ALBERT

Description: Key Points Mutational trajectories are defined by complex patterns of molecular heterogeneity in MDS, including lower-risk cases. Therapeutic intervention dynamically reshapes mutational patterns often resulting in branched or independent evolution of MDS clones.

Description: Introduction Recently, Erythroferrone (ERFE) was discovered as a new regulator of hepcidin in the context of hematopoietic stress and erythropoietin (EPO) stimulation (Kautz et al., Nature Genetics 2014). ERFEhas been shown to be expressed by erythroprogenitor cells of the bone marrow in response to increased erythroid activity induced by phlebotomy, EPO treatment or simulation of infectious situations in mice. It induces increased iron availability by downregulation of hepcidin in the liver and therefore represents an important new factor in iron homeostasis to be explored as a potential diagnostic or therapeutic target in the context of anemia and iron overload. Myelodysplastic Syndromes (MDS) are a group of heterogeneous malignant hematologic diseases characterized by inefficient hematopoiesis, severe anemia and deregulated iron homeostasis. In order to determine the specific role of ERFE in MDS, we analyzed the gene expression of ERFE in different hematopoietic compartments of MDS patients and healthy controls and correlated the differential expression data with clinical parameters and survival. Methods CD71+ erythroprogenitor cells (n=198 samples) were immunomagnetically purified from mononuclear bone marrow (BM) cells of a total of n=148 MDS and n=18 sAML patients. Chronological samples were available in n=21 cases. For controls, CD71+ BM cells were analyzed from n=35 healthy donors. In addition to CD71+ cells, CD61+, CD15+ , CD34+, selected from BM, as well as CD3+ selected peripheral blood (PB) cells were immunomagnetically collected from three MDS patients as well as two healthy young and two healthy old volunteers. After total RNA extraction using the AllPrep DNA/RNA Mini kit (Qiagen), cDNA was transcribed from RNA via Quantitect cDNA synthesis kit (Qiagen). Subsequently, ERFE expression was quantified from cDNA by quantitative PCR. Results In comparative expression analyses of different hematopoietic BM progenitor fractions (CD34+, CD15+, CD61+ and CD71+), ERFE was almost exclusively expressed in the erythropoietic CD71+ compartment. ERFE expression profiles in the CD71+ subset revealed a highly significant overexpression of this gene in MDS IPSS-low/int-1-risk (fold change (FC)=4.3, p

Description: Introduction Deletion of chromosome 5q (del(5q)) defines a distinct clinical subtype of myelodysplastic syndromes (MDS) and qualifies patients to specific treatment with Lenalidomide (LEN). Therefore, detection and monitoring of this deletion is an important element in routine clinical diagnostics for determining molecular response. Current methodologies for performing these analyses consist of cytogenetics, fluorescence in situ hybridization (FISH) or microarrays. All of these methods have downsides due to the high demands to the input material, i.e. viable cells or necessity for large amounts of high quality genomic DNA (gDNA). To perform quantitative assessment of cytogenetic lesions in low quantity or residual material we here present the establishment of a PCR-based assay for interrogation of del(5q) in MDS, based on the allelic loss at heterozygous short tandem repeat (STR) loci within deleted regions. Methods Genomic DNA was isolated from bone marrow (BM) and peripheral blood (PB) of n=86 MDS del(5q) patients. 49 non-del(5q) MDS patients were used as controls. Serial chronological BM samples (n=95) following treatment with LEN from n=40 del(5q) patients, who were enrolled in the LEMON-5 trial from the German MDS study group, were analysed. Using 10ng DNA, 12 fluorochrome-labelled PCR amplicons of STR loci located between chromosomal bands 5q21 and 5q31 were amplified in a single optimized multiplex-PCR reaction. Subsequently, amplicon fragment analysis was carried out via capillary electrophoresis and allele size quantification of heterozygous STR loci was performed. Finally, the degree of skewing in the allelic ratios of all informative STR markers was averaged and translated into an allelic burden of del(5q). Results Paired quantitative correlation of clone sizes using STR-PCR and interphase FISH was carried out in n=34 samples and revealed highly concordant results with r²=0.924. The diagnostic accuracy of the PCR assay was evaluated by receiver operating characteristic (ROC) analysis and revealed an area under the curve of 0.989 (sensitivity and specificity of 0.977 and 0.948, respectively). Prior to treatment with LEN, clone sizes as determined by STR-PCR were heterogeneous (mean: 57%, range: 11-91%). During follow-up analysis, while cytogenetic analyses failed (e.g. metaphase failure) in 7/40 (18%) cases, our STR-PCR assay successfully generated estimates of del(5q) cell burden in all available samples. Upon LEN treatment, n=12 patients achieved major cytogenetic remission (absence of del(5q)-positive metaphases). The mean clone size carrying del(5q) determined by STR-PCR in that group was 7% (range 3 - 10%) and significantly increased compared with n=15 patients who reached minor cytogenetic response (defined as 50% reduced aberrant metaphases, mean 13%, range 5 - 39%, p=0.025). Intriguingly none of n=6 patients without cytogenetic response achieved a del(5q) clone size of less than 35% as determined by STR-PCR (mean 46%, range 35 - 66%), highlighting the correlation of PCR based follow-up analysis with currently used cytogenetic methods for response evaluation. Finally in n=93 matched PB and BM samples a correlation of del(5q)-frequency in BM versus PB showed r²=0.81. Moreover, in 96% of samples in which the BM still showed clone sizes 〉10%, we reliably detected del(5q) in corresponding PB cells with a robust sensitivity of 5% deleted cells. Discussion We present a highly adaptable tool for precise measurement of large chromosomal deletions, requiring only minute amounts of genomic DNA. It shows a very good quantitative correlation with established methods and good diagnostic accuracy. Most importantly, this PCR based assay does not require dividing cells so it can be performed from PB, which shows a sufficient correlation with clone sizes in BM and rarely involves the risk of underrepresentation of del(5q)-clones in PB, possibly allowing the use of PB as a regular specimen for clone size monitoring. Thus, especially in the context of serially monitored patients this assay represents an alternative method for less invasive tracking of cytogenetically aberrant clones. Disclosures Platzbecker: Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Boehringer: Research Funding. Schlenk:Janssen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria; Teva: Honoraria, Research Funding; Arog: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees. Bug:TEVA Oncology, Astellas: Other: Travel Grant; NordMedica, Boehringer Ingelheim, Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene, Novartis: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Description: Introduction: Myelodysplastic Syndrome (MDS) can occur in young people but it is mainly a disease of the elderly with a dramatic increase of incidence in the decades above 60 years. Accordingly, the factor age may be an important gateway to the understanding of the molecular pathogenesis of MDS. Insights into the molecular changes of aging hematopoiesis in healthy organisms have found molecular changes, which often parallel the observations in MDS such as increase of clonality with age, change of epigenetic profiles, skewed lineage commitment toward the myeloid compartment and reduced regenerative capacity after stress. The development of MDS is often suggestive of an accelerated extrapolation of molecular changes, which also occur in normal aging hematopoiesis. Beyond this, increasing evidence is suggesting that MDS hematopoiesis is highly dependent on support of the bone marrow (BM) stroma, which has been shown to display aberrant transcriptomic profiles as compared to healthy BM stroma. To this end, we aimed to test the hypothesis whether the emergence of MDS may be associated with a continuity of molecular changes in BM stroma cells during aging. Therefore, we performed explorative RNA sequencing in a set of MSCs collected from healthy young, healthy old and patients with MDS with a highly standardized pre-analytical work-up algorithm. Methods: We collected BM samples from voluntary healthy young adults (age = 24 - 25 years, female n=3, male n=3), healthy old adults (age 66 - 79 years, female n=3, male n=3) and patients with very low - intermediate risk MDS (age 51 - 87 years, female n=3, male n=3). After isolation of BM mononuclear cells by Ficoll gradient centrifugation, 5x106 mononuclear BM cells were seeded into 25cm² flasks and cultured using StemMACS human MSC Expansion Media (Miltenyi Biotec) with weekly media exchange to select for MSCs. These were expanded and harvested in passage 2. Absence of residual hematopoietic cells was controlled by FACS with anti CD45, CD31, and CD146. Whole transcriptome RNA-sequencing on all samples was carried out from 150ng of high quality RNA using the TruSeq stranded total RNA protocol and 100bp paired end sequencing (Illumina). The bio-informatical pipeline consisted of mapping using hisat2 and cufflinks for calculation of differentially expressed genes. Results: RNA-sequencing generated a mean of 94 million reads per sample. Between the groups "healthy young" and "healthy old" 331 differentially regulated genes were identified. Between "healthy old" and "MDS" 514 genes were differentially regulated (fold change 〉 1.5, false discovery rate, FDR 〈 0.05). Among these, 197 genes were differently expressed between all three groups. With these parameters, a total of 17 genes showed a continuous and significant increase of expression from healthy young over healthy old toward MDS. Among these were Kit ligand (KITLG) but also a cluster of membrane based cell adhesion molecules such as Cadherin-6 (CDH6), Laminin Subunit Alpha 2 (LAMA2) and Laminin Subunit Gamma 2 (LAMC2) and others. Conversely, 5 genes showed a continuous and significant decrease of expression from healthy young over healthy old toward MDS, among these Leukocyte-specific protein 1 (LSP1), a gene implicated in regulation of T-cell migration. Gene set enrichment analysis revealed that MDS MSCs exhibited a significant depletion of genes involved in early adipogenic differentiation and enrichment of gene sets associated with extracellular matrix remodeling (FDR 〈 0.05, normalized enrichment score 〉 1.7). Although cells were cultured under normoxic conditions, MDS-MSCs displayed marked intrinsic feature of hypoxia. Conclusion: By integrating transcriptomic data from BM stroma cells from healthy individuals during aging and comparison to BM stroma cells from MDS patients we have identified gene sets that are significantly differentially expressed per continuitatem. On the background of the hypothesis that molecular changes in the microenvironment of MDS are an exacerbation of changes also taking place during normal aging in the bone marrow, these genes, which are accumulated in the context of extracellular matrix and cell adhesion are promising candidates to further elucidate a BM stroma based pathogenesis of MDS. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal myeloid malignancies characterized by ineffective hematopoiesis and an increased risk of transformation into acute myeloid leukemia. Particularly early stage MDS are at least in part characterized by an increased apoptosis of myeloid and erythroid progenitors that causes peripheral cytopenia. APG101 is a glycosylated fusion protein consisting of the extracellular domain of human CD95 (Fas receptor) and the Fc domain of human IgG1. APG101 effectively binds to the CD95 ligand (CD95L) expressed on effector cells as well as to functionally active ligand in solution, by that blocking the interaction between CD95 and its ligand. The aim of our study was to evaluate whether APG101 treatment of primary CD34+ reduces the apoptotic rate and improves the differentiation capacity of these cells. Methods Bone marrow cells were obtained during routine bone marrow aspiration after all patients gave their written informed consent. Isolated primary CD34+ cells from 11 MDS patients were cultured in complete supplemented IMDM medium for 6 days with increasing concentrations of APG101 (1 µg/mL, 3 µg/mL, 10 µg/mL, 30 µg/mL, 100 µg/mL, 200 µg/mL, 300 µg/mL). After incubation time, cells were multicolor- stained with the following dye combination: Annexin-FITC + CD235a-PE + CD34-PECy7 + CD71-APC + 7-AAD and analyzed immediately on a flow cytometer (FACSCanto, BD Bioscience, Heidelberg, Germany). Analysis of raw FACS data was done with the FACSDiva software. To analyze the differentiation capacity of CD34+ progenitors, methylcellulose assays were performed in parallel to the aforementioned experiments. However, due to limited cell numbers, colony assays were performed on 9 MDS patients only. Cells were cultured in triplicates with increasing concentrations of APG101 for 14 days. Colonies were counted and the mean number of colonies was determined. Results Treatment of differentiating CD34+ cells with APG101 led to a decreased apoptosis in both CD34+ cells and CD71+ cells, respectively, indicated by decreased Annexin-FITC fluorescence. Interestingly, this effect was particularly seen at low APG101 concentrations (maximum of 10 µg/ml), while the effect was abrogated at higher APG101 concentrations. The anti-apoptotic effect was more pronounced in low risk MDS patients compared to high risk MDS patients. No effect was seen when the CD235a+ fraction of cells was analyzed. With regard to colony formation, an improvement of erythroid differentiation, indicated by an increase in CFU-E, was found in 3 out of 4 low risk patients (less than 5% blasts in the bone marrow). No effect was seen on erythroid differentiation in high risk patients (more than 5% blasts in the bone marrow). Conclusion APG101 shows promising in vitro activity in viable CD34+ cells with regard to inhibition of apoptosis and promotion of differentiation. The observation that the anti-apoptotic effect was more pronounced in low risk MDS patients as compared to high risk MDS patients supports the concept of increased apoptosis particularly in early stage MDS progenitors. Although the numbers in the differentiation experiments are small, we found a promising effect of APG101 on CFU-E formation at lower doses in patients with less than 5% bone marrow blasts. Moreover, the same dose-dependent effect was observed in the apoptosis assays. Since the activation of the CD95 pathway seems not only to be involved in apoptosis induction, but is also required for terminal erythroid differentiation in normal hematopoiesis, this dose-dependent effect might particularly reflect these ambivalent roles of CD95 and its ligand in both MDS and healthy hematopoiesis, repsectively. Disclosures: Kunz: APOGENIX GmbH: Employment. Fricke:APOGENIX GmbH: Employment.

Description: Background Myelodysplastic Syndromes (MDS) likely arise from an evolutionary process involving accumulation of somatic mutations and selection that occurs at the level of hematopoietic stem cells. Using next generation sequencing (NGS), several groups found recurrent somatic mutations to be associated with MDS. However, the history of stepwise molecular progression is still poorly defined. Likewise, little is known about patient-specific subclonal compositions, which may well contribute to the tremendous heterogeneity observed both in terms of clinical manifestations and response to treatment. Therefore, we sought to reconstruct patient-specific clonal hierarchies and decipher their dynamic evolution during long-term disease monitoring in order to better understand MDS pathogenesis and aid in adapting targeted therapeutic options in the future. Methods Bone marrow (BM) or CD34+ cells from patients with MDS were subjected to mutational screening by whole exome sequencing (WES, n=44) or targeted NGS (n=28) interrogating up to 17 recurrently mutated genes. Mesenchymal stromal cells (MSCs) were used as germline control. Allelic burdens were quantified with custom pyrosequencing assays and interphase-FISH in FACS purified myeloid, erythroid, lymphoid and stem cells isolated from both patients’ BM and corresponding xenografts in NSG mice. For unbiased follow up analysis, BM or CD34+ cells from two distant time points (median: 3 years, range 0.5-4.0 years) were analyzed for 13 patients using WES and these data were used to design patient-specific mutational panels. These panels contained amplicons representing mutational events uniquely present at one time point as well as others present at both time points. The panels were then subjected to ultra-deep-sequencing (UDS) to accurately quantify mutational burdens in all follow up (FU) samples. Results Integrative mutational data allowed us to reconstruct patient-specific mutational hierarchies in 35 cases, which revealed both linear and branching evolution in MDS. The data is also in support of the notion that potential founder lesions are highly enriched in genes involved in RNA splicing and epigenetic regulators, which we further show to be frequently detected in primary and/or xenografted lymphocytes. In contrast, we clearly demonstrate that large scale cytogenetic lesions (e.g. monosomy 7, trisomy 8, del(5q)) occur as late mutational events in at least 85% of the cases analyzed (n=17/20, 3 unresolved cases). Moreover, we could readily demonstrate the existence of subclonal heterogeneity in the patients’ BM, with variable contribution to different lineages and also cases showing lineage restriction of specific sub-clones. Most importantly, UDS analysis of FU samples from 13 independent patients (median FU 3.3 years, range FU 0.5-11.8 years and median of 5 samples per patient) with detailed clinical data revealed a highly dynamic clonal evolution during the course of the disease and dramatic shifts in the composition of mutational (sub-)clusters during therapy. Treatment often resulted in complete disappearance of specific sub-clones and most importantly, simultaneous outgrowth of previously undetectable subclones that subsequently dominated the marrow. Conclusion By reconstructing patient-specific mutational hierarchies we gained invaluable insights into the dynamic clonal composition and the molecular progression of human MDS. Our study shows that acquisition of large scale cytogenetic lesions appears to be a rather late event, which likely indicates that such lesions might not be tolerated by healthy stem cells. Most importantly, WES and subsequent deep sequencing analysis of FU samples demonstrated that patient-specific clonal diversity is highly dynamic and modulated during the course of the disease. This is especially true upon response to treatment, where concomitant disappearance and emergence of new pathogenic subclones was observed. Our work unravels two major paths of mutational acquisition by which MDS cells evade therapeutic pressure: (1) linear evolution of the previously sensitive subclone or (2) by branching evolution of an earlier independent founder clone. Our findings therefore strongly emphasize the importance of a genetically unbiased disease monitoring and the development of novel therapeutic strategies aiming at targeting founder lesions that are present in all ensuing subclones. Disclosures Nolte: Celgene Corp., Novartis Pharma: Honoraria, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Description: Introduction: MDS with isolated deletion 5(q) accounts for approximately 5% of all MDS cases. A recent retrospective analysis has found a cumulative progression rate of 18% after 5 years in patients with MDS deletion 5(q) without an increased medullary blast count[1]. Retrospective analyses have indicated that mutations in TP53 have an adverse impact on the clinical course of affected patients and their response to Len treatment. Here we report on the results of the German multi-center, prospective Le-Mon-5 trial that investigated the safety and efficacy of lenalidomide (Len) in patients with MDS and isolated deletion 5(q). Methods: Le-Mon-5 is a trial of Len in patients with MDS with isolated 5q abnormality, a blast count of 〈 5% in the bone marrow, a platelet count 〉 50.000/µl and absolute neutrophil counts of 〉 500/µl in the peripheral blood. Patients were treated with the standard dose of 10 mg Len for 21 days q28 days. The primary endpoint was safety. Secondary endpoints included response according to IWG criteria (2000), time to response, duration of transfusion independency and incidence and time to AML-transformation, respectively. All patients gave written informed consent to the trial including additional molecular genetic analyses. Central cytologic, cytogenetic and histologic review was performed. Mutational analysis of TP53 was done by next generation sequencing (NGS). For generation of PCR amplicon libraries TP53 primer plate assays were used and technically validated within the IRON-II (Interlaboratory Robustness Of Next generation sequencing) study network. Amplicon sequencing of TP53 was performed on a Roche 454 GS Junior system. Mean coverage of sequenced exons was about 800-fold allowing an approximate detection sensitivity of 2% mutational burden. Results: A total of 91 patients were enrolled into the trial. Of those, 71 patients (male, n=13) were analyzed for TP53 mutations. Median age was 71 years (range 40-88 years). TP53 mutations prior to treatment initiation with Len were found in 7 patients (10%). There was no difference between the TP53 mutated (TP53mut) versus TP53 wildtype patients (TP53WT) with regard to age, IPSS risk, hemoglobin levels, absolute neutrophil counts and platelet counts at baseline. Transfusion independence was achieved in a significantly lower proportion of patients in the TP53mut group versus TP53WT group (43% vs. 62%, p=0.036). Moreover, median survival was significantly shorter in the TP53mut group as compared to TP53WT group (533 days vs. not reached, p=0.0002). No difference was seen with regard to cytologic and cytogenetic response. Data on evolution into AML are currently being collected. Of 2 patients we had follow-up samples available. Both patients showed no difference with regard to the mutation frequency after a follow-up of 4-17 months on Len treatment (27% and 51%, respectively), although one of the patients achieved a complete cytogenetic response during Len treatment. Conclusions: Using the NGS technique on a routine basis, we achieved high quality runs with a high mean coverage of analyzed exons of TP53. Presence of TP53 mutations adversely affected response to Len with regard to transfusion independence. Moreover, TP53mut patients had a shorter overall survival as compared to TP53WT patients underlining the prognostic relevance of TP53 mutations in this patient cohort. Therefore, mutation analysis of TP53 might guide treatment decisions in the future, e.g. consideration of combination regimens. Since the TP53 clone obviously prevails during Len treatment, careful monitoring for signs of transformation should be performed. Disclosures Platzbecker: Celgene Corp.: Honoraria, Research Funding. Götze:Celgene Corp, Novartis Pharma: Honoraria. Schlenk:Celgene Corp.: Research Funding, Speakers Bureau. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Bug:Celgene: Honoraria, Research Funding. Germing:Celgene Corp.: Honoraria, Research Funding. Nolte:Celgene Corp., Novartis Pharma: Honoraria, Research Funding.

Description: Introduction: Thrombocytopenia is a common complication among MDS patients. Thus, many patients are dependent on platelet (PLT) transfusions, which give short-term therapeutic relief but are also associated with considerable clinical risks. In this context, thrombopoietin receptor agonists (TRAs) are under investigation as alternative treatment option, albeit with the concern that these substances may promote adverse events in MDS. However, beside potential positive effects on thrombopoiesis in MDS patients the TRA Eltrombopag (EPAG) has also been shown to exert positive disease modifying effects in vitro (Roth et al., Blood 2012). Using a MDS xenograft model, we here investigate the efficacy of EPAG and its influence on clonal composition on primary patient derived MDS xenografts and present data from an ongoing study. Methods: Currently, samples from n=18 MDS patients (MDS del(5q)=2, MDS-MLD=6, MDS-RS-MLD=1 MDS-EB-1=2, MDS-EB-2=7) have been xenografted into NSG mice by intrafemoral co-injection of CD34+ hematopoietic stem cells and mesenchymal stromal cells using a modified protocol according to Medyouf et al., Cell Stem Cell 2014. Long term engraftment is assessed 12 weeks post-transplant by intrafemoral bone marrow (BM) biopsy and mice with positive human engraftment are subsequently treated with either EPAG (50mg/kg) or vehicle control for 18 weeks. During that time, the mice are bled every two weeks and BM aspiration is performed every six weeks. Human hematopoietic cells are FACS sorted. In peripheral blood, human PLTs are specifically and absolutely counted with a FACS assay based on hCD41+ cells and beads. To track clonal composition of MDS samples upon xenografting and EPAG treatment in comparison to placebo control, the original patient sample and the final MDS xenograft sample are being whole exome sequenced (WES). Interspersed time points are analyzed with a patient individual amplicon based deep sequencing approach (Mossner et al., Blood 2016) to calculate dynamics of variant allele frequencies (VAF) in dependency of treatment. Results: To date, n=12 patient samples have been analyzed for human engraftment after 12 weeks post-transplant. Of these, n=7 (58%) have shown positive human engraftment and are being treated with EPAG versus placebo. To this end, one case has been completely followed up, including final molecular analysis. This MDS high risk case (MDS-EB-2) with a clinical PLT count of 29x109 PLT/L was transplanted into n=3 NSG mice. While two mice treated with EPAG survived the complete duration of the experiment, the placebo mouse died prematurely due to severe weight loss after 6 weeks of treatment. Further, EPAG treatment led to an initial rise of human PLT levels, while the placebo treated mouse presented a continuous decline of human PLTs, showing the efficacy of EPAG on human xenografts in the model. This observation has been confirmed in another case currently still under treatment. Molecular tracking by WES confirmed MDS patient specific molecular lesions in the MDS xenograft such as monosomy 7 and the disease related mutations CBL, DNMT3A and EZH2 with VAFs of 83%/43%/23% respectively. The monosomy 7 was detectable in all mice. CBL and DNMT3A exhibited similar VAFs in mouse EPAG1 (VAF=100%/54%), EPAG2 (VAF=100%/34%) and placebo (VAF=100%/50%). The EZH2 mutation was only detected in mouse EPAG2 (VAF=11%). Interestingly, the placebo mouse acquired a de novo mutation of U2AF1 (VAF=10%), which was not detectable in the initial patient sample or the EPAG treated mice. This spliceosomal mutation is associated with a higher risk of transformation to AML and shorter survival (Graubert et al., Nat Genet 2012; Makishima et al., Blood 2012). Conclusions: Our data show first proof of principle results that new treatment options can be tested successfully in a preclinical murine xenograft model of primary MDS patient samples in a placebo controlled experimental setting. This approach allows the performance of patient individual substance testing that can segregate substance specific effects from natural disease progression in the same patient. Clinical parameters such as human PLT production and molecular clonal composition can be measured with a high confidence in vivo. Our current data show preliminary support for the hypothesis that EPAG may be efficacious in increasing PLT production in MDS patients without adversely influencing the underlying clonal composition. Disclosures Nowak: Novartis: Research Funding.

Description: Background Beside cytogenetic aberrations, additional gene mutations are powerful predictors of outcome in myeloid diseases. Moreover, myelodysplastic syndromes with isolated deletion (5q) (MDS del(5q)) have been regarded as one of the most favorable entities among MDS. However, a substantial proportion of MDS del(5q) patients experience transformation into AML soon after diagnosis (Germing et al. Leukemia. 2012;26:1286-1292). Mutations of TP53 gene have early been recognized as an unfavorable prognostic biomarker in MDS in general and recent data suggest a role of TP53 mutations in the transformation of MDS del(5q) into AML. Lenalidomid (Len) is now approved in the US as well as in Europe for the treatment of MDS del(5q) it is of particular interest whether Lenalidomide can alter the course of pretreatment TP53 mutated MDS del(5q). Methods The Le-Mon-5 trial investigated the safety and efficacy of Len in patients with MDS and isolated deletion (5q). All patients gave their written informed consent to the clinical trial and to additional molecular genetic analyses. Bone marrow aspirates were performed at screening prior treatment initiation and during follow-up every 6 months. Only freshly extracted, high-quality DNA from ficollized mononuclear cells was used for next-generation deep-sequencing analysis. For generation of PCR amplicon libraries TP53 oligonucleotide primer plate assays were used and technically validated within the IRON-II (Interlaboratory Robustness Of Next generation sequencing) research study network. Amplicon deep-sequencing of TP53 (exons 4-11) was performed on a Roche 454 GS Junior system. Mean coverage of sequenced exons was about 800-fold allowing an approximate detection sensitivity of 2% mutational burden. Results Central cytological, histological and cytogenetic review was performed in all patients establishing the diagnosis of MDS with isolated deletion (5q). A total of 68 patients (male: n=9) were analyzed with a median age of 71 years (range 41-88 years). TP53 mutations prior to treatment initiation with Len were found in 7 patients (10%). Mean mutation frequency was 38%. Notably, we did not find mutation frequencies lower than 15%. Of 4 evaluable patients, three patients became transfusion independent within 4 months of Len treatment. Of 2 patients we had follow-up samples available. Both patients showed no difference with regard to the mutation frequency after a follow-up of 4 and 17 months on Len treatment (27% and 51%, respectively). Noteworthy, one the two patients achieved a complete cytogenetic remission despite maintaining his TP53 mutation frequency. Conclusion Using freshly extracted DNA we achieved high-quality NGS results with a high mean coverage of the relevant coding region of TP53. However, prevalence of TP53 mutations in our patient cohort was lower as compared to previously published data and we did not find low-level allele burdens as published by other groups, which might be due to the different sample sources used. Transfusion independence as well as cytogenetic remissions can be achieved in patients with TP53 mutations who are treated with Lenalidomide. Disclosures: Platzbecker: Celgene: Honoraria. Giagounidis:Celgene: Consultancy, Honoraria. Götze:Celgene Corp.: Honoraria. Haase:Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Bug:Celgene: Honoraria, Research Funding. Hofmann:Celgene: Research Funding. Germing:Celgene: Honoraria, Research Funding. Nolte:Celgene: Honoraria, Research Funding.

Description: Introduction:Particularly in low risk MDS, a pro-apoptotic milieu has been described with increased levels of apoptosis-promoting factors such as tumor necrosis factor and fas ligand (CD95 ligand). Evidence exists that in low risk MDS increased apoptosis of erythroid progenitors mediated via CD95 activation results in peripheral cytopenia. APG101 is a fusion protein consisting of the extracellular domain of human CD95 and the Fc domain of human IgG1. APG101 binds to CD95 ligand on effector cells as well as to soluble ligand, thus blocking the interaction between CD95 and its ligand. Here we report on results of a phase I study in transfusion-dependent low risk MDS patients treated with APG101. Primary objectives were safety and tolerability of APG101. Secondary objectives were hematologic, cytologic and cytogenetic response rates using modified International Working Group (IWG) response criteria (Cheson et al., 2006), incidence and time to leukemic progression as well as overall survival (OS) at 37 weeks. Furthermore, this study was focused on the depiction of molecular and functional changes underlying the stem cell defect in MDS including age related changes and high-throughput molecular profiling to evaluate the biological effect of APG101 in patients with lower risk MDS. Methods: The study was performed according to ICH-GCP guidelines and the Declaration of Helsinki as an open-label, non-randomized study for up to 37 weeks including an initial 12 weeks treatment phase with APG101 and a follow-up period of 25 weeks. 400 mg of APG101 were given every week as an intravenous (i.v.) infusion to the first 6 patients, 100 mg APG101 were given to all further patients. Bone marrow aspirates were obtained during the screening period (baseline), at the end of treatment (EOT, week 13), at week 25 (follow up) and at week 37 (End of Study) with subsequent comprehensive cytologic, cytogenetic, molecular and immunophenotypic analyses. In order to track clonal development and heterogeneity, methylcellulose assays on isolated CD34+cells from bone marrow aspirates at the different time points were performed and subjected to amplicon deep sequencing to detect gene mutations prior to APG101 dosing and to follow the mutational allele burden during and after treatment. Results: A total of 29 patients were screened for this study. Twenty patients received at least one dose of APG101, 17 patients completed the treatment phase and 15 patients completed the entire study including the follow-up phase. Median age was 74.5 years (range 56-82). WPSS categories were low in 19 patients and intermediate in 1 patient, respectively. Except for two patients with RCMD-RS and RARS, all others were classified as RCMD (WHO 2008). According to the exclusion criteria, none of the patients showed a medullary blast count ≥ 5%. Quantification of the allele burden of pre-existing mutations (mutations of ASXL1, SF3B1, TTBK, del(ETV6) and IDH2, SRSF2, SPEG2, BRCC3, NF1) showed no expansion of the mutated clones during the course of treatment. One patient, however, was reported with leukemic progression and AML transformation. One patient died from sepsis due to pre-existing neutropenia. Ex vivo differentiation analyses revealed an increase in CFU-E/BFU-E forming capacity. With regard to efficacy, an improvement in Hb level was observed in 6 patients at various assessment periods in this study. In 8 patients, a decrease in transfusion frequency from treatment until EOS was observed. Conclusions: APG101 is a very well tolerable compound in transfusion-dependent lower risk MDS patients comprising mostly of elderly patients. Risk of AML progression was not elevated. Deep amplicon sequencing did not show any signs of expansion of preexisting malignant clones during therapy, i.e. APG101 did not provide these clones with a survival benefit. Analysis of efficacy as secondary objective revealed that APG101 has the potential to improve hematopoiesis in MDS patients. Therefore, APG101 is an interesting compound for further clinical studies. Disclosures Fricke: Apogenix AG: Employment. Kunz:Apogenix AG: Employment.