Search

Despite the increasing role of molecular markers, differential counts and morphology of hematopoietic cells in the bone marrow (BM) remain essential diagnostic criteria in hematological diseases. However, the respective reference values for BM myelogram commonly used came from small series with limited numbers of healthy individuals. We evaluated the myelograms of 236 healthy individuals who underwent unrelated bone marrow donation. Health check-ups were performed 4 weeks prior to harvest. Samples for this study, taken from the first aspiration, were stained according to the standard Pappenheim method. Three experienced investigators assessed cellularity, megakaryopoiesis, and differential counts independently. The median donor age was 31 (range 18–51) years. Predonation tests did not reveal any relevant morbidity. Thirty-seven out of 42 hypocellular marrow samples were from younger donors up to 39 years. Content of megakaryocytes was normal in 210 specimens (89%). Gender and body mass index had significant impact on hematopoiesis, whereas age had not. The number of erythroblasts was higher (about 32%) and the proportion granulopoiesis slightly lower (about 50%) compared with previous studies. Differential counts showed also some differences with respect to individual maturation stages in these lines. Interrater comparisons showed greater reliability for the assignment of cells to the different hematopoietic cell lines than for single-cell diagnoses. This study largely confirms the results for cell counts in normal human bone marrow available from previous reports and provides some insights into factors that affect individual cell populations. It also reveals substantial variability among even experienced investigators in cytological diagnoses.

Background Double induction using two subsequent 7+3 regimens of cytarabine plus anthracycline is commonly performed in AML patients with an adequate performance status in order to maximize dose intensity upfront. However, for patients with a good early response at day 15 of first induction, there is no prospective randomized evidence on the necessity or value of a second induction cycle. Aims In order to answer the question if good responders of the first 7+3 induction could be spared a second induction cycle, we set up randomized-controlled SAL DaunoDouble trial. The study prospectively assesses the outcome of patients with a good early response with respect to the number of induction cycles (single versus double). We assumed non-inferiority of single induction in terms of complete remission (CR/CRi) rate, based on a margin of 7.5%. Here, we present the results of the planned interim analysis. Methods Patients (pts) 18-65 years with newly diagnosed AML, normal cardiac and organ function received a first induction cycle with seven days of cytarabine plus three days of daunorubicin ("7+3"). Response assessment in bone marrow was done on day 15 after the initiation of chemotherapy and confirmed by central review. A blast count Results Between 2014 and 2020, 624 evaluable pts were enrolled and received the first induction cycle with 7+3. A marrow blast clearance below 5% on day 15 was achieved in 298 pts (48%), providing eligibility for randomization. Of these patients, 150 were randomized into arm S and 148 into arm D, respectively. Median age was 52 years, 92% had de novo AML, NPM1 mutation was present in 53%, FLT3-ITD in 25% of pts. Favorable, intermediate and adverse risk (ELN 2017) were present in 56%, 34% and 10% of pts, respectively. CR/CRi rates at the end of induction were 86% after single induction and 85% after double induction. The CR/CRi rates in 224 pre-defined per-protocol pts were 88% versus 91%, resulting in a CR difference of 3% (95%-CI -0.047-0.111; p for non-inferiority test 0.145). After a median follow-up time of 24 months, RFS was slightly but not significantly lower after single induction with a 3-year RFS of 53% versus 64% (HR 1.4, p=0.125), whereas no differences were seen in 3-year OS, with a of rate of 74% versus 75% (HR 1.1, p=0.645) after single versus double induction. Conclusion The interim analysis results show that in good responders, the difference between CR rates after single versus double induction was even smaller than the predefined 7.5% margin, suggesting a trend for non-inferiority of single induction, although statistical significance was not reached. The trial continued recruitment. These findings suggest that in good responders, it may be safe to omit a second induction cycle if a second cycle poses a high risk. Figure. CR + CRi, RFS and OS after randomization to single versus double induction. Disclosures Alakel: Pfizer: Consultancy. Jost:Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; JAZZ: Other: travel support; Celgene: Other: travel support. Novak:Roche: Consultancy; Janssen: Other: Travel expenses; Takeda: Consultancy; Amgen: Consultancy, Other: Travel expenses; Pfizer: Consultancy; Novartis: Consultancy. Krause:Takeda: Honoraria; Celgene: Other: Travel Support; MSD: Honoraria; Pfizer: Honoraria; Siemens: Research Funding; Gilead: Other: Travel Support. Held:Roche: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding; BMS: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding; MSD: Consultancy; Acrotech: Research Funding; Spectrum: Research Funding; Amgen: Research Funding. Platzbecker:AbbVie: Consultancy, Honoraria; Amgen: Honoraria, Research Funding; Geron: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria. Thiede:AgenDix GmbH: Other: Co-owner and CEO. Müller-Tidow:Daiichi Sankyo: Research Funding; Pfizer: Research Funding, Speakers Bureau; BiolineRx: Research Funding; Janssen-Cilag GmbH: Speakers Bureau.

Inflammation-induced thrombosis represents a severe complication in patients with myeloproliferative neoplasms (MPN) and in those with kidney dysfunction. Overlapping disease-specific attributes suggest common mechanisms involved in MPN pathogenesis, kidney dysfunction, and thrombosis. Data from 1420 patients with essential thrombocythemia (ET, 33.7%), polycythemia vera (PV, 38.5%), and myelofibrosis (MF, 27.9%) were extracted from the bioregistry of the German Study Group for MPN. The total cohort was subdivided according to the calculated estimated glomerular filtration rate (eGFR, (mL/min/1.73 m2)) into eGFR1 (≥90, 21%), eGFR2 (60–89, 56%), and eGFR3 (&lt, 60, 22%). A total of 29% of the patients had a history of thrombosis. A higher rate of thrombosis and longer MPN duration was observed in eGFR3 than in eGFR2 and eGFR1. Kidney dysfunction occurred earlier in ET than in PV or MF. Multiple logistic regression analysis identified arterial hypertension, MPN treatment, increased uric acid, and lactate dehydrogenase levels as risk factors for kidney dysfunction in MPN patients. Risk factors for thrombosis included arterial hypertension, non-excessive platelet counts, and antithrombotic therapy. The risk factors for kidney dysfunction and thrombosis varied between MPN subtypes. Physicians should be aware of the increased risk for kidney disease in MPN patients, which warrants closer monitoring and, possibly, early thromboprophylaxis.

Cancers 13(12), 2905 (2021). doi:10.3390/cancers13122905, Published by MDPI, Basel

Early results of the randomized placebo-controlled SORAML trial showed that, in patients with newly diagnosed acute myeloid leukaemia (AML), sorafenib led to a significant improvement in event-free (EFS) and relapse-free survival (RFS). In order to describe second-line treatments and their implications on overall survival (OS), we performed a study after a median follow-up time of 78 months. Newly diagnosed fit AML patients aged ≤60 years received sorafenib (n = 134) or placebo (n = 133) in addition to standard chemotherapy and as maintenance treatment. The 5-year EFS was 41 versus 27% (HR 0.68; p = 0.011) and 5-year RFS was 53 versus 36% (HR 0.64; p = 0.035). Allogeneic stem cell transplantation (allo SCT) was performed in 88% of the relapsed patients. Four years after salvage allo SCT, the cumulative incidence of relapse was 54 versus 35%, and OS was 32 versus 50%. The 5-year OS from randomization in all study patients was 61 versus 53% (HR 0.82; p = 0.282). In conclusion, the addition of sorafenib to chemotherapy led to a significant prolongation of EFS and RFS. Although the OS benefit did not reach statistical significance, these results confirm the antileukaemic activity of sorafenib.

In patients with relapsed or refractory (r/r) acute myeloid leukemia (AML), allogeneic hematopoietic stem cell transplantation (HSCT) is considered to be the only treatment providing long-term disease control. The BRIDGE trial studied the safety and efficacy of a clofarabine-based salvage therapy before HSCT in patients with r/r AML. Here, we report the long-term follow-up of this phase II multicenter trial and exploratory analyses on the impact of comorbidity on outcome. Eighty-four patients with a median age of 61 years (range, 40 to 75) were enrolled. Patients were scheduled for at least 1 cycle of salvage therapy with CLARA (clofarabine 30 mg/m2; cytarabine 1 g/m2, days 1 to 5). Chemo-responsive patients with a donor received HSCT after first CLARA. The conditioning regimen consisted of clofarabine 30 mg/m2, day −6 to −3, and melphalan 140 mg/m2 day −2. The Eastern Cooperative Oncology Group (ECOG) score, the hematopoietic cell transplantation–specific comorbidity index (HCT-CI), and the Cumulative Illness Rating Scale were obtained at study enrollment as well as before HSCT. Sixty-seven percent of the patients received HSCT within the trial. After a median follow up of 40 months, the estimated 3-year overall survival (OS) for all enrolled patients and those with HSCT within the trial was 40% and 55%, respectively. Relapse-free survival for patients who underwent transplantation with a complete remission afterwards (n = 50) was 48%, calculated from the day of transplantation. In multivariate analysis, both the HCT-CI and ECOG score had a statistically significant impact on OS with a hazard ratio of 1.22 (P = .025)and 1.72 (P = .001), respectively. Using a clofarabine-based salvage therapy combined with early allogeneic HSCT, we were able to achieve good long-term results for patients with r/r AML. In this cohort, both the HCT-CI and the ECOG scores gave prognostic information on OS, showing the feasibility and clinical relevance of comorbidity evaluation at the time of diagnosis of r/r AML patients.

Extramedullary (EM) disease in patients with acute myeloid leukemia (AML) is a known phenomenon. Since the prevalence of EM AML has so far only been clinically determined on examination, we performed a prospective study in patients with AML. The aim of the study was to determine the prevalence of metabolically active EM AML using total body 18Fluorodesoxy-glucose positron emission tomography/computed tomography (18FDG-PET/CT) imaging at diagnosis prior to initiation of therapy. In order to define the dynamics of EM AML throughout treatment, PET-positive patients underwent a second 18FDG-PET/CT imaging series during follow up by the time of remission assessment. A total of 93 patients with AML underwent 18FDG-PET/CT scans at diagnosis. The prevalence of PET-positive EM AML was 19% with a total of 65 EM AML manifestations and a median number of two EM manifestations per patient (range, 1-12), with a median maximum standardized uptake value of 6.1 (range, 2-51.4). When adding those three patients with histologically confirmed EM AML who were 18FDG-PET/CT negative in the 18FDG-PET/CT at diagnosis, the combined prevalence for EM AML was 22%, resulting in 77% sensitivity and 97% specificity. Importantly, 60% (6 of 10) patients with histologically confirmed EM AML still had active EM disease in their follow up 18FDG-PET/CT. 18FDG-PET/CT reveals a high prevalence of metabolically active EM disease in AML patients. Metabolic activity in EM AML may persist even beyond the time point of hematologic remission, a finding that merits further prospective investigation to explore its prognostic relevance. (Trial registered at clinicaltrials.gov identifier: 01278069.)

Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPN) comprise a heterogeneous group of chronic hematologic malignancies. The quality of life, morbidity, and mortality of patients with MPN are primarily affected by disease-related symptoms, thromboembolic and hemorrhagic complications, and progression to myelofibrosis and acute leukemia. Major bleeding represents a common and important complication in MPN, and the incidence of such bleeding events will become even more relevant in the future due to the increasing disease prevalence and survival of MPN patients. This review discusses the causes, differential diagnoses, prevention, and management of bleeding episodes in patients with MPN, aiming at defining updated standards of care in these often challenging situations.

The optimal timing of allogeneic hematopoietic stem cell transplantation (HCT) in acute myeloid leukemia (AML) is controversial. We report on 1179 patients with a median age of 48 years who were randomized upfront. In the control arm, sibling HCT was scheduled in the first complete remission for intermediate-risk or high-risk AML and matched unrelated HCT in complex karyotype AML. In the experimental arm, matched unrelated HCT in first remission was offered also to patients with an FLT3-ITD (FMS-like tyrosine kinase 3-internal tandem duplication) allelic ratio >0.8, poor day +15 marrow blast clearance and adverse karyotypes. Further, allogeneic HCT was recommended in high-risk AML to be performed in aplasia after induction chemotherapy. In the intent-to-treat (ITT) analysis, superiority of the experimental transplant strategy could not be shown with respect to overall survival (OS) or event-free survival. As-treated analyses suggest a profound effect of allogeneic HCT on OS (HR 0.73; P=0.002) and event-free survival (HR 0.67; P

Philadelphia-negative myeloproliferative neoplasms (MPN) comprise a heterogeneous group of chronic hematological malignancies with significant variations in clinical characteristics. Due to the long survival and the feasibility of oral or subcutaneous therapy, these patients are frequently treated outside of larger academic centers. This analysis was performed to elucidate differences in MPN patients in three different health care settings: university hospitals (UH), community hospitals (CH), and office-based physicians (OBP). The MPN registry of the Study Alliance Leukemia is a non-interventional prospective study including adult patients with an MPN according to WHO criteria (2008). For statistical analysis, descriptive methods and tests for significant differences were used. Besides a different distribution of MPN subtypes between the settings, patients contributed by UH showed an impaired medical condition, a higher comorbidity burden, and more vascular complications. In the risk group analyses, the majority of polycythemia vera (PV) and essential thrombocythemia (ET) patients from UH were classified into the high-risk category due to previous vascular events, while for PV and ET patients in the CH and OBP settings, age was the major parameter for a high-risk categorization. Regarding MPN-directed therapy, PV patients from the UH setting were more likely to receive ruxolitinib within the framework of a clinical trial. In summary, the characteristics and management of patients differed significantly between the three health care settings with a higher burden of vascular events and comorbidities in patients contributed by UH. These differences need to be taken into account for further analyses and design of clinical trials.

Summary Background Preclinical data and results from non-randomised trials suggest that the multikinase inhibitor sorafenib might be an effective drug for the treatment of acute myeloid leukaemia. We investigated the efficacy and tolerability of sorafenib versus placebo in addition to standard chemotherapy in patients with acute myeloid leukaemia aged 60 years or younger. Methods This randomised, double-blind, placebo-controlled, phase 2 trial was done at 25 sites in Germany. We enrolled patients aged 18–60 years with newly diagnosed, previously untreated acute myeloid leukaemia who had a WHO clinical performance score 0–2, adequate renal and liver function, no cardiac comorbidities, and no recent trauma or operation. Patients were randomly assigned (1:1) to receive two cycles of induction therapy with daunorubicin (60 mg/m 2 on days 3–5) plus cytarabine (100 mg/m 2 on days 1–7), followed by three cycles of high-dose cytarabine consolidation therapy (3 g/m 2 twice daily on days 1, 3, and 5) plus either sorafenib (400 mg twice daily) or placebo on days 10–19 of induction cycles 1 and 2, from day 8 of each consolidation, and as maintenance for 12 months. Allogeneic stem-cell transplantation was scheduled for all intermediate-risk patients with a sibling donor and for all high-risk patients with a matched donor in first remission. Computer-generated randomisation was done in blocks. The primary endpoint was event-free survival, with an event defined as either primary treatment failure or relapse or death, assessed in all randomised patients who received at least one dose of study treatment. We report the final analysis. This trial is registered with ClinicalTrials.gov, number NCT00893373, and the EU Clinical Trials Register (2008-004968-40). Findings Between March 27, 2009, and Nov 28, 2011, 276 patients were enrolled and randomised, of whom nine did not receive study medication. 267 patients were included in the primary analysis (placebo, n=133; sorafenib, n=134). With a median follow-up of 36 months (IQR 35·5–38·1), median event-free survival was 9 months (95% CI 4–15) in the placebo group versus 21 months (9–32) in the sorafenib group, corresponding to a 3-year event-free survival of 22% (95% CI 13–32) in the placebo group versus 40% (29–51) in the sorafenib group (hazard ratio [HR] 0·64, 95% CI; 0·45–0·91; p=0·013). The most common grade 3–4 adverse events in both groups were fever (71 [53%] in the placebo group vs 73 [54%] in the sorafenib group), infections (55 [41%] vs 46 [34%]), pneumonia (21 [16%] vs 20 [14%]), and pain (13 [10%] vs 15 [11%]). Grade 3 or worse adverse events that were significantly more common in the sorafenib group than the placebo group were fever (relative risk [RR] 1·54, 95% CI 1·04–2·28), diarrhoea (RR 7·89, 2·94–25·2), bleeding (RR 3·75, 1·5–10·0), cardiac events (RR 3·46, 1·15–11·8), hand-foot-skin reaction (only in sorafenib group), and rash (RR 4·06, 1·25–15·7). Interpretation In patients with acute myeloid leukaemia aged 60 years or younger, the addition of sorafenib to standard chemotherapy has antileukaemic efficacy but also increased toxicity. Our findings suggest that kinase inhibitors could be a useful addition to curative treatment for acute myeloid leukaemia. Overall survival after long-term follow-up and strategies to reduce toxicity are needed to determine the future role of sorafenib in treatment of this disease. Funding Bayer HealthCare.

In patients with relapsed or refractory (r/r) acute myeloid leukemia (AML), long-term disease control can only be achieved by allogeneic hematopoietic stem cell transplantation (HSCT). We studied the safety and efficacy of clofarabine-based salvage therapy. The study was designed as phase II, multicenter, intent-to-transplant (ITT) study. A total of 84 patients with r/r AML were enrolled. All patients received at least one cycle of CLARA (clofarabine 30 mg/m(2) and cytarabine 1 g/m(2), days 1-5). Chemo-responsive patients with a donor received HSCT in aplasia after first CLARA. Generally, HSCT was performed as soon as possible. The conditioning regimen consisted of clofarabine (4 × 30 mg/m(2)) and melphalan (140 mg/m(2)). The median patient age was 61 years (range 40-75). On day 15 after start of CLARA, 26% of patients were in a morphologically leukemia-free state and 79% exposed a reduction in bone marrow blasts. Overall, 67% of the patients received HSCT within the trial. The primary end point, defined as complete remission after HSCT, was achieved by 60% of the patients. According to the ITT, overall survival at 2 years was 43% (95% confidence interval (CI), 32-54%). The 2-year disease-free survival for transplanted patients was 52% (95% CI, 40-69%). Clofarabine-based salvage therapy combined with allogeneic HSCT in aplasia shows promising results in patients with r/r AML.

Acute graft-versus-host disease (aGvHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Steroid-resistant aGvHD is associated with poor outcome, and no commonly accepted salvage therapy is available for its treatment. Here, we report 58 adult patients treated with mesenchymal stromal cells (MSCs) as salvage therapy for steroid-refractory aGvHD. Third-party MSCs expanded in platelet lysate-containing medium were transfused at a median dose of 0.99 × 106 cells per kg b.wt. A median of two MSC infusions were administered to each patient. Median time between the onset of aGvHD and the first infusion of MSCs was 12 days (range, 6–62 days). Most patients (79%) had grade IV aGvHD. Five patients showed complete response, five showed very good partial response, 17 showed partial response, and 31 showed no response. The estimated probability of survival after 1 year was 19%, and median survival was 69 days. Overall survival was not significantly different from that of a historical cohort of patients receiving alternative salvage therapy and no MSC infusions. In conclusion, MSC treatment on top of conventional immunosuppression was associated with an overall response rate of 47% but improved outcome in terms of survival remains to be shown.

Background: The addition of sorafenib to standard induction and consolidation therapy in newly diagnosed patients (pts) ≤60 years (yrs) with acute myeloid leukemia (AML) led to significant prolongation of event-free survival (EFS) and relapse-free survival (RFS) in the randomized placebo-controlled SORAML trial (NCT00893373). After a median follow-up of 3 yrs, a benefit for sorafenib treated pts was observed also in overall survival (OS), but this difference was not significant. Here, we present updated survival data and information on relapse treatment and outcome. Methods: In the SORAML trial, 267 newly diagnosed untreated fit AML pts up to 60 yrs of age and irrespective of FLT3 mutation status received two cycles of induction chemotherapy with DA (daunorubicin 60 mg/m2 days 3-5 plus cytarabine 100 mg/m2 cont. inf. days 1-7), followed by three cycles of high-dose cytarabine consolidation (3 g/m2 b.i.d. days 1, 3, 5). Allogeneic stem cell transplantation (SCT) was scheduled for all intermediate-risk pts in first complete remission (CR) with a sibling donor and for all high-risk pts with a matched related or unrelated donor. At study inclusion, pts were randomized to receive either sorafenib (2x400 mg/day) or placebo as add-on to standard treatment in a double blinded fashion. Study medication was given on days 10-19 of DA I+II, from day 8 of each consolidation until 3 days before the start of the next consolidation and as maintenance for 12 months (mos) after the end of consolidation. The primary endpoint of the trial was EFS. The results after follow-up of 3 yrs were presented at ASH 2014 (Röllig et al., Blood 2014; 124: 6) and fully published (Röllig et al., Lancet Oncol 2015; 16: 1691-9). Here, we present the results after prolonged follow-up. For this analysis, information on remission and survival status, mode and outcome of relapse treatment including SCT were collected for all randomized pts and analyzed by standard statistical methods. Results: Of 267 treated pts, 134 were randomized in the sorafenib arm and 133 in the placebo arm with a resulting CR rate of 60% and 59%, respectively. After a median observation time of 78 mos, the primary study endpoint EFS in the placebo vs sorafenib arm was 9 mos vs 26 mos (HR 0.68, p=0.01) in univariate Kaplan Meier analysis. The beneficial effect of sorafenib on EFS was confirmed in multivariate Cox regression analysis with a HR of 0.61 (p=0.005). Median RFS in the placebo vs sorafenib arms was 22 vs 63 mos, corresponding to a HR of 0.64 (p=0.033). Exploratory analyses were performed in the 70 relapsing pts (40 after placebo vs 30 after sorafenib treatment). Among relapsing pts, 82% vs 73% achieved a second CR. In these two groups, 88% and 87% of pts received a SCT as part of salvage treatment. A lower proportion of pts in the placebo arm received a second SCT as salvage treatment (5% vs 13%). In the context of salvage SCT, the proportion of haploident donors in the placebo and sorafenib group was 3% vs 15% and the incidence of Grade 3/4 GVDH was 17% vs 0%. SCT-related non-relapse mortality (NRM) was similar in both groups, but the cumulative incidence of second relapse (CIR) was higher in the sorafenib group (35% vs 54% after 48 mos). Therefore, median OS from relapse in the placebo vs sorafenib groups were 27 mos vs 10 mos, corresponding to a HR of 1.68 (p=0.098). The projected median OS from randomization is 83 mos in the placebo arm and was not reached for the sorafenib arm, corresponding to a 5-year OS of 52% vs 61% (HR 0.81, p=0.263). Conclusion: Mature follow-up data confirms the antileukemic efficacy of sorafenib in younger AML pts with and without FLT3 mutation. The addition of sorafenib to standard chemotherapy resulted in a significantly longer EFS and clinically relevant 36% risk reduction for relapse or death. Five pts need to be treated (NNT) to prevent one relapse or death at 3 years and six pts at 5 yrs. Exploratory analyses in relapsing pts show that survival after relapse is shorter after sorafenib which might be due to i) a higher rate of second SCTs and a higher incidence of haploidentical SCT despite the lower frequency of severe GVHD, most likely by chance and not explainable by systematic reasons and ii) a lower response to salvage treatment after sorafenib therapy. Despite these observations, primary sorafenib treatment led to an OS benefit with a 19% risk reduction for death which was not statistically significant since this phase II trial was not adequately powered to detect OS differences. Figure Figure. Disclosures Rollig: Bayer: Research Funding; Janssen: Research Funding. Hüttmann: Gilead, Amgen: Other: Travel cost; Bristol-Myers Squibb, Takeda, Celgene, Roche: Honoraria. Giagounidis: Acceleron: Consultancy; Celgene: Consultancy. Mackensen: AMGEN: Research Funding. Hänel: Roche: Honoraria; Novartis: Honoraria. Thiede: Roche: Consultancy; Novartis: Consultancy, Speakers Bureau; Bayer: Consultancy, Speakers Bureau; Agendix: Employment. Schetelig: Sanofi Aventis: Consultancy, Research Funding; Roche: Honoraria; Abbvie: Honoraria; Janssen: Consultancy, Honoraria.

Reply to: ‘Flow cytometric identification of acute myeloid leukemia with limited differentiation and NPM1 type A mutation: a new biologically defined entity’ by Kern et al.

[Figure][1] In March 2012, a 31-year-old male patient was diagnosed with alveolar rhabdomyosarcoma (ARMS) manifesting within the left sinus maxillaris with local lymphonodular metastases. No further metastases could be found by positron emission tomography–magnetic resonance imaging. He

In patients with relapsed or refractory (r/r) Acute Myeloid Leukemia (AML), allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is considered to be the only treatment providing long-term disease control for fit patients. The BRIDGE trial studied the safety and efficacy of a clofarabine-based salvage therapy prior to HSCT in patients with r/r AML. Here, we report the long-term follow up of this Phase II, multi-center, Intent-To-Transplant study and the impact of comorbidity on outcome. Eighty-four patients with a median age of 61 years (range 40 - 75) were enrolled. Patients were scheduled for at least one cycle of salvage therapy with CLARA (clofarabine 30 mg/m2 and cytarabine 1 g/m2, days 1-5). Chemo-responsive patients with a donor received HSCT after first CLARA. In the event of a prolonged donor search, HSCT was performed as soon as possible. The conditioning regimen consisted of clofarabine 30 mg/m2, day -6 to -3, and melphalan 140 mg/m2 on day -2. The ECOG score, hematopoietic cell transplantation-specific comorbidity index (HCT-CI) and Cumulative Illness Rating scale (CIRS) were obtained at study enrolment as well as prior to HSCT. Sixty-seven percent of the patients received HSCT within the trial. After a median follow up of 40months (95% CI, 38-49 months), the estimated 4-year OS (Figure 1) for all enrolled patients was 38% (95% CI, 28-50%) and Disease-Free Survival for transplanted patients was48% (95% CI, 36-64%). The CIR at four years was 30% (95% CI, 17-43%) and the NRM 22% (95% CI, 10-33%).Those patients who received an allogeneic HSCT within the trial had a median HCT-CI at the time of study enrollment of 1 (range, 0 - 6) compared to a median of 2 (range, 0 - 6) for those who did not proceed to allogeneic HSCT (p = .17). Corresponding figures for the CIRS were a median of 2 (range, 0 - 9) compared to 4 (range, 0 - 8) (p = .09). The median ECOG score was 1 (range, 0 - 3) in both groups. Compared to the time point of study enrollment, both the HCT-CI as well as the CIRS increased to a median of 2 (observed range of score, 0 - 7) and a median of 4 (observed range of score, 0 - 12), respectively, at the time of start of the conditioning regimen. This was almost exclusively due to an increase in infectious complications (Figure 2). Inmultivariate analysis, both the baseline HCT-CI and the ECOG score had a statistically significant impact with a HR of 1.22 (p = .025) and 1.72 (p = .001), respectively, on OS. Using a clofarabine-based salvage therapy combined with early allogeneic HSCT we were able to achieve good long-term results for patients with r/r AML. In this cohort, both the HCT-CI and the ECOG score gave prognostic information on OS, showing feasibility of comorbidity evaluation at the time of diagnose of r/r AML. Figure 1 OS of all enrolled patients Figure 1. OS of all enrolled patients Figure 2 Changes of the HCT-CI at baseline to HSCT Figure 2. Changes of the HCT-CI at baseline to HSCT Disclosures Middeke: Sanofi: Honoraria. Rösler:Janssen: Consultancy, Other: Travel/Accommodation/Expenses. Thiede:AgenDix: Employment, Other: Ownership. Schetelig:Sanofi: Honoraria.

According to WHO 2008 guidelines, the required percentage of cells manifesting dysplasia in the bone marrow to qualify as significant is 10% or over in one or more hematopoietic cell lineages, but this threshold is controversial. No 'normal' values have been established. Therefore, we investigated dyshematopoiesis in bone marrow aspirate squash preparations of 120 healthy bone marrow donors.Bone marrow squash slides of 120 healthy unrelated bone marrow donors were examined independently by 4 experienced morphologists. Samples were taken from the first aspiration during the harvest. Bone marrow preparation and assessment were performed according to WHO recommendations and ICSH guidelines.More than 10% dysmyelopoiesis could be detected in 46% of bone marrow aspirate squash preparations with 26% in 2 or more cell lineages and 7% in 3 cell lineages in healthy bone marrow donors. Donors under the age of 30 years exhibited more dysgranulopoietic changes and dysmegakaryopoietic changes (P0.001) compared to the older donors. Female donors showed more dysgranulopoietic changes than male donors (P = 0.025). The concordance rate between the 4 investigators was modest in dysgranulopoiesis but poor in dyserythropoiesis and dysmegakaryopoiesis.The poor reliability of the 10% cut off was partly related to the proximity of the current criteria to the observed cut-off mean values of the normal population. These findings question the current WHO threshold of the 10% or over necessary for the percentage of cells manifesting dysplasia to be considered significant, and suggest that either a higher threshold would be more appropriate or different thresholds should be set for each lineage.

Introduction: Acute myeloid leukemia (AML) may present with either concomitant or isolated extramedullary (EM) AML. Data on the prevalence of EM AML are based on retrospective and clinical analyses which rely on findings from physical examination only or on coincidental findings in standard imaging procedures and range from 2.5 to 9.1% (Bakst et al., Blood 2011;118(14):3785-3793). Previous studies identified EM AML as a prognostic factor in patients with AML. 18Fluorodesoxy-Glucose Positron Emission Tomography – Computed Tomography (18FDG/PET-CT) is able to detect highly metabolic tissue and has proven efficacy in imaging studies for various types of malignant diseases. In a pilot study in patients with histologically proven EM AML, we were able to demonstrate a sensitivity of 90% using 18FDG/PET-CT imaging and found additional EM sites in 60% of the patients (Stölzel et al., Haematologica 2011;96(10):1552-1556). The aim of this prospective, observational study was to determine the prevalence of EM AML in patients with newly diagnosed or relapsed AML using 18FDG/PET-CT (ClinicalTrials.gov Identifier: NCT01278069). Patients and Methods: A total of 94 patients with AML (newly diagnosed AML, n = 85 and relapsed AML, n = 9) underwent total body 18FDG/PET-CT scans at diagnosis after giving informed consent. Patients with EM diagnosed by 18FDG/PET-CT underwent a second 18FDG/PET-CT scan after induction chemotherapy. The median age of all patients was 61 years (range, 27 to 79 years). Patients were included only if a delay of ≤ 5 days of initiation of induction- or re-induction chemotherapy was clinically justifiable to perform the study. 18FDG/PET-CT scans were performed using a Siemens Sensation 16 as part of a biograph (Siemens, Knoxville, TN, USA) with i.v. application of 18FDG (range of activity 223 to 433 MBq) and 120 ml i.v. contrast media Ultravist 370 (Bayer Schering Pharma, Leverkusen, Germany). Adverse reactions due to the application of i.v. 18FDG and i.v. contrast media did not occur. Results: Total body 18FDG/PET-CT imaging detected highly metabolic manifestations suggestive for EM AML in 21 patients (22%). During follow-up after induction chemotherapy, 18FDG-avid EM was still observed in 50% of all PET-positive patients. In comparison with PET-negative patients, those with PET-positive EM AML had a higher bone marrow blast count, a higher prevalence of FAB M5 morphology, higher peripheral WBC, higher serum LDH, and less frequent FLT3-ITD mutations. Sites of EM AML were connective tissue (n=4), parenchymal tissues (n=8), and lymph nodes (n=15). In patients with clinically overt EM AML (n=8) additional EM manifestations were detected in 62% (n=5). In 13 patients, samples from EM sites were obtained for histology review – in 10 patients histology confirmed the occurrence of EM AML in these sites, indicating a specificity of 77%. Importantly, in the remaining patients in whom histology could not confirm EM AML, concomitant malignant tumors were found. Extrapolating these results on the entire cohort, the prevalence of EM AML in newly diagnosed and relapsed AML is 17%. Conclusions: Our study is the first to prospectively evaluate 18FDG/PET-CT imaging for the diagnosis of EM AML. According to our results, 18FDG/PET-CT is a useful and safe tool in order to detect EM AML with a high specificity. We found a prevalence of EM AML of 17%. This is two- to eightfold higher, than in previously reported clinical studies. Integration of 18FDG/PET-CT-based imaging procedures as adjunct for response assessment in these patients seems to be important. Further development of other PET-based imaging procedures and integration in the setting of relapse after allogeneic hematopoietic stem cell transplantation might be necessary and will contribute to a better understanding of the biology and prognostic role of EM AML and the development of targeted treatment strategies in patients with AML. Disclosures No relevant conflicts of interest to declare.

Background: Sorafenib is a multi-kinase inhibitor with activity against several oncogenic kinases that may play a role in the pathogenesis of acute myeloid leukemia (AML). In-vitro data and results from non-randomized clinical trials suggest that sorafenib might be an effective drug for the treatment of AML. We present the results of the randomized placebo-controlled SORAML trial testing sorafenib versus placebo as add-on to standard induction and consolidation treatment in AML patients ≤60 years. Patients and Methods: Between March 2009 and October 2011, 276 patients from 25 centers were enrolled in the SORAML trial (NCT00893373). The main eligibility criteria were newly diagnosed AML, age from 18 to 60 years and suitability for intensive therapy. The treatment plan for all patients included two cycles of induction with DA (daunorubicin 60 mg/m2 days 3-5 plus cytarabine 100 mg/m2 cont. inf. days 1-7), followed by three cycles of high-dose cytarabine consolidation (3 g/m2 b.i.d. days 1, 3, 5). Patients without response after DA I received second induction with HAM (cytarabine 3 g/m2 b.i.d. days 1-3 plus mitoxantrone 10 mg/m2 days 3-5). Allogeneic stem cell transplantation was scheduled for all intermediate-risk patients in first complete remission with a sibling donor and for all high-risk patients with a matched related or unrelated donor. At study inclusion, patients were randomized to receive either sorafenib (800 mg/day) or placebo as add-on to standard treatment in a double blinded fashion. Block randomization at a ratio of 1:1 was performed within cytogenetic and molecular risk strata, allocation was concealed and treatment was double blinded. Study medication was given on days 10-19 of DA I+II or HAM, from day 8 of each consolidation until 3 days before the start of the next consolidation and as maintenance for 12 months after the end of consolidation. The primary endpoint of the trial was event-free survival (EFS) with an event being defined as either failure to achieve a complete remission (CR) after induction, relapse or death. Secondary endpoints were relapse-free survival (RFS), overall survival (OS), CR rate and incidence of adverse events (AE). We present the results of the final analysis of the primary endpoint EFS (intent to treat) after the occurrence of 134 events. Results: Out of 276 enrolled patients, 267 received study treatment, 134 in the sorafenib arm and 133 in the placebo arm. Demographic and disease characteristics were equally distributed between the two arms; the incidence of FLT3-ITD was 17%. The median cumulative dose of administered study medication was similar in both arms. The CR rates were 59% versus 60% in the placebo versus sorafenib arm (p=0.764). After a median observation time of 36 months, the median EFS was 9.2 months in the placebo arm and 20.5 months in the sorafenib arm, corresponding to a 3-year EFS of 22% versus 40% (p=0.013). Median RFS after standard treatment plus placebo was 23 months and not yet reached after sorafenib treatment, corresponding to a 3-year RFS of 38% and 56%, respectively (p=0.017). The median OS had not been reached in either arm; the 3-year OS was 56% with placebo versus 63% with sorafenib (p=0.382). In 46 FLT3-ITD positive patients, no difference in EFS, but a trend for prolonged RFS and OS in favor of sorafenib was observed. The most common reported AEs Grade ≥3 were fever (40%), infections (22%) and bleeding events (2%). The risk for fever, bleeding events and hand-foot syndrome was significantly higher in the sorafenib arm while the incidence of all other AEs showed no significant differences. Conclusions: In younger AML patients, the addition of sorafenib to standard chemotherapy in a sequential manner is feasible and associated with antileukemic efficacy. We observed a higher incidence of infections and bleeding events under sorafenib. Whereas OS in both treatment arms was similar, sorafenib treatment resulted in a significantly prolonged EFS and RFS. Figure 1: Event-free survival Figure 1:. Event-free survival Disclosures Off Label Use: sorafenib for treatment of aml. Serve:Bayer HealthCare: Research Funding. Ehninger:Bayer HealthCare: Research Funding.

Background: In relapsed or refractory acute myeloid leukemia (AML), long-term disease-free survival may only be achieved with allogeneic hematopoietic stem cell transplantation (HSCT). Within the BRIDGE Trial, the safety and efficacy of a clofarabine salvage therapy as a bridge to HSCT was studied. Here, we report long-term survival data and the impact of donor availability at the time of study enrollment. The BRIDGE trial (NCT 01295307) was a phase II, multicenter, intent-to-transplant study. Patients and Methods: Between March 2011 and May 2013, 84 patients with relapsed or refractory AML older than 40 years were enrolled. Patients were scheduled for at least one cycle of induction therapy with CLARA (clofarabine 30 mg/m2 and cytarabine 1 g/m2, days 1-5). Patients with a donor received HSCT in aplasia after first CLARA. In case of a prolonged donor search, HSCT was performed as soon as possible. The conditioning regimen consisted of clofarabine 30 mg/m2, day -6 to -3, and melphalan 140 mg/m2 on day -2. In patients with partially matched unrelated donors, ATG (Genzyme) at a cumulative dose of 4.5 mg/kg was recommended. GvHD prophylaxis consisted of CsA and mycophenolate mofetil. Results: Forty-four patients suffered from relapsed AML and 40 patients had refractory disease. The median patient age was 61 years (range 40 – 75). According to the current ELN risk stratification 17% of pts were classified as favorable risk, 35% as intermediate I, 17% as intermediate II and 20% as adverse risk. The overall response rate assessed at day 15 after start of CLARA was 80% (defined as at least a marked reduction in BM blasts or BM cellularity and absence of blasts in the peripheral blood) with 31% of patients having less than 5% BM blasts at that time. Seventeen patients did not respond to CLARA, and were subsequently treated off-study. Due to early death, three patients were not evaluable for treatment response. Overall, 66% of the patients received HSCT within the trial. Donors were HLA-identical siblings in eight cases (14%), HLA-compatible unrelated donors in 30 cases (55%) and unrelated donors with one mismatch in 17 cases (31%). Treatment success was defined as complete remission (CR), CR with incomplete recovery (CRi) or CRchim (BM donor chimerism >95% and absolute neutrophil count >0.5/nL) on day 35 after HSCT. Treatment success was achieved in 61% of the patients. With a median follow up of 25 months, the OS for all enrolled patients at two years was 42% (95% CI, 32% to 54%). (Figure 1) The Leukemia-free survival at two years for those 51 patients who achieved the primary endpoint was 52% (95% CI, 40% to 69%). (Figure 2) At the time of enrollment, 14% of patients had a related donor and 33% had an unrelated donor available. In 46% of the patients, donor search was initiated at the time of enrollment. For 7% of patients, donor search was unsuccessful prior to enrollment and reinitiated. The OS at 2 years for patients with a related or an unrelated donor available was 75% (95% CI, 54% to 100%) and 47% (95% CI, 31% to 71%), respectively, while it was 29% (95% CI, 18% to 48%) for patients for whom donor search was initiated at time of enrollment (p = .09). Conclusions: Salvage therapy with CLARA, and subsequent conditioning with clofarabine and melphalan prior to allogeneic HSCT, provides good anti-leukemic activity in patients with relapsed or refractory AML. Fast unrelated donor search and work up, with conditioning in aplasia allowed a high rate of successful HSCTs. The leukemia-free survival for this group of elderly, high risk AML patients is very promising. Figure 1 Figure 1. Overall survival for all patients, n=84 Figure 2 Figure 2. Leukemia-free survival for all patients with primary treatment success, n=51 Disclosures Middeke: Genzyme: Speakers Bureau. Off Label Use: Clofarabine for AML. Schetelig:Genzyme: Research Funding; DKMS German Bone Marrow Donor Center: Employment.

Introduction CMV-infection is a serious complication in patients after allogeneic stem cell transplantation (SCT) where immunosuppressive therapy and impaired T cell reconstitution result in a high risk for viral infections. Monitoring of CMV-virus load by PCR and preemptive therapy are important tools to prevent CMV disease. However, CMV specific cytotoxic T cells (CMV-CTLs) are needed to successfully control CMV-infections. CMV-specific multimers composed of the patients HLA Class I molecule bound to CMV pp65 epitopes give the possibility to monitor CMV-CTLs. Here, we present the case of CMV-reactivation following SCT for AML. Methods The percentage of CMV-specific CD8+ T cells was determined by flow cytometry and mapped to clinical and laboratory parameters of the patient. CD8+ T cells were detected using CD8-fluorescein isothiocyanate (FITC, Beckman Coulter) antibody and CD3 as a T-cell marker was labeled with CD3-allophycocyanin (APC, MACS Miltenyi Biotec) antibody. CMV-specific CD8+ T cells were detected using the CMV major histocompatibility complex (MHC) with Strep-Tactinphycoerythrin (PE) conjugate (Streptamers, IBA GmbH). Case A 60 years old male patient was diagnosed with acute myeloid leukemia (AML) with 95% myeloid blasts in the bone marrow and extramedullary AML manifestations at the time of diagnosis. Following induction therapy the patient was transplanted from a matched unrelated donor. The stem cell recipient as well as his donor had been tested sero-positive for CMV prior to SCT. Within the first month following transplantation, the patient developed an effective CMV specific immunity as seen by high levels of CMV-specific T cells (Figure 1). About three months following transplantation the patient was diagnosed with intestinal GVHD requiring high-dose glucocorticoid treatment. Following steroid exposure, levels of CMV-CTLs dropped and shortly thereafter rising CMV-copy numbers were observed which was accompanied by clinical signs of CMV enteritis. With the administration of antiviral treatment the CMV specific virus load decreased. However, levels of CMV-CTLs remained low, presumably as a result of ongoing steroid exposure. Discussion High levels of CMV-CTLs appeared to control CMV, as seen by a non-detectable virus load in standard PCR testing. The close correlation between the drop in CMV-CTL count and CMV activation highlights the potential of this method to monitor and understand immune responses to CMV following SCT. Of note, early presence of high frequencies of CMV-CTLs did not guarantee CMV-control under steroid exposure as seen in our case. Previous reports have suggested that high dose glucocorticoids may impact CMV-CTLs survival. This is supported by our case, where we see a rapid drop in CMV-CTLs following glucocorticoid exposure. However, the exact molecular mechanisms and more importantly, the predictive value of this finding remain elusive. Furthermore, these data suggest, that patients with ongoing high steroid exposure may not benefit from a transfer of CMV-specific T-cells to control CMV disease. Conclusion Further investigations to clarify the potential of CMV-CTL measurements and to understand the effect of steroid exposure at the functional level are warranted. Studies to correlate CMV-CTL counts with the level of immunosuppression and their influence on controlling CMV-disease will follow. In future, this tool could provide a chance to select patients at high risk of CMV reactivation who could profit from an individualized monitoring and early treatment. Disclosures: No relevant conflicts of interest to declare.

Introduction Patients with MDS harboring a del(5q) often display characteristic morphological features in the bone marrow. For the first time we have recently demonstrated a strong association of the presence of a cytogenetic abnormality - del(5q) - with a specific 5-parameter immunophenotypic profile measured by flow cytometry (FCM). The aim of this prospective study was to test, if this FCM profile could also be applied for monitoring response to treatment with disease-modifying drugs like lenalidomide and azacitidine. Patients and methods Overall, 137 FCM investigations were performed in 52 well-characterized patients with MDS and a del(5q) (IPSS low/int-1: n=30, int-2/high: n=22) including 34 patients with isolated del(5q): 53 measurements before treatment and 84 during treatment (lenalidomide=47 and/or azacitidine=35; chemotherapy and/or allogeneic transplantation=6). Nineteen of 41 thereupon evaluated patients showed a concomitant TP53 mutation. An 8-color/5-tube FCM diagnostic procedure (lyse-stain-wash) was performed on a FACS-Canto II cytometer. Thus, the typical del(5q) FCM profile includes the following 5 parameters: percentage of myeloid progenitors (myPC > 2%) = 3 points, CD45 MFI ratio (lymphocytes : myPC ≤ 7.0) = 10 points, SSC ratio (granulocytes : lymphocytes < 6.0) = 2 points, CD71 (≤ 20%) on granulocytes = 1.5 points, and female gender = 1.5 points. The weighting of these parameters has been done using a logistic regression model. Results In 48/53 (92%, mean score=16.5±2.0) of the FCM measurements before therapy del(5q) was predictable with the 5-parameter FCM score. In the few FCM inconclusive cases the mutational analysis showed a TP53 mutation. Remarkably, in all 18 cases being in cytogenetic complete remission (CCR) in response to therapy the disappearance of del(5q) by conventional cytogenetic analyses was accompanied by the disappearance of the del(5q) FCM profile (specificity=100%; mean score=13.0±1.0). On the other hand, in patients without CCR during treatment the presence of del(5q) could be predicted by the FCM-score (≥ 15) with a sensitivity of 65% (43/66; mean score=15.5±3.0). Of note, in most of those inconclusive measurements (85%) a mutation of the TP53 gene and/or a deletion of chromosome (17p) could be detected suggesting a modification of the immunophenotype mediated by these molecular changes. Interestingly, the complexity of karyotypic abnormalities appeared to play a minor role in modifying the immunophenotype because in two-thirds of the FCM inconclusive cases del(5q) was present as a single abnormality. Conclusion The proposed 5-parameter del(5q) FCM profile can serve as a surrogate for the presence of a del(5q) and could therefore be used as a rapid tool for response monitoring during treatment with disease-modifying drugs. The role of TP53 mutations in modifying the FCM-profile in this context should be the purpose of further investigations. Disclosures: No relevant conflicts of interest to declare.

Background In relapsed or refractory acute myeloid leukemia (AML) long-term disease-free survival may only be achieved with allogeneic stem cell transplantation (HSCT). However, only about 40% of patients (pts) with relapsed AML receive HSCT. A number of factors contribute to this low rate, among them, a moderate activity of currently available salvage regimens and accumulating toxicity of chemotherapy. Clofarabine is considered to have a favorable risk-benefit ratio in this indication and has been successfully used in conditioning regimens. Our goal was to study the safety and efficacy of a clofarabine salvage therapy as a bridge to HSCT. Here, we report the results of the BRIDGE trial (NCT 01295307), a phase II, multicenter, intent-to-transplant study. Patients and Methods Between March 2011 and May 2013, 84 pts with relapsed or refractory AML older than 40 years were enrolled. Pts were scheduled for at least one cycle of induction therapy with CLARA (clofarabine 30 mg/m2 and cytarabine 1 g/m2 days 1-5). Pts with a donor received HSCT in aplasia after first CLARA. In case of a prolonged donor search HSCT was performed as soon as possible. The conditioning regimen consisted of clofarabine 30 mg/m2 day -6 to -3 and melphalan 140 mg/m2 on day -2. In pts with partially matched unrelated donors ATG (Genzyme) at a cumulative dose of 4.5 mg/kg was recommended. GvHD prophylaxis consisted of CsA and mycophenolate mofetil. Results Median age was 61 years (range 40 – 75). Forty-four pts suffered from relapsed AML and 40 pts had refractory disease. According to the current ELN risk stratification 17% of pts were classified as favorable risk, 35% as interm. I, 17% as interm. II and 20% as adverse risk. Complex and monosomal karyotypes were present in only 12% and 10% of pts, respectively. FLT3, NPM1 and CEPBA mutations were found in 16%, 24%, and 4% of the pts. The mean value of the HCT-CI score was 1.6 (range 0 - 7) at the time of study enrollment and 2.3 (range 0 - 7) at the time of conditioning. The overall response rate assessed at day 15 after start of CLARA was 80% (46% good response defined as less than 10% blast in the bone marrow (BM) and 33% moderate response with at least a marked reduction in BM blasts or BM cellularity and absence of blast in the peripheral blood). Seventeen pts did not respond to CLARA and were subsequently treated off study. Due to early death, three pts were not evaluable for treatment response. Overall, 66% of the pts received HSCT within the trial. Donors were HLA-identical siblings in eight pts (14%), HLA-compatible unrelated donors in 30 pts (55%) and unrelated donors with one mismatch in 17 pts (31%). Treatment success defined as complete remission, CR with incomplete recovery or >95% BM donor chimerism and an absolute neutrophil count >0.5 /nL on day 35 after HSCT was achieved in 62% of the pts. Disease-free survival (DFS) is shown in Figure 1. With a median follow up of 16 months the OS for all enrolled patients at one year is 51% (95% CI, 39% to 63%). At the time of enrollment, 14% had a related donor and 33% had an unrelated donor. In 46% of the pts donor search was initiated at the time of enrollment. For 7% of pts donor search was not successful. Time from study entry to HSCT was remarkably low with a median of 33 days (range 19 – 116 days). Of note, time interval did not differ between related and unrelated donors (Figure 2). Day 30 and day 100 mortality, which covered salvage therapy and HSCT, was 9% and 27%, respectively. Six out of seven pts who died within the first 30 days hat refractory AML and thus entered the trial already with a history of long-lasting neutropenia. Liver toxicity was the most frequent adverse event. Fifty percent of the pts had transiently elevated liver enzymes CTCAE grade III considered to be related to clofarabine. Twenty-one patients developed CTCAE grade III – IV sepsis throughout the study treatment. GvHD grade II – IV and III-IV until day 100 after HSCT occurred in 36% and 21% of the pts, respectively. Conclusions This intent-to transplant study allows for a realistic estimate for the outcome of elderly pts with relapsed or refractory AML. We demonstrate a high rate of leukemia-control by CLARA. Fast unrelated donor search and work up and conditioning with clofarabine and melphalan in aplasia allowed for a high rate of successful HSCTs. While the long-term results require longer follow-up the overall results are promising. Disclosures: Middeke: Genzyme: Speakers Bureau. Schetelig:Genzyme: Research Funding. Off Label Use: Clofarabine, not approved for AML.

Introduction Acute Graft-versus-Host Disease (aGvHD) remains the major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). So far, corticosteroids are the only first-line treatment proven to be effective. Steroid-resistance is associated with poor outcome and no commonly accepted second-line salvage therapy is available until now. Mesenchymal stromal cells (MSC) have shown promising immunomodulatory effects and have been suggested as cell-based treatment option in patients with steroid-refractory acute GvHD. Here, we report our experience from a large cohort of patients treated with MSC. Patients, Materials and Methods Fifty-eight patients with steroid-refractory acute GvHD after HSCT were treated with MSC at our centre between 2007 and 2012. MSC were obtained from volunteer third-party donors and expanded in platelet-lysate containing medium. Median age at transplantation was 55 years (range 19-71). In 25 patients AML was diagnosed initially. Further diagnoses were CLL (n=9), ALL (n=5), MDS (n=5) and others (n=14). For transplantation, patients received peripheral blood stem cells (n=56) or bone marrow (n=2) from HLA-identical (n=43) or HLA mismatched donors (n=15) after varying reduced-intensity conditioning regimens (n=50) or myeloablative conditioning (n=8). Eight-teen patients received anti-thymocyte globulin as part of their conditioning. GvHD prophylaxis consisted mostly of cyclosporine A (CsA) plus methotrexate, CsA alone or CsA plus mycophenolate mofetile. The majority of patients suffered from aGvHD grade IV (79%), median interval from HSCT to onset of GvHD was 36 days. Involvement of gastrointestinal tract, liver and skin was observed in 91%, 43% and 41% of patients, respectively. Most patients (64%) had involvement of 2 or 3 organs at the same time. Besides corticosteroids, 48 patients (83%) received at least one additional immunosuppressive agent before the first MSC infusion. Response was assessed 28 days after initiation of MSC treatment and overall survival of the MSC treated cohort was compared to a historic patient cohort (n=36) with steroid-refractory aGvHD not receiving MSC. Results Median time between onset of aGvHD and first application of MSC was 12 days (range 6-62). Altogether 139 doses of MSC were transfused at a median dosage of 0.99x106cells/kg bodyweight (range 0.448 -2.077). A median number of 2 MSC infusions were given per patient (range 1-6). During MSC treatment, 39 patients needed further escalation of immunosuppression due to persistence or progression of GvHD. No side-effects directly related to MSC infusions were observed. Four weeks after first MSC application, 9% (n=5) of patients showed a complete response (CR), 9% (n=5) exhibited a very good partial response (VGPR), 29% (n=17) experienced partial response (PR) whereas 53% (n=31) were classified as non-responders. There were no significant differences in organ specific response. One-year and 2-year-survival after onset of aGvHD was 19% [95% CI: 9-29%] and 17% [95% CI: 7-26%], respectively. Median survival was 69 days [95% CI: 38-100 days]. Causes of death were aGvHD (54%), infectious complications (29%), relapse of the underlying disease (4%) and others (13%). Median follow up was 1689 days and at the end of follow-up, 8 patients were still alive (median survival after HSCT 58 months). Of those eight, 6 were initially classified as responders (CR n=4, VGPR n=1, PR n=1). Responders showed higher survival compared to non-responders (hazard ratio 0.38; p=0.004). Overall survival in the MSC treated cohort did not differ significantly to that of the historic cohort not receiving MSC. Conclusions MSC in combination with further immunosuppressive strategies resulted in a response rate of 47% in patients with steroid-refractory aGvHD and should therefore be considered as a valuable treatment option in a difficult clinical situation. However, compared to our historic cohort, treatment with MSC did not lead to an improvement of survival. Future studies need to define which patient subsets are likely to benefit from MSC therapy and whether certain MSC preparations from specific donors may have a more pronounced and long-lasting immunosuppressive effect. Therefore, further insights into MSC biology are urgently needed to optimize the translation into clinical practice. Disclosures: No relevant conflicts of interest to declare.

Abstract 144 Background: Sorafenib is a multi-kinase inhibitor with activity against several oncogenic kinases, which may play a role in the pathogenesis of acute myeloid leukemia (AML). In-vitro data and results from non-randomized clinical trials suggest that sorafenib might be an effective drug for the treatment of AML. So far, no randomized-controlled data are available for treatment of newly diagnosed AML patients up to the age of 60 years. We present the first results from the randomized placebo-controlled SORAML trial of the Study Alliance Leukemia (SAL). Patients and Methods: Between March 2009 and October 2011, 276 patients from 25 centers were enrolled in the SORAML trial (NCT00893373). The main eligibility criteria were: newly diagnosed AML, age from 18 to 60 years and suitability for intensive therapy. The treatment plan for all patients included two cycles of induction with DA (daunorubicin 60 mg/m2 days 3–5 plus cytarabine 100 mg/m2 cont. inf. days 1–7), followed by three cycles of high-dose cytarabine consolidation (3 g/m2 b.i.d. days 1, 3, 5). Patients without response after DA I received second induction with HAM (cytarabine 3 g/m2 b.i.d. days 1–3 plus mitoxantrone 10 mg/m2 days 3–5). Allogeneic stem cell transplantation was scheduled for all intermediate-risk patients in first complete remission with a family donor and for all high-risk patients with a matched donor. At study inclusion, patients were randomized to receive either sorafenib (800 mg/day) or placebo as add-on to standard treatment. Block randomization at a ratio of 1:1 was performed within cytogenetic and molecular risk strata, allocation was concealed and treatment was double blinded. Study medication was given on days 10–19 of DA I+II or HAM, from day 8 of each consolidation until 3 days before the start of the next consolidation and as maintenance for 12 months after the end of consolidation. The primary endpoint of the trial is event-free survival (EFS) with an event being defined as either failure to achieve a complete remission (CR) after induction, relapse or death. Secondary endpoints were overall survival (OS), CR rate and incidence of adverse events (AE). We present the results of the planned interim analysis (intent to treat) after the occurrence of 50% of EFS events. The O'Brien/Fleming adjusted significance level was set at p=0.0052. Results: Out of 276 randomized patients, 264 were evaluable for EFS, 132 in each arm. Demographic and disease characteristics were equally distributed between the two arms; the FLT3-ITD incidence was 16%. The median cumulative dose of administered study medication was equal in both arms. The CR rates were 56% versus 60% in the placebo versus sorafenib arm (p=0.622). By the time of analysis, a total number of 100 events had occurred. After a median observation time of 18 months, the median EFS was 12.2 months in the placebo arm and was not reached in the sorafenib arm, corresponding to a 1-year EFS of 50% versus 64% (p=0.023). The median OS had not been reached in both arms, the 2-year OS was 66% versus 72% in placebo and sorafenib arms, respectively (p=0.367). The most common reported AEs CTC Grade ≥3 were infectious complications including fever and pneumonia, followed by bleeding events, cardiac and hepatic toxicity, hypertension, skin toxicity and headache. The risk for hepatic toxicity (relative risk 6.2, p=0.025) and bleeding events (relative risk 3.6, p=0.016) was significantly higher in the sorafenib arm while the incidence of all other AEs showed no significant differences. Conclusions: In younger AML patients, the addition of sorafenib to standard chemotherapy is feasible but associated with a higher risk of liver toxicity and bleeding events. Sorafenib treatment resulted in a marked EFS prolongation; this difference is not significant according to the adjusted significance level of this interim analysis. Results from the final analysis including post-hoc exploration of molecularly defined subgroups are necessary for drawing final conclusions on the efficacy of sorafenib. Disclosures: Off Label Use: sorafenib for the treatment of acute myeloid leukemia. Serve:Bayer: Research Funding. Ehninger:Bayer: Research Funding.

Abstract 886 The diagnosis acute myeloid leukemia (AML) describes a heterogeneous group of myeloid stem cell disorders. Based on current concepts of disease development, the combination of at least two mutations is necessary for transformation, typically affecting transcription factors (e.g. RUNX1) blocking normal differentiation and growth promoting genes, e.g. receptor tyrosine kinases like FLT3. This model has been challenged by more recent results of genome-wide mutational analysis, which revealed a typical load of 8–12 mutations affecting several additional pathways, e.g. epigenetic regulation (DNMT3A, TET2 or IDH1). However, the sequence of acquisition and the individual impact of these mutations are largely unknown because these aspects are difficult to study. Here we describe a unique case of a donor cell leukemia giving unexpected insights into the development of AML in man. Case report and methods: In May 2004, a 51-year old male (P1) with an 8-year history of B-CLL received G-CSF mobilized peripheral blood stem cells after dose reduced conditioning from his HLA-identical sister, because he had relapsed after several lines of conventional therapy. He rapidly engrafted and showed complete donor chimerism (DC). In February 2012, he was admitted to the hospital with elevated WBC counts and circulating blasts. Bone marrow (BM) aspiration and morphology revealed an infiltration of the BM with 94% myeloid blasts (FAB M1). Cytogenetic and standard molecular assessment showed a normal female karyotype and NPM1 and FLT3-ITD mutations. STR-based analysis also revealed a persistent, 100% DC, thus the diagnosis of a donor cell AML was made, which developed almost 8 years after SCT. Interestingly, his sister, the donor (P2) had been also diagnosed with AML (FAB M2, cytogenetics: 47, XX,+8; NPM1 and FLT3-ITD neg.) only 3 months before her brother in Nov. 2011. Currently, P1 is in CR after re-SCT from an unrelated donor, whereas P2 relapsed and is scheduled for SCT after reinduction. Since DNA material of both individuals was available and due to this unique constellation, we performed next generation sequencing of whole exome enriched material using an Illumina HiSEQ 2000 platform after obtaining informed consent to compare both AMLs. Identified mutations were then confirmed using conventional Sanger sequencing and traced back by 454-based amplicon deep sequencing in a pre-SCT sample of the donor/P2 as well as several post SCT samples collected from P1 for the documentation of chimerism. Results: Comparison of the two AML-samples with a pre-SCT donor sample and a sample taken after 1.SCT as well as the HG19 and dbSNP135 releases revealed more than 100 unknown SNPs. Confirmation focused on cancer related changes or genes in critical pathways. In P1, in addition to the known NPM1 and FLT3-ITD mutations, we found somatic changes in CLCA1, PKHD1 and TET2, whereas in P2, we identified and confirmed somatic mutations in CDCA2, CBL, IDH1, NEK9 and PHF6. In addition, a typical DNMT3A R882C mutation was found in both leukemias. Interestingly, this mutation was also detectable by conventional Sanger sequencing in the pre-SCT sample of P2, but not in P2-germline DNA derived from buccal swaps. As shown in the figure, the 454-amplicon sequencing revealed a gradual increase of the TET2 (R1167G) mutational load over time in P1, and showed also that this mutation was present at low levels (4%) already in the pre-SCT sample of P2, but not in her final AML. FLT3-ITD and NPM1 mutations were detectable only in the AML-sample of P1, but not at any prior time points or in P2. Conclusions: These data indicate that mutations like the DNMT3A R882C can be present in normal appearing hematopoiesis at high levels years before the development of AML. The presence of the mutation in the absence of overt leukemia or MDS indicates that these mutations might not have a direct effect on the development of the disease, but favor the development of aberrant clones which then acquire additional changes in a latency phase. Other mutations (e.g. TET2) might give a small clonal advantage, but only the final acquisition of abnormalities like NPM1 and FLT3-ITD might transform this latency phase into a rapidly proliferating status, consistent with the “driver” status of these aberrations. The divergent mutational pattern found in the two AMLs emerging from the same DNMT3A-starting clone points to the high clonal diversity which might develop even within a single individual. Disclosures: Thiede: AgenDix GmbH: Employment, Equity Ownership.

Abstract 1481 Introduction Acute erythroleukemia (AEL) represents a rare type of acute myeloid leukemia accounting for less than 5% of all cases. So far, according to WHO classification this AML entity is thought to have a poor prognosis in itself. Design 3267 patients with newly diagnosed AML were treated according to the protocols of the AML96, AML2003 or AML60+ studies of the Study Alliance Leukemia (SAL). 116 of these patients had acute erythroleukemia (AEL). The median age both for patients with AEL and non-AEL was 57 years. We assessed the influence of relevant clinical and demographic parameters, FLT3-ITD, NPM1 status and cytogenetics on complete remission rates (CR), overall survival (OS) and event free survival (EFS) separately in AEL and non-AEL patients. Results Compared to non-AEL AML, significantly more AEL were due to secondary causes than non-AEL AML (31% versus 20.4%; p=0.024). NPM1 mutations were found in 11.1% (out of 99) of the patients with AEL and in 32.8% (out of 2693) of the patients with non-AEL AML (p 0.8 were found less frequently in AEL (FLT3-ITD mutation in 4.9% vs 23.3%; p=0.001; FLT3-ITD ratio >0.8 in 0% vs. 33.4%; p The cytogenetic aberrations +8, −7 and complex aberrant karyotype (>/= 3 independent aberrations) were found more often in the AEL cohort (14.8%, 10.3% and 26.7%) than in the non-AEL AML cohort (8.9%, 5% and 13.2%) (p=0.036, p= 0.01 and p Despite these differences, no significant differences in CR rates, OS and EFS were found between both groups. This finding was confirmed in a multivariate analysis including cytogenetics, molecular markers and clinical parameters (LDH, WBC, blast count, platelet count and ECOG). According to the analysis, ALE morphology was not an independent prognostic factor for OS and EFS. With the AEL group, patients with monosomy 7 (n=12) had a higher median blast count (NEC) of 62% and shorter median OS with 5.7 months compared to patients with AEL and no monosomy 7 (n=104) with a median blast count (NEC) of 43% (p=0.013) and an median OS with 15.7 months (p=0.016). A complex aberrant karyotype was found more often in patients with secondary AEL than in patients with de novo AEL (p=0.037). Significant differences were also seen in patients with AEL and complex aberrant karyotype compared to AEL without complex aberrant karyotype with respect to CR rates (54.8% versus 77.6%; p=0.016), OS (6.2 versus 17.4 months; p In patients with AEL and abn17p/-17 (n=15), lower median platelet counts (31 versus 48 ×106/μl; p=0.017), a worse OS (6.4 vs 15.7 months; p=0.011) and EFS were observed (1.7 vs 5.7 months; p=0.046). Conclusions According to our data, the characteristic morphological features of acute erythroleukemia do not confer an unfavorable prognosis in itself. Furthermore, we can confirm the relevant influence of established prognostic factors such as cytogenetics and disease status within the AEL subgroup. Disclosures: Off Label Use: sorafenib for the treatment of acute myeloid leukemia.

Abstract 2017 Background In relapsed or refractory AML allogeneic transplantation (HCT) is considered to be the only chance to achieve long-term disease-free survival. However, only about 40% of younger patients with relapsed AML receive allogeneic HCT. A number of factors contribute to this low rate. Among them, a moderate activity of currently available salvage regimens and accumulating toxicity of chemotherapy may prevent from transplantation. Clofarabine is considered to have a favorable risk-benefit ratio in this indication. Therefore, our goal was to study the safety and efficacy of a clofarabine salvage therapy prior to allogeneic HCT (NCT 1295307). Here, we report data from patients of stage I of a two-stage phase II study. Patients and Methods Patients above the age of 40 with relapsed or refractory AML who were fit for allogeneic HCT were eligible to participate in this multicenter, single-arm study. All patients received at least one cycle of clofarabine 40 mg/m2 followed by intermediate dose cytarabine 1 g/m2 days 1–5 (CLARA). Patients with a donor who exposed at least a reduction of leukemic blasts were scheduled for allogeneic HCT in aplasia after CLARA. Patients without a donor should receive consolidation therapy with CLARA. The conditioning regimen started earliest on day 15 after CLARA and consisted of clofarabine 30 mg/m2 on days -6 to -3 and melphalan 140 mg/m2 on day -2 prior to HCT. GvHD prophylaxis consisted of cyclosporine in combination with MMF. In patients with partially matched unrelated donors the administration of a cumulative dose of 4.5 mg/kg ATG (Genzyme) was recommended. Primary endpoint was treatment success defined as a complete remission (CR, CR(i)) six weeks after completion of therapy. Toxicities were graded according to CTCAE Version 3.0. Results Twenty-six patients were enrolled into stage I of this trial. Median age was 60 years (range, 40 to 74 years). Fifty percent of the patients each had refractory or relapsed AML. At early response assessment on day 15 after CLARA-1 13 patients (50%) had less than 10% marrow blasts. Ten patients (38%) showed a reduction in marrow cellularity or blast percentage. Two patients did not respond to CLARA and were subsequently treated off study. One patient died on day 18 after the first cycle from septic multi-organ failure. Twenty-two patients (85%) received allogeneic HCT within this trial. Donors were HLA-identical siblings in 5 patients (23%), HLA-compatible unrelated donors in 11 patients (50%) and partially matched unrelated donors with one mismatch in 6 patients (27%). All 26 patients have been evaluated for the primary endpoint. Sixteen patients had a CR (62%) and 6 patients a CRi (23%) at final response evaluation, all after allogeneic HCT. One patient responded to chemotherapy but needed surgical intervention due to a pulmonary aspergillosis and could not proceed with therapy within the projected timelines and was therefore considered as treatment failure. Liver toxicity was the most frequent adverse event. Seventeen patients (65%) developed grade III liver enzyme elevation considered to be at least possible related to the study drug. Grade IV liver toxicity was observed in 1 patient. Liver injury indicated by elevated transaminases was transient and did not lead to persistent liver damage or hepatic sinusoidal obstruction syndrome. Due to frequently required per-protocol dose reductions of clofarabine prompted by grade III elevations of transaminases the study protocol was amended to a reduced dose of clofarabine 30 mg/m2 in CLARA chemotherapy. Five patients developed grade I/II elevation of creatinine and 2 patients developed renal failure (grade IV) in the context of septic organ failure. Grade I/II hand-foot syndrome occurred in 5 patients. With a maximum follow up of 17 months 14 patients have died (5 patients died after relapse). At present 12 patients are alive, 7 of these are relapse-free, 3 patients are alive with relapse and 2 patients are currently receiving hypomethylating agents for molecular relapse. Conclusion Salvage therapy with CLARA and subsequent conditioning with clofarabine and melphalan prior to allogeneic HCT provides good anti-leukemic activity in patients with relapsed or refractory AML. The complete remission rate of the first 26 patients was evaluated favorably and the trial is currently recruiting to reach the total number of 82 patients. Longer follow up is needed to evaluate relapse-free survival. Disclosures: Middeke: Genzyme: Honoraria. Off Label Use: Clofarabine, not approved for AML. Bornhäuser:Genzyme: Honoraria. Schetelig:Genzyme/Sanofi: Honoraria, Research Funding.

[Figure][1] An 83-year-old woman with chronic gastrointestinal problems developed retinal vein thrombosis. Laboratory tests revealed an erythrocyte sedimentation rate of 45, renal dysfunction (GFR 25 mL/min), and mild anemia (hemoglobin 10 G/dL) with normal white cells and platelets. β2

Abstract 2534 Introduction Cytomorphological evaluation of bone marrow remains a fast and easy accomplished tool in patients with suspected acute myeloid leukemia (AML). Some cytogenetic aberrations can be predicted by specific morphological features like abnormal eosinophils associated with inv(16)/t(16;16) and faggot cells with t(15;17). But implementations of cytogenetic and molecular tests nearly diminished the interest in studying cytomorphological features that might be associated with chromosomal aberrations. Common morphological characteristics often found in AML associated with t(8;21) are large blasts with abundant basophilic cytoplasm, containing numerous azurophilic granules, perinuclear clearing or hofs and single long and sharp Auer rods. But not all AML with t(8;21) show these characteristic signs. During our cytomorphological review for patients with AML within the German prospective multicenter randomized AML96 and AML2003 trials we observed that a focal non-specific esterase stain for blasts in t(8;21) appears to be another specific feature for this subentity. With this study we tried to analyze its predictive value for this chromosomal aberration. Materials and methods Bone marrow (BM) aspirates and peripheral blood smears of 156 eligible patients from the Study Alliance Leukemia (SAL) AML96 and AML2003 trials were investigated by conventional cytochemical methods (May-Giemsa, myeoloperoxidase, alpha-naphthyl butyrate esterase and iron staining). AML classification was performed according to FAB and WHO 2001. Diagnosis of t(8;21)/RUNX1-RUNX1T1 was made by FISH, chromosomal banding and RT-PCR analysis. Bone marrow samples from patients with AML associated with t(8;21)/RUNX1-RUNX1T1 were evaluated and reviewed by an independent and experienced morphologist. Blast morphology was evaluated for granulation, perinuclear clearing or hofs, focal positivity for non-specific esterase (NSE) and the presence of Auer rods, pseudo-Pelger neutrophils and eosinophils. Besides that we investigated its correlation for aberrant expression of CD19 and CD56 in immunophenotyping and the KIT exon-17 mutation. All statistical calculations were performed using the software program PASW statistics 17 (SPSS, Chicago, IL, USA) and R 2.10.1 (The R Foundation for Statistical Computing, 2010). Results Of 156 eligible patients, 72 did not have adequate bone marrow samples to assess the amount of blasts with NSE positivity; therefore, the final study cohort consisted of data sets from 84 patients. The median age was 44 years (range, 19–85 years). The distribution of gender was 39 women (46%) and 45 men (54%). The majority of cases (81%) were subclassified in FAB M2. Cytomorphology showed a focal NSE positivity in 82 patients (98% of cases). Chromosome banding analyses were performed in all cases and showed t(8;21) in all cases. Forty-three patients demonstrated additional abnormalities with 29 (35%) cases showing single additional aberrations and 14 (17%) cases having two or more additional aberrations. Focal positivity of non-specific-esterase stain was found in 25% in 11 (13%), 50% in 29 (35%), 75% in 22 (26%) and 100% in 20 (24%) of bone marrow blasts. The presence of focal NSE-positivity demonstrates a significant association with the cytogenetic aberration t(8;21) (p < 0.001). Focal NSE-positivity was present even in the absence of further morphological features associated with t(8;21). No correlation between focal NSE stain and the aberrant expression CD19 and CD56 and KIT-Mutation could be seen. In AML other than t(8;21) no focal positivity for NSE could be seen (n=100). Conclusion Our investigations could prove that focal positivity for NSE is a reliable surrogate marker to predict the chromosomal aberration t(8;21). Disclosures: No relevant conflicts of interest to declare.

Abstract 255 Introduction: Standard treatment of acute myeloid leukemia (AML) comprises one or two cycles of chemotherapy to induce complete remission (CR) followed by postremission treatment in order to prevent relapse of the disease (consolidation therapy). In 2003, we initiated a prospective multicenter randomized trial to investigate the impact of different consolidation strategies on long-term outcome in AML patients ≤ 60 years. Consolidation options comprised upfront allogeneic stem cell transplantation (allo SCT) in aplasia after induction therapy, autologous SCT, and three cycles of standard high-dose-cytarabine-based consolidation. For patients receiving high-dose cytarabine, the main study aim was to evaluate the benefit of adding additional mitoxantrone and amsacrine to cytarabine consolidation. Design: From 2003 to 2009, 1182 patients (median age, 48 years; range 16–60 years) with untreated AML were randomly assigned at diagnosis to different consolidation strategies after classical 7+3 induction. According to the risk-adapted treatment strategy of the trial, cytogenetically or molecular intermediate-risk (IR) and adverse-risk (AR) patients should receive an allo SCT as consolidation treatment if an HLA-identical-sibling donor (IR) or HLA-matched related or unrelated donor (AR) was available. IR and AR patients with no available donor should receive autologous SCT. All favorable risk patients and patients with no available donor were scheduled for high-dose cytarabine based consolidation. Half of the patients were randomized for high dose cytarabine based consolidation. Half of the patients were randomized for high dose cytarabine alone while the other half received high dose cytarabine with the addition of amsacrine and mitoxantrone. Standard chemotherapy consisted of three cycles with high dose cytarabine (2 × 3 g/m2, day 1,3,5) whereas combined consolidation contained two cycles of MAC (cytarabine 2 × 1g/m2, day 1–6, mitoxantrone 10 mg/m2, day 4–6) plus one cycle of MAMAC (cytarabine 2 × 1 g/m2, day 1–5, amsacrine 100 mg/m2, day 1–5). In order to evaluate the effect of the two cytarabine based consolidation strategies, we determined overall survival (OS) and event free survival (EFS) using the method of Kaplan Meyer. Survival distributions were compared using the log rank test. Results: 1182 patients were randomized for further intervention (Arm A+B: n=582, 49.3%; Arm C+D: n=600, 50.7 %). Median follow-up was 41.4 months (95%-CI 39.3–43.6). A total number of 375 patients received allogeneic (n=322) or autologous SCT (n=53) and 807 patients were consolidated with cytarabine. Of these patients, 407 were randomized for cytarabine alone and 400were randomized to receive cytarabine plus mitoxantrone and amsacrine (MAC/MAC/MAMAC). Complete remission rate (CR) after second induction therapy was 59.1% (n=698). Between the four arms, there were no significant differences of the CR rates. Five-year OS of patients receiving high dose cytarabine alone was 47.1% (95%-CI 42.0–52.2%), for patients receiving MAC/MAMAC as consolidation therapy it was 46.8% (95%-CI 42.3–51.3%; p = 0.610). Three-year event free survival (EFS) was also not significant with 30.5% (95%-CI 26.6–34.4%) for patients receiving high dose cytarabine alone and 35.6% (95%-CI 31.7–39.5%; p = 0.059) for patients receiving MAC/MAMAC. Conclusions: According to our data, the addition of mitoxantrone and amsacrine to high dose cytarabine consolidation confers no benefit for treatment outcome in younger AML patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 5038 Case Report We report of a 53 year old woman who presented repeatedly with syncopes over a period of 3 months. Laboratory results revealed severe pancytopenia (Hb 4.6 mmol/l (7.4 g/dl), WBC 1.22 Gpt/L, platelet count 13 Gpt/L) and severe hemolysis (haptoglobin < 0.20 g/l, normal range 0.3–2.0 g/l; LDH 33.1 μmol/s*l, normal range 2.25–3.55 μmol/s*l). Routine examination of the bone marrow aspirate showed typical features of megaloblastic erythropoiesis, which could be confirmed by a low serum cobalamin level (53 pg/ml, normal 211–911 pg/ml) and the presence of anti-intrinsic factor antibody (16.84 U/ml). Additionally, atrophic gastritis was seen in biopsies taken of gastric mucosa. The diagnosis of pernicious anemia was suspected and the patient treated with cobalamin. Except for hemoglobin, the peripheral blood counts recovered within one week. Meanwhile, cytogenetics from the bone marrow revealed metaphases with del(3p) and histopathological results were suspicious of an increased number of blast cells with highly expression of CD163 possibly mimicking MDS or (acute) monocytic leukemia. Therefore, bone marrow examination was repeated two weeks after recovery, which still showed dysplastic changes paralleling hematopoietic recovery but no increased number of blast cells. Additionally, the cytogenetic aberration had disappeared. Discussion Diagnostic work-up for megaloblastic anemia rarely includes cytogenetic analysis of bone marrow cells. Therefore, the finding of a transient cytogenetic aberration has possibly not reported frequently before in the literature. In our case, the initial finding of del(3p) appears to be due to ineffective hematopoiesis caused by vitamin B12 deficiency which leads to impaired DNA synthesis and genomic instability. This might be an explanation for this cytogenetic abnormality which disappeared after substitution of cobalamin. CD163 is exclusively expressed on monocytes and macrophages and with signs of (slightly) increased blast counts might mimic (acute) monocytic leukemia. However, in pernicious anemia with severe hemolysis as seen in this case it might reflect an acute phase reaction, as CD163 represents a signal-inducing macrophage receptor that scavenges haemoglobin by mediating endocytosis of haptoglobin-hemoglobin complexes. In conclusion, vitamin B12 deficiency might be associated with cytogenetic abnormalities and thus in addition to the bone marrow morphology feign certain haematological diseases. Disclosures: No relevant conflicts of interest to declare.

Abstract 4006 Background: According to WHO, the recommended requisite percentage of cells manifesting dysplasia in the bone marrow to qualify as significant is ≥ 10% in one or more hematopoietic cell lineages. However, routine diagnostic of bone marrow smears often reveals atypical hematopoietic cell morphology in non-malignant disease, systemic infection or inflammation as well as a physiological phenomenon. Normal values of differential counts of bone marrow aspirates are based on the analysis of very few healthy subjects. Therefore the threshold of 10% is controversial. Methods: We investigated dyshematopoiesis in bone marrow (BM) smears of 120 healthy bone marrow donors, who had undergone thorough investigation prior to a harvest in general anesthesia. Samples were taken from the first aspiration during the harvest. Bone marrow preparation and assessment of myelodysplasia was performed as recommended by the WHO (Vardiman et al. 2008) and according to ICSH guidelines (Lee, Erber et al. 2008). The bone marrow smears were examined independently by four experienced morphologists. Each investigator had to do a 200-cell differential count of all nucleated cells, and 100 cells of erythropoiesis, 100 cells of granulopoiesis and 30 cells of megakaryopoiesis were assessed for the degree of dysplasia. Finally, the individual results were averaged. Subgroups were defined by gender (63 male, 57 female donors), age groups (40 years old with the oldest donor being 51 years), smoking status and regular alcohol intake. T-Tests for independent samples were used for analysis of difference of means between two groups. If more than two groups were compared analysis of variance was performed with additional post-hoc adjustment for all group comparisons. Results: The peripheral blood counts of all donors were in the normal range. Mean results for hemoglobin were 9.0 ± 0.8 mmol/l and for serum ferritin 67.9 ± 65.2 μg/dl. The proportion of cells with dyserythropoietic changes was 8.2 ± 4.4 %, with 32% of all donors showing > 10% dysplastic changes (33% of the female and 32% of the male donors). None of the donors had >50% dyserythropoiesis. Ring sideroblasts could not be found within the iron stain. Mean dysgranulopoietic changes were found in 13.9 ± 5.5 % the cases, with 51% of all donors showing > 10% dysgranulopoiesis (59% of the female and 43% of the male donors). 5% of the female donors showed > 50% dysgranulopoiesis. No auer rods or excess of blasts were found. Mean dysmegakaryopoietic changes were 8.3 ± 5.6 %, with 29% of all donors showing >10% (36% of the female and 22% of the male donors; none > 50%). There were no significant differences of dyserythropoiesis according to age. In contrast, donors below the age of 30 years showed significantly more dysgranulopoietic cells (18.3% versus 11%, p=0.05) compared to donors above the age of 40 years. In parallel, donors below the age of 30 years had significant more dysmegakaryopoietic cells compared to donors above the age of 40 years (10.4% versus 5.3%, p10% dysplastic changes in 2 (33% of the female and 29% of the male donors) and 6.5 % in 3 cell lineages (8% of the female and 5% of the male donors) respectively. No significant differences in dyshematopoiesis could be detected regarding sex, smoking status or regular alcohol intake. 85% of the BM smears were normocellular and 15% hypocellular. No significant differences were found with an uncorrected Chi-square-Test comparing bone marrow cellularity in the respective age groups. Summary and conclusion: More than 10% dysmyelopoiesis could be detected in 37% of the bone marrow smears with 31% in ≥ 2 cell lineages (6,5% in 3 cell lineages) in healthy bone marrow donors. These findings suggest that the threshold of ≥ 10% for the percentage of cells manifesting dysplasia in bone marrow to qualify as significant should be questioned, especially when other MDS-related changes (e.g. cytogenetic abnormalities) are lacking. Unexpectedly, donors below the age of 30 years exposed more dysgranulopoietic and dysmegakaryopoietic changes compared to the elder donors. Disclosures: No relevant conflicts of interest to declare.

Abstract 1326 Introduction: Assessment of the individual risk of mortality after allogeneic hematopoietic cell transplantation (HCT) is essential for decision-making and patient counseling. For patients with AML several risk factors have been identified, including patient age, comorbidity, unfavorable cytogenetics, prior history of myelodysplasia and the remission status at HCT. The hematopoietic cell transplantation – specific comorbidity index (HCT-CI) has been designed and validated to predict mortality by concurrent disease and organ dysfunction prior to HCT at the Fred Hutchinson Cancer Research Center. Local data-dependence may affect the transportability of a prognostic system. Cross center validation of prognostic systems is therefore essential. In order to check the performance of the HCT-CI for our center, we applied the HCT-CI in a well defined cohort of AML patients. Our unit represents a large German transplant center characterised by referral of a large proportion of elderly patients with advanced myeloid leukemias. Methods: We performed a retrospective cohort analysis in patients with AML according to the WHO definition who were transplanted between January 2000 and December 2008. Second allogeneic HCTs and haploidentical HCTs were excluded. Baseline variables were extracted from the local database by a trained data manager. Supplementary data were collected by comprehensive review of medical records by the investigator. For each patient we assessed the HCT-CI prior to transplant. Overall survival (OS) was estimated using the Kaplan-Meier method. Non-relapse mortality (NRM) was calculated with cumulative incidence statistics. The effect of the HCT-CI on the OS and NRM was analyzed in a multivariate Cox regression model, considering age, Karnofsky score, type of AML, cytogenetic risk (according to the ELN classification), treatment and remission status, sex match, CMV match, donor type, and conditioning intensity. Results: 340 patients were eligible for the analysis. The median age of our patients was 53 years (range, 11 to 76 years). Eighty-six patients (26%) were in CR-1 of standard risk AML and 45 patients (14%) were in CR-1 of high risk AML while the remainder had more advanced disease. The median HCT-CI was 4 (range, 0 to 10). Donors were HLA-identical siblings in 116 patients (34%), matched unrelated donors in 130 patients (38%) and partially matched unrelated donors in 94 patients (28%). Overall survival at 2 years was 41±12%, 49±6% and 49±3% in the low, intermediate and high-risk HCT-CI groups (p=0.7), respectively. The corresponding NRM at 2 years was 33±23%, 26±10% and 23±5% (p=0.6). In multivariate analysis the HCT-CI failed to predict OS and NRM. In multivariate regression modeling neither its inclusion as a categorical variable (low, intermediate or high risk) nor as a numerical variable demonstrated adequate discrimination. Comparing our population with the original validation cohort of the index hepatic impairment (51% vs. 20%, p=0.06), infection (47% vs. 4%, p= Conclusion: We found no predictive value of the HCT-CI for OS and NRM in our patients. Our results suggest that different working definitions for the assignment of comorbidities had been applied. Refined definitions could therefore help to improve the performance of the index. Until its validity has been shown across different patient populations, its use as decision making tool for transplantation eligibility needs to be reconsidered. Disclosures: Platzbecker: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

In about 30% of patients with AML the predominantly nucleolar protein nucleophosmin (NPM1) is dislocated into the cytoplasm (NPM1c), caused by mutations in exon 12 of the NPM1 gene. NPM1-mutations are associated with a specific subgroup of AML with particular clinical, cytogenetic, and prognostic characteristics, and NPM1-mutations are part of the new WHO classification, which makes the detection an important diagnostic aspect. Thus far, NPM1 mutations can be detected either using molecular techniques or immunocyto- or histochemistry. We reasoned that by the cytoplasmic dislocation, the protein might be accessible also for rapid diagnostics using intracellular flow cytometry. The aim of the present work was to evaluate this procedure in vitro and prospectively in newly diagnosed patients with AML. Therefore, cell lines and in addition bone marrow (BM) or peripheral blood of 134 newly diagnosed AML patients, enrolled in two different treatment protocols, were prospectively investigated by a lyse-wash flow cytometric procedure including an anti-NPM monoclonal antibody labelled with Zenon labelling technology. Bone marrow of 23 healthy donors served as control. Cytoplasmic expression of NPM was measured after fixation and permeabilisation. Mean fluorescence intensity (MFI), assessing the ratio between the intensity of the specific antibody and the isotype matched control, was evaluated. All patients samples were routinely analyzed for NPM1-exon 12 mutations using PCR followed by high resolution fragment analysis. Cell lines with known mutational status (mutant: OCI-AML3 and wt: MV4-11) showed a clear separation (MFI: 44.6 vs. 3.7). In healthy BM the median MFI for the NPM expression was 3.8 ± 1.7. Thus, the threshold MFI for NPM positivity was set to 8.0. In overall in 93% of samples (125/134) both methods showed concordant results – positivity in 33% (44) and negativity in 61% (81) of patients. Thus, the MFI was significantly higher in NPM1c cases than in PCR negative patients and BM donors (MFI: 19.0 vs. 3.3 vs. 3.8; p