ALBERT

Description: Abstract 1155 Introduction. Anterior spinal artery thrombosis (AST) results in sudden, often irreversible sensory and motor loss, with partial or complete paraplegia. This is recognized as a complication of: prothrombotic states; right to left shunting; trauma and spinal surgery. Both venous and arterial thrombosis, including the central nervous system, are well-recognized risks in sickle cell disease (SCD) but AST has not been described. In thalassemia disorders (Thal), cerebral infarction, often silent, is increasingly recognized (Mussalam et al, Thromb Res, Aug 2012). However AST is not a recognized complication. Here we describe four cases of acute onset AST in adults (3 Thal major, 1 SCD), leading to severe neurological irreversible or partially reversible deficits. Patients and Methods. Patients described were attending outpatients for monitoring drawn from two large adults clinics in the UK (UCLH, Whittington, over 1500 hemoglobinopathy patients) and Italy (Ospedale Maggiore Policlinico, Milan, 400 patients). All events occurred within a 3 year period. Results. Presenting symptoms and Magnetic Resonance Imaging (MRI) findings are shown in the Table. All cases presented acutely with a sensory level on examination and with bladder dysfunction. Three presented with motor weakness of both lower limbs (1 case initially in one limb). Case 3 presented with a sensory deficit affecting the sacral region but no motor deficit. Partial reversibility occurred in cases 1 and 3. In Thal cases, no prodromal syndrome and no prior history of thrombosis were seen. MRI showed changes consistent with acute cord ischemia (delayed in case 4 until 5 days). Extra-medullary hematopoiesis was demonstrated by MRI only in case 2, but was not sufficient to cause cord compression. Cerebrospinal fluid analysis was normal in all cases. Concomitant risk factors such as autoimmune markers, active hepatitis, trauma, or demonstrable prothrombotic markers other than those expected in SCD or Thal were not detected. In none of the Thal cases was a thrombotic history elicited but the SCD patient had a history of retinal artery and renal artery thrombosis (1 year previously). Discussion. A case series of this serious complication has not been previously reported. The known prothrombotic tendency in SCD and Thal is the most likely risk factor as other risk factors were absent. Thal cases were transfusion dependent, where thrombosis risk is generally about a quarter of that in non transfusion dependent Thal (Cappellini, Blood Reviews, 265, 2012, S20–23). In the SCD case, the prior history of arterial thrombosis, consistent with an embolic etiology, led us to examine whether a patent foramen ovale (PFO) was present, which was confirmed by bubble jet studies. This was subsequently closed. In patients with a history of embolic thrombosis, the presence of PFO should be sought and closure considered. Two cases were treated with Methylprednisolone soon after presentation. Two cases were commenced on Aspirin 75mg once a day to limit extension and as secondary prevention. The use of thrombolytic agents such as tissue thromboplastin activator have not been described in AST. In conclusion, spinal cord ischemia should be considered when facing a Thal or SCD patient with acute neurological symptoms affecting legs or bladder. This may be more common in hemoglobin disorders than is apparent from the literature. Disclosures: Cappellini: Novartis Pharmaceuticals: Research Funding.

Description: Abstract 642 HSCs are rare immature cells capable of reconstituting all blood cell lineages throughout the life of an individual. We have previously shown that intermittent treatment with PTH is sufficient to increase the number of HSCs in the marrow of mice. This PTH effect is blocked in vitro with inhibition of gamma-secretase, the mediator of a required step in Notch signaling. Osteoblastic cells are a critical component of the HSC niche and are likely mediators of the PTH-induced increase in HSCs. Specifically the Notch ligand Jag 1 is expressed on osteoblasts and is therefore implicated as a mechanism through which PTH acts on HSCs. Therefore we investigated in vivo the role of osteoblastic Jag 1 in the PTH-dependent increase in HSCs. We utilized the 2.3kb collagen 1 promoter driven cre recombinase to specifically excise Jag 1 from osteoblastic cells in mice (OBJag1 mice). As we previously reported treatment of wild type (WT) controls with PTH 3 times daily for 10 days resulted in a significant increase in phenotypic HSC populations including Lin-Sca1+cKit+CD48-CD150- short-term HSCs (ST-HSCs) (VEH/PTH 0.0405±0.001 vs 0.0650±0.0038, p≤0.0001) and Lin-Sca1+cKit+CD48-CD150+ long-term HSCs (LT-HSCs) (VEH/PTH 0.0077±0.0008 vs 0.0125±0.00096, p≤0.01) as determined by flow cytometric analysis. In contrast treatment of OBJag1 mice did not result in a phenotypic increase in these populations. Despite the lack of a phenotypic increase in HSCs in OBJag1 mice, when HSC function was assessed by competitive repopulation assay, OBJag1 marrow cells demonstrated the same increased repopulating ability as WT mice (WT: VEH/PTH 12.16±2.7 vs 22.32±2.4, p≤0.01, OBJag1: VEH/PTH 13.6±1.8 vs 31.6±5.9, p≤0.01). Upon secondary transplantation however, HSCs from OBJag1 donors treated with PTH resulted in a lower engraftment rate than VEH treated controls (VEH/PTH 14.61±3.8 vs 4.38±0.9, p≤0.05). This result suggests that osteoblastic Jag 1 is necessary for the increase in phenotypic HSCs resulting from PTH treatment and is required to maintain LT-HSC self-renewal. However these data also suggest an osteoblastic Jag 1 independent mechanism that mediates a transient increase in repopulating ability. Decreased apoptosis is a potential mechanism by which PTH may functionally increase HSCs in the absence of increased self-renewal. To determine if PTH treatment decreases the apoptosis rate of HSCs, WT mice were treated intermittently with PTH once a day for 7 days. Despite a lack of increased HSCs by phenotypic analysis at 7 days, marrow from PTH treated mice displayed an increase in LT-HSC function as measured by competitive transplantation. We determined to measure the effect of PTH on apoptotic rates of HSCs using Annexin V membrane expression. By the 7th day of PTH treatment, LT-HSC apoptotic rates were decreased in the PTH treated group (VEH/PTH 10.482±2.25 vs. 6.27±1.93, p≤0.01) suggesting that changes in apoptotic rate of LT-HSCs precedes the HSC increase. These results were confirmed by flow cytometric measurement of activated caspase 3. PTH treatment decreased the percentage of LT-HSCs that were positive for activated caspase 3 (VEH/PTH 4.3±0.5 vs. 2.4±0.3, p≤0.01). PTH induced micro-architectural changes in trabecular bone at day 7 of treatment suggesting bone involvement despite the lack of an increase in bone volume. These results suggest for the first time that PTH may exert its beneficial effect on bone marrow reconstitution through both Jag 1 dependent and independent effects. Additionally, HSCs demonstrate decreased apoptotic rates and increased reconstitution ability prior to a demonstrable phenotypic increase, mimicking the effect seen in the absence of osteoblastic Jag 1. Together these results suggest that the decreased apoptotic rate may be mediated by an osteoblastic Jag 1 independent mechanism. Whether osteoblasts are required for the observed osteoblastic Jag 1 independent effects remains to be seen as these effects could be mediated by a Jag 1 independent osteoblastic mechanism or by an altogether different cellular component of the HSC niche. Further, since stressful manipulation of HSCs ex vivo is essential for their use in transplantation, defining factors regulating and decreasing their apoptosis may improve their engraftment efficiency, expanding their clinical use when their numbers are limited. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3265 Introduction: The clinically significant forms of thalassemia are associated with transfusional and non-transfusional iron overload (IO). IO contributes to cirrhosis, cardiac failure and endocrinopathies, amongst other complications. Despite advances in iron load monitoring and chelation therapy, patients still continue to be at risk of iron-associated complications. Free iron leads to production of reactive oxygen species and oxidative damage of lipids, proteins, and DNA, resulting in apoptosis and organ damage. Tools that allow for early detection of iron associated changes and toxicities may allow for earlier clinical intervention. Thalassemia major (TM) patients have increased levels of the lipid peroxidation marker malondialdehyde (MDA), which decreases with chelation therapy. The DNA oxidative damage marker, 8-hydroxy-2'-deoxyguanosine (8-OHdG) has not been studied in thalassemia patients. Iron associated oxidative damage has been shown to affect cell and organelle membranes, affect glucose and lipid homeostasis, and contribute to inflammation, fibrosis and organ damage. We hypothesized that metabolomics technologies that assess changes in global metabolite profiles (metabolism of sugars, lipids, amino acids etc) may have a role to play as biomarkers of iron load, organ damage, or therapeutic response. Aims: 1) To use metabolomics technologies to determine if we can identify metabolite profile differences between thalassemia patients and control individuals, 2) To examine 8-OHdG levels as a marker of DNA oxidative damage in thalassemia, 3) To use metabolomics technologies to investigate for correlations between metabolite profiles and markers of iron load and oxidative damage. Methods: 24 subjects were enrolled with a mean age of 34 years (7 TM, 2 thalassemia intermedia (TI), 2 hemoglobin H disease (HbH) and 1 pure red cell aplasia, and 12 age and sex matched controls). Iron load was assessed with serum ferritin, non-transferrin bound iron (NTBI), LIC by magnetic resonance imaging (MRI), and cardiac MRI T2*. Serum and urine samples were collected at baseline, as well as 6 and 12 months. Serum MDA and urinary 8-OHdG were measured and correlated with iron load markers. Mass spectrometry metabolomics data of serum samples were analyzed using supervised multivariate regression techniques to identify relations to markers of iron load, oxidative damage and disease. Results: Serum ferritin and NTBI were significantly higher in the IO group compared to controls (p 〈 0.05). The mean LIC was 8.42 +/− 6.17 mg iron/gm dry weight and mean cardiac T2* was 31 +/− 17.03 ms in the study group. Serum MDA (0.021 +/− 0.01 vs 0.012 +/− 0.003 mmol/g protein, p 〈 0.001) and urinary 8-OHdG (17.26 +/− 8.61 vs 2.49 +/− 1.13 ng/mg Cr, p 〈 0.001) were significantly increased in iron-overloaded patients. Metabolite profiling of baseline serum revealed a significant difference in the IO group compared to controls (p = 0.006, R2 = 0.929). Significantly distinct metabolite profiles reflected the 3 distinct types of thalassemias (TM, TI, HbH) and different chelation therapy regimens. There was a significant relationship between serum metabolite profiles and markers of iron load including serum ferritin, LIC, and cardiac T2* (p 〈 0.05). A significant relationship was seen between serum metabolite profiles and, i) MDA (p = 0.007) and ii) urinary 8-OHdG (p = 0.01). Predictive models identified a profile of 19 different features that were predictive of serum MDA levels, and a profile of 52 unique features that were predictive of serum 8OHdG levels. The relationship of metabolite profiles to oxidative damage markers was more robust when reanalyzed based on chelator status (deferasirox vs non-deferasirox). Conclusion: Markers of oxidative damage are increased in iron overloaded thalassemia patients, with the first report of elevated 8-OHdG in this population. Metabolomics identifies different metabolite profiles between iron overloaded thalassemia patients and controls. Unique serum metabolite profiles show relationships with iron load, markers of lipid and DNA oxidative damage, type of thalassemia, and chelation therapy. These metabolite profiles may have the potential to serve as biomarkers for diagnosis, prognosis, organ damage, and therapeutic response in iron loaded thalassemia patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3168 Background: Peripheral neuropathy (PN) is the dose-limiting toxicity of bortezomib in MM patients, affecting 〉30% patients, including painful PN. Treatment is mostly supportive with narcotics, antidepressants, and anticonvulsants. Acupuncture has been reported to be effective in treating neuropathic pain in diabetic and cancer patients. Methods: We initiated a pilot study to assess the feasibility, safety and efficacy of acupuncture in reducing the severity of BIPN in MM patients. Patients had acupuncture twice weekly for 2 weeks, weekly for 4 weeks, and then biweekly for 4 weeks. All patients continued their prescribed PN medications. Clinical Total Neuropathy Score (TNSc), Functional Assessment of Cancer Therapy/Gynecologic Oncology Group-Neurotoxicity (FACT/GOG-Ntx) questionnaire and Neuropathy Pain Scale (NPS) were used to assess patients at study entry and week 1, 2, 3, 4, 5, 6, 8, 10 during and after 4 weeks of completion of therapy. Serum was obtained from the patients at baseline, right after first acupuncture and before acupuncture session on week 2, 4, 8, and at follow up visit on week 14. Serum samples were used to measure cytokines concentrations. Before and after acupuncture treatments, a complete neurological examination was performed. Nerve conduction studies at baseline and end of study were conducted. Treatment included insertion of disposable sterile acupuncture needles, which retained for 25 minutes, at the following points: bilateral ear points: shen men, point zero, two auricular points where electro-dermal signal was detected, and bilateral body acupuncture points: LI4, SJ5, LI11, ST40, and Ba Feng in upper and lower extremities. Results: From May 17, 2011 to February 28, 2012, 27 MM patients were enrolled in the study. Thirteen patients were women; median age was 63 years (range: 49–77); nine were black, one Asian, 17 Caucasian, and eight had diabetes. All patients had grade 2–4 sensorimotor PN: grade 2 (n=12), grade 3 (n=14), and grade 4 (n=1); 8 had painful PN. All patients had persistant PN despite discontinuation of bortezomib for a median of 19 months (range: 1–83). Eight patients were enrolled within 6 months of stopping bortezomib, all of them had grade 3–4 painful PN. Median time from diagnosis of MM to study entry was 30 months (range: 5–178). Nineteen patients were in remission and eight had progressive disease. MM therapy at study entry and through the follow up period included: revlimid (n=12), carflizomib (n=2). Therapy of PN included narcotics (n=13), gabapentin (12), amitriptyline (n=3), pregabalin (n=2), and duloxetine (n=2). Two patients withdrew after first acupuncture treatment. Twenty-five patients were evaluable for response after completion of 3 weeks (n=2), 6 weeks (n=2), 9 weeks (n=1) and 10 weeks of acupuncture (n=20). There were no adverse events associated with the acupuncture treatment. All but five patients maintained the same dose of pain medications throughout the study. Fourteen patients (56%) reported improved daily functions (e.g. walking and coordination). Ten patients (40%) reported 〉 50% decrease in average NPS during treatment (week 1–10). Seven patients reported 50% reduction in FACT/GOG-Ntx total scores. The decrease in NPS and FACT/GOG-Ntx, was statistically significant between baseline assessments through week 14; p-values are 0.001 and 10% increase in motor nerve amplitude but there was no correlation observed between symptoms/function improvements and nerve conduction studies. At week 8, one of the measured cytokines: Brain-Derived Neurotrophic Factor (BDNF) significantly increased from baseline (p=0.043). This increase was no longer significant at week 14 follow up. Conclusions: Acupuncture is safe and effective in treating persistent moderate to severe BIPN, with improvements of patient reported outcomes, pain and function. The increased BDNF during acupuncture treatment suggests that acupuncture may work through promoting survival and growth of neurons, a lengthy process that may explain the lag of significant objective clinical improvement in the short follow up of our patients. The mechanism of acupuncture working through BDNF to treat PN warrants further evaluation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2600 Background: Older patients diagnosed with acute lymphoblastic leukemia (ALL) receiving intensive induction therapy often suffer from poor outcomes due to therapy-related toxicity and high rates of relapse. We previously reported that patients age ≥60 diagnosed with ALL and treated at our institution with either hyperfractionated cyclophosphamide, vincristine, doxorubicin and dexamethasone alternating with high-dose methotrexate and cytarabine (hyperCVAD) or daunorubicin, vincristine and prednisone (DVP) induction therapies achieved comparable rates of survival. Now with longer follow-up and further analysis, we describe additional outcomes and factors predictive of survival in this patient population. Methods: Thirty-seven patients diagnosed with ALL at age ≥60 and treated at the University of Pennsylvania between July 2003 and June 2011 who received induction therapy with either hyperCVAD (≥1 A+B cycle) or DVP (phase I+II) were analyzed. HyperCVAD was administered as first described at the MD Anderson Cancer Center and DVP per the ECOG 2993/UKALL XII protocol. Therapy adjustments and bone marrow biopsy to confirm remission were performed at the discretion of the treating physician. Almost all Philadelphia chromosome (Ph) positive patients received a tyrosine kinase inhibitor. Event-free survival (EFS) was defined as the time from diagnosis to either relapse or death from any other cause. Results: Table 1 describes baseline characteristics. Table 2 describes outcomes. If achieved, morphologic remission was recognized upon the first bone marrow assessment performed while on therapy, which occurred after a median of 4 (2 A+B) cycles of hyperCVAD and by completion of phase II induction of DVP. EFS and overall survival (OS) trended in favor of DVP. HyperCVAD patients were more likely to complete intensive therapy but less likely to receive maintenance therapy, and more likely to relapse with the majority of relapses occurring off active treatment. Primary reasons for not starting or stopping maintenance therapy were infections and cytopenias. Relapsed disease was the most frequent cause of death. Table 3 describes univariate Cox regression analysis. Receipt of maintenance therapy demonstrated the strongest association with survival (p=0.0001, hazard ratio 0.06 for EFS; p=0.0002, hazard ratio 0.05 for OS). Valid multivariate analysis could not be performed due to small sample size. Conclusions: In older ALL patients treated with aggressive induction therapies and achieving remission, receipt of maintenance therapy appears to be most predictive of EFS and OS. Outcomes in the DVP and hyperCVAD groups were similar although a trend towards prolonged survival in the DVP group was seen, which may be explained by a lower rate of relapse due to a higher likelihood of remaining on therapy over time. Our findings suggest that these patients may benefit from attenuated courses of intensive initial therapy in order to avoid developing toxicities that may prohibit tolerance of prolonged maintenance therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2124 Background: Without effective iron chelation therapy (ICT), patients with transfusional iron overload are at risk of excess iron-related cardiac complications. Cardiac iron accumulation can be measured using T2* magnetic resonance (normal 〉20 ms, high risk 0.9. Results: From 925 screened patients, 197 patients (mean age 19.8 ± 6.4 yrs) were randomized. Mean time since start of transfusions was 19.3 and 18.4 yrs in deferasirox and DFO patients, respectively. All patients had received previous ICT. At BL, Gmean cardiac T2* was 11.4 ms; mean ± SD LIC was 29.8 ± 17.5 mg Fe/g dw in deferasirox patients and 30.3 ± 17.9 mg Fe/g dw in DFO patients; median (range) serum ferritin level was 5062 (613–15331) and 4684 (677–13342) ng/mL, respectively. 160 (81.2%) patients completed 1 yr. Mean actual dose of deferasirox was 36.7 ± 4.2 mg/kg/d and DFO was 41.5 ± 8.7 mg/kg/d for 7 d/wk. Overall, Gmean cardiac T2* increased by 12% with deferasirox and 7% with DFO after 1 yr (Fig A). The Gmean ratio between the two arms was 1.0557 (95% CI 0.9981, 1.1331). Lower limit of the 95% CI was 〉0.9, demonstrating non-inferiority of deferasirox vs DFO, with a trend towards superiority (P=0.0567). Trends toward increases were observed in patients with severe or mild/moderate cardiac iron (Fig B, C). In patients with BL LIC upper limit of normal (ULN). Overall, 14.6% of deferasirox patients and 3.3% of DFO patients had ALT levels 〉5xULN and 〉2xBL. One death (arrhythmia) in the deferasirox arm was considered unrelated to study drug. One death (meningitis) in a DFO patient was suspected to be related to DFO. Discussion: CORDELIA, the first randomized controlled trial comparing deferasirox with DFO for cardiac iron removal, met its primary endpoint in demonstrating non-inferiority of deferasirox vs DFO, with a trend for superiority. There was a trend toward more pronounced improvements in cardiac T2* with deferasirox vs DFO in patients with BL LIC

Description: Abstract 1023 Introduction. Pulmonary hypertension (PH) is a complication associated with thalassemia syndromes, particularly thalassemia intermedia. There are limited data on the safety and efficacy of selective pulmonary vasodilators in this at-risk population. Methods. We evaluated the safety and efficacy of a 12-week prospective, phase 1/2 pilot scale study of sildenafil (100 mg TID) in ten b-thalassemia patients with elevated TRV on Doppler-echocardiography ≥ 2.5 m/s suggestive of PH risk. Patients were evaluated at baseline and at weeks 2, 4, 8 and 12 of sildenafil therapy, and six minute walk distance (6MWD), biomarkers of hemolysis, coagulation, inflammation and adhesion were assessed. Results. Our study population had an average age of 37±12.3 years, 8/10 were male, and 50% were thalassemia intermedia. Splenectomy prevalence was high (90%), and only 30% of patients were transfused since infancy. The mean pre-transfusion hemoglobin was 10.4±1.5 g/dL. A 12-week open-label trial resulted in a significant decrease in TRV by 13.3% (3.0±0.7 vs. 2.6±0.5m/s, p=0.04, Figure 1), improvements in left ventricular end systolic/diastolic volume (p≤0.02), diffusion capacity for carbon monoxide (DLCO, p=0.003) and a trend towards a reduced Borg Dyspnea Score and improved NYHA Functional class. No significant change in 6MWD was noted, although 6MWD correlated strongly with DLCO (ρ=0.72, p=0.03) suggesting that oxygen diffusion across the alveolar-capillary membrane was an important determinant of exercise capacity. Sildenafil was generally well tolerated, but most patients experienced anticipated transient headaches and visual/color disturbances associated with sildenafil use. One patient withdrew from the study due to worsening dyspnea. No other serious adverse events were reported. A strong direct correlation between total dose of sildenafil (mg) taken and % change in plasma NO metabolite concentration was observed (ρ =0.80, p=0.01). A significant increase in plasma and erythrocyte arginine concentration occurred, without an associated change in plasma arginase activity/concentration, nitric oxide metabolites or vascular endothelial growth factor. However arginase concentration was elevated in this cohort similar to prior reports, and correlated inversely to hemoglobin (ρ=-0.41, p=0.01), and directly to ALT (ρ=0.40, p=0.004), AST (ρ=0.38, p=0.04), left ventricular end systolic volume (ρ=0.77, p=0.001), and end-diastolic volume (ρ=0.79, p=0.001). Conclusion. Our study suggests that sildenafil is safe and may improve cardiopulmonary hemodynamics in patients at risk for PH, however improved exercise capacity as reflected by an improved 6MWD was not observed. The reduction in left ventricular dimensions is promising, and could reflective of either increased inotropy or chronotropy, or decreased systemic afterload. This is also the first report of an influence of sildenafil on diffusion capacity of the lungs in patients with thalassemia and the first description of increased plasma and erythrocyte arginine concentration after sildenafil therapy. Given the association of arginine bioavailability with long-term survival in cardiovascular disease, this is an unexpected effect of sildenafil that warrants further investigation. These data support the need for further clinical trials evaluating the use of sildenafil in thalassemia. Disclosures: Taher: Novartis: Research Funding, Speakers Bureau.

Description: Abstract 2379 The enzymatic activity of the protein methyltransferase (PMT) DOT1L has been shown to be a driver of cell proliferation in MLL-rearranged leukemia. Our group has previously reported the design of potent and selective aminonucleoside inhibitors of DOT1L [Daigle et al. (2011) Cancer Cell 20: 53–65; Basavapathruni et al. (2012) Chem. Biol. Drug Design, in press]. Structure-guided design, together with robust biochemical and biological assays, was used to optimize the potency, selectivity and pharmacological features of the aminonucleosides, resulting in the compound EPZ-5676. EPZ-5676 is an S-adenosyl methionine (SAM) competitive inhibitor of DOT11L that displays a Ki value of 80 pM and a drug-target residence time of 〉 24 hours. The compound is highly selective for DOT1L, demonstrating 〉 37,000-fold selectivity against all other PMTs tested. Crystallographic studies reveal that the high affinity, durable inhibition of DOT1L by EPZ-5676 has its origin in a conformational adaptation of the protein that attends inhibitor binding, extending the compound binding pocket to include novel recognition elements beyond the SAM binding active site. Treatment of leukemia cells with EPZ-5676 results in concentration- and time-dependent reduction of H3K79 methylation without effect on the methylation status of other histone sites. The reduction of H3K79 methylation leads to inhibition of key MLL target genes and selective, apoptotic cell killing in MLL-rearranged leukemia cells, but has minimal impact on non-rearranged cells. EPZ-5676 is highly soluble in aqueous solution and can thus be formulated for intravenous administration. The effective pharmacokinetic half-life of EPZ-5676 in systemic circulation has been measured to be 0.25 and 1.5 h in rats and dogs, respectively. A nude rat subcutaneous xenograft model of MLL-rearranged leukemia has been established. Continuous intravenous infusion of EPZ-5676 for 21 days in this model leads to dose-dependent anti-tumor activity. At the highest dose, complete tumor regressions are achieved with no regrowth for up to 32 days after the cessation of treatment (Figure 1). Figure 1. EPZ-5676 causes complete and sustained tumor regression in a MV4–11 nude rat xenograft model of MLL-rearranged leukemia. No significant weight loss or obvious toxicity was observed in rats treated with EPZ-5676 during this efficacy study. EPZ-5676 is thus a potent, selective inhibitor of DOT1L that demonstrates strong efficacy in a rat xenograft model of MLL-rearranged leukemia. Details of the preclinical characterization of this compound will be presented. Figure 1. EPZ-5676 causes complete and sustained tumor regression in a MV4–11 nude rat xenograft model of MLL-rearranged leukemia. . / No significant weight loss or obvious toxicity was observed in rats treated with EPZ-5676 during this efficacy study. EPZ-5676 is thus a potent, selective inhibitor of DOT1L that demonstrates strong efficacy in a rat xenograft model of MLL-rearranged leukemia. Details of the preclinical characterization of this compound will be presented. Disclosures: Pollock: Epizyme: Employment, Equity Ownership. Daigle:Epizyme: Employment, Equity Ownership. Therkelsen:Epizyme: Employment, Equity Ownership. Basavapathruni:Epizyme: Employment, Equity Ownership. Jin:Epizyme: Employment, Equity Ownership. Allain:Epizyme: Employment, Equity Ownership. Klaus:Epizyme, Inc.: Employment, Equity Ownership. Raimondi:Epizyme: Employment, Equity Ownership. Porter Scott:Epizyme: Employment, Equity Ownership. Chesworth:Epizyme: Employment, Equity Ownership. Moyer:Epizyme: Employment, Equity Ownership. Copeland:Epizyme Inc.: Employment, Equity Ownership. Richon:Epizyme, Inc.: Employment, Equity Ownership. Olhava:Epizyme: Employment.

Description: Abstract 3576 Eltrombopag, a non-peptide, small molecule, thrombopoietin receptor agonist, has been shown to increase platelet counts in patients with idiopathic thrombocytopenic purpura (ITP), hepatitis C associated thrombocytopenia and severe aplastic anemia. Pre-clinical studies show that eltrombopag stimulates megakaryopoiesis in bone marrow samples from patients with acute myelogenous leukemia (AML) and inhibits leukemic cell growth. The clinical use of eltrombopag in the AML patient population has not been previously explored. We report the results of an investigator-initiated, phase I dose escalation clinical trial to evaluate the safety and efficacy of eltrombopag monotherapy in older patients with AML. Eligible patients were ≥ 60 years old with non-M3 AML and a baseline platelet count ≤75,000/ul. A standard 3+3 design was used employing dose escalation in 4 sequential cohorts of 50mg, 100mg, 200mg and 300mg of eltrombopag daily. Subjects who did not complete at least one cycle of therapy (4 weeks) or experience a dose-limiting toxicity (DLT) were replaced. The primary endpoint was safety. Secondary endpoints included platelet and disease response. Bone marrow biopsies to evaluate for disease response and fibrosis were planned at one month, 3 months and 6 months. Twenty-three subjects were enrolled from June 2010 to February 2012 at the Hospital of the University of Pennsylvania and received at least one dose of study drug. The median patient age was 73 (range 60–86). Twenty-one of 23 subjects were relapsed or refractory after prior therapy. Two of 23 subjects (ages 85 and 86) were previously untreated. Nineteen subjects were platelet transfusion-dependent. Thirteen subjects had a history of antecedent myelodysplastic syndrome (MDS) and 6 were requiring hydroxyurea at time of study entry. Fourteen of 23 subjects completed the first cycle of therapy. Three remained on study for the entire 6-month study period. At the time of this report one subject remains on eltrombopag in an extension phase (8+ months of therapy). Indications for drug cessation included: possible DLT=1; progressive disease=5; death due to sepsis=5; lack of response=6, loss of response=1; and pursuit of hospice or alternative therapy=4. Overall eltrombopag was well tolerated at all doses studied and a maximally tolerated dose (MTD) was not reached. One subject at the 300mg dose level developed Grade 4 hepatic laboratory abnormalities possibly related to study drug, which met criteria for a DLT. The subject had concurrent polymicrobial sepsis and subsequently died. No other grade 3 or greater liver function abnormalities were observed. Asymptomatic skin discoloration with associated abnormal serum pigmentation was noted in subjects at the 200mg and 300mg dose levels. There were no platelet responses in the 50 mg or 100 mg cohorts (7 subjects total). Two of 7 subjects treated at the 200mg dose level and 2 of 9 subjects treated at the 300mg dose level achieved a platelet response (25% platelet response rate at these higher dose levels). All 4 of these subjects became platelet transfusion independent. The duration of platelet responses were 5 weeks, 12 weeks, 6 months and 8+ months (ongoing at time of this report). One subject with primary refractory AML, associated with monosomy 7, obtained a complete morphologic and cytogenetic response by 3 months of therapy at the 300mg dose level. This response was sustained at most recent bone marrow biopsy at 6 months of therapy and the patient remains on study drug. Increased reticulin fibrosis was also observed in this subject over time. No other subjects experienced a PR or CR. 6 patients had stable disease (SD) at one month, which was maintained at 3 months for 4 patients. Our study shows that eltrombopag is well tolerated in patients with AML at doses well above those typically used to treat ITP. At higher doses, we observed clinically meaningful platelet and antileukemic responses. Further exploration of eltrombopag as both a platelet agonist and disease-modifying agent in the treatment of AML is justified. Disclosures: Frey: GSK: Consultancy, Research Funding. Porter:Novatis: Patents & Royalties; Celgene: Honoraria; Genentech: Employment; Pfizer: Research Funding. Perl:Astellas: Consultancy. Carroll:GlaxoSmithKlein: Research Funding.

Description: Abstract 143 Background: Mammalian target of rapamycin (mTOR) inhibitors enhance cytotoxic chemotherapy effects in primary acute leukemia cells in preclinical assays. This prompted a multi-center evaluation of a combination of mTOR inhibitor plus induction chemotherapy in AML. As mTOR is frequently but not uniformly activated in primary AML samples, it is unclear which patients benefit from this targeted approach. Thus, we sought to monitor mTOR kinase activity during therapy to determine whether target activation and/or inhibition predicted clinical response. We previously reported our preliminary experience monitoring pS6 in AML blasts by flow during clinical trials combining sirolimus and AML induction chemotherapy (Kasner et al, ASH 2011, #230). Here we provide the final clinical and pharmacodynamic results from this cohort of subjects. Methods: Subjects had relapsed/refractory AML or untreated AML with unfavorable risk factors (e.g. therapy-related, prior MDS or MPN, or age 〉60 without favorable karyotype) with a median age of 60.5 years (range 32–77). Subjects received oral sirolimus (12 mg on day 1, then 4 mg daily on days 2–9) plus MEC (mitoxantrone 8 mg/m2/day, etoposide 100 mg/m2/day, cytarabine 1 gm/m2/d on days 4–8) on one of two successive clinical trials. Clinical response was assessed at hematologic recovery or day 42 using IWG criteria (CR, CRp, PR vs. non-response). Pharmacodynamic samples were collected from blood or marrow at baseline, 2 hours post-sirolimus dose on days 1 and 4, and at trough on day 4 (prior to chemotherapy administration). Concurrent blood rapamycin concentration was measured by immunoassay or HPLC. Whole blood/marrow fixation was performed using published methods (Perl, et al. Clin. Cancer Res. 2012). Positive gates for pS6 were created by comparing blasts in ex vivo stimulated (phorbol ester/PMA) and inhibited (rapamycin) conditions and/or autofluorescence (FMO) controls. Results: We enrolled 52 subjects in 2 consecutive trials; 51 were evaluable for clinical response. Toxicity was similar to published MEC data. 3 infectious deaths occurred (6%). Prolonged aplasia was not observed. 24/51 (47%) subjects responded, with 18 CR (35%), 1 CRp, and 5 PR's observed. Mean peak and trough rapamycin concentrations on day 4 were 22.0 and 8.9 ng/ml, respectively, and did not differ among clinically responding or non-responding subjects. Median survival time for the whole group was 243 days (longest follow up 1584 days). Among the 24 subjects achieving CR or PR, median duration of time to the first event (relapse or death) was 261 days. 20 subjects were able to proceed to a stem cell transplant following therapy. Serial flow cytometric analysis was performed in 46 subjects, of which 37 provided paired day 1 and day 4 flow samples and were evaluable for clinical response at count recovery. The overall response rate (ORR) among subjects with baseline constitutive pS6 was 14/27 (52%, 9 CR, 1 CRp, 4 PR). The ORR for subjects without constitutive pS6 was 4/10 (40%, 3 CR, 1 PR). Subjects with 〉50% reduction in pS6 positive blasts on day 4 were considered to be biochemically sensitive to rapamycin, while subjects with