Abstract: Abstract 3816 Background Conventional chromosome banding (CCB) analyses of bone marrow (bm) metaphases represent the gold standard of cytogenetic diagnostics in myelodysplastic syndromes (MDS), but they are not suitable for frequent follow-up analyses. Most aberrations can also be detected by fluorescence in situ hybridisation (FISH), and they are provable in CD34+ cells from peripheral blood (pb). In our prospective multicenter German diagnostic study “Screening and genetic monitoring of patients with MDS under different treatment modalities by cytogenetic analyses of circulating CD34+cells” (ClinicalTrails.gov NCT01355913) we followed MDS pts by sequential FISH analyses. Methods CD34+ pb cells were enriched by immunomagnetic cell sorting (MACS®) and analysed by FISH using a “Superpanel” (D7/CEP7, EGR1, CEP8, CEP XY, D20, TP53, IGH/BCL2, TEL/AML1, RB1, MLL, 1p36/1q25, CSF1R, all Abbott® Products) at initial screening, every 12 months during follow-up and in case of suspected disease progression and a “Standardpanel” (EGR1, D7/CEP7, CEP8, TP53, D20, TEL/AML1, CEP XY, plus -if necessary- another informative probe) every 2 months in the 1st and every 3 months in the 2nd and 3rd year. If bm aspirate was available, additional CCB and FISH analysis of CD34+ and native bm cells were performed. Cut-off values for each FISH probe were evaluated in our lab. Cytogenetics, bm morphology, clinical course and therapies were documented in a database. All pts gave their written informed consent. The study was approved by all local ethic committees. Results After 3 years of study time, 361 patients (25 AZALE (University of Dresden), 110 LEMON5 (University of Duesseldorf), 226 CD34+FISH) have been included in the study, resulting in a total number of 19,516 FISH analyses: Median age, gender distribution and MDS subtypes were typical for the disease, median follow-up at the time of analysis was 8.2 (1–36) months. Chromosomal aberrations could be detected by FISH of CD34+ pb cells in 71.5% of pts (55% of CD34+FISH-cohort, 99% of LEMON5-trial pts, 100% of AZALE-trial pts). FISH and CCB were highly correlated: p 〈 0.01 for CD34+ pb FISH vs CCB and p 〈 0.01 for CD34+ bm vs CCB. The clone sizes were significantly larger in CD34+ cells compared to native pb (p 〈 0.01). Discussion Our interim results demonstrate that FISH analysis of circulating CD34+ pb cells provides relevant cytogenetic informations. It is a reliable novel method for screening and cytogenetic monitoring of MDS pts during the course of disease and under different therapies, and helps in cases where a bm biopsy is not possible or not successful. Disclosures: Braulke: Celgene: This study was supported by Celgene. Other. Götze:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bug:Celgene: Honoraria, travel support, advisory board Other; Novartis: Honoraria, travel support, advisory board, travel support, advisory board Other; Boehringer Ingelheim: Honoraria, travel support, advisory board, travel support, advisory board Other. Schafhausen:Novartis: Honoraria, travel support Other; BMS: Honoraria, travel support, travel support Other; Roche: Honoraria, travel support, travel support Other; Celgene: Honoraria, travel support, travel support Other; Alexion: Honoraria, travel support Other. Haase:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract: Somatic mutations of the receptor tyrosine kinase Flt3 consisting of internal tandem duplications (ITD) occur in 20% of patients with acute myeloid leukemia. They are associated with a poor prognosis of the disease. In this study, we characterized the oncogenic potential and signaling properties of Flt3 mutations. We constructed chimeric molecules that consisted of the murine Flt3 backbone and a 510-base pair human Flt3 fragment, which contained either 4 different ITD mutants or the wild-type coding sequence. Flt3 isoforms containing ITD mutations (Flt3-ITD) induced factor-independent growth and resistance to radiation-induced apoptosis in 32D cells. Cells containing Flt3-ITD, but not those containing wild-type Flt3 (Flt3-WT), formed colonies in methylcellulose. Injection of 32D/Flt3-ITD induced rapid development of a leukemia-type disease in syngeneic mice. Flt3-ITD mutations exhibited constitutive autophosphorylation of the immature form of the Flt3 receptor. Analysis of the involved signal transduction pathways revealed that Flt3-ITD only slightly activated the MAP kinases Erk1 and 2 and the protein kinase B (Akt) in the absence of ligand and retained ligand-induced activation of these enzymes. However, Flt3-ITD led to strong factor-independent activation of STAT5. The relative importance of the STAT5 and Ras pathways for ITD-induced colony formation was assessed by transfection of dominant negative (dn) forms of these proteins: transfection of dnSTAT5 inhibited colony formation by 50%. Despite its weak constitutive activation by Flt3-ITD, dnRas also strongly inhibited Flt3-ITD–mediated colony formation. Taken together, Flt3-ITD mutations induce factor-independent growth and leukemogenesis of 32D cells that are mediated by the Ras and STAT5 pathways.

Abstract: Recombinant human granulocyte colony-stimulating factors (G-CSFs) are indicated for the prevention of chemotherapy-induced febrile neutropenia in patients receiving myelosuppressive chemotherapy. Filgrastim is a 175-amino-acid, short-acting, recombinant methionyl form of human G-CSF (r-metHuG-CSF) with a molecular weight of ∼19 kDa. The addition of polyethylene glycol (PEG) via PEGylation is a well-established technology that improves the physicochemical profile, enhances the solubility, and prolongs the half-life of therapeutic proteins. Pegfilgrastim, a PEGylated version of filgrastim, is currently the only available long-acting G-CSF, requiring fixed-dose subcutaneous (SC) administration only once per chemotherapy cycle. A new long-acting G-CSF, lipegfilgrastim, is under investigation for the prevention of chemotherapy-induced neutropenia. The rational design and development of lipegfilgrastim, and its novel manufacturing process, may provide a valuable fixed-dose, once-per-cycle, alternative long-acting G-CSF option. This analysis compares the design, protein chemistry, and physicochemical properties of lipegfilgrastim and pegfilgrastim, and evaluates the clinical implications of their differences. Methods The rational design of lipegfilgrastim focused on refining the PEGylation process. A novel technology platform was developed that enabled selective addition of PEG to a previously enzymatically attached glycan moiety instead of directly to the amino acid (as in standard PEGylation). Sequon scanning was used to scan the G-CSF protein to identify the best sites for glycosylation and glycoPEGylation that would have the least predicted effect on protein biologic activity. In recombinant G-CSF derived from Escherichia coli (E. coli), this was determined to be the single, natural, non-utilized O-glycosylation site. Addition of an O-glycan at this site was achieved by enzymatic activity of a selective, truncated N-acetylgalactosaminyltransferase isoform 2 fused with maltose-binding protein at the threonine residue within the chosen site. A 20-kDa PEG-sialic acid derivative was enzymatically transferred to the O-glycan with a sialyltransferase. Thus, the glycoPEGylation process modified only the O-glycosylation site of the G-CSF protein, resulting in a therapeutic product with an extended half-life. Results The resultant glycoPEGylation product, lipegfilgrastim, is a long-acting, site-specific glycoPEGylated r-metHuG-CSF (molecular weight ∼38 kDa) produced by conjugation of a single 20-kDa PEG to the natural O-glycosylation site of G-CSF expressed in E. coli. In comparison, pegfilgrastim is produced by conjugation of a 20-kDa PEG to the N-terminal of G-CSF expressed in E. coli, and PEGylation results in a heterogeneous product with multiple PEGylated isoforms requiring further purification during the manufacturing process. Potency and receptor-binding studies have shown no apparent difference between lipegfilgrastim and pegfilgrastim (Scheckermann C, 2013, EHA, Abstract P1024). However, glycoPEGylation appears to provide lipegfilgrastim with different pharmacokinetic and pharmacodynamic profiles. In phase I studies in healthy volunteers, lipegfilgrastim 6 mg SC had ∼64% greater cumulative exposure and ∼36% higher peak exposure compared with pegfilgrastim 6 mg SC (Kohler E, 2012, MASCC/ISOO, Abstract A-445-0013-00997). Lipegfilgrastim also had a longer half-life compared with that of pegfilgrastim (geometric means, 32.4 hours vs 27.2 hours, respectively). In a double-blind, randomized, phase III trial, lipegfilgrastim was shown to be noninferior to pegfilgrastim in the duration of severe neutropenia in patients with breast cancer (Udo M, 2012, EHA, Abstract 1375). Conclusions GlycoPEGylation is an advanced and efficient process that permits the manufacture of a novel, customized, homogenous therapeutic protein, lipegfilgrastim, for the prevention of chemotherapy-induced neutropenia. The manufacturing process is simple, controllable, and scalable. Lipegfilgrastim has an extended half-life, permitting once-per-cycle SC administration and improved bioavailability and exposure and appears to be an effective alternative long-acting G-CSF. Disclosures: Mahlert: Merckle Biotec GmbH (Teva Pharmaceuticals, Inc. Company): Employment. Schmidt:BioGeneriX GmbH (Teva Pharmaceuticals Inc. company): Employment. Allgaier:Merckle GmbH, Ulm, Germany (Teva Pharmaceuticals, Inc. company): Employment. Liu:Teva Pharmaceuticals, Inc: Employment. Müller:Teva Pharmaceuticals, Inc: Employment. Shen:Teva Pharmaceuticals, Inc: Employment.

Abstract: Arsenic trioxide (ATO) is regarded as the treatment of choice for relapsed PML-RARA+ acute promyelocytic leukemia (rAPL). In 2008, a European online registry of rAPL based on uniform CRFs was established under the auspices of the European LeukemiaNet (ELN) to gain insights in the clinical and biological characteristics of rAPL treated with ATO and to allow an assessment of the different options for postconsolidation therapy. Methods Eligibility criteria for prospective or retrospective registration were PML-RARA+ 1st or successive molecular or clinical relapse of APL from the year 2003 onwards. The treatment should be based on the European recommendations for treatment of rAPL (www.leukemia-net.org/content/), which offer induction and consolidation therapy with ATO and several options for post-consolidation therapy including autologous or allogeneic stem cell transplantation or chemotherapy consolidation followed by various modifications of maintenance therapy ± ATO - to be selected depending on several variables including patient age, performance status, PCR status after consolidation, type of frontline therapy, first CR duration and donor availability. Results By 30 June 2013, of 220 registered cases, 198 were evaluable (172 in 1st, 26 in ≥2nd relapse). Of these, 149 patients (pts) in 1st relapse received ATO-based salvage therapy after standard frontline therapy based on all-trans retinoic acid (ATRA) and anthracyclines (98 hematological (hematol) relapses of bone marrow combined with CNS relapse in 5 pts, 40 molecular (mol), 11 isolated extramedullary, mainly CNS). Clinical characteristics: median age at relapse 44 years (y) (4 to 81), 67% males, Sanz Risk Score at 1st diagnosis: low 23%, intermediate 48% and high 29% of pts. Median duration of first remission was 565 d (105 d to 7.0 y). The median treatment duration of ATO (0,15 mg/kg/day) plus ATRA (44% of pts) for remission induction (ind) was 30 days (d) and for consolidation (cons) 25 d. CNS relapses received ATO and additional intrathecal chemotherapy ± irradiation. WBC white blood cell count; Leuko.: leukocytosis requiring hydroxyurea; ADS: APL differentiation syndrome; 1 median; 2 hematological; 3 RT-PCR of PML-RARA negative. In non-hematological relapses, no early deaths occurred and no major side effects of ATO were seen. Median follow up of the 149 pts was 2.8 y (6 d to 10 y). Three-year overall survival (OS) was 70%, 95%CI [61;79] and 6-year OS 56% [42;70] . Three-year OS of hematological, molecular and extramedullary relapse was 69% [58;80], 66% [48;84] and 90% [81;99], respectively (p=0.2). Concerning outcome after transplantation, 3-year OS after autologou s was 82% [70;94] (n=55), after allogeneic 75% [58;92] (n=32) and without transplantation 66% [48;84] (n=55) (p=0.4). Conclusions With ATO-based salvage therapy over 50% of patients in 1st relapse of APL can probably be cured. Pts in molecular relapse have a lower rate of early complications and death. Long-term survival is possible with transplantation-free approaches, but transplantation seems to improve the outcome. Disclosures: Lengfelder: TEVA/ Cephalon: Research Funding. Lo-Coco:TEVA: Speakers Bureau.

Abstract: Introduction: Advanced follicular lymphoma (FL) is a clinically and molecularly heterogeneous disease. About 20% of patients have early progression of disease (POD) and short overall survival (OS). We have previously shown that integration of lymphoma-specific gene mutations and clinical factors improves pretreatment risk stratification (Pastore, 2015) and prediction of early POD (i.e., within 24 months, POD24; Jurinovic, 2016). Recently, we have shown that high-dose therapy (HDT) followed by autologous stem-cell transplantation (ASCT) is an effective treatment option for eligible patients with high-risk disease as defined by POD24 (Jurinovic, 2018). Here, we aimed to explore whether HDT/ASCT is an effective frontline therapy for patients identified to be high-risk by clinical (i.e., FLIPI) or clinicogenetic risk models (i.e., m7-FLIPI, POD24-PI). Methods: We performed targeted DNA deep-sequencing of 〉 150 genes in available diagnostic FL biopsies from 165 patients ≤60 years with advanced FL from the GLSG2000 trial who uniformly received R-CHOP as frontline treatment. Of these, 87 patients (53%) were randomized to receive consolidative HDT/ASCT, 78 (47%) were randomized to interferon maintenance. We performed intention-to-treat (ITT) survival and regression analyses to explore whether known clinical and clinicogenetic risk factors can be overcome by ASCT. Results: The HDT/ASCT and no-HDT/ASCT cohorts were balanced regarding age (48 vs 50 years), sex (49% vs 64% male patients), high-risk FL International Prognostic Index (FLIPI; 25% vs 29%), Eastern Cooperative Oncology Group Performance Score 〉 1 (6% vs 5%) and mutation status of EZH2 (23% vs 18%) and TP53 (3% vs 3%). The incidence of POD24 was not significantly lower in the HDT/ASCT cohort (8% vs 14%, p=0.32). After a median follow-up of 7.5 years, 5-year failure-free survival (FFS) rates in the HDT/ASCT and no-HDT/ASCT cohorts were 77% and 69% (HR 0.7, p=0.16), 5-year OS rates were 95% and 90% (HR 0.6, p=0.21), respectively. The high-risk cohorts identified by FLIPI, m7-FLIPI, and POD24-PI comprised 27% (n=45), 18% (n=29) and 22% (n=37) of patients, respectively (Fig. A). The m7-FLIPI reclassified 10% (n=16) of patients from high-risk FLIPI to low-risk m7-FLIPI. The POD24-PI reclassified 5% (n=9) of patients from high-risk FLIPI to low-risk POD24-PI; one patient was reclassified from low-risk FLIPI to high-risk POD24-PI. Patients identified to be high-risk by all three indices had shorter FFS (FLIPI: HR 2.8, p=0.0002; m7-FLIPI: HR 3.0, p=0.0003; POD24-PI: HR 2.5, p=0.0013), but OS was not different (FLIPI: HR 1.4, p=0.47; m7-FLIPI: HR 1.5, p=0.45; POD24-PI: HR 1.5, p=0.47). The risk to develop POD24 was increased in high-risk patients (FLIPI: OR 4.4, p=0.007; m7-FLIPI: OR 4.8, p=0.005; POD24-PI: OR 4.3, p=0.008). Consolidative HDT/ASCT did not prolong FFS in high-risk patients as defined by FLIPI (HR 1.2, p=0.67), m7-FLIPI (HR 1.2, p=0.70; Fig. B) and POD24-PI (HR 1.3, p=0.63; Fig. B). Similarly, OS was not significantly improved in all three high-risk cohorts (FLIPI: HR 0.2, p=0.13; m7-FLIPI: HR n/a, p 〉 0.99; and POD24-PI: HR 0.3, p=0.22). In low-risk patients, HDT/ASCT was associated with a non-significant trend towards prolonged FFS (FLIPI: HR 0.5, p=0.061; m7-FLIPI: HR 0.6, p=0.16; POD24-PI: HR 0.5, p=0.068; Fig. B), but again OS was not significantly different (FLIPI: HR 0.8, p=0.69; m7-FLIPI: HR 0.8, p=0.66; and POD24-PI: HR 0.7, p=0.52). Conclusions: Our ITT-analysis confirms that consolidative HDT/ASCT should not be offered to unselected cohorts of patients with previously untreated, advanced FL after R-CHOP. Also, our current clinicogenetic risk models are not optimized to select high-risk patients who may benefit from frontline HDT/ASCT. The fraction of patients identified to be high-risk by FLIPI, m7-FLIPI and POD24-PI is low when applied to younger, medically fit patients. Moreover, the fraction of patients being reclassified by integrating gene mutation data is low in this patient cohort. Therefore, we are developing specific stratification algorithms for younger, medically fit patients who are eligible for dose-intensified approaches. Figure. Figure. Disclosures Klapper: F.Hoffman-La Roche: Honoraria, Research Funding; HTG Molecular Diagnostics, Inc.: Research Funding; Regeneron: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Takeda: Honoraria, Research Funding. Schmitz:Riemser: Honoraria, Other: Travel grants; Kite/Gilead: Honoraria, Other: Travel grants; Novartis: Honoraria, Other: Travel grants; Celgene: Other: Travel grants; Roche: Honoraria. Hess:CTI: Research Funding; Celgene: Consultancy, Honoraria, Other: travel expenses, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Unterhalt:F. Hoffman-La Roche: Other: Travel support. Dreyling:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sandoz: Membership on an entity's Board of Directors or advisory committees. Schmidt:Gilead: Honoraria, Other: Travel Grants; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Grants; Celgene: Honoraria. Hoster:F. Hoffman-La Roche: Other: Travel support, Research Funding; Roche Pharma AG: Other: Travel support, Research Funding. Hiddemann:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding. Weigert:Roche: Research Funding; Novartis: Research Funding.

Abstract: Largest prospective trial for adult Burkitt lymphoma/leukemia patients. Substantial cure rates and high treatment-realization rates in all age groups.

Abstract: Somatic mutations of the receptor tyrosine kinase Flt3 consisting of internal tandem duplications (ITD) occur in 20% of patients with acute myeloid leukemia. They are associated with a poor prognosis of the disease. In this study, we characterized the oncogenic potential and signaling properties of Flt3 mutations. We constructed chimeric molecules that consisted of the murine Flt3 backbone and a 510-base pair human Flt3 fragment, which contained either 4 different ITD mutants or the wild-type coding sequence. Flt3 isoforms containing ITD mutations (Flt3-ITD) induced factor-independent growth and resistance to radiation-induced apoptosis in 32D cells. Cells containing Flt3-ITD, but not those containing wild-type Flt3 (Flt3-WT), formed colonies in methylcellulose. Injection of 32D/Flt3-ITD induced rapid development of a leukemia-type disease in syngeneic mice. Flt3-ITD mutations exhibited constitutive autophosphorylation of the immature form of the Flt3 receptor. Analysis of the involved signal transduction pathways revealed that Flt3-ITD only slightly activated the MAP kinases Erk1 and 2 and the protein kinase B (Akt) in the absence of ligand and retained ligand-induced activation of these enzymes. However, Flt3-ITD led to strong factor-independent activation of STAT5. The relative importance of the STAT5 and Ras pathways for ITD-induced colony formation was assessed by transfection of dominant negative (dn) forms of these proteins: transfection of dnSTAT5 inhibited colony formation by 50%. Despite its weak constitutive activation by Flt3-ITD, dnRas also strongly inhibited Flt3-ITD–mediated colony formation. Taken together, Flt3-ITD mutations induce factor-independent growth and leukemogenesis of 32D cells that are mediated by the Ras and STAT5 pathways.

Abstract: There is a strong negative association between comorbidities at diagnosis and overall survival. There is no negative effect of comorbidities on remission rates and progression to advanced phases in CML.

Abstract: The Bcr-Abl oncogene is present in 30-40% of adult patients with acute lymphoblastic leukemia (ALL). Therapy with imatinib has become standard for Ph+ ALL but resistance to the tyrosine kinase inhibitor occurs for the majority of patients. In about 80% of patients with acquired resistance mutations in the tyrosine kinase domain (TKD) have been found. In contrast, primary resistance to imatinib appears to be multifactorial and precise mechanisms have been incompletely elucidated. We have established an imatinib-resistant cell line (SupB15RT) which was derived from the previously well characterized SupB15 cell line (SupB15WT) by gradually increasing the exposure to imatinib. We found that several commonly implicated mechanisms of imatinib resistance, i.e. Bcr-Abl gene amplification, point mutations in the TKD, Bcr-Abl overexpression, up-regulation of multidrug resistance gene proteins or ineffective inhibition of Bcr-Abl tyrosine phosphorylation do not play a role in conferring the imatinib-resistant phenotype in SupB15RT cells. Thus, the SupB15RT cells represent a suitable model for the analysis of resistance mechanisms in Ph+ ALL with primary imatinib resistance. Interestingly, SupB15RT cells show cross-resistance to the second generation Abl kinase inhibitors Nilotinib and Dasatinib. Analysis of signal transduction pathways downstream of Bcr-Abl revealed that imatinib exposure was not associated with down-regulation of pSTAT-5 and pErk in the imatinib-resistant SupB15RT cells, in contrast to SupB15WT. Phosphorylation of Akt was inhibited by 0.5μM imatinib in SupB15WT cells, whereas imatinib in concentrations up to 5μM failed to suppress Akt phosphorylation in SupB15RT cells, indicating constitutive activation of Akt kinase during imatinib treatment. By comparative gene expression analysis of SupB15WT vs. SupB15RT cells using Affimetrix-Microarrays, we identified 29 differentially regulated (at least 3-fold) genes. One of the most highly up-regulated genes in imatinib-resistant SupB15RT cells was Autotaxin (ATX), a nucleotide pyrophosphatase/ phosphodiesterase 2. This exo-enzyme was originally identified as a tumor cell autocrine motility factor, which is involved in tumor progression and migration in various tumor cell types. ATX is a lysophospholipase D which is involved in the synthesis of lysophosphatidic acid (LPA), a signaling molecule that promotes survival, growth, differentiation, and motility. We investigated if LPA imparted imatinib resistance in SupB15WT cells by modulation of growth, survival and migration. When SupB15WT cells were treated with LPA, alone or in combination with imatinib, SupB15WT cell proliferation was increased both in the absence as well as in the presence of imatinib. The dose-dependent increase of proliferation after LPA treatment was 1.9–2.6-fold (1–10μM LPA) in the presence of 1μM imatinib. In addition we performed migration experiments using Transwell assays. We detected a 3-fold increase in migration of SupB15RT vs. SupB15WT cells. We found no influence on apoptosis in imatinib treated SupB15WT cells treated with LPA compared with cells not treated with LPA. Taken together, our results indicate a role of ATX in imatinib-resistant SupB15RT cells, preferentially by stimulating proliferation and migration through LPA signaling via LPA receptors and activation of PI3K and Akt.