ALBERT

Description: The past decade has shown a marked increase in the use of high-throughput assays in clinical research into human cancer, including acute myeloid leukemia (AML). In particular, genome-wide gene expression profiling (GEP) using DNA microarrays has been extensively used for improved understanding of the diagnosis, prognosis, and pathobiology of this heterogeneous disease. This review discusses the progress that has been made, places the technologic limitations in perspective, and highlights promising future avenues

Description: Abstract 1689 Poster Board I-715 Introduction The use of the proteasome inhibitor bortezomib has demonstrated activity in multiple myeloma and lymphomas. The HDAC inhibitor romidepsin is being evaluated in CTCL and PTCL, though its activity in B-cell lymphomas is less clear. We hypothesized that the combination of bortezomib and romidepsin would result in synergistic apoptosis in different B-cell NHL cell lines based upon the observed activity of this combination in more mature B-cell malignancies such as myeloma. Experimental Design Daudi, HT, Ramos and SUDHL-4 cell lines were exposed to different concentrations of bortezomib and romidepsin, separately, concurrently, and sequentially. Cell viability was assessed using MTT-assay, induced apoptosis was evaluated using Annexin V and PI staining from 24-48 hours. Apoptosis was also evaluated using western blot analysis of caspases and PARP cleavage. LC3 and HDAC6 level expressions were performed to determine if the effect of the combination was a result of the aggresome or autophagy pathway. Cell cycle studies were also performed to study if there were any changes after treating cells with the combination. Results The combination of bortezomib and romidepsin resulted in synergistic B-cell apoptosis as measured by MTT-assay with combination indices of 〈 0.5. This was associated with increased caspases and PARP cleavage as early as 24 hours after exposure. Order of addition experiments demonstrated definite sequence specificity. When romidepsin was added first, and 6 hours later followed by bortezomib, apoptosis was enhanced, compared to both agents being given concurrently or when bortezomib was administered first. Cell cycle analysis studies demonstrated that pretreatment of cells with romidepsin for 6 hours followed by the addition of bortezomib arrested the cells in G2M phase. HDAC6 expression was significantly reduced following combination therapy, and LC3-I was cleaved to LC3-II in treated cells suggesting that the combination affected aggresome formation and autophagy. Conclusion The combination of romidepsin and bortezomib at low nanomolar concentrations suggests that this may be an important clinical combination to test in patients with relapsed or refractory B-cell malignancies. Sequence of administration data is currently being tested to determine if the effect is a result of autophagy inhibition as is seen in myeloma cell lines. Additional mechanistic studies will be presented with the goals of identifying predictors of response that can then be validated in prospective clinical trials. Disclosures Lechowicz: Gloucester: Consultancy. Kaufman:Millennium: Consultancy; Genzyme: Consultancy; Celgene: Consultancy; Merck: Research Funding; Celgene: Research Funding. Lonial:Gloucester: Research Funding; Novartis: Consultancy; BMS: Consultancy; Millennium: Consultancy, Research Funding; Celgene: Consultancy. Flowers:Millennium: Research Funding.

Description: Abstract 3701 Poster Board III-637 Background Romidepsin is an anti-neoplastic agent that has been identified as a novel pan-HDAC inhibitor with single-agent activity in T-cell lymphoma. In a combined analysis of 167 patients (pts) with cutaneous T-cell lymphoma (CTCL) from 2 clinical studies (GPI and NCI studies), the overall response rate was 35%, including 10 pts with a complete clinical response (CCR). Median duration of response was 13.8 months and 42% of pts with advanced disease (stage ≥IIB) responded [Demierre et al. J Clin Oncol 27:15s, 2009 (suppl; abstr 8546)]. The most common hematologic abnormalities in these pts included anemia (41%), thrombocytopenia (34%), neutropenia (27%), and lymphopenia (26%). Most hematologic toxicities were Grade 1 or 2, although 'Grade 3 events were observed. These events were reversible and a small portion of the patients discontinued the study drug because of these events (2%). This report details an analysis of platelet counts in pts receiving romidepsin and an investigation into the mechanism of thrombocytopenia in nonclinical studies. Methods Pts with CTCL who received ≥1 prior systemic therapy failure and had an ECOG PS of 0-2 were enrolled in 2 single-arm, open-label, multicenter and international clinical studies. Treatment with QTc prolonging therapies or CYP34A inhibitors was prohibited and pts with significant cardiovascular abnormalities were excluded. Romidepsin at 14 mg/m2 was administered as a 4-hr IV infusion on days 1, 8, and 15 of a 28 day cycle. Nonclinical studies were conducted in mice to investigate the mechanism of romidepsin effects on platelets. Romidepsin was administered to female BALB/c mice at doses of 1 or 4 mg/kg by tail-vein injection on days 1, 5 and 9. Blood samples were collected every 2 days from alternating groups of mice to minimize effects of bleeding on platelet counts. Results In clinical studies, there is a mean decrease in platelet counts during the treatment period of each cycle, and a return to baseline levels or above between cycles observed in both clinical studies as described in the table below. No clinically meaningful change has been observed in the central tendency over 4 cycles of treatment in both studies. In the mouse studies, dose-dependent effects were seen on both WBC and platelet counts. Day 2 WBC counts dropped to 45% and 10% of normal at the 1 and 4 mg/kg doses, respectively. WBC counts remained low until after the dosing period in the 1 mg/kg romidepsin group, but recovered more quickly in the 4 mg/kg group. Day 2 platelet counts were 70% of normal at the 1 mg/kg dose and remained near this level until day 10, followed by recovery to normal at day 15. At the 4 mg/kg dose, profound thrombycytopenia was induced, with platelet counts only 20% of normal on days 4-6. Platelet counts slowly recovered to 70% of normal by day 15. Plasma thrombopoietin levels were normal throughout the experiment for the 1 mg/kg group, and showed a large increase to 275% of normal on day 6 in the 4 mg/kg group, which is the expected response to thrombocytopenia as a signal to increase platelet production and indicates that platelet reduction is not attributable to defective TPO production. Bone marrow megakaryocyte populations are being examined to determine the effects of romidepsin on these platelet-producing cells. Conclusions Following romidepsin administration, a saw tooth pattern is observed in the reduction and recovery of platelets. Recovery of platelets appears to occur more rapidly in humans than in mice; however, the effects are reversible after dosing in clinical studies and in murine models. In the clinical data the recovery pattern suggests that the transient effects are direct and are not effects on bone marrow. Disclosures: Whittaker: Gloucester Pharmaceuticals: Research Funding. Prince:Gloucester Pharmaceuticals: Consultancy. Demierre:Gloucester Pharmaceuticals: Consultancy, Honoraria. Lonial:Gloucester Pharmaceuticals: Honoraria. Kim:Gloucester Pharmaceuticals: Consultancy, Honoraria. Nichols:Gloucester Pharmaceuticals: Employment, Equity Ownership. Nix:Gloucester Pharmaceuticals: Employment.

Description: Precursor T-cell acute lymphoblastic leukemia (T-ALL) in children represents a clinical challenge, because relapses are usually fatal. It is thus necessary to identify high-risk patients as early as possible to effectively individualize treatment. We aimed to define novel molecular risk markers in T-ALL and performed array-based comparative genomic hybridization (array-CGH) and expression analyses in 73 patients. We show that DNA copy-number changes are common in T-ALL and affect 70 of 73 (96%) patients. Notably, genomic imbalances predicted to down-regulate the TGF-β or up-regulate the PI3K-AKT pathways are identified in 25 of 73 (34%) and 21 of 73 (29%) patients, suggesting that these pathways play key roles in T-ALL leukemogenesis. Furthermore, we identified a deletion at 6q15-16.1 in 9 of 73 (12%) of the patients, which predicts poor early treatment response. This deletion includes the CASP8AP2 gene, whose expression is shown to be down-regulated. The interaction of CASP8AP2 with CASP8 plays a crucial role in apoptotic regulation, suggesting a functional link between the clinical effect of the deletion and the molecular mode of action. The data presented here implicate the TGF-β and PI3K-AKT pathways in T-ALL leukemogenesis and identify a subgroup of patients with CASP8AP2 deletions and poor early treatment response.

Description: Abstract 1955 Poster Board I-978 The detection of chromosome abnormalities in mature B-cell neoplasms by conventional cytogenetics remains difficult, mainly due to the low proliferative rate of mature lymphoid cells. The current FISH panel for chronic lymphocytic leukemia (CLL) is designed to detect some of the more common abnormalities of prognostic significance in CLL [i.e., del(6q), del(11q), +12, del(13q), del(17p)]. This CLL FISH panel has improved the detection rate of these markers by making it possible to obtain cytogenetic information from interphase cells; however, as it is limited to only these 5 markers, it cannot detect all abnormalities associated with CLL. More importantly, the impact of other chromosome abnormalities on prognosis and disease progression, with and without the presence of these 5 prognostic markers, is not known. CpG-oligodeoxynucleotides (ODNs) such as DSP30 activate cells of the immune system in a sequence-dependent manner and promote proliferation of CLL cells in vitro [Decker et al. Blood 2000;95:999-1006]. They also upregulate costimulatory molecules and potential target antigens during immunotherapy. The use of DSP30 in combination with interleukin 2 (IL2) has proven effective in increasing the detection of chromosome abnormalities in CLL [Dicker et al. Blood 2006;108:3152-60] and other mature B-cell lymphoid malignancies by conventional cytogenetics [Struski et al. Leukemia 2009;23:617-9], when compared to the well-established B-cell mitogens. In our extensive clinical experience of incorporating DSP30/IL2 into our culture media, this cocktail has significantly increased the detection of chromosome abnormalities in CLL by conventional cytogenetics, from 55% to greater than 80%. We thus decided to investigate if various other lymphoid malignancies would respond to the mitogen activity of DSP30/IL2 as well as or better than CLL. Specifically, we evaluated 812 cases of mature B-cell lymphoid malignancies that were abnormal by flow cytometry, morphology, or cytogenetic analysis. All samples (bone marrow or blood) were cultured for approximately 72 hours using the DSP30/IL2 mitogen cocktail. Of these 812 cases, 746 (91%) provided sufficient mitotic index and quality for a complete cytogenetic analysis and interpretation. Of the CLL cases (n=509), 79 were initially interpreted as normal by conventional cytogenetic analysis, but were later interpreted as abnormal by FISH for deletion 13q only. In view of the known cryptic nature of this deletion in CLL, these cases were not included in the study, leaving a total of 430 CLL cases, and thus bringing the total number of cytogenetically successful study cases to 667. In addition to the 430 CLL cases, there were 14 variant CLLs; 36 diffuse large B-cell lymphomas (DLBCLs); 35 follicular lymphomas; 34 non-Hodgkin lymphomas (not further specified); 29 marginal zone B-cell lymphomas of splenic type (sMZBCL); 27 mantle cell lymphomas (MCLs), of which 8 were blastoid; 16 MZBCL of MALT type; 13 hairy cell leukemias (HCLs); 12 lymphoproliferative disorders (not further specified); 10 lymphoplasmacytic lymphomas (LPLs); 6 Burkitt lymphomas; 3 Hodgkin lymphomas; and 2 B-cell prolymphocytic leukemias (PLLs). Of particular interest is the fact that we detected clonal abnormalities in 100% of HCLs, blastoid MCLs, variant CLLs, and B-cell PLL, as well as in 97% of sMZBCLs, 89% of DLBCLs, and 80% of LPLs This is of great importance since HCLs and LPLs are rarely abnormal by conventional cytogenetics using the more traditional combinations of mitogens making it difficult to identify markers of prognostic significance. In conclusion, our findings demonstrate that the DSP30/IL2 cocktail induces proliferation of various B-cell mature lymphoid disorders and that its mitogenic action is not limited to CLL. We are continuing to develop our understanding of the considerable response of specific lymphoid malignancies to the DSP30/IL2 cocktail by correlating additional clinical data, and hope that the end result will open new avenues in regards to prognostic outcome and therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

Description: Higher levels of procoagulant factors and factor XII deficiency may be risk factors for first venous thromboembolism (VTE). We studied associations of coagulation factors IX through XIII with risk of future VTE in 2 general population samples. Using a nested case-control study combining the 21 860 participants of the Atherosclerosis Risk in Communities study and the Cardiovascular Health Study, we determined antigenic levels of these coagulation factors in primarily pre-event blood samples from 462 participants who subsequently developed VTE and 1047 participants who remained free of VTE. Only elevated levels of factors IX and XI were associated with increased risk of VTE after adjustment for age, sex, race, and study. For factor IX, the odds ratio (OR) was 1.4 (95% confidence interval [CI], 1.0-2.0) comparing the top to bottom quintile. The OR for factor XI was higher: 2.0 (95% CI, 1.4-2.9). With further adjustment for body mass index and diabetes, only elevated factor XI remained associated with VTE risk: OR 1.8 (95% CI, 1.3-2.7). Associations were similar by study and whether the thrombosis was idiopathic or secondary. Factor XII deficiency was not related to VTE risk. Among these procoagulant factors, only elevated factor XI was a risk factor for VTE.

Description: The concept of endothelial progenitor cells (EPCs) has attracted considerable interest in cardiovascular research, but despite a decade of research there are still no specific markers for EPCs and results from clinical trials remain controversial. Using liquid chromatography–tandem mass spectrometry, we analyzed the protein composition of microparticles (MPs) originating from the cell surface of EPC cultures. Our data revealed that the conventional methods for isolating mononuclear cells lead to a contamination with platelet proteins. Notably, platelets readily disintegrate into platelet MPs. These platelet MPs are taken up by the mononuclear cell population, which acquires “endothelial” characteristics (CD31, von Willebrand factor [VWF], lectin-binding), and angiogenic properties. In a large population-based study (n = 526), platelets emerged as a positive predictor for the number of colony-forming units and early outgrowth EPCs. Our study provides the first evidence that the cell type consistent with current definitions of an EPC phenotype may arise from an uptake of platelet MPs by mononuclear cells resulting in a gross misinterpretation of their cellular progeny. These findings demonstrate the advantage of using an unbiased proteomic approach to assess cellular phenotypes and advise caution in attributing the benefits in clinical trials using unselected bone marrow mononuclear cells (BMCs) to stem cell-mediated repair.

Description: Abstract 18 Introduction: Long-term survival in pediatric relapsed AML is only 20-30%. Optimal reinduction therapy is unknown, and there is a concern about cardiotoxicity with repeated anthracycline use at relapse. Preclinical in vitro and animal studies, and limited clinical data suggest that liposomal daunorubicin (DaunoXome®, DNX) is less cardiotoxic. These considerations lead to a phase III study, in the setting of the International BFM Study Group. Materials and methods: FLAG was randomised against FLAG/DNX in the 1st reinduction course. The conventional 5-days FLAG only was recommended as the 2nd course. DNX was dosed at 60 mg/m2/day on days 1, 3 and 5. After induction, allogeneic stem cell transplantation was generally recommended, but time-to-transplant could be bridged by high- or low-intensive consolidation therapy. Primary endpoint of the study was early treatment response, based on bone marrow examination shortly before reinduction course 2, and defined as either good (≤20% leukemic blasts) or poor (〉20% leukemic blasts). This endpoint was chosen because of its prognostic value in earlier relapsed AML BFM-trials, and because compliance with an extended protocol guideline was likely to be suboptimal within the context of a highly multinational and multicenter AML Relapse protocol. However, secondary endpoints were defined, including the CR2 rate determined after 2 courses, long-term survival, and toxicity. Patients with AML M3 and those 〉18 years of age at initial diagnosis were ineligible. The study opened in most countries in 2002/2003. The study closed for accrual on April 1, 2009 when the required 360 fully eligible and evaluable patients had been randomized. Early and late relapsed AML was defined as a relapse within or after 1 year from initial diagnosis, but this only influenced treatment in that early relapsed AML patients were eligible for haploidentical SCT, while late relapsed AML patients were eligible for autologous SCT, if a matched or partly mismatched transplant was not possible. Thirteen groups from 20 countries and 〉100 centers have enrolled patients, with informed consent and after approval of the study by regulatory authorities. Data are presented according to intention-to-treat, with a median follow-up of 2.7 years for patients at risk. Results: Overall 4-year probability of survival (pOS) was 35% SE 2%, the overall CR2 rate 62%. The good early responders had a 4-year pOS of 45% SE 3% versus 10% SE 3% for poor responders (p

Description: Abstract 634 FLT3, a transmembrane receptor tyrosine kinase constitutively activated via mutation in blasts of patients (pts) with AML, is an important therapeutic target. Blasts from approximately 25% of pts have a length or internal tandem duplication (ITD) mutation in the juxtamembrane region or tyrosine kinase domain (TKD1) of FLT3, which is associated with reduced disease-free survival and overall survival (OS), particularly in pts with normal cytogenetics. Blasts from 5–10% of pts have a point mutation (typically D835Y) in the tyrosine kinase domain (TKD); the effect of this mutation on prognosis is uncertain. Midostaurin (PKC412) is a multi-targeted kinase inhibitor with demonstrated clinical activity in FLT3-mutant (FLT3–mut) and FLT3-wild-type (FLT3–wt) AML (peripheral blood blast reduction in 70% and 30% of pts, respectively) but rarely produces complete remissions). Preclinical studies demonstrated synergy between FLT3 inhibitors and chemotherapy. We conducted a Phase 1b trial to investigate the feasibility of administering daunorubicin (60 mg/m2 IV, days 1–3) and cytarabine (100 mg/m2 IVCI, days 1–7) induction and high-dose cytarabine post-remission therapy (3 gm/m2 over 3h every 12h, days 1, 3, and 5 for 3 cycles) plus oral midostaurin at 100 mg or 50 mg each twice daily on days 8–21 (sequentially) or days 1–7, 15–21 (concomitantly) with all chemo cycles in newly diagnosed pts under age 61 with de novo AML. Whereas 100 mg of midostaurin plus induction chemotherapy was poorly tolerated due to nausea and vomiting, the 40 pts who received 50 mg of midostaurin orally twice daily ( 20 each on the sequential and concomitant schedules; 27 FLT3–wt; 13 FLT3–mut [9 with an ITD]), tolerated the combination well. Median midostaurin exposure was 133 days (range 21–975) for the FLT3–mut pts and 90 days (range 7–1016) for FLT3–wt pts. Maintenance therapy with midostaurin was allowed with investigator discretion and was received by 5 pts (3 FLT3–mut, 2 FLT–wt). The median ages for the FLT3–wt and FLT3–mut pts were 50 years (range 25–60) and 46 years (range 20–65), respectively. 77% of the FLT3–mut pts displayed normal, 15% adverse and 8% other intermediate cytogenetics compared with 18.5%, 26%, and 26%, respectively, for FLT3-wt (also 18.5% favorable; 11% unknown). Complete response occurred in 32/40 (80%) of all pts (20/27 [74%] of FLT3–wt patients, 12/13 [92%] of FLT3–mut pts). Patients were censored at the last date they were known to be alive with a median post treatment follow-up for FLT3-mut pts of 1059 days and 1086 days for FLT3-wt. Even accounting for their differing cytogenetics and ages, the OS of the FLT3–mut subgroup was expected to be inferior to that of the FLT3–wt subgroup. However, we report that the 1 and 2 year OS for the pts with FLT3–mut AML was 85% and 62%, respectively, and was comparable to that of the FLT3–wt subgroup (81% and 59%, respectively). Although based on small numbers and not stratified for type of FLT3 mutation (TKD, ITD, ITD length, location, or allelic ratio), these long-term results suggest that combination therapy with a FLT3 inhibitor and chemotherapy might be effective enough to obviate the perceived need for allogeneic stem cell transplantation for FLT3–mut AML pts in first complete remission. Moreover, these data support the rationale for the ongoing international phase 3 study of induction, post-remission intensification, and maintenance with midostaurin (50 mg po bid) or placebo. Disclosures: Stone: Novartis: Research Funding, ad hoc consultancy; Cephalon: ad hoc consultancy. Off Label Use: midostaurin with chemothereapy for AML. Paquette:Novartis: Honoraria, Research Funding, Speakers Bureau. Schiller:Novartis: Research Funding, Speakers Bureau; Millenium: Research Funding, Speakers Bureau; Genzyme: Research Funding; Vion: Research Funding; Centocor: Research Funding; Eli Lilly: Research Funding; Celgene: Research Funding. Schiffer:Novartis: Consultancy, Research Funding; Genzyme: Consultancy. Ehninger:Novartis: Honoraria, Research Funding. Cortes:Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Wyeth: Research Funding. Kantarjian:Novartis: Research Funding. DeAngelo:Bristol-Myers Squibb: Speakers Bureau; Celgene: Speakers Bureau; Enzon: Speakers Bureau; Novartis: Speakers Bureau. Huntsman-Labed:Novartis: Employment, Equity Ownership. Dutreix:Novartis: Employment, Equity Ownership. Rai:Novartis: Employment, Equity Ownership. Giles:Novartis: Research Funding; Merck: Research Funding; Bristol-Myers Squibb: Research Funding; Vion: Research Funding.

Description: Abstract 2168 Poster Board II-145 The deregulated tyrosine kinase associated with t(9;22) fusion protein BCR-ABL is efficiently targeted by tyrosine kinase inhibitors (Imatinib, Nilotinib and Dasatinib). It is generally believed that most patients have residual disease. The inability to eliminate the refractory leukemia stem cell in chronic myeloid leukemia (CML) is not well understood. The refractory stem cell is present despite effective inhibition of the BCR-ABL kinase in these cells, and dissociation between kinase inhibition and cell death implies lack of dependency on BCR-ABL aberrant kinase activity, unlike the one seen in more mature CML cells. Thus the oncogene addictive dependency of these leukemia stem cells may be overcome by intrinsic and/or extrinsic factors/pathways. One such pathway involved in hematopoeitic stem cell maintenance is the Wnt/β-catenin signaling pathway. BCR-ABL signaling is known to directly activate the Wnt/β-catenin pathway. The loss of function of a negative regulator of the Wnt/β-catenin pathway, known as GSK3β is associated with CML progression from chronic phase to the blast phase. Lastly, the loss of β-catenin in murine models impairs the self renewal capacity of both the normal and the BCR-ABL+ leukemia stem cell. Thus this pathway is important during various stages of the disease. We explored the effect of Wnt inhibition using a novel Wnt pathway inhibitor (AG-214, University of Michigan). AG-214 is structurally related to a previously reported Wnt-inhibitor. AG-214 is able to antagonize β-catenin/TCF in luciferase reporter assays, and expression of Wnt targets in colon cancer cell lines. We utilized a serum free culture system with 5 added cytokines and treated both CML blast crisis CD34+ as well as chronic phase CD34+ cells with AG-214. Our in vitro experiments show that primary blast crisis CD34+ cells are induced to undergo apoptosis at an IC-50 of approximately 2 μM. Furthermore, combination of 1.25 μM of AG-214 with 2 μM Imatinib achieved greater than 50% apoptosis. Analysis of chronic phase CML CD34+ cells showed that targeting of Wnt/β-catenin pathway requires higher concentration of the Wnt-inhibitor (〉2.5 μM), and that the addition of Imatinib can cooperate to enhance the apoptotic response. Furthermore, chronic phase progenitors (Lin-CD38+/CD34+) are more sensitive to lower concentrations of AG-214 whereas 5 μM is required to induce significant apoptosis of ∼70% in the primitive leukemia stem cell (Lin-/CD38-/CD34+) population, and addition of 2 μM Imatinib increased their apoptotic response to ∼84%. Normal CD34+ cells do not undergo significant apoptosis with 5 μM of AG-214 (∼10%) even when combined with 2 μM Imatinib (∼20%). Targeting of the Wnt/β-catenin pathway enhances apoptosis in both blast crisis and chronic phase CML progenitors and leukemia stem cells. Disclosures: No relevant conflicts of interest to declare.