ALBERT

Description: Abstract 886 The diagnosis acute myeloid leukemia (AML) describes a heterogeneous group of myeloid stem cell disorders. Based on current concepts of disease development, the combination of at least two mutations is necessary for transformation, typically affecting transcription factors (e.g. RUNX1) blocking normal differentiation and growth promoting genes, e.g. receptor tyrosine kinases like FLT3. This model has been challenged by more recent results of genome-wide mutational analysis, which revealed a typical load of 8–12 mutations affecting several additional pathways, e.g. epigenetic regulation (DNMT3A, TET2 or IDH1). However, the sequence of acquisition and the individual impact of these mutations are largely unknown because these aspects are difficult to study. Here we describe a unique case of a donor cell leukemia giving unexpected insights into the development of AML in man. Case report and methods: In May 2004, a 51-year old male (P1) with an 8-year history of B-CLL received G-CSF mobilized peripheral blood stem cells after dose reduced conditioning from his HLA-identical sister, because he had relapsed after several lines of conventional therapy. He rapidly engrafted and showed complete donor chimerism (DC). In February 2012, he was admitted to the hospital with elevated WBC counts and circulating blasts. Bone marrow (BM) aspiration and morphology revealed an infiltration of the BM with 94% myeloid blasts (FAB M1). Cytogenetic and standard molecular assessment showed a normal female karyotype and NPM1 and FLT3-ITD mutations. STR-based analysis also revealed a persistent, 100% DC, thus the diagnosis of a donor cell AML was made, which developed almost 8 years after SCT. Interestingly, his sister, the donor (P2) had been also diagnosed with AML (FAB M2, cytogenetics: 47, XX,+8; NPM1 and FLT3-ITD neg.) only 3 months before her brother in Nov. 2011. Currently, P1 is in CR after re-SCT from an unrelated donor, whereas P2 relapsed and is scheduled for SCT after reinduction. Since DNA material of both individuals was available and due to this unique constellation, we performed next generation sequencing of whole exome enriched material using an Illumina HiSEQ 2000 platform after obtaining informed consent to compare both AMLs. Identified mutations were then confirmed using conventional Sanger sequencing and traced back by 454-based amplicon deep sequencing in a pre-SCT sample of the donor/P2 as well as several post SCT samples collected from P1 for the documentation of chimerism. Results: Comparison of the two AML-samples with a pre-SCT donor sample and a sample taken after 1.SCT as well as the HG19 and dbSNP135 releases revealed more than 100 unknown SNPs. Confirmation focused on cancer related changes or genes in critical pathways. In P1, in addition to the known NPM1 and FLT3-ITD mutations, we found somatic changes in CLCA1, PKHD1 and TET2, whereas in P2, we identified and confirmed somatic mutations in CDCA2, CBL, IDH1, NEK9 and PHF6. In addition, a typical DNMT3A R882C mutation was found in both leukemias. Interestingly, this mutation was also detectable by conventional Sanger sequencing in the pre-SCT sample of P2, but not in P2-germline DNA derived from buccal swaps. As shown in the figure, the 454-amplicon sequencing revealed a gradual increase of the TET2 (R1167G) mutational load over time in P1, and showed also that this mutation was present at low levels (4%) already in the pre-SCT sample of P2, but not in her final AML. FLT3-ITD and NPM1 mutations were detectable only in the AML-sample of P1, but not at any prior time points or in P2. Conclusions: These data indicate that mutations like the DNMT3A R882C can be present in normal appearing hematopoiesis at high levels years before the development of AML. The presence of the mutation in the absence of overt leukemia or MDS indicates that these mutations might not have a direct effect on the development of the disease, but favor the development of aberrant clones which then acquire additional changes in a latency phase. Other mutations (e.g. TET2) might give a small clonal advantage, but only the final acquisition of abnormalities like NPM1 and FLT3-ITD might transform this latency phase into a rapidly proliferating status, consistent with the “driver” status of these aberrations. The divergent mutational pattern found in the two AMLs emerging from the same DNMT3A-starting clone points to the high clonal diversity which might develop even within a single individual. Disclosures: Thiede: AgenDix GmbH: Employment, Equity Ownership.

Description: Background: Acute myeloid leukemia (AML) is the second most common form of leukemia and the most frequent cause of leukemia-related deaths in the United States. While complete response rates can be as high as 80% in patients undergoing initial induction chemotherapy, the majority of AML patients will relapse. Refractory or relapsed patients have a bleak prognosis. As there is currently no standard regimen for these patients, a great clinical need exists for new treatment options. Selinexor, an oral first-in-class Selective Inhibitor of Nuclear Export (SINE), inhibits XPO1 mediated nuclear export inducing cytotoxicity in cells with genomic damage. Preclinical data with Ara-C and selinexor significantly prolonged the survival of leukemic mice. A phase I clinical study demonstrated encouraging results in AML patients. The role of selinexor as mono-therapy or in combination is currently under investigation in phase II trials of AML. Aims: This phase II trial investigates the efficacy & tolerability of Ara-C and Idarubicin in combination with selinexor in patients with relapsed or refractory AML. Methods: Patients with relapsed/refractory AML were treated with Ara-C (100 mg/m2, continuous infusion, day 1-7), idarubicin (10 mg/m2, day 1, 3, 5) every 4 weeks. Selinexor was administered twice weekly orally starting on day 2 (40 mg/m2). A small cohort of patients received selinexor after registration and before first induction cycle for correlative studies. The primary endpoint was percentage of patients achieving a complete remission or complete remission without normalization of peripheral blood counts (CRi). Secondary endpoints were partial remission rate, percentage of patients undergoing subsequent allogeneic stem cell transplantion, early death rate, overall survival (OS), event-free survival and toxicity. Results: As of June-16-2015, 21 patients with AML have been enrolled at 3 sites in Germany (NCT02249091) after obtaining informed consent of which 20 had received ≥ 1 induction cycle and were evaluable for efficacy and toxicity. Median age was 60 (range 34-78) years. Seven patients had a complex and 6 a normal karyotype (unknown in 7 cases). On average, patients had received 3.5 (range 1-6) prior therapies. 2 patients had primary refractory disease and 11 patients an early relapse and 7 patients a late relapse. 7 patients had prior allogeneic transplantation. Overall response rate was 60% (25% of patients achieved CR, 25% of patients achieved CRi, 10% of patients achieved PR). Sixty percent of patients received or were planned for stem cell transplantion or donor lymphocyte infusion. The most frequent non-hematologic AES were vomiting, diarrhoea, nausea, fatigue, anorexia and neutropenic fever. One treatment-related death occurred. The patient developed subarachnoid haemorrhage during thrombocytopenia grade 4 and died. Summary / Conclusion: The prognosis of relapsed/refractory AML patients is remarkably poor. Our findings suggest that Ara-C and Idarubicin in combination with selinexor resulted in a remarkable response rate and is a promising regimen in this particularly unfavourable cohort of patients without unexpected toxicities enabling the majority of them to proceed to first or second allogeneic stem cell transplantation. Disclosures Fiedler: Teva, Amgen, Astellas: Other: Travel Grant; Pfizer, Amgen, Kolltan: Research Funding; Karyopharm: Research Funding. Off Label Use: Selinexor has been used in combination with AraC and idarubicin in relapsed/refractory AML patients.. Chromik:Karyopharm: Research Funding. Theile:GSO Hamburg: Employment. Kranich:GSO Global: Employment. Heuser:Karyopharm: Research Funding.

Description: Background: Sorafenib is a multi-kinase inhibitor with activity against several oncogenic kinases that may play a role in the pathogenesis of acute myeloid leukemia (AML). In-vitro data and results from non-randomized clinical trials suggest that sorafenib might be an effective drug for the treatment of AML. We present the results of the randomized placebo-controlled SORAML trial testing sorafenib versus placebo as add-on to standard induction and consolidation treatment in AML patients ≤60 years. Patients and Methods: Between March 2009 and October 2011, 276 patients from 25 centers were enrolled in the SORAML trial (NCT00893373). The main eligibility criteria were newly diagnosed AML, age from 18 to 60 years and suitability for intensive therapy. The treatment plan for all patients included two cycles of induction with DA (daunorubicin 60 mg/m2 days 3-5 plus cytarabine 100 mg/m2 cont. inf. days 1-7), followed by three cycles of high-dose cytarabine consolidation (3 g/m2 b.i.d. days 1, 3, 5). Patients without response after DA I received second induction with HAM (cytarabine 3 g/m2 b.i.d. days 1-3 plus mitoxantrone 10 mg/m2 days 3-5). Allogeneic stem cell transplantation was scheduled for all intermediate-risk patients in first complete remission with a sibling donor and for all high-risk patients with a matched related or unrelated donor. At study inclusion, patients were randomized to receive either sorafenib (800 mg/day) or placebo as add-on to standard treatment in a double blinded fashion. Block randomization at a ratio of 1:1 was performed within cytogenetic and molecular risk strata, allocation was concealed and treatment was double blinded. Study medication was given on days 10-19 of DA I+II or HAM, from day 8 of each consolidation until 3 days before the start of the next consolidation and as maintenance for 12 months after the end of consolidation. The primary endpoint of the trial was event-free survival (EFS) with an event being defined as either failure to achieve a complete remission (CR) after induction, relapse or death. Secondary endpoints were relapse-free survival (RFS), overall survival (OS), CR rate and incidence of adverse events (AE). We present the results of the final analysis of the primary endpoint EFS (intent to treat) after the occurrence of 134 events. Results: Out of 276 enrolled patients, 267 received study treatment, 134 in the sorafenib arm and 133 in the placebo arm. Demographic and disease characteristics were equally distributed between the two arms; the incidence of FLT3-ITD was 17%. The median cumulative dose of administered study medication was similar in both arms. The CR rates were 59% versus 60% in the placebo versus sorafenib arm (p=0.764). After a median observation time of 36 months, the median EFS was 9.2 months in the placebo arm and 20.5 months in the sorafenib arm, corresponding to a 3-year EFS of 22% versus 40% (p=0.013). Median RFS after standard treatment plus placebo was 23 months and not yet reached after sorafenib treatment, corresponding to a 3-year RFS of 38% and 56%, respectively (p=0.017). The median OS had not been reached in either arm; the 3-year OS was 56% with placebo versus 63% with sorafenib (p=0.382). In 46 FLT3-ITD positive patients, no difference in EFS, but a trend for prolonged RFS and OS in favor of sorafenib was observed. The most common reported AEs Grade ≥3 were fever (40%), infections (22%) and bleeding events (2%). The risk for fever, bleeding events and hand-foot syndrome was significantly higher in the sorafenib arm while the incidence of all other AEs showed no significant differences. Conclusions: In younger AML patients, the addition of sorafenib to standard chemotherapy in a sequential manner is feasible and associated with antileukemic efficacy. We observed a higher incidence of infections and bleeding events under sorafenib. Whereas OS in both treatment arms was similar, sorafenib treatment resulted in a significantly prolonged EFS and RFS. Figure 1: Event-free survival Figure 1:. Event-free survival Disclosures Off Label Use: sorafenib for treatment of aml. Serve:Bayer HealthCare: Research Funding. Ehninger:Bayer HealthCare: Research Funding.

Description: The BCR-ABL oncogene activates several signaling pathways, most notably by constitutive phosphorylation of the signal transducer and activator of transcription protein 5 (STAT5). After phosphorylation and nuclear translocation, STAT5 transcriptionally activates numerous genes responsible for proliferation, survival and differentiation of hematopoietic stem and progenitor cells. Among the STAT5 target genes are suppressor of cytokine signaling (SOCS) proteins. SOCS proteins inhibit JAK kinases by multiple mechanisms, thereby terminating cytokine signaling in a classical negative feedback loop. The SOCS family of proteins comprises eight members: cytokine-inducible SRC homology 2 (SH2) domain protein (CIS) and SOCS1–SOCS7. SOCS1 is frequently silenced by hypermethylation in multiple myeloma and inactivating mutations have been found in Hodgkin lymphoma with consecutive increase in JAK2 kinase activity. More recently, we identified SOCS1 as a “conditional oncogene” in the context of the FLT3-ITD oncogene (Reddy et al, Blood 2012): SOCS1 significantly enhanced FLT3-ITD-mediated myeloid transformation, both in vitro and in vivo. We hypothesized that this may be a more general mechanism of transformation and therefore analyzed the role of SOCS proteins in BCR-ABL mediated transformation and leukemogenesis. First, we investigated gene expression levels of SOCS proteins in BCR-ABL positive (versus BCR-ABL negative) cell lines and primary ALL long term-cultured cells. Upon treatment with the BCR-ABL inhibitor imatinib, mRNA expression levels of CIS and SOCS1-4 were reduced. SOCS5-7 did not exhibit any changes and were non-responsive to ABL-kinase inhibition. In lineage-depleted primary murine bone marrow retrovirally transduced with BCR-ABL, high induction of CIS and SOCS1-3 mRNA was detected, while SOCS4-7 showed only minor changes. When overexpressed in IL-3 dependent cell lines, SOCS1 led to a very rapid cell death within few days. Similar effects were demonstrated for CIS and SOCS2 overexpression, however, with a slower kinetics. In contrast, BCR-ABL transduced cells were insensitive to SOCS1 overexpression. In colony formation assays performed with primary hematopoietic cells, expression of SOCS1 led to a significant decrease of colony numbers. Interestingly, co-expression of SOCS1 and BCR-ABL (hereafter abbreviated as SOCS1/BCR-ABL) also lowered colony numbers compared to cells expressing BCR-ABL alone. However, when cells were subjected to interferon alpha or interferon gamma treatment, SOCS1/BCR-ABL positive cells displayed higher colony numbers and gained a growth advantage over BCR-ABL expressing cells, since anti-proliferative effects of the cytokines were inhibited by the presence of SOCS1. A careful analysis of the downstream signaling cascade of BCR-ABL and SOCS1/BCR-ABL expressing cells did not demonstrate any differences in the phosphorylation of AKT, ERK1/2 and STAT5. However, when BCR-ABL was inhibited by imatinib, STAT5 phosphorylation was significantly decreased in SOCS1/BCR-ABL transduced cells. Finally, the influence of SOCS1 in BCR-ABL mediated leukemia was investigated in a murine bone marrow transplantation model. BCR-ABL or SOCS1/BCR-ABL expressing cells led to disease formation with a chronic myeloid leukemia-like phenotype. Interestingly, the co-expression of SOCS1 and BCR-ABL prolonged disease latency, as opposed to the phenotype observed with FLT3-ITD (where SOCS1 co-expression shortened latency). In this setting SOCS1 acts as a tumor suppressor, protecting BCR-ABL transformed cells from rapid disease development, and a molecular analysis will be presented. Disclosures: No relevant conflicts of interest to declare.

Description: Up to 30% of patients with acute myeloid leukemia (AML) harbor internal tandem duplications (ITD) within the FLT3 gene, a tyrosine kinase receptor. The signalling pathways and downstream targets are only partly understood. We demonstrated that the presence of Flt3-ITD constitutively activated Akt (PKB), a key serine-threonine kinase within the PI3-kinase pathway. The constitutive activation of Akt induced phosphorylation and transcriptional inhibition of the transcription factor Foxo3a. Also, forced Foxo3a activity reversed Flt3-ITD-mediated growth properties. Using a novel Akt inhibitor, we showed that Flt3-ITD-mediated Foxo3a phosphorylation was dependent on Akt. Conditional Akt activation targeted to the cell membrane conferred cytokine-independent survival, proliferation and transformation of myeloid progenitor cells. Induction of Akt activity in vivo promoted the development of a myeloproliferative disease in a syngeneic mouse model. Importantly, phosphorylation of Akt was frequently found in primary myeloid blasts, supporting that this signalling pathway may be important in the development of AML. Furthermore, activation of Akt constitutively activated the downstream kinase mammalian target of rapamycin (mTOR). mTOR has several important biological functions, notably in nutrient sensing and protein translation. Treatment with rapamycin, a specific inhibitor of mTOR, inhibited mTOR kinase activity at 1nM in all examined cell systems. Rapamycin caused cell cycle arrest, apoptosis and completely inhibited transformation in FLT3-ITD-positive cells. These effects were most pronounced in the low nanomolar range, which are therapeutic concentrations of rapamycin-treated patients. The biological advantage in Akt-expressing cells was fully reversed by treatment with rapamycin. In all examined primary blasts from AML patients, mTOR was inhibited by rapamycin. In short term proliferation assays, twelve out of 18 (67%) examined showed marked sensitivity with an IC50 below 10nM rapamycin, while one third were found to be more resistant. Taken together, increased survival, proliferation and leukemic transformation by Flt3-ITD is mediated by Akt and mTOR, and can be inhibited by rapamycin. We conclude that rapamycin may be an attractive substance for the treatment of patients with AML.

Description: Background: We recently showed that the combination of ATRA and arsenic trioxide (ATO) is at least not inferior and possibly superior to standard ATRA and chemotherapy (CHT) in the front-line management of low/intermediate risk APL (Italian-German APL 0406 trial; Lo-Coco et al., NEJM 2013). We report herein on the extended and final series of 276 patients (162 were in the previous report) with the last case being enrolled into the study in January 2013. Methods: The APL0406 study was a prospective, open-label, randomized intergroup trial conducted by the Italian GIMEMA and the German SAL and AMLSG study groups. Eligible patients were adults aged 18-

Description: Abstract 1054 Activating mutations of FLT3 such as FLT3-ITD are frequently found in AML patients and confer poor clinical prognosis. It is unclear how leukemic blasts escape cytokine control, which regulates normal hematopoiesis. We have recently demonstrated that FLT3-ITD, when localized to the endoplasmic reticulum, aberrantly activates STAT5. Here we show that one of the target genes induced by STAT5 is SOCS1, a potent negative regulator of cytokine signaling and known tumor suppressor gene. Importantly, a significantly increased SOCS1 expression was found in FLT3-ITD+ AML. While SOCS1 expression in murine bone marrow severely impaired cytokineinduced colony growth, it failed to inhibit FLT3-ITD supported colony growth, indicating resistance of FLT3-ITD to SOCS1. Furthermore, SOCS1 co-expression inhibited interferon-γ signaling and protected FLT3-ITD hematopoietic cells from interferon-γ mediated growth inhibitory effects. In a murine bone marrow transplantation model, the co-expression of SOCS1 and FLT3-ITD significantly shortened the latency of a myeloproliferative disease compared to FLT3-ITD alone (p