ALBERT

Description: Abstract 2773 Introduction: For patients with high-risk myelodysplastic syndromes an epigenetic therapy with hypomethylating agents is considered standard of care. Intensive chemotherapy can be offered to a subset of patients; however, data about the long-term outcome of MDS patients receiving intensive chemotherapy are scarce. Methods: For this evaluation, 104 adult patients with IPSS intermediate-2 or high-risk MDS with at least 10% bone marrow blasts of all age groups treated within the AMLCG1999 trial were included. Patients were randomized upfront to receive 1. double induction therapy with either standard-dose containing TAD - versus high-dose containing HAM–HAM, 2. TAD consolidation therapy followed by either a monthly maintenance therapy for 3 years after achievement of CR or an autologous stem cell transplantation (patients aged ≥ 60 years were all assigned to maintenance therapy), and 3. blast priming with filgastrim starting on day -1 of chemotherapy in selected centers. Results: Fifty-four patients had IPSS Score intermediate-2 and 50 patients were IPSS high risk. Median bone marrow blast count at diagnosis was 15%. The median age was 63.5 years (range: 27–76 years), 39 patients (37.5 %) were female. Median lactate dehydrogenase (LDH) serum level was 296 U/l, median leukocyte count at diagnosis was 5,950 per μl. The cytogenetic risk groups were as follows: favorable 3, intermediate 57, unfavourable 37, missing 7. Among 38 patients with normal karyotype, NPM1/FLT3 mutational status was available for 22 with 5 patients having the combination NPM1 mutated/FLT3 wildtype. Comparison with 2051 patients with de novo AML within the same trial revealed the following significant differences: patients with MDS were older, had a higher male to female ratio, a lower LDH serum level at diagnosis, a lower leukocyte count at diagnosis and were more likely to have adverse cytogenetic risk. Compared to 636 patients with secondary AML after MDS, cytotoxic therapy or irradiation, the cohort of patients with MDS did not display any significant differences except the sex distribution. Patients with MDS displayed a CR rate of 48% (50/104 patients), which was significantly lower than de novo AML patients (67%) and not different to secondary AML patients (47%). Median overall survival in MDS patients was 320 (95% CI: 236 to 505) days with a 2-year and 5-year survival of 33.4% (95% CI: 23.6% to 43.2%) and 22.7% (95% CI: 13.5% to 31.9%), respective, which was significantly (p=0.03) lower than in patients with de novo AML (median 484, 95% CI 435 to 541 days) and comparable to patients with secondary AML (median 282, 95% CI 224 to 311 days, p=0.13). Median relapse-free survival in responding MDS patients was 536 (95% CI: 264 to 1299) days with no significant differences of RFS compared to de novo or secondary AML patients. In multivariate analyses, the diagnosis of MDS remained an independent prognostic factor for CR probability but had no independent influence on survival compared with de novo AML patients. Nine patients proceeded to allogeneic stem cell transplantation in first complete remission of whom six remain in first complete remission between 1354 and 1911 days after achievement of CR. In addition, 16 patients remained in CR for more than one year without allogeneic transplantation. Discussion: Taken together, outcome of patients with intermediate-2 or high-risk MDS after intensive chemotherapy is comparable to the outcome of patients with secondary AML. Adjustment for known risk factors such as age, cytogenetic risk and LDH revealed that inferior outcome of MDS patients compared to patients with de novo AML is attributable to the higher incidence of adverse risk factors. CR-rates appear to be higher compared to hypomethylating therapy and a fraction of MDS patients experiences long-term survival by intensive chemotherapy. Allogeneic transplantation can improve long-term survival for patients achieving remission. Disclosures: Krug: MedA Pharma: Honoraria; Novartis: Honoraria; Alexion: Honoraria; Boehringer Ingelheim: Research Funding; Sunesis: Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Description: In the IPSS the variables bone marrow blasts, cytogenetics and cytopenias were found to be most relevant for clinical outcome by multivariate analysis. Comparing cytogenetics and blasts scoring points were assigned as follows: 0 (good cytogenetics and less than 5% blasts), 0.5 points (intermediate cytogenetics, and 5–10% blasts), 1.0 points (poor cytogenetics), 1.5 points (11–20% blasts), 2.0 (21–30% blasts). In order to examine the correctness of weighting of cytogenetics in comparison to blast counts we compared the survival curves, median survival times (mst) and differences of mst (mst diff.) related to the mst of 37.5 months (mo) of our entire study population (on the basis of 2124 pts. with MDS from our German-Austrian database) between patient subgroups. The results in the subgroups were as follows: Blasts below 5% (n=609) (mst: 58 mo, mst diff.: +20.5 mo), good cytogenetics (n=768) (mst: 55.3 mo, mst diff.: +17.8 mo), blasts 5–10% (n=231) (mst: 28.0 mo, mst. diff.: −9.5 mo), intermediate cytogenetics (n=222) (mst: 28.0 mo, mst diff.: −9.5 mo), blasts: 11–20% (n=160) (mst: 16.5 mo, mst diff.: −21 mo), poor cytogenetics (n=212) (mst: 11.1 mo, mst diff.: −26.3 mo), blasts 21–30% (n=92) (mst: 11.7, mst diff.: −25.7 mo). Our results clearly show that within the IPSS poor cytogenetics are significantly underweighed. Referring to the survival data unfavorable cytogenetics resulted in a survival disadvantage at the same scale as compared to 21–30% blasts. Thus, in a revised IPSS this cytogenetic feature should get the same scoring points as compared to 21–30% blasts when using the FAB-classification. For a scoring system based on the WHO-classification unfavorable cytogenetics should get an even higher scoring value as compared to the maximum blast count of 19%. Further statistical analyses are on the way to substantiate our conclusions.

Description: The 5q deletion syndrome is described as having a favourable outcome in MDS. However, little data is available concerning del(5q) and additional karyotype anomalies or taking into account bone marrow blast count. We screened the MDS registries of Düsseldorf, Germany and Lausanne, Switzerland in order to obtain data on patients with karyotype anomalies including del(5q). In both registries 1073 patients were karyotyped at diagnosis. 198 patients with del(5q) with or without other karyotype anomalies and 105 patients with complex karyotype anomalies without del(5q) were analysed. 106 patients showed del(5q) as single anomaly, 23 had 1 additional karyotype anomaly, 69 had 2 or more additional anomalies and were therefore classified as complex karyotype. In the group with del(5q) only, mean survival was 76 months as compared to 42 months in patients with a normal karyotype. In the group with 1 additional anomaly mean survival was 47 months (p=0.02), in the group with 2 or more additional anomalies 7 months (p=0.0005). Survival differed significantly in each subgroup when patients presenting less than 10% of bone marrow blasts where compared to patients with 10% or more. Patients with del(5q) only show a median survival of 12 months when presenting with more than 10% medullary blasts. Patients with a complex karyotype including del(5q) with less than 5% medullary blasts had a median survival of 12 months, but median survival was only 6 months in patients with elevated medullary blasts. By multivariate analyses, we found that additional karyotype anomalies, followed by an elevated medullary blast count above 10% are the most important independent prognostic parameters. Furthermore, we compared the subgroups with del(5q) and 2 or more additional karyotype anomalies (n=69) to patients with complex karyotype anomalies not including del(5q) (n=105). Median survival of the group with complex karyotype anomalies including del(5q) was 7 months as compared to 12 months in the group without del(5q) (p=0.02). 12 months after diagnosis, 30% of the group with del(5q) in a complex karyotype was alive as compared to 45% of the group with a complex karyotype without del(5q). Disease related death was noted in 77% of patients with a complex karyotype and del(5q) and 73% without del(5q). Therefore, our data show that the prognosis of survival in patients with del(5q) is highly dependent on the number of additional karyotype anomalies as well as medullary blast count. The prognosis of MDS patients with del(5q) is associated with an extremely poor prognosis when diagnosed within a complex karyotype. Although there are no substantial differences in clinical, haematological and morphological data between patients with or without del(5q), the median survival differs significantly. Figure Figure

Description: Lenalidomide is the first drug to consistently induce transfusion independence and complete cytogenetic remissions in MDS patients with deletion of 5q. However, recently concerns were raised whether lenalidomide treatment may increase the risk of leukemic transformation. The selective activity of lenalidomide against the deletion 5q clone may allow pre-existing aberrant clones to expand if the dominant deletion 5q clone is eradicated in a circumstance in which few normal stem cells remain. This conditional selection of pre-existing clones (M. Cazzola, Haematologica2008; 93:967–72) could represent a potential mechanism for leukemic transformation. We therefore analyzed 22 European patients with transfusion-dependent anemia due to low- or intermediate-1-risk deletion 5q MDS treated with lenalidomide who were enrolled in the MDS-003 (CC-5013-MDS-003) study between 12/2003 and 2/2004. For these patients, sufficient cytogenetic, morphologic and clinical follow-up data were available. Ten of these patients achieved a complete (n = 6, 3/6 transiently) or partial (n = 4, 3/4 transiently) cytogenetic remission and 11 patients (5/11 transiently) achieved an erythroid response resulting in transfusion independence. Seven of the 22 patients (32%) developed complex clones containing typical aberrations like der(7q), +8, del(12p), del(17p) and +21 within 5 to 42 months (median 19 months) after study entry, and within 15 and 72 months (median 44 months) after diagnosis. Among these 7 patients, the findings at baseline included that 2 patients had the deletion 5q plus additional chromosomal aberrations, i.e. del(12p) and trisomy 21, respectively, 2 patients had an isolated deletion 5q karyotype and low-risk MDS, and 3 patients had an elevated blast count ≥ 5%. Six of the these 7 patients progressed to AML, and one patient lost the complex clone with continued lenalidomide treatment, showing the isolated deletion 5q again, and achieved a partial remission from RAEB-1 to RCMD. Both patients with low risk MDS and isolated deletion 5q developed complex clones 12 and 9 months after study entry and 19 and 24 months after diagnosis, respectively, and progressed into AML. Moreover, two patients without cytogenetic response progressed to AML 5 and 6 months after study entry, and 15 and 72 months after diagnosis, respectively. In total, 8 patients (36%), 2 of them with 5q- syndrome, 4 with RCMD and 2 with RAEB-1 according to the WHO classification, progressed into AML. FISH analyses were performed retrospectively to clarify whether complex karyotypes were already present at the time of diagnosis. In no case were such aberrations detected. The FISH data also showed that 13% to 84% (median 50%) of the cells contained a del (5q) abnormality, demonstrating that in all patients who underwent karyoptypic evolution, a substantial number of normal cells was present in the bone marrow. Overall, these data show that karyotypic evolution of 5q- clones into complex clones plays a significant role in the progression of low and int-1 MDS with 5q- into AML. No evidence of conditional selection by FISH analyses was observed. However, the presence of small complex clones at baseline may have been missed because FISH has a detection limit of up to 10%. It remains unclear whether the development of complex clones occurs during the natural course of the disease, whether lenalidomide treatment increases chromosomal instability or whether pre-existing clones are selected during treatment. Clearly, these findings warrant careful follow-up of MDS patients treated with lenalidomide and show that a multi-center, double-blind, placebo-controlled study with a long observation time is needed to define long-term safety and efficacy of lenalidomide.

Description: Introduction: Lenalidomide is a novel immunomodulatory drug that is highly effective in patients with transfusion-dependent MDS with del(5q.31) chromosomal abnormality. In the recent MDS-003 study of lenalidomide, there was a 76% erythroid response rate, including 67% transfusion independence. Cytogenetic complexity or bone marrow blast percentage did not affect this response rate. A number of patients experienced disease progression to higher FAB subtypes or AML. We questioned whether lenalidomide might promote disease progression in del(5q) MDS and performed a retrospective analysis to identify risk profiles. Methods: Fifty patients from three institutions were included in this analysis. They were partly treated within the Lenalidomide-MDS003-study. Patients were treated with an initial lenalidomide dose of 10 mg po daily. In case of grade 〉2 neutropenia, G-CSF and antibiotics were administered. Results: The median age was 71 years; 31 patients were female and 20 were male. Disease progression to a higher FAB subtype or to AML occurred in 13 patients (29.5%). 7 of the 13 patients had RAEB at the first lenalidomide dose. In addition, 3 of these had additional chromosomal aberrations (2, trisomy 21; 1, complex karyotype). Of the remaining 6 patients, 2 had a complex karyotype at the first lenalidomide dose, 1 had an additional inv(9)(p11q12), and 1 had hypocellular bone marrow so no FAB subtype could be assigned. Only 2 of 50 patients (4.3%) with 5q-syndrome progressed to AML; both patients developed acute erythroid leukemia (FAB M6). Conclusion: Within the del(5q) MDS subgroup, patients with an isolated del(5q) chromosomal aberration and a bone marrow blast count of 5% bone marrow blasts or additional chromosomal anomalies. Lenalidomide does not seem to increase the risk of transition of del(5q) MDS to higher stages of disease.

Description: All-trans-retinoic acid (ATRA) alone or in combination with cytokines and vitamins has shown erythroid remitting capacities in low-grade myelodysplastic syndromes (MDS). We performed a phase II study on 29 patients with MDS and isolated del(5q) including bands 5q31–5q33 to determine the efficacy and safety of ATRA in combination with tocopherol-α. All patients had low/intermediate-1 risk MDS according to the international prognostic scoring system. Inclusion criteria were isolated del(5q), medullary blast count of

Description: Abstract 4008 Introduction: Loss of the Y chromosome has been reported to be associated with hematopoietic diseases (Wiktor et al., 2000), but it was also described as an age-related phenomenon in males (UKCCG, 1992). Determination of clonality and prediction of prognosis and treatment outcome might benefit from a differentiation between age- and MDS-associated Y loss. The aim of this study was to evaluate if Y loss was an age- and/or MDS-associated phenomenon by retrospectively analyzing our multicenter, international DACH-, ICWG- and IMRAW-database and by testing the established hypotheses in an experimental study. Patients and Methods: In our multicenter MDS-database of 2901 patients, 101 primary, untreated MDS patients (3.5%) with loss of the Y were identified. We analyzed them according to age, clone size, and the presence or absence of additional chromosomal aberrations and assessed the prognostic relevance of the aberrations using univariate and multivariate models. Additionally, by immunomagnetic cell sorting, we enriched clonal CD34+ cells and CD3+ T-cells not belonging to the MDS clone from peripheral blood of three patients and compared the percentage of cells with -Y using FISH. Results: Isolated loss of Y was observed in 65.3% (n=66) of the 101 patients identified in the multicenter MDS-database, 14.9% (n=15) of the patients displayed one additional aberration and in 19.8% (n=20) -Y occurred as part of complex abnormalities. Overall survival of patients with -Y as a sole change was significantly better compared to patients with a normal karyotype (60.8 vs. 47.4 months; hazard ratio = 0.50, p

Description: The myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic malignancies. We have used Affymetrix microarray technology to determine the gene expression profiles in CD34+ cells of 84 MDS patients (25 RA, 28 RARS and 31 RAEB) and 16 healthy controls. Twenty-five of 84 patients had a del(5q). CD34+ cells were isolated from bone marrow samples using MACS magnetic columns. Extracted total RNA was amplified using the Two-Cycle Target Labelling kit (Affymetrix) and samples were hybridized to Affymetrix U133 Plus2.0 chips (representing 39,000 human genes). Cell intensity calculation and scaling was performed using GeneChip Operating Software and data analysis using GeneSpring 7.3. The expression profiles of MDS CD34+ cells showed many similarities to reported interferon-γ induced gene expression in normal CD34+ cells. Indeed the two most up-regulated genes, IFIT1 and IFITM1, are interferon-stimulated genes. IFIT1 and IFITM1 were up-regulated by 〉2-fold in 58/84 and 53/84 MDS patients respectively. Genes down-regulated by 〉2-fold in the majority of MDS patients include the putative tumor suppressor gene Gravin/AKAP12, ARPP-21, CD24 and MME. The association of distinct gene expression profiles with specific FAB and cytogenetic groups was determined using data from 55 MDS patients as a training set. Hierarchical clustering performed using 457 significantly different genes between different FAB subtypes showed that MDS patients with RARS constitute a homogeneous group, while MDS patients with RA and RAEB show more overlap. CD34+ cells from patients with RARS showed up-regulation of mitochondrial-related genes, and in particular of those of heme synthesis (e.g. ALAS2). Statistical analysis showed that 889 probe-sets could discriminate MDS patients with a del(5q) from those without a del(5q). MDS patients with the del(5q) showed distinctive down-regulation of genes mapping to chromosome 5q, and up-regulation of the histone HIST1 gene cluster at chromosome 6p21 and of genes related to the actin cytoskeleton. In order to identify genes differentially expressed between early and advanced MDS, a comparison was made between the 18 patients with RA and the nine MDS patients with RAEBII. 762 significantly different probe sets were identified that could group together MDS patients with RAEBII. The most significant genes identified include CASP3 and FLT3, and represent potential prognostic markers or markers of disease progression. The remaining 29 MDS patients were used as a test set for class prediction using support vector machines. The FAB subtype was correctly predicted for 83% of the test samples. The presence or absence of a del(5q) was predicted correctly for 93% of the test samples. Finally, 94% of the test samples were predicted correctly as RA or RAEBII. This study provides important and new insights into the pathophysiology of MDS.

Description: Myelodysplasia (MDS) is a heterogeneous group of clonal disorders of hematopoietic stem cells characterised by ineffective hematopoiesis and a variable risk of transformation to acute myelogenous leukaemia. We have used Comparative Genomic Hybridisation (CGH) microarray analysis, a technology that represents a significant improvement in resolution over conventional cytogenetic analysis, to screen genomic DNA from MDS patients for the identification of genome-wide Copy-Number Changes (CNCs). We have studied genomic DNA obtained from the neutrophil population of 48 MDS patients and 40 normal controls. Of the 48 MDS patients 10 had the 5q- syndrome, 32 were assigned normal karyotype and 6 had complex karyotypes. Comparative Genomic Hybridisation (CGH) microarray analysis was performed using microarrays containing 3500 BAC clones at 1Mb intervals over the whole human genome. Furthermore we used a whole genome tiling-path (27 000 overlapping BAC clones) array to profile 9 5q-syndrome patients and for 3 of those patients the T-cell DNA were also profiled to act as constitutional control. The patient DNA and a pool of normal reference DNA was labelled with different fluorochromes and cohybridised to the microarray. The normalised ratio of signal intensities was calculated and log2 ratios between −0.4 and 0.4 were considered normal. Ratios below or above the normal range were interpreted as loss or gain of genetic material, respectively. The deletions on chromosome 5q were precisely mapped by array-CGH in the patients with the 5q- syndrome but no additional CNCs were detected. One of the 5q deletions, however, displayed a discontiguous pattern with the tiling resolution array. Copy-number changes (CNCs) that escaped conventional cytogenetic detection were identified in the MDS patients originally reported with normal bone marrow karyotypes. 8 out of those 32 patients displayed CNCs that were not detected in the 40 normal controls and as such were considered as disease-related changes (non-polymorphic). Many of those CNCs were single-clone abberrations that were validated by dye-swap experiments and some were confirmed by quantitative PCR. Microarray CGH data confirmed all abnormalities reported by conventional cytogenetic analysis in the MDS patients with complex karyotypes and previously undetected abnormalities were uncovered. Several genes involved in either the initiation or progression of hematological malignancies are known to map within the cryptic abnormalities identified in the patients studied. For example, one patient with an apparently normal karyotype showed a small deletion at 17q11 which encompasses the NF1 gene. Further work will determine whether particular abnormalities detected by microarray CGH are recurrent and the nature of the genes involved. However, the promise of microarray CGH in the diagnostic work up of MDS particularly in those patients with normal karyotypes is clear.

Description: After recent reports addressed prognostic factors and outcome in older age AML (Burnett et al. Blood106:162a,2005; Wheatley et al. Blood106:199a,2005; Appelbaum et al. Blood107:3481–5,2006; Farag et al. Blood108:63–73,2006) we evaluated 764 patients of 60–85 (median 66) years reduced to those with de-novo AML, known karyotype, and identical consolidation-maintenance chemotherapy, who were part of the 1992 and 1999 multicenter randomized trials by the German AMLCG (Buchner et al. J Clin Oncol21:4496–504,2003;24:2480–9,2006). 521 patients were 60 -〈 70 (median 64) and 243 patients were 70–85 (median 73) years of age. 64% and 50% patients respectively went into complete remission, 24% and 29% remained with persistent AML, 12% and 21% succumbed to early and hypoplastic death (p