Abstract: Biallelic mutations of the CEBPA gene (CEBPAbi) define a distinct entity associated with favorable prognosis; however, the role of monoallelic mutations (CEBPAsm) is poorly understood. We retrospectively analyzed 4708 adults with acute myeloid leukemia (AML) who had been recruited into the Study Alliance Leukemia trials, to investigate the prognostic impact of CEBPAsm. CEBPA mutations were identified in 240 patients (5.1%): 131 CEBPAbi and 109 CEBPAsm (60 affecting the N-terminal transactivation domains [CEBPAsmTAD] and 49 the C-terminal DNA-binding or basic leucine zipper region [CEBPAsmbZIP] ). Interestingly, patients carrying CEBPAbi or CEBPAsmbZIP shared several clinical factors: they were significantly younger (median, 46 and 50 years, respectively) and had higher white blood cell (WBC) counts at diagnosis (median, 23.7 × 109/L and 35.7 × 109/L) than patients with CEBPAsmTAD (median age, 63 years, median WBC 13.1 × 109/L; P & lt; .001). Co-mutations were similar in both groups: GATA2 mutations (35.1% CEBPAbi; 36.7% CEBPAsmbZIP vs 6.7% CEBPAsmTAD; P & lt; .001) or NPM1 mutations (3.1% CEBPAbi; 8.2% CEBPAsmbZIP vs 38.3% CEBPAsmTAD; P & lt; .001). CEBPAbi and CEBPAsmbZIP, but not CEBPAsmTAD were associated with significantly improved overall (OS; median 103 and 63 vs 13 months) and event-free survival (EFS; median, 20.7 and 17.1 months vs 5.7 months), in univariate and multivariable analyses. Additional analyses revealed that the clinical and molecular features as well as the favorable survival were confined to patients with in-frame mutations in bZIP (CEBPAbZIP-inf). When patients were classified according to CEBPAbZIP-inf and CEBPAother (including CEBPAsmTAD and non-CEBPAbZIP-inf), only patients bearing CEBPAbZIP-inf showed superior complete remission rates and the longest median OS and EFS, arguing for a previously undefined prognostic role of this type of mutation.

Abstract: Abstract 2060 Poster Board II-37 Introduction: The Flt3-internal tandem duplication can be found in up to 30% of all acute myeloid leukemia (AML) patients and confers a poor risk status characterized by an increased relapse rate and poor overall survival. Moreover, Flt3-ITD-positive AML patients relapsing after allogenic stem cell transplantation (SCT) have very limited therapeutic options. Sorafenib is a multikinase inhibitor that is approved for the treatment of metastatic renal cell and hepatocellular carcinoma. Besides targeting Raf, the platelet derived growth factor receptor (PDGFR) and the vascular endothelial growth factor receptor (VEGFR) it has also significant inhibitory activity against the Flt3 receptor tyrosine kinase, and, specifically the mutated variant of Flt3, Flt3-ITD. It has previously been shown that sorafenib monotherapy may have considerable activity in relapsed Flt3-ITD positive AML. Nevertheless, clinical experience is still limited. Here we report compassionate use experience on 18 relapsed or refractory Flt3-ITD positive AML patients treated with sorafenib monotherapy. Methods: A questionnaire was developed and sent to 28 centers in Germany in order to obtain more insight into the clinical efficacy and tolerablilty of sorafenib monotherapy in Flt3-ITD positive AML. Forms were returned from 13 centers, reporting 26 patients. Among them, eight had to be excluded from further analysis. Five of them were Flt3-ITD mutation negative and three received contemporary chemotherapy. Available patient information included age, FAB-classification, karyotype, type and response to prior therapy, sorafenib dosing, tolerability, treatment duration, and response. Results: Of the 18 patients (12 male, 6 female), five were primary refractory to induction chemotherapy and 13 received sorafenib in first (n=11) or second (n=2) relapse. Eight of 18 patients relapsed after SCT and were treated with sorafenib. One patient was treated for steadily increasing Flt3-ITD copy numbers, that is, in molecular relapse after SCT. Patients received between 200mg and 800mg sorafenib p.o. daily. The median treatment duration was 98 days (range, 16-425 days). All patients achieved a hematological response (HR) characterized by complete (n=17) or near complete peripheral blast clearance (n=2). Of the 18 patients the documented best response to sorafenib were: HR in 9 cases, bone marrow response (HR and blast reduction in marrow) in 4 cases, complete remission (normalization of peripheral blood counts and bone marrow blasts 〈 5%) in one case and complete molecular remission (molecular negativity for Flt3-ITD) in 4 patients. After a median treatment duration of 180 days (range, 82-270 days) 7 of the 18 (39%) patients developed clinical sorafenib resistance: two of eight (25%) of the SCT-group and 5 of 10 (50%) of the non-SCT group. Sorafenib was generally well tolerated. Pancytopenia or thrombocytopenia grade III and IV were the most significant side effects, observed in 13 patients. Other reported side effects such as diarrhea, exanthema were documented from the centers as being minor. Conclusion: Sorafenib monotherapy has significant clinical activity in Flt3-ITD positive relapsed and refractory AML and may be particularly effective in the context of allo-immunotherapy where 3 CMR could be seen. Disclosures: Enghofer: Bayer Schering Pharma: Employment. Off Label Use: sorafenib, used to treat Flt3-ITD positive AML patients.

Abstract: Overcoming disease persistence in chronic myeloid leukemia (CML) patients is of considerable importance to the issue of potential cure. Here we asked whether autocrine signaling contributes to survival of BCR/ABL positive cells in presence of imatinib mesylate (IM, Gleevec™) and nilotinib (AMN107, NI), a novel more selective Abl inhibitor. Conditioned media (CM) of IM-resistant LAMA84-cell clones (R-CM) was found to substantially protect IM-naïve LAMA cells and primary CML progenitors from IM- or NI-induced cell death. This was due to an increased secretion of the granulocyte-macrophage colony stimulating factor (GM-CSF) which was identified as the causative factor mediating IM resistance in R-CM. GM-CSF elicited IM and NI drug resistance via a BCR/ABL-independent activation of the JAK-2/STAT-5 signaling pathway in GM-CSF-receptor alpha receptor (CD116) expressing cells, including primary CD34+/CD116+ GM-progenitors (GMP). In a cell based resistance induction assay, GM-CSF enabled the evolution of IM- and NI-resistant colonies. When analysing GM-CSF expression in over 120 patient samples of first diagnosis CML patients and resistant patient, elevated mRNA and protein levels of GM-CSF were detected exclusively in IM-resistant patient samples, suggesting a contribution of GM-CSF secretion for IM and NI resistance in vivo. Importantly, inhibition of JAK-2 with AG490 abrogated GM-CSF-mediated STAT-5 phosphorylation and NI resistance in vitro. Together, this suggests that adaptive autocrine secretion of GM-CSF and cytokines in general may be a novel IM and NI resistance mechanism, which may also contribute to CML-persistence. GM-CSF protects CML-progenitors via a BCR/ABL-independent activation of the anti-apoptotic JAK-2/STAT-5 pathway. Inhibition of JAK-2 overcomes GM-CSF-induced IM and NI progenitor cell resistance providing a rationale for the application of JAK-2 inhibitors to eradicate residual disease in CML.

Abstract: Abstract 339 Initial reports that high dose imatinib results in better responses more rapidly than standard dose imatinib remain controversial. The German CML Study Group therefore compared imatinib 800 mg (IM 800) with standard dose imatinib +/- IFN (IM 400, IM 400 + IFN) in newly diagnosed, not pretreated CML with regard to molecular response at 12 months and survival in a randomized clinical trial. By April 30, 2009, 1026 chronic phase CML patients have been randomized (326 for IM 400, 338 for IM 800, 351 for imatinib + IFN). Comparison was for molecular and cytogenetic remissions, overall (OS) and progression free (PFS) survival and toxicity. 1015 patients were evaluable at baseline, 904 for survival analysis (294 for IM 400, 286 for IM 800, 324 for IM 400+IFN), 790 for cytogenetic (analysis of at least 20 metaphases required) and 823 for molecular response. The three treatment groups were similar regarding median age, sex, median values of Hb, WBC, platelets and distribution according to the EURO score. Median follow-up was 25 months in the imatinib 800 mg arm and 42 months in the imatinib 400 mg +/-IFN arms. The difference is due to the fact that at first the IM 800 arm was designed for high risk patients only and opened up to all risk groups in July 2005. The median daily doses of imatinib were 626 mg (209- 800 mg) in the IM 800 arm and 400 mg (184- 720 mg) in the IM 400 +/- IFN arms. Of 218 patients receiving imatinib 800 mg and evaluable for dosage at 12 months, 100 (45.9%) received more than 700 mg/day, 27 (12.4%) 601-700 mg, 37 (17.0%) 501-600 mg, 48 (22.0%) 401-500 mg and only 6 (2.8%) 400 mg/day or less. The cumulative incidences at 12 months of complete cytogenetic remission (CCR) were 52.3%, 64.9% and 50.6%, and of major molecular remission (MMR) 30.2%, 54.3% and 34.6% with IM 400, IM 800 and IM 400 +IFN, respectively. The cumulative incidences of achieving CCR and MMR with IM 400, IM 800 and IM 400+IFN at 6, 12, 18 and 24 months after start of treatment are summarized in the table. MMR at 12 months was reached faster with IM 800 than with IM 400 (p=0.0003) or IM400+IFN (p=0.0131). Optimal molecular response (OMR= 〈 0.01% BCR-ABL according to the international scale) was reached with IM 800 after a median of 31.3 months vs. 47.5 and 42.5 months with IM 400 +/- IFN. Also CCR was reached faster with IM 800 (p 〈 0.01). The more rapid achievement of MMR with IM 800 was observed in low and intermediate risk patients with little or no difference in high risk patients. In an analysis “as treated” patients receiving more than 600 mg/day reached remissions faster than those receiving lower dosages (CCR after a median of 7.8 vs. 8.9 months, MMR after a median of 10.4 vs. 12.9 months). At the time of this evaluation, OS (92% at 5 years) and PFS (88% at 5 years) showed no difference. Type and severity of adverse events (AE) at 12 months did not differ from those expected (all grades and grades III/IV). Hematologic (thrombocytopenia 7% vs. 4%) and non-hematologic AEs (gastrointestinal 35% vs. 15-24% and edema 29% vs. 16-19%) were more frequent with IM 800, fatigue (14% vs. 7-13%) and neurological problems (15% vs. 6-7%) more frequent with IM 400 + IFN (all grades). These data show a significantly faster achievement of MMR at 12 months with IM 800 as compared to IM 400 +/-IFN. So far, this faster response rate did not translate into better OS or PFS. Hence IM 400 should still be considered as standard of care. With some individual dose adjustments tolerability of IM 800 was good. Longer observation is required to determine whether this more rapid achievement of MMR and CCR will have a long term impact or not. Disclosures: German CML Study Group: Deutsche Krebshilfe: Research Funding; Novartis: Research Funding; European LeukemiaNet: Research Funding; Kompetenznetz Leukämie: Research Funding; Roche: Research Funding; Essex: Research Funding.

Abstract: Abstract 782 Introduction: Current evidence indicates that acquired genetic instability in chronic myeloid leukemia (CML) as a consequence of the t(9;22)(q34;q11) and the resulting BCR-ABL fusion causes the continuous acquisition of additional chromosomal aberrations (ACA) and mutations and thereby progression to accelerated phase and blast crisis (BC). Around 10 –12% of patients in chronic phase (CP) CML have ACA already at diagnosis. During the course of the disease this number rises to 80% in BC. Acquisition of ACA during treatment is considered as a poor prognostic indicator, whereas the impact of ACA at diagnosis is controversial. Patients and methods: Clinical and cytogenetic data of 1151 out of 1311 patients with Philadelphia and BCR-ABL positive CP CML randomized until 2009 to the German CML-Study IV were investigated in a prospective study. There were 459 females (40%) and 692 males (60%). Median age was 53 years (range, 16–88). All patients were treated with imatinib alone or in combination with interferon alpha or araC. The impact of ACA at diagnosis on time to complete cytogenetic and major molecular remission (CCR, MMR) and progression-free and overall survival (PFS, OS) was investigated. Written informed consent was obtained from all patients prior to entering the study. Results: At diagnosis 1003/1151 patients (87%) had the standard t(9;22)(q34;q11) only and 69 patients (6.0%) had a variant t(v;22). In 60 of 69 patients with t(v;22), only one further chromosome was involved in the translocation, in 7 patients two, and in 2 patients three further chromosomes were involved. Seventy-nine patients (6.9%) had ACA. Of these, 38 patients (3.3%) lacked the Y chromosome (-Y) and 41 patients (3.6%) had ACA except -Y. Sixteen of the 41 patients had major-route ACA (+8, i(17)(q10), +der(22)t(9;22)(q34;q11), ider(22)(q10)t(9;22)(q34;q11)) and 25 minor-route ACA [e.g. t(3;12), t(4;6), t(2;16), t(1;21)]. In patients with major-route ACA, trisomy 8 was the most frequent additional alteration (n=9). +der(22)t(9;22)(q34;q11) was observed in six patients, isochromosome (17)(q10) in five patients and ider(22)(q10)t(9;22)(q34;11) in three patients. After a median observation time of 5.3 years for patients with t(9;22), t(v;22), -Y, minor- and major-route ACA median times to CCR were 1.01, 0.95, 0.98, 1.49 and 1.51 years, to MMR 1.40, 1.58, 1.65, 2.49 and 〉 7 years, 5-year PFS 90%, 81%, 88%, 96% and 50% and 5-year OS 92%, 87%, 91%, 96% and 53%, respectively. In patients with major-route ACA times to CCR and MMR were longer. PFS and OS were shorter (p 〈 0.001) than with standard t(9;22)(q34;q11). Loss of Y chromosome had no influence on time to CCR or MMR, PFS and OS. Conclusion: We conclude that the prognostic impact of additional cytogenetic findings at diagnosis of CML is heterogeneous and consideration of their types may be important. Major-route ACA identify a small group of patients with significantly poorer prognosis as compared to all other patients requiring early and more intensive intervention such as stem cell transplantation. Disclosures: Hochhaus: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kneba:Hoffmann La Roche: Honoraria.

Abstract: BCR-ABL overexpression and stem cell quiescence supposedly contribute to the failure of imatinib mesylate (IM) to eradicate chronic myeloid leukemia (CML). However, BCR-ABL expression levels of persisting precursors and the impact of long-term IM therapy on the clearance of CML from primitive and mature bone marrow compartments are unclear. Here, we have shown that the number of BCR-ABL–positive precursors decreases significantly in all bone marrow compartments during major molecular remission (MMR). More importantly, we were able to demonstrate substantially lower BCR-ABL expression levels in persisting MMR colony-forming units (CFUs) compared with CML CFUs from diagnosis. Critically, lower BCR-ABL levels may indeed cause IM insensitivity, because primary murine bone marrow cells engineered to express low amounts of BCR-ABL were substantially less sensitive to IM than BCR-ABL–overexpressing cells. BCR-ABL overexpression in turn catalyzed the de novo development of point mutations to a greater extent than chemical mutagenesis. Thus, MMR is characterized by the persistence of CML clones with low BCR-ABL expression that may explain their insensitivity to IM and their low propensity to develop IM resistance through kinase point mutations. These findings may have implications for future treatment strategies of residual disease in CML.

Abstract: Overcoming imatinib mesylate (IM) resistance and disease persistence in patients with chronic myeloid leukemia (CML) is of considerable importance to the issue of potential cure. Here we asked whether autocrine signaling contributes to survival of BCR/ABL+ cells in the presence of IM and nilotinib (NI; AMN107), a novel, more selective Abl inhibitor. Conditioned media (CM) of IM-resistant LAMA84 cell clones (R-CM) was found to substantially protect IM-naive LAMA cells and primary CML progenitors from IM- or NI-induced cell death. This was due to an increased secretion of the granulocyte-macrophage colony-stimulating factor (GM-CSF), which was identified as the causative factor mediating IM resistance in R-CM. GM-CSF elicited IM and NI drug resistance via a BCR/ABL-independent activation of the janus kinases 2 (JAK-2)/signal transducer and activator of transcription 5 (STAT-5) signaling pathway in GM-CSF receptor α receptor (CD116)–expressing cells, including primary CD34+/CD116+ GM progenitors (GMPs). Elevated mRNA and protein levels of GM-CSF were detected in IM-resistant patient samples, suggesting a contribution of GM-CSF secretion for IM and NI resistance in vivo. Importantly, inhibition of JAK-2 with AG490 abrogated GM-CSF–mediated STAT-5 phosphorylation and NI resistance in vitro. Together, adaptive autocrine secretion of GM-CSF mediates BCR/ABL-independent IM and NI resistance via activation of the antiapoptotic JAK-2/STAT-5 pathway. Inhibition of JAK-2 overcomes GM-CSF–induced IM and NI progenitor cell resistance, providing a rationale for the application of JAK-2 inhibitors to eradicate residual disease in CML.

Abstract: Purpose Mutations of the isocitrate dehydrogenase-1 (IDH1) and IDH2 genes are one of the most frequent alterations in acute myeloid leukemia (AML) and can be found in ~20% of patients at diagnosis. Several IDH inhibitors are currently in late stage clinical development with Enasidenib, an IDH2 inhibitor, being recently approved by the FDA. Previous analyses have reported differential impact on response to chemotherapy and outcome, depending on the IDH-mutation type, co-occurring mutations and cytogenetic abnormalities, as well as the variant allele frequency (VAF) of IDH mutations. In order to better understand its prognostic role, we analyzed newly diagnosed AML patients enrolled in prospective trials of the Study Alliance Leukemia (SAL) to investigate the impact of IDH1/2 mutations on outcome. Patients and Methods All AML patients consecutively enrolled into intensive AML treatment protocols of the SAL or into the SAL registry were included in this analysis. Next-generation sequencing (NGS) on an Illumina MiSeq-system was performed to detect IDH1/2 mutations using pre-treatment samples. Overall survival (OS) and response to therapy were analyzed for all patients with intensive treatment and according to the mutational status. Results Overall, samples of 3898 patients were analyzed. The median follow-up was 91 months (95% CI 87.2 - 93.9). Patients' characteristics are shown in Tbl.1. Three-hundred twenty-nine patients (8.4%) had IDH1 mutations and 423 (11%) had IDH2 mutations; both mutations were found in 12 pts, so the overall mutation rate in IDH1 and 2 was 19% (740/3898 patients). Of the IDH1 variants, the most common ones were the R132C found in 143 patients (43%) and R132H in 137 patients (42%). For IDH2, 324 patients had the R140Q (77%) and 80 patients the R172K (19%) variant. According to the two main variants of the more common IDH2 mutations, as reported before, the IDH2 R172K was mutually exclusive with NPM1 and/or FLT3-ITD mutations. Overall, there was a trend for increased OS for patients with IDH2 R172K (26 vs. 15 months) as compared to those with R140Q. Considering only patients with a normal karyotype and no NPM1/FLT3-ITD mutation, these patients (n=27) had a highly significant better OS than patients with IDH2 R140Q (46.3 vs. 13.1 months, p=.012), supporting the findings published by Papaemmanuil et al. (NEJM 2016). In IDH1-mutated patients, we observed statistically significant differences in baseline characteristics between the two most common mutation types, IDH1 R132C and R132H. Patients carrying the R132C mutation were older (62 vs. 55 years, p=.001), had lower WBC (3.6 vs. 21 Gpt/L, p≤.001) and were less likely to have a normal karyotype (43% vs. 66%, p=.002), NPM1 (23% vs. 66%, p= 〈 .001), and FLT3-ITD mutations (8% vs. 27%, p 〈 .001) than those with the R132H variant. In univariate testing, the CR rate was also statistically significant lower in patients with IDH1 R132C (53% vs. 72%, p≤.001), with a median OS of 12.9 months compared to 17.4 months for patients with R132H variant (p=.08). In multivariate analysis including age, WBC, NPM1 and FLT3 status, and ELN risk, the CR rate was significantly lower in patients with the IDH1 R132C variant (p=.038). The median IDH VAF was 38% (range, 0.1 - 58) with no difference according to the different types of mutation. Patients with a VAF 〉 30% had a significantly higher BM blast count (73% vs 40% for VAF≤5%) and WBC (21.2 Gpt/L vs. 3.7 Gpt/L) at baseline, but there was no clear impact on CR rate or OS found in multivariate analysis. Conclusion In this large cohort of AML patients with IDH1/2 mutations, we found significant and so far not reported differences for one of the two most prominent mutations types of IDH1. The R132C variant was associated with increased age, lower WBC, and lower NPM1 and/or FLT3 co-mutation rate. Further, these patients had lower CR rates and a trend for shorter OS. For IDH2 we were able to reproduce findings on co-mutations and showed a favorable outcome for intensively treated patients with a normal karyotype and no NPM1/FLT3-ITD mutation and the IDH2 R172K variant, providing additional evidence for classification as a separate AML entity. Disclosures Middeke: Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees. Rollig:Bayer: Research Funding; Janssen: Research Funding. Kramer:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; Daiichi Sankyo: Consultancy. Scholl:Alexion: Other: Travel support; Abbivie: Other: Travel support; Novartis: Other: Travel support; Deutsche Krebshilfe: Research Funding; Carreras Foundation: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; MDS: Other: Travel support; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees. Hochhaus:Incyte: Research Funding; Pfizer: Research Funding; Takeda: Research Funding; Bristol-Myers Squibb: Research Funding; Novartis: Research Funding. Brümmendorf:Takeda: Consultancy; Pfizer: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Novartis: Consultancy, Research Funding. Burchert:Novartis: Research Funding; Pfizer: Honoraria; Bristol Myers Squibb: Honoraria, Research Funding; AOP Orphan: Honoraria, Research Funding; Bayer: Research Funding. Krause:Novartis: Research Funding. Hänel:Amgen: Honoraria; Novartis: Honoraria; Roche: Honoraria; Takeda: Honoraria. Platzbecker:Celgene: Research Funding. Mayer:Johnson & Johnson: Research Funding; Roche: Research Funding; Eisai: Research Funding; Affimed: Research Funding; Novartis: Research Funding. Serve:Bayer: Research Funding. Ehninger:Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; Bayer: Research Funding; GEMoaB Monoclonals GmbH: Employment, Equity Ownership. Schetelig:Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria; Janssen: Consultancy, Honoraria; Roche: Honoraria; Sanofi: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding. Stoelzel:Neovii: Speakers Bureau.