizing the readily available paraffin-embedded clot sections as a source of patient samples. Microarray probes to predicted miRNAs were also identified which distinguished the three patients who progressed to acute myeloid leukemia within 24 months from the seven that did not. Conclusions A miRNA signature was identified which distinguishes early cases of MDS from normal controls. These miRNAs are predicted to regulate many of those mRNAs identified by traditional transcriptional profiling. Ultimately, it is hoped that assessment of miRNAs or their downstream targets will provide practicing pathologists with new markers for the identification of MDS and provide clinicians with additional prognostic information to guide the use of therapeutic interventions. Disclosures Carroll: Sanofi Aventis Corp: Research Funding; Cephalon Oncoloy: Consultancy.

Description: Abstract 4129 Several immunophenotypic subgroups within AML are associated with specific disease characteristics, e.g., CD19-positive AML is associated with t(8;21)(q22;q22) and CD56 expression is associated with adverse outcome. CD33 is a sialic acid-binding immunoglobulin-like lectin (Siglec) located on chromosome 19q13.3. It is highly expressed on early multilineage hematopoietic progenitors but absent from the pluripotent hematopoietic stem cells. It contains two tyrosine phosphorylation motifs whose phosphorylation depends on Src and is the target of gemtuzumab ozogamicin. We asked if lack of CD33 expression (

Description: Recent reports describe hematopoietic abnormalities in mice with targeted instability of the mitochondrial genome. However, these abnormalities have not been fully described. We demonstrate that mutant animals develop an age-dependent, macrocytic anemia with abnormal erythroid maturation and megaloblastic changes, as well as profound defects in lymphopoiesis. Mice die of severe fatal anemia at 15 months of age. Bone-marrow transplantation studies demonstrate that these abnormalities are intrinsic to the hematopoietic compartment and dependent upon the age of donor hematopoietic stem cells. These abnormalities are phenotypically similar to those found in patients with refractory anemia, suggesting that, in some cases, the myelodysplastic syndromes are caused by abnormalities of mitochondrial function.

Description: Abstract 3113 Poster Board III-50 Blastic transformation of myeloproliferative neoplasms (MPN) is still poorly understood. Mutations of JAK2V617F were described in the majority of MPN patients but only about half of those undergoing blastic transformation continue to harbor that mutation. We describe a cohort of 23 patients from Roswell Park Cancer Institute (RPCI) and discuss 90 additional cases from the English literature for whom biologic features were described. We also screened our cases for JAK2V617F, JAK2T875N and MPL515L/K. We initially compared our 23 patients to the 90 cases from the literature. Our population had significantly less patients with prior history of polycythemia vera (22% vs. 53%; P

Description: Studies on the etiology of MDS have been hampered by the lack of relevant animal model systems. Numerous murine models of MDS/MPD overlap syndromes have been described, but MDS/MPD overlap syndromes are now felt to be clinically distinct from MDS. Likewise, a number of murine models of myeloid leukemia have been developed in which dysplastic features arise during conversion to frank leukemia. Here we describe the hematopoietic phenotype of the PolgD257A mitochondrial mutator mouse and demonstrate that this model system fulfills the myeloid dysplasia criteria outlined in the Bethesda Proposals (Blood, 100, 238, 2002). The PolgD257A allele eliminates the proofreading activity of Polg, the sole mitochondrial DNA (mtDNA) polymerase, and PolgA/A mice rapidly accumulate mtDNA mutations. Starting at 9 months of age, PolgA/A mice display multiple features of accelerated aging and develop a progressive and ultimately lethal anemia (Science, 309, 481, 2005). In data obtained at time of necropsy for other studies, the hemoglobin of young PolgA/A mice is identical to that of controls but by 10 months of age begins to fall below 10 g/dL. Overall, while Polg+/+ mice maintain a hemoglobin of greater than 10 g/dL through 30 months of age, the hemoglobin of PolgA/A drops below 5 g/dL as the mice succumb to their anemia at 13–16 months of age. As the hemoglobin of aging PolgA/A mice falls the MCV demonstrates a striking increase, from 53.6 fL to 69.8 fL. In order to confirm these intial findings, complete blood counts have been assessed prospectively in mice of all three genotypes (Polg+/+, Polg+/A, and PolgA/A; 5 mice per genotype) every other month up to 8 months of age and monthly thereafter. Full data up to 11 months of age, as well as 12 month data from all 5 PolgA/A mice, will be presented. As expected, up to eight months of age no significant difference in hemoglobin levels are apparent, but by eight months PolgA/A mice demonstrate a significant increase in MCV (51.5 fL) compared to either Polg+/A (44.9, p=0.0015) or Polg+/+ (44.5, p=0.0010) littermate controls. The disparity in MCV between PolgA/A mice and the other genotypes is progressive, with the MCV at 9 months increasing to 53.4 fL in PolgA/A mice (N=5), compared to 44.8 (p

Description: Abstract 4128 Aberrant expression of the B lymphoid marker, CD19, in acute myeloid leukemia (AML) has frequently been associated with t(8;21)(q22;q22). However, AML cases lacking this translocation may occasionally express CD19. We identified eight such cases at Roswell Park Cancer Institute. Three of these cases had noticeable CD19-positive leukemic subsets with the following karyotypic abnormalities. The first case had monosomy X in 6 cells and normal karyotype in 14, the second case had trisomy 22 in 19 cells and normal karyotype in one, and the third case had monosomy 7 in 18 cells and trisomy 18 in two. We therefore asked if CD19 expression is restricted to the karyotypically abnormal leukemic cells. Bone marrow cells were sorted according to the specific immunophenotype. First, a generous mononuclear sorting region was drawn in the bivariate display of forward versus side scatter to exclude debris and aggregates. Next, the CD45-negative to CD45-dim, CD19-positive and CD34-positive leukemic cells were sorted. Fluorescent in situ hybridization (FISH) studies were carried out on the sorted samples using the following commercially available probes: the Centromere enumeration probe CEP X (SpectrumGreen) in the first case; the locus specific BCR (22q11) (SpectrumGreen)/ABL1 (9q34) (SpectrumOrange) dual color, dual fusion, translocation probe in the second, and the locus specific D7S486 (7q31) SpectrumOrange/CEP 7 SpectrumGreen probe in the third. A total of 200 nuclei were assessed in each case. The results are displayed in the Table below. There were no significant differences in the signal pattern between the CD19-positive and CD19-negative leukemic populations. These results indicate that aberrant CD19 expression in t(8;21)-negative AML is not restricted to leukemic cells that harbor specific karyotypic abnormalities. Table FISH results based on CD19 expression Case # Probe Signal Pattern CD19-positive CD19-negative 1 CEP X 1 Green (-X) 20/200 30/200 2 BCR/ABL 3 Green/2 Orange (+22) 163/200 170/200 3 CEP 7 1 Green/1 Orange (-7) 200/200 200/200 Disclosures: No relevant conflicts of interest to declare.

Description: The myelodysplastic syndromes (MDS) are a diverse group of primary bone marrow disorders characterized by persistent cytopenias and a variable rate of progression to acute myelogenous leukemia (AML). A consistent finding in the bone marrow of patients with MDS is increased apoptosis and a defect in terminal differentiation of maturing cells. We hypothesized that MDS cells are unable to tolerate the increasing oxygen tension experienced during hematopoiesis as cells move out of the hypoxic stem cell niche and this intolerance leads to increased apoptosis. In order to test this hypothesis, we have studied the growth in methylcellulose colonies of bone marrow progenitors in eight samples from patients with MDS at normoxia (21% O2) and at hypoxia (3% O2). In 5/8 samples there is a greater than 8-fold (range 8.5–15.9) increase in colony number under hypoxic conditions. The remaining three MDS samples, while only demonstrating an average increase in colony number of 2.4-fold, demonstrated significantly larger colonies of various lineages. Comparatively, in six normal patient samples the augmentation averaged 1.5-fold and never exceeded 2-fold, with a statistically significant (p=0.01) difference in the mean fold-change between normal and MDS marrows. These results suggest that MDS cells have an impaired ability to tolerate increasing levels of oxygen and also suggests a methodology for expansion of MDS cells for further biochemical analysis of this phenotype. The cellular response to hypoxia is modulated in part through the mitochondrion and significant genetic data suggests that a mitochondrial abnormality is associated with MDS. In order to examine mitochondrial potential in MDS progenitor cells, we have adapted a multiparameter flow cytometric strategy for the simultaneous analysis of mitochondrial potential (ψm) in defined hematopoietic progenitors using the potentiometric dye tetramethyl rhodamine ethyl ester (TMRE). These studies confirm that in normal bone marrow samples, ψm increases with development from the hematopoietic stem cell (HSC) to differentiated progenitor cells. Analysis of marrow from a patient with early MDS (refractory cytopenia with multilineage dysplasia, IPSS Low) and a 10-fold increase in colony number under hypoxic conditions demonstrates the absence of this progressive increase in ψm through myeloid development when compared to normal controls. Additionally, two samples from patients with progression of their MDS to frank AML demonstrated a markedly elevated ψm in the blast cells, consistent with the elimination of the mitochondrial pathology during the genesis of the leukemic clone. Analysis of mitochondrial potential in additional MDS samples is ongoing and will be presented. These data suggest that a defect in the mitochondrial adaptation to increasing oxygen tension experienced during hematopoietic differentiation may explain the impaired growth of MDS progenitor cells.