Acute Myeloid Leukemia Regimens Research Articles

CD33 Targeted Immunoliposomal Delivery of OSU-2S, a Non-Immunosuppressive FTY720 Derivative, Mediates Selective Cytotoxicity in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an incurable disease with 5 year survival rates of 10% in patients over 60 years. Poor tolerance to chemotherapy, chemo resistance and high rate of relapse warrants less toxic and more effective regimens in AML. OSU-2S is a novel non-immunosuppressive derivative of FTY720, a sphingosine analogue. The promising in-vitro and in-vivo activity of OSU-2S against a number of leukemias and lymphomas, and other malignancies such as hepatocellular carcinoma, impelled us to evaluate the activity of OSU-2S in AML. The potent cytotoxic activity of the OSU-2S (5µM, 24hrs) in AML cell lines HL-60, MV411 and MOLM13 (n=3; HL-60: p=0.008; MV411: p=0.04; MOLM13: p=0.0094) encouraged us to evaluate the effect of OSU-2S in primary leukemic cells from AML patients. OSU-2S (5µM, 24 and 48 hrs) demonstrated significant cytotoxic activity against AML cells, including high risk FLT3-ITD mutated AMLs, (n=13, p<0.0001, mean difference in viability= -65.46) in a dose dependent manner (dose trend p<0.0001 at 24 and 48hrs). While OSU-2S induced caspase activation in primary AML cells as evidenced by Poly (ADP-ribose) polymerase cleavage, its cytotoxic effect is independent of caspase activation (n=8, p=0.0016), as demonstrated by comparable cytotoxicity even in the presence of a broad spectrum caspase inhibitor Q-VD-OPH. Moreover, OSU-2S also significantly increased the levels of reactive oxygen species production in primary AML cells (n=8, p=0.003, mean difference in ROS production= 33.5).Interaction of AML cells with the bone marrow stromal environment plays a critical role in leukemic cell survival and proliferation, thus contributing to resistance to chemotherapy. To effectively mimic this interactive environment which would be encountered in the patients, bone marrow stromal cells (MSCs) were cultured and expanded from AML patients to develop autologous co-culture systems with AML cells. The autologous stromal cells mediated significant protective effect on AML cells (n=3, p=0.022, mean difference in viability with MSC=21.05), which was compromised in the presence of OSU-2S (5µM) (n=3, p=0.01, mean difference in viability= -64.36), indicating lack of stromal protection mediated resistance to OSU-2S. To circumvent any unintended off target effects of OSU-2S on normal cells, we synthesized an immunoliposomal (ILP) nanoparticle formulation targeting CD33 (SIGLEC-3), a transmembrane receptor which is highly expressed on myeloid progenitor cells and AML. OSU-2S encapsulated immunoliposomes (OSU-2S-CD33-ILP) showed significant cytotoxicity as compared to empty-CD33-ILPs in primary cells (n=5 AML, p=0.0005, mean difference in viability= -48.63) as well as AML cell lines (n=3, MOLM13: p=0.002; MV411: p=0.004). Importantly, OSU-2S-CD33-ILP selectively depleted CD33 positive myeloid population (n=3, p=0.0011, mean difference in % viable population= -43.39) without compromising the CD33 negative non targeted lymphoid population (n=3, p=0.013, mean difference in % viable population= 29.56) in primary AML cell cultures. Similarly, selective cytotoxicity ablated CD33+ MOLM13 and MV411 AML cell lines but not CD33 non targeted Jurkat cell line, which is sensitive to the free (naked) drug. In summary, OSU-2S mediated potent cytotoxic activity against primary AML cells that is not compromised in the presence of autologous bone marrow stromal cells from AML patients. Further, CD33 targeted delivery of OSU-2S has promising selective activity against CD33+ but not CD33- cells. Ongoing studies are evaluating the efficacy of free OSU-2S and OSU-2S-CD33-ILP formulations in-vivo. [This work was supported by NIH-R01-CA197844-01, P50-CA140158, Lauber Funds for Immunotherapy in AML and Robert J. Anthony Leukemia Fund] DisclosuresChen:The Ohio State University: Patents & Royalties: OSU-2S patent.

A Novel Regimen for Acute Myeloid Leukemia with MLL Partial Tandem Duplication: Results of a Phase 1 Study NCI 8485

Background: MLL partial tandem duplication (MLL PTD) occurs in approximately 5-8% of patients with acute myeloid leukemia (AML) and is associated with an adverse prognosis. Our group has published that the MLL wild type (WT) allele is epigenetically silenced in MLL PTD; we showed that re-expression of this gene can be induced with methyltransferase (DNMT) and/or histone deacetylase (HDAC) inhibitors. Further, re-expression of MLL WT following combined decitabine and HDAC inhibitor treatment sensitized MLL PTD myeloid leukemia cells to chemotherapy in vitro. We hypothesized that epigenetic silencing of the MLL WT contributes to MLL PTD-associated leukemogenesis and that its pharmacologic re-expression with DNMT and HDAC inhibitors would activate apoptotic mechanisms important for chemo-response in the clinic. We aimed to develop a regimen to be tested in this unique molecular subset of disease. Because of the relatively low frequency of MLL PTD AML, this dose finding study was conducted in relapsed/refractory (R/R) AML regardless of molecular subtype but was enriched for MLLPTD.Methods:In this phase 1 study, adults aged 18-59 years with R/R AML, ECOG 0-2, and preserved organ function were enrolled. Patients received decitabine 20mg/m2 daily on days 1-10 and vorinostat 400mg daily on days 5-10 for all dose levels. Dose-escalated cytarabine was given on days 12, 14, 16 (six doses) according to the following schedule: dose level (DL) 1, 1.5g/m2/q12hr; DL 2, 2g/m2/q12hr; DL 3, 2.5g/m2/q12hr; and DL 4, 3g/m2/q12hr. Standard method 3+3 phase I design was used with the primary objective to determine the maximum tolerated dose and define specific toxicities with this combination therapy, in order to ultimately develop a regimen for MLLPTD AML.Results:Seventeen adults with R/R AML and median age of 46 years (range, 21-59 years) enrolled. The median number of prior induction therapies was 2 (range, 1-4). European LeukemiaNet (ELN) genetic risk classification frequencies were: favorable (n=2), intermediate-I (n=3), intermediate-II (n=5), and adverse (n=7), respectively. Four patients had MLL PTD. A total of 6 patients were treated at DL 1 with no non-hematologic dose limiting toxicity (DLT). DL 1 was expanded after two patients experienced prolonged but uncomplicated myelosuppression. Since both patients achieved complete remission (CR) shortly after passing the day 42 DLT cut off for hematologic recovery, the protocol was amended to allow further time for count recovery (up to 56 days). Three patients each were treated on DL 2 and 3; 5 patients were treated on DL 4 with no other DLTs observed. Diarrhea, nausea, fatigue, febrile neutropenia, and elevated ALT were the most common toxicities (any grade, regardless of attribution), occurring in 41%, 29%, 29%, 35%, and 35% of patients, respectively, but none were DLTs. In regards to ≥ Grade 3 toxicities, febrile neutropenia and catheter-related infections were most common at 35% and 24%. Significant mucositis was not observed. DL 4 was the recommended phase 2 dose (RP2D). CR or CR with incomplete count recovery (CRi) was observed in 6/17 patients (35%, 5 with CR). Of 6 responders, all (n=4) patients with abnormal karyotype achieved cytogenetic remission. The median number of prior therapies for patients achieving CR/CRi was 2 (range, 1-3). Four patients subsequently received allogeneic transplantation. Of the four patients known to have MLL PTD mutations, two responded (1 with CR and 1 with CRi). It is interesting to note that the two patients with MLL PTD mutations who did not respond to treatment also had FLT3-ITD mutations, while this mutation was absent in the two responding patients who had MLLPTD.Conclusions: We successfully determined the RP2D for this novel treatment regimen. The regimen had modest toxicities beyond uncomplicated (though prolonged) myelosuppression, and we propose that the study provides a framework for larger efficacy studies for AML patients with the uncommon but biologically distinct molecular feature of MLLPTD.Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number U01CA076576. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Also supported by P30 CA016058/CA/NCI. DisclosuresNo relevant conflicts of interest to declare.

In Vitro Expanded γδ T Cells Derived from Cord Blood Induce Potent and Specific Cytotoxicity Against Human Acute Myeloid Leukemia Cells

Conventional chemotherapeutic regimens for acute myeloid leukemia (AML) patients have demonstrated unsurpassed efficacy in the past decades, but are far from optimal with many patients experiencing multiple disease recurrence or intolerant of the intensive chemotoxicity. Variations in the treatment scheme as well as the use of alternative, targeted agents have been pursued with limited success. This is partly ascribed to the highly heterogeneous nature of the disease comprising a dynamic repertoire of evolving leukemic clones that are both molecularly and biologically diverse, making it difficult to achieve complete disease eradication without inducing adverse off-target effects. In this regard, cellular immunotherapy has emerged as a plausible alternative, leveraging on the diversity and degeneracy of the tumor antigen-recognizing receptor complex expressed by immune cells. In particular, there is a growing interest in the specific anti-leukemia efficacy of the innate-like γδ T cells, prompted by the association of an increased number of donor derived γδ T cells (specifically the Vδ1+ subtype) in allogeneic hematopoietic stem cell transplant (HSCT) patients with improved disease control in the absence of significant graft-versus-host disease (GvHD). We therefore hypothesize that these allogeneic γδ T cells exhibit potent leukemia specific cytotoxicity and serve as an effective treatment for AML.Given the rapid availability and widespread use of cord blood (CB) as an alternative for allogeneic HSCT, we first characterized and explored the potential of expanding CB-derived γδ T cells in vitro. Compared to mobilized peripheral blood (mPB), there is a significantly lower level of γδ T cell within CB mononuclear cells (MCs) (0.61% ± 0.36% in CB vs 4.95% ± 3.83% in mPB, p<0.001). However, the fraction of Vδ1+ subset within the γδ T cells in CB is >3.5-fold higher than that in mPB (56.05% ± 9.49% in CB vs 14.54% ± 12.2% in mPB, p<0.001). Importantly, while >90% of the Vδ1+ T cells in CB are of naive or central memory phenotype, more than 40% of these cells in mPB show effector memory expression. We established that optimal in vitro expansion of CB-derived γδ T cells requires direct contact to a mixture of irradiated PBMCs and Epstein-Barr virus-transformed lymphoblastoid cell line (EBV-LCL) at a fixed ratio in the gas-permeable G-Rex culture flask. Under these conditions, we were able to achieve up to 5,200-fold expansion of the starting γδ T cells over a period of 21 days. These cells exhibit potent in vitro cytotoxicity against a range of human AML cell lines, including K562, MOLM-14, MV4-11 and NOMO-1, as well as primary patient samples in a dose dependent manner. In contrast, there is minimal in vitro cytotoxicity against CD34+ cells isolated from allogeneic CB samples even at the highest effector-to-target cell (E:T) ratio tested. Infusion of the expanded γδ T cells into NOD/SCID/IL2Rγ-/- (NSG) mice at 3 weeks post-transplantation of a FLT3-ITD+ AML patient sample (P1) resulted in a significant decrease in leukemic cell engraftment in 40% of the γδ T cells-treated mice (87.46 ± 2.25% in non-treated vs 74.85 ± 1.55% in γδ T cells-treated mice, p=0.022). In a separate experiment, infusion into NSG mouse that was engrafted with low level (0.1%) of a different FLT3-ITD+ AML patient sample (P2) maintained the leukemic cell level low at 0.1% at 4 weeks post-infusion, as opposed to the >15-fold increase in leukemic burden (1.76%) seen in the untreated mouse. Consistent with our in vitro finding, infusion of up to 5 x 108 expanded CB derived γδ T cells/kg failed to induce severe GvHD symptoms in NSG mice engrafted with allogeneic human CB cells up to 8 weeks post-infusion, with no significant effect on the level of in vivo regenerated human myeloid and lymphoid cells, as well as colony-forming cells (CFCs).In summary, our data demonstrates that in vitro expanded CB derived γδ T cells show potent AML-specific cytotoxicity both in vitro and in vivo, making it a promising alternative cell source for immunotherapy. Further investigations to enhance the mechanistic understanding would be needed to seed for future clinical translation. DisclosuresHwang:Pfizer: Honoraria, Other: Travel support; MSD: Honoraria, Other: Travel support; BMS: Honoraria, Other: Travel support; Novartis: Honoraria, Other: Travel support; Celgene: Honoraria, Other: Travel support; Roche: Honoraria, Other: Travel support; Janssen: Honoraria, Other: Travel support; Sanofi: Honoraria, Other: Travel support.

Comparable Outcomes after Allogeneic Stem-Cell Transplantation with Intravenous Busulfan or Treosulfan-Based Reduced-Intensity and Reduced Toxicity Regimens in Acute Myeloid Leukemia. a Study on Behalf of the Acute Leukemia Working Party of EBMT

▪Allogeneic stem cell transplantation (SCT) is a potentially curative therapy for patients with acute myeloid leukemia (AML). Myeloablative conditioning (MAC) is associated with prohibitive rates of non-relapse mortality (NRM) in older and less medically fit patients. Several reduced intensity conditioning regimens (RIC) and more recently the more dose-intensive reduced toxicity myeloablative (RTC) regimens were designed to replace MAC in this setting. The backbone of these regimens is usually fludarabine with busulfan and more recently also with treosulfan, but there is no clear data on the comparative outcomes with these different regimens in the different SCT settings. The current study included 3561 patients with AML given a first allogeneic SCT from an HLA-matched sibling (n=1683) or a 10/10 matched unrelated donor (n=1878) between the years 2000-2014 and reported to the acute leukemia working party (ALWP) of EBMT. Only patients given fludarabine with either intravenous busulfan (ivBu), (FB, n=2990) or treosulfan (FT, n=571) alone were analyzed. Fludarabine and ivBu at 6.4 mg/kg (n=1457) or treosulfan at 30-36 gr/m2 (n=168) were considered RIC regimens while fludarabine with ivBu at a total dose of 9.6-12.8 mg/kg (n=1533) or treosulfan at 42 gr/m2(n=403) were considered RTC regimens according to EBMT criteria. The median age of FB and FT recipients was 55.5 and 58.3 years, respectively (P< 0.0001). The status at SCT was 72.5% CR1, 15.0% CR2 and 12.5% advanced disease in the FB group compared to 55.0%, 20.3% and 24.7% in the FT group, respectively (P<0.0001). More FT recipients had SCT from unrelated donors (64.8% Vs. 50.4%, P<0.0001) but less had in-vivo T-cell depletion (58.4%Vs 70.5%, P<0.0001). Cytogenetic subgroup distribution was similar between the groups. Ninety percent had peripheral blood stem cell grafts in both groups. The median follow-up was 19 and 43 months after FB and FT, respectively. Using univariant analysis, the 2-year relapse incidence (RI) was 32.7% and 35.5%, respectively (P=0.49). NRM was 17.6% and 19.4%, respectively (P=0.09). Leukemia-free survival (LFS) and overall survival (OS) were 49.5% and 54.8% after FB and 45.1% and 52.6% after FT, respectively (P=0.04, P=0.17). Acute GVHD grade II-IV and chronic GVHD were 23.1% and 35.7% after FB and 18.8% and 39.8% after FT, respectively (P=0.03, P=0.04). In all, the GVHD/ relapse-free survival (GRFS) was 36.5% and 31.5%, respectively (P=0.08). After adjusting for the differences in patient characteristics, there was no difference between the FB and FT groups in RI, NRM, LFS, OS and GRFS. However, acute GVHD grade (II-IV) was higher after FB (HR, 1.49, P=0.0004). The same observations were seen when the analysis was limited to RIC or RTC regimens only, or when only patients in remission were analyzed. However, when analyzing only the 516 patients with advanced disease at SCT, 2-year OS was 29.7% and 43.0% after FB and FT (P=0.002) and this difference remained significant in the multivariant analysis (HR, 1.50, p=0.003). Among the entire group, the factors associated with reduced survival were advanced age (HR 1.01, P<0.0001), secondary AML (HR 1.19, P=0.005), CR2 (HR 1.21, P=0.007) and advanced disease (HR 2.02, P<0.0001) compared to CR1, and female donor to male recipient (HR 1.15, P=0.03). Conditioning type and intensity, donor type, CMV status and in vivo T-cell depletion were not significant. Relapse was lower and NRM was higher with RTC compared with RIC, but OS was similar. The same factors predicted for GRFS, a surrogate for quality of life, with the only difference been the positive role of in vivo T-cell depletion (HR 0.8, P=0.0002). In conclusion, RIC and RTC regimens with ivBu or treosulfan-based regimens are associated with similar transplantation outcomes. OS is primarily affected by disease factors such as status of disease at SCT and secondary leukemia. Treosulfan- based conditioning is associated with a lower rate of acute GVHD, but with similar rates of chronic GVHD, NRM and GRFS. Treosulfan conditioning may have some advantage in patients with advanced disease at SCT. DisclosuresNo relevant conflicts of interest to declare.

Precision Medicine Assays Detect Novel Targetable FLT3 Fusions Amenable to Therapeutic Intervention in a Patient with Refractory Acute Myeloid Leukemia

Context:The 5-year survival rate for acute myeloid leukemia (AML) is 26.6%. The prognosis of patients with adverse events such as older age or unfavorable risk cytogenetics remains poor, even for those who undergo allogeneic hematopoietic cell transplant. AML is a heterogeneous disease and novel N-of-1 clinical trial designs may offer benefit to individuals compared to conventional clinical trials by offering improved utilization of investigational chemotherapy regimens. Precision medicine based assays that reveal a deeper understanding of the cancer biology and potential for novel therapeutics may improve survival in the future. Objective:The goal of the clinical trial on which this patient was enrolled, Individualized Treatment for Relapsed/Refractory Acute Leukemia Based on Chemosensitivity and Genomics/Gene Expression Data (ClinicalTrials.gov Identifier:NCT01872819) was to determine feasibility of a study that utilized results of comprehensive mutation analysis and an in vitrohigh throughput functional assay to choose treatment for individual patients with refractory AML. Feasibility was defined as initiating chosen treatment within 21 days. A secondary objective was to achieve a response (cytoreduction or at least partial response) greater than that expected for comparable refractory patient populations with other salvage regimens. Design, Setting, and Patient:A single center enrolled individuals who had failed at least 2 inductions at initial diagnosis or >1 salvage regimen for relapsed AML. Patients could receive any FDA approved drug or combination regimen based on molecular analysis and high throughput drug sensitivity assay. A 58 year old female was enrolled into this protocol with MECOM (EVI1) rearranged, Monosomy 7 refractory AML. Methods and Main Outcome Measures:The patient had various assays performed on her samples, including next generation sequencing and a high throughput in vitro assay that analyzed enriched blast samples for sensitivity to 150 drugs or drug combinations. MyAML™, a next generation sequencing panel, analyzed 194 genes including breakpoint hotspot loci with long paired end sequencing and high depth that optimized detection of large insertion and deletions and other structural variants found in AML at low variant allele frequency.Results:The MyAML assay detected multiple variants including: NRAS:c.38G>A; p.Gly13Asp VAF = 100%, RUNX1:c.494_495ins; p.165_R166ins VAF = 42%, WT1:c.1149_1150ins; p.E384Pfs*5 VAF=38%. Fusions were also detected: t(13;17)(q12.2;q11.2), t(8;13)(q21.13;q12.2), and t(9;12)(q32;p13.2) which involved FLT3 novel fusions. FLT3 internal tandem duplications (ITD) or tyrosine kinase domain (TKD) variants were not detected. The assay also detected the Monosomy 7 and confirmed the t(2;3) as a THADA-MECOM (EVI1) fusion to the nucleotide breakpoint. These variants involved activated signaling, myeloid transcription factor and DNA demethylation pathways.The high throughput drug sensitivity assay identified sensitivity to kinase inhibitors such as the MEK inhibitor selumetinib (IC50 8.1 X 10e-9 M), Flt3 inhibitor staurosporine (IC50 1.4 X 10e-8 M) and Abl kinase inhibitor ponatinib (IC50 2.7 X 10e-8 M). Insurance coverage for these agents was not able to be obtained as this indication would be considered off label. However, based on the FLT3 fusion we identified using MyAML, we decided to treat the patient with the tyrosine kinase inhibitor sorafenib that we were able to obtain through a patient assistance program.This novel case is the first demonstration of a FLT3 fusion detection with a drug sensitivity assay suggesting that kinase inhibitors with multiple targets might be suitable for this type of variant.Conclusion:Specialized assays designed to identify clonal and subclonal architecture of genes associated with specific diseases can reveal variants that present therapeutic options not currently utilized for high risk patients, suggesting broader use of this approach could improve current clinical outcomes. DisclosuresPatay:Invivoscribe, Inc: Consultancy. Carson:Invivoscribe, Inc: Employment. Becker:GlycoMimetics: Research Funding.

Preclinical Evidence That Trametinib Enhances the Response to Tyrosine Kinase Inhibitors in Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is the most common type of acute leukemia in adults and the second in children. The overall survival is less than 35% and 60% for adults and children respectively. Activating mutations of FLT3 are now recognized as the most common molecular abnormality in this disease, and the poor prognosis of patients harboring these mutations renders FLT3 an obvious target of therapy. Although different tyrosine kinase inhibitors (TKI) have been used for this purpose, the ability of these drugs to extend progression-free and overall survival in this patient population is limited by drug resistance. This strategy could be improved by rationally combining TKIs with other agents. In this work, we have explored by phosphoproteomics the alternative pathways activated after TKI treatment in vitro and ex vivo.The phosphoproteome profile of the bone marrow from a FLT3-AML patient before and after TKI treatment, studied by LC-MSMS after IMAC enrichment, suggested the activation of Ras-Raf-MEK-ERK1/2 pathway as a possible mechanism for TKI resistance, which could be avoided by dual inhibition using the MEK inhibitor trametinib. Therefore, we characterized the effect of trametinib in combination with the TIK pazopanib and sorafenib by the in vitro cell viability assay using WST8in the FLT3-ITD AML cell line MOLM13. As it is presented in figure 1a, trametinib showed an IC50 value in the low-nanomolar range (5.4 nM) and this MEKI produced a strong synergy (0.5<Ci) with the two TKI tested (figure1b). Moreover, when we analized the activation of the three main pathways downstream the FLT3 receptor by western blot (figure 1c), we observed that the combination of trametinib with both pazopanib and sorafenib showed a complete inhibition of phospho-ERK1/2 compared to the DMSO control (P≤0.0001). Interestingly, we also observed some differences between the two combinations: while trametinib in combination with sorafenib inhibited STAT5 phosphorilation (P≤0.05), the MEKI combination with pazopanib produced a significant decrease on phospho-AKT levels (P≤0.01). In conclusion, we provide preclinical evidence that combining a TKI, such as sorafenib or pazopanib, with a MEKI, such as trametinib, is a rational and efficacious treatment regimen for AML. As trametinib has previously shown good results when combined with pazopanib in clinical trials for other kind of tumors, we expect similar results in AML. On the other hand, trametinib+sorafenib could offer an optimal combination in those patients with elevated levels of phospho-STAT5, as it has been described for patients which present FLT3-ITD mutations.M.L. holds a postdoctoral Fellowship of the Spanish Ministry of Economy and Competitiveness (FPDI-2013-16409) y ML.M. holds a Fellowship of the Spanish Ministry of Education, Culture and Sport (REF-91442). [Display omitted] DisclosuresMartínez-López:Novartis: Honoraria, Speakers Bureau.

Acute Myeloid Leukemia Induction with Cladribine: Effects of Age and Leukemia Risk

Background: Acute myeloid leukemia (AML) induction traditionally includes seven days of cytarabine and three days of an anthracycline (7+3). Co-administration of cladribine may increase efficacy of cytarabine. This rationale directed Saint Louis University (SLU) to adopt a regimen derived from the Polish Adult Leukemia Group (Holowiecki, JCO 2012) which adds five days of cladribine to idarubicin based 7+3 (IAC). We report the SLU experience with toxicity and survival following IAC induction. Additionally, we discuss the effects of IAC in patients 60 and older as an area of particular interest.Methods: Retrospective analysis of patients with newly diagnosed AML (APL excluded) and high-risk MDS who received IAC from January 2009 to September 2015. IAC was initiated as cytarabine 200mg/m2 by continuous infusion for seven days, idarubicin 12mg/m2 daily for three days, and cladribine 5mg/m2 for five days. Mortality and disease response were analyzed, with stratification by age and NCCN leukemia risk classification.Results: Of 107 patients, 57 patients (53%) were age 60 and older. By NCCN cytogenetic and molecular stratification, 21 (20%) had favorable risk; 43 (40%) intermediate risk; and 43 (40%) poor risk leukemia. Complete response (CR) occurred in 75 (70%) patients and CRi in an additional 9 patients (8%) for an overall response rate of 79% (see Table 1). At one year, 58% of patients were still alive with 47% (27/57) of patients ≥60 years and 72% (36/50) of those <60 years surviving (OR 2.8, 95% CI 1.3-6.4). When stratifying by NCCN risk, no deaths at one year occurred among the favorable risk group (0/21), but 16/43 (37%) and 28/43 (65%) occurred in intermediate and poor-risk strata, respectively (p<0.001 by chi-squared). Median overall survival was 17.2 months with mean follow-up of 32.8 months. Cox proportional hazard ratio (HR) for death were significant for age ≥60 compared to age <60 (HR 2.2, 95% CI 1.3-3.7). Additionally, HR for intermediate risk leukemia was 6.2 (95% CI 1.8-21) and for poor risk leukemia it was 11 (95% CI 3.4-36), when each were compared to favorable risk. Thirty-three (31%) of patients were known to have received an allogeneic transplant, of which 42% (14/33) were 60 and older.Adverse events: Overall, the treatment related mortality (TRM) (defined as death within the first 28 days) was 11%. Eleven of 12 TRM events occurred in patients ≥60 years (OR 12, 95% CI 1.5-94). Diarrhea occurred in 71% of patients with 46% enduring mucositis. Intensive care unit admission occurred in 23% overall, and infections were documented in 73% of patients. Most common infectious etiology was coagulase negative staph in 29% (23/78). For patients surviving to discharge (90/107), median hospitalization was 30 days post induction.Conclusions: Cladribine combined with cytarabine and idarubicin is an effective induction regimen for AML with high rates of CR, even in patients 60 and older. Our data show that NCCN leukemia risk is a better predictor of survival compared to age and that patients aged ≥60 years have better survival (lower HR for death) compared to those with intermediate or poor risk leukemia. Additionally, CR and 1 year overall survival with IAC are better than expected in the older cohort, however, TRM is elevated among those 60 and older. Higher intensity regimens in older patients may be justified in select patients, especially given the greater life expectancies enjoyed in the modern era. Several ongoing prospective trials investigating the safety and efficacy of cladribine in older patients will provide additional information to inform the use of IAC in older patients. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Outcome of Standard Induction Therapy with Daunorubicin and Ara-C (3+7) in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a clonal, malignant disease of hematopoietic tissue.Main variables precluding cure are the treatment-related mortality and relapse rates.The most common induction chemotherapy regimens in AML involve the use of cytarabine (Ara-C) and an anthracycline. It was observational study carried out in the inpatient Department of Hematology, Dhaka Medical College Hospital, Dhaka, from July 2009 to June 2014.76 newly diagnosed cases of AML patients between 15 year to 60 yearwere enrolled in this study. All patient received standard induction therapy with Daunorubicin 45mg/m2 I/V for 3 days and Ara-C 100mg/m2 I/V over 24 hours for 7 days. A bone marrow aspirate to assess remission status was carried out at day 28+, after the peripheral recovery.The mean age of the patients were 35.12±12.45 years, ranging from 17 to 55 years. 68% were male and 32% cases were female. Total 34(44.7%) patient achieved complete remission (CR), out of them 17% had t(8:21), 5.8% had inv16 and only 2.9% was FLT3 positive. 26(34%) patient achieved partial remission (PR), 11% had t(8:21) and 3.8% inv16. 13.1% patient failed to achieve any remission (Non responder, NR) and 6 patient (7.8%) died during induction therapy. Age, performance status and cytogenetics had significant influence (P&lt;0.025, P&lt;0.030 and P&lt;0.003 respectively) on the outcome. The presenting peripheral blast percent (%) also had a significant influence (P=0.012) on the outcome after induction therapy.Complete remission rate is significantly lower in our center comparing discussed study. This is probably due to lack of modern hospital facility and socio-economic condition of our patients.Cytogenetic analysis provides the most powerful independent predictor of disease outcome. To improve outcome, all the supportive measures have to be improved and more tools are required in identifying the good prognostic groups.J Dhaka Medical College, Vol. 24, No.2, October, 2015, Page 141-145

The impact of HLA-matching on reduced intensity conditioning regimen unrelated donor allogeneic stem cell transplantation for acute myeloid leukemia in patients above 50years-a report from the EBMT acute leukemia working party.

BackgroundData comparing fully matched and mismatched-unrelated-donor (M- and mM-URD) allogeneic hematopoietic stem cell transplant (allo-SCT) following reduced intensity conditioning regimens for acute myeloid leukemia are limited.MethodsWe retrospectively compared the outcome of 3398 patients above the age of 50 years who underwent 10/10 M-URD (n = 2567), 9/10 (n = 723), or 8/10 (n = 108) mM-URD allo-SCT for acute myeloid leukemia after reduced intensity conditioning regimen between 2000 and 2013. The Kaplan-Meier estimator, the cumulative incidence function, and Cox proportional hazards regression models were used where appropriate.ResultsHLA matching had no impact on engraftment (p = 0.31). In univariate analysis, in comparison to 10/10 M-URD, mM-URD was associated with higher incidence of grade II–IV acute graft-versus-host disease (GVHD) (p = 0.0002), similar rates of chronic GVHD (p = 0.138) but increased incidence of its extensive form (p = 0.047). Compared to 10/10 M-URD, patients transplanted in the first complete remission (CR1) with a 9 or an 8/10 mM-URD had decreased 2-year leukemia free (LFS) (p = 0.005) and overall survivals (OS) (56.7, 46.1, and 50.2 %, respectively, p = 0.005), while outcomes were comparable between all groups for patients transplanted beyond CR1. In multivariate analysis, 9/10 versus 10/10 URD was associated with higher non-relapse mortality (HR 1.34, p = 0.001), similar risk of relapse and chronic GVHD and inferior LFS (HR 1.25, p = 0.0001), and OS (HR 1.27, p = 0.0001). There was no difference in adjusted transplant outcomes between 9/10 and 8/10 mM-URD.ConclusionsReduced intensity conditioned allo-SCT with a 10/10 M-URD remains the preferable option for AML patients above the age of 50 years. The use of a 9/10 or an 8/10 mM-URD in patients not having a fully matched donor represents an alternative therapeutic option that should be compared to other alternative donor transplant strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0295-9) contains supplementary material, which is available to authorized users.

Long-Term Outcomes after Treatment with Clofarabine ± Fludarabine with Once-Daily Intravenous Busulfan as Pretransplant Conditioning Therapy for Advanced Myeloid Leukemia and Myelodysplastic Syndrome

Pretransplant conditioning regimens critically determine outcomes in the setting of allogeneic stem cell transplantation (allo-SCT). The use of nucleoside analogs such as fludarabine (Flu) in combination with i.v. busulfan (Bu) has been shown to be highly effective as a pretransplant conditioning regimen in acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and myelodysplastic syndrome (MDS). Because leukemia relapse remains the leading cause of death after allo-SCT, we studied whether clofarabine (Clo), a nucleoside analog with potent antileukemia activity, can be used to complement Flu. In a preliminary report, we previously showed the safety and efficacy of Clo ± Flu with i.v. Bu in 51 patients with high-risk AML, CML, and MDS. The study has now been completed, and we present long-term follow-up data on the entire 70-patient population, which included 49 (70%), 8 (11%), and 13 (19%) patients with AML, MDS, and CML, respectively. Thirteen patients (19%) were in complete remission, and 41 patients (59%) received matched unrelated donor grafts. Engraftment was achieved in all patients. Sixty-three patients (90%) achieved complete remission. There were no deaths reported at day +30, and the 100-day nonrelapse mortality rate was 4% (n = 3). Thirty-one percent of patients (n = 22) developed grades II to IV acute graft-versus-host disease, and the median overall survival and progression-free survival times were 2.4 years and .9 years, respectively. Our results confirm the safety and overall and progression-free survival advantage of the arms with higher Clo doses and lower Flu doses, which was most prominent in the AML/MDS group.

A comparative study of idarubicin 12 mg/m(2) and 8 mg/m(2) combined with cytarabine as the first induction regimen for adult acute myeloid leukemia patients.

ObjectiveThis study aimed to explore a suitable dose of idarubicin (IDA) combined with cytarabine for the initial induction regimen for acute myeloid leukemia (AML) patients.Patients and methodsA total of 100 adult patients with de novo AML aged between 14 years and 80 years were enrolled in the current study and randomized into two arms for the initial induction: an IDA 12 mg/m2 arm and an IDA 8 mg/m2 arm. All patients received the same consolidation chemotherapy. The follow-up period was January 1, 2009, to December 31, 2014. Overall survival (OS), disease-free survival (DFS), and morphology leukemia relapse (hematological and/or extramedullary) were recorded.ResultsThe complete remission rates were 80% and 75% in the IDA 12 mg/m2 and IDA 8 mg/m2 arms, respectively, after initial induction. High-dose IDA (12 mg/m2) resulted in a higher complete remission rate after two courses of induction therapy (96.4% vs 76.5%) in the cytogenetic intermediate-risk group (P=0.026). There were no differences in the number of units of infused red blood cells, agranulocytosis time, or infection rates between the two arms. Patients in the IDA 12 mg/m2 arm received more platelet transfusions (P=0.047). In the intention-to-treat analysis, after a median follow-up of 13 months, high-dose IDA (12 mg/m2) resulted in improved OS (median OS, 54.0 months vs 26.7 months, P=0.021) and DFS (median DFS, 54.0 months vs 18.3 months, P=0.031), particularly in the cytogenetic intermediate-risk group (median OS, 54.0 months vs 29.5 months, P=0.009; median DFS, 54.0 months vs 15.3 months, P=0.014). IDA 12 mg/m2 significantly improved OS and DFS in the cytogenetic intermediate-risk group (P=0.009 and P=0.018).ConclusionOur results suggest that a high dose of IDA (12 mg/m2) combined with cytarabine is a suitable and safe initial remission induction regimen that results in superior long-term survival of adult AML patients, particularly patients in the cytogenetic intermediate-risk group.

Association between early promoter-specific DNA methylation changes and outcome in older acute myeloid leukemia patients

Treatment options for older patients with acute myeloid leukemia (AML) range from supportive care alone to full-dose chemotherapy. Identifying factors that predict response to therapy may help increase efficacy and avoid toxicity. The phase II SWOG S0703 study investigated the use of hydroxyurea and azacitidine with gemtuzumab ozogamicin in the elderly AML population and found survival rates similar to those expected with standard AML regimens, with less toxicity. As part of this study, global DNA methylation along with promoter DNA methylation and expression analysis of six candidate genes (CDKN2A, CDKN2B, HIC1, RARB, CDH1 and APAF1) were determined before and during therapy to investigate whether very early changes are prognostic for clinical response. Global DNA methylation was not associated with a clinical response. Samples after 3 or 4 days of treatment with azacitidine showed significantly decreased CDKN2A promoter DNA methylation in patients achieving complete remission (CR) compared to those who did not. Samples from day 7 of treatment showed significantly decreased RARB, CDKN2B and CDH1 promoter DNA methylation in responders compared to nonresponders. Gene-specific DNA methylation analysis of peripheral blood samples may help early identification of those older AML patients most likely to benefit from demethylating agent therapy.

Alvocidib Potentiates the Activity of Cytarabine and Mitoxantrone through the Targeting of MCL-1 When Administered in a Time Sequential Regimen in AML

Alvocidib has demonstrated a significant improvement in the complete response rates of newly diagnosed acute myeloid leukemia (AML) patients when administered before cytarabine and mitoxantrone (FLAM regimen) in a randomized Phase 2 study compared to 7+3, the current standard of care. Although the mechanism of action of alvocidib as a single agent is documented, the mechanism underlying synergy found in the FLAM regimen is still not fully understood. The FLAM regimen was originally developed based on the perceived benefit of time-sequential cell cycle arrest (alvocidib) followed by release of the cells from cell cycle arrest and inhibition of DNA replication (cytarabine) during S-phase. However, recent reports suggest that the transcriptional repression of key anti-apoptotic proteins (eg., MCL-1) mediated by alvocidib's CDK9 inhibition, drive the activity in the FLAM regimen. We, therefore, hypothesized that MCL-1 transcriptional repression constitutes the primary mechanism for the synergism observed with the treatment of the FLAM regimen. Here, we demonstrate that treatment with alvocidib, followed by treatment with cytarabine and mitoxantrone, is synergistic in vitro and correlates with the downregulation of MCL-1 expression. The FLAM regimen results in significant increases in caspase activity in comparison to any single agent within the combination. As has been previously reported, we also observe that increased activity of cytarabine in alvocidib-treated cells corresponds with progression into the S-phase of the cell cycle, following the washout of alvocidib. However, this observation accounts for only a small portion of the inhibition of cell proliferation. This is further confirmed by the observation that CDK4/6 (cell cycle) specific inhibitors, such as palbociclib, do not show synergistic increases in caspase activity following treatment in the same setting. In various AML cell lines treated with MCL-1 siRNA, followed by cytarabine and mitoxantrone treatment, we also observe a synergistic increase in the inhibition of cell proliferation. Therefore, considering our earlier work showing that MCL-1 dependence predicts AML patient response to the FLAM regimen, we propose that MCL-1 repression is the primary mechanism of alvocidib's biological activity and also a primary mechanism conferring resistance to cytarabine. We also conclude that the FLAM regimen is an effective regimen, clinically, in treating patients with high-risk AML, as a consequence of its inhibition of transcription via CDK9. DisclosuresKim:Tolero Pharmaceuticals: Employment. Bahr:Tolero Pharmaceuticals: Employment. Soh:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Lee:Tolero Pharmaceuticals: Employment. Peterson:Tolero Pharmaceuticals: Employment. Whatcott:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Weitman:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment.

A Comparison of Clofarabine-Based (GCLAC) and Cladribine-Based (CLAG) Salvage Chemotherapy for Relapsed/Refractory AML

Introduction: Salvage chemotherapy regimens for patients with relapsed/refractory acute myeloid leukemia (AML) are associated with complete response rates of 30 - 60%. Determining the superiority of one treatment over another is difficult due to the lack of comparative data. There are no data comparing treatments with cladribine and clofarabine based salvage regimens to each other. Therefore, we conducted a retrospective study of GCLAC (clofarabine 25 mg/m2 IV days 1-5, cytarabine 2 gm/m2 IV days 1-5, and G-CSF) and CLAG (cladribine 5 mg/m2 IV days 1-5, cytarabine 2 gm/m2 IV days 1-5, and G-CSF).Methods: We identified 41 consecutive patients with pathologically diagnosed relapsed or refractory AML who received either GCLAC or CLAG between 2011 and 2014. The primary outcome was the complete response rate (CRp or CR) as defined by the International Working Group. Secondary outcomes included the percentage of patients who underwent allogenic stem cell transplant, relapse free survival (RFS), and overall survival (OS). Fisher's exact and Wilcoxon Rank Sum tests were used to compare patient characteristics and response rates. The Kaplan Meier method and Log Rank tests were used to evaluate RFS and OS.Results: We found no significant differences in the baseline characteristics of patients treated with GCLAC (n=22) or CLAG (n=19) including age, race, gender, organ function, or cytogenetic risk group (table 1). There were also no significant differences in the percentage of relapsed patients (36% vs. 21%), the average duration of the previous remission (28.6 vs. 19.4 months) or in their previous therapy. An anthracycline-based "7+3" regimen was given to 82% of the GCLAC patients and to 90% of the CLAG patients.The outcomes with these two regimens were also not significantly different. Patients treated with GCLAC had a 64% CR/CRp rate compared with 47% for CLAG patients (p= 0.36). 45% GCLAC patients underwent allogeneic stem cell transplant compared with 26% of CLAG patients (p= 0.32). The median RFS on GCLAC and CLAG respectively was 1.59 years [0.41, non-estimable (NE)] and 1.03 years [0.49, 1.03], (p= 0.75). The median OS was 1.03 years [0.52, NE] and 0.70 years [0.28, 1.11], (p= 0.08).Given the similarities of these regimens, we combined the data sets to compare the OS for patients with refractory AML to relapsed AML. The OS for patients with refractory AML was not significantly worse than patients with relapsed AML (0.94 years [0.36, 1.3] vs.1.11 years [0.46, not evaluable]; p=0.49).Conclusion: We find no significant differences in outcomes using GCLAC or CLAG for relapsed/refractory AML patients. The trends in outcome that favored GCLAC are likely explained by trends in patient populations (e.g. longer first remission for GCLAC patients). Since our results are similar to the published reports describing these regimens, we feel the choice of regimen can be based on other considerations such as cost. We do find the efficacy of both regimens in refractory AML to be encouraging. However, we recognize that overall survival of one year is not acceptable and that most relapsed/refractory patients should be entered into clinical trials. Table 1Baseline CharacteristicsGCLAC (n=22)CLAG (n=19)p ValueAge (years)54.75 ± 11.552.9 ± 12.50.69Race (C vs Non C)18 (82%)12 (63%)0.21Gender (M)11 (50%)11 (58%)0.76Risk groupFavorable4 (19%)2 (11%)0.48Int-12 (10%)4 (22%)Int-27 (33%)3 (17%)Adverse8 (38%)9 (50%)Salvage attempt120 (91%)15 (79%)0.39>12 (9%)4 (21%)Relapse vs RefractoryRelapse8 (36%)4 (21%)0.32Primary Refractory14 (64%)15 (79%)OutcomesGCLAC (n=22)CLAG (n=19)p ValueCRp or CR14 (64%)9 (47%)0.36Transplant9 (45%)5 (26%)0.32Median RFS (years)1.59 (0.41,NE)1.03 (0.49, 1.03)0.75Median OS (years)1.03 (0.52, NE )0.70 (0.28, 1.11)0.083RelapseRefractoryp ValueOS (years) of relapse vs refractory patients*1.11 (0.46, NE)0.94 (0.36, 1.34)0.49*All GCLAC and CLAG patients combined DisclosuresFoster:Celgene: Research Funding.

A Phase 1/2, Open-Label, Dose-Escalation Study of Midostaurin in Pediatric Patients (Pts) with Relapsed or Refractory (R/R) Acute Leukemia: Final Results of Study ITCC-024 (CPKC412A2114)

Introduction Outcomes for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) have improved, but the prognosis for pts with R/R disease remains poor. Childhood AML and MLL-rearranged ALL (MLL r-ALL) often overexpress or have activating mutations in the FLT3 tyrosine kinase (TK). Midostaurin (PKC412) is an orally available TK inhibitor that targets FLT3, KIT, and other kinases. Data in adults for single-agent midostaurin, including from studies CPKC412A2104 and CPKC412A2106, showed that 100 mg bid (≈ 60 mg/m2 bid) was adequately tolerated, elicited leukemic blast reduction, and inhibited FLT3 autophosphorylation. Here, we present the safety and preliminary clinical activity of midostaurin in children with R/R acute leukemia.Methods In this multicenter dose-escalation study, children (3 mo to < 18 y) with FLT3 -mutated AML or MLL r-ALL refractory to standard induction therapy or in second or subsequent relapse received oral midostaurin solution (NCT00866281). The primary objective was to estimate the maximum tolerated dose (MTD) or recommended dose for expansion (RDE) for 2 age groups: younger (3 mo to 2 y) and older (> 2 y to < 18 y). The secondary objective was to evaluate response, time to relapse, overall survival (OS), pharmacokinetics (PK), pharmacodynamics, safety, and tolerability. In the dose-escalation phase (starting dose, 30 mg/m2 bid), each cohort received increasing doses until a maximum dose of 60 mg/m2 bid was reached. Dose escalation was determined using a Bayesian logistic regression design, allowing separate dose escalation in each age group. Dose-limiting toxicity (DLT) was defined as a grade 3/4 nonhematologic adverse event (AE), abnormal drug-related laboratory value, or drug discontinuation within 14 days of study start.Results This study enrolled 22 pts (7 at 30 mg/m2 bid [2 younger, 5 older]; 15 at 60 mg/m2 bid [9 younger, 6 older]) starting on Sep 21, 2009. The median age was 2 y (range, 0.5-17 y); 9 pts (9 older) had AML and 13 pts (11 younger, 2 older) had ALL. The dose-determining set included 17 pts.No DLTs occurred at 30 mg/m2 bid; thus, in each age group, the dose was escalated directly to 60 mg/m2 bid. One DLT (increased alanine aminotransferase [ALT]) occurred in a younger pt receiving 60 mg/m2 bid; no older pts had a DLT. The most common AEs regardless of study drug relationship (any grade/grade 3/4) were vomiting (68%/5%), pyrexia (41%/14%), and thrombocytopenia (41%/36%). Grade 3/4 AEs (particularly thrombocytopenia, elevated ALT, and anemia) were more common with the higher dose. Consistent with the safety profile of midostaurin in adults, the most frequent drug-related any-grade AEs were vomiting (55%), nausea (32%), and diarrhea (18%). The MTD was not reached.Median OS was 3.7 mo (95% CI, 2.7-8.3 mo) in pts with AML and 1.4 mo (95% CI, 1.0-2.9 mo) in pts with ALL. All 5 on-treatment deaths (ie, death while on study drug or within < 28 days of final dose) were due to underlying AML (n = 4) or ALL (n = 1); 16 additional pts (4 with AML, 12 with ALL) died ≥ 28 days after the final dose. One pt was alive at trial end.Midostaurin demonstrated limited single-agent clinical activity in this study. Five pts (56% [95% CI, 21%-86%]) with AML achieved a clinical response. The best response was a morphological complete remission with incomplete count recovery on day 14. The pt remained in remission until stopping treatment on day 64, received a stem cell transplant (SCT) on day 76, and was alive at day 960 after completing the per-protocol survival follow-up. Three pts (23% [95% CI, 5%-54%]) with ALL achieved a clinical response; all were peripheral blood blast responses.All pts discontinued treatment; reasons were disease progression (64%), start of new cancer therapy (including SCT [18%]), withdrawn consent (14%), or AEs (5%). Median duration of exposure was 15.5 days (range, 3-64 days).Time-dependent PK and exposures of midostaurin and its 2 active metabolites (CGP52421 and CGP62221) at the selected doses were similar to adult data.Conclusions Single-agent midostaurin was adequately tolerated at 60 mg/m2 bid, the highest dose evaluated. Only limited clinical activity was demonstrated; thus, future trials in children with FLT3 -mutated AML should be in combination with established chemotherapies. The RDE of midostaurin for such studies is 30 mg/m2 bid due to a higher frequency of grade 3/4 AEs in the 60 mg/m2 bid dose group and known toxicities of existing standard pediatric AML regimens. DisclosuresZwaan:Janssen: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Söderhäll:Novartis: Other: Investigator in clinical trials within my duties or ordinary salary at the hospital; Merck: Other: Investigator in clinical trials within my duties or ordinary salary at the hospital. Sternberg:Novartis Oncology: Employment, Equity Ownership. Besse:Novartis: Employment. Dutreix:Novartis: Employment. Ho:Seattle Genetics (employment started after completion of involvement in presented study): Employment. Dufour:Pfizer: Consultancy.

Prospective comparison of early bone marrow evaluation on day 5 versus day 14 of the "3 + 7" induction regimen for acute myeloid leukemia.

Evaluation of early response during induction therapy for acute myeloid leukemia (AML) is used for prognostication and re-induction strategy, yet the optimal evaluation time point is unknown. Clearance of bone marrow (BM) blasts by day 14 of therapy does not ensure remission; thus, some patients requiring re-induction are neglected. This study aimed to examine the role of earlier BM evaluation during induction for predicting remission and overall survival. Results of BM testing on the 5th and 14th day of intensive induction were prospectively compared in 127 adult patients with AML. Re-induction was given, based on Day 14 results, to 25 patients. Reduction of the BM blast count to <5% as early as by the fifth day of induction was more specifically associated with the achievement of remission compared to Day 14 (88.2% vs. 60%, respectively). Rapid responders have a better 3-year overall survival (OS). Day 5 results are a stronger predictor of OS by multivariate analysis and better segregate long-term survivors than the Day 14th BM count (66% vs. 30%, P = 0.0001 and 48% vs. 37%, respectively, P = 0.04). The Day 5 evaluation of BM carries significant clinical information. The benefit of prescribing re-induction based on such early evaluation should be prospectively studied.

Incidence, Risk Factors, and Outcomes of Febrile Neutropenia in Thai Hematologic Malignancy Patients Receiving Chemotherapy: A 6-year Retrospective Cohort Study.

A 6-year retrospective cohort study was conducted among Thai hematologic malignancy (HM) patients receiving intensive chemotherapy. Of the 145 eligible patients receiving 893 chemotherapy sessions, 46.9% were female, median age was 52 years, and the most common HM diagnosis was diffuse large B-cell lymphoma (46.2%). Febrile neutropenia (FN) occurred in 14.9% of chemotherapy sessions with an incidence of 24.8 per 1,000 chemotherapy cycles per year. Independent factors associated with FN were receiving the first chemotherapy cycle [adjusted hazard ratio (aHR) 4.1], having hemoglobin ≤100 g/L (aHR 3.7) and platelet ≤140,000/μL (aHR 2.7) on chemotherapy day and receiving acute myeloid leukemia regimens (aHR 20.8). Granulocyte colony stimulating factor was significantly associated with reduced rate of FN when given in those receiving CHOP regimen. With the median follow-up time of 16 months, the overall survival time was significantly longer in patients without FN than those with FN (61.7 vs. 20.8 months; p<0.001).

Abstract 3139: CD33 directed chimeric antigen receptor T cell therapy as a novel regimen prior to allogeneic stem cell transplantation in acute myeloid leukemia

Abstract Allogeneic stem cell transplantation is the only option in relapsed acute myeloid leukemia (AML). However, patients are often excluded due to refractory disease. The successful translation of Chimeric Antigen Receptor (CAR) T cell therapy to AML would constitute a vertical advance in the field. We developed a 2nd generation CAR using the anti CD33 scFv of Gemtuzumab ozogamicin, 41BB costimulation, CD3ζ signaling domain, and a lentiviral (LV) vector. In vitro, CART33 resulted in robust functions when incubated with the CD33+ cell line MOLM14. In NSGS mice (NOD SCID γc-/-(NSG), transgenic for stem cell factor, IL3 and GM-CSF) engrafted with primary AML, CART33 eradicated AML and led to a long term disease free survival (Table 1). Activity of CART33, compared to control untransduced T cellsCART33UTDP-value% Degranulation (4 hr incubation with MOLM14)98%1.3%&amp;lt;0.0001% Specific Lysis, 24 hr incubation with MOLM14 (E:T 0.625:1)65.7%30%&amp;lt;0.0001CFSE based proliferation (5-day incubation with MOLM14)91.6%3%&amp;lt;0.0001IL-6 (24-hour incubation with MOLM14, median, pg/ml)22.655.73&amp;lt;0.0001GM-CSF (24-hour incubation with MOLM14, median, pg/ml)1324816.9&amp;lt;0.0001MIP-1b (24-hour incubation with MOLM14, median, pg/ml)81809.15&amp;lt;0.0001IFN-g (24-hour incubation with MOLM14, median, pg/ml)249890.35&amp;lt;0.0001IL-2 (24-hour incubation with MOLM14, median, pg/ml)93000.39&amp;lt;0.0001Overall survival at 200 days (NSG-S mice engrafted with primary AML blasts and treated with CART-33 or UTD)100%0%&amp;lt;0.0001Median AML burden, measured by bioluminescence (photons/sec) in NSG mice engrafted with MOLM14, 6 weeks after treatment7×10(7)2×10(9)0.01 Since CD33 is also expressed on normal myeloid progenitors, we assessed the potential myelotoxicity of CART33 in humanized immune system (HIS) mice (NSG mice engrafted with human fetal liver CD34+ cells). CART33 resulted in human lineage cytopenias and in a significant reduction of CD34+CD38- hematopoietic stem cells and CD34+CD38+ myeloid progenitors. Both the anti-leukemic activity and myeloablative potential of CART33 could be used as a novel conditioning regimen in refractory AML. However, the effect of the CAR T cells needs to be terminated to avoid engraftment failure. We therefore designed a “biodegradable” mRNA modified CAR33. T cells were electroporated with this construct and expressed CAR for up to six days. When compared with LV-CART33, RNA-CART33 resulted in comparable in vitro functions that declined over time post electroporation. In NSG mice engrafted with MOLM14, treatment with cyclophosphamide (for lymphodepletion) plus RNA-CART33 resulted in deeper and longer leukemic response compared to cyclophosphamide plus control T cells. In conclusion, our preclinical studies show potent activity of CART33 in AML. Biodegradable CART33 could be used as a novel anti-leukemic cellular conditioning in patients with relapsed AML Note: This abstract was not presented at the meeting. Citation Format: Saad S. Kenderian, Marco Ruella, Olga Shestova, Michael Klichinsky, John Scholler, Decheng Song, David L. Porter, Martin Carroll, Carl H. June, Saar Gill. CD33 directed chimeric antigen receptor T cell therapy as a novel regimen prior to allogeneic stem cell transplantation in acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3139. doi:10.1158/1538-7445.AM2015-3139

Targeting Mitochondria with Avocatin B Induces Selective Leukemia Cell Death.

Treatment regimens for acute myeloid leukemia (AML) continue to offer weak clinical outcomes. Through a high-throughput cell-based screen, we identified avocatin B, a lipid derived from avocado fruit, as a novel compound with cytotoxic activity in AML. Avocatin B reduced human primary AML cell viability without effect on normal peripheral blood stem cells. Functional stem cell assays demonstrated selectivity toward AML progenitor and stem cells without effects on normal hematopoietic stem cells. Mechanistic investigations indicated that cytotoxicity relied on mitochondrial localization, as cells lacking functional mitochondria or CPT1, the enzyme that facilitates mitochondria lipid transport, were insensitive to avocatin B. Furthermore, avocatin B inhibited fatty acid oxidation and decreased NADPH levels, resulting in ROS-dependent leukemia cell death characterized by the release of mitochondrial proteins, apoptosis-inducing factor, and cytochrome c. This study reveals a novel strategy for selective leukemia cell eradication based on a specific difference in mitochondrial function.

Prognostic significance of day+14 plasma cell count after initial induction therapy in Acute Myeloid Leukemia.

e18056 Background: Induction therapy regimens for acute myeloid leukemia (AML) target attainment of complete remission. Bone marrow biopsy is done usually after two to three weeks after the start o...