ALBERT

Description: Key Points In AML with bialleleic CEBPA-mut relapse-free survival was improved by allogeneic and autologous hematopoietic stem cell transplantation. In relapsed patients second complete remission rate was high and survival was favorable after an allogeneic transplantation.

Description: Monitoring of measurable residual disease (MRD) provides prognostic information in patients with Nucleophosmin1 mutated (NPM1mut) acute myeloid leukemia (AML) and represents a powerful tool to evaluate treatment effects within clinical trials. We determined NPM1mut transcript levels (TL) by RQ-PCR and evaluated the prognostic impact of NPM1mut MRD and the effect of gemtuzumab ozogamicin (GO) on NPM1mut TL and the cumulative incidence of relapse (CIR) in patients with NPM1mut AML enrolled in the randomized phase III AMLSG 09-09 trial. 3733 bone marrow (BM) and 3793 peripheral blood (PB) samples from 469 patients were analyzed. NPM1mut TL log10 reduction ≥3 and achievement of MRD negativity in BM and PB were significantly associated with a lower CIR rate, after two treatment cycles and at end of treatment (EOT). In multivariate analyses, MRD positivity consistently revealed as poor prognostic factor in BM and PB. With regard to treatment effect, the median NPM1mut TL were significantly lower in the GO-Arm across all treatment cycles, resulting in a significantly higher proportion of patients achieving MRD negativity at EOT (56% vs 41%; P=.01). The betterreduction of NPM1mut TL after two treatment cycles in MRD-positive patients by the addition of GO led to a significantly lower CIR rate (4-year CIR 29.3% vs 45.7%, P=.009). In conclusion, the addition of GO to intensive chemotherapy in NPM1mut AML resulted in a significantly better reduction of NPM1mut TL across all treatment cycles leading to a significantly lower relapse rate. The AMLSG 09-09 trial was registered at www.clinicaltrials.gov as #NCT00893399.

Description: Background: Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 occur in roughly 25% of younger adult patients (pts) with acute myeloid leukemia (AML), implicating FLT3 as a potential target for kinase inhibitor therapy. The multi-targeted kinase inhibitor midostaurin shows potent activity against FLT3 as a single agent but also in combination with intensive chemotherapy. Aims: To evaluate the feasibility and efficacy of midostaurin in combination with intensive induction therapy and as single agent maintenance therapy after allogeneic hematopoietic stem cell transplantation (alloHSCT) or high-dose cytarabine (HIDAC). Methods: The study includes adult pts (age 18-70 years (yrs)) with newly diagnosed FLT3-ITD positive AML enrolled in the ongoing single-arm phase-II AMLSG 16-10 trial (NCT: NCT01477606). Pts with acute promyelocytic leukemia are not eligible. The presence of FLT3-ITD is analyzed within our diagnostic study AMLSG-BiO (NCT01252485) by Genescan-based fragment-length analysis (allelic ratio 〉0.05 required to be FLT3-ITD positive). Induction therapy consists of daunorubicin (60 mg/m², d1-3) and cytarabine (200 mg/m², continuously, d1-7); midostaurin 50 mg bid is applied from day 8 onwards until 48h before start of the next treatment cycle. A second cycle is optional. For consolidation therapy, pts proceed to alloHSCT as first priority; if alloHSCT is not feasible, pts receive three cycles of age-adapted HIDAC in combination with midostaurin from day 6 onwards. In all pts maintenance therapy for one year is intended. This report focuses on the first cohort of the study (n=149) recruited between June 2012 and April 2014 prior to the amendment increasing the sample size; the amendment to the study is active since October 2014. Results: At study entry patient characteristics were median age 54 years (range, 20-70, 34% ≥ 60 yrs); median white cell count (WBC) 48.4G/l (range 1.1-178G/l); karyotype, n=103 normal, n=3 t(6;9), n=2 t(9;11), n=20 intermediate-2 and n=7 high-risk according to ELN recommendations, n=14 missing; mutated NPM1 n=92 (62%). Data on response to first induction therapy were available in 147 pts; complete remission (CR) 58.5%, partial remission (PR) 20.4%, refractory disease (RD) 15% and death 6.1%. A second induction cycle was given in 34 pts. Overall response after induction therapy was CR 75% and death 7.5%. Adverse events 3°/4° reported during the first induction cycle were most frequently gastrointestinal (n=34) and infections (n=81). During induction therapy midostaurin was interrupted, dose-reduced or stopped in 55% of the pts. Overall 94 pts received an alloHSCT, 85 in first CR (n=65 age

Description: AML and high risk MDS are two myeloid malignancies with poor prognosis. Epigenetic changes such as silencing of tumor suppressor genes by promoter hypermethylation play a role in pathogenesis and disease progression. There is no consensus regarding the prognostic implications of epigenetic alterations in AML. We analyzed 108 de novo AML (median age 62 years, range 23–85), and 47 patients with MDS-related disease (19 MDS with IPSS INT-2 or High, 28 MDS-AML, median age 77 years, range 54–83), with respect to clinical and prognostic parameters and hypermethylation of the promoter region of three tumor suppressor genes; HIC, CDKN2b(p15) and CDH. All patients were eligible for and received standard induction chemotherapy. Methylation analysis was performed by Denaturing Gradient Gel Electrophoresis (DGGE) following bisulfite treatment and promoter specific PCR. A complete remission (CR) was achieved by 81 and 43 percent in the de novo AML group and the MDS group respectively. Hypermethylation of CDH or of multiple genes has previously been reported to correlate negatively to the probability of CR after intensive chemotherapy in the MDS cohort (p=0.008 and p=0.05, respectively). This was not true for the de novo AML patients (p=0.748 and p=0.681, respectively). The incidence of hypermethylation was significantly higher in de novo AML where 89% had at least one methylated gene compared to 66% in MDS (p=0.006). The mean number of methylated genes were 1.8 in de novo AML compared to 1.0 in MDS (p=0.002). In de novo AML the number of methylated genes decreased with increasing age (p=0.041) and an opposing trend was seen in MDS (p=0.15). Hypermethylation of all three genes was associated with higher age in MDS (p=0.040) whereas a trend towards the opposite was seen in de novo AML (p=0.084). In de novo AML the occurrence of FLT3-TKD was associated with hypermethylation of all three genes (p=0.011). Kaplan-Meier curves showed that hypermethylation of CDH and p15 together was associated with better overall survival (OS) in the de novo AML patients (p=0.001) (fig 1A). Median OS was 1.4 months for cases with no methylated genes and 13.5 and 14.5 months for those with one or two methylated genes, respectively. In contrast, in the MDS cohort, hypermethylation of CDH and p15 was a negative prognostic factor (p=0.002) (fig 1B). Median OS was 2.9 months for cases with two methylated genes and 12.7 and 9.4 for 0–1 methylated genes, respectively. The significance of these findings were retained after correction for age in a Cox regression analysis, HR 0.20 (p=0.001) for de novo AML and HR 2.96 (p=0.015) for MDS. We conclude that de novo AML and high risk MDS/MDS-AML show significant differences with respect to DNA promoter hypermethylation. Also, the correlation between hypermethylation and age as well as the effect on OS and complete remission rate differs between the de novo AML and the MDS cohort, respectively. This indicates epigenetic differences that may explain some of the clinical and morphological differences between the diseases. We also for the first time describe a relationship between FLT3-TKD and hypermethylation in AML. Fig 1A: Kaplan Meier curve showing the effect of methylation of p15 and CDH on OS (months) in de novo AML. Fig 1B: Kaplan Meier curve showing the effect of methylation of p15 and CDH on OS (months) in high risk MDS. Fig 1A:. Kaplan Meier curve showing the effect of methylation of p15 and CDH on OS (months) in de novo AML. . / Fig 1B: Kaplan Meier curve showing the effect of methylation of p15 and CDH on OS (months) in high risk MDS.

Description: Most patients with acute myeloid leukemia (AML) become resistant to chemotherapy at some point in their course and succumb to their disease. It is necessary to prevent chemo-resistance or enhance chemosensitivity in a selective fashion to lead to a higher cure rate and a lower toxic burden. The B-cell leukemia /lymphoma 2 (BCL-2) protein acts to prevent commitment to programmed cell death initiated in the mitochondrion. Inhibiting the function of this protein is an attractive approach to cancer therapy, but it remains a challenge to identify those tumors that will best be treated with such a strategy. Here, we test the sensitivity of AML cell lines and primary patient samples to the selective BCL-2 inhibitor, ABT-199, and test for correlation between ABT-199 sensitivity with BCL-2 dependence measured by BH3 profiling. We hypothesized that cells that are more dependent on BCL-2 will be more sensitive to inhibition by ABT-199. To this end, we tested the sensitivity of seven AML cell lines and 34 primary patient samples to ABT-199. We found that both cell lines and primary patient samples were sensitive to ABT-199 (Figure 1). In the case of primary patient cells, the median EC-50 was approximately 20 nM, and cell death could be seen within 2 hours. Importantly, we found that sensitivity to ABT-199 was independent of cytogenetics and NPM1 and FLT3 mutations of the primary patient cells suggesting that treatment with ABT-199 could be useful for a variety of AML patients, even those with an intrinsically poor prognosis. We also measured dependence on BCL-2-mediated apoptosis in cell lines and primary patient samples by BH3 profiling. BH3-profiling is a method to determine the mitochondrial priming level of a cell by exposing cellular mitochondria to standardized amounts of peptides derived from the BH3 domains of BH3-only proteins and determining the rate of cytochrome c release. BH3-profiling can also identify which anti-apoptotic species are critical in mediating cell death in a given cell type. For instance, the BAD BH3-only protein binds with high affinity to BCL-2, BCL-XL and BCL-W. Thus, release of cytochrome c following BAD peptide incubation suggests an anti-apoptotic dependency on BCL-2, BCL-XL or BCL-W. Therefore, we tested whether ABT-199 sensitivity correlates with functional dependence on BCL-2 in both cell lines and primary patient samples. We found that cells that released cytochrome c following incubation with the BAD peptide were more sensitive to ABT-199 treatment (Figure 2). This demonstrates that ABT-199 functions on target at the mitochondria since the ABT-199 mitochondrial activity correlates well with ABT-199 cytotoxicity data. Since ABT-199 does not inhibit MCL-1, increased expression of MCL-1 could be a potential source of upfront resistance to BCL-2 inhibition. Therefore, we asked if there was a correlation between MCL-1 dependence and ABT-199 sensitivity. We observed a weak anti-correlation between mitochondrial sensitivity to ABT-199 and sensitivity to the MCL-1 selective peptide NOXA BH3. This suggests that there is a minor tendency for MCL-1 dependent mitochondria to be less sensitive to ABT-199. The ex-vivo sensitivity of AML cells to ABT-199, which appears to be BCL-2 specific, is similar to that observed in CLL, a disease for which ABT-199 has demonstrated consistent activity in clinical trials. Moreover, the BH3 profiling studies demonstrate ABT-199 activity at the mitochondrion that correlates very well with cytotoxicity, supporting a mitochondrial mechanism of action. Our BH3 profiling studies will be undertaken to determine if the results will serve as a predictive biomarker in an upcoming phase II clinical trial of ABT-199 in AML. Disclosures: Letai: AbbVie: Consultancy; Dana-Farber Cancer Institute: Patents & Royalties.

Description: To identify cooperating lesions in core-binding factor acute myeloid leukemia, we performed single-nucleotide polymorphism-array analysis on 300 diagnostic and 41 relapse adult and pediatric leukemia samples. We identified a mean of 1.28 copy number alterations per case at diagnosis in both patient populations. Recurrent minimally deleted regions (MDRs) were identified at 7q36.1 (7.7%), 9q21.32 (5%), 11p13 (2.3%), and 17q11.2 (2%). Approximately one-half of the 7q deletions were detectable only by single-nucleotide polymorphism-array analysis because of their limited size. Sequence analysis of MLL3, contained within the 7q36.1 MDR, in 46 diagnostic samples revealed one truncating mutation in a leukemia lacking a 7q deletion. Recurrent focal gains were identified at 8q24.21 (4.7%) and 11q25 (1.7%), both containing a single noncoding RNA. Recurrent regions of copy-neutral loss-of-heterozygosity were identified at 1p (1%), 4q (0.7%), and 19p (0.7%), with known mutated cancer genes present in the minimally altered region of 1p (NRAS) and 4q (TET2). Analysis of relapse samples identified recurrent MDRs at 3q13.31 (12.2%), 5q (4.9%), and 17p (4.9%), with the 3q13.31 region containing only LSAMP, a putative tumor suppressor. Determining the role of these lesions in leukemogenesis and drug resistance should provide important insights into core-binding factor acute myeloid leukemia.

Description: Background: The Wilms’ tumor 1 gene (WT1) encodes a zinc finger protein that functions as a transcriptional regulator. Although its role in haematopoiesis is still not clarified yet, disruption of WT1 function is discussed to promote stem cell proliferation and to induce a block in differentiation, the hallmarks of the two complementation groups of gene mutations that have been postulated for the development of acute myeloid leukemia (AML). In two small studies WT1 mutations were detected in 11% of cytogenetically normal (CN) AML and were associated with a lower complete remission (CR) rate and a higher rate of resistant disease (RD). Aims: To evaluate the incidence and clinical impact of WT1 mutations in the context of NPM1, FLT3-ITD (internal tandem duplication)/TKD (tyrosine kinase domain mutations at codon 835), CEBPA, MLL-PTD (partial tandem duplication), and NRAS mutation status in CN-AML. Methods: Bone marrow or peripheral blood samples from 279 younger adult patients (pts) 16 to 60 years of age with CN-AML (n=242 de novo, n=32 secondary, n=5 therapy-related) were studied. Pts had been entered on three AMLSG treatment trials (AML HD93, AML HD98A, AMLSG 07–04). WT1 gene mutation screening was performed using standard PCR-based direct sequencing of exons 1 to 10; mutation analyses for the other genes were performed as previously described. Results: WT1 mutations were identified in 37/279 (13%) CN-AMLs, predominantly clustering in exon 7 (25/37) and exon 9 (6/37), but also occurring in exons 1, 2, 3, and 8. Most of them were frameshift mutations resulting from insertions or deletions; 33 pts had heterozygous and 4 pts had homozygous mutations. Correlation of WT1 to the FLT3-ITD/TKD, NPM1, CEBPA, MLL-PTD, and NRAS mutation status revealed mutant (mut) WT1 to be significantly associated with FLT3-ITDpos (p=0.003) and CEBPAmut (p=0.02). In a multivariable logistic regression model on induction success, the genotype WT1mut was not significant (p=0.89); the only significant variable was the NPM1mut/FLT3-ITDneg genotype (p=0.003). In univariable analyses for overall (OS), event-free (EFS) and relapse-free (RFS) survival, there was no difference between the WT1wt and the WT1mut group. Multivariable Cox proportional hazard models for OS, EFS and RFS also revealed no significant impact of the genotype WT1mut (p=0.4, p=0.71, and p=0.81, respectively); significant variables were the genotypes NPM1mut/FLT3-ITDneg (p=0.007, p=0.0001, and p=0.01) and CEPBAmut (p=0.01, p=0.0003, and p=0.03); and in addition logarithm of white blood cell count for OS (p=0.02). Conclusion: In our study on a large series of molecularly well characterized CN-AML, WT1 mutations occurred in 13% of the pts and were significantly associated with the genotypes FLT3-ITDpos and CEBPAmut. However, in both univariable and multivariable analyses WT1 mutations did not impact on the prognosis of pts with CN-AML. Additional pts have to be investigated to determine whether WT1 mutation status might have an impact within distinct genotypes.

Description: Background: Activating mutations in receptor tyrosine kinases like FLT3 (FLT3mut) lead to an aberrant signal transduction thereby causing an increased proliferation of hematopoietic cells. Internal tandem duplications (FLT3-ITD) or mutations in the tyrosine kinase domain (FLT3-TKD) occur in about 25% of younger adult patients (pts) with acute myeloid leukemia (AML), with FLT3 -ITD being associated with an unfavourable outcome. FLT3mut present an excellent target for small molecule tyrosine kinase inhibitors (TKI). The multi-targeted kinase inhibitor midostaurin (PKC412) is currently under investigation as a FLT3-inhibitor in combination with intensive chemotherapy. Monitoring of the efficacy of such a targeted therapy and correlation of the results with clinical outcome will be of major importance. The plasma inhibitor activity (PIA) assay allows the visualization of the level of dephosphorylation of the target under TKI therapy. Preliminary data suggest a correlation between the grade of dephosphorylation, as a marker for the activity of the TKI, and clinical outcome. Aims: To individually measure the level of FLT3 dephosphorylation by PIA analysis in a large cohort of FLT3-ITD AML pts treated within our AMLSG16-10 trial (NCT: NCT01477606) which combines midostaurin with intensive chemotherapy, and to correlate the results with clinical outcome. Methods: Plasma samples from pts (age 18-70 years) with newly diagnosed FLT3-ITD AML were obtained at different time points for PIA analysis. All pts were enrolled on the ongoing AMLSG 16-10 trial applying intensive therapy in combination with midostaurin (50mg twice a day). For consolidation therapy, pts proceeded to allogeneic hematopoietic stem cell transplantation (alloHSCT) as first priority; pts not eligible for alloHSCT were intended to receive 3 cycles of age-adapted high-dose cytarabine (HiDAC) in combination with midostaurin from day 6 onwards. In all pts one year of maintenance therapy with midostaurin was intended. PIA analyses were performed at defined time points (day 15 of induction, each consolidation cycle, at the end of each treatment cycle, every 3 months during maintenance therapy) as previously described (Levis MJ, et al. Blood 2006; 108:3477-83). Results: So far, PIA analyses were performed in 63 pts (median age, 51.6 years; range, 20-70 years) during (n=63) and after (n=73) first and second induction cycle, during (n=40) and after (n=53) consolidation therapy with HiDAC as well as during maintenance therapy (n=82). During and after induction therapy median levels of phosphorylated FLT3 (p-FLT3) were 46.6% (4.5-100%,

Description: Background Progress in the treatment of acute myeloid leukemia (AML) in older patients (pts) is still limited with poor complete remission (CR) rate and overall survival (OS). This is attributed to a variety of reasons including an inherently poor biology, especially a higher incidence of poor-risk karyotypes, co-morbidities, and an age-related functional impairment. In our randomized AML HD98B trial, the addition of all-trans retinoic acid (ATRA) to intensive chemotherapy resulted in an increased CR rate, event-free (EFS) and OS (Schlenk et al Leukemia 2004). More recent reports on in vitro studies indicated a synergistic action of the histone deacetylase inhibitor valproic acid (VPA) when associated with ATRA plus cytarabine and anthracyclines. In the randomized AMLSG 06-04 trial, therefore, VPA was evaluated in combination with intensive induction therapy plus ATRA in older pts (〉60 years) with newly diagnosed AML. In first analyses, the addition of VPA did not provide a significant advantage in OS and EFS after a median follow-up of 47 months (Tassara et al, ASH 2010, abstract #185). This was mainly due to increased hematological toxicity by VPA after the second induction therapy. Here we provide updated analyses especially on survival outcome data based on mature follow-up. Aims To evaluate VPA in combination with intensive induction therapy and ATRA in older patients with newly diagnosed AML. Methods Between August 2004 and February 2006 186 patients were randomized (standard-arm, n=93; experimental-arm, n=93) in the AMLSG 06-04 study (ClinicalTrials.gov Identifier: NCT00151255); median age was 68 years (60-84). The first 77 pts were randomized to receive 2 induction cycles (idarubicin 12 mg/m2 i.v. days 1-3, cytarabine 100 mg/m2 cont. i.v. days 1-5, ATRA 45 mg/m2 days 3-5 and 15 mg/m2 days 6-28) with or without VPA (days 1-28; started at 400 mg bid and then adapted in order to obtain a serum level of 60-150 mg/l). After an interim analysis the study was amended; for the following 109 patients idarubicin was dose-reduced to day 1 and 3 and VPA only added during the first induction cycle. All patients were intended for consolidation. Molecular diagnostics were performed as previously published (Schlenk et al, Haematologica 2009) Results Details of the response rate and toxicity of the induction treatment have already been presented (Tassara et al, ASH 2010, abstract #185). To summarize, CR rates after double induction were in trend higher in the standard-arm (52% vs. 40%; p=0.10), and early death rate higher in the experimental-arm (14% vs. 26%; p=0.06). The main toxicities attributed to VPA were grade 3/4 infections and delayed hematologic recovery (leukocytes, neutrophils and platelets) observed after the second induction cycle. Therapy (i.e. double induction and consolidation) was completed by 37/93 (40%) of patients in the standard arm and 19/93 (20%) in the experimental arm (p=0.01) After a median follow up of 84 months, analysis of the primary endpoint EFS revealed no differences between the two arms (EFS at 5 years, standard arm 2.3%, experimental arm 7.6%; p=0.95); similarly OS was not different (OS at 5 years, standard arm 11.7%, experimental arm 11.4%; p=0.57). However, pts in the experimental arm had a significantly better relapse-free survival (RFS at 5 years, standard arm 6.4%, experimental arm 24.0%, p=0.02). In explorative subset analyses superior RFS (p=0.03) and OS (p=0.03) of CR-patients were observed in AML patients with mutated NPM1 randomized into the experimental arm (RFS at 5 years, standard arm 8%, experimental arm 42%; OS at 5 years, standard arm 37%, experimental arm 52%). In contrast no differences were seen in AML patients with NPM1 wild-type for RFS (p=0.13) and OS (p=0.87) of CR-patients (RFS at 5 years, standard arm 7%, experimental arm 20%; OS at 5 years, standard arm 15%, experimental arm 22%). Due to a low frequency of FLT3-ITD (9/72) in this patient subset meaningful analyses were not possible. Conclusion In older patients with AML, the addition of VPA to standard induction treatment was associated with severe hematological toxicity as well as higher rates of infections and did not improve EFS and OS. However, after a long follow-up VPA was associated with a significantly improved RFS, which might be related to the mutated NPM1 genotype. Disclosures: Schlenk: Celgene: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Chugai: Research Funding; Amgen: Research Funding; Novartis: Research Funding; Ambit: Honoraria.