Treatment Of AML Patients Research Articles

Intradermal Vaccination with Vididencel in MRD+ AML-Patients Leads to Increase in Antigen Presenting Cells and T-Cells to the Injection Site, Visualized Using Imaging Mass Cytometry, Showing Local Immune Cell Interactions Leading to Systemic Immune Responses

Background. Vididencel is an allogeneic leukemia-derived dendritic cell vaccine being developed as maintenance treatment in AML patients. Intradermal vaccination using vididencel is known to induce a local skin reaction, with local antigen processing by skin dendritic cells and direct presentation to T cells as well as dendritic cells, crawling out of the skin and presentation of the antigen in the draining lymph nodes for a full-blown immune response. It was previously shown that vididencel is able to induce an influx of immune cells in the skin using standard immunohistochemistry (van de Loosdrecht, 2018). In a phase 2 study vididencel was investigated as AML maintenance treatment after induction of CR1 with chemotherapy (ADVANCE-II, Clintrials.gov: NCT03697707). Using imaging mass cytometry up to 43 markers can be assessed in the same tissue section, to allow in depth analysis of the immune cells on a single cell level infiltrating in the dermis at the injection site. Methods. AML patients, in first complete remission with measurable residual disease (MRD +) and not planned for HSCT at inclusion, received 4 biweekly doses of vididencel followed by 2 booster doses at week 14 and 18. Skin biopsies of the injection site were taken 2 days after the 1 st, 4 th and 6 th vaccination. Control samples were collected from unvaccinated/unaffected skin in two patients and from healthy controls. Samples were fixed and embedded in paraffin, and stained by standard immunohistochemistry for immune cell markers like CD4, CD8 and CD68 in the central lab. For patients treated at Haukeland University Hospital (N=6), tissue sections were prepared, mounted and stained with the panel of 29 antibodies labelled with heavy metal isotopes for use in the Hyperion mass cytometry imaging. For each sample a region of interest (ROI) of 2.8mm 2 was scanned based on the localization of the immune infiltrates, guided by standard H&E staining from neighboring slides. Results In 88.5% of patients injection site reactions were reported in this phase 2 study, characterized by redness, swelling and warmth at the site of intradermal injection, which were mild (maximum grade 1 or 2), and were of short duration (generally resolved within 12 days). Skin biopsies of a subset of 6 patients were analyzed using Hyperion. In standard single stain immunohistochemistry a large influx of CD4, CD8 and CD68 cells was observed in contrast to normal skin. Imaging mass cytometry confirmed these dense immune infiltrations. Using additional markers to characterize monocytes, dendritic cells and T-cells revealed that these cells are in close proximity and suggestive for cell-to-cell interaction (Figure 1). At sites of dense immune infiltration, influx of GranzymeB expressing CD8 T-cells and CD38 positive CD4 T-cells were observed as well as CD1c+ or CD141+ dendritic cells with high HLA-DR expression. These T-cells and dendritic cells are in close contact suggestive for patients' dendritic cells migrating from blood to skin, inducing a local immune response. Conclusion/discussion. Vaccination with vididencel triggered an inflammatory skin reaction in the majority of patients, with redness, warmth and swelling, of short duration, indicative for an active immune response at the site of injection. Skin biopsies using standard immunohistochemistry showed influx of activated CD4 and CD8 T cells as well as antigen presenting cell, including dendritic cells.Extended analysis with multiparameter imaging mass cytometry showed specific immune cell subsets infiltrating in these site of injection sites. The observed co-localization of the different immune subsets following vaccination supports the hypothesis of local antigen capturing by blood derived and local antigen presenting cells (Zuo et al, 2021). The local immune response may ultimately leads to a systemic immune response and disease control.

Development of a Fast and Quantitative FLT3-TKD Mutation Prototype Using Lab in a Cartridge™ Technology

Introduction Acute myeloid leukemia (AML) is a heterogenous hematologic malignancy characterized by the rapid proliferation of abnormal myeloid cells in the bone marrow and blood. Fast and reliable diagnostic tests are imperative for the advancement of targeted therapies in the treatment of AML. FLT3 (FMS-like tyrosine kinase3) is one of the most frequent genetic alterations in AML and is present in approximately one-third of newly diagnosed patients. Considering the targeted therapies, such as midostaurin and gilteritinib available for FLT3 mutated AML, accurate and timely diagnosis of FLT3 mutations is vital for guiding treatment decisions and improving patient outcomes. The current diagnostic methods to detect FLT3-TKD mutations require a series of complex steps including blood sample handling, plate-based PCR, enzyme digestion and capillary electrophoresis or sequencing which can be time-consuming and labor-intensive. We have developed and evaluated the performance of a FLT3-TKD prototype using Cepheid's automated cartridge technology that demonstrates significantly less hands-on time and faster turnaround. Objectives We have developed a novel, fast and simplified qPCR-based FLT3-TKD prototype using Cepheid's GeneXpert® automated platform for detecting 14 mutations, including 7 mutations present in codon D835 and another 7 mutations in I836. The FLT3-TKD prototype is an easy-to-use sample in/result out assay with minimal hands-on time and time to results in less than 3 hours, and without the use of restriction enzymes or capillary electrophoresis. This assay is designed for quantitative detection of FLT3-TKD mutated DNA (D835 & I836) using ABL1 as an endogenous control in peripheral blood (PB) of AML patients. This assay utilizes the special chemistry, special design primers, and an optimized wild-type (WT) blocker to specifically detect SNPs and indels. With the combination of WT blocker and unique design of special design primers we were able to selectively amplify the rare mutations in the presence of abundant closely related WT fragments. Results Using this prototype, we were able to detect and quantify all 14 mutations (7 of D835 and 7 of I836) with an estimated LoD of AR 0.02 (Ratio of TKD mutation copies/ABL copies in PB background) in PB samples spiked-in with positive templates. ABL1 serves as a reference gene to normalize the WT DNA present in the background. Plasmid DNA with individual mutations was generated and spiked into the PB to assess the dynamic range of the test. We observed good alignment of dCt-AR (deltaCt-Allelic ratio) regression curve among all 14 mutations within the dynamic range tested (0.02-0.33). With pre-defined thresholds, our assay specificity has been above 95% among 20 screened EDTA donor blood samples. To further assess the sensitivity of this prototype, we also evaluated a CRISPR-CAS engineered cell line for the D835 G>A mutation. Using this cell line, we were successful in detecting the mutation down to AR of 0.008 in a PB background. For quantification analysis we prepared a linear panel of the most prevalent mutation D835(G>T) in the PB background and were able to quantify all mutations within the range of AR 0.008-to 0.83. Conclusions Here we describe the development and evaluation of an automated cartridge-based FLT3-TKD prototype that can detect 14 FLT3-TKD mutations. The cartridge prototype is preloaded with sample processing reagents and qPCR reagents with a turn-around time of less than 3 hours including offboard peripheral blood processing. When tested with contrived samples generated with either plasmid DNA or engineered cell lines spiked into blood, all 14 tested FLT3-TKD mutations were detected within a dynamic range of 0.02 to 0.33 AR with a specificity of > 95% in healthy donor whole blood background. With optimized primer and WT blocker design this prototype showed high sensitivity & specificity. We tested a broader dynamic range for the most prevalent mutation, D835 G>T, and were able to detect from AR of 0.008 to 0.83 AR. Additionally, since the prototype uses the GeneXpert® platform and current universal lysate sample prep procedure, the sample can potentially be used across all Cepheid Hematology Oncology products. This prototype significantly reduces the hands-on time with future potential to provide clinicians with fast and accurate detection of FLT3 mutation status that can aid prognostic evaluation and treatment in AML patients.

Real-Life Comparative Analysis of Prognostic Risk Classification According to ELN 2010, 2017 and 2022 in AML Patients

Background Prognostic risk stratification is crucial to individualize treatment in AML patients. The 2010 European Leukemia Net (ELN) classification grouped patients into 4 risk categories based on genetic alterations, which were redefinedinto 3 prognostic groups in 2017 taking into account new molecular data (TP53, RUNX1 and ASXL1). Recently, ELN has proposed a new update considering additional prognostic mutations (such as SF3B1, SRSF2 and U2AF1)and removing ratio for FLT3-ITD. These ELN 2022 recommendations have not been validated yet in large cohorts of AML patients in real life. Aims 1. Analyze the mutational landscape detected by Next Generation Sequencing (NGS) in consecutive real-life AML patients 2. Analyze whether the application of ELN 2022 risk model improves prognostic risk stratification compared to ELN 2010and 2017 and Medical Research Council (MRC). Methods We included 112 adult AML patients diagnosed in our center between June 2017 and December 2021 (Table 1) and who were studied at diagnostic using the Spanish PLATAFOLMA PETHEMA. The prognostic risk was established according to MRC, ELN 2010, ELN 2017 and ELN 2022 classification. NPM1 and FLT3-ITD were determined by melting curve analysis and standard PCR-EC technique according to Thiede et al (Blood 2002) in ABI 3130 Analyzer (Thermofisher). For NGS, the commercial panel Myeloid SolutionTM (Sophia Genetics) KAPA Kit amplification libraries and sequencing on ILUMINA Myseq platform were used. Variant analysis was performed using DDM software (Sophia Genetics). Results The majority of patients (97.1%) had at least one mutation at diagnosis detected by NGS. The median number of mutations was 2.4 (0-6). Grouped by functional genes, the most frequent were those related to DNA methylation (44.6 %) and signaling/kinase pathways (37.5%). The most prevalent were FLT3 (29 %), IDH1/IDH2 (27%) and TP53 (19.6%) followed by NPM1 and RUNX1 (17%). Patients older than 60 years, presented higher percentage of unfavorable ASXL1 (18.6%), RUNX1 (20%) and TP53 (25.7%) mutations with respect to younger (4.8%, 11.9% and 9.5%) being these differences statistically significant in ASXL1 and TP53 (P=0.044 and P=0.047, respectively). NPM1 (28.6%) and FLT3 (40.5%) mutations were significantly more frequent in younger patients (P=0.017 and P=0.013, respectively), than in >60 years (10% and 17.1%, respectively). Re-stratifying according to ELN2022, 32.4% of patients categorized within the intermediate risk group according to ELN 2017 change to adverse prognostic group and 13.2% in adverse risk group change to intermediate group due to the lack of impact of ratio for FLT3-ITD. In the global series, the overall survival (OS) analysis shows statistically significant differences taking into account either ELN 2022, ELN 2017 or ELN 2010 and MRC (P=0.001,P=0.010, P=0.049 and P=0.012, respectively) (Figure 1). However, the greater differences between intermediate and unfavorable groups are found according to ELN 2022 compared to other classifications, and are also confirmed in patients receiving intensive treatment (P=0.021).Remarkably, patients receiving allotransplantation reverse adverse prognosis and enhance survival similar to intermediate group. In our series, patients harboring mutations in signaling pathways genes (including FLT3) show better OS than those lacking, reaching statistical significance in allo-HSCT patients (P=0.037). Summary/Conclusion 1. Our results validate the prognostic significance of the ELN2022 classification in real life, for the overall series and for candidates to intensive treatment. 2.ELN 2022 establishes greater survival differences between the unfavorable and intermediate group than ELN 2017or prior classifications. However, patients in adverse group undergoing allo-HSCT improve OS similar to intermediate group 3. FLT3 mutated allo-HSCT patients showed a strikingly low risk of relapse, partly due to the treatment with FLT3 inhibitors. 4. New studies including larger number of patients are needed to confirm our results. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Phase Ib Study of the Safety and Efficacy of Venetoclax When Combined with Escalating Doses of Omacetaxine in Patients with Relapsed/Refractory Acute Myeloid Leukemia Failing Treatment with Venetoclax-Containing Regimens (VEN-OM)

Background: Venetoclax (VEN) combined with hypomethylating agents (HMA) are increasingly used as first line (1L) treatment in AML patients (pt) intolerant of intensive chemotherapy, due to the high complete remission (CR) rate (66.4%), tolerance of therapy and a median duration CR of 17.5 months. However, >60% of pts discontinued VEN/HMA (n=286) during the VIALE-A trial, mostly due to disease progression (DiNardo et al.N Engl J Med2020; 383:617-29). Outcomes for such pts receiving 2L and 3L therapies for relapsed/refractory (R/R) AML are poor and may be due to development of resistance to BCL-2 inhibition through mechanisms including the upregulation of the anti-apoptotic proteins BCL-XL or MCL1. In vitro studies demonstrate that VEN-resistant leukemia stem cells (LSC) are re-sensitized to BCL-2 inhibition when MCL-1 is also inhibited (Ramsey et al. Cancer Discov 2018; 8:1566-81). Novel MCL-1 inhibitors are thus in phase 1 clinical trials in R/R AML (e.g. NCT02675452), however there have been concerns for cardiotoxicity. Metabolic studies (Stevens et al. Nat Commun 2018: 9, 3694) indicate that the CD123+ malignant stem cell compartment of pts with high-grade MDS rely on upregulation of the protein synthesis machinery and mitochondrial oxidative phosphorylation (OxPhos). Omacetaxine (OM), a protein translation inhibitor both inhibits OxPhos and down-regulates (other) proteins with a short half-life such as MCL1. Treatment of a xenograft AML model with a combination of OM and VEN indicates in vivo synergistic effects in impairing mitochondrial respiration, ATP production, BCL-2 inhibition and MCL-1 downregulation (Fig 1A). We hypothesize this combination will prove effective in pts with R/R AML including those resistant to VEN-containing regimens. A similar drug combination is being studied by Dinardo et al. in R/R AML and MDS pts with RUNX1 mutations. Methods: VEN-OM is a single arm, open-label, phase Ib dose-escalation study in R/R AML pts (18-75y). The trial utilizes an innovative Bayesian Optimal Interval (BOIN) adaptive design (Yuan et al. Clin Cancer Res 2016)to determine the maximum tolerated dose (MTD), with a target dose-limiting toxicity (DLT) rate of 2%, and 4 pre-specified dose levels of OM, involving 24-30 pts. Major eligibility: ECOG 0-2, adequate organ function, R/R AML having progressed on ≥ 1L of therapy, which must have included a VEN-containing regimen. Exclusion criteria: Prior use of OM, peripheral blast count > 25 × 109/L, unresolved ≥ grade 2 clinically significant nonhematologic toxicities from prior anticancer therapy, unresolved ≥ grade 2 disseminated intravascular coagulation (DIC), other active malignancy within 1 year before study entry, or active infections. Induction Phase: Beginning with the first pt treated at the lowest dose of OM (0.625 mg/m2 q12h), OM dose escalation and de-escalation will involve comparison of the observed DLT rate at the current dose with a pair of fixed, pre-specified boundary rates. After a VEN ramp-up phase (days 1-3), the VEN dose is fixed at 400mg daily on days 4-28. OM doses are given days 1-7. OM dose will be escalated for each subsequent pt until the first DLT occurs, whereupon the current dose cohort is expanded to 3. In simulations of these conditions with a target DLT rate of 20%, the percentage correct selection of the MTD is between 60% with enrollment of 20 pts and 70% with 24 pts. Consolidation Phase: OM dose will be as per induction dose for days 1-7 of each cycle. VEN dose is 400mg days 1-21. Regimen comprises 3 cycles: Induction cycle x 1 and Consolidation cycles x 2 (Fig 1B). Primary endpoint is MTD of the combination of VEN and OM. Secondary endpoints include overall response rate after 3 cycles, characterization of adverse effects, and estimates of EFS and OS at 12 months. Exploratory objectives include measurement of validated markers of early renal injury including urine TIMP-2 x IGFBP7 and KIM-1 to identify pts susceptible to OM-induced nephrotoxicity and potential correlation with OM efficacy. Our previous experience with OM added to 7+3 ("AML-02", ASH 2022 abstract submitted) suggests that quantification of BM leukemia blast protein synthesis rates (Walker et al. Clin Cancer Res 2021, 27:819-30) and 'BH3 profiling' at presentation predicts outcomes, and these correlative studies will also be performed. The study recently opened, with the first patient completing induction without complications. Trial registered as (NCT04926285). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PD1 inhibitor in combination with 5-azacytidine and low-dose DLI for the successful treatment of AML patients who relapsed after transplantation.

PD1 inhibitor in combination with 5-azacytidine and low-dose DLI for the successful treatment of AML patients who relapsed after transplantation.

Abstract 6532: HFB9-2: A novel Gal-9 neutralizing antibody to reverse immune suppression in the tumor microenvironment

Abstract Although monoclonal antibodies targeting immune checkpoints have demonstrated clinical success in a range of tumor types, sustained responses are only observed in a fraction of patients due to primary or secondary resistance to treatment. Recent evidence has implicated the pleiotropic immunosuppressive modulator Galactoside-binding lectin Galectin 9 (Gal-9) as a key factor present in the tumor microenvironment that renders tumors resistant to current immunotherapies. High Gal-9 expression has been reported in different types of cancer including hematological malignancies such as AML and ALL, and multiple solid tumors. We hypothesize that targeting Gal-9 may represent a valuable strategy to overcome resistance and improve clinical response in selected cancer patients. We present a monoclonal antibody, HFB9-2, that specifically binds to human Gal-9 with sub-nanomolar affinity, recognizes recombinant Gal-9 and Gal-9 produced by human tumor cells, and is cross-reactive with mouse and monkey Gal-9 orthologs. HFB9-2 blocks the interaction of Gal-9 with its receptors TIM3 and CD44 in a dose dependent manner. These two receptors have been described to mediate Gal-9-immunosuppressive signals in effector and regulatory T cells. Treatment of human PBMCs from healthy donors with HFB9-2 prevents Gal-9-induced Th1 cell apoptosis and suppresses the expansion of regulatory T cells. A humanized variant of HFB9-2, HFB9-2hz11, was generated and further characterized for its stability and pharmacokinetic profile. HFB9-2hz11 has a favorable developability profile. It demonstrated stability for at least 14 days at 40°C, as well as for several hours at low pH, and following several freeze-thaw cycles. High plasma exposures following a single dose administration to mice were observed. Furthermore, several preclinical in vivo studies are ongoing to demonstrate the efficacy of HFB-2hz11. Gal-9 has been reported to play a dual role in AML as both a self-renewal factor for leukemic stem cells and a suppressor of anti-cancer immunity, suggesting that Gal-9 neutralization represents an attractive therapeutic approach for treatment of AML patients. To explore this hypothesis, we have initiated a predictive biomarker discovery effort, using HiFiBiO's proprietary Drug Intelligent Science (DIS™) platform, using AML primary patient cells and integrating single-cell technology to identify patient subpopulations likely to respond to HFB9-2hz11. Potential novel biomarkers for patient stratification, that could be applied to other tumor indications, will be identified for AML patients. Altogether, the data presented here provide evidence that neutralization of Gal-9 with HFB9-2hz11 blocks key immunosuppressive mechanisms known to limit the efficacy of current immunotherapies and position HFB9-2hz11 as a drug candidate for clinical exploration in AML and other indications. Citation Format: Germain Margall, Rachel Pacherie, Julie Prigent, He Zhou, Francisco Adrian, Liang Schweizer, Andreas Raue, Roshan Kumar, Nicola Beltraminelli, Stéphanie Beq. HFB9-2: A novel Gal-9 neutralizing antibody to reverse immune suppression in the tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6532.

Evaluation of BECN1 Gene Expression in Remission-Induction Therapy of Acute Myeloid Leukemia

Background: Acute myeloid leukemia is a type of hematological malignancy. Chemotherapy is the first-line treatment for AML. The main aim of treatment is to achieve complete remission in patients. Although complete remission is achieved in most patients, still the rate of relapse remains high. Recent studies revealed that drug resistance is an important cause of treatment failure in AML patients. Autophagy as a conserved catabolic process has a significant role in drug resistance of AML. Autophagy can act as a chemo-resistance mechanism in response to chemotherapy in AML patients. However, the role of this pathway in response to treatment of AML patients is not yet fully clarified. Objectives: The aim of this study was to investigate the effect of the BECN1 gene, as a key regulator of autophagy on remission and response to chemotherapy in AML patients. Methods: The BECN1 gene expression was evaluated in 30 AML patients at diagnosis stage, on 18 patients with complete remission and 15 controls using qRT-PCR. Results: The results showed that BECN1 gene expression level was significantly higher in AML patients than controls at the rate of 5/3 fold, P < 0.0001. We found that expression level of BECN1 was significantly reduced in patients with complete remission in comparison with newly diagnosed AML patients at the rate of 0.73 fold, P = 0.004. Conclusions: Low expression of BECN1 gene might be associated to complete remission. Therefore, perhaps BECN1 gene can be used as a biomarker to assess remission status. Moreover, targeting BECN1 can be used as a potential strategy to improve treatment in AML patients.

Maintenance 5-Azactidine May Improve Outcomes after Allogeneic Stem-Cell Transplantation in High-Risk AML and MDS Patients

Introduction: Allogeneic stem-cell transplantation (SCT) is curative therapy in patients with AML or MDS. However, disease recurrence is the major cause of treatment failure. 5-Azacytidine has been used in the standard treatment of MDS and also in the treatment of AML patients not eligible for standard chemotherapy. There is limited data on the safety and efficacy of Azacytidine after SCT. We explored the use of low-dose Azacytidine maintenance therapy for the prevention of relapse of AML/MDS after SCT. Methods: Patients considered to be at high risk for relapse after SCT were given Azacytidine at 32-50 mg/m2 for 5 days every 28 days for a planned duration of 2 years. Transplantation outcomes were compared with a historical group matched for patient age, disease, status at SCT, cytogenetics, conditioning and donor type. To reduce bias, control patients were required to be alive and in remission for at least the time until the start of Azacytidine prophylaxis in their counterparts. Patients relapsing after SCT and achieving second CR with chemotherapy were also eligible for secondary Azacytidine prophylaxis. The control patients for these patients were also matched for the time from SCT to relapse. Results: Forty patients, median age 62 years (25-74), 24 male and 16 female, were given azacytidine after SCT from HLA-matched sibling (n=8) or matched-unrelated (n=32) donors. Diagnosis was AML (n=32) or MDS (n=8). The conditioning regimen was myeloablative (n=20) or reduced-intensity (n=20). The status at SCT was untreated MDS/ secondary AML (n=10), CR1 (n=19), CR2 (n=4), refractory AML (n=7). 15 had normal karyotype, 9 complex karyotype and 16 other cytogenetic abnormalities (12 of them high-risk). Fifteen patients were in CR after SCT but at high-risk for relapse due to active disease (n=7) or positive MRD (n=3) at SCT, or poor-risk cytogenetics (n=5). Eight patients were given Azacytidine as secondary prevention after achieving CR with chemotherapy for post-transplant relapse. 17 patients were given Azacytidine pre-emptively for mixed-chimerism (n=9) or positive measurable residual disease (n=8) after SCT. The estimated leukemia-free survival (LFS) in this group of patients is less than 20%. Patients started Azacytidine in a median of 2.2 months (1.2-6.9) after SCT and received a median of 14 courses (1-50). Three patients were also given donor-lymphocyte infusions concomitantly with Azacytidine. 18 patients are still on therapy, 16 died or progressed, 3 stopped after long remission, 2 stopped due to regulatory issues and only 1 had to discontinue due to toxicity. 12 of 23 patients given prophylactic Azacytidine remained in CR, 11 relapsed and 7 of them died. 15 of 17 patients given Azacytidine preemptively achieved a response (10 CR) and 7 remained in CR. 9 died of disease progression and one of non-relapse cause. With a median follow-up of 22.5 months (10.2-45.2), the estimated 3-year overall survival (OS) and LFS rates are 53.9% (95%CI, 29.5-78.4) and 40.0% (95%CI, 21.2-58.6), respectively. In the matched control group, 8 patients are alive, 26 died of relapse and 6 of non-relapse causes. The estimated 3-year OS and LFS rates are 27.1% (95%CI, 13.2-41.0, P=0.002, Figure 1) and 19.7% (95%CI, 7.9-32.1, P=0.005, Figure 2), respectively. The relapse rates were 57.6% (95%CI, 41.6-79.8) and 65.3% (95%CI, 52.0-82.0) in the Azacytidine and control groups, respectively (P=0.04, Figure 3). The median time to relapse was 9.1 months (0.7-25.5) and 4.9 months (1.1-35.9), respectively. The non-relapse mortality (NRM) rates were 2.5% (95%CI, 0.3-17.3) and 15.0 (7.1-31.4), respectively (P=0.01). Conclusions: Azacytidine is safe and effective therapy when used prophylactically or preemptively in high-risk AML/MDS patients to prevent relapse. Azacytidine maintenance may reduce but more often delay relapse in this high-risk patient group. Azacytidine may also reduce NRM and improve survival in this patient group and should be further explored, possibly in combination with other methods to prevent relapse. Figure 1 Disclosures No relevant conflicts of interest to declare.

Phase I Study of the PTEFb Inhibitor BAY 1251152 in Patients with Acute Myelogenous Leukemia

Introduction: The critical role of cyclin-dependent kinases in cell division and gene transcription has made them targets for anti-cancer therapies. Positive transcription elongation factor b (PTEFb)/cyclin-dependent kinase 9 (CDK9) plays a key role in transcription of short-lived anti-apoptotic survival proteins and oncogenes such as MYC and MCL-1. BAY 1251152 (BAY) is a potent and highly selective second generation PTEFb/CDK9 inhibitor. We report here the results of a phase I study to determine the safety, tolerability, pharmacokinetics (PK), maximum tolerated dose and preliminary anti-tumor activity of BAY in patients with AML.Methods: Patients with confirmed AML who are refractory to or have exhausted all available therapies were eligible. A minimum of 3 and a maximum of 9 patients were to be enrolled per dose level in a sequential dose escalation design. BAY was administered intravenously over 30 minutes once weekly for 21-day cycles. PK was assessed at cycle-1, day 1/D8/D15. Dose-limiting toxicities (DLTs) for hematologic events were defined as prolonged grade (G) 4 neutropenia and/or G3 thrombocytopenia and/or any G5 event. DLTs for non-hematological toxicity were any G3-G5 event not clearly resulting from underlying malignancy, among others. Responses were evaluated using Cheson (2003) criteria. Adverse events (AEs) were assessed using CTCAE v4.03.Results: A total of 30 patients were enrolled, 9 of whom failed screening. Twenty one patients received treatment with BAY in 4 dose cohorts: 5, 10, 20, and 30 mg. The median age was 66 (range 20-79), with 62% being male. The median number of prior lines of therapy was 3 (range 1 - 6). Four patients were treated per dose cohorts 1-3 without occurrence of DLTs. Nine patients were treated in cohort 4 (30 mg) and were evaluable for safety, with 7 evaluable for response. Nine patients (43%) discontinued treatment due to clinical progression, 19% due to withdrawal by subject, 19% due to lack of efficacy, 14% due to an AE associated with disease progression. A MTD was not defined. PK assessment indicated that the mean elimination half-life was 3-9 hours with no accumulation over multiple doses. The mean AUC of the 30 mg cohort was within the expected therapeutic exposure range based on preclinical studies. Interestingly, pharmacodynamic assessment of MYC, MCL-1 and PCNA mRNA in whole blood indicated that BAY treatment led to significant, but short-lived, reductions; highest degree of down-regulation was achieved 0.5-4 hours post-dose for MYC, 1-3 hours post-dose for MCL-1, and 1-4 hours post-dose for PCNA. However, no objective responses were observed, with only modest reductions in bone marrow blasts in some patients. In the 30 mg cohort, the longest treatment duration was 63 days. Single patients in each of cohorts 1-3 exceeded this duration but only minimally. Overall treatment-emergent G3/G4 AEs recorded in >10% of patients included anemia (G3 26.7%/G4 6.7%), lung infection (G3 23.3%), neutrophil count decreased (G4 20%), febrile neutropenia (G3 13.3%), and leukocytosis (G3 10%). Clinical laboratory assessments in cohort 4 indicated that 7 of 9 patients (78%) had G4 neutrophil count decreased and G3 anemia, and 5 of 9 (56%) had platelet count decreased. G5 events included 2 each for sepsis and cardiac arrest, and 1 patient with leukocytosis; none of the G5 events were considered related to the study treatment.Conclusions: Treatment of AML patients with BAY did not result in objective responses despite achieving drug levels in the expected therapeutic range and evidence of target engagement. Given the good tolerability and on-target activity, BAY 1251152 might be a good candidate for future combination therapies. DisclosuresOttmann:Celgene: Consultancy, Research Funding; Amgen: Consultancy; Pfizer: Consultancy; Incyte: Consultancy, Research Funding; Takeda: Consultancy; Fusion Pharma: Consultancy, Research Funding; Novartis: Consultancy. Scholz:Bayer: Employment. Ince:Bayer: Employment. Valencia:Bayer: Employment. Souza:Bayer: Employment.

Clinical Significance of Lymphoid Enhancer-Binding Factor 1 (LEF-1) Expression in Acute Myeloid Leukemia

Background: Lymphoid enhancer-binding factor-1 (LEF1) is a member of the LEF/T-cell factor family of transcription factors and a key mediator of the Wingless-type (Wnt) pathway. It mediates Wnt signals through recruiting β-catenin and its co-activators to Wnt response elements of target genes. It plays crucial roles in normal hematopoiesis, not only in the development of B and T lymphocytes but also in granulopoiesis. Objectives: The aim of this study was to evaluate of LEF1 expression levels in patients with AML and correlate this expression with clinical data. Methodology: The present study was conducted on 30 de novo adult AML patients and 10 age and sex-matched controls with non-malignant hematological disorders (e.g.: hypersplenism, megaloblastic anemia, immune thrombocytopenic purpura) who attended Hematology/Oncology unit of Ain-Shams University Hospitals during the period from May 2017 till January 2018, after taking the approval of the Scientific and Ethical Committee of Ain- Shams University. LEF1 expression levels were measured by quantitative real-time PCR. Results: The present study showed that there was highly statistically significant association between patients and control group in LEF1 expression level. There was positive significant correlation between LEF1 expression level and Hb level and PLT count. While negative significant correlation was found with age, TLC count and peripheral blood blast %. No correlation was found with bone marrow blasts %. There was statistically significant difference between LEF1 expression level and both hepatosplenomegaly. No statistically significant difference was found between LEF1 expression level and Sex or lymphadenopathy. Conclusion: Our study has shown that Lymphoid Enhancer-Binding Factor 1 (LEF-1) is over expressed in AML patients. LEF1 expression might be involved in the process of disease progression, and possibly can serve as a molecular parameter for risk assessment and/or monitoring of treatment in AML patients. Thus, molecular assessment of LEF1 expression at diagnosis may be of value to add to the prognostic work of AML patients.

Sensitivity of AML Cells with FLT3- to PKC412 In Vitro

To explore the clinical value of PKC412 (midostaurin) in treatment of AML patients with FLT3-. The bone marrow or peripheral blood were collected and heparinized from 21 newly diagnosed FLT3- AML patients, then the mononuclear cells from bone marrow or peripheral blood were isolated by density-gradient method. The sensitivity of leukemia cells to PKC412 of 8 concentration in vitro was detected by ATP-bioluminescence-tumor chemosensitivity assay (ATP-TCA), and the relationship among sensitivity results in vitro, risk stratification and therapeutic efficacy was analyzed. The leukemia cells of 21 patients with AML displayed different sensitivities to PKC412 in vitro. The rate of sensitivity in vitro was 42.9%, and sensitive concentration in vitro were between 1 µmol/L and 5 µmol/L. There was no significant relationship between risk stratification and sensitivity results of PKC412 in vitro. There was also no significant relationship between clinical efficacy and sensitivity results of PKC412 in vitro. The survival of patients in low-risk and intermediate-risk groups was better than that of patients in high-risk groups (P=0.015). PKC412 can be one of the effective therapeutic method for AML patients without FLT3 mutation. The sensitivity of leukemia cells to PKC412 may become a prognostic marker for evaluating clinical efficacy of PKC412, which is independent of other factors.

Sequential treatment of AML patients with alvocidib followed by cytarabine and mitoxantrone is highly effective through a mechanism dependent on MCL1 expression and function

Multiple phase I/II studies have shown alvocidib to be highly effective in both frontline and relapsed/refractory AML when sequentially administered before cytarabine and mitoxantrone (ACM). In frontline patients, ACM resulted in a complete remission (CR) rate of 70% versus 46% CR with ara-C and daunorubicin (7+3). The clinical activity of alvocidib in AML is significantly correlated with inhibition of cyclin-dependent kinase-9 (CDK9) and disruption CDK-9 mediated transcription. The MCL1 gene is regulated by CDK-9 transcriptional control and its expression is tightly regulated by alvocidib. Studies with AML cell lines to model the sequential treatment of the ACM regimen have shown that MCL1 expression and apoptosis are tightly connected to treatment with alvocidib. To further investigate the correlation of MCL-1 mediated survival, mitochondrial profiling (BH3 priming) was conducted on 63 archived ACM-treated samples taken directly from patients bone marrow or circulating blasts. Analysis of the BH3 priming states in AML clinical samples revealed NOXA priming was significantly higher in CR bone marrow samples (44.5% primed) compared with samples from non-responders (NR) (5.2% primed) (p=0.006). NOXA is known to interact directly with MCL1, suggesting that the AML samples that are most responsive to ACM treatment may have a high survival dependence on MCL1. The correlation between NOXA and ACM response is distinct from priming states predicting response to other ara-C regimens in samples from AML patients. This work reveals a potential biomarker for identification of patients likely to respond to ACM and this biomarker is currently being prospectively tested in a phase II clinical trial.The successive treatment initially presented and broadly concentrated in Italy has continually shown higher destruction rates than the traditional triple treatment. Consecutive treatment is another fourfold treatment, comprising of 10-day treatment in which a PPI in addition to amoxicillin is allowed for 5 days followed by a PPI in addition to clarithromycin and a nitroimidazole (metronidazole or tinidazole) for an additional 5 days. Strangely, this methodology of the consecutive treatment may even work in territories of high clarithromycin obstruction. Inside the initial 5 days of treatment, clarithromycin-safe strains get killed by PPI–amoxicillin treatment, and in the staying 5 days the leftover strains are disposed of by the triple treatment. The consecutive treatment has now been assessed in different randomized preliminaries and helpful achievement was affirmed generally as for the standard triple therapy. This routine gives high annihilation rates (>90%) in different nations and mainlands around the globe, in spite of the fact that not generally in controlled trials. However, it must be noticed that it may not work in every geographic territory as an ongoing, not completely distributed preliminary from Korea found no predominance of the successive treatment contrasted and standard triple treatments. This might be identified with higher paces of double obstruction here. Besides, past examinations showed fundamentally lower destruction rates with the metronidazole-based routine contrasted and the tinidazole-based routine.

Oncogenic NRAS Primes Primary Acute Myeloid Leukemia Cells for Differentiation.

RAS mutations are frequently found among acute myeloid leukemia patients (AML), generating a constitutively active signaling protein changing cellular proliferation, differentiation and apoptosis. We have previously shown that treatment of AML patients with high-dose cytarabine is preferentially beneficial for those harboring oncogenic RAS. On the basis of a murine AML cell culture model, we ascribed this effect to a RAS-driven, p53-dependent induction of differentiation. Hence, in this study we sought to confirm the correlation between RAS status and differentiation of primary blasts obtained from AML patients. The gene expression signature of AML blasts with oncogenic NRAS indeed corresponded to a more mature profile compared to blasts with wildtype RAS, as demonstrated by gene set enrichment analysis (GSEA) and real-time PCR analysis of myeloid ecotropic viral integration site 1 homolog (MEIS1) in a unique cohort of AML patients. In addition, in vitro cell culture experiments with established cell lines and a second set of primary AML cells showed that oncogenic NRAS mutations predisposed cells to cytarabine (AraC) driven differentiation. Taken together, our findings show that AML with inv(16) and NRAS mutation have a differentiation gene signature, supporting the notion that NRAS mutation may predispose leukemic cells to AraC induced differentiation. We therefore suggest that promotion of differentiation pathways by specific genetic alterations could explain the superior treatment outcome after therapy in some AML patient subgroups. Whether a differentiation gene expression status may generally predict for a superior treatment outcome in AML needs to be addressed in future studies.

Effect of autologous peripheral blood stem cell transplantation in 13 patients with AML1/ETO (+) acute myeloid leukemia

This study was purposed to analyse the clinical efficacy of autologous peripheral blood stem cell transplantation (APBSCT) in 13 Patients with AML1/ETO (+) acute myeloid leukemia, and to evaluate the role of quantitative detecting the AML1/ETO gene in treatment of AML patients. A total of 13 patients with AML1/ETO (+) acute myeloid leukemia treated with APBSCT from August 2007 to November 2012 were retrospectively analyzed. The median follow-up time was 26 (7.8-75.8)months. Kaplan-Meier analysis was used to calculate the overall survival (OS), leukemia-free survival (LFS) and cumulative relapse rate (RR). Log rank method was used to perform univariate analysis. The results showed that the 3 year-OS, LFS, and RR were (70.5 ± 15.3)%, (51.3 ± 16.7)%, 48.7%, respectively. The AML1/ETO expression level in 4 cases out of 5 relapsed patients was quantified during and after therapy, and the result showed that AML1/ETO expression level significantly increased before morphological relapse. In univariate analysis, there was no statistic significance in terms of age, sex, count of white blood cells at diagnosis, interval from diagnosis to transplantation, count of MNC for infusion. It is concluded that APBSCT has good therapeutic effect on AML1/ETO (+) AML, and regular quantitative monitoring of AML1/ETO expression level can predict early recurrence. Allogeneic hematopoietic stem cell transplantation after relapse may contribute to obtain opportunity to achieve the long-term survival for intermediate and high risk patients.

Decitabine.

Besides 5-azacytidine (azacitidine, Vidaza®), 5-aza-2'-deoxycytidine (decitabine, Dacogen®) is the most widely used inhibitor of DNA methylation, which triggers demethylation leading to consecutive reactivation of epigenetically silenced tumor suppressor genes in vitro and in vivo. Although antileukemic activity of decitabine is known for almost 40 years, its therapeutic potential in hematologic malignancies has only recently led to its approval in higher-risk MDS patients and as first-line treatment in AML patients>65 years who are not candidates for intensive chemotherapy. Several clinical trials showed promising activity of low-dose decitabine also in CML and hemoglobinopathies, whereas its efficacy in solid tumors is very limited. Clinical responses appear to be exerted both by epigenetic alterations and by induction of cell-cycle arrest and/or apoptosis. Recent and ongoing clinical trials investigate new dosing schedules, routes of administration, and combination of decitabine with other agents, including histone deacetylase inhibitors.

Abstract 3516: AZD1208 PIM kinase inhibitor - Preliminary evidence of target pathway inhibition in Phase I clinical trials of AML.

Abstract PIM kinases have been shown to play a key role as downstream effectors of growth factor signalling pathways including Flt3 and the Jak-STAT signalling pathways in AML, NHL and other solid tumors. AZD1208 is a novel, orally bioavailable, highly selective PIM kinase inhibitor with single nanomolar potency against all three PIM kinases and is currently undergoing Phase I testing and dose escalation studies in AML. Here we describe a multiplexed biomarker strategy measuring pBAD, p4EBP1 and p-p70S6K as downstream pharmacodynamic biomarkers for PIM kinase inhibition in clinical trials. Patient bone marrow aspirates and peripheral blood samples were collected pre- and post-AZD1208 treatment in AML patients during the Phase I dose escalation and expansions. Primary patient samples were analyzed for quantitative changes in pBAD, p4EBP1 by NanoPro and MesoScale assay platforms as well as a qualitative evaluation of p-p70S6K and other exploratory endpoints. Preclinical PK-PD modeling data with AZD1208 had suggested that greater than a 50% decrease in the levels of one of these phosphorlyated substrates would be indicative of efficacy and PIM pathway inhibition. Following a single dose of AZD1208 at 120mg, the first dose level, approximately 60-70% inhibition of pBAD, S112 was seen in the bone marrow and peripheral blasts. Taken together, the data presented here provide evidence for single agent AZD1208 activity in AML patients based on quantitative reduction in these biomarkers. Correlations of these biomarker endpoints with Phase I pharmacokinetic data underscore the therapeutic potential of Pim kinase inhibition by AZD1208 for the treatment of AML, and strongly support further investigation of this agent in other indications where PIM signaling may play a role in tumorigenesis and survival. Citation Format: Kristen A. McEachern, Yichen Cao, Rachel DuPont, Lourdes Pablo, Patricia McCoon, Jorge E. Cortes, Daniel J. DeAngelo, Mark D. Minden, Becker Hewes, Jeffrey L. Brown, Carl Barrett. AZD1208 PIM kinase inhibitor - Preliminary evidence of target pathway inhibition in Phase I clinical trials of AML. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3516. doi:10.1158/1538-7445.AM2013-3516

Expression levels of nucleoside transporter hENT1 and dCK enzyme, as prognostic factors in patients with AML treated with cytarabine.

6599 Background: Cytarabine has been set as the main drug included in different schemas for the treatment of AML patients. This drug requires inflow to the cell by the nucleoside transporter hENT1 and its activation rate is limited by dCK enzyme. Therefore decreased expression levels of these genes may influence response rate to this drug. Methods: AML patients > 15 years, without previous treatment were included. After informed consent signature, peripheral blood was obtained and DNA was extracted by standard methodology. Amplification of hENT1 and dCK genes was done by RT-PCR. Clinical parameters were registered. All patients received cytarabine + doxorrubicine as induction regimen (7 + 3 schema). Descriptive statistics was used. Uni- and multivariate analysis was done to determine factors influencing on response and overall survival. This protocol was approved by local IRB. Results: From January till December 2011, 22 patients have been included. Median age was 36 years (15-72 y), 52.3 % were male. According with FAB classification, M2 subtype was the most frequent (28.5 %). All patients had an adequate performance status (ECOG 1 & 2 = 34 & 66 %, respectively). Cytogenetic risk was considered as intermediate risk in 38 %, followed by unfavorable in 28 %. Complete response was achieved in 58 %. Higher hENT1 expression levels was the only factor influencing on response and survival by Spearmen correlation analysis. Conclusions: These are preliminary results but are highly suggestive that pharmacogenetic analysis regarding cytarabine inflow may be decisive in AML patients. Our results require to be confirmed with a larger sample, and a longer follow-up.

Identification of N-(5-tert-Butyl-isoxazol-3-yl)-N′-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea Dihydrochloride (AC220), a Uniquely Potent, Selective, and Efficacious FMS-Like Tyrosine Kinase-3 (FLT3) Inhibitor

Treatment of AML patients with small molecule inhibitors of FLT3 kinase has been explored as a viable therapy. However, these agents are found to be less than optimal for the treatment of AML because of lack of sufficient potency or suboptimal oral pharmacokinetics (PK) or lack of adequate tolerability at efficacious doses. We have developed a series of extremely potent and highly selective FLT3 inhibitors with good oral PK properties. The first series of compounds represented by 1 (AB530) was found to be a potent and selective FLT3 kinase inhibitor with good PK properties. The aqueous solubility and oral PK properties at higher doses in rodents were found to be less than optimal for clinical development. A novel series of compounds were designed lacking the carboxamide group of 1 with an added water solubilizing group. Compound 7 (AC220) was identified from this series to be the most potent and selective FLT3 inhibitor with good pharmaceutical properties, excellent PK profile, and superior efficacy and tolerability in tumor xenograft models. Compound 7 has demonstrated a desirable safety and PK profile in humans and is currently in phase II clinical trials.

Granulocyte Colony Stimulating Factor Increases Drug Resistance of Leukaemic Blast Cells to Daunorubicin

Acute leukaemia is known as the most common cancer in childhood. Febrile neutropenia is a common serious side effect of the cytostatic treatment of malignancies. The clinical use of Granulocyte Colony Stimulating Factor (G-CSF) has become widespread to minimize chemotherapy-induced myelosuppression and febrile neutropenia in childhood solid tumors, acute lymphoid leukaemia (ALL) and in several trials with AML. In case of ALL this seems to be reasonable because, due to the absence of G-CSF receptor (G-CSFR) on the surface of normal lymphoid cells, G-CSF does not have any influence on the pathways of proliferation and differentiation of lymphoid lineage cells. It has been suggested, however, that ALL blasts with B or T cell surface antigens as well as biphenotypic leukaemia cells express G-CSFR, and they are able to respond to exogenously added G-CSF with proliferation. In this study we investigated how G-CSF might influence the sensitivity of leukemic cells to daunorubicin induced cell death using MTT assay, flow cytometry and Western blot analysis. After pretreatment of KG-1 leukaemic cells with G-CSF a moderate increase in the resistance of these cells to daunorubicin could be observed. These results draw attention to the risk of G-CSF application as an adjuvant therapy of childhood ALL. In addition, adjuvant treatment of AML patients with G-CSF in order to prevent neutropenia, or its use in priming regimens might result resistance to daunorubicin.

Autologous Peripheral Blood Stem Cell Transplantation Using G-CSF Combined Conditioning in AML Patients

Background: The possibility that G-CSF recruits leukemic cells from the G0 to S phase, which may lead to a greater susceptibility to cytotoxic drugs, such as ara-C, has been presented in Harada’s study. Methods: In this study, we referred to the protocol of Harada et al 1 to try G-CSF combined marrow-ablative chemotherapy and autologous PBSCT, for the treatment of AML patients in CR1 status. Between January 1997 and March 1998, six AML patients (3: children, 3: adults) in CR1 status were autografted and followed up to 3 years. Results: The major regimen related toxicity was composed of mucositis and diarrhea without death. The time of ANC recovery to 500/L and 1,000/L was 11~48 and 16~81 days, respectively. The mean time of platelet recovery to 20,000/L and 50,000/L was 21~233 and 35~370 days, respectively. The platelet recovery time to 50,000/L was markedly prolonged for more than 100 days in four patients (66.7%). Moreover, four patients (66.7%) experienced a relapse of leukemia after transplantation, with a mean interval of 147.5 days after PBSCT. Two patients were in CR status for 53 and 51 months after PBSCT, respectively. Conclusion: The G-CSF combined marrow-ablative chemotherapy and autologous PBSCT resulted in a markedly delayed platelet recovery and no advantages for decreasing the relapse rate of AML. But, further studies will be warranted. (Immune Network 2002;2(1):49-52)