Glutaminase Inhibition Selectively Slows the Growth of Primary Acute Myeloid Leukemia (AML) Cells with Isocitrate Dehydrogenase (IDH) Mutations.

Abstract Abstract 2624 Introduction: The incidence of mutations in IDH1 and IDH2 (mIDH) in de novo AML is 10–15%. These mutations are enriched in normal karyotype AML, and their presence carries an unfavorable prognostic factor, according to some studies. Furthermore, mutations in IDH1/2 genes have been identified in approximately 5% of myelodysplastic syndromes and 10% of myeloproliferative neoplasms. Although wild-type IDH in cytosol and mitochondria catalyze the conversion of isocitrate to α-ketoglutarate (α-KG) with the production of NADPH, altered amino acids in mIDH1 and mIDH2 reside in the catalytic pocket and result in a neoenzymatic activity, converting α-KG to 2-hydroxyglutarate with the consumption of NADPH. The primary source for α-KG for these cells is glutamine, which is first converted to glutamate by glutaminase and subsequently to α-KG. Because glutamine is the primary source for α-KG, we hypothesized that cells with mIDH are in essence addicted to glutamine via glutaminase activity, such that depletion of glutamine or interruption of its metabolism would be deleterious to cellular metabolism and survival. The aim of this study was to investigate whether inhibition of glutaminase by a small molecule, BPTES (bis-2-[5-(phenylacetamido)-1,3,4-thiadiazol-2-yl]ethyl sulfide), could selectively kill primary AML cells with mIDH1, but not IDH-wild type AML cells. We and others have previously demonstrated that BPTES inhibits glutaminase effectively. Method: Two independent sets of experiments were performed by two separate research groups. One group was blinded to mutant versus wild type IDH status. The other group was blinded to drug identity including solvent versus BPTES and to various BPTES concentrations. Primary AML cells from patients were cultured in RPMI-1640 medium with 20% fetal bovine serum, 20% 5637 cell-conditioned medium and 1% antibiotics with no drug, DMSO control (0.1% concentration) and 20 or 40 microM BPTES. Cells were counted manually on days 2, 4 and 6. Growth curves were generated for viable cells as assessed by trypan blue exclusion. Experiments were performed in triplicates. Results: Growth curves of primary AML cells (with mutation status indicated) with no drug and with DMSO or BPTES (20 or 40 microM) are shown in Figure 1. Cells #2, #3, #5 and #10 carried IDH1 mutations. Cells #4 and #9 were wild type. On day 4, there was approximately a two-fold decrease in the growth of all IDH-mutant AML cells exposed to 20 microM BPTES compared to DMSO. No significant difference in activity was observed between 20 and 40 microM of BPTES. There was no difference in cell growth between exposure to no drug and to DMSO. The growth of wild type AML cells was not significantly affected by the glutaminase inhibitor. Results were consistent between the two research groups. Conclusions: Although IDH mutations are frequently found in AML, a therapeutic strategy targeted at these mutations has not been reported. To the best of our knowledge, this is the first report of a targeted approach to the treatment of IDH-mutant AML. We found that inhibition of glutaminase by a small molecule, BPTES, preferentially slows the growth of primary AML cells with mutant IDH1 versus those AML cells with wild type IDH. Further investigation in xenograft models and pharmacologic studies are ongoing. Disclosures: No relevant conflicts of interest to declare.

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Disruption of the ASXL1 Gene Is Prevalent In PMF: Analysis of Molecular Genetics and Clinical Phenotypes

Blood ◽

10.1182/blood.v116.21.3074.3074 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3074-3074

Author(s):

Brady L Stein ◽

Donna M Williams ◽

Michael A McDevitt ◽

Christine L. O'Keefe ◽

Ophelia Rogers ◽

...

Keyword(s):

Molecular Genetics ◽

De Novo ◽

Conflicts Of Interest ◽

Single Nucleotide Polymorphism Array ◽

Myeloproliferative Neoplasms ◽

Direct Sequencing ◽

Snp Array ◽

Uniparental Disomy ◽

Jak2 V617f ◽

Wild Type

Abstract Abstract 3074 Background: The myeloproliferative neoplasms, PV, ET and PMF, share phenotypic features and molecular lesions, yet PMF distinguishes itself by its unfavorable natural history and rate of leukemic evolution. These distinctions may occur as a result of cooperating genomic lesions specific to PMF compared to PV or ET. We performed single nucleotide polymorphism array (SNP-A)-based karyotyping in 210 MPN patients and identified 20q11 deletions in 10% of PMF cases and in none of the PV or ET cases. The 20q11 deletion region spanned 1,662 KB and encompassed 37 genes, of which ASXL1 was included. To test whether ASXL1 contained lesions in the MPN cohort at large, we directly sequenced key regions of the ASXL1 gene in 65 PMF, 11 PV and 14 ET cases, as well as 7 controls from the SNP-array cohort. Genomic DNA from neutrophils and in select cases, purified CD34+ cells was used for both SNP-A and direct sequencing. Clinical parameters were correlated with genomic findings and the quantitative JAK2 V617F neutrophil allele burden Molecular genetics: 26/65 (40%) of PMF cases had abnormalities in ASXL1 (4 deletions, 22 mutations) whereas none of the 32 PV, ET or control cases had such lesions. The majority of ASXL1 sequence variations were nonsense lesions including the previously reported 1934dupG which comprised 30% of all of the mutations. The residual ASXL1 allele in all 20q11 deletion cases containing the ASXL1 gene was intact. In three PMF cases, more than one distinct ASXL1 mutation was identified, and cloning experiments on two of those cases indicated that the lesions were biallelic. Using banked samples, we observed the acquisition of an ASXL1 lesion over time, and established that ASXL1 lesions detected in 2 post ET-MF cases were also detected at low levels in the ET phase of the MPN. Genotype/Phenotype Correlations: ASXL1 deletions and mutations were prevalent in de novo PMF (37%), post PV-PMF (20%) post ET-PMF (62%) and in PMF/AML (33%). ASXL1 mutations did not associate with chemotherapy exposure as the prevalence of hydroxyurea use was similar in patients with and without mutations, and ASXL1 –mutation positive cases were present in patients who had never received any form of chemotherapy. There was no dependence upon JAK2 status as the presence of ASXL1 mutations were identified in JAK2 V617F-negative cases (9/26); JAK2 V617F-heterozygous cases (10/26); and JAK2 V617F-homozygous cases (7/26). Based on results of SNP-A, patients with ASXL1 mutations were equally as likely to have uniparental disomy (involving 9p or other regions) and loss/gain abnormalities (>1MB) compared to those without ASXL1 mutations. There were no differences in sex, age, or disease duration between PMF patients with and without ASXL1 mutations. In the ASXL1-mutant group, there was a trend toward a lower median white blood cell count (8 vs. 12.5 k/cu mm; p=0.3) and hemoglobin (9.7 vs. 11 g/dl; p=0.3) compared to ASXL1-wild-type patients. Furthermore, those PMF patients with ASXL1 mutations were significantly more likely to have received anemia-directed therapy (transfusion, erythropoietin, immunomodulating agents, steroids) compared to those without mutations (15/26 (58%) vs. 11/39 (23%); p=0.02). Post ET-MF patients comprised 31% (8/26) of ASXL1-mutant cases, compared to only 10% (4/39) ASXL1- wild-type cases (p=0.03). However, the presence of an ASXL1 mutation did not associate with an accelerated transition rate from ET to MF; among the 12 post ET-MF cases in the cohort, the median time of transition from ET to MF was 15.5 years in those with ASXL1 mutations compared to 7 years in those with ASXL1 wild-type status (p=0.02). Conclusion: Disruption of the ASXL1 gene occurs in 40% of PMF cases. The association of ASXL1 lesions, due to either mutation or deletion, suggests that ASXL1 haplo-insufficiency is associated with a PMF phenotype in the context of other known and unknown lesions, and that disruption of ASXL1 function may directly contribute to the pathophysiology and clinical complications of primary and secondary myelofibrosis. These data support the concepts that cooperative lesions in addition to JAK2 V617F are critical in generating PMF, that PMF is molecularly more complex than either PV or ET, and that the transition of PV or ET to PMF is associated with the acquisition of genomic lesions, such as ASXL1, that are present in PMF at large. Disclosures: No relevant conflicts of interest to declare.

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Aurora A Kinase Is Required for Hematopoiesis

Blood ◽

10.1182/blood.v120.21.1201.1201 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1201-1201

Author(s):

Benjamin Goldenson ◽

Jeremy Q Wen ◽

John Crispino

Keyword(s):

Bone Marrow ◽

Steady State ◽

Small Molecule ◽

Dna Content ◽

Colony Formation ◽

Conflicts Of Interest ◽

Aurora A Kinase ◽

Aurora A ◽

Wild Type ◽

Significant Difference

Abstract Abstract 1201 In acute megakaryocytic leukemia (AMKL), there is a failure of megakaryocytes to differentiate, become polyploid and stop dividing. We used an integrated screening approach that included chemical, proteomic and genetic screens to identify small molecules and their targets that control polyploidization and differentiation of normal and malignant megakaryocytes. We identified several small molecule inducers of polyploidy and used siRNA and proteomic target ID approaches to determine the cellular targets of the lead small molecule dimethylfasudil (diMF). Aurora Kinase A (AURKA) was identified as one of the top targets of diMF. AURKA is an attractive target in AMKL for several reasons. First, AURKA is overexpressed in AMKL cells. Second, at the biologically effective doses used in our cell-based assays, AURKA inhibition was selective for the megakaryocyte lineage. Third, AURKA inhibition by diMF or the selective AURKA inhibitor MLN8237 increased MK polyploidy, induced features of differentiation, blocked proliferation of AMKL blasts, and improved survival in an AMKL mouse model. AURKA is important in mitotic spindle assembly, mitosis, chromosomal alignment and segregation. Moreover, it is required for embryonic development, as Aurka−/− embryos fail to grow beyond the blastocyst stage. However, the extent to which AURKA is necessary for steady state hematopoiesis in adults is unknown. To investigate the necessity of AURKA in hematopoiesis, we utilized a conditionally targeted strain of mice (Aurkaflox/flox). To delete AURKA in megakaryocytes ex vivo, Aurkaflox/flox bone marrow cells were expanded, transduced with a retrovirus expressing Cre and GFP, and then cultured in the presence of THPO for 72 hours. We found that deletion of AURKA resulted in increased CD41 and CD42 expression as well as increased DNA content. Assays for apoptosis by Annexin V staining of Aurkaflox/flox cells infected with Cre also showed increased apoptosis in AURKA-deleted cells at 24 and 48 hours. To delete AURKA in vivo, we crossed Aurkaflox/flox mice to MX1-Cre mice and injected wild-type, heterozygous and homozygous floxed mice expressing MX1-Cre with pIpC every other day for six days. We found that deletion of AURKA in hematopoietic progenitors leads to pancytopenia, profound bone marrow defects and death within two weeks. Colony formation assays showed significantly decreased myeloid, erythroid and megakaryocyte colony formation with AURKA deficiency. Bone marrow histology displayed markedly hypocellular marrow, but curiously, flow cytometry revealed a significant increase in the percentage of CD41 and CD42 positive cells. This observation suggests that AURKA normally acts to restrain terminal differentiation of megakaryocytes and is consistent with the CD41 and CD42 inducing ability of AURKA inhibitors. To confirm that AURKA is the key target of our recently identified polyploidy inducers, we assayed the effects of diMF and MLN8237 on Aurka+/+, Aurka+/− and Aurka−/− megakaryocytes. 300 nM diMF and 100 nM MLN8237, concentrations that strongly induce polyploidy, did not increase MK polyploidization in Aurka−/− MKs. diMF and MLN8237 treatment increased polyploidy in Aurka+/− MKs with no significant difference in comparison to Aurka+/+ MKs. We also assayed the ability of wild-type or the T217D mutant of AURKA, which is resistant to inhibition by MLN8237, to reduce the induction of polyploidy caused by diMF and MLN8237 upon overexpression. CMK cells were infected with viruses harboring wild-type or T217D AURKA, treated with DMSO, 3 μM diMF or 30 nM MLN8237 for 72 hours, and then evaluated for DNA content. The increase in polyploidization induced by both compounds was significantly decreased in cells overexpressing the T217D mutant of AURKA. With overexpression of the wild-type AURKA, there was a trend towards reduction in polyploidy, but more variable effects and no significant difference. Thus, AURKA T217D overexpression reduced the ability of diMF and MLN8237 to induce polyploidization, consistent with our conclusion that diMF targets AURKA. Together, our data support a role of AURKA in megakaryocyte polyploidization and differentiation and show that AURKA is required for steady state hematopoiesis. The results also show that AURKA is the key target of diMF in the induction of polyploidization of megakaryocytes and support the development of Aurora A kinase inhibitors in clinical trials for AMKL. Disclosures: No relevant conflicts of interest to declare.

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Prognostic Significance Of ASXL1 and IDH Mutations inPhiladelphia Negative Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v122.21.5260.5260 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5260-5260

Author(s):

Ipek Yonal ◽

Aynur Daglar Aday ◽

Aysegul Basak Akadam ◽

Ceylan Yilmaz ◽

Meliha Nalcaci ◽

...

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Prognostic Significance ◽

Jak2v617f Mutation ◽

Bleeding Complications ◽

Study Group ◽

Wild Type ◽

Leukemic Transformation ◽

Sequence Variations ◽

Idh Mutations

Abstract Introduction ASXL1 gene mutations and IDH1/IDH2 mutations were described in Ph-negative MPN in 2009 and 2010, respectively. We aimed to study frequency of aferomentioned mutations and their clinical importance, prevelance and potential prognostic significance in Ph-negative MPN. Methods Study group consisted of 107 ET (58 females, 49 males) and 77 PMF (43 females, 34 males) patients diagnosed according to WHO 2008 criteria. Exon 12 of ASXL1 was amplified from genomic DNA and bidirectionally sequenced. High-resolution melting was performed for IDH1 mutations (R130) and IDH2 mutations (R140 and 172) followed by confirmatory bidirectional sequence analysis. To our knowledge, this is the largest cohort of ET studying ASXL1 mutations. Results Frequency of ASXL1 mutations (exon 12) was significantly higher in PMF (24.7%) compared to ET patients (8.4%; p=0.005). Our analysis identified 29 different mutations of ASXL1 in 28 out of a total 184 patients, (15.2%) (one PMF patient presented with two simultaneous mutations). Nonsense mutations, including frameshift mutations were detected in 19 patients (10.3%). Mutant IDH was detected in 2 ET (1 IDH1 R140Q, 1 IDH1 R132C) and 5 PMF patients (3 IDH1 R123C, 1 IDH1 R132S, 1 IDH2 R140Q). The ET patient with IDH2 mutation (R140Q) harbored JAK2V617F mutation with an allele burden of 5% while the other patient with IDH1 mutation (R132C) did not harbor JAK2V617F mutation. 3 of 5 IDH mutated PMF patients (1 IDH1 R132C, 1 IDH1 R132S and 1 R140Q) concurrently carried JAK2V617F mutation with an allel burden of 31-50%, 5-12.5% and 31-50%, respectively. Frequency of IDH mutation for ET and PMF patients was 1.9% and 6.5%, respectively (p=0.131). We observed shortened OS in ASXL1-mutated PMF patients as compared to ASXL1 wild-type PMF patients(mean 108 months; 95% CI: 62-153 and 202 months; 95% CI: 123-282, respectively; p=0.025). (Figure 1). Also, trend towards shorter OS was observed in presence of ASXL1 nonsense sequence variations with respect to other sequence variations (p=0.09). Multivariate survival analysis confirmed independent prognostic value of ASXL1 mutations (OR: 2.75; 95% CI:1.37-5.5; p=0.033) but not ASXL1 nonsense sequence variations (p=0.131). Although number of IDH mutated PMF patients was small, we observed significantly shorter LFS in IDH mutated patients as compared to IDH wild-type patients (p=0.024). Conclusion In our study group, 7% of JAK2V617F-negative ET and 26.3% of JAK2V617F-negative PMF patients showed ASXL1 mutations. Based on our findings, it may be deduced that analysis of ASXL1 genes provided an additional 7% increase in ET and a 26.3% increase in PMF patients with a detected molecular marker of clonality. In our relatively large cohort of PMF patients, ASXL1 and IDH mutations were associated with shortened OS and LFS, respectively. One of 5 IDH mutated PMF patients harboring IDH mutations showed leukemic transformation. Thus, it can be suggested that IDH mutations may play a role in prediction of leukemic transformation. ASXL1 mutated PMF patients did not show an increase in poor karyotype features. In PMF, death and bleeding complications were associated with combined ASXL1 and IDH mutations. Additional studies are needed to clearly assess impact of mutation combinations on disease course and severity. An important characteristic feature of IDH mutated PMF in this study was the paucity of abnormal or unfavorable karyotype. All IDH mutated PMF patients had normal karyotype. Thus, it seems that ASXL1 mutations and IDH mutations represent independent prognostic biomarkers in PMF. Our findings support previous observations that mutations affecting epigenetic regulation might be prognostically more relevant than those involving JAK-STAT signaling in PMF. Disclosures: No relevant conflicts of interest to declare.

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Negative Effects of GM-CSF Signaling In t(8;21)-Induced Leukemia

Blood ◽

10.1182/blood.v116.21.3163.3163 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3163-3163

Author(s):

Shinobu Matsuura ◽

Ming Yan ◽

Eun-Young Ahn ◽

Miao-Chia Lo ◽

David Dangoor ◽

...

Keyword(s):

Myeloid Leukemia ◽

Sex Chromosome ◽

De Novo ◽

Conflicts Of Interest ◽

Hematopoietic Cells ◽

Receptor Activation ◽

Pseudoautosomal Region ◽

Wild Type ◽

Gm Csf

Abstract Abstract 3163 The t(8;21)(q22;q22) translocation is one of the most common chromosomal translocations in de novo acute myeloid leukemia (AML). The 8;21 translocation is often associated with additional cytogenetic abnormalities. The loss of the sex chromosome (LOS) is by far the most frequent abnormality found in association with the t(8;21) leukemia, accounting for 32–59% of patients, in contrast to other types of AML in which the LOS occurs in less than 5% of patients. To evaluate the role of sex chromosome deletion in t(8;21)-related leukemogenesis, hematopoietic cells from a mouse line with only one sex chromosome were used in retrovirus-mediated t(8;21) (AML1-ETO) expression and transplantation assays. The absence of leukemia in those animals suggested that a gene present in the pseudoautosomal region of sex chromosomes in humans but not in mice may be the target gene in LOS. The granulocyte-macrophage colony-stimulating factor receptor α (GM-CSFRα) gene is one such gene and is also known to be involved in myeloid cell survival, proliferation and differentiation. The GM-CSFRα gene is specifically down-regulated in AML patients with t(8;21), but not in other common translocations (Valk PJM et al, NEJM, 2004). The GM-CSFR complex is composed of α and βc subunits that assemble into a complex for receptor activation and signaling. To investigate the role of GM-CSFR signaling in t(8;21)-mediated leukemogenesis, GM-CSFR common β subunit knockout (GM-CSFRβc-/-) mice were used in our studies as a model for deficient GM-CSFR signaling. Transduction of AML1-ETO in hematopoietic cells from GM-CSFRβc-/- resulted in myeloid leukemia of a median survival time of 225 days, high percentage of blasts in peripheral blood and bone marrow, anemia, thrombocytopenia, hepatomegaly and splenomegaly. Comparison of wild-type and GM-CSFRβc-/- cells in the same transplantation resulted in development of AML1-ETO-induced leukemia at higher penetrance in GM-CSFRβc-/- cells (28.5% vs 100%). Moreover, the latency of leukemia was shorter in GM-CSFRβc-/- cells than in wild-type cells after transduction of AML1-ETO9a. Analysis of the hematopoietic compartment of healthy GM-CSFRβc-/- mice detected no significant abnormalities in the immature hematopoietic compartment (LSK, CMP, GMP, MEP), suggesting that AML1-ETO expression is required for leukemia to occur. In vitro, expression of AML1-ETO alone is sufficient for the immortalization of normal hematopoietic cells, as demonstrated by serial replating capacity of cells in methylcellulose colony assay. Addition of mGM-CSF to the basic cytokine cocktail (mIL-3, hIL-6, mSCF, hEPO) did not significantly affect number, type, size, and cell composition of colony cells. In contrast, the addition of mGM-CSF eliminated the replating capacity of AML1-ETO expressing cells, although they survived longer than control vector-infected cells. The results suggest that activation of GM-CSF signaling can specifically abrogate the self-renewal ability of potential leukemic stem cells in the early immortalization phase. These results support a possible tumor suppressor role of GM-CSF in leukemogeneis by AML1-ETO and may provide clues to understand how AML1-ETO corrupts normal GM-CSF signals to its own advantage for leukemogenic transformation. Disclosures: No relevant conflicts of interest to declare.

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Analysis of ASXL1, IDH1, IDH2, c-CBL, and WT1 Mutations in De Novo Acute Myeloid Leukaemia

Blood ◽

10.1182/blood.v118.21.1450.1450 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1450-1450

Author(s):

Mariam Ibañez ◽

Esperanza Such ◽

Jose Cervera ◽

Irene Luna ◽

Sandra Dolz ◽

...

Keyword(s):

De Novo ◽

Prognostic Impact ◽

Significant Implication ◽

Wild Type ◽

Idh Mutations ◽

Flt3 Mutations ◽

Exon 4 ◽

De Novo Aml ◽

Cebpa Mutations ◽

Acute Myeloid

Abstract Abstract 1450 The clinical relevance and prognostic implications of some recently identified mutations in acute myeloid leukemia (AML) is not yet well established. Among them, we have selected to be analyzed those affecting the following genes: Additional Sex Combs-Like 1 (ASXL1), Isocitrate Dehydrogenase (IDH1 and IDH2), Casitas B-lineage Lymphoma (c-CBL), and Wilms Tumor 1 (WT1). They have been previously reported with a variable incidence: ASXL1 mutations in 10.8% patients with normal karyotype (NK), IDH1 and IDH2 mutations in 8 – 33% of de novo AML, c-CBL mutations in 2% of de novo AML, and WT1 mutations in 5–12% of de novo AML patients. In order to know the incidence and prognostic impact of these mutations and their possible cooperative role in leukemogenesis, we have screened for ASXL1, IDH1, IDH2, c-CBL, WT1, FLT3, NPM1 and CEBPa, mutations in a cohort of de novo AML patients from a single centre. We studied 174 de novo AML patients [98M/76F; median age: 62 yr. (range: 16 – 88); favourable (n= 13), intermediate (n= 86) and high (n= 51) cytogenetic risk classification by the MRC group]. DNA was isolated from bone marrow samples obtained at diagnosis. In order to determine cooperating mutations, we developed a new combination of high-resolution melting (HRM) assays on a LightCycler® 480 and lastly direct sequencing, to detect somatic mutations for ASXL1 (exon 12), IDH1 (exon 4), IDH2 (exon 4), WT1 (exons 7, 8 and 9) and c-CBL (exons 8 and 9). All mutations reported in this study were confirmed al least twice. FLT3 (ITD and D835Y), NPM1 (exon 12) and CEBPa were performed as described previously by standard methods. Sequence analysis was checked by its corresponding GeneBank Accession Number. The number of patients found to carry mutations in our series was: 16 patients with ASXL1 mutations (9.2%), 16 patients with IDH mutations (2.9% had a IDH1R132, 12.6% the SNP rs11554137 and 6.3% IDH2R140), 5 patients with WT1 mutations (2.9%), 37 patients with FLT3 mutations (21.3%), 44 patients with NPM1 mutations (25,3%) and 8 patients with CEBPa mutations (4.6%). No mutations where found in c-CBL. We could not found a pattern of cooperating mutations in the studied group of genes. WT1, FLT3 and NPM1 were associated with leukocyte count >30 × 109/L at diagnosis (80% vs. 31% for WT1, P =0,022; 68% vs. 22% for FLT3, P= 0.001; and 50% vs. 24% for NPM1, P= 0.002; in mutated vs. wild-type patients, respectively). WT1 was also associated with a platelet count > 50 × 109/L at diagnosis (100% vs. 57% in mutated vs. wild-type patients, respectively; P =0,048). Besides, FLT3 and NPM1 mutations were more frequent in the intermediate cytogenetic risk group (82% and 74%; P =0.004 and P =0.047; respectively). ASXL1 and IDH mutations were not correlated with any of the clinical and biological features studied. In univariate analysis, only age and cytogenetics had an impact on overall survival (OS, median of 12mo vs. 3mo, for patients < and ≥65 yr., P <0.001 and 24mo, 11mo and 3mo for favourable, intermediate and high risk, P =0.005). Mutational status of ASXL1, IDH1, IDH2, WT1, FLT3, NPM1 and CEBPa did not impact on outcome in the whole series. However, when the analysis was restricted to patients with intermediate cytogenetic risk, patients with FLT3 mutations had a shorter OS (19mo vs. 8mo, wild-type vs. mutated patients; P =0.047) and those with WT1 mutations showed a trend towards an inferior OS (11mo vs. 1mo, wild-type vs. mutated patients; P = 0.066). In multivariate analysis in patients with intermediate cytogenetic risk, the age [HR (95% CI) = 3.3 (1.9 − 5.9) P <0.001], and FLT3 status [HR (95% CI) = 2.2 (1.2–3.9) P =0.008] retained an independent adverse significance for OS. In terms of relapse free survival any of the variables showed a significant implication. To sum up, the incidence found for the studied genes was lower than the previously reported: ASXL1, 9.2%; IDH1R132, 2.9%; IDH2R140, 6.3%; WT1, 2.9%; and c-CBL, 0%. We were unable to find a pattern of cooperating mutations in the studied group of genes or any impact of these mutations on the outcome. Disclosures: No relevant conflicts of interest to declare.

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A Meta-Analysis of CAG (cytarabine, aclarubicin, G-CSF) Regimen for the Treatment of 1045 Patients with Leukemia in China and Japan,

Blood ◽

10.1182/blood.v118.21.3638.3638 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3638-3638

Author(s):

Guoqing Wei ◽

Wanmao Ni ◽

Dicky Chiao ◽

He Huang ◽

Zhen Cai ◽

...

Keyword(s):

Acute Leukemia ◽

Fixed Effects ◽

De Novo ◽

Conflicts Of Interest ◽

Meta Analysis ◽

Early Death ◽

Newly Diagnosed ◽

Significant Difference ◽

China And Japan ◽

Better Than

Abstract Abstract 3638 Background: CAG regimen (cytarabine, aclarubicin, G-CSF) has been commonly used in China and Japan for the treatment of AML and MDS. This study is to summarize the data and to analyze the efficacy as well as the toxic effects of CAG regimen in acute leukemia (AL) and MDS pts. Methods: The databases of PubMed, Wanfang Data, as well as American Society of Hematology (ASH) annual meeting abstracts were searched for articles published in English, Chinese and Japanese languages between January 1995 and December 2010. Eligible studies were relevant clinical trials on AL and MDS pts treated with CAG regimen. Complete remission (CR) rates and odds ratio (OR) were compared through a meta-analysis using a random-effects or fixed-effects model. Results: 37 trials with a total of 1045 AL and MDS pts were included for analysis. Among the 1045 pts treated with CAG, 819 pts were AML, 215 pts were de novo MDS or transformed AML (MDS/tAML), 6 pts were ALL, and 5 pts were biphenotypic acute leukemia (BAL). The AML CR rate of CAG from 29 studies was 58.0% (95% CI, 53.1%-62.7%). The MDS/t-AML CR rate from 12 studies was 45.7% (95% CI, 39.0%-52.4%). The AML CR rate was significantly better than that of MDS /tAML (Q=8.072, p<0.01). Among 819 AML pts, 327 pts were newly diagnosed, 370 pts were relapsed/refractory (R/R) AML. The AML status was not specified in the rest 122 pts. Interestingly, no significant difference in CR rates was noted between the newly diagnosed (57.0%, 95% CI 51.5%-62.3%) and R/R AML pts (60.1%, 95% CI 50.5%-68.9%) (Q=0.312, p>0.05). The CR rate for the 367 elderly AML pts was 52% (95% CI 51.5%-62.3%). The CR rate was also significantly higher in pts with favorable (64.5%, 95% CI 38.8%-83.9%) and intermediate (69.6%, 95% CI 60.4%-77.5%) cytogenetics than those with unfavorable one (29.5%, 95% CI 19.7%-41.8%) (p<0.05). There were 7 trials that compared the CR rates of CAG regimen with those of other induction regimens in AML pts. Surprisingly, the CR rate of CAG was significantly higher than those of other induction regimens (OR 2.425, 95% CI, 1.515–3.880). CAG regimens were well tolerated with cardiotoxicity in 0.42% cases (4/954) and early death occurred in 4.40% cases (44/1000). Conclusions: CAG regimen induced significantly higher CR rates in AML than in MDS pts. The CR rates of CAG regimen was significantly better than those of other induction regimens in AML pts. This regimen was well tolerated with low cardiotoxicity and early death rate. Disclosures: No relevant conflicts of interest to declare.

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5-Azacytidine (AZA) Compared to Intensive Chemotherapy in Elderly Acute Myeloid Leukemia (AML) Patients: Results of a Retrospective Single Centre Matched Analysis,

Blood ◽

10.1182/blood.v118.21.3620.3620 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3620-3620 ◽

Cited By ~ 1

Author(s):

Elsa Lestang ◽

Sameh Ayari ◽

Patrice Chevallier ◽

Thierry Guillaume ◽

Fanny Rialland ◽

...

Keyword(s):

Elderly Patients ◽

De Novo ◽

Conflicts Of Interest ◽

Intensive Therapy ◽

Subcutaneous Administration ◽

Intensive Chemotherapy ◽

Single Centre ◽

Secondary Aml ◽

Elderly Aml ◽

Significant Difference

Abstract Abstract 3620 AML in elderly patients is characterized by a poor prognosis, especially in those patients aged >70, and/or in frail patients with comorbidities or poor performance status (PS). Moreover, several studies already suggested that elderly AML patients with unfavorable karyotype may not benefit from intensive chemotherapy. With this background, and using a matched analysis, this report aimed to assess the outcome of a single centre series of elderly AML patients who received either non intensive therapy by hypomethylating agents, or standard induction with intensive therapy. All patients were aged over 60 and had de novo or secondary AML. For the purpose of this comparison, the cohort was divided in two distinct groups. Group A included 36 cases treated by intensive chemotherapy between 1995 and 2005 according to the GOELAMS AML-SA2002 or SA3&4 protocols (5+7 induction with idarabicine 5 mg/m2/d and cytarabine 100 mg/m2/d). In this group, patients who could achieve CR received either 3 or 6 consolidation courses delivered over 1 or 2 years (according the protocol AML-SA2002 versus SA3&4). Group B included another 36 patients who were treated between 2006 and 2010 with AZA according to the recommendations of the “compassionate use program” authorized by the French Health Agency (one cycle of AZA = 7 days of subcutaneous administration 75mg/m2 every 28 days until progression).In this group, response was assessed after 3 cycles and qualified using IWG criteria. These two groups were matched based on cytogenetic features and age. The median age for the total cohort was 72 years (range, 60–86). Groups were comparable for WBC, % marrow blasts infiltration, WHO subtypes, and cytogenetic features at diagnosis. A higher rate of secondary AML was observed in the AZA arm. CR and CR with incomplete hematological recovery (CRi) rates were significantly higher in the intensive vs. AZA arm (63% vs. 28%, p<0.0001). However, there was a trend for a higher rate of partial remission (PR) in the AZA Arm (25% vs. 5%, p=0.02). With a median follow-up of 13.3 months (range, 5–80) from diagnosis, median overall survival (OS) was comparable between the two arms: 10.4 vs. 10.3 months, p=0.3) In multivariate analysis for OS including treatment strategy, the strongest prognostic factors were an unfavorable karyotype (HR=2.05, 95%CI, 1.09–3.85; p=0.03), PS status (0 vs. 1–2; HR=2.04 95%CI, 1.16–3.58; p=0.01) and platelets number at diagnosis (analyzed as a continuous variable) (HR=1, 95%CI, 0.99–1.00; p=0.04). Of note, the treatment arm was not found to be a significant determinant for OS: (AZA vs intensive chemotherapy.; HR=1.86, 95%CI, 0.86–3.16; p=0.13). This analysis suggests that the use of AZA as an alternative to intensive chemotherapy in elderly patients with de novo or secondary AML may lead to similar OS, despite a significant difference in terms of CR and CRi rate. The different mechanism of action of AZA in comparison to conventional chemotherapy, and the higher rate of PR that can be achieved after AZA therapy might contribute to improved OS through relatively long lasting disease control. These results set the frame for a prospective controlled trial to test AZA as an ambulatory alternative to standard intensive chemotherapy in elderly AML patients, especially those patients with unfavorable karyotype or poor PS and comorbidities. Disclosures: No relevant conflicts of interest to declare.

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Adverse Prognostic Impact of GAS6 Expression in De Novo Cytogenetically Normal Acute Myeloid Leukemia (CN-AML) (CALGB 8461, 9665, 20202; Alliance)

Blood ◽

10.1182/blood.v120.21.1293.1293 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1293-1293

Author(s):

Susan Whitman ◽

Jessica Kohlschmidt ◽

Kati Maharry ◽

Deedra Nicolet ◽

Sebastian Schwind ◽

...

Keyword(s):

De Novo ◽

Conflicts Of Interest ◽

Expression Profiles ◽

White Blood Count ◽

Categorical Variables ◽

Prognostic Impact ◽

Age Group ◽

Wild Type ◽

Aberrant Expression ◽

Expression Status

Abstract Abstract 1293 Receptor tyrosine kinases (RTKs) constitutively activated by gene mutation, overexpression and/or autocrine activation via ligand expression have been shown to negatively impact on outcomes of AML patients (pts). AXL, a member of the TAM (TYRO3, AXL, MERTK) RTK gene family was reported to be overexpressed and associated with poor survival in AML (Rochlitz, et al, Leukemia, 1999: 13:1352–8). No AXL mutations have been described, suggesting its activation may occur via aberrant expression in leukemic blasts of a TAM RTK ligand, GAS6. GAS6 was shown to be overexpressed in AML (Dirks, et al Leuk. Res. 23:643–51); yet its prognostic relevance is unknown. We report clinical and molecular associations and prognostic impact of aberrant GAS6 expression, in the context of TAM RTKs and known prognostic markers in de novo CN-AML pts (n=270; aged 18–83 y) treated with cytarabine/anthracycline-based therapies. Sixty-nine (26%) pts expressed GAS6 (>background signal; derived from microarray gene expression profiles of AML samples). TYRO3 expression status [positive (+) vs negative (–)] was similar in GAS6+ and GAS6– pts (P=.74), while AXL+ (P<.001) and low expression MERTK (P=.02) were more frequent in GAS6+ pts. Compared to GAS6– pts, GAS6+ pts were older (P=.02), had more platelets (P=.03), lower % blood blasts (P=.01) and increased frequency of hepatomegaly (P=.006); were more often NPM1 (P<.001) and CEBPA (P=.02) wild-type, and RUNX1 (P<.001) and ASXL1 (P=.002) mutated and expressed higher MN1 levels (P=.05). In univariable analyses, none of the TAM RTKs associated with complete remission (CR) and only TYRO3+ associated with reduced disease-free (DFS; P=.005), overall (OS; P=.005) and event-free survival (EFS; P=.008). GAS6+ vs GAS6– pts had lower CR rates (P<.001), shorter DFS (P=.03), OS (P=.004) and EFS (P<.001). While no TAM RTK entered the CR multivariable (MVA) model, GAS6+ expression status remained an independent marker for lower CR rate after adjusting for NPM1 status, white blood count (WBC) and age group (Table). In the DFS, OS and EFS models (Table), there was an interaction between GAS6 and the combined dual receptor (TYRO3/AXL) variable. GAS6 independently associated with shorter survival in TYRO3–/AXL– pts but not TYRO3+/AXL+ pts after adjusting for other variables. We show for the 1st time that GAS6 expression is an independent prognostic marker in CN-AML; negatively impacting on CR attainment, independent of TAM RTKs and on survival endpoints in pts lacking TYRO3 and AXL expression, regardless of MERTK expression. Our results suggest that GAS6 expressed by AML blasts plays a role in chemotherapy resistance. As GAS6 is expressed but not its RTKs in a subgroup of pts with poor outcome, this may lead to the hypothesis that the prognostic impact of GAS6 in those patients is mediated by the encoded ligand acting on cells other than AML blasts including, for example, natural killer cells, where activation of AXL RTK is reported to suppress innate immunity. Table. MVA models Variable CR DFS OS EFS P OR (95% CI) P HR (95% CI) P HR (95% CI) P HR (95% CI) GAS6 expression, + v – .02 0.46 (0.24, 0.88) .03* 1.78 (1.07, 2.96) .05* 1.59 (1.00, 2.52) .03* 1.59 (1.06, 2.41) NPM1, mut v wt .001 2.97 (1.53, 5.77) – – – – .006 0.64 (0.46, 0.88) FLT3-ITD, present v absent – – .003 1.66 (1.19, 2.33) – – .005 1.55 (1.14, 2.11) WT1, mut v wt – – – – <.001 3.42 (1.97, 5.96) .03 1.84 (1.06, 3.18) RUNX1, mut v wt – – – – .002 2.00 (1.29, 3.10) – – ASXL1, mut v wt – – – – – – .003 1.66 (1.05, 2.60) DNMT3A  R882 mut v wt  Non-R882 v wt .006 .21 1.65 (1.15, 2.36) 1.33 (0.85, 2.08) WBC, continuous, 50 unit increase <.001 0.56 (0.41, 0.76) – – – – <.001 1.25 (1.12, 1.39) Age group, ≥ 60 y v < 60 y .01 0.40 (0.19, 0.83) <.001 2.09 (1.44, 3.03) <.001 2.64 (1.80, 3.87) <.001 2.16 (1.54, 3.02) OR, odds ratio; HR, hazard ratio; CI, confidence interval; mutated, mut; wild-type, wt. ORs > (<) 1.0 mean higher (lower) CR rate, and HRs > (<) 1.0 mean higher (lower) risk for relapse or death (DFS, EFS), respectively, for the higher values of the continuous variables and the first category listed for the categorical variables. Variables significant at α =.20 in univariable models were considered, although all considered variables are not shown. *There are interactions between GAS6 and TYRO3/AXL dual receptor status for DFS (P=.16), OS (P=.06) and EFS (P=.12). The P-values, HRs and CIs are for comparisons of GAS6+ v GAS6– pts within the TYRO3–/AXL– subset. Disclosures: No relevant conflicts of interest to declare.

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Serum 2-Hydroxyglutarate Levels Predict Isocitrate Dehydrogenase Mutations and Clinical Outcome in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v120.21.2481.2481 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2481-2481

Author(s):

Courtney D. DiNardo ◽

Ross L. Levine ◽

Kathleen J Propert ◽

Alison W. Loren ◽

Elisabeth Paietta ◽

...

Keyword(s):

Research Funding ◽

De Novo ◽

Mutational Analysis ◽

Eastern Cooperative Oncology Group ◽

Elevated Serum ◽

Wild Type ◽

Serum Samples ◽

Prognostic Information ◽

Idh Mutations ◽

De Novo Aml

Abstract Abstract 2481 Purpose: Cancer-associated IDH mutations produce the metabolite 2-hydroxyglutarate (2HG), but the clinical utility of serum 2HG measurements has not been previously established. We studied whether 2HG measurements in AML patients correlate with the presence of IDH mutations and whether diagnostic or remission 2HG measurements predict survival. Patients and Methods: Serum samples from 223 previously untreated adults (≤ 60 years of age) with de novo AML from the Eastern Cooperative Oncology Group E1900 clinical trial (62 IDH mutated, 161 IDH wild-type) were analyzed for 2HG concentration by reverse-phase liquid chromatography coupled to mass spectrometry (GC-MS). Results: Pretreatment 2HG levels ranged from 10 to 30000 ng/ml and were significantly elevated in IDH-mutant samples (median 3004.1 ng/ml), as compared to the wild-type cohort (median 61.2 ng/ml) (p < 0.0005). 2HG levels did not differ among the specific IDH1 or IDH2 allelic variants. In ROC analysis, a discriminatory level of 700 ng/ml segregated patients with and without IDH mutations with 86.9% sensitivity and 90.7% specificity. On repeat mutational analysis of 13 IDH wild-type samples with 2HG levels >700 ng/ml, IDH mutations were identified in nine samples, most often at low allele burden. IDH mutant patients with 2HG levels ≤ 200 ng/ml at complete remission experienced improved overall survival compared to those with higher 2HG levels (HR 3.5, p = 0.02) (Figure 1). Conclusion: We establish a firm association between IDH mutations and elevated serum 2HG concentration in AML. These data confirm that peripheral blood measurement of an oncometabolite provides useful diagnostic and prognostic information for cancer therapy, and furthermore can inform patient selection of IDH mutant targeted therapies. Disclosures: Levine: Agios Pharmaceuticals: Research Funding. Straley:Agios Pharmaceuticals: Employment. Yen:Agios Pharmaceuticals: Employment. Agresta:Agios Pharmaceuticals: Employment. Carroll:Agios Pharmaceuticals: Research Funding.

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Incidence and Outcomes of Myeloproliferative Neoplasms (MPN) in Adolescents and Young Adults (AYAs).

Blood ◽

10.1182/blood.v120.21.2845.2845 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2845-2845 ◽

Cited By ~ 1

Author(s):

Naveen Pemmaraju ◽

Hagop M Kantarjian ◽

Jorge E. Cortes ◽

Alfonso Quintas-Cardama ◽

Sherry A. Pierce ◽

...

Keyword(s):

Conflicts Of Interest ◽

Systemic Mastocytosis ◽

Myeloproliferative Neoplasms ◽

Hypereosinophilic Syndrome ◽

Hematologic Malignancies ◽

Kaplan Meier ◽

Female Predominance ◽

Significant Difference ◽

Baseline Characteristics ◽

Better Than

Abstract Abstract 2845 Background: The MPNs are a family of chronic hematologic malignancies that typically affect pts ages 60–70s. However, there is growing awareness of hematologic malignancies developing in young pts, particularly a unique subset of pts known as AYAs. Little is known about incidence and outcomes of AYA pts with MPNs. Objectives: To determine the incidence and outcomes of MPN AYA pts. Results: We retrospectively reviewed charts of 1,616 MPN pts evaluated at our institution from 1986–2011. A total of 171 MPN pts (11%) were identified whose ages were 16–39, defined as AYA pts (per NCCN guideline recommendations on AYA cancers). Breakdown by MPN subtype: Essential Thrombocytosis, ET (n=78, 46% of AYA MPNs), Polycythemia Vera, PV (n=25, 15%), Myelofibrosis, MF(n=24, 14%), [3 post-ET, 1 post-PV, 20 primary MF], Hypereosinophilic syndrome, HES (n=22, 13%), and Systemic Mastocytosis, SM (n=22, 13%). Baseline characteristics of AYA MPN pts detailed in Table 1. Only significant difference among AYA and non-AYA pts in terms of baseline CBC parameters was WBC: 6.4 (0.4–108.4) in AYA MF vs 10.1 (0.6–361) in non-AYA MF, p=0.0049. Analysis of median overall survival (OS), by Kaplan-Meier method, compared by age group (AYA vs non-AYA pts) and broken down by MPN subtype, shown in Table 2. Conclusion: MPN AYA pts constitute 11% of MPN pts at our institution. Overall, their 5- and 10- yr OS were significantly better than their older counterparts in the 3 major MPN subtypes (ET, PV, MF) and trended towards better survival but not statistically significant in HES and SM. Among AYA MPN pts, female predominance was noted among ET and SM pts and only 1 transformation event was noted among all MPN AYA pts in this analysis. Disclosures: No relevant conflicts of interest to declare.

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