High-risk Acute Myelogenous Leukemia Research Articles

Clofarabine Followed By Lenalidomide for Treatment of High Risk Myelodysplastic Syndromes and Acute Myeloid Leukemia- Preliminary Clinical Outcomes and Potential Immune-Modulating Effects

Introduction:High risk myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) commonly affect individuals of advanced age with multiple co-morbidities unsuited for curative standard treatments with allogeneic stem cell transplantation (allo-SCT) or intensive chemotherapy (ICT). New reduced intensity therapeutic approaches bringing improved life expectancy are needed for such patients. We conducted a phase I clinical trial to evaluate the safety and activity of sequential therapy with low-dose clofarabine followed by lenalidomide in older high risk MDS or AML. We hypothesized that lympho-depletion by clofarabine could "reboot" lymphocyte reactivity against the malignancy and promote favorable immune modulation by lenalidomide. Here we report preliminary clinical outcomes and associated cellular and molecular immune profiles.Methods:Four subjects (median age 68 years, range-60-79) with relapsed or refractory high-risk MDS (IPSS risk score>intermediate 2) or AML were enrolled to the protocol (12-H-0146, clinicaltrials.gov ID: NCT01629082). All subjects had been previously treated with at least one hypomethylating agent. Subjects received a single course of intravenous low-dose clofarabine 5mg/m2/d for five days, followed by consolidation therapy with oral lenalidomide with dose escalation from 25 mg daily up to 50 mg daily for 2 cycles. In the absence of dose limiting toxicity (DLT) or disease progression, subjects received lenalidomide maintenance 10 mg daily in 28 day cycles, with dose adjustments, for up to 12 cycles. Blood and marrow samples were collected before starting clofarabine, before consolidation with lenalidomide, and during maintenance. Multiparameter flow cytometric analysis was performed to characterize T cell subsets (memory T cells, T regs), natural killer (NK) cells, with functional markers representing T cell exhaustion (PD-1, LAG-3, TIM-3, PDL-1), activating and inhibitory NK cell receptor (KIR, LIR1, NKG2A, CD57). Relative changes in RNA expressions of target genes related to cancer immunology were evaluated by PCR array.Results:Of four subjects enrolled, two subjects showed responses at 28 days post clofarabine and at 28 days post first cycle of lenalidomide. These two responders tolerated subsequent maintenance lenalidomide for 4-6 months. The other two subjects were non-responders. Three subjects were taken off study due to disease progression on day 70, 162, and 206, and 1 subject for DLT on day 69. The clinical trial is now closed without dose escalation beyond the first cohort for reasons of poor accrual and lack of long term responses. In responders immunophenotype analysis showed a decrease in CD4+ %PD-1 expression in blood or bone marrow (0.87±0.29 fold change) after clofarabine and lenalidomide treatment, rising again after lenalidomide discontinuation in one responder (Figure). In contrast, CD4+ %PD-1 expression was increased in non-responders (1.23±0.19 fold change). In pre-treatment samples terminally differentiated CD57+ NK cell with high LIR1 expression (34.6±23.5%) were increased. In responders, both the levels of CD57 and LIR1 expression fell with reciprocal increase in CD56brightNK cells, suggesting restoration of NK cell repertoires after clofarabine. RNA expression profiles in one responder on maintenance lenalidomide showed significant up-regulation of gene expressions in IL-1A, FASLG, ICAM1, IL-27, WT1 (p<0.01) in comparison to the pre-treatment sample.Conclusion:Sequential treatment of low-dose clofarabine and lenalidomide is feasible for elderly patients with high risk MDS and AML. However individuals vary in the lenalidomide dose tolerated. Immune reconstitution profiles supports a potential immune-rebooting effect of clofarabine leading to restoration of CD4 cell and NK cell repertoires with fewer exhaustion or inhibitory markers. Lenalidomide may further promote a favorable anti-leukemic immune milieu. Immune profiling of T and NK cells can help to guide the application of these immune-modifying agents in high risk MDS and AML. [Display omitted] DisclosuresBattiwalla:NIH/NHLBI: Employment.

HLA-Mismatched Stem Cell Microtransplantation Compared to Matched-Sibling Donor Transplantation for Acute Myeloid Leukemia in First Complete Remission

HLA-mismatched stem cell microtransplantation is a new transplantation which has been reported in recent years. We treated 41 patients with intermediate- or high-risk acute myelogenous leukemia (AML) in first complete remission (CR1) undergoing HLA-mismatched stem cell microtransplantation and compared with 56 patients undergoing HLA-matched sibling donor (MSD) transplantation at the same period in our center from July 2012 to August 2015. The estimated leukemia-free survival (LFS) at 2-year was 65.6% ± 9.5% in the MSD group and 27.6% ± 14.0% in the microtransplantation group (P = 0.011). The estimated overall survival (OS) at 2-year was 73.6% ± 8.6% in the MSD group and 46.2% ± 15.6% in the microtransplantation group (P = 0.056). The cumulative incidences of relapse were 22.5% and 58.7% among MSD and microtransplantation groups (P = 0.011). The cumulative incidence of nonrelapse mortality was 7.8% in MSD group, and no nonrelapse mortality in microtransplantation group (P = 0.265). Hematopoietic recovery time was shorter in the microtransplantation group than MSD group (P < 0.05). The infection rate of MSD group was higher than in the microtransplantation group (91.1% vs 75.6%, P = 0.037). The preliminary results suggest that LFS of microtransplantation is inferior to MSD transplantation and a trend toward the OS of microtransplantation was lower than MSD transplantation for intermediate- or high-risk AML in CR1. DisclosuresNo relevant conflicts of interest to declare.

TP53 Mutations Determine Outcome of Patients (pts) with High-Risk Myelodysplastic Syndrome (HRMDS) and Acute Myelogenous Leukemia (AML) Regardless of Karyotype and ELN Risk Stratification

Background: Cytogenetic and molecular abnormalities are the backbone of risk stratification for pts with AML/MDS. Yet, the current stratification systems still fail to identify some pts at high risk for treatment failure. TP53 mutations have been linked to poor outcome and treatment resistance in many malignancies. We conducted a retrospective case-control study of pts with HRMDS and AML with TP53 mutations (TP53 m) to define disease characteristics, response, and outcome after transplant (HSCT).Methods: Twenty-one consecutive pts with HRMDS or AML were treated between January 2014 and March 2016 who wereTP53 m. The pts were compared to a control cohort of 46 pts with wild type TP53 (TP53 wt). Mutation analysis was performed using next generation sequencing. All AML pts were treated with a standard 3+7 induction protocol. Pts were excluded for incomplete cytogenetic and molecular data.Results: A total of 21 patients were identified with TP53 m (14 HRMDS, 11 AML). The median age of the 21 pts was 64 years (range 32-84). Pts with TP53 m were older (median age 64 vs 55 years, p=.0256) and exhibited a higher rate of adverse cytogenetics (71% vs 30.4%, p=.043) when compared to TP53 wt. Secondary AML was also more common with TP 53 m (57% vs 31%, p=.05). Pts with TP53 m were less likely to achieve CR (33% vs 74%, p=.009). Three pts with TP53 m proceeded to transplant (2 with HRMDS and 1 with AML).With a median follow up of 3.4 months (range 0.3-73) the estimated PFS and OS at 18 months was superior in TP 53 wt compared to TP 53 m (17% vs 69%, p=.037 and 17% vs 50%, p=.0095 respectively). HSCST was associated with a 70% reduction in mortality, but this was not statistically significant (p=.067).Conclusion: TP 53 mutations in pts with HRMDS and AML are associated with advanced age, high risk cytogenetic abnormalities, and secondary AML. Our findings demonstrate that TP53 m in HR MDS and AML is associated with poor clinical outcome including lower CR rates, and lower PFS and OS. Different treatment strategies need to be devised for these pts. DisclosuresMcCloskey:Incyte: Consultancy; Ariad: Consultancy, Speakers Bureau; Amgen: Speakers Bureau; Novartis: Speakers Bureau. Koprivnikar:Alexion: Consultancy, Speakers Bureau. Depadova:Amgen: Speakers Bureau. Stanislaus:Novartis: Consultancy, Speakers Bureau. Goldberg:COTA Inc: Employment; Bristol Myers Squibb, Novartis: Speakers Bureau; Neostem: Equity Ownership; Novartis: Consultancy; Pfizer: Honoraria. Faderl:Celgene: Consultancy, Research Funding; JW Pharma: Consultancy; BMS: Research Funding; Ambit Bioscience: Research Funding; Karyopharm: Consultancy, Research Funding; Celator Pharmaceuticals: Research Funding; Astellas: Research Funding; Pfizer: Research Funding; Seattle Genetics: Research Funding; Amgen: Speakers Bureau.

Haploidentical Natural Killer Cells Infused before Allogeneic Stem Cell Transplantation for Myeloid Malignancies: A Phase I Trial

Allogeneic stem cell transplantation is an effective treatment for high-risk myeloid malignancies, but relapse remains the major post-transplantation cause of treatment failure. Alloreactive natural killer (NK) cells mediate a potent antileukemic effect and may also enhance engraftment and reduce graft-versus-host disease (GVHD). Haploidentical transplantations provide a setting in which NK cell alloreactivity can be manipulated, but they are associated with high rates of GVHD. We performed a phase I study infusing escalating doses of NK cells from an HLA haploidentical–related donor—selected for alloreactivity when possible—as a component of the preparative regimen for allotransplantation from a separate HLA-identical donor. The goal of infusing third-party alloreactive NK cells was to augment the antileukemic effect of the transplantation without worsening GVHD and, thus, improve the overall outcome of hematopoietic transplantation. Twenty-one patients with high-risk acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or chronic myelogenous leukemia refractory or beyond first remission received a preparative regimen with busulfan and fludarabine followed by infusion of apheresis-derived, antibody-selected, and IL-2–activated NK cells. Doses were initially based on total nucleated cell (TNC) content and later based on CD56+ cells to reduce variability. CD56+ content ranged from .02 to 8.32 × 106/kg. IL-2, .5 × 106 units/m2 subcutaneously was administered daily for 5 days in the final cohort (n = 10). CD3+ cells in the NK cell product were required to be < 105/kg. Median relapse-free, overall, and GVHD-free/relapse-free survival for all patients enrolled was 102, 233, and 89 days, respectively. Five patients are alive, 5 patients died of transplantation-related causes, and 11 patients died of relapse. Despite the small sample size, survival was highly associated with CD56+ cells delivered (P = .022) and development of ≥ grade 3 GVHD (P = .006). There were nonsignificant trends toward higher survival rates in those receiving NK cells from KIR ligand–mismatched donors and KIR-B haplotype donors. There was no association with disease type, remission at time of transplantation, or KIR content. GVHD was not associated with TNC, CD56+, or CD3+ cells infused in the NK cell product or the stem cell product. This trial demonstrates a lack of major toxicity attributable to third-party NK cell infusions delivered in combination with an HLA-compatible allogeneic transplantation. The infusion of haploidentical alloreactive NK cells was well tolerated and did not interfere with engraftment or increase the rate of GVHD after allogeneic hematopoietic transplantation. Durable complete remissions occurred in 5 patients at high risk for disease recurrence. This approach is being further developed in a phase I/II trial with ex vivo–expanded NK cells to increase the NK cell dose with the objective of reducing relapse and improving the outcome of allogeneic hematopoietic transplantation for AML/MDS.

Molecular Pathways: Mitochondrial Reprogramming in Tumor Progression and Therapy.

Small-molecule inhibitors of the phosphoinositide 3-kinase (PI3K), Akt, and mTOR pathway currently in the clinic produce a paradoxical reactivation of the pathway they are intended to suppress. Furthermore, fresh experimental evidence with PI3K antagonists in melanoma, glioblastoma, and prostate cancer shows that mitochondrial metabolism drives an elaborate process of tumor adaptation culminating with drug resistance and metastatic competency. This is centered on reprogramming of mitochondrial functions to promote improved cell survival and to fuel the machinery of cell motility and invasion. Key players in these responses are molecular chaperones of the Hsp90 family compartmentalized in mitochondria, which suppress apoptosis via phosphorylation of the pore component, Cyclophilin D, and enable the subcellular repositioning of active mitochondria to membrane protrusions implicated in cell motility. An inhibitor of mitochondrial Hsp90s in preclinical development (gamitrinib) prevents adaptive mitochondrial reprogramming and shows potent antitumor activity in vitro and in vivo. Other therapeutic strategies to target mitochondria for cancer therapy include small-molecule inhibitors of mutant isocitrate dehydrogenase (IDH) IDH1 (AG-120) and IDH2 (AG-221), which opened new therapeutic prospects for patients with high-risk acute myelogenous leukemia (AML). A second approach of mitochondrial therapeutics focuses on agents that elevate toxic ROS levels from a leaky electron transport chain; nevertheless, the clinical experience with these compounds, including a quinone derivative, ARQ 501, and a copper chelator, elesclomol (STA-4783) is limited. In light of this evidence, we discuss how best to target a resurgence of mitochondrial bioenergetics for cancer therapy.

Azatidine Treatment in Very Elderly Patients Diagnosed with Acute Myelogenous Leukemia (AML) or High Risk Myelodysplastic Syndrome (MDS)

Background: The use of azatidine (AZT) is the agent of choice for first line treatment of elderly (age >74 years) patients (pts) with high risk MDS or AML (2015 National Comprehensive Cancer Network Guidelines). The Hematology consultant is frequently involved in the care of very elderly (age >79 years) individuals. Our aim is to report our experience with 3 patient aged 80 or above that received treatment with AZT.Methods: Retrospective reviewResults: We reviewed the medical records of patients treated for high risk MDS or AML at our institution between July 2009 and March 2012. A total of 3 pts aged 80 or above were identified. All three pts were males, and all received AZT 100 mg/m2/day x 7 days subcutaneously in every cycle of 28 days. Patient 1 (aged 81) had 12 % of blasts in the bone marrow (BM), was diagnosed with Refractory Anemia with Excess Blasts-2 (RAEB-2) applying the 2008 World Health Organization Classification (WHO) of MDS. Patient 2 (aged 90) had AML with multilineage dysplasia and 38% blasts in BM. Patient 3 (aged 80) had AML with multilineage dysplasia and 30% blasts in BM. Pts characteristics and results are summarized in Table 1.Conclusions: In pts diagnosed with high risk MDS or AML, advanced chronological age (> 79 years) alone should not preclude the use of effective and well tolerated therapy. Identification of pts in this geriatric subgroup who may benefit from AZT merits additional research.Table 1DiagnosisAge(years)Baseline laboratory Peripheral blood%Blast in Bone MarrowNumber of AZT cycles receivedResponseSurvival(months)Patient 1RAEB-281Hb 14 g/dl Platelets 56x10e9/L Leucocytes 1.90x10e9/L (Neutrophils 0.81x10e9/L)1211CR* in BM after 6 cycles17Patient 2AML90Hb 8.06 g/dl Platelets 20x10e9/L Leucocytes 1.30x10e9/L(Neutrophils 0.64x10e9/L)3815CR in BM after 7 cycles22Patient 3AML80Hb 7.50 g/dl Platelets 30x10e9/L Leucocytes 6.00x10e9/L(Neutrophils 1.44x10e9/L)3019CR in BM after 6 cycles23*CR: complete remission (<5% blast in aspirate with spicules) DisclosuresNo relevant conflicts of interest to declare.

Preliminary Results of a Phase 1/2 Clinical Trial of Cndo-109-Activated Allogeneic Natural Killer Cells in High Risk Acute Myelogenous Leukemia Patients in First Complete Remission

BACKGROUNDWe previously reported that resting human peripheral blood NK cells can be primed to kill NK-resistant tumor cells by co-incubation with a lysate of the leukemia cell line CTV-1. CNDO-109 is a clinical-grade CTV-1 lysate that primes NK cells ex-vivo to kill NK-resistant acute myeloid leukemia (AML) cells. CNDO-109-activated NK cells can be cryopreserved and remain primed when thawed. Incubation or treatment with IL-2 is not required. We report preliminary safety, outcome and NK chimerism data from an ongoing Phase 1/2 transitional clinical trial of CNDO-109-NK cells.METHODSA 3x3 dose escalation phase 1 trial was opened in 2013 for patients with AML in 1st CR with high-risk disease and no conventional treatment options. Patients were given preparative chemotherapy of cyclophosphamide and fludarabine on Study Days -6 to -2, followed on day 0 by a single dose of CNDO-109-activated NK cells at the following doses; cohort 1 = 3×105, cohort 2 = 1×106, cohort 3 = up to 3×106 cells/kg recipient body weight. The MTD will set the dose for the transitional phase 2 trial.CNDO-109-NK cells were generated from a single apheresis collection from HLA-haploidentical related donors. NK cells were isolated with anti-CD56 microbeads (CliniMACS, Miltenyi Biotec) and co-incubated with CNDO-109 lysate (Coronado Biosciences) overnight under cGMP conditions. After lysate removal cells were cryopreserved in dosed aliquots and released for infusion. Exogenous IL-2 was not used. Quality control testing of the final product included NK purity (>50%), viability (> 70%), potency, sterility, mycoplasma and endotoxin. Residual T cell contamination (<104 cells/kg patient body weight) was a lot release safety criterion. Patients were assessed for safety and efficacy whilst expansion, proliferation and persistence of donor NK cells were assayed by molecular-based chimerism techniques, and by flow cytometry, where informative markers were available. Pre- and post-treatment samples from cohort 3 patients are being tested for flow cytometric and molecular MRD (University of Washington - Walter, R.B.2013 and NextGene Sequencing).RESULTSSeven eligible patients have been enrolled. All products met lot release and contained activated NK cells as determined by killing of NK resistant Raji cells as well as increased expression of CD69 & CD25: TableBaseline(Resting NK Cells)Activated NK CellsChangep-valueRaji Cell Lysis15.2%+/- 10.0%29.7% +/- 13.9%0.008CD69+ Expression5.9% +/- 2.9%42.6% +/- 12.5%0.008CD25+ Expression3.1% +/- 1.8%5.8% +/- 1.9%0.008No infusional toxicity, adverse events attributed to NK therapy, GvHD nor deaths have been reported. As expected, all patients experienced transient myelosuppression (approx. 2 weeks). 3 patients suffered early relapse post-treatment (2 in cohort 1, 1 in cohort 2; average time to relapse from CR1 for these 3 patients was 104 days).In 5 of 7 evaluable patients, persistence of donor activated NK cells was observed from Day +7 post-infusion (chimerism = 1%-84%) to as late as day +56 in one patient. Flow cytometric comparison of donor NK cells and patient NK cells in the same sample by selective gating on the mismatched HLA allele showed that circulating donor NK cells typically differed from recipient NK cell in higher expression of NKG2A (e.g. 63.5% vs 2.7%), CD57 (e.g. 80.5% vs 13.2%) and CD69 (e.g. 11.6% vs 2.52%) suggesting a more normal, mature and activated phenotype than the endogenous host NK population.Even after loss of circulating donor primed NK cells, 2 of the 3 patients tested showed persistence of low levels of activated autologous NK cells (~10-20% of circulating NK) exceeding the numbers circulating pre-CNDO-109 NK treatment, out to Day +56, suggesting that the therapy may induce endogenous NK activation to enhance the patients’ innate immunity to AML.CONCLUSIONSThis establishes proof of concept that CNDO-109-NK can persist transiently in patients with lasting microchimerism for > 1 month and can induce activation of endogenous NK cells in patients treated without cytokine administration. To date 4 of the 7 patients enrolled remain relapse free (max. = 410 days post CR1 date). These results highlight the potential of CNDO-109-NK cells in the treatment of AML and other malignancies. DisclosuresMiller:Coronado Biosciences: Consultancy. Hillman:Coronado Biosciences: Employment. Silver:Coronado Biosciences: Employment. Szarek:Coronado Biosciences: Consultancy. Lowdell:Coronado Biosciences: Consultancy, Patents & Royalties. Gorelik:Coronado Biosciences: Employment. Rowinsky:Coronado Biosciences: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

The Challenge of t (6;9) and FLT3-Positive Acute Myelogenous Leukemia in a Young Adult.

Translocation t(6;9) is a rare cytogenetic abnormality found in fewer than 5% of pediatric and adult cases of acute myelogenous leukemia (AML). The outcomes of t(6;9) AML are generally poor, with low five-year overall survival and increased risk for relapse. Furthermore, FLT3-ITD is one of the most common molecular abnormalities found in AML that is associated with increased risk of treatment failure and mortality. Allogeneic hematopoietic cell transplantation (HCT) with the best available donor is a standard treatment option for these cases once remission is achieved.We report a challenging case of t(6;9) and FLT3-positive AML in a young adult male. After failing multiple standard induction regimens, morphologic remission was eventually achieved with a FLT3 inhibitor (sorafenib) and a hypomethylating agent (azacytidine). However, despite allogeneic HCT and re-initiation of sorafenib in the post-HCT setting, he experienced early relapse with the original [FLT3-ITD and t(6;9)] and new (FLT3-D835 and +8) molecular and cytogenetic markers, respectively. This case highlights the need for improved strategies in the post-HCT setting for high-risk AML.

High-risk acute myelogenous leukemia: treatment today … and tomorrow

High-risk acute myelogenous leukemia (AML) constitutes a distinct subset of disease based on clinical and biological characteristics and comprises a significant percentage of all cases of adult AML. Biologic features such as distinct clonal cytogenetic and molecular abnormalities identify a subgroup of AML patients characterized by poor response to induction chemotherapy and poor long-term survival after treatment with consolidation chemotherapy. Clinical variables that predict for poor response include AML relapsed after less than 1 year of remission and AML characterized by resistance to conventional agents. We review here our understanding of the defining biologic subtypes of AML and discuss how adequate initial evaluation can be used to inform the choice of treatment. By defining high-risk biologic and clinical variables, a strong case can be made for treating patients with investigational agents, with treatment directed at distinct cytogenetic or molecular abnormalities. Allogeneic transplantation is the only form of therapy available outside of the setting of a clinical trial that may offer a chance for long-term survival for patients with high-risk AML.

Hematopoietic Cell Transplantation in High-Risk Childhood Acute Myelogenous Leukemia

In this issue of Biology of Blood and Marrow Transplantation, Burke et al. present their findings on the efficacy of allogeneic hematopoietic stem cell transplantation (HCT) in patients with high-risk childhood acute myelogenous leukemia (AML). This limited single-institution study demonstrates that patients with high-risk disease (as defined by cytogenetics/molecular markers) who received allogeneic HCT in first compete remission (CR) have a favorable outcome, similar to those with standard-risk disease. The role of HCT as consolidation therapy for childhood AML has evolved significantly since Woods et al. first demonstrated that children who received allogeneic HCT from related donors had an improved outcome compared with chemotherapy recipients [1]. This study further demonstrated that pretransplantation chemotherapy can affect posttransplantation survival; patients who received more intensive induction chemotherapy had a significantly lower rate of relapse and more favorable survival after HCT, suggesting that quality of remission (lower residual disease) was an important determinant of post-HCT survival. In this study, although HCT carried higher treatment-related mortality, lower relapse resulted in more favorable outcomes in HCT recipients. This seminal study led to the change in the standard of care in childhood AML in the United States, where patients with newly diagnosed AML with suitably matched related donors were allocated to receive HCT in first CR. Subsequent studies (Children’s Cancer Group 2961 and Children’s Oncology Group 03P1 pediatric AML trials) allocated all patients with a matched family donor to receive HCT in first CR [2,3]. With the improvements in HLA typing and supportive care, and with broader availability of suitable donors, unrelated HCT has been more broadly used in patients in first CR. Further, reevaluation of the role of stem cell transplantation in AML in the context of emerging prognostic factors has changed the utility of HCT from a biologic allocation based on donor availability to a more risk-appropriate therapeutic allocation aimed at improving outcome while minimizing risk and toxicity. In this risk-based allocation to HCT, patients with

Maintenance Hypomethylating Therapy Post Allogeneic Stem Cell Transplantation Provides Favorable Progression Free Survival in High Risk Acute Myelogenous Leukemia or Non Responsive Myelodysplastic Syndrome

Maintenance Hypomethylating Therapy Post Allogeneic Stem Cell Transplantation Provides Favorable Progression Free Survival in High Risk Acute Myelogenous Leukemia or Non Responsive Myelodysplastic Syndrome

Maintenance Hypomethylating Therapy Post Allogeneic Stem Cell Transplantation Provides Favorable Progression Free Survival in High Risk Acute Myelogenous Leukemia or Non Responsive Myelodysplastic Syndrome

Abstract 4468 IntroductionPatients with acute myelogenous leukemia (AML) or non responsive myelodysplastic syndrome (MDS) who fail to achieve a complete remission, have unfavorable cytogenetics or are refractory to therapy have a poor prognosis. Allogeneic HSCT is frequently considered a salvage option for these patients although remissions are short-lived. New strategies are needed for maintaining remission in this extremely high risk patient group. We hypothesized hypomethylating agents post allogeneic HSCT would enable patients to remain in complete remission. MethodsEleven patients were treated. Patient characteristics: median age, 48 years (range, 18–70 years), PBSC (n=9) or bone marrow (n=2); Diagnoses AML= 10, Non- responsive MDS=1, CIBMTR disease risk category: High [n=8], Intermediate [n=1], Low [n=2]; 36% of the patients had primary induction failure, 54% had complex molecular abnormalities with 18% of the patients having deletion 11q 23 molecular abnormality. Donors were unrelated (36%) and related (64%). All but 1 donor-recipient pairs were fully matched. Preparative regimen- busulfan based (16mg/kg or equivalent) (82%), TBI ≥ 12 Gy (9%) based or reduced-intensity haplo-identical regimen (9%). The median number of prior chemotherapy regimens was 2.5 (range, 2–6). Azacitidine (n=10) or decitabine (n=1) was selected at physician discretion as a planned prophylactic regimen post allogeneic HSCT engraftment. Initial starting doses were based on hematological conditions at the time of therapy initiation. ResultsTen patients received azacitidine subcutaneously daily for seven days for a median of 5 cycles (range, 1–10 cycles) and one patient received decitabine 20mg/m2 intravenously for 5 days for 3 cycles. Median initial Azacitidine dose of 50 mg/m2 (range, 25–75mg/m2) daily were given at a median of 40 days (range, 38–101 days) post HSCT engraftment. Azacitidine doses were increased based on counts to a maximum dose of 75mg/m2. With a median follow-up of 24 months (range, 1.7–49 months) 6 patients are alive in complete remission. Twenty-four month progression free survival is 51% with overall survival of 76%. ConclusionGiven the dismal results of progression free survival and overall survival following allogeneic HSCT in high risk AML or non responsive MDS, hypomethylating agents given post transplantation provide a valuable approach to prolonging remission. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Unrelated Donor Transplants for Older AML Patients Using a Low Dose TBI Containing Regimen Are As Efficacious As Related Donor Transplants, and Exhibit Favorable Outcomes for Intermediate Risk Disease.

Abstract 3119Hematopoietic stem cell transplants (HCT) for older patients with acute myelogenous leukemia (AML) have increased significantly over the past decade, associated with the use of reduced intensity conditioning (RIC) regimen. In the unrelated donor (URD) setting, anti-thymocyte globulin (ATG) has routinely been used to improve engraftment and decrease the incidence and severity of graft versus host disease (GVHD). We hypothesized that replacing ATG with low dose total body irradiation (TBI) during conditioning would provide sufficient immunosuppression to permit engraftment in older patients undergoing unrelated donor transplants for AML. Outcome data, including engraftment and GVHD rates were then compared to similar patients undergoing matched related donor (MRD) HCT for AML. Methods:Between 2007–2012, 50 patients (55–70 years, median 61 years) with AML in CR1 (n=41) or >CR1 (n=9) were prospectively enrolled on an IRB-approved clinical trial using a RIC regimen. Donors were either MRD (n=23), or URD (n=27) if a suitable MRD was not available. HLA matched donors were available in 39 of the 50 patients, with single antigen mismatched donors used in 11 (41%) URD cases. All patients were conditioned with fludarabine (160 mg/m2) + busulfan (6.4 mg/m2) [FluBu2]; URD recipients additionally received 200cGy TBI on day -1 pre-transplant. GVHD prophylaxis was tacrolimus and mycophenolate. Patients exhibited high risk (n=21) or intermediate risk (n=29) disease at initial diagnosis, as defined by cytogenetic and molecular criteria. High risk (HR) patients included those with either a FLT3 mutation, monosomy 5 or 7, 5q-, 7q-, t(6:9) or complex cytogenetics (> 3 abnormalities). Patients with t(8:21), t(16:16) or inv 16 were excluded from analysis. Stem cell sources included peripheral stem cells (n=49) or marrow (n=1). Results:For the entire cohort, the 1y and 3y relapse free survival (RFS) was 56% (95% CI: 43–69) and 43% (95% CI: 28–58) respectively, with 1y and 3y overall survival (OS) rates 58% (95% CI:46–74) and 44% (95% CI: 27–62%). There was no significant difference in 1y RFS between URD and MRD recipients [62±9.5% vs 48±11%] or 3y RFS [44±11% vs 40±12%], p = 0.42 (Figure). There was no difference in outcome by age, with 3y RFS 42.5±11% for patients 55–59 yrs (n=20) and 40±12% for patients 60–70 yrs (n=30), p =0.54. The median donor age was 37 yrs (range 19–50) for URD and 58 yrs (range 42–70) for MRD recipients. The median CD34 cell dose infused was 5.0 × 10(6)/kg in both cohorts [range URD: 1.2 –8.3 × 10(6), MRD: 1.9 –9.8 × 10(6)]. There was no difference in time to neutrophil engraftment, with both cohorts engrafting a median 12 days post-transplant. Primary graft failure occurred in 1 (3.4%) URD recipient, who received an HLA mismatched graft. No cases of secondary graft failure occurred in either cohort. Day 100 CD33 chimerism was all donor in all URD cases, and in 75% of MRD recipients. Grade 2–4 acute GVHD developed in 44% URD and 30% MRD respectively, with grade 3–4 acute GVHD in 7% URD and 10% MRD. Patients with intermediate risk disease experienced 1y and 3y RFS of 74±8.5% and 3-year RFS 62±10.5%, respectively. By comparison, transplant outcomes were poor for patients with high risk disease, with 1y and 3y RFS only 31±10% and 13±10% (Figure). Conclusion:Replacing ATG with low dose TBI in older AML patients undergoing RIC URD does not appear to compromise engraftment or increase GVHD rates. Survival is similar to that experienced by MRD recipients who are transplanted without either ATG or TBI. RFS for patients with high risk AML was poor, but patients with intermediate risk AML may benefit from this strategy when compared to historical results with non-transplant approaches for this patient population. [Display omitted] [Display omitted] Disclosures:Talpaz:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; B.M.S.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Teva: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Couriel:Therakos: Speakers Bureau; Merck: Speakers Bureau.

BCL-2 Inhibition with ABT-737 Prolongs Survival in an NRAS/BCL2-Mediated MOUSE MODEL of Myelodyspastic Syndrome (MDS) Progressing to ACUTE Myelogenous Leukemia (AML) by Targeting Leukemia Initiating CELLS

AbstractAbstract 678 Background and aims:BCL-2 activation plays a role in the progression of MDS to AML and BCL 2 inhibition may represent a therapeutic target in such patients. Using our double transgenic mouse model MRP8[NRASD12/BCL2], in which the transgenes induce MDS progressing to AML with dysplasia (Omidvar Cancer Res, 2007), we assessed the effect of ABT-737, a small molecule and mimetic inhibitor binding the BH3 domain of the BCL-2 family of proteins, on survival and leukemia initiating cells (LIC) in this mouse model. Methods:In this MRP8[NRASD12/hBCL2] double transgenic mouse model 2-week old mice have MDS with a mean of 6% bone marrow (BM) blasts (compared with 3% in the normal wild type littermates), while adult mice have AML, with a mean of 60% marrow blasts. In the present study, double transgenic mice were treated just after weaning and genotyping at 3 weeks of age with 75 mg/kg dose of ABT-737 (MDA & Abbott) 3 times weekly for 30 days. A cohort of mice (60 untreated and 35 treated) was followed for survival. Mice were sacrificed and BM harvested after treatment and Giemsa stained for BM analysis by microscopy (n=6 in each group), for LICs characterized as part of the lineage negative (Lin-)/Sca1+/cKit+ (LSK) population by flow cytometry (in 8 treated and 12 untreated mice), and for progenitor assays (n=4 in each group). Hematoxylin and eosin stained liver sections were examined and apoptosis assessed by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assays of liver sections (n=4 per group). RNA was extracted from Sca+ enriched spleen cells from untreated and treated mice (n=3 from each group) and assayed for gene expression profiling using exon specific arrays (Affymetrix). Results:Survival from birth of 35 treated mice was significantly longer than for 60 untreated mice (p<0.0001) underscoring drug efficacy and tolerance (Fig. 1). This correlated with a reduction of bone marrow blasts (19%±7% in treated versus 60%±6% in untreated mice, p<0.0001). After ABT-737 treatment, the proportion of BM LSK cell population decreased to nearly normal levels (normal wild type littermates mean of 3%, n=4) (12.9±1.4 in untreated versus 6.4±1.1 in treated, p<0.005)) with a complete restoration of colony growth to normal range (40±10 in wild type normal mice, 79.6±7.0 in untreated versus 48.6±13.5 in treated, p<0.01). Decreased invasion of the liver and spleen was observed due to increased apoptosis (3±2 in untreated to 50±12% in treated, p<0.001). Exon specific gene expression arrays with Sca1+ enriched splenocytes showed that 997 genes were differentially expressed between the treated and the untreated mice; 764 and 233 genes were upregulated and downregulated respectively amongst which were upregulated genes important for stem cell development, maintenance and differentiation such as TCF712 (or TCF4), PIWII2, BRMPR1a and Spp1. This may reflect the partial restoration of normal stem cell function, which is consistent with the reduced LSK and progenitor numbers. Downregulation of anti-apoptotic genes such as BCL-2a1b or upregulation of pro-apoptotic genes such as PARP4, CALPAIN2, TNFR, and CARD was observed, consistent with the TUNEL data. Restoration of normal hematopoiesis was confirmed by the upregulation of myeloid differentiation genes (CD14, CSF1, RARalpha) and down regulation of genes implicated in cell cycle (Hsp60, MYC and E2F1). Conclusions:ABT-737 extends lifespan in NRASD12/BCL2 transgenic mice, a preclinical model of high risk MDS/AML. ABT-737 targets the leukemia initiating cell, and regulates, amongst several, cell cycle (proliferation), differentiation and apoptosis pathways. These data suggest that clinical trials in high risk MDS and AML patients are warranted. [Display omitted] Disclosures:Grange:Genosplice Technology: Employment.

High Alloreactivity of Low-Dose Prophylactic Donor Lymphocyte Infusion in Patients with Acute Leukemia Undergoing Allogeneic Hematopoietic Cell Transplantation with an Alemtuzumab-Containing Conditioning Regimen

The value of prophylactic donor lymphocyte infusion (pDLI) is unclear and differs among diseases and transplantation protocols. Experience with this approach in patients with acute leukemia undergoing hematopoietic cell transplantation (HCT) with an alemtuzumab-incorporating conditioning protocol is lacking. We conducted a single-center prospective study to investigate the applicability and efficacy of prophylactic donor lymphocyte infusion (pDLI) in patients with leukemia undergoing HCT with a low-dose alemtuzumab-containing conditioning regimen. Inclusion criteria were high-risk acute myelogenous leukemia, acute lymphoblastic leukemia, or increasing mixed chimerism. All patients included were tapered off of immunotherapy. Exclusion criteria were a history of ≥grade II or active graft-versus-host disease (GVHD). Of the 56 consecutive patients who underwent HCT with an alemtuzumab-containing regimen, 15 patients (8 with acute myelogenous leukemia and 7 with acute lymphoblastic leukemia) met the study inclusion criteria and received prophylactic DLI (total of 45 infusions) from 7 sibling donors and 8 unrelated donors. The first infusion was given at a median of 162 days posttransplantation. The median number of DLIs was 3, and the median cumulative CD3+ cell dose was 2 × 106cells/kg. Six of the 8 patients (75%) who received pDLI while in mixed chimerism converted to stable, complete donor chimerism. Some 47% of DLI recipients developed GVHD (4 acute GVHD and 3 with chronic GVHD) after a median cumulative dose of 2 × 106 CD3+ cells/kg. After a median follow-up of 575 days, 11 (73%) pDLI recipients were alive. All 4 deaths were due to GVHD-related causes. None of the patients who received pDLIs relapsed. Patients with leukemia who received low-dose pDLI after conditioning with alemtuzumab are at low risk for relapse; however, this approach is associated with a relatively high incidence of severe GVHD.

Comparison of Allogeneic Stem Cell Transplantation from Familial-Mismatched/Haploidentical Donors and from Unrelated Donors in Adults with High-Risk Acute Myelogenous Leukemia

To weigh the pros and cons of familial-mismatched/haploidentical transplantation (FMT) in patients with high-risk acute myelogenous leukemia, we assessed outcomes of 23 patients who underwent FMT, using reduced-intensity conditioning with total body irradiation 800 cGy/busulfan/fludarabine/antithymocyte globulin without ex vivo T cell depletion, compared to 33 patients who underwent well-matched unrelated donor transplantation (WM-UDT) and 13 who underwent partially matched unrelated donor transplantation (PM-UDT) during the same period. The FMT patients had not only a similar pattern of engraftment and immune reconstitution as the WM-UDT and PM-UDT patients but also comparable incidences and severity of acute and chronic graft-versus-host disease. The FMT patients did not experience any form of engraftment failure. However, the cumulative incidence of cytomegalovirus DNAemia was significantly higher in the FMT group compared with the other groups (P = .036). After a median follow-up of 28 months, overall survival, disease-free survival, relapse, and nonrelapse mortality were 83%, 74%, 20%, and 7%, respectively, for WM-UDT; 51%, 51%, 31%, and 18% for PM-UDT; and 66%, 64%, 26%, and 10% for FMT. This demonstrates a trend for favorable survival outcomes of WM-UDT over FMT and of FMT over PM-UDT. However, we found no significant statistical differences in survival according to donor type. These data need to be interpreted cautiously because of limited power calculations due to the small number of each donor group. This pilot study suggests the feasibility of FMT using our novel regimen with careful evaluation of CMV DNAemia compared with WM-UDT and PM-UDT. Further trials with larger numbers of patients, comparing FMT directly with transplantation with other donor types, are needed.

5-Azacitidine in aggressive myelodysplastic syndromes regulates chromatin structure at PU.1 gene and cell differentiation capacity

Epigenetic 5-azacitidine (AZA) therapy of high-risk myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) represents a promising, albeit not fully understood, approach. Hematopoietic transcription factor PU.1 is dynamically regulated by upstream regulatory element (URE), whose deletion causes downregulation of PU.1 leading to AML in mouse. In this study a significant group of the high-risk MDS patients, as well as MDS cell lines, displayed downregulation of PU.1 expression within CD34+ cells, which was associated with DNA methylation of the URE. AZA treatment in vitro significantly demethylated URE, leading to upregulation of PU.1 followed by derepression of its transcriptional targets and onset of myeloid differentiation. Addition of colony-stimulating factors (CSFs; granulocyte-CSF, granulocyte-macrophage-CSF and macrophage-CSF) modulated AZA-mediated effects on reprogramming of histone modifications at the URE and cell differentiation outcome. Our data collectively support the importance of modifying the URE chromatin structure as a regulatory mechanism of AZA-mediated activation of PU.1 and induction of the myeloid program in MDS.

A Phase I Study of Gemtuzumab Ozogamicin (GO) in Combination with Busulfan and Cyclophosphamide (Bu/Cy) and Allogeneic Stem Cell Transplantation in Children with Poor-Risk CD33 + AML: A New Targeted Immunochemotherapy Myeloablative Conditioning (MAC) Regimen

Children with high-risk acute myelogenous leukemia (AML) (induction failure [IF], refractory relapse [RR], third complete remission [CR3]) have dismal outcomes. Over 80% of AML patients express CD33, a target of gemtuzumab ozogamicin (GO). GO is an active drug in childhood AML but has not been studied in a myeloablative conditioning regimen. We sought to determine the safety of GO in combination with busulfan/cyclophosphamide (Bu/Cy) conditioning before allogeneic hematopoietic stem cell transplantation (alloSCT). GO was administered on day -14 at doses of 3.0, 4.5, 6.0, and 7.5 mg/m(2), busulfan on days -7, -6, -5, -4 (12.8-16.0 mg/kg), and cyclophosphamide on days -3 and -2 (60 mg/kg/day). GVHD prophylaxis consisted of tacrolimus and mycophenolate mofetil. We enrolled 12 patients: 8 IF, 3 RR, 1 CR3; median age: 3 years (1-17); median follow-up: 1379 days (939-2305). Nine received umbilical cord blood (UCB), 2 matched unrelated donors (MUDs) and 1 HLA-matched sibling donor: 3 patients each at GO doses of 3.0, 4.5, 6.0, or 7.5 mg/m(2). No dose-limiting toxicities secondary to GO were observed. Day 100 treatment-related mortality (TRM) was 0%. Myeloid and platelet engraftment was observed in 92% and 75% of patients at median day 22 (12-40) and 42 (21-164), respectively. Median day +30 donor chimerism was 99% (85%-100%). The probability of grade II-IV acute graft-versus-host disease (aGVHD) was 42% and chronic GVHD (cGVHD) was 28%. One-year overall survival (OS) and event-free survival (EFS) was 50% (95% confidence interval [CI], 20.8-73.6). GO combined with Bu/Cy regimen followed by alloSCT is well tolerated in children with poor-risk AML. GO at 7.5 mg/m(2) in combination with Bu/Cy is currently being tested in a phase II study.

Upfront Allogeneic Blood Stem Cell Transplantation for Patients with High-Risk Myelodysplastic Syndrome or Secondary Acute Myeloid Leukemia Using a FLAMSA-Based High-Dose Sequential Conditioning Regimen

Patients suffering from high-risk myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML) secondary to MDS (sAML) are characterized by poor response to conventional cytotoxic chemotherapy. The purpose of our prospective single-center study was to examine the safety and efficacy of an allogeneic hematopoietic stem cell transplantation (HSCT) following a sequential conditioning regimen as first-line therapy for previously untreated patients with high-risk MDS or sAML. Between November 2003 and June 2010, 30 patients (20 high-risk MDS, 10 sAML) received fludarabine (4 × 30 mg/m(2)), amsacrine (4 × 100 mg/m(2)), and Ara-C (4 × 2 g/m(2), FLAMSA). After 2 to 3 days of rest, patients received high-dose melphalan alone (200 mg/m(2) for patients with an age <50 years, 150 mg/m(2) for patients with an age between 50 and 60 years, and 100 mg/m(2) for patients with an age >60 years; n = 24) or melphalan and thiotepa (10 mg/kg, Mel/Thio, n = 6). Following these high-dose conditioning regimens, a median number of 7.7 × 10(6) CD34(+) cells/kg body weight (range: 2.9 × 10(6)-17.2 × 10(6)) were transplanted from 13 related or 17 unrelated donors. Antithymocyte globulin (Fresenius 30-60 mg/kg) as well as tacrolimus and mycophenolate mofetil were used for graft-versus-host disease (GVHD) prophylaxis. All patients except 1 with primary graft failure achieved complete remission after HSCT. After a median follow-up time of 28 months (range: 7-81), 21 patients (70%) were alive and free of disease. Overall, 4 patients relapsed. At 2 years, overall survival, event-free survival, and treatment-related mortality were 70%, 63%, and 30%, respectively. Because of undue toxicity, thiotepa is no longer part of the conditioning regimen. Our results add to the body of evidence that a FLAMSA-based sequential conditioning therapy is effective for previously untreated patients with high-risk MDS or sAML.

Abstract 2625: MGCD0103, an oral isotype-selective HDAC inhibitor, has antitumor activity via the induction of the expression of Annexin A1 on AML1/ETO AML in vitro and in vivo

Abstract MGCD0103 is a non-hydroxamate and an orally available isotype-selective HDAC inhibitor (HDACi) whose clinical activity has been shown in multiple phase II trials in patients with relapsed or refractory Hodgkin lymphoma, high-risk myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). It has previously been shown that FK-228, class I selective HDACi, and SAHA, isotype-nonselective HDACi, activate apoptosis through induction of pro-apoptotic protein annexin A1 on t(8;21) translocated AML cells which produce AML1-ETO fusion protein (AML1/ETO AML). To determine whether the existence of t(8;21) translocation in AML affect on the efficacy of MGCD0103, we explored the action of MGCD0103 on anti-tumor activity in AML1/ETO AML and AML1/ETO-negative AML xenograft models in mice in vivo and investigated the putative mechanism of the efficacy.MGCD0103 was orally administrated to the NOD SCID mice implanted with AML1/ETO AML xenograft or AML1/ETO-negative AML xenograft using an intermittent dosing regimen (3 times a week) for 3 weeks. Tumor volume was calculated during the treatment and tumor weight at final date was measured for final evaluation. In addition, we measured not only the activity of apoptosis related factor, caspase-3/7, but also the expression of pro-apoptotic protein annexin A1 that induced by MGCD0103 in vitro and in vivo. Further understanding of the mechanism of MGCD0103, we assessed the acetylated histone in the promoter region of annexin A1 using ChIP assay in vitro whether its induction is derived from the histone acetylation activity of MGCD0103. MGCD0103 showed a strong growth inhibition of the tumor in the AML1/ETO AML xenograft. There were significant decreases in the AML1/ETO AML tumor volume and weight in the MGCD0103 treated group compared with the control group, but not in the AML1/ETO-negative AML xenograft. Caspase-3/7 was strongly activated and the expression of annexin A1 was significantly induced in the MGCD0103 treated AML1/ETO AML cells. In addition, histone of annexin A1 promoter region in AML1/ETO AML tended to be acetylated more than the AML1/ETO-negative AML cells by MGCD0103. These data possibly indicate that the strong anti-tumor effect was derived from the direct effect of MGCD0103 that leads apoptosis of the AML1/ETO AML cells through the induction of acetylation of annexin A1 promoter. Selection of AML patients with t(8;21) translocation may leads good therapeutic outcome of MGCD0103 in AML treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2625. doi:10.1158/1538-7445.AM2011-2625