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Introduction: Multiple myeloma (MM) is a B-cell malignancy characterized by an abnormal proliferation and infiltration of malignant plasma cells in the bone marrow (BM). Mesenchymal stromal cells (MSCs) represent a crucial component of the BM niche and mediate essential signalling via cytokines and cell-cell interactions. The interplay of MM cells and BM-MSC is complex and relies on multiple signaling pathways leading to MM progression and therapeutic resistance. Objectives: MM remains an incurable disease so far. Distinctive for this disease is a long-lasting polarization of the BM niche influencing MM progression and prognosis. We, therefore, focussed on MSCs to identify enrichment for different hallmark gene sets and their aberrant signaling contributing to the pathogenesis of the disease, therapy response and to further identify novel therapeutic strategies. Methods: BM-MSCs were isolated from patients with MM at diagnosis (MM-D-MSC) and in remission (MM-R-MSC) as well as from donors with other malignant diseases (CTR-MSC). RNA sequencing and Western Blot were used for examination of enriched pathways. Various functional assays for proliferation, apoptosis and cell cycle were performed either using a mono-culture or co-culture protocol of MSC and the MM-cell lines MM.1S and SKMM2 treating the cells with the pan-PI3K-inhibitor GDC-0941. Results: MM-D-MSCs supported the growth of myeloma cell lines better (3 fold, p We confirmed these findings on a proteomic level. We found evidence for the upregulation of PI3Kα, AKT, pAKT and mTOR in MM-D-MSC comparing to the other MM-R- and CTR-MSCs (p As stated MM-D-MSC supported the growth of myeloma cells better than other MSC types. However, upon GDC-0941 treatment, the proliferation of MM-D-MSCs was significantly reduced compared to the other MSC-types. In addition, the inhibition of proliferation of myeloma cell lines MM1S and SKMM2 was much more pronounced when they were cocultured with MM-D-MSC (32 and 34 %, p=0.04) compared to the growth of myeloma cells in coculture with MSC types, either in remission or other malignancies. Conclusion: We here identified functionally distinct differences in MM-D-MSCs compared to MM-R-MSCs or CTR-MSCs. Our data further provides a deeper insight into the molecular signature of MM-MSCs, a predictive of patient prognosis and treatment outcome. Targeting MSCs as a crucial part of the MM-BM niche by using PI3K-inhibitors could contribute to novel therapeutic strategies to effectively block MM-MSC interaction improving overall patient survival. Disclosures Raab: Roche: Consultancy; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Khandanpour: BMS/Celgene: Honoraria; Sanofi: Honoraria, Research Funding; Pfizer: Honoraria; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria; GSK: Honoraria.

Tumor heterogeneity plays a significant role in the development of therapy resistance in multiple myeloma (MM). Focal lesions (FLs), which are nodular accumulations of MM cells, have been shown to be hotspots of genetic spatial tumor heterogeneity, which is characterized by unique tumor sub-clones at different sites in the bone marrow (BM). However, little is known about the mechanisms leading to mutations in FLs, the architecture of the tumor microenvironment (ME) at these sites, and the link between FL sub-clones and relapse. We applied whole genome sequencing (WGS) to CD138 + MM cells from paired FL and iliac crest random BM aspirates (RBMA) of 15 newly diagnosed MM (NDMM) patients. For 7 of these patients, single cell (sc) analyses were performed, including sc gene expression (scRNA) and T-cell receptor (TCR)-sequencing and sc assay for transposase-accessible chromatin (ATAC)-sequencing for paired BM CD138 + MM and CD138 - ME, as well as peripheral blood mononuclear cells (PBMC). WGS data was analyzed using inhouse pipelines. Mutations, copy-number-variations and mutational signatures were called using mpileup, ACESeq and mmsig. Neoantigen epitopes were predicted using NeoPredPipe. Sc data was generated using the 10X Genomics platform. Pre-processing and analysis of the sc data was performed with CellRanger and the R-packages Seurat, ArchR and inferCNV. In 13/15 patients we found significant differences in chromosomal and mutational profiles between FLs and paired RBMAs, with major unshared mutations (mutation seen in > 60% cells) being enriched at the FL site (mean 310 vs. 123, p On average, 23 (range 0-83) site-unique baseline mutations were predicted to be neoantigens. Thus, we hypothesized that spatial tumor heterogeneity could be associated with heterogeneity in the tumor ME. We did not observe expansion of site-unique T cell clones, but some of the clones were enriched up to 10-fold at one of the two sites. These clones were typically seen in the PB at low frequency. Expanded T-cells clones were almost exclusively found in the CD8 +-compartment, with 65% and 27% of expanded T-cell clones being CD45RO +/CD57 +-memory- and CD69 +-effector-T-cells, respectively. Besides differences in the T-cell clonality, we observed changes in proportions of other cell types, including a depletion of CD14+- and CD16+-macrophages in FLs (p In conclusion, our results strengthen the concept of MM as a spatially heterogeneous disease, suggest that reactive oxygen species result in site-specific mutagenesis, and support the hypothesis that FLs are the origin of aggressive disease. We demonstrate spatial heterogeneity at single-cell level in the BM immune ME for the first time, which implies that understanding the complex biology of FLs could be important in the context of novel immune therapies such as bispecific antibodies and CAR-T-cells. Disclosures John: Janssen: Consultancy. Müller-Tidow: Janssen Cilag: Consultancy, Research Funding; Bioline: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Goldschmidt: Takeda: Consultancy, Research Funding; Sanofi: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Dietmar-Hopp-Foundation: Other: Grant; Novartis: Honoraria, Research Funding; Mundipharma: Research Funding; MSD: Research Funding; Molecular Partners: Research Funding; Johns Hopkins University: Other: Grant; Janssen: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Incyte: Research Funding; GSK: Honoraria; Chugai: Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; BMS: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Celgene: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Adaptive Biotechnology: Consultancy; Amgen: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding. Raab: Janssen: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees. Weinhold: Sanofi: Honoraria.

Introduction: Treatments incorporating autologous transplantation (ASCT), proteasome inhibitors (PIs), and immunomodulatory drugs (IMIDs) induce deep and durable remissions in most multiple myeloma (MM) patients, resulting in prolonged survival. Yet, patients who suffer from early relapse within 2 years of treatment initiation or become refractory to PIs and IMIDs still have a dismal outcome. The mutational landscape in early relapsed MM (ERMM) and relapsed/refractory MM (RRMM) has been comprehensively described. Several aberrations are associated with these two types of high-risk disease but little is known about the biological difference between them. To this end, we have comparatively and sequentially analyzed whole genome and RNA sequencing data from ERMM and RRMM patients. Methods: We included 32 patients who had relapsed within 2 years of first-line therapy (ERMM group, 30 after ASCT). Samples were collected at first relapse. Paired baseline samples were available from 17 patients. For the RRMM group, we included 43 patients with a median of 5 prior lines of therapy (range 2 - 13; 88% with ASCT), who had relapsed after PIs and IMiDs. For 22 of them consecutive tumor samples were available. Sequencing data were pre-processed using in-house pipelines. Mutations, indels, translocations, and copy number variants were called using Platypus, SOPHIA and ACESeq. Mutational signatures were identified with MMSig and subclonal structures with SciClone. Differential gene expression was assessed using DESeq, gene set enrichment analysis was performed with hypeR, and gene fusions were detected with Arriba. Results: Nonsynonymous mutations occurred more frequently in RRMM (median=180) compared to ERMM at first relapse (median=62, p Conclusions: According to our results ERMM and RRMM are biologically distinct entities of MM. While ERMM is characterized by inactivation of tumor suppressors and upregulation of gene sets associated with hypoxia and glycolysis, RRMM shows mutations in multiple gene networks, upregulation of ribosomal protein pseudogenes with unknown function and a signature linked to defective DNA repair, suggesting multifactorial mechanisms that lead from first relapse to end-stage relapsed refractory disease. Comparing paired samples, we did not observe major difference in evolution patterns between ERMM and RRMM. Yet, the low prevalence of the melphalan MM1 signature in ERMM suggests selection of pre-existing clones in this entity. In contrast, single tumor cells exposed to melphalan are often the precursors of clones dominating at the RRMM stage, indicating that first-line ASCT has a long-term effect on MM evolution. Disclosures John: Proteona: Research Funding. Mueller-Tidow:Janssen-Cilag Gmbh: Membership on an entity's Board of Directors or advisory committees; Deutsche Forschungsgemeinschaft: Research Funding; Deutsche Krebshilfe: Research Funding; Daiichi Sankyo: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; BiolineRx: Research Funding; Bayer AG: Research Funding; Jose-Carreras-Siftung: Research Funding; Wilhelm-Sander-Stiftung: Research Funding; BMBF: Research Funding. Goldschmidt:Johns Hopkins University: Other: Grants and/or provision of Investigational Medicinal Product; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Incyte: Research Funding; Molecular Partners: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Novartis: Honoraria, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma GmbH: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; University Hospital Heidelberg, Internal Medicine V and National Center for Tumor Diseases (NCT), Heidelberg, Germany: Current Employment; GlaxoSmithKline (GSK): Honoraria; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product, Research Funding; Chugai: Honoraria, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Dietmar-Hopp-Foundation: Other: Grants and/or provision of Investigational Medicinal Product:; Merck Sharp and Dohme (MSD): Research Funding. Raab:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Heidelberg Pharma: Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Introduction: Multiple myeloma (MM) is a heterogeneous malignancy of clonal plasma cells that accumulate in the bone marrow (BM). Despite new treatment approaches, in most patients resistant subclones are selected by therapy, resulting in the development of refractory disease. While the subclonal architecture in newly diagnosed patients has been investigated in great detail, intra-tumor heterogeneity in relapsed/refractory (RR) MM is poorly characterized. Recent technological and computational advances provide the opportunity to systematically analyze tumor samples at single-cell (sc) level with high accuracy and througput. Here, we present a pilot study for an integrative analysis of sc Assay for Transposase-Accessible Chromatin with high-throughput sequencing (scATAC-seq) and scRNA-seq with the aim to comprehensively study the regulatory landscape, gene expression, and evolution of individual subclones in RRMM patients. Methods: We have included 20 RRMM patients with longitudinally collected paired BM samples. scATAC- and scRNA-seq data were generated using the 10X Genomics platform. Pre-processing of the sc-seq data was performed with the CellRanger software (reference genome GRCh38). For downstream analyses the R-packages Seurat and Signac (Satija Lab) as well as Cicero (Trapnell Lab) were used. For all patients bulk whole genome sequencing (WGS) data was available, which we used for confirmatory studies of intra-tumor heterogeneity. Results: A comprehensive study at the sc level requires extensive quality controls (QC). All scATAC-seq files passed the QC, including the detected number of cells, number of fragments in peaks or the ratio of mononucleosomal to nucleosome-free fragments. Yet, unsupervised clustering of the differentially accessible regions resulted in two main clusters, strongly associated with sample processing time. Delay of sample processing by 1-2 days, e.g. due to shipment from participating centers, resulted in global change of chromatin accessibility with more than 10,000 regions showing differences compared to directly processed samples. The corresponding scRNA-seq files also consistently failed QC, including detectable genes per cell and the percentage of mitochondrial RNA. We excluded these samples from the study. Analysing scATAC-seq data, we observed distinct clusters before and after treatment of RRMM, indicating clonal adaptation or selection in all samples. Treatment with carfilzomib resulted in highly increased co-accessibility and >100 genes were differentially accessible upon treatment. These genes are related to the activation of immune cells (including T-, and B-cells), cell-cell adhesion, apoptosis and signaling pathways (e.g. NFκB) and include several chaperone proteins (e.g. HSPH1) which were upregulated in the scRNA-seq data upon proteasome inhibition. The power of our comprehensive approach for detection of individual subclones and their evolution is exemplarily illustrated in a patient who was treated with a MEK inhibitor and achieved complete remission. This patient showed two main clusters in the scATAC-seq data before treatment, suggesting presence of two subclones. Using copy number profiles based on WGS and scRNA-seq data and performing a trajectory analysis based on scATAC-seq data, we could confirm two different subclones. At relapse, a seemingly independent dominant clone emerged. Upon comprehensive integration of the datasets, one of the initial subclones could be identified as the precursor of this dominant clone. We observed increased accessibility for 108 regions (e.g. JUND, HSPA5, EGR1, FOSB, ETS1, FOXP2) upon MEK inhibition. The most significant differentially accessible region in this clone and its precursor included the gene coding for krüppel-like factor 2 (KLF2). scRNA-seq data showed overexpression of KLF2 in the MEK-inhibitor resistant clone, confirming KLF2 scATAC-seq data. KLF2 has been reported to play an essential role together with KDM3A and IRF1 for MM cell survival and adhesion to stromal cells in the BM. Conclusions: Our data strongly suggest to use only immediately processed samples for single cell technologies. Integrating scATAC- and scRNA-seq together with bulk WGS data showed that detection of individual clones and longitudinal changes in the activity of cis-regulatory regions and gene expression is feasible and informative in RRMM. Disclosures Goldschmidt: John-Hopkins University: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; John-Hopkins University: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Research Funding; Molecular Partners: Research Funding; Dietmar-Hopp-Stiftung: Research Funding; Janssen: Consultancy, Research Funding; Chugai: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees.

Introduction: Despite significant improvements in therapy during the last decade, most multiple myeloma (MM) patients develop refractory disease over time. Treatment of refractory MM is a major challenge, likely due to the still poorly characterized inter- and intratumor heterogeneity at this stage of the disease, and the complex interplay of MM cells with the microenvironment (ME). In particular, there is an urgent need to unravel how these features of MM are linked to molecular mechanisms of drug resistance. Methods: We resolved the cellular composition, underlying transcriptional inter- and intra-patient heterogeneity and molecular treatment response of relapsed/refractory MM by single cell RNA sequencing (scRNA-seq). Using droplet-based microfluidics, ~230,000 single cell gene expression profiles from bone marrow (BM) aspirates of 21 patients sorted into CD138+and CD138- fractions were acquired, allowing for a comprehensive analysis of both MM cells as well as their ME. Patients had a median of 4 prior lines of therapy including both a proteasome inhibitor and an immunomodulator and were refractory to their immediate prior line of therapy at time of sampling. In addition, paired samples before either pomalidomide- or carfilzomib-based therapies were analyzed for 16/21 patients. Genomic aberrations in individual patients were mapped by interphase fluorescence in situ hybridization. Cells were clustered and CD138+ MM subtypes as well as immune cell-types of the ME were identified from their single cell transcriptomes and a copy number variation (CNV) analysis. As a reference for non-malignant cells and to construct a developmental B-cell trajectory the Human Cell Atlas BM scRNA-seq reference dataset was used. To characterize interactions of MM cells with their ME, the correlated expression of ligand-receptor pairs was exploited. Results: The analysis of inter- and intra-tumor heterogeneity of molecular MM subgroups revealed distinct transcriptome signatures with contributions that could be assigned to differences in heavy and light chain immunoglobulin expression as well as known genomic alterations, including t(11;14), t(4;14) and hyperdiploidy. MM cells from individual patients largely maintained a plasma cell specific gene expression profile but a partial loss of plasma cell identity was detected based on mapping to a developmental B-cell trajectory. It was characterized by the upregulation of subgroup transcriptome signatures associated with earlier stages of B-cell development in almost 50% of patients, such as a pre-B or mature B cell-like phenotype. Within individual samples, subclonal MM cell populations with specific gene expression programs were resolved based on the CNV analysis and included those characterized by expression of the immune-activator CD27 and the modulator of WNT signaling FRZB. The analysis of longitudinally collected samples revealed both changes in the cell subtype cluster structure as well as drug-specific adaptation of gene expression programs in distinct subpopulations persisting or emerging at relapse. These profile changes were characterized by e.g. downregulation of Myc target genes upon pomalidomide treatment or induction of heat shock proteins under carfilzomib. Within the ME of refractory MM patients, we observed that the fraction of B cells and CD4+ T cells was strongly reduced while CD14+ and CD16+ monocytes as well as dendritic cells expanded. Notably, the immune checkpoint protein PD-1H (aka VISTA) that inhibits T cell activation was highly expressed in cell types from the myeloid compartment in contrast to healthy donors. Further, a ligand-receptor analysis revealed that MM cells displayed the strongest interactions with monocytes, which were mediated by MIF, BAFF and other cytokines. Conclusions: Our study demonstrates the value of scRNA-seq analysis for identifying crucial transcriptome features that classify refractory MM subtypes and their evolution in response to treatment including regulation of drug resistance associated signaling pathways. Our data suggest that refractory MM cells shape the myeloid compartment in the BM to generate an immune suppressive ME. Understanding the evolution of MM cell heterogeneity and the bone marrow milieu in refractory disease will lead to novel treatment approaches and eventually improve patient outcome. Disclosures Müller-Tidow: MSD: Membership on an entity's Board of Directors or advisory committees. Goldschmidt:John-Hopkins University: Research Funding; Molecular Partners: Research Funding; Amgen: Consultancy, Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Dietmar-Hopp-Stiftung: Research Funding; Janssen: Consultancy, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; John-Hopkins University: Research Funding; Chugai: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Research Funding.

Abstract 667 Aim. The aim of this study was to prove in a large prospective multicenter trial the tolerance and efficacy of short-intensive chemotherapy combined with the antibody Rituximab directed against CD20 for patients with Burkitt Non-Hodgkin lymphoma (B-NHL) and Burkitt leukemia (B-L). Background. In adult Burkitt lymphoma/leukemia with short-intensive chemotherapy regimen - mostly derived from pediatric protocols - a complete remission (CR) rate of 83% and an overall survival (OS) of 62% (both weighted mean) could be achieved. Further intensification of chemotherapy apparently did not improve the overall outcome. This was the rationale to integrate the monoclonal anti-CD20 antibody Rituximab in B-NHL / leukemia patients with a CD20 expression of >90%. Patients and Methods. 363 adult patients (229 B-NHL and 134 B-L), 15 years or older (without age limit) were recruited from 98 centers in the German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia (GMALL) B-ALL/NHL 2002 protocol, initiated in 8/2002 until 06/2011. Median age of the Burkitt lymphoma cohort was 40 years (16–79) and for the Burkitt leukemia cohort 47 years (16–85). CNS involvement was observed in 6% / 18%. In the Burkitt lymphoma cohort, 6% had mediastinal tumor, 53% had stage III/IV and IPI>2 of 35%. Treatment. The treatment consisted of 6 five-day chemotherapy cycles with high-dose methotrexate (HD-MTX) 1500 mg/m2 (total 6 doses), high-dose cytosine arabinoside (HD-AraC) 2000 mg/m2 (total 4 doses), cyclophosphamide, etoposide, ifosphamide and corticosteroids, and a triple intrathecal therapy (MTX, AraC, Dexa). Elderly patients >55 years received reduced drug doses (500 mg/m2), particularly no C-cycles with HD-AraC among other drugs. Rituximab was given d ⦵1 before each cycle and twice at 4 week intervals thereafter, for overall 8 doses. Total treatment duration was 28 weeks (figure 1). Results. CR rate in B-NHL patients was 91% (182/229) and 86% (162/182) in B-L patients. For the B-NHL cohort the results were excellent with an OS of 88%, and a progression-free survival (PFS) of 83% at >7 yrs, with no significant difference in OS for adolescents 15-≤25 yrs with 91%, adults 26-'55 yrs with 91% or elderly >55 yrs with 80%. In Burkitt leukemia the OS for adolescents was also very promising with 90%, for adults OS was 71%, but inferior for elderly patients with 46%. Therefore two cycles C, including high dose AraC, were added for older patients in an amendment. Prognostic factors. In B-NHL patients the age adapted International Prognostic Index (aIPI) was the only significant prognostic factor for OS (p = 0.02) whereas in B-L patients the factors age 15-≤25, 26-≤55 and >55 yrs (p = 0.0007) and a lower platelet count Conclusion. In the largest prospective trial for adult Burkitt NHL/leukemia, overall survival and progression-free survival could be substantially improved by a combination of short-intensive chemotherapy with Rituximab with manageable toxicity. Even with lower doses of HD-MTX outcome of B-NHL was excellent in all age groups, including elderly. Disclosures: No relevant conflicts of interest to declare.

Abstract 494 Several randomised pediatric trials have demonstrated that intensification of Asparaginase (ASP) treatment in ALL can contribute to improved outcome. In adult ALL few data are availabe and optimal ASP preparation, schedule and intensity with respect to efficacy and tolerability have to be defined. The optimisation of ASP treatment is therefore an essential aim of the GMALL. Treatment: Induction treatment of the ongoing study 07/2003 consists of dexamethasone, vincristine, daunorubicine, pegylated asparaginase (PEG-ASP) (phase I), mercaptopurine, cyclophosphamide and cytarabine (phase II) as previously described (Brueggemann et al, Blood 2006: 107; 1116). During the study the dose for PEG-ASP was increased from 1000 to 2000 U/m2 in induction and from 500 to 2000 U/m2 in consolidation (combined with HDMTX and MP) for pts aged between 15 and 55 years. 1 application for high risk and 7 applications for standard risk (SR) were scheduled during the first year and the aim was improvement of overall survival (OS) and remission duration (RD). Patients: From more than 100 centers in Germany 1226 pts with a median age of 35 (15-55) yrs were evaluable. 826 pts were treated with 1000 U/m2 (cohort 1) and 400 pts with 2000 U/m2 (cohort 2) and both groups were comparable regarding major entry criteria. The analysis was restricted to pts who received one of the scheduled PEG-ASP doses during induction. Outcome: CR rate after induction was 91% vs 91% in cohort 1 and 2 resp., with comparable rates for early death (4% vs 5%) and failure (5% vs 4%). Data on molecular response (MRD below 10−4) after induction are available in a subset and showed no difference between both cohorts after induction (79% vs 82%). OS after 3 years was improved in cohort 2 (60% vs 67%; p>.05). The positive effect was specifically evident in SR patients (N=407 vs 190) with respect to OS (68% vs 80%; p=.02) and RD (61% vs 74%; p=.02). It was demonstrated in younger pts (15-45 yrs) (71% vs 82%; p=.02) and older pt (45-55 yrs) (56% vs 74%; p>.05). Excellent results were achieved in young adults (15-25 years) with respect to OS (77% vs 86%; p>.05) and RD (60% vs 78%; p>.05). Toxicity: The analysis of toxicity was focused on grade III-IV events during induction with potential correlation to PEG-ASP (764/382 pts in cohort 1/cohort 2)). Incidences are as follows: GOT or GPT (30%/30%), bilirubine (10%/16%), thrombosis (5%/5%) and hypersensitivity ( Bilirubine °III/IV occurred median 16d after PEG-ASP during phase II of induction. In univariate analysis it was correlated to dose (10% vs 16%; p=.004), age <> 45 yrs (11% vs 17%; p=.005), BMI <> 30 (12% vs 18%; p=.04) and rituximab application (11% vs 18%; p=.009). Hepatomegaly, infections or imatinib application had no significant effect. In multivariate analysis dose and age remained independent significant prognostic factors. Bilirubine increase during induction was associated with treatment delays and inferior prognosis. Conclusions: This is the largest cohort of adult ALL treated with PEG-ASP. Due to prolonged activity fewer applications are required which is a pre-requisite for realisation of ASP intensification in the context of an intensive multidrug chemotherapy for adult ALL. Although CR rate and molecular CR were not significantly improved PEG-ASP intensification was associated with an improved OS and RD. The improvement was specifically evident in SR pts treated with up to 7 doses of PEG-ASP. Overall intensified PEG-ASP was feasible. The rate of grade III-IV bilirubine elevation increased after dose escalation and led to treatment delays in individual pts which were prognostically relevant. It would be an important goal to identify parameters to predict severe ASP related toxicity. Further intensification of ASP by additional applications would be of interest. Supported by Deutsche Krebshilfe 70–2657-Ho2 and partly BMBF 01GI 9971 and Medac GmbH. Disclosures: Goekbuget: Medac: Consultancy, Research Funding, Speakers Bureau. Hoelzer: Medac: Speakers Bureau.

Several conflicting options regarding management of adult ALL are currently discussed. One major issue is the indication for SCT depending on risk factors and age, and the other is the recommendation to use unmodified pediatric protocols for “young” adults. Decision making on SCT is generally based on conventional risk factors – mainly disease characteristics – available at diagnosis, and decision making for “pediatric” chemotherapy on age. It is essential to develop more sophisticated criteria – also to reduce the risk of selection in clinical trials. In order to enhance prognostic models and to better address individual patient characteristics and the course of disease we reanalysed conventional prognostic factors together with new patient specific factors in a large cohort of adult ALL patients. A total of 1657 well characterised pts (15–55 yrs) included in the risk stratified protocols of the German Multicenter Study Group (GMALL) 06/99 and 07/03 was analysed. Treatment and risk stratification have been described (Brüggemann, Blood2006: 107;1116). Age remained a highly significant factor for CR, survival (OS) and disease free survival (DFS). OS ranged from 58% for 15–25 yrs, 52% for 26–35 yrs, 43% for 36–45 yrs to 32% for 45–55 yrs (p=.0001). Poorer outcome with increasing age was mainly due to early death (ED) and death in CR. CR, OS (45% c/pre-B, 45% pro-B, 38% early T, 47% mature T and 64% thymic T;p30) was associated with higher ED (p=.04). BMI (low vs normal vs high) had a significant impact on OS (43% vs 48% vs 39%;p=.003). Additional prognostic factors were identified during therapy. OS was sign.inferior for pts with infections in induction phase II compared to others (39% vs 52%;p 30.000/μl, late CR, Ph/BCR-ABL, proB in B-precursor and early or mature T-ALL in T-lineage ALL are poor prognostic factors. These factors identify pts for SCT. Age has a high prognostic relevance and is not used for this purpose but for selection of age adapted treatment approaches. For the first time other new prognostic factors were described. Poor general condition identifies pts who need specific attention e.g. stabilisation before treatment start. Furthermore complications during induction – particularly infections and liver toxicity – as well as treatment delays in general affect outcome adversely. These findings support the hypothesis that a) results in adult ALL can be improved by better supportive care, protocol compliance and subgroup adjusted treatment and b) these and additional individual factors e.g. comorbidity should be utilised to refine prognostic models and decision making on intensive chemotherapy and/or SCT including dose-reduced SCT.

Asparaginase (ASP) has a unique role in the treatment of ALL, and several pediatric studies have demonstrated that modifications of schedule, preparation and dose of ASP may have an impact on overall outcome. The optimisation of ASP treatment is therefore an important aim of the German Multicenter Study Group for Adult ALL (GMALL). In 1999 a pilot trial for adult ALL was started (GMALL 06/99), followed by the ongoing main trial 07/2003. Induction comprised dexamethasone, vincristine, daunorubicine and i.th. methotrexate. One dose of pegylated ASP (PEG-ASP) 1000 U/m2 (500 U/m² if > 55yrs) was given instead of 7 doses of 5000 U/m2 native E.coli asparaginase (ASP) given over 14 days in previous GMALL studies. In consolidation one dose PEG-ASP (500 U/m2) replaced one dose of E.coli ASP (10000 U/m2) in combination with high-dose methotrexate (1500 U/m²) and mercaptopurine. ASP activity was above 100 U/l at day 10 in 80% of the pts after 1000U/m2 and in only 42% after 500 U/m2. Based on this correlation between dose and duration of activity in study 07/2003 (amendment II in 12/2006) the dose for PEG-ASP was increased to 2000 U/m2 in induction and consolidation for pts < 55 yrs and to 1000 U/m2 for pts > 55 yrs in order to improve treatment efficacy. Details of the protocol have been reported (Brüggemann, Blood2006: 107;1116). Patients: 959 pts with a median age of 36 yrs are evaluable. 8% (N=82) were older than 55 yrs. Clinical characteristics were representative for adult ALL and similar in the cohorts. 766 pts were treated before the amendment with 1000 U/m2 (cohort 1) and 117 pts after the amendment with 2000 U/m2 (cohort 2), with the respective reductions for pts > 55yrs. 76 pts did not receive ASP in induction due to various reasons (cohort 3). Efficacy: In cohort 1 and 2 91% and 90% achieved CR after induction (80% in cohort 3). Data on molecular response, defined as MRD below 10 −4,after induction are available in a subset of both cohorts. There is a trend towards earlier and higher molecular CR rate in cohort 2 (82% after induction) compared to cohort 1 (70%). Survival after 1 yr is similar in cohort 1 (79%) compared to cohort 2 (77%) and inferior in cohort 3 (66%). Probability of continuous CR after 1 year shows a trend to improvement with 87% in cohort 2 vs 77% in cohort 1. Toxicity in induction: Toxicity reported here is focused on potentially ASP related oIII–IV (WHO scale) events. 676 pts are evaluable for cohort 1 and 107 pts for cohort 2. Incidences for both cohorts are as follows: GOT or GPT (30%/27%), bilirubine (12%/14%), thrombosis (5%/2%), bleeding (2%/0%) and hypersensitivity (1%/1%). Details on adverse events of all degrees are available in a subset of pts showing an increase of any WHO grade in 81% for bilirubine, 80% for GPT, 52% for amylase, 29% for lipase and 51% for glucose. Data on substitution of clotting factors were available in 84 and 61 pts: 73% vs 93% required substitution (25%/12% FFP, 37%/46% ATIII concentrate and 37%/42% both). Conclusions: This is the largest cohort of adult ALL pts treated with PEG-ASP so far. Overall intensified PEG-ASP was feasible in the context of intensive multidrug induction. Coagulation disturbances occurred frequently and substitution was extensive, but bleeding or thrombosis were rare events. Although substitution of clotting factors was clinically effective it remains open whether it is clinically necessary. The rate of severe hepatotoxicity was stable after dose escalation however lead to significant treatment delays in individual pts. Lab value changes e.g. liver occured in a large proportion of pts; it remains open to what extent they are clinically relevant and require interruption of further chemotherapy. It would be an important goal to identify parameters to predict severe ASP related toxicities e.g. by pharmacogenomics. The molecular CR rates after dose escalation are promising and will hopefully turn out into an improved overall survival. Supported by supported by Deutsche Krebshilfe 70-2657-Ho2 and partly BMBF 01GI 9971 and Medac GmbH