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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