Acute Myelogenous Leukemia Therapy Research Articles

Predictive Factors for Complete Remission and Survival in AML Patients Failing to Achieve Adequate Bone Marrow Day 14 Blast Eradication

Introduction: Complete remission (CR) is an important endpoint after cytarabine plus anthracycline [7+3] induction therapy in Acute Myelogenous Leukemia (AML). Even though CR is observed in about 50-90% of unselected European Leukemia Network 2017 (ELN-2017) patients (pt), factors such as age >65 years, complex karyotype, and adverse mutations (RUNX1, ASLX1, TP53, FLT3 ITD high, KMT2A, secondary AML), leads to inadequate blast eradication. Early response to induction is a predictor of subsequent complete remission (CR). Bone marrow day 14 [D14] inform pt with early inadequate leukemia eradication [blast >10%] who are suitable for re-induction, a strategy that seeks to facilitate conversion to CR or CR with incomplete hematologic recovery (CRi), secure potential allogenic transplantation and reproduce superior outcomes. In this study, we investigated the clinical outcome of an unselected ELN-2017 AML cohort with inadequate D14 marrow response. From the subgroup of patients exhibiting sub-optimal response (SOR), we examined the odds and predictors for subsequent achievement of CR/CRi for those patients who did not receiving immediate re-induction therapy. Additionally, we evaluated the effect of subsequent CR/CRi achievement on survival.Methods: With prior IRB approval, 160 AML pt diagnosed with AML from 1995 to 2017 within Baylor College of Medicine institutions were evaluated. Kaplan-Meier method was used to estimate overall survival (OS) among pt achieving D14 <> 10% blast in an unselected ELN-2017 AML cohort and pt exhibiting >10% blast in D14 marrow with and without CR/CRi. Logistic and cox regression analysis in SOR pt [1] attaining subsequent CR/CRi and [2] OS, respectively, was performed to investigate multiple independent variables with predictive value for the 2 above outcomes.Results: 68/160 (42.5%) of pt had available D14 [early assessment] and sequential day 30 marrow for CR/CRi evaluation. Among 68 unselected ELN-2017 AML pt with D14 marrow for CR/CRi assessment, 42/68 (61.7%) and 26/68 (38.2%) had D14 marrow blasts < and > 10%. Median age was 57 y (range 27-73) and 59 y (range 24-89), respectively, p= 0.74. OS was 459 d vs 169 d in pt with D14 marrow <10% and >10%, at day 14 (p=0.001 95% CI 0.2-0.9) [Fig 1A]. CR/CRi was observed in 36/42 (90%) and 10/26 (38.7%) of pt with D14 marrow <10% vs >10%, respectively, p=0.0005. After controlling for traditional high-risk factors including WBC, age, platelet count, RDW, de novo v secondary AML, only ELN-2017 classification [fav vs unfav and intermediate vs unfav, p= 0.0026 and p=0.01] retained impact on survival. In pt with SOR, we performed second analysis to investigate survival among pt with and without subsequent CR/CRi achievement who did not receive re-induction [Fig 1B]. 16/26 (62.5%) of pt with SOR failed to achieved CR/CRi. OS was 333 d vs 109 d for pt with CR/CRi vs those without CR/CRi [p=0.002, 95% CI 2.6-3.4]. Logistic regression identified in pt with CR/CRi vs those without CR/CRi that: [a] age [63.1 vs 43.6 y-p=0.001]; [b] lower platelet count [47.1 vs 83.1 K/uL-p=0.03]; [c] higher absolute monocyte count (AMC) [3.7 vs 0.41 K/uL-p=0.04]; [d] increased RDW [18.3 vs 14.7-p=0.004] and [e] high BMI [31 vs 24.1-p=0.0003] were significantly associated with failure to achieve CR/CRi. Typical complex karyotype and initial marrow blast % were not associated with subsequent CR/CRi achievement. However, in pt with SOR, lack of high-risk mutations [P53, RUNX, FLT3-ITD, U2AF1] was significant associated with CR/CRi, [40% v 62.5%, p= 0.0004]. Cox proportional regression model showed significant impact on survival for high-risk mutations and higher BMI in survival.Conclusion: In our retrospective study, despite 38.7% of patients with detectable D14 residual leukemia achieved CR/CRi without re-induction, failure to attain CR/CRi was frequently observed after SOR. Advanced age, lower platelet count, higher AMC, RDW and BMI predict failure to achieve CR/CRi status in patients exhibiting initial SOR. Lack of high-risk mutation was a strong predictor for CR/CRi achievement. Our study is novel by suggesting that a combination of pre-induction and “early post-induction” variables facilitate recognition of high-risk AML subgroups requiring re-induction or alternative novel therapy via clinical trials. [Display omitted] DisclosuresYellapragada:Takeda: Research Funding; Novartis: Employment; Celgene: Research Funding.

Outcomes of Allogeneic Stem Cell Transplantation for AML and MDS Based on Pre-Transplant MRD Status By Next-Generation Sequencing

BackgroundAfter induction therapy for acute myelogenous leukemia (AML), the presence of minimal residual disease (MRD) by targeted next-generation sequencing (NGS) during complete remission (CR) predicts relapse and survival, particularly after exclusion of pre-leukemic mutations. MRD assessment is not routinely performed for AML prior to transplant, partly because consensus regarding assay methodology, appropriate timing, interpretation of results, and therapeutic value prior to SCT is lacking. We therefore sought to describe the rates of mutational clearance and correlate these with relapse rates post-transplant.MethodsWe conducted a retrospective review of sequential AML or myelodysplastic syndrome (MDS) patients undergoing allogeneic hematopoietic cell transplant (alloHCT) at our institution between 2014 and 2017. There were 119 patients with AML/MDS who were treated with either myeloablative or reduced intensity conditioning regimens. Of the 119 patients transplanted, 60 had both pre- and post-treatment NGS results and were included in the analysis. 56 patients had somatic mutations on initial NGS and were therefore eligible for mutational clearance analysis. Twelve patients were in active disease and excluded from further analyses. The remaining patients (n=44) represent the core dataset.Blood and/or marrow specimens were analyzed via a clinical NGS panel targeting 68 leukemia-associated genes. Median coverage (across 88 samples) was 2817 reads. Mutations were considered persistent if present at variant allele frequencies (VAF) ≥ 1% for single nucleotide variants (SNV) or ≥ 2 copies for insertions and deletions (indels). Validated laboratory reporting practice at our institution reports VAF > 4% for SNVs and ≥ 1% for indels with a minimum of 250 total reads. We therefore defined three levels of mutational clearance on the basis of the VAF of residual mutations: VAF for SNV <1% (and/or indels ≤1 copy), between 1-4% (and/or indels <1% and ≥ 2 copies), and >4% (and/or indels > 1%). Patients with ≥ 1 mutation meeting these thresholds were designated NGS(-), NGS-low and NGS(+), respectively. The median follow-up was 332 days.ResultsOn review of NGS data, 120 mutations were present in initial sequencing, with 64 mutations persistent in pre-transplant samples from 26 patients. The most commonly mutated genes from initial samples were FLT3 (18), ASXL1 (11), TET2 (10), NPM1 (9), RUNX1 (8), SRSF2 (8), and DNMT3A (7) (Figure 1A). Mutational clearance varied widely, with the putative pre-leukemic genes DNMT3A, TET2, and ASXL1 (DTA) demonstrating low rates of mutational clearance (Figure 1A). Mutations persisting below the validated reporting threshold were present in 20 patients, including 10 patients otherwise negative by NGS.There were 16 patients categorized as NGS(+), 10 NGS-low, and 18 NGS(-), with relapse rates of 31%, 22%, and 30%, respectively. No difference in relapse risk was observed between NGS(-) and NGS-low subgroups (p = 0.72), and no RFS benefit was observed for patients without persistent mutations > 4% relative to the NGS(+) subgroup (p = 0.56, Figure 1B). Recent work has shown a survival benefit in AML patients in CR without persistent mutations that is enhanced when DTA genes were excluded from the analysis (Jongen-Lavrencic, NEJM 2018). In our cohort, after exclusion of DTA mutations, 6 patients were reclassified by mutational clearance status, and 2 were excluded from the analysis as they had only DTA mutations in pre-treatment samples. Similar to the more comprehensive cohort, no RFS benefit based on NGS status was observed in the post-transplant period (p = 0.42, Figure 1C).ConclusionsThere were similar outcomes regardless of molecular MRD findings by NGS for patients with advanced myeloid malignancies who were in morphologic CR prior to alloHCT. These results contrast with those in the published literature that address a more uniform patient population of clinical trial participants, not all of whom went on to transplant. Further detailed analyses from larger more homogeneous populations will be useful to determine the prognostic significance of MRD by NGS prior to allogeneic HCT. [Display omitted] DisclosuresFrey:Servier Consultancy: Consultancy; Novartis: Consultancy. Perl:Novartis: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Actinium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; NewLink Genetics: Membership on an entity's Board of Directors or advisory committees; Arog: Consultancy; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy; Daiichi Sankyo: Consultancy. Stadtmauer:Takeda: Consultancy; Celgene: Consultancy; AbbVie, Inc: Research Funding; Amgen: Consultancy; Janssen: Consultancy. Porter:Genentech: Other: Spouse employment; Kite Pharma: Other: Advisory board; Novartis: Other: Advisory board, Patents & Royalties, Research Funding. Gill:Extellia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Carisma Therapeutics: Equity Ownership; Novartis: Research Funding.

Isolated Bilateral Ear and Scalp Rash After Cytarabine Therapy for Acute Myelogenous Leukemia

Waseem, Husnain MBBS1; Henriquez Felipe, Carlos MD1; Hashmi, Arsalan Talib MBBS1; Ansar, Maham MBBS2; Batool, Asiya MBBS2; Saverimuthu, Angela MD3; Kamholz, Stephan L. MD1 Author Information

Results of second salvage therapy in 673 adults with acute myelogenous leukemia treated at a single institution since 2000.

The prognosis is poor for patients who have relapsed-refractory acute myelogenous leukemia (AML). Most published reports analyzed results from therapies in first-salvage AML or in studies that were conducted before 2000. Several novel agents and strategies are being tested for potential approval as treatment for patients with relapsed-refractory AML in second salvage. Therefore, it is important to establish the historic results of anti-AML therapies in this setting in the modern era. The objective of the current study was to analyze the results from second salvage therapies in patients with AML since 2000 with regard to response and survival. In total, 673 patients who received second salvage therapies for AML since 2000 were analyzed. Their median age was 60 years (range, 18-89 years). Salvage therapy consisted of cytarabine-based regimens in 267 patients, noncytarabine combinations in 37, hypomethylating agent-based regimens in 136, and phase 1 and 2 single agents in 233. Eighty-six of the 673 patients (13%) achieved a complete response (CR) or a CR with low platelet count (CRp). The median duration of CR-CRp was 7.2 months. The median survival was 4.4 months (95% confidence interval, 4.0-4.8 months), and the 1-year survival rate was 16% (95% confidence interval, 14%-19%). Multivariate analysis identified the following as independent adverse factors for achievement of CR-CRp: platelets < 50 × 109 /L (P < .001), complex karyotype with ≥3 chromosomal abnormalities (P = .02), regimens that did not include cytarabine or hypomethylating agents (P = .014), and no prior CR lasting ≥12 months with frontline or salvage 1 therapies (P < .001). The independent adverse factors associated with worse survival were age ≥60 years (P = .01), platelets < 50 × 109 /L (P = .02), peripheral blasts ≥ 20% (P = .03), albumin ≤ 3 g/dL (P = .04), and complex karyotype (P = .003). The authors also applied and validated, in the current population, the 2 multivariate-derived prognostic models for CR and survival developed in their previous study of 594 patients who received treatment for second salvage AML from the previous 2 decades. This large-scale analysis establishes the modern historic results of second salvage therapy in AML and validates the prognostic models associated with outcome. These data could be used to analyze the differential benefits of current or future investigational strategies under evaluation in this setting and for the purpose of potential approval of new agents in the United States and the world. Cancer 2018;124:2534-40. © 2018 American Cancer Society.

Combination of the low anticoagulant heparin CX-01 with chemotherapy for the treatment of acute myeloid leukemia

AbstractRelapses in acute myelogenous leukemia (AML) are a result of quiescent leukemic stem cells (LSCs) in marrow stromal niches, where they resist chemotherapy. LSCs employ CXCL12/CXCR4 to home toward protective marrow niches. Heparin disrupts CXCL12-mediated sequestration of cells in the marrow. CX-01 is a low-anticoagulant heparin derivative. In this pilot study, we combined CX-01 with chemotherapy for the treatment of AML. Induction consisted of cytarabine and idarubicin (7 + 3) with CX-01. Twelve patients were enrolled (median age, 56 years; 3 women). Three, 5, and 4 patients had good-, intermediate-, and poor-risk disease, respectively. Day 14 bone marrows were available on 11 patients and were aplastic in all without detectable leukemia. Eleven patients (92%) had morphologic complete remission after 1 induction (CR1). Eight patients were alive at a median follow-up of 24 months (4 patients in CR1). Three patients received an allogeneic stem cell transplant in CR1. Median disease-free survival was 14.8 months. Median overall survival was not attained at the maximum follow-up time of 29.4 months. No CX-01-associated serious adverse events occurred. Median day to an untransfused platelet count of at least 20 × 109/L was 21. CX-01 is well tolerated when combined with intensive therapy for AML and appears associated with enhanced count recovery and treatment efficacy.

Oroxylin A, a natural compound, mitigates the negative effects of TNFα-treated acute myelogenous leukemia cells.

Tumor necrosis factor alpha (TNFα) is a complicated cytokine which is involved in proliferation and differentiation of acute myelogenous leukemia (AML) cells through a poorly understood mechanism. Mechanistic studies indicate that TNFα induced binding of PI3K subunit p85α to N-terminal truncated nuclear receptor RXRα (tRXRα) proteins, and activated AKT. The activated PI3K/AKT pathway negatively regulated differentiation of AML cells through the upregulation of c-Myc. In addition, TNFα also induced activation of nuclear factor κB (NF-κB), a nuclear transcription factor which was shown to promote cell proliferation. The present study demonstrates that oroxylin A, a natural compound isolated from Scutellariae radix, sensitizes leukemia cells to TNFα and markedly enhances TNFα-induced growth inhibition and differentiation of AML cell including human leukemia cell lines and primary AML cells. Activation of PI3K/AKT pathway could be inhibited by oroxylin A through inhibiting expression of tRXRα in NB4 and HL-60-resistant cells. Furthermore, we found that oroxylin A inhibited the activation of NF-κB and the DNA binding activity by TNFα proved by EMSA in these two AML cell lines. Moreover, in vivo studies showed that treatment with oroxylin A in combination with TNFα decreased AML cell population and prolonged survival in NOD/SCID mice with xenografts of primary AML cells. Overall, our results indicate that oroxylin A is able to inhibit the negative effects of TNFα for AML therapy, suggesting that combination of oroxylin A and TNFα have the potential to delay growth or eliminate the abnormal leukemic cells, thus representing a promising strategy for AML treatment.

Timed sequential therapy for acute myelogenous leukemia: Results of a retrospective study of 301 patients and review of the literature

Timed sequential therapy (TST) aims to improve outcomes in acute myelogenous leukemia (AML) by harnessing drug-induced cell cycle kinetics of AML, where a second drug is timed to coincide with peak leukemia proliferation induced by the first drugs. We analyzed outcomes in 301 newly diagnosed AML patients treated from 2004–2013 with cytarabine, anthracycline, and etoposide TST induction. Median age was 52 (range 20–74) and complete remission rate 68%. With median follow-up 5.8 years, 5-year DFS and overall survival (OS) were 37% (95% CI 31–45%) and 32% (95% CI 27–38%), respectively. In multivariate analysis, older age, unfavorable cytogenetics, and WBC≥50×109/L resulted in worse OS. Among patients not undergoing blood and marrow transplant, a propensity score analysis, which reduces imbalance in baseline characteristics, showed consolidation with TST compared with 1 or more cycles high-dose cytarabine trended toward lower DFS and post-remission survival with hazard ratio (HR) 1.9 (95% CI 0.9–4.0), and 1.6 (95% CI 0.7–3.6), respectively. Our results demonstrate the efficacy and feasibility of TST induction for newly diagnosed patients with AML, with results comparable to that seen in clinical trials with other TST therapies and 7+3.

The Multi-kinase Inhibitor Debio 0617B Reduces Maintenance and Self-renewal of Primary Human AML CD34+ Stem/Progenitor Cells.

Acute myelogenous leukemia (AML) is initiated and maintained by leukemia stem cells (LSC). LSCs are therapy-resistant, cause relapse, and represent a major obstacle for the cure of AML. Resistance to therapy is often mediated by aberrant tyrosine kinase (TK) activation. These TKs primarily activate downstream signaling via STAT3/STAT5. In this study, we analyzed the potential to therapeutically target aberrant TK signaling and to eliminate LSCs via the multi-TK inhibitor Debio 0617B. Debio 0617B has a unique profile targeting key kinases upstream of STAT3/STAT5 signaling such as JAK, SRC, ABL, and class III/V receptor TKs. We demonstrate that expression of phospho-STAT3 (pSTAT3) in AML blasts is an independent prognostic factor for overall survival. Furthermore, phospho-STAT5 (pSTAT5) signaling is increased in primary CD34+ AML stem/progenitors. STAT3/STAT5 activation depends on tyrosine phosphorylation, mediated by several upstream TKs. Inhibition of single upstream TKs did not eliminate LSCs. In contrast, the multi-TK inhibitor Debio 0617B reduced maintenance and self-renewal of primary human AML CD34+ stem/progenitor cells in vitro and in xenotransplantation experiments resulting in long-term elimination of human LSCs and leukemia. Therefore, inhibition of multiple TKs upstream of STAT3/5 may result in sustained therapeutic efficacy of targeted therapy in AML and prevent relapses. Mol Cancer Ther; 16(8); 1497-510. ©2017 AACR.

Targeting the pyrimidine synthesis pathway for differentiation therapy of acute myelogenous leukemia

One of the key features of acute leukemia is the differentiation arrest at early progenitor stage of hematopoietic maturation. This has led to the search for therapeutic agents that can overcome this differentiation block to promote terminal differentiation. The first and most notable clinical success to date has been treatment with all-trans retinoic acid (ATRA) for acute promyelocytic leukemia (APL), which accounts for 10% of acute myeloid leukemias (AML) (1).

Pilot Initiative of Fitness Testing for Treatment Selection in Elderly Acute Myelogenous Leukemia (AML)

Introduction: The optimal treatment of older patients with Acute Myeloid Leukemia (AML) is unclear. Traditional induction chemotherapy is less effective in older patients and often more toxic. Clinical trials for elderly AML patients feature inclusion/exclusion criteria based on "fitness" for chemotherapy that often include subjective assessments and variable enrollment criteria. Determining "fitness" remains controversial, and there are few validated objective markers to assist in determining whether a patient is "fit" or "unfit" for intensive induction chemotherapy. Previously published work identified a Short Physical Performance Battery (SPPB) score < 9 and a Modified Mini-Mental Status (3MS) score <77 as independent predictors of worse survival in patients treated with intensive therapy in AML (Klepin et al Blood 2013). Other data suggests using objective measures that include SPPB and 3MS scores to categorize risk in elderly AML patients as "fit", vulnerable or frail (Klepin et al Hematology 2014). Use of the geriatric assessment and quality of life measurements has been suggested to be incorporated in the initial evaluation of AML patients in order to help predict which patients will have poor outcomes with intensive therapy (Deschler et al Haematologica 2013). Based on these publications, a pilot initiative was begun to establish the feasibility of incorporating the SPPB and 3MS tests into practice as objective measures to assist in determination of "fitness" for intensive chemotherapy.Methods: Beginning December 2015, patients age ≥ 60 who presented with new or relapsed AML were evaluated using the 3MS and SPPB tests. To increase objectivity, the 3MS and SPPB tests were performed by a non-treating provider when possible. Patients who were determined to be a fall risk by history had their SPPB test performed by a specially trained physical therapist. Scores were made immediately available to the treating physician and were used as a guide to determine if the patient was "fit" for intensive therapy or "unfit". If "unfit" the patient was evaluated for a clinical trial for "unfit" patients, hypomethylating agents or best supportive care (BSC). Scores were taken into context with age and comorbidities for treatment decisions.Results: Testing was feasible using the SPPB and 3MS and easy to implement, with testing taking approximately 15 minutes. A total of 21 new or relapsed AML patients have been evaluated. The median age was 72, ranging from age 60-83. There were10 male and 11 female patients. Three patients had relapsed disease. Fifteen patients had evaluable data >60 days. Seven were determined "fit" for intensive chemotherapy. Median SPPB and 3MS scores were 9 and 95, respectively. Six achieved remission following intensive induction therapy. One patient underwent allogeneic stem cell transplant and remains in remission. Three relapses occurred at days 44, 50 and 54 from initial induction therapy. Two deaths occurred from relapsed disease at days 148 and 175 from initial induction and one death from infection at day 54 during consolidation therapy.Eight patients were determined to be "unfit" for intensive chemotherapy. Median SPPB and 3MS scores were 5 and 93, respectively. Two patients received BSC based on clinical judgement and died at days 32 and 50 from initial diagnosis. Both were the only patients of 21 evaluated to have a 3MS score < 77. Four patients were eligible for clinical trials at our institution. Two deaths occurred 95 and 52 days from initiation of clinical trial, and 2 remain on trial at 61 and 65 days from initial therapy with one in complete remission. Two patients were given decitabine. One died at 34 days from initial therapy due to infectious complications and the other remains on treatment.Conclusion: Performance of the SPPB and 3MS tests on patients age ≥ 60 is feasible and should be considered at diagnosis for elderly AML patients. Our group found that the use of objective measures was most helpful in identifying the two patients who had impaired cognition at baseline with 3MS scores < 77. The use of a second provider as an examiner allowed for thoughtful discussion regarding choice of initial therapy. Based on this pilot, further investigation and validation of SPPB and 3MS testing for "fitness" prior to determining choice of therapy in patients with new or relapsed AML age ≥ 60 should be pursued. DisclosuresNo relevant conflicts of interest to declare.

Thwarting PTEN Expression by siRNA Augments HL-60 Cell Differentiation to Neutrophil-Like Cells by DMSO and ATRA.

Abnormal cell differentiation, in particular suppression of terminal cell differentiation, exists in all tumors. Therapeutic interventions to restore terminal differentiation ("differentiation therapy") are a very attractive way to treat cancer, especially leukemia. A variety of chemicals stimulates differentiation of leukemic cells, such as dimethyl sulfoxide (DMSO) and all-trans retinoic acid (ATRA). Tumor suppressor genes have a vital role in the gateway to terminal cell differentiation. In this study, we inhibited PTEN tumor suppressor gene expression by siRNA to investigate the effect of potentiating cell survival and inhibiting apoptosis on HL-60 cell differentiation by DMSO and ATRA. Our results show that PTEN siRNA increases HL-60 cell differentiation in the presence of DMSO and ATRA. At the same time, the presence of siRNA hampers accumulation of apoptotic cells during incubation. Our study suggests that manipulation of PTEN could hold promise for enhancing efficacy of differentiation therapy of acute myelogenous leukemia.

Chemotherapy Options for Poor Responders to Neoadjuvant Chemotherapy for Orbital Granulocytic Sarcoma.

Granulocytic sarcoma (GS) is a rare manifestation of myeloid proliferation, characterized by formation of a mass comprised of immature cells of myeloid origin. Orbital granulocytic sarcoma is rarer still, with only a small fraction of GS patients having orbital involvement. Given the rarity of orbital GS, no unified therapy plan has been identified, as large prospective trials are not feasible, but it is widely accepted that patients with GS ought to be treated with systemic intensive chemotherapy consistent with standard of care regimens for acute myelogenous leukemia (AML) or chronic myelogenous leukemia (CML). Development of a treatment plan for GS in poor responders involves a systemic leukemia plan as novel therapeutics have not been investigated for treatment GS per se, but used more widely for AML. GS is most commonly associated with AML and thus will be addressed in that context in this review. Patients with GS associated with CML should receive CML-specific therapy. When conventional and traditional cytotoxic GS/AML chemotherapy regimens are insufficient, patients often require a combination of novel therapeutics, stem cell transplantation (SCT), and radiation. Much of the recent advancement in AML therapy, as well as in AML translational research, has been in targeting molecular facets of the disease and enabling more specificity with treatment. The aim of treating patients for whom conventional treatment was unsuccessful with personalized therapy has not yet been realized, but many of the novel therapeutics reviewed below have demonstrated promise and are cause for optimism. In our center, when a GS/AML patient is refractory to frontline therapy, we rely on novel chemotherapy therapeutic options as outlined below.

Targeting Notch-1 positive acute leukemia cells by novel fucose-bound liposomes carrying daunorubicin.

Complete remission by induction therapy in acute myelogenous leukemia (AML) can be achieved due to improvements in supportive and optimized therapy. However, more than 20% of patients will still need to undergo salvage therapy, and most will have a poor prognosis. Determining the specificity of drugs to leukemia cells is important since this will maximize the dose of chemotherapeutic agents that can be administered to AML patients. In turn, this would be expected to lead to reduced drug toxicity and its increased efficacy. We targeted Notch-1 positive AML cells utilizing fucose-bound liposomes, since activation of Notch-1 is required for O-fucosylation. Herein, we report that intravenously injected, L-fucose-bound liposomes containing daunorubicin can be successfully delivered to AML cells that express fucosylated antigens. This resulted in efficient tumor growth inhibition in tumor-bearing mice and decreased proliferation of AML patient-derived leukemia cells. Thus, biological targeting by fucose-bound liposomes that takes advantage of the intrinsic characteristics of AML cells could be a promising new strategy for Notch-1 positive-AML treatment.

The role of the gastrointestinal microbiome in infectious complications during induction chemotherapy for acute myeloid leukemia.

Despite increasing data on the impact of the microbiome on cancer, the dynamics and role of the microbiome in infection during therapy for acute myelogenous leukemia (AML) are unknown. Therefore, the authors sought to determine correlations between microbiome composition and infectious outcomes in patients with AML who were receiving induction chemotherapy (IC). Buccal and fecal specimens (478 samples) were collected twice weekly from 34 patients with AML who were undergoing IC. Oral and stool microbiomes were characterized by 16S ribosomal RNA V4 sequencing using an Illumina MiSeq system. Microbial diversity and genera composition were associated with clinical outcomes. Baseline stool α-diversity was significantly lower in patients who developed infections during IC compared with those who did not (P = .047). Significant decreases in both oral and stool microbial α-diversity were observed over the course of IC, with a linear correlation between α-diversity change at the 2 sites (P = .02). Loss of both oral and stool α-diversity was associated significantly with the receipt of a carbapenem P < 0.001. Domination events by the majority of genera were transient (median duration, 1 sample), whereas the number of domination events by pathogenic genera increased significantly over the course of IC (P = .002). Moreover, patients who lost microbial diversity over the course of IC were significantly more likely to contract a microbiologically documented infection within the 90 days after IC neutrophil recovery (P = .04). The current data present the largest longitudinal analyses to date of oral and stool microbiomes in patients with AML and suggest that microbiome measurements could assist with the mitigation of infectious complications of AML therapy. Cancer 2016;122:2186-96. © 2016 American Cancer Society.

HDAC Inhibition Induces microRNA-182 Which Targets Rad51 Protein and Impairs Homologous Recombination Repair to Sensitize Cells to the Double Strand Break Inducing Nucleoside Analog, Sapacitabine in AML

Acute myelogenous leukemia (AML) is characterized by multiple genetic and epigenetic abnormalities including a profound dysregulation of microRNA expression.Effective clinical treatment of AML has largely depended on a class of antimetabolites - the nucleoside analogs. Of these, sapacitabine is a nucleoside analog prodrug that is in development for the therapy of AML. It is converted to its active metabolite 2-C-cyano-2-deoxy-1-β(-D-arabino-pentafuranosyl) cytosine (CNDAC), which interferes with DNA synthesis by initially causing a single stranded DNA break that is converted into a double strand break in the subsequent replicative cycle. Such double strand breaks are primarily repaired by the homologous recombination repair (HR) pathway. Consequently, efficient HR may offer a potential resistance mechanism to therapy with sapacitabine. Rad51 is a protein plays a critical role in HR, and high levels of Rad51 are linked to resistance to DNA damaging therapies.Histone deacetylases (HDACs) are chromatin modulating agents that decrease levels of acetylation of histones, repress gene expression. HDAC inhibitors (HDACis) function by modifying chromatin to epigenetically reverse gene silencing of coding and non-coding genes such as the microRNAs (miRs). miRs are endogenous noncoding RNAs 19-25 nucleotides in length that bind to complimentary sequences in target RNA to either destabilize it or prevent its transcription.In this study, we determined that determined primary AML blasts and cell lines express low levels of microRNA-182. Recruitment of HDAC1 and its co-repressors were linked to the epigenetic silencing of miR-182 in AML. Conversely, HDAC inhibition led to accumulation of activating chromatin modifications followed by the upregulation of miR-182 in AML blasts and cell lines. The HDACi-induced increases in miR-182 were linked to decreases in the levels of Rad51, an inhibition in the ability of cells to conduct homologous recombination repair as measured by the Homologous recombination directed repair (HDR) assay, persistent levels of DNA damage as measured by the levels of Ɣ-H2AX and sensitization to sapacitabine. We then mechanistically defined the relation between miR-182 and Rad51. Ectopic expression of miR-182 in AML cell lines identified that Rad51 was a target of miR-182. An assay with luciferase constructs bearing full length or mutated Rad51 3'UTR indentified that Rad51 was a direct target of miR-182. We also determined that ectopic expression of miR-182 attenuated the ability of AML cells to conduct homologus repair as measured by the Homologous recombination directed repair (HDR) assay which resulted in sensitizing AML cells to the cytotoxic action of CNDAC as measured by colony forming assays.In conclusion, our data show that HDAC inhibitors target Rad51 via miR-182 to compromise HR repair to result in higher levels of residual DNA damage and sensitize AML cells to double strand damaging agents such as CNDAC. DisclosuresNo relevant conflicts of interest to declare.

MEN1112/OBT357, an Anti Bst1/CD157 Humanized Antibody Inducing Acute Myelogenous Leukemia (AML) Blast Depletion in an Autologous Ex Vivo Assay: A Potential New Targeted Therapy for AML

Acute myeloid leukemia (AML) is a disease with a poor outcome and novel approaches are needed to improve survival and decrease toxicity of current therapies. Bst1/CD157 is a protein belonging to the ADP-ribosyl-cyclase family expressed on monocytes and neutrophils. This antigen was shown to be also expressed in peripheral blood (PB) and bone marrow (BM) blasts of acute myeloid leukemia (AML) patients either at primary diagnosis or at relapse(1,2,3). MEN1112/OBT357 is a humanized, de-fucosylated antibody targeting Bst1/CD157 with high affinity and developed to generate antibody dependent cell-mediated cytotoxicity (ADCC) response against AML blasts. Peripheral blood (PB) and bone marrow (BM) samples of 38 AML patients (29 at diagnosis, 6 at relapse, 3 resistant), have been analyzed for the expression of Bst1/CD157 on AML blast cells by fluorescence-activated cell sorting (FACS) using a PE conjugated form of MEN1112/OBT357. Bst1/CD157 expression has been confirmed in 91% and 96% of PB and BM AML samples, respectively. Furthermore, statistical analysis demonstrated that monocyte-oriented blasts are characterized by a brighter expression of Bst1/CD157 compared to blasts of non-monocytic lineage. The efficacy of MEN1112/OBT357 in depleting AML blasts was evaluated through FACS analysis in an autologous ex vivo assay performed on whole blood. The assay was set up using blood from healthy donors exposed to 10 μg/ml Rituximab for 18 hours to induce B cell depletion. In the same conditions, the ability of 10 μg/ml MEN1112/OBT357 to induce blasts depletion was tested.In whole PB,MEN1112/OBT357 was able to deplete AML blasts in 15/32 evaluable cases (46%). In BM, MEN1112/OBT357 induced blast depletion in 9/24 evaluable cases (36%). Interestingly, higher depletion rate was observed in relapse/refractory patients. When CD16A-158Phe/Val polymorphisms were analyzed utilizing a sequence based typing (SBT) assay, it was demonstrated that AML blast depletion was independent by FcRg polymorphism. Furthermore, no significant shedding of Bst1/CD157 antigen was observed in sera from AML patients, compared to the sera from patients with other hematologic diseases or healthy donors.In summary, we confirmed the frequent expression of Bst1/CD157 on blasts from AML patients, with the brightest pattern of positivity observed in cases belonging to monocytic differentiation lineage. MEN1112/OBT357 also induced a promising ADCC against AML blasts in an autologous setting, which is independent from FcR g phenotype. Since in vivo the exposure of AML blasts to MEN1112/OBT357 largely exceeds the incubation time of the depletion assay, we expect a further improvement of its anti-leukemic effect in the clinical setting. Based on these results, a phase I study in patients with relapsed or refractory AML has been initiated in December 2014. DisclosuresBellarosa:Menarini Ricerche: Employment. Bressan:Menarini Ricerche: Employment. Wilson:Oxford Biotherapeutics: Employment. Manzini:Menarini Ricerche: Employment. Capriati:Menarini Ricerche SpA: Employment. Simonelli:Menarini Ricerche SpA: Employment. Binaschi:Menarini Ricerche: Employment.

Abstract 5416: Activity of the pan-PIM kinase inhibitor INCB053914 in models of acute myelogenous leukemia

Abstract The PIM family of serine-threonine protein kinases (PIM1, PIM2 and PIM3) was initially identified as preferential integration sites of the Moloney murine leukemia virus in Eμ-myc mice, resulting in perinatal lymphomagenesis. Molecular characterization has revealed that PIM kinases drive cell proliferation and survival in a number of hematological malignancies beyond lymphomas by mediating responses to cytokines, growth factors and cellular stress. Overexpression of various PIM kinase family members in these malignancies has been associated with poor overall survival and with resistance to chemotherapeutic agents. Therefore, development of a pan-PIM inhibitor may be useful in the treatment of hematological malignancies, both as a single agent and in combination with chemotherapy or targeted agents. The in vitro and in vivo activity of INCB053914, a pan-PIM kinase inhibitor, was determined in a panel of acute myelogenous leukemia (AML) cell lines. Greater than half of all AML cell lines tested were sensitive to single agent INCB053914, with anti-proliferative IC50 potencies &amp;lt;100 nM. Consistent with recent findings, the majority of these sensitive cell lines expressed higher levels of CD25, which is co-regulated with PIM through the JAK/STAT signaling pathway and which may serve as a biomarker for PIM sensitivity in AML. INCB053914 suppressed the phosphorylation of multiple PIM kinase substrates and, in a compensatory manner, increased PIM kinase expression in both cell lines and primary AML patient samples. The potency of INCB053914 to suppress BAD phosphorylation in vivo in MOLM16 tumors is consistent with its potency in blocking BAD phosphorylation in MOLM16 cells spiked into human whole blood in vitro. Dose dependent tumor growth inhibition (TGI) was seen in mice bearing MOLM16 tumors, with maximal TGI achieved with 12-20 hours of MOLM16 WB IC50 coverage. Synergistic activity was seen in vivo in the KG1 model when INCB053914 was administered with cytarabine, a standard of care therapy for AML. Further studies were undertaken to assess the potential for combined PIM inhibition with other targeted agents. Because the PIM kinases have been shown to regulate c-myc expression levels, agents whose activity has been shown to be influenced by c-myc levels or to regulate c-myc expression were examined. The combination of INCB53914 with the pan-BET inhibitor INCB54329 in MOLM16 xenografts synergistically decreased pBAD and c-myc levels in tumors, resulting in enhanced efficacy. Synergy was also seen when INCB53914 was combined with the pan-FGFR inhibitor INCB54828 in KG1 xenografts harboring the FOP-FGFR1 translocation, suggesting that this combination might be useful in this rare patient population as well as other diseases driven by oncogenic FGFR activation. Taken together, these data support the utility of PIM inhibition in AML patients as monotherapy and in combination with other targeted agents. Citation Format: Holly Koblish, Niu Shin, Leslie Hall, Sybil O'Connor, Qian Wang, Kathy Wang, Lynn Leffet, Maryanne Covington, Krista Burke, Jason Boer, Kevin Bowman, Ke Zhang, Hao Feng, Chu-Biao Xue, Yun-Long Li, Wenqing Yao, Reid Huber, Kris Vaddi, Peggy Scherle. Activity of the pan-PIM kinase inhibitor INCB053914 in models of acute myelogenous 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 5416. doi:10.1158/1538-7445.AM2015-5416

DNMT3A Mutational Status Affects the Results of Dose-Escalated Induction Therapy in Acute Myelogenous Leukemia.

DNA methyltransferase 3A (DNMT3A) is one of the commonly mutated genes in acute myelogenous leukemia (AML). Reports on the prognostic significance of DNMT3A mutations have been inconsistent, and most of the data are available only for patients 60 years of age or younger. We hypothesized that this inconsistency is due to an interaction between the dose of anthracycline used in induction therapy and DNMT3A status. We studied whether patients with DNMT3A-mutated AML treated with standard dose anthracyclines had an inferior survival compared with patients with other mutation profiles or those who received high-dose therapy. A total of 152 patients in this retrospective cohort study (median age, 54 years) with de novo AML underwent induction therapy and next-generation sequencing of 33 commonly mutated genes in hematologic malignancies, including DNMT3A, FLT3-ITD, NPM1, and IDH1/2. Cox regression was used to know whether those with DNMT3A mutations who were treated with standard dose anthracycline had inferior survival. DNMT3A mutations, found in 32% of patients, were not associated with an inferior survival. Dose escalation of anthracycline in the induction regimen was associated with improved survival in those with DNMT3A mutations but not those with wild-type DNMT3A. Patients with DNMT3A mutations who received standard dose induction had shorter survival time than other patient groups (10.1 months vs. 19.8 months, P = 0.0129). This relationship remained significant (HR, 1.90; P = 0.006) controlling for multiple variables. Patients with DNMT3A-mutated AML have an inferior survival when treated with standard-dose anthracycline induction therapy. This group should be considered for high-dose induction therapy.

Immunotherapy with Long-Lived Anti-CD123 × Anti-CD3 Bispecific Antibodies Stimulates Potent T Cell-Mediated Killing of Human AML Cell Lines and of CD123+ Cells in Monkeys: A Potential Therapy for Acute Myelogenous Leukemia

CD123 (IL-3 receptor alpha) is highly expressed on acute myeloid leukemia stem cells and blasts, and represents a promising target of antibody therapies for AML. Anti-CD123 antibodies such as CSL-362 and KHK2823 are currently in clinical development; however, a limitation of these molecules is that they are unable to stimulate T cell-mediated killing of CD123+ AML cells. To exploit the potent activity inherent to T cell immunotherapy while maintaining the favorable dosing regimen of a therapeutic antibody, we have designed a novel bispecific antibody that recruits T cells to attack CD123+ AML stem and blast cells. Such antibodies act via a mechanism known as “redirected T cell-cytotoxicity” (RTCC), because they stimulate targeted T cell-mediated killing regardless of T cell antigen specificity. Unlike other bispecific formats, these antibodies possess a full Fc domain and spontaneously form stable heterodimers that are readily manufactured. Their Fc domain was also engineered to abolish binding to Fcγ receptors (to reduce the potential for nonselective T cell activation), yet preserve binding to human FcRn (to maintain long serum half-life).We first generated a library of humanized and affinity-optimized anti-CD123 × anti-CD3 bispecific antibodies and assessed their potency using RTCC assays, in which bispecifics stimulated killing by human T cells of the CD123+ AML cell lines KG-1a and TF-1. From this cell-based screen, we selected the bispecific antibody XmAb14045 for testing in animal models. This antibody has 0.1 nM affinity for human CD123, and a T cell-engaging domain with 8 nM affinity for human CD3. XmAb14045 stimulated T cell-mediated killing of KG-1a and TF-1 cells with an EC50 < 1 ng/ml (8 pM). In contrast, XmAb14045 had no cytotoxic activity against the CD123− Raji B cell line, demonstrating target specificity of the T cells.XmAb14045 had a prolonged serum half-life in mice of 6.2 days, in marked contrast to non-Fc domain-containing bispecific formats. Because this antibody was optimized for human CD123 and CD3 targets and does not crossreact with mouse antigens, we evaluated efficacy in cynomolgus monkeys. We treated 3 monkeys per group with a single dose of XmAb14045 at 0.01, 0.1, or 1 mg/kg. We quantified CD123+ cell numbers, including basophils and plasmacytoid dendritic cells (pDC) as CD123+ surrogate populations for AML stem and blast cells. Within 4 hours of dosing, XmAb14045 strongly activated T cells and stimulated depletion of over 99% of circulating CD123+ cells within 1 hr, particularly at the 0.1 and 1 mg/kg doses. Basophil and pDC counts fell to baseline within 4 hr and remained low for several weeks. Circulating CD4+ and CD8+ T cells were activated immediately after dosing and this was sustained for 48 hr, as measured by markedly increased levels of the activation markers CD25 and CD69. Notably, XmAb14045 induced rapid margination of CD4+ and CD8+ T cells from the circulation, with blood T cell populations returning to baseline within several days. Bone marrow CD123+ cells were depleted by over 95% at all doses, and these cell populations had not recovered by 8 days after treatment.Our results demonstrate that bispecific antibodies can recruit and activate T cells to efficiently kill CD123+ cells not only in the circulation but also in the bone marrow. Results in monkeys also suggest that changes in basophil and/or plasmacytoid dendritic cell numbers are readily quantifiable in peripheral blood, and thus these populations may serve as biomarkers for clinical efficacy. Our preclinical data provide a rationale for clinical assessment of anti-CD123 × anti-CD3 bispecific antibodies in patients with acute myeloid leukemia. DisclosuresChu:Xencor: Employment, Equity Ownership. Pong:Xencor, Inc.: Employment, Equity Ownership. Chen:Xencor, Inc.: Employment, Equity Ownership. Phung:Xencor, Inc.: Employment, Equity Ownership. Chan:Xencor, Inc.: Employment, Equity Ownership. Endo:Xencor, Inc.: Employment, Equity Ownership. Rashid:Xencor, Inc.: Employment, Equity Ownership. Bonzon:Xencor, Inc.: Employment, Equity Ownership. Leung:Xencor, Inc.: Employment, Equity Ownership. Muchhal:Xencor, Inc.: Employment, Equity Ownership. Moore:Xencor, Inc.: Employment, Equity Ownership. Bernett:Xencor, Inc.: Employment, Equity Ownership. Szymkowski:Xencor, Inc.: Employment, Equity Ownership. Desjarlais:Xencor, Inc.: Employment, Equity Ownership.

Assessing the Mechanism of Cytarabine-Induced Killing in Acute Leukemia

The nucleoside analogue cytarabine is part of standard therapy for acute myelogenous leukemia (AML) and is also widely used in various acute lymphocytic leukemia (ALL) regimens. During a 7-day infusion at 100 mg/m2/d, which is part of the “7 + 3” AML induction regimen, steady-state cytarabine concentrations in the 100-200 nM range are achieved. Previous literature suggesting the mechanism of cytarabine-induced killing in AML examined cytarabine concentrations in the micromolar range, which might be less pertinent to routinely achieved drug exposure. To better understand the mechanism of cytarabine-induced killing, AML and ALL cell lines were exposed to varying cytarabine concentrations (25-200 nM) for 24-48 hours and assayed for cell death using a variety of assays. Dose- and time-dependent induction of apoptosis was observed in both AML and ALL cell lines, as detected by phosphatidylserine externalization (Annexin V staining), DNA fragmentation (propidium iodide staining for subdiploid cells), nuclear morphological changes, and cleavage of caspase substrates. Jurkat T cell ALL variants lacking critical components of the death receptor pathway (FADD or procaspase 8) underwent apoptosis with kinetics indistinguishable from parental cells, arguing against involvement of the death receptor pathway. Immunoblotting for Bcl-2 family members in AML and ALL cell lines revealed upregulation of the BH3-only proteins Bim, Puma and Noxa across multiple lines. In further experiments, small interfering RNA (siRNA)-mediated knockdown of Bim, Puma, and Noxa or the mitochondrial proteins Bax and Bak markedly diminished the apoptotic response to cytarabine. These results suggest that cytarabine at nanomolar concentrations kills cells through the mitochondrial apoptotic pathway but, in contrast to more targeted agents, does so by upregulating multiple BH3-only Bcl-2 family members simultaneously. Additional studies to determine the mechanisms of Bim, Puma, and Noxa upregulation are underway. (Supported in part by R01 CA166741 and predoctoral fellowships from the Mayo Foundation for Education and Research.) DisclosuresNo relevant conflicts of interest to declare.