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Anti-CD38 monoclonal antibodies have consistently shown increased efficacy when added to standard of care for patients with multiple myeloma. We aimed to assess the efficacy of isatuximab in addition to lenalidomide, bortezomib, and dexamethasone in patients with newly diagnosed transplantation-eligible multiple myeloma.This open-label, multicentre, randomised, active-controlled, phase 3 trial was done at 67 academic and oncology practice centres in Germany. This study is ongoing and divided into two parts; herein, we report results from part 1. Eligible patients were aged 18-70 years; had a confirmed diagnosis of untreated multiple myeloma requiring systemic treatment and a WHO performance status of 0-2; and were eligible for induction therapy, high-dose melphalan and autologous haematopoietic stem-cell transplantation, and maintenance treatment. Patients were randomly assigned (1:1) to receive three 42-day cycles of induction therapy either with isatuximab plus lenalidomide, bortezomib, and dexamethasone (isatuximab group) or lenalidomide, bortezomib, and dexamethasone alone (control group) using a web-based system and permuted blocks. Patients in both groups received lenalidomide (25 mg orally on days 1-14 and 22-35), bortezomib (1·3 mg/mBetween Oct 23, 2018, and Sep 22, 2020, 660 patients were included in the ITT analysis (331 in the isatuximab group and 329 in the control group). 654 (99%) patients were White, two were African, one was Arabic, and three were Asian. 250 (38%) were women and 410 (62%) were men. The median age was 59 years (IQR 54-64). MRD negativity after induction therapy was reached in 166 (50%) patients in the isatuximab group versus 117 (36%) in the control group (OR 1·82 [95% CI 1·33-2·48]; p=0·00017). Median follow-up time from start to end of induction therapy was 125 days (IQR 125-131) versus 125 days (125-132). At least one grade 3 or 4 adverse event occurred in 208 (63%) of 330 patients versus 199 (61%) of 328 patients. Neutropenia of grade 3 or 4 occurred in 77 (23%) versus 23 (7%) patients and infections of grade 3 or 4 occurred in 40 (12%) versus 32 (10%) patients. Among 12 deaths during induction therapy, one death due to septic shock in the isatuximab group and four deaths (one cardiac decompensation, one hepatic and renal failure, one cardiac arrest, and one drug-induced enteritis) in the control group were considered treatment-related.Addition of isatuximab to lenalidomide, bortezomib, and dexamethasone for induction therapy improved rates of MRD negativity with no new safety signals in patients with newly diagnosed transplantation-eligible multiple myeloma.Sanofi and Bristol Myers Squibb (Celgene).

Patients with cancer are at high risk of severe coronavirus disease 2019 (COVID-19), with high morbidity and mortality. Furthermore, impaired humoral response renders severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines less effective and treatment options are scarce. Randomized trials using convalescent plasma are missing for high-risk patients. Here, we performed a randomized, open-label, multicenter trial (https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-001632-10/DE) in hospitalized patients with severe COVID-19 (n = 134) within four risk groups ((1) cancer (n = 56); (2) immunosuppression (n = 16); (3) laboratory-based risk factors (n = 36); and (4) advanced age (n = 26)) randomized to standard of care (control arm) or standard of care plus convalescent/vaccinated anti-SARS-CoV-2 plasma (plasma arm). No serious adverse events were observed related to the plasma treatment. Clinical improvement as the primary outcome was assessed using a seven-point ordinal scale. Secondary outcomes were time to discharge and overall survival. For the four groups combined, those receiving plasma did not improve clinically compared with those in the control arm (hazard ratio (HR) = 1.29; P = 0.205). However, patients with cancer experienced a shortened median time to improvement (HR = 2.50; P = 0.003) and superior survival with plasma treatment versus the control arm (HR = 0.28; P = 0.042). Neutralizing antibody activity increased in the plasma cohort but not in the control cohort of patients with cancer (P = 0.001). Taken together, convalescent/vaccinated plasma may improve COVID-19 outcomes in patients with cancer who are unable to intrinsically generate an adequate immune response.

Background Patients with hematological malignancy or other cancers as well as immunosuppression bear a high risk for severe COVID-19. Monoclonal antibodies (mAb) are efficient at early stages of the disease but may lose potency with new variants. Trials on plasma from convalescent donors in unselected patients have not shown clinical benefit. No randomized trials focussing on patients with underlying disease have been published. Methods We conducted an open-label, multicenter, randomized controlled trial to evaluate efficacy of plasma (CVP - convalescent or after vaccination) in patients with COVID-19 at high risk for adverse outcome in Germany. We assessed the effect of high-titer CVP (2 units from different donors, 238-337 ml each, on subsequent days). Patients with hematological or other malignancy (group 1), immunosuppression (group 2), age >50 and ≤75 years and lymphopenia and/or high D-dimers (group 3) or age >75 years (group 4) who were hospitalized with confirmed SARS-CoV-2 infection and with an oxygen saturation ≤94% were included. Primary outcome measure was time to clinical improvement on a seven-point ordinal scale, secondary outcome was mortality (Janssen et al. Trials 2020 Oct 6;21(1):828). Results Overall, 133 patients were randomized, 68 received CVP with an additional 10 patients as a crossover on day 10. Median age (range) was 68 years (39-95) in the CVP group and 70 (38-90) in controls. For the entire cohort, no significant difference was seen in time to improvement (median days: CVP 12.5 vs. control 18; HR 1.24 (95% confidence interval (CI) 0.83-1.85), p=0.29). Subgroup analysis (group 1+2) revealed shortened time to improvement (median days CVP 13 vs. control 32; HR 2.03 (95%CI 1.17-3.6), p=0.01) and mortality was reduced (mortality CVP n=6 (18%) vs. control n=10 (29%). No significant differences in time to improvement were observed in group 3 or 4 (HR 0.72 (95%CI 0.41-1.28), p=0.26). No relevant adverse events were observed. Conclusion CVP improves time to clinical improvement and mortality for COVID-19 patients with underlying hematological disease/cancer or other reasons of impaired immune response. Even with new variants, high-titer CVP may offer a widely available and inexpensive therapy option in high-risk groups. Funding BMBF FKZ 01KI20152; EudraCT 2020-001632-10. Disclosures Uta Merle, MD, Gilead: Sponsored congress travel and accommodation Markus Weigand, MD, Bbraun: Speakers fee/ad boards fee|Biotest: Speakers fee/ad boards fee|Eumedica: Speakers fee/ad boards fee|Gilead: Speakers fee/ad boards fee|MSD: Speakers fee/ad boards fee|Pfizer: Speakers fee/ad boards fee|Shionogi: Speakers fee/ad boards fee|SOBI: Speakers fee/ad boards fee Martin Bornhäuser, MD, Alexion: Honoraria|Jazz Pharmaceuticals: Honoraria|MSD: Honoraria|Novartis: Honoraria Nael Alakel, MD, Amgen: personal fee, travel grant|Gilead: personal fee, travel grant|MSD Sharp and Dohme GmbH: personal fee, travel grant|Pfizer: personal fee, travel grant Timo Wolf, MD, Gilead Sciences: Lecture fee, travel grant|Janssen Pharmaceuticals: Lecture fee, travel grant|Merck Sharp Dome: Lecture fee, travel grant Maria Vehreschild, Prof. Dr., 3M: speaker fee|Astellas: Advisor/Consultant|Astellas: speaker fee|biologische heilmittel heel gmbh: Grant/Research Support|BioNtech: Grant/Research Support|EUMEDICA: Advisor/Consultant|Farmak International Holding: Advisor/Consultant|Ferring: Advisor/Consultant|Ferring: Speaker fee|Gilead Sciences: Advisor/Consultant|Immunic AG: Advisor/Consultant|MaaT: Advisor/Consultant|Merck: Advisor/Consultant|Merck: speaker fee|MSD: Advisor/Consultant|MSD: Grant/Research Support|MSD: speaker fees|Pfizer: speaker fee|Roche Molecular Systems: Grant/Research Support|Roche Molecular Systems: speaker fees|SocraRTec R&D GmbH: Advisor/Consultant|Takeda California: Grant/Research Support Hanns-Martin Lorenz, MD, Abbvie: Advisor/Consultant|Abbvie: Honoraria|Actelion: Advisor/Consultant|Actelion: Honoraria|Alexion: Advisor/Consultant|Alexion: Honoraria|Amgen: Advisor/Consultant|Amgen: Grant/Research Support|Astra Zeneca: Advisor/Consultant|Astra Zeneca: Honoraria|Baxter: Advisor/Consultant|Baxter: Advisor/Consultant|Baxter: Honoraria|Baxter: Honoraria|Bayer Vital: Advisor/Consultant|Bayer Vital: Honoraria|Biogen: Advisor/Consultant|Biogen: Honoraria|BMS: Advisor/Consultant|BMS: Honoraria|Boehringer Ingelheim: Advisor/Consultant|Boehringer Ingelheim: Honoraria|Celgene: Advisor/Consultant|Celgene: Honoraria|Fresenius: Advisor/Consultant|Fresenius: Honoraria|Genzyme: Advisor/Consultant|Genzyme: Honoraria|Gilead/Galapagos: Advisor/Consultant|Gilead/Galapagos: Honoraria|GSK: Advisor/Consultant|GSK: Honoraria|Hexal: Advisor/Consultant|Hexal: Honoraria|Janssen-Cilag: Advisor/Consultant|Janssen-Cilag: Honoraria|Lilly: Advisor/Consultant|Lilly: Honoraria|Medac: Advisor/Consultant|Medac: Honoraria|MSD: Advisor/Consultant|MSD: Honoraria|Mundipharm: Advisor/Consultant|Mundipharm: Honoraria|Mylan: Advisor/Consultant|Mylan: Honoraria|Novartis: Advisor/Consultant|Novartis: Honoraria|octapharm: Advisor/Consultant|octapharm: Honoraria|Pfizer: Advisor/Consultant|Pfizer: Honoraria|Roche/Chugai: Advisor/Consultant|Roche/Chugai: Honoraria|Sandoz: Advisor/Consultant|Sandoz: Honoraria|Sanofi: Advisor/Consultant|Sanofi: Honoraria|Shire: Advisor/Consultant|Shire: Honoraria|SOBI: Advisor/Consultant|SOBI: Honoraria|Thermo Fisher: Advisor/Consultant|Thermo Fisher: Honoraria|UCB: Advisor/Consultant|UCB: Honoraria.

BackgroundOverall survival remains poor in older patients with acute myeloid leukemia (AML) with less than 10% being alive after five years. In recent studies, a significant improvement in event-free, relapse-free and overall survival was shown by adding gemtuzumab ozogamicin (GO), a humanized antibody-drug conjugate directed against CD33, to intensive induction therapy once or in a sequential dosing schedule. Glasdegib, the small-molecule inhibitor of smoothened (SMO), also showed improved overall survival in patients not eligible for intensive chemotherapy when combined with low-dose cytarabine compared to low-dose cytarabine alone. These findings warrant further investigations in the phase III GnG trial.Methods/DesignThis is a randomized phase III trial with measurable residual disease (MRD) after induction therapy and event-free survival (EFS) as primary endpoints. The two research questions are addressed in a 2 by 2 factorial design. Patients age 60 years and older are upfront randomized 1:1 in one of the two induction arms: GO administered to intensive induction therapy on days 1,4 and 7 versus GO administered once on day 1 (GO-147 versus GO-1), and double-blinded 1:1 in one of the subsequent treatment arms glasdegib vs. placebo as adjunct to consolidation therapy and as single-agent maintenance therapy for six months. Chemotherapy backbone for induction therapy consists of standard 7+3 schedule with cytarabine 200mg/m² continuously days 1 to 7, daunorubicin 60mg/m² days 1, 2 and 3 and high-dose cytarabine (1g/m², bi-daily, days 1,2,3) for consolidation therapy. Addressing two primary endpoints, MRD-negativity after induction therapy and event-free survival (EFS), 252 evaluable patients are needed to reject each of the two null hypotheses at a two-sided significance level of 2.5% with a power of at least 85%. ETHICS AND DISSEMINATION: Ethical approval and approvals from the local and federal competent authorities were granted. Trial results will be reported via peer-reviewed journals and presented at conferences and scientific meetings.TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT04093505; EudraCT Number: 2019-003913-32.TRIAL REGISTRATION DATE: October 30, 2018

Trials 22(1), 765 (2021). doi:10.1186/s13063-021-05703-w, Published by BioMed Central, London

In contrast to the evidence regarding azacitidine (Aza), there is limited knowledge about the combination of decitabine (DAC) and donor lymphocyte infusions as salvage therapy for relapse after allogeneic stem cell transplantation (allo-SCT) so far. We retrospectively analyzed data of 36 patients with hematological (n = 35) or molecular relapse (n = 1) of acute myeloid leukemia (AML, n = 29) or myelodysplastic syndrome (MDS, n = 7) collected from 6 German transplant centers. Patients were treated with a median of 2 cycles DAC (range, 1 to 11). DAC was the first salvage therapy in 16 patients (44%), whereas 20 patients (56%) had previously received 1 to 5 lines of salvage therapy including 16 of them had been treated with Aza. In 22 patients (61%), a median of 2 DLI per patient (range, 1 to 5) was administered in addition to DAC. As a result, overall response rate was 25% including 6 complete remissions (CR, 17%) and 3 partial remissions (PR, 8%). Three patients within the first-line group achieved CR, while also 3 patients receiving DAC as second-line treatment reached CR including 2 patients with previous Aza failure. Median duration of CR was 10 months (range, 2 to 33) and no patient relapsed so far. The 2-year OS rate was 11% (± 6%) without any difference between first-line and pretreated patients. Incidence of acute and chronic graft-versus-host disease was 19 and 5%. Taken together, DAC exerts clinical efficacy in patients with AML or MDS relapsing after allo-SCT and is able to induce durable remissions in individual patients suggesting that DAC may be an alternative to Aza or even a second choice after Aza failure.

Background: Chronic neutrophilic leukemia (CNL) is a rare BCR/ABL negative myeloproliferative neoplasm (MPN) characterized by sustained, predominantly mature neutrophil proliferation, bone marrow granulocytic hyperplasia, and hepatosplenomegaly. CSF3R mutation is a central diagnostic criterion. Most patients are over the age of 60 and the median survival is estimated at 2 years. No effective standard therapy has been established yet. Case-report: In March 2019 a 29 y/o male presented with cough, arthralgias, and hepatosplenomegaly. His initial WBC was 47.000/µl with 80% neutrophils, few neutrophil progenitors, no monocytosis. Bone marrow was consistent with a myeloproliferative syndrome, particularly CML, however BCR/ABL was negative. Flow cytometry suggested a myelodysplastic syndrome (MDS Score ↑) und with the preliminary diagnosis of mixed MPN/MDS he received one course of azacytidine without response. After this course we received the results of molecular genetic analysis which showed CSF3R c.1853C>T (also pos. for DNMT3A, IDH2, neg. for JAK2, CALR, PDGFRα/β, FGFR1). WBC increased further to 77.000/µl and we initiated hydroxyurea and - after insurance granted coverage - switched to ruxolitinib (initially 10 mg BID, then 15 mg BID). His hepatosplenomegaly responded and WBC came down slowly. In August he started to lose response, WBC rose again, and he needed blood transfusions. Because of his young age we had planned stem cell transplantation from the very beginning. The main purpose of ruxolitinib had been to reduce pre-transplant leukemic burden. Bone marrow from March 2019 showed 2% CD34+ cells, in Sept. 4%, Jan 2020 20-25%, consistent with transformation to AML. There was no family donor. In March 2020 he underwent allogeneic stem cell transplantation from a matched female unrelated donor (MUD allo-SCT 10/10 match) which he tolerated well. Conditioning was busulfan based. His further clinical course was complicated starting d+40 after SZT with °IV gastrointestinal GvHD (no CMV or other infectious agent identified). Immunosuppression had to be escalated using steroids (1 mg/kg BW BID) + ruxolitinib + photopheresis. Eventually AML recurred and the patient deceased on d +81 due to multi organ failure induced by refractory GvHD and AML recurrence. Conclusion: As most described cases of CNL occur in elderly patients reports on stem cell transplantation are rare. Here we present the course of a 29 y/o male who had only a short response to ruxolitinib and hydroxyurea. Both agents did not alter the bone marrow leukemic burden, therefore he proceeded to alloSCT. He did not achieve a sustained remission. We decided to present this case despite the unfortunate outcome because - to our knowledge - there has been no prior report on alloSCT in such a young patient with CNL. Disclosures Matzdorff: Novartis Oncology: Consultancy, Other: Honoraria paid to institution; Amgen GmbH: Consultancy, Other: Honoraria paid to institution; Grifols Deutschland GmbH: Consultancy, Other: Honoraria paid to institution; Swedish Orphan Biovitrium GmbH: Consultancy, Other: Honoraria paid to institution; UCB Biopharma SRL: Consultancy, Other: Honoraria paid to institution; Roche Pharma AG: Other: Family stockownership.

Myelodysplastic syndromes (MDS) are characterized by dyserythropoiesis resulting in anemia. This pathological hallmark is incompletely understood. Notch signaling has been linked to impaired erythropoietic and megakaryopoietic development of CD34+ progenitor cells, but its role in MDS is unclear. We have analyzed the transcriptional activity of Notch pathway elements and its association with the key erythroid factor globin transcription factor 1 (GATA1) and the apoptosis regulatory gene B-cell lymphoma-xl (BCLxl) in MDS. The methylation of GATA1 erythroid promoter CpG dinucleotides flanking cis-regulatory elements, including an N-box suppressor binding site for HES1 and a GATA-box binding site, was examined in normal and MDS erythropoiesis. We have generated a kinetic in vitro model of MDS erythropoiesis using CD34+ bone marrow cells from healthy donors (n = 7) and patients with MDS (low risk: RA/n = 6, RARS/n = 3; high risk: RAEB/n = 4, RAEB-T/n = 2). RNA expression of GATA1, BCLxl, DLK1, Notch1, HES1, and HERP2 was measured by real-time RT-PCR (qPCR). DNA methylation at seven CpG dinucleotides of the GATA1 gene promoter was quantitatively analyzed by pyrosequencing of bisulfite-treated genomic DNA at any specific time point. For the Notch pathway elements, no conclusive expression differences were found between MDS and normal erythropoiesis. But we found steadily up-regulated RNA expression of GATA1 and of BCLxl during late normal erythropoietic differentiation. In contrast, during MDS, erythropoiesis a loss of typical up-regulation of GATA1 and BCLxl was observed. Hypermethylation of CpG dinucleotides flanking the repressor HES1 binding site within the 5' region of GATA1 was detected particularly during late MDS erythropoiesis. Interestingly, decremental GATA1 promotor methylation values were seen during normal erythropoiesis matching GATA1 RNA up-regulation. Our data show that the critical erythropoietic transcription factor GATA1 as well as the antiapoptotic molecule BCLxl fails to be normally up-regulated during MDS erythropoiesis. The higher residual 5'-GATA1 methylation values in MDS erythropoiesis but decremental loss thereof in normal erythropoiesis suggest a gene dose effect for GATA1 during erythropoiesis being finely tuned by CpG methylation. Its dysregulation may contribute to the ineffective erythropoiesis observed in MDS.

Objective Recently, the "epigenetic molecular clock hypothesis" linked increasing DNA methylation in a distinct CpG island in the cardiac-specific homeobox gene ( CSX ) gene to relative mitotic cell age. To determine mitotic cell age in hematopoietic cells of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients, we assessed differential C SX methylation patterns in these diseases vs age-adjusted healthy controls. Materials and Methods We performed bisulfite pyrosequencing to analyze CSX methylation in CD34 + and bone marrow (BM) cells from 53 MDS, 62 AML, 77 ALL patients, and 37 controls. Results Analysis of MDS CD34 + and BM cells revealed significantly increasing methylation of CSX in controls CSX methylation between the CD34 + vs the unselected BM compartment were detected in matched MDS low-risk but not high-risk and AML samples. ALL samples displayed highly elevated CSX methylation levels as compared to controls. Conclusions Assessment of mitotic cell age by CSX methylation analysis could reveal novel insights into the distinct progression of hematologic diseases.

Aberrant promoter methylation may contribute to the hematopoietic disturbances in myelodysplastic syndromes (MDS). To explore a possible mechanism, we therefore analyzed expression of DNA methyltransferase (DNMT) subtypes kinetics and aberrant promoter methylation of key regulatory genes during MDS hematopoiesis. An in vitro model of MDS lineage-specific hematopoiesis was generated by culturing CD34+ cells from healthy donors (n=7) and MDS patients (low-risk: RA/n=6, RARS/n=3; high-risk: RAEB/n=4, RAEB-T/n=2) with EPO, TPO and GCSF. Promoter methylation analysis of key genes involved in the control of apoptosis (p73, survivin, DAPK), DNA-repair (hMLH1), differentiation (RARb, WT1) and cell cycle control (p14, p15, p16, CHK2) was performed by methylation specific PCR of bisulfite-treated genomic DNA. Expression of DNMT1, DNMT3a and DNMT3b was analyzed and correlated with gene promoter methylation for each lineage at different time points. DNMT expression (all isoforms) was increased during thrombopoiesis whereas elevated DNMT1 level were seen during erythropoiesis. Associations between aberrant promoter methylation and DNMT expression were found in high-risk MDS for all lineages and during erythropoiesis. Hypermethylation of p15, p16, p73, survivin, CHK2, RARb and DAPK were associated with elevated DNMT isoform expression. No general overexpression of DNMT subtype was detected during MDS hematopoiesis. However a negative association of DNMT3a and 3b expression with MDS disease risk (IPSS) could be observed. Our data indicate that all mammalian DNMT isoforms may be involved in the aberrantly methylated phenotype in MDS but seem also to be essential for the differentiation of normal hematopoietic stem cells. In particular elevated DNMT1 expression may in particular contribute to ineffective erythropoiesis in MDS.

Deregulated epigenetic mechanisms are likely involved in the pathogenesis of myelodysplastic syndromes (MDSs). Which genes are silenced by aberrant promotor methylation during MDS hematopoiesis has not been equivalently investigated. Using an in vitro differentiation model of human hematopoiesis, we generated defined differentiation stages (day 0, day 4, day 7, day 11) of erythro-, thrombo- and granulopoiesis from 13 MDS patients and seven healthy donors. Promotor methylation analysis of key regulatory genes involved in cell cycle control (p14, p15, p16, CHK2), DNA repair (hMLH1), apoptosis (p73, survivin, DAPK), and differentiation (RARb, WT1) was performed by methylation-specific polymerase chain reaction. Corresponding gene expression was analyzed by microarray (Affymetrix, HG-U133A). We provide evidence that p16, survivin, CHK2, and WT1 are affected by promotor hypermethylation in MDSs displaying a selective International Prognostic Scoring System risk association. A methylation-associated mRNA downregulation for specific hematopoietic lineages and differentiation stages is demonstrated for survivin, CHK2, and WT1. We identified a suppressed survivin mRNA expression in methylated samples during erythropoiesis, whereas WT1 and CHK2 methylation-related reduction of mRNA expression was found during granulopoiesis in all MDS risk types. Our data suggest that lineage-specific methylation-associated gene silencing of survivin, CHK2, and WT1 in MDS hematopoietic precursor cells may contribute to the MDS-specific phenotype.

Background Investigation of X-chromosome inactivation patterns (XCIP) by determination of differential CpG-methylation has been widely applied for investigation of female cell clonality. Using this approach the clonal origin of various tumours has been corroborated. Controversially, strong age-related increase of peripheral blood (PB) cell clonality in haematologically healthy female subjects was reported. Recently, transcriptional XCIP ratio analysis challenged these results and questioned the suitability of methylation based clonality assays. Methods To reinvestigate XCIP-skewing in CD34, lowdensity mononuclear bone marrow (BM) as well as PB cells from healthy female subjects and patients with myelodysplastic syndromes (MDS), we established a transcriptional assay using pyrosequencing technique for quantification of single nucleotide polymorphism allele frequencies, representative for XCIP ratios. Results Our assay provides high sensitivity for XCIP ratio assessment as determined by standard curves, reproducibility, inter-marker correlation as well as correlation with the DNA-methylation based human androgen receptor (HUMARA) assay. Notably, in agreement with most studies investigating this issue, significant age-related increase of XCIP skewing in PB cells from healthy elderly female subjects was confirmed. Moreover, XCIP ratio analysis suggests even stronger clonal manifestation in BM and CD34 cells. In MDS, XCIP skewing levels were distinctively elevated as compared with controls of similar age and higher degrees were associated with poor clinical outcome. Conclusions Transcriptional clonal profiling via pyrosequencing allows accurate assessment of XCIP ratios, confirms the validity of the DNA-methylation based HUMARA assay and reveals important insights into ageing healthy and myelodysplastic haematopoiesis.

Background: During the last years, based on its efficacy and favourable toxicity profile the hypomethylating agent (HMA) Azacitidine (Aza) has proven to be a valuable treatment option for patients with AML or MDS who relapse after allo-SCT. In contrast to Aza, reports on the use of Decitabine (DAC), the second HMA approved in Europe for the treatment of AML, as salvage therapy for relapse after allo-SCT are scarce covering a total of 9 patients so far. This prompted us to perform a retrospective survey in order to gather more experience on the use of DAC after allo-SCT. Patients and Methods: Retrieving information from the EBMT Med-A form and a study-specific questionnaire we were able to analyze data of 36 patients (median age 56 years, range 21-72 years) from 6 German transplant centers who had received at least one cycle of DAC for the treatment of relapse of AML (n=29) or MDS (n=7) after allo-SCT. Median time to haematological (n=34) or molecular (n=2) relapse was 370 days (range 43-2623 days). Results: Overall, DAC was the first treatment for relapse in 16 pts (44%), whereas 20 pts (56%) had previously received one (n=14), two (n=2) or three (n=4) lines of salvage therapy for relapse after allo-SCT. This included 16 pts treated with Aza, 3 pts with intensive chemotherapy and 2 pts with radiation. Five pts had received a second allo-SCT and 9 pts donor lymphocyte infusions (DLI) before DAC therapy. Patients received a median of 2 DAC cycles (range, 1-10) with 24 pts (67%) treated with the approved dose of 20 mg/m2 for 5 days and 12 pts (33%) treated with 20 mg/m2 for 10 days based on the local policy of the individual transplant center. In addition to DAC, DLI (median number of DLI =1, range: 1-5) were administered to 22 pts (61%). Following treatment with DAC +/- DLI the median survival was 5 months (range 1 - 40 months). Six pts achieved a complete remission (CR, 17%) and 3 pts achieved a partial remission (PR, 8%) leading to an overall response rate of 25%. Median time to documentation of CR was 157 days (range: 47-255 days) and 4 DAC cycles (range: 1-8 cycles). Of 6 patients achieving CR after DAC, 3 had received DAC as first salvage therapy and 3 had previously received Aza, including 2 pts not responding to Aza and 1 patient switched to DAC therapy due to Aza intolerability. With a median follow-up of 12 months (range: 5-40 months), 3 of 6 patients remain in ongoing remission for 4, 23, and 33 months respectively without any further antileukemic therapy, while the other 3 patients died in remission due to infectious complications after second transplant. The 2-year overall survival rate of the entire group as calculated from the start of DAC therapy was 9%. Incidence and severity of acute GvHD (overall: 19%, grade I: 3%, grade II: 8%, grade III: 5%, grade IV: 0%, missing: 3%) and chronic GvHD (overall: 6%, limited 6%, extensive 0%) were low and mild. Conclusion: Our analysis shows, that also the second HMA DAC exerts relevant clinical efficacy in patients with AML or MDS relapsing after allo-SCT and can induce durable remissions in individual pts. Given the heterogeneity of our patient group and the limitations of a retrospective analysis this asks for confirmation in a prospective trial. Disclosures Platzbecker: Onconova, Teva, Celgene, Janssen, Novartis, Amgen: Honoraria, Research Funding. Kobbe:Jansen: Honoraria, Other: travel support; Celgene: Honoraria, Other: travel support, Research Funding.

Myelodysplastic syndromes (MDS) are characterized by dyserythropoiesis resulting in anemia. This pathological hallmark is incompletely understood. Notch signaling has been linked to impaired erythropoietic and megakaryopoietic development of CD34+ progenitor cells, but its role in MDS is unclear. We have analyzed the transcriptional activity of Notch pathway elements and its association with the key erythroid factor globin transcription factor 1 (GATA1) and the apoptosis regulatory gene B-cell lymphoma-xl (BCLxl) in MDS. The methylation of GATA1 erythroid promoter CpG dinucleotides flanking cis-regulatory elements, including an N-box suppressor binding site for HES1 and a GATA-box binding site, was examined in normal and MDS erythropoiesis. We have generated a kinetic in vitro model of MDS erythropoiesis using CD34+ bone marrow cells from healthy donors (n = 7) and patients with MDS (low risk: RA/n = 6, RARS/n = 3; high risk: RAEB/n = 4, RAEB-T/n = 2). RNA expression of GATA1, BCLxl, DLK1, Notch1, HES1, and HERP2 was measured by real-time RT-PCR (qPCR). DNA methylation at seven CpG dinucleotides of the GATA1 gene promoter was quantitatively analyzed by pyrosequencing of bisulfite-treated genomic DNA at any specific time point. For the Notch pathway elements, no conclusive expression differences were found between MDS and normal erythropoiesis. But we found steadily up-regulated RNA expression of GATA1 and of BCLxl during late normal erythropoietic differentiation. In contrast, during MDS, erythropoiesis a loss of typical up-regulation of GATA1 and BCLxl was observed. Hypermethylation of CpG dinucleotides flanking the repressor HES1 binding site within the 5' region of GATA1 was detected particularly during late MDS erythropoiesis. Interestingly, decremental GATA1 promotor methylation values were seen during normal erythropoiesis matching GATA1 RNA up-regulation. Our data show that the critical erythropoietic transcription factor GATA1 as well as the antiapoptotic molecule BCLxl fails to be normally up-regulated during MDS erythropoiesis. The higher residual 5'-GATA1 methylation values in MDS erythropoiesis but decremental loss thereof in normal erythropoiesis suggest a gene dose effect for GATA1 during erythropoiesis being finely tuned by CpG methylation. Its dysregulation may contribute to the ineffective erythropoiesis observed in MDS.

Abstract 977 X-chromosome inactivation occurs in female cells leading to stochastically random silencing of one X-chromosome. Accordingly, investigation of X-chromosome inactivation patterns (XCIP) in healthy female tissues revealed similar ratios of cells carrying either an active maternal or paternal X-chromosome. Genomic XCIP markers like the HUMARA locus have been used to elucidate clonality of myelodysplastic syndromes (MDS). However, analysis of healthy female peripheral blood (PB) cells has shown a strong age-related increase in skewing of XCIP suggesting the frequent manifestation of clonal hematopoiesis in the elderly. Since the occurrence of clonality in blood cells from healthy individuals remains controversially discussed (Swierczek et al, Blood 2008), we developed a novel highly accurate transcription based approach using Pyrosequencing to detect clonal hematopoiesis. In addition, we present the first comprehensive XCIP analysis of CD34 purified (CD34+) and unfractioned bone marrow (BM) cells of MDS patients and healthy females. BM, CD34+ and PB cells were obtained from patients with MDS (IPSS-low/int-1-risk BM: n=25, CD34+: n=13, PB: n=21, IPSS-int-2/high-risk BM: n=16, CD34+: n=9, PB: n=12) and age related healthy donors (BM: n=19, CD34+: n=15, PB: n=105) after informed consent. Genomic DNA SNP genotyping was carried out in order to screen for heterozygous clonality marker genes located on the X-chromosome, namely BTK, FHL1, IDS, MPP1 and G6PD. After PCR-amplification of informative marker loci from cDNA transcripts, SNP allele ratios, representative for XCIP, were quantified using the PyroMark ID system (Qiagen, Hilden, Germany). Clonality was assumed for samples exhibiting skewed XCIP ratios >80% (allelic ratio of >4:1). Standard curves from pyrosequencing reactions with predefined allelic ratios revealed strong correlations for assessment of XCIP ratios in all markers (R2>0.99). Furthermore, excellent inter-marker correlations of XCIP ratios from individuals with multiple informative markers were achieved ranging from R2>0.83 to R295% in 56% of BM, 62% of CD34+ and 57% of PB cells from MDS low/int-1-risk and 50% of BM, 67% of CD34+ and 67% of PB cells from MDS int-2/high-risk patients but only in 22% of BM, 29% of CD34+ and 3% of PB cells from healthy elderly subjects. Our detection of highly clonal cells in MDS patients in 3 hematopoietic cell compartments and frequent detection of extreme XCIP skewing provides strong evidence for the manifestation of a malignant MDS clone even in early progenitor cells. Moreover, detection of non-clonal PB cells in patients with clonal CD34+ cells indicates the presence of residual healthy hematopoietic cells in the peripheral blood. Notably, in our hands, age related increases of XCIP skewing have been detected in PB cells and, for the first time, in CD34+ and BM cells of healthy females suggesting the clonal outgrowth of hematopoietic cells in the early progenitor compartment of healthy elderly. In conclusion, pyrosequencing based clonality assessment has proven to be suitable for quantitative monitoring of clonal and potentially malignant cell populations in hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Abstract 1760 Poster Board I-786 Myelodysplastic syndromes (MDS) are clonal hematologic malignancies with molecular defects most probably arising in the hematopoietic stem or progenitor compartment. However, due to a frequent lack of trackable cytogenetic aberrations in a large proportion of MDS patients the capability to monitor the manifestation and origin of malignant MDS clones remains limited. To elucidate clonal dominance in a given cell population, the analysis of skewed X-chromosome inactivation patterns in females, based on the methylation analysis of X-chromosomal HUMARA alleles has been used widely. However, this method has several technical and biological drawbacks as methylation changes can be induced with increasing age leading to inaccuracy of the method in this context. Recently, the application of a quantitative PCR-based method to accurately detect single nucleotide polymorphism (SNP) allele-specific RNA transcription from the X-chromosome (Swierczek et al, Blood, 2008) has shown robust results for reliable calculation of X-chromosome allelic ratios. In our study we employed this method to assess clonality in CD34+ selected and unselected bone marrow cells derived from MDS patients and provide evidence for distinct clonal manifestations of MDS clones in hematopoietic progenitor cells of all analysed MDS samples as compared to healthy controls. Bone marrow (BM) cells were obtained from patients with MDS (IPSS-low/int-1-risk n=9, IPSS-int-2/high-risk n=9) after informed consent. Immunomagnetic selection of CD34+ cells was performed from the BM samples of MDS patients (IPSS-low/int-1-risk n=8, IPSS-int-2/high-risk n=10) and age related healthy donors (n=6) served as controls for normalization. Genomic DNA SNP genotyping using Taqman SNP Genotyping Assays (Applied Biosystems, Foster City, CA) was carried out in order to screen for informative clonality marker genes located on the X-chromosome, namely BTK, FHL1, IDS and MPP1. RNA transcripts from different alleles were quantified using SNP/allele-specific primers in a Taqman based quantitative PCR approach. Individual allelic ratios were calculated as previously described. Clonality values were assigned to 0 % according to an allelic ratio of 50/50 (polyclonal) up to 100 % for a ratio of 100/0 (clonal). All clonality values are presented as mean. Our analyses revealed a remarkably elevated proportion of clonal cells in all purified CD34+ cells from MDS low/int-1-risk (88 %) and MDS int-2/high-risk patients (98 %) compared to the cells from healthy donors (16 %, p Our observation of specific clonality in both unselected bone marrow and purified CD34+ cells of MDS patients as compared to healthy controls underlines the proliferative manifestation of malignant MDS clones even in early hematopoietic progenitor cells. Furthermore, the high degree of clonality in all purified CD34+ cells suggests a clonal involvement of not only myeloid but also lymphoid lineages. Interestingly, we also identified 2 patients harboring polyclonal cells in the unselected bone marrow. In these cases differentiating cells in the bone marrow may be sustained by residual healthy hematopoietic progenitor cells. The determination whether patients with this constellation have a different clinical prognosis from patients with clonality of the complete bone marrow may be interesting to pursue. In summary, determination of clonality levels in distinct cell populations of hematologic malignancies using quantitative PCR appears to be highly suitable for monitoring the manifestation and origin of a malignant hematopoietic stem/precursor cell. Disclosures No relevant conflicts of interest to declare.

Objective Identification of genomic lesions in progenitor cells of patients with myelodysplastic syndrome (MDS) could lead to the discovery of new disease-specific genes and may be of prognostic value. Materials and Methods We carried out a genome-wide mapping of DNA from CD34+ cells of MDS patients with high-resolution 500K single nucleotide polymorphism arrays and a concomitant integration with global gene expression analysis. Thirteen MDS patients were analyzed. Results Copy number and loss of heterozygosity analyses detected heterozygous deletions on chromosomes 2, 9, 13, 16, 17, and 20 ranging in size from 0.1 megabases (Mba) to 2.1 Mba. Additionally, numerous regions with significant uniparental disomy were detected. Integration of the genomic data with gene expression analysis showed that genes, which were downregulated at least 1.5-fold in regions of significant deletion and uniparental disomy were exclusively downregulated in those samples displaying the aberration. Genomics and gene expression data were confirmed by real-time polymerase chain reaction and variable number tandem repeat analysis. Conclusion High-density genomic mapping of CD34+ bone marrow cells from patients with MDS identifies cryptic genetic lesions and offers new opportunities for the discovery of target genes in MDS by integration with gene expression analysis.

Introduction: Disturbed proliferation and differentiation are the most crucial oncogeneic factors leading to malignant turnover of hematopoiesis in myeloid malignancies. Therefore, estimating the lifetime proliferation status of malignant hematopoietic cells is critical. Recently the hypothesis of an epigenetic molecular clock has been corroborated. Depending on the accumulation of CpG methylation errors throughout life after each cell division it is possible to measure an increased DNA methylation of formerly unmethylated CpG islands and subsequently relate it to the mitotic cell age. In order to elucidate the importance of disturbed proliferation in hematologic diseases we initiated a novel approach for profiling mitotic ages of hematopoietic cells in myelodysplastic syndrome and acute leukemia. Patients & Methods: Bone marrow (BM) cells of patients with myelodysplastic syndrome (MDS, IPSS-low/int-1-risk n=23, IPSS-int-2/high-risk n=27), acute myeloid leukemia (AML, n=55), acute lymphoblastic leukemia (ALL, T-lineage n=40, B-lineage n=8), and of age matched healthy individuals (n=24) were analyzed. In addition, selection of CD34+ cells was performed in MDS (n=43), in AML (n=10) as well as in healthy BM samples (n=31). CD19+ peripheral blood cells from healthy donors (n=13) served as an additional control. Genomic DNA was isolated and bisulfite converted using standard TRIZOL technique (Invitrogen, Carlsbad/CA, USA) followed by EpiTect-Bisulfite-Kit conversion (Qiagen, Hilden, Germany). PCR amplification of a CpG rich 3′ site of the Cardiac Specific Homeobox gene (CSX), considered as an epigenetic molecular clock locus, was performed as previously reported. DNA methylation was quantitative measured using the PyroMark ID Pyrosequencing system (Biotage, Uppsala, Sweden). Quantitative DNA methylation data are presented with mean ± S.E.M. Results: In MDS int-2/high-risk specific DNA methylation of BM (26.6 ± 1.8 %) and CD34+ (28.6 ± 2.7 %) was significant higher compared to low/int-1-risk MDS (BM: 19.2 ± 1.6 %, p=0.0047, CD34+: 18.7 ± 2.4 %, p=0.0093) and healthy donors (BM: 17.8 ± 0.5 %, CD34+: 17.0 ± 0.4 %, p Discussion: The significant higher CSX methylation in AML compared to int-2/high-risk and in int-2/high-risk compared to low/int-1-risk MDS or healthy individuals could possibly be considered as a disease stage related molecular marker. The intra-individual similarity of CSX methylation levels between BM and CD34+ cells in int-2/high-risk MDS patients supports the theory of a stem cell origin of this disease subgroup, whereas low/int-1-risk MDS samples reveal higher differences possibly pointing to an origin in a more differentiated progenitor cell. However, the observation of higher mitotic ages in T-lineage but not B-lineage ALL raises questions about the role of cell proliferation in distinct lymphoblastic leukemias. In summary, the determination of mitotic cell ages by quantitative DNA methylation analysis could contribute to the molecular classification of hematological malignancies and may further be used for riskassessment in patients with MDS.

Myelodysplastic syndromes (MDS) are mainly characterized by dyserythropoiesis resulting in anemia. So far, this pathological hallmark is incompletely understood. Notch signalling has recently been linked to impaired erythropoiesis and megakaryopoiesis of CD34+ progenitor cells, however its role in MDS is unclear. On the other hand, in MDS demethylating therapy results in decreased transfusion dependency, indicating aberrant methylation of key erythroid genes. Therefore, we have analyzed Notch pathway elements and its association with the key erythroid factors GATA1 and BCLxl and examined the methylation of CpGs flanking cis-regulatory elements (including N-box suppressor binding site for HES1 and GATA box) of the GATA1 erythroid promoter in differentiating CD34+ cells selected from MDS patients. We have generated an in-vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ bone marrow cells from healthy donors (n=7) and MDS patients (low risk: RA/n=6, RARS/n=3; high risk: RAEB/n=4, RAEB-T/n=2) with EPO. Cell harvest was at days 0, 4, 7 and 11. RNA-expression of GATA1, BCLxl, DLK1, Notch1, HES1 and HERP2 was measured by real time RT-PCR (qPCR). GPI was used as a housekeeping gene. DNA methylation at 7 CpGs of the GATA1 gene promoter was quantitatively analyzed by Pyrosequencing (Pyro Mark ID, Biotage, Uppsala, Sweden) of bisulfite treated genomic DNA at any specific time point. In normal erythropoietic cells, RNA expression of GATA1 and of BCLxl was steadily up regulated, particularly during late erythropoietic differentiation. In contrast, during MDS erythropoiesis a loss of typical up regulation of GATA1 (day 11: 2.08 vs. 0.11; p=0.001) and BCLxl (day 11: 7.46 vs. 0.16; p=0.0005) was observed. Notch ligand DLK1 showed increased expression during erythropoiesis particularly in high risk MDS as compared to normal controls (days 4–11: 0.38 vs. 0.03; p=0.02). Furthermore, expression of HES1 was increasing during the course of normal erythropoietic differentiation but not in lineage specific cells from MDS patients (day 11: 0.01 vs. 0.006; p=0.1). No hypomethylation of CpGs flanking repressor HES1 binding site within the 5′-GATA region was detected in MDS erythropoiesis. Interestingly, decremental GATA1 promotor methylation values were seen during normal erythropoiesis matching GATA1 RNA up regulation in contrast to MDS erythropoiesis (day 11: 26% vs. 58%; p=0.00004). Our data show that the critical erythropoietic transcription factor GATA1 as well as the antiapoptotic molecule BCLxl fail to be up regulated during MDS erythropoiesis. The higher residual 5′-GATA1 methylation values in MDS erythropoiesis but decremental loss thereof in normal erythropoiesis suggests a gene dose effect for GATA1 during erythropoiesis being finely tuned by CpG methylation. Its dysregulation may contribute to the ineffective erythropoiesis observed in MDS. However, a transcriptional activation of the Notch pathway leading to increased expression of the GATA1 repressor HES1 and a hypomethylation of its binding site could not be detected in MDS.

Notch signals have recently been shown to inhibit erythroid and megakaryocytic differentiation of hematopoietic progenitor cells. In myelodysplastic syndrome (MDS) its role in dyserythropoiesis has not been fully elucidated. Therefore we asked whether dysregulation of Notch pathway elements might be associated with impaired GATA1 and BCLxl expression and ineffective erythropoiesis being a hallmark of MDS hematopoiesis. We have generated an in-vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ bone marrow cells from healthy donors (n=7) and MDS patients (low risk: RA/n=6, RARS/n=3; high risk: RAEB/n=4, RAEB-T/n=2) with EPO and TPO. Cell harvest was at days 0, 4, 7 and 11. Expression of GATA1, BCLxl, DLK1, Notch1, HES1 and HERP2 was measured by real time RT-PCR (qPCR). RNA expression of GATA1 and of BCLxl was steadily upregulated, particularly during late normal erythropoiesis. During normal megakaryopoiesis expression of both genes was up to 50 times lower as compared to normal erythropoiesis. In contrast, during MDS erythropoiesis a loss of typical late upregulation of GATA1 and BCLxl was observed. DLK1 expression during erythropoiesis showed increased expression particularly in high risk MDS vs. normal controls. Expression of HES1 was increasing during the course of normal erythropoietic and megakaryopoietic differentiation but not in lineage specific cells from MDS patients. In conclusion our data show that the central erythropoietic transcription factor GATA1 and the associated antiapoptotic molecule BCLxl are markedly downregulated during MDS erythropoiesis which may contribute to the ineffective erythropoiesis seen in this disease. Increased DLK1 expression in differentiated stem cells from high risk MDS patients was seen. However, an upregulation of the Notch pathway leading to increased expression of the GATA1 repressor HES1 could not be detected.

Identification of common genomic lesions in progenitor cells of MDS Patients could lead to the discovery of new target genes in this disease and may be of prognostic value. Therefore, we carried out a detailed genome-wide mapping of genomic DNA from highly purified CD34+ progenitor cells from MDS patients and healthy individuals with high-resolution single nucleotide polymorphism (SNP) microarrays which scan 500,000 SNPs with a median inter-SNP distance of approximately 2.5 kb. Bone marrow aspirates were obtained from 14 MDS patients (IPSS low risk n=6, high risk n=8) and 6 healthy individuals after informed consent. CD34+ cells were purified by high gradient magnetic cell separation. Genomic DNA and RNA were extracted with standard TRIZOL technique and quality controlled with the Agilent Bioanalyzer 2100 and Nanodrop ND-1000 systems. 500 ng of each of the genomic DNA were processed according to the protocol of the Affymetrix 500 k NspI and StyI genomic mapping protocol, hybridized to 500 k NspI/StyI chip sets and scanned on an Affymetrix GeneChip scanner 3000. The median SNP call rate of analysed samples was 88.6% and ranged from 76.3% to 95.4%. One sample from the MDS patients and two samples from the healthy donors were excluded from analysis due to insufficient call rates. Raw signal intensity data was generated by the GCOS 4.0 software and imported into Partek Genomics 6.2 software. The control samples of healthy individuals were assigned a copy number of two and used as a reference baseline to calculate copy numbers in MDS samples. On the calculated values genomic smoothing was performed with a window width of 0.5 Mbps and a Gaussian width at half maximum 50% of window width. Significant regions of copy number alterations were calculated with a test region width of 0.5 Mbp and contiguous regions set to contain at least 1 Mbp (p

Recent findings suggest that in myelodysplastic syndrome (MDS) several key regulatory genes are affected by aberrant promotor methylation. To explore the molecular basis of this impairment we have generated an in vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ cells from healthy donors (n=7) and MDS patients (low risk: RA/n=6, RARS/n=3; high risk: RAEB/n=4, RAEB-T/n=2) with EPO, TPO and GCSF. Cell harvest was at days 0, 4, 7 and 11. Promotor methylation analysis of key genes involved in the control of apoptosis (p73, survivin, DAPK), DNA-repair (hMLH1), differentiation (RARb, WT1) and cell cycle control (p14, p15, p16, CHK2) was performed by methylation specific PCR of bisulfite treated genomic DNA for each lineage at each time point. In addition, expression of DNMT1 (maintenance DNA methyltransferase), DNMT3a and DNMT3b (both de novo DNA methyltransferase) was analyzed by real time RT-PCR and correlated with gene promotor methylation at any time point. DNMT1 expression was increased during erythropoiesis in both, normal controls and MDS patients. On the other hand, expression of de novo DNMTs was elevated during thrombopoiesis at all time points. During erythropoiesis hypermethylation of p73, hMLH1 and RARb was associated with elevated DNMT1, hypermethylation of p15, p16, p73 and survivin was positively associated with increasing DNMT3 expression. Interestingly, DNMT1 was only elevated in low risk MDS, but not further increased in high risk MDS patients. Surprisingly, MDS specific survivin promotor methylation was inverse correlated with DNTM1 and DNMT3a expression. However, a negative correlation of DNMT3a with survivin expression was found in low risk MDS but not in high risk MDS. In summary our data indicate that all mammalian DNMT isoforms may be involved in the aberrant DNA-methylation phenotype in MDS. Elevated DNMT1 expression may in particular contribute to ineffective erythropoiesis in low risk MDS. DNMT3a and 3b were elevated during megakaryopoiesis and their expression was inversely correlated with MDS disease risk (IPSS). We conclude that the knowledge about distinct expression patterns of DNMT isoforms in hematopoiesis may be of help for further strategies to implicate DNMT-inhibitors in the treatment of patients with MDS.

Survivin has recently been shown to critically regulate erythroid versus megakaryocytic differentiation in mouse hematopoietic stem cells. Its role in human hematopoiesis has not been fully elucidated yet. To answer the question whether dysregulation of expression of survivin and its protein stabilisator HSP90 contributes to the ineffective erythropoiesis in myelodysplastic syndrome (MDS), we have generated an in vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ cells from healthy donors (n=7) and MDS patients (low risk: RA/n=6, RARS/n=3; high risk: RAEB/n=4, RAEB-T/n=2) with EPO, TPO and GCSF. Cell harvest was at days 0, 4, 7 and 11. Expression analysis of survivin splicing variants (wt, 2a, d3ex) and of HSP90 was done by real time RT-PCR (qPCR) for each lineage at each time point. Furthermore, epigenetic analysis of key regulatory genes by methylation specific PCR and qPCR to detect DNMT1 (maintenance methylation) and DNMT3a and 3b (de novo methylation) expression was performed for each of the experimental conditions. RNA expression of all survivin isoforms and of HSP90 was continously upregulated during normal erythropoiesis. During late megakaryopoiesis of normal hematopoietic stem cells a significant downregulation was seen with elevated expression values for survivin wt and survivin 2b during early thrombopoiesis. In contrast, during MDS erythropoiesis a significant downregulation of expression of all survivin isoforms and HSP90 during late erythropoiesis was observed. Lower expression for survivin wt, survivin 2b and HSP90 was seen during early MDS megakaryopoiesis. Promotor methylation analyisis revealed MDS specific aberrant methylation for survivin, particularly during MDS high risk erythropoiesis (p=0.02). However, expression of DNMT1 was only elevated during erythropoiesis and in low risk MDS. A significant inverse correlation between RNA expression of DNMT enzymes and survivin splicing variants could not be detected. In conclusion our data support the essential role for survivin in regulation of erythropoietic versus megakaryopoietic differentiation of hematopoietic precursor cells. The dysregulation of survivin expression during MDS erythropoiesis may therefore account for the ineffective erythropoiesis known in MDS. Furthermore, downregulation of survivin (protein) stabilisator HSP90 might additionally contribute to this effect. Although an inverse correlation with overexpression of DNMTs and downregulation of survivin splicing variants was not seen, the aberrant survivin promoter methylation during MDS erythropoiesis indicates that epigenetic dysregulation results in low survivin expression.

Promotor demethylation of oncogenes has been associated with transcriptional activation in cancer cells. The proto-oncogene HOX11/TLX1 has been found to be aberrantly expressed in up to 30% of adult T-ALL patients. In few, a translocation between the HOX11 locus at 10q24 and the T-cell receptor locus has been identified. In the majority of cases the mechanism leading to HOX11 reactivation remains unclear. It had been proposed that an epigenetical modification by demethylation of the proximal HOX11 promotor could be responsible for an aberrant expression of HOX11. To test this hypothesis we have correlated the methylation status of CpG residues in the proximal HOX11 promotor with the gene expression status of HOX11 in adult T-ALL samples from the German Multicenter ALL Study (GMALL) 5/93 and 6/99. HOX11 expression was measured in 286 pretreatment peripheral blood and bone marrow blasts by comparative real-time RT-PCR as described previously. The methylation status was then randomly analyzed after bisulphite treatment by methylation-specific PCR (MSP) in 53 T-ALL samples with HOX11 expression (HOX11 positive) and 102 samples without HOX11 expression (HOX11 negative). Of the 150 analyzed patients only 4% of HOX11 positive patients (n=53) and 10% of HOX11 negative patients were methylated (M) in the analyzed promotor area. HOX11 negative patients were significantly more common associated with an unmethylated status (U) then HOX11 positive patients (57% vs 32%, p=0.003). The most prominent methylation phenotype in HOX11 positive patients compared to HOX11 negative samples was a mixed (MU) methylation status (55% vs. 27%, p=0.001). Interestingly, remission duration was significantly higher in pt. with the MU methylation status (M=49%, U=54% vs. MU=76%, p-logrank=0.0362). This translated also into a significant difference in the overall survival (M=55%, U=49%, MU=75%, p-logrank= 0.0202). However, in a multivariate analysis the methylation status could not be confirmed as an independent prognostic factor. The promotor-associated CpG methylation status was found to be remarkably heterogenous in the analyzed adult T-ALL patients with a predominantly unmethylated status in HOX11 negative samples and a mixed methylated/unmethylated picture in HOX11 positive samples. These findings contrast with our initial hypothesis that HOX11 expression is silenced in normal tissue by a promotor-associated CpG methylation and aberrantly reexpressed in leukemia cells by demethylation. However, various CpG residues associated with the HOX11 promotor might be of variable importance for the gene expression and intrinsic limitations of methylation-specific PCR might have influenced our analysis. Bisulphite sequencing techniques as well as the newly developed genome-wide cytosine methylation array could help overcome these issues. Understanding the role of promotor-associated methylation in HOX11 expression could clarify pathways in leukemogenesis and provide a valuable tool for better risk stratification in T-ALL.

The molecular pathogenesis of myelodysplastic syndromes (MDS) is poorly understood. To detect epigenetic alterations of key regulatory genes during lineage-specific hematopoietic differentiation, CD34+ cells from healthy donors (n=7) and MDS patients with low risk (LR; RA/n=6, RARS/n=3) and high risk (HR; RAEB/n=4, RAEB-T/n=2) MDS according to IPSS were in vitro differentiated with EPO, TPO or GCSF and harvested at days 0, 4, 7 and 11. For each lineage at each time point, promotor methylation analysis of genes involved in cell cycle control (p14, p15, p16, CHK2), DNA-repair (hMLH1), apoptosis (p73, survivine, DAPK) and differentiation (RARb, WT1) was performed by methylation specific PCR (MSP) of bisulfite converted genomic DNA. Corresponding gene expression was analyzed by oligonucleotide microarrays. Remarkably in CD34+/d0 cells p16 was methylated in 0% controls in contrast to 22% in MDS LR- and 50% in MDS HR-patients. Likewise in CD34+/d0 cells WT1 methylation was found in 0% controls but in 62% MDS LR- and 33% MDS HR-patients. During differentiation (d4, d7, d11) survivine promoter methylation was found in all 3 hematopoietic lineages in MDS only (LR/EPO: 22%, LR/TPO: 11%, LR/GCSF: 55%; HR/EPO: 67%, HR/TPO: 33%, HR/GCSF: 67%). WT1 promotor methylation during differentiation was almost absent in controls (EPO: 0%, TPO: 0%, GCSF: 29%) in contrast to MDS (LR/EPO: 22%, LR/TPO: 11%, LR/GCSF: 55%; HR/EPO: 17%, HR/TPO: 17%, HR/GCSF: 67%). CHK2 methylation during differentiation was not found in controls but in MDS patients (LR/EPO: 44%, LR/TPO: 22%, LR/GCSF: 22%; HR/EPO: 33%, HR/TPO: 33%, HR/GCSF: 50%). Interestlingly, mRNA expression of survivine was significantly reduced in methylated samples during erythropoiesis (p=0,04). Unexpectedly p73 RNA-expression was lower during granulopoiesis (p=0,052) in unmethylated samples. Similarly p16 mRNA showed significantly reduced expression during erythropoiesis (p=0,034) and on day 10 (p=0,064) and day 14 (p=0,035) during lineage specific differentiation in general in unmethylated specimen. Furthermore in low risk MDS WT1 RNA-expression was significantly reduced for methylated samples (p=0,01). P16 hypermethylation of CD34+/d0 cells shows a trend to correlate with MDS IPSS, but higher p16 mRNA expression values were found in methylated cases. Similarly a trend for IPSS risk correlation is shown for survivine promotor methylation during lineage specific differentiation, making it a potential marker of MDS progression, which leads to reduced mRNA expression particularly in erythropoiesis. For WT1 and CHK2 methylation patterns specific for MDS were identified, corresponding to reduced expression in WT1 promotor methylated LR-MDS. The detailed exploration of epigenetic alterations of these genes during early hematopoesis in MDS may give new insights into the pathophysiology of MDS. In particular this study identified survivine and WT1 as genes with lower methylation associated expression in MDS hematopoietic precursor cells which may ultimately contribute to the clonal expansion of the malignant cell clone.

hCDC4 (FBW7, FBXW7) is a new potential tumor suppressor gene which provides substrate specificity for SCF (Skp–Cullin–F-box) ubiquitin ligases and thereby regulates the degradation of potent oncogenes such as cyclin E, Myc, c-Jun and Notch. Mutations in the hCDC4 gene have been found in several solid tumors such as pancreas, colorectal or endometrial cancer. We carried out a mutation analysis of the hCDC4 gene in 35 samples of patients with Acute Myeloid Leukemia (AML) to elucidate a possible role of hCDC4 mutations in this disease. By direct DNA sequencing and digestion with Surveyor nuclease one heterozygous mutation in the 5' untranslated region of exon 1, transcript variant 3 was detected. Additionally, we could identify a new intronic SNP downstream of exon 10. The new variation was present in 20% of AML samples and was furthermore confirmed in a panel of 51 healthy individuals where it displayed a frequency of 14%. In conclusion we provide first data that in contrast to several solid tumors, mutations in the hCDC4 gene may not play a pivotal role in the pathogenesis of AML. Furthermore, we describe a new intronic polymorphism with high frequency in the intron sequence of the hCDC4 gene.