ALBERT

Description: Background: Understanding health-related quality of life (HRQOL) profile, including functional aspects and symptom burden, of yet untreated patients with myelodysplastic syndromes (MDS) is important to help clinicians to better identify subgroup of patients in need of special attention from the very beginning of therapy. Aims: The primary objective of this study was to investigate baseline (i.e., pretreatment) HRQOL profile of untreated patients with lower-risk MDS, examining differences by age, gender, risk score category and comorbidity. A secondary objective was to provide age and sex baseline reference HRQOL values, according to the EORTC QLQ-C30 questionnaire, to be used as benchmark comparisons in future MDS studies. Methods: This analysis is based on 443 newly diagnosed adult MDS patients with International Prognostic Scoring System (IPSS) risk score of low (46 %) or intermediate-1 (54%), enrolled in an international prospective cohort observational study. Median age was 75 years (range 32-94), with 261 men (59%) and 182 (41%) women. HRQOL was assessed by the EORTC QLQ-C30 questionnaire at study entry, before any treatment (except for transfusions). This well validated questionnaire consists of five functioning scales: physical, role, emotional, cognitive and social; three symptom scales: fatigue, nausea/ vomiting and pain; six single item scales: dyspnoea, sleep disturbance, appetite loss, constipation, diarrhea and financial impact; and the global health status/HRQOL scale. The items were scaled and scored using the recommended EORTC procedures. At the time of baseline HRQOL assessment, 111 (25%) patients had received at least one red blood cell transfusion. We used Wilcoxon-Mann-Whitney and Kruskal-Wallis tests for all comparisons. We used the false discovery rate approach to account for multiple testing, with a nominal α-level=0.05. In addition to statistical significance, clinically relevant HRQOL differences were also evaluated based on previously published criteria (Cocks K, et al, J Clin Oncol 29:89-96, 2011). Results: There were not statistically significant differences in any of the HRQOL scales measured by the EORTC QLQ-C30, by the specific IPSS risk category (i.e., low risk vs intermediate-1 risk score). Overall, women reported worse HRQOL scores than men, with clinically relevant differences for physical (Δ=-7.1, P=0.002), role (Δ=-9.9, P=0.002) and emotional functioning, (Δ=-10, P

Description: Abstract 3511 Introduction: In order to allow a better understanding of acute myeloid leukemia (AML) and develop more promising therapeutic strategies the establishment of functional and reproducible in vivo models is widely pursued. Of available model systems, xenografts in immunodeficient mice reproduce the clinical situation best. Here, we performed extensive analysis of AML engraftment in NOD/SCID-IL2-receptor-gamma-chain−/− (NSG) mice comparing tail vein versus intratibial injection and growth behavior of AML patient-derived bone marrow versus peripheral blood cells. Furthermore, tumor growth characteristics in the murine host were correlated with the disease stage and the molecular risk factor profile of the individual donors. Methods: Bone marrow and peripheral blood cells from 17 AML patients were injected intratibially into NSG mice (n=4–8/patient, 82 mice in total). As controls, 14 mice received bone marrow from three different donors and 5 mice were mock-injected. Tumor growth was monitored via a) determination of overall survival, b) fluorescence-based in vivo imaging (IVI, Kodak FX, Alexa750 labeled anti-human CD45 or CD33 and c) confirmation of IVI data by histological and immunohistochemical examination of bone marrow and spleen. When highly positive IVI signals and/or the overall condition of individual mice indicated enlarged tumor burden, the respective animals were sacrificed and the human AML cells transferred to another animal. In parallel the engraftment pattern of AML cells 2–4 weeks posttransplant was correlated with clinical disease activity, application route and origin of the particular tumor cells. Results: Patients included in the present study represent multiple different French-American-British (FAB) subtypes, various karyotypes and molecular features in terms of the mutational status of NPM1 and FLT3. All patient-derived specimens were capable of recapitulating the disease in NSG mice at 4–6 weeks after transplantation. Over a period of 13 months 12 out of 17 xenografts could be passaged once and 9 at least twice. Up to six passages were performed for an individual AML xenograft. In contrast, engraftment of healthy donor bone marrow cells could be determined merely until day 56 after implantation. The human bone marrow cells of the healthy donors did not engraft in serial passages. Mean survival time of AML bearing animals ranged between 21 and 82 days for a respective xenograft. No differences could be determined between engraftment capacities of peripheral blood or bone marrow cells of one patient. Neither karyotype, FAB classification nor leucocyte count or the percentage of monomorphic blasts in the bone marrow seemed to have an impact on engraftment capacity in the murine organism. However, mice bearing AML xenografts with mutations in FLT3 as well as in NPM1 showed particular short overall survival times and high tumor cell engraftment determined by IVI. This phenomenon became more obvious along the different passages. The intratibial approach proved to be superior in comparison to the intravenous application as cells of an individual patient engrafted faster when injected directly into the bone marrow microenvironment. Determination or tumor load via IVI permits to closely monitor not only the growth behavior but also the homing characteristics of the human cells over time. The positive IVI signals in bone marrow and spleen could be confirmed by histological examination as well as by immunohistochemistry specific for human CD45 and CD33. Conclusions: Our xenografts show a close resemblance to the AML-disease regarding the level of dissemination and organ involvement. Collection of whole-body IVI data proved to be a time- and animal-saving analysis that allows to closely monitor AML growth. As AML is characterized by an increasing number of molecular subtypes with completely different therapeutic options it seems to be extremely worthwhile to develop patient derived xenograft models representing as many AML subtypes as possible. Our results suggest that this model reflects the heterogeneity and important clinical characteristics of the disease, and thus may serve as a tool for preclinical drug testing and investigation of the pathophysiology of AML. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1861 Introduction: Total or partial Monosomy 7 (-7/del(7q)) is one of the most frequent cytogenetic abnormalities in MDS, occurring in about 11% of abnormal cases in patients (pts) with primary MDS. The cytogenetic module of the IPSS defines any abnormality of chromosome 7 as unfavourable and classifies them, combined with complex abnormalities, into the poor risk cytogenetic subgroup. However, in previous publications from other groups, the prognosis of isolated -7/del(7q) was described as intermediate. The aim of the present study was to re-analyze the prognostic impact of -7/del(7q) as a single anomaly based on a large, international MDS database which was previously presented at the 2009 ASH-meeting (Schanz et al. abstract #2772). Materials and Method: Patients with -7/del(7q), derived from the international MDS database were examined. The large international data collection contains 2901 patients with MDS, originating from the German-Austrian (GA)-, the International MDS Risk Analysis Workshop (IMRAW)- and the Spanish Cytogenetic Working group (GCECGH) and the International Cytogenetics Working Group of the MDS Foundation (ICWG). Inclusion criteria for the study were defined as follows: Primary MDS, age 〉=16, and bone marrow blasts

Description: Background: The translocation (8;21) is the most common chromosomal rearrangement in AML, resulting in the expression of the fusion protein AML1/ETO. We have developed an ecdysone-inducible U937 model, in which AML1/ETO is expressed in response to treatment with Ponasterone (Pon) A (Fliegauf et al, Oncogene 2004). This model system was used to determine the cellular effects of AML1/ETO and to identify its target genes in U937 cells. Methods: Effects of AML1/ETO expression upon cell growth, viability, cell cycle and apoptosis were analyzed by trypan blue exclusion, FACS analysis using propidium iodide and DiOC6 staining, DNA laddering and Western blot for PARP cleavage, respectively. The gene expression profile of U937 with and without conditional AML1/ETO expression was assessed using Affymetrix U133A microarrays. Wild-type U937 cells with and without PonA treatment as well as AML1/ETO-negative and AML1/ETO-positive myeloid cell lines served as controls. Northern and Western Blotting were used for validation of expression changes. Results: Induction of AML1/ETO expression in U937 resulted in reduced cell growth, G1 arrest and in apoptosis beginning 48–72 hours after PonA treatment. To investigate the underlying mechanisms, microarray analysis was performed. Expression profiles of AML1/ETO-positive and AML1/ETO-negative cell lines formed distinct clusters. Based on stringent criteria, 191 different genes were found upregulated, whereas 37 were downregulated upon expression of AML1/ETO in U937. The identified genes were screened for genes with known functions in cell cycle and apoptosis by automated and manual review and included 13 apoptosis-related genes. Among them, the CDK inhibitor p21/WAF/CIP1 was upregulated 19-fold upon induction of AML1/ETO, whereas the apoptosis regulator MCL-1 was induced 2.5-fold. Based on our criteria, no differential expression of other transcriptionally-controlled apoptosis regulators (such as BCL2, BAX, BAK1, BAD or c-flip) was noted. Northern and Western Blot analysis confirmed the strong induction of p21/WAF/CIP1 that paralleled the expression of AML1/ETO 10 hours after PonA treatment. Induction of p21/WAF/CIP1 was independent of the tumor suppressor protein p53 (Dou et al., Proc. Natl. Acad. Sci. 1995), and by Western blot, p53 was undetectable in U937. Northern Blot analysis revealed a higher expression of p21/WAF/CIP1 in the AML1/ETO-positive cell lines Kasumi-1 and SKNO-1 than in the AML1/ETO-negative cell lines HL-60, KG-1 and U937, supporting our finding that AML1/ETO may induce p21/WAF/CIP1. Conclusions: AML1/ETO expression resulted in increased expression of p21/WAF/CIP1, which might contribute to the observed growth arrest and induction of apoptosis caused by the conditional expression of AML1/ETO.

Description: Introduction: In older patients with AML in whom conventional chemotherapy is not indicated (comorbidities, performance status, poor cytogenetics etc.), treatment with low-dose azanucleoside DNA demethylating agents may be a less intensive alternative. In MDS, these drugs need to be administered over a prolonged time period in order to gain full benefit, since delayed responses are common, and thus occur not infrequently after more than 6 months of treatment. In a phase II multicenter trial (00331) of low-dose decitabine (DAC, 135 mg/m2 administered over 3 days in 9 intravenous 3-hour infusions, with 15 mg/m2/infusion, repeated every 6 weeks) patients benefiting from this treatment were offered an outpatient maintenance with the drug given at an even lower dose and as one-hour infusions on 3 consecutive days. Patients and Methods: Patients having completed 4 courses of DAC according to the study protocol and being in complete or partial remission (CR, PR), having achieved an antileukemic affect or stable disease were offered continued DAC treatment with 20 mg/m2 given intravenously over one hour on 3 consecutive days, repeated every 6 to 8 weeks. Maintenance treatment was continued until relapse or progression. Results: Of the 235 patients included in the 00331 study, 57 (25%) received the 3-day DAC maintenance. Median age of these 57 patients before study inclusion was 71 years (range 60 – 81), the median white blood count 4200/μl (range 0.2–285,000/μl), 59% had preceding MDS, with a median of 16 months duration. Performance status before initial treatment with DAC: ECOG 0, 1 and 2 in 29%, 58% and 13% of the patients, respectively. 60% had intermediate-risk cytogenetics, 28 % poor-risk cytogenetics, no metaphases were obtained or cytogenetics not done in 12 %. Remission status at maintenance start, i.e. response to 4 courses of DAC, was CR+PR in 71 % of the patients. A median number of 4 maintenance courses was administered (range, 1–16), with 24 patients (42 %) receiving 5 or more courses. Treatment was overall very well tolerated, with dose reductions because of cytopenia necessary in

Description: Introduction: Azanucleoside DNA demethylating agents upregulate large sets of genes in vitro. Cancer testis antigens (CTAs) are a growing group of immunogenic proteins that provide attractive targets for cancer-specific immunotherapy in solid tumors. In contrast, only a very limited number of studies has been performed on epigenetic regulation of CTAs in myeloid leukemia. A single report suggested specific in vivo induction of CTA mRNAs in blasts from AML and MDS pts treated with intermediate-dose Decitabine (DAC; Sigalotti et al., Blood101:4644–6, 2003). We wished to extend this finding by examining kinetics and spectrum of CTA and leukemia-associated antigen (LAA) regulation by DAC in myeloid cell lines and primary AML blasts. Materials and Methods: Myeloid cell lines were treated over 72 hrs with 50–200 nM DAC. RNA was isolated from peripheral blood blasts of AML pts treated with DAC (135mg/m2 over 72 hours within the 00331 phase II trial) before and during early phase of the treatment (= days 2, 5 from start of DAC) when circulating blasts were still 〉40% (mean 75%). Expression analyses were performed by Western Blot (NY-ESO-1), qPCR (NY-ESO-1, MAGEA1, MAGEA3, Myeloblastin), and by HG-U133plus 2.0 mRNA microarray. Results: The five myeloid cell lines Kasumi-1, U937, HL60, K562 and NB4 did not express NY-ESO-1 protein. Upon treatment with DAC, NY-ESO-1 was markedly de-repressed in 3/5 cell lines (Kasumi-1, U937, HL60). Induction of expression was dose- and time-dependent, with maximum induction at day 6, and reversible with prolonged culturing up to day 21. No derepression was seen when U-937 and HL-60 were treated with equitoxic low concentrations of cytarabine (not a Dnmt inhibitor). MAGEA1, MAGEA3 and the LAA Myeloblastin (MBN) were also induced by DAC in Kasumi-1, with the strongest effect observed for NY-ESO-1 and the least for MBN (high baseline expression). Expression analyses were extended to all 67 CTAs present on the U133plus array (45 located on the X-chromosome, 22 autosomally), of which 11/67 (17%) were induced 〉2 fold in Kasumi-1. Notably, all 11 CTA genes were X-chromosomal, whereas 0/22 autosomal CTAs showed reinduction (p= 0.008 by Fisher-Exact test). 2/9 LAA genes (22%) were also reinduced. In primary AML blasts from 9 pts, DAC treatment resulted in 〉2fold induction of 3 CTAs and 5 LAAs. qPCR demonstrated NY-ESO-1 upregulation in 2/5 patients, with a maximum at day 5 from start of treatment. Conclusions: Marked derepression of NY-ESO-1 and other CTAs by treatment of myeloid cell lines with an azanucleoside DNA demethylating agent occurred preferentially with genes residing on the X chromosome, in line with a major role of DNA methylation for their regulation. Derepression by DAC was also observed in vivo, albeit less marked and occurring early during treatment. One of the therapeutic effects of low-dose DNA demethylating agents on leukemic myeloid blasts may be mediated via upregulation of antigens rendering the malignant cells more immunogenic.

Description: p16 and p15, 2 inhibitors of cyclin-dependent kinases, are frequently hypermethylated in hematologic neoplasias. Decitabine, or 5-Aza-2′-deoxycytidine, reverts hypermethylation of these genes in vitro, and low-dose decitabine treatment improves cytopenias and blast excess in ∼50% of patients with high-risk myelodysplastic syndrome (MDS). We examined p15and p16 methylation status in bone marrow mononuclear cells from patients with high-risk MDS during treatment with decitabine, using a methylation-sensitive primer extension assay (Ms-SNuPE) to quantitate methylation, and denaturing gradient gel electrophoresis (DGGE) and bisulfite-DNA sequencing to distinguish individually methylated alleles. p15 expression was serially examined in bone marrow biopsies by immunohistochemistry. Hypermethylation in the 5′ p15 gene region was detected in 15 of 23 patients (65%), whereas the 5′ p16 region was unmethylated in all patients. Among 12 patients with hypermethylation sequentially analyzed after at least one course of decitabine treatment, a decrease in p15 methylation occurred in 9 and was associated with clinical response. DGGE and sequence analyses were indicative of hypomethylation induction at individual alleles. Immunohistochemical staining for p15 protein in bone marrow biopsies from 8 patients with p15 hypermethylation revealed low or absent expression in 4 patients, which was induced to normal levels during decitabine treatment. In conclusion, frequent, selectivep15 hypermethylation was reversed in responding MDS patients following treatment with a methylation inhibitor. The emergence of partially demethylated epigenotypes and re-establishment of normal p15 protein expression following the initial decitabine courses implicate pharmacologic demethylation as a possible mechanism resulting in hematologic response in MDS.

Description: The chromosomal translocation (8;21) fuses the AML1 gene on chromosome 21 and the ETO gene on chromosome 8 in human acute myeloid leukemias, resulting in expression of the chimeric transcription factor AML1/ETO. AML1/ETO-mediated dysregulation of target genes critical for hematopoietic differentiation and proliferation is thought to contribute to the leukemic phenotype. Several mechanisms, including recruitment of histone deacetylases (HDACs) to AML1 target genes, may be responsible for altered gene expression. We used an ecdysone-inducible expression system in the human monoblastic U-937 cell line to isolate genes that were differentially expressed upon induction of AML1/ETO expression. By representational difference analysis (cDNA-RDA), we identified 26 genes whose expression levels were significantly modulated following AML1/ETO induction for 48 hours. None of these genes has previously been described as a target of AML1, ETO or AML1/ETO. One gene down-regulated by AML1/ETO in vitro, Williams Beuren Syndrome critical region 5 (WBSCR5), was expressed in primary t(8;21) negative AML blasts but not in primary t(8;21) positive AML blasts, strongly implying a role of this gene in the phenotype of t(8;21) positive AML. WBSCR5 is part of the critical region located on chromosome 7q11.23 that is deleted in the Williams Beuren syndrome (OMIM 194050), an autosomal dominant disorder comprising vascular, neurological, behavioral and skeletal abnormalities. WBSCR5 has recently been shown to have a role in the activation and differentiation of B cells (Brdicka et al., J. Exp. Med. 196:1617, 2002) and thus was also termed Non-T cell activation linker.. WBSCR5 as well as seven other regulated genes were further studied using all-trans-retinoic acid (ATRA), an inducer of differentiation of U-937 cells, and Trichostatin A (TSA), an HDAC inhibitor. WBSCR5 and two other out of these eight genes were regulated during ATRA-induced monocytic differentiation of U-937 cells, however none of them antagonistically, upon both ATRA-treatment and AML1/ETO-induction. Since repression of WBSCR5 might be mediated by recruitment of HDACs through the fusion gene, cells were treated with TSA prior to transgene induction. However, the AML1/ETO-associated dysregulation of WBSCR5 gene expression (as well as that of the other seven genes studied) was not mediated by a TSA-sensitive mechanism. The identified genes provide a useful model to study the mechanism by which the AML1/ETO fusion protein exerts its function in transcriptional dysregulation in acute myeloid leukemia. The role of WBSCR5 in malignant hematopoietic cells warrants further study.

Description: INTRODUCTION: Myelodysplastic syndromes are dynamic diseases affecting the stem cell compartment in bone marrow presenting with different clinical courses ranging from stable, indolent disease to rapid progression to acute myeloid leukemias. So far, only 3 studies on karyotype analysis in MDS with a minimum of 30 patients have been published. Most knowledge about genetic evolution in MDS is based on the description of parallely existing subclones within one single examination. Thus, little is known about the real frequency, the time spans and the clinical impact of karyotype evolution. MATERIALS AND METHODS: So far, data from 322 patients with MDS or secondary AML and at least two successfully performed classical cytogenetic analyses are available from four centres of the Competence Network Acute and Chronic Leukemias. As yet, we retrospectively examined 268 patients out of this data set. Karyotype evolution (KE) was defined as acquisition of additional aberrations, expansion of an aberrant clone (〉20%) or development of a completely aberrant karyotype after an initial mosaic karyotype. RESULTS: In 44 cases (16%) KE was observed. In the mean 2.8 (range 2–9) cytogenetic examinations have been performed. In 27 cases additional aberrations occurred and in 17 cases the abnormal clone expanded in a subsequent analysis. Compared to stable courses, patients with KE had a tendency towards a shorter survival (p=0.15). In the group of patients with expansion of the aberrant clone the most frequent karyotypes were −7/7q- (4x), complex (3x), 5q- (3x) and +8 (3x). The most frequent karyotypes in which during the course of the disease additional aberrations occurred were complex (4x) and karyotypes with two miscellaneous aberrations (4x). The most frequent additional aberrations were 5q- (3x) and −17/17p- (3x). CONCLUSIONS: In sequential cytogenetic examinations KE is a frequent event. Patients with KE tend to have a shortened survival. In our collective no long-term survivor could be observed in the group displaying KE regardless of the therapy strategies (excluding allogeneic transplantation). In this multicentric study which encompasses the largest data base on sequential analyses in MDS to date, frequency, evolution patterns and prognostic relevance of karyotype changes have been studied allowing a better insight into the genetic dynamics of MDS.

Description: The chromosomal translocation (8;21) results in the expression of the chimeric transcription factor AML1/ETO, the most frequent fusion gene in acute myeloid leukemia. The AML1/ETO fusion protein acts as a transcriptional repressor by mediating epigenetic silencing through recruitment of histone deacetylases. Recently, it was shown that it also mediates gene silencing by associating with DNA methyltransferase (Dnmt). We therefore hypothesized that cells expressing AML1/ETO might be preferentially sensitive to the effects of an inhibitor of Dnmt activity, and might provide a superior model for in vitro demethylation and reactivation of the promoter of the p15/INK4b gene (encoding a negative regulator of the cell cycle) that is frequently methylated and silenced in AML and MDS. The 3 myeloid cell lines Kasumi-1 cells (AML1/ETO-positive), KG-1, and KG-1a (both AML1-ETO-negative) are all bearing a heavily methylated p15/INK4b promoter. They were treated with 50 – 1000 nM 5-aza-2′-deoxycytidine (DAC) for three pulses of 24 hrs each. After 6 days, cell growth and viability were determined and FACS analysis performed after propidium iodide staining. Kasumi-1 showed the highest sensitivity to DAC treatment (growth inhibition at 500 nM DAC: Kasumi-1 74.28 %, KG-1 69.16 %, KG-1a 62.38 %). In addition, DAC treatment (500 nM) led to a stronger increase in the sub-G1 fraction in Kasumi-1 (30.46%) compared to KG-1a (20.84 %). Regional p15/INK4b promoter methylation was assessed quantitatively by bisulfite sequencing of ≤10 individual cloned alleles (containing 21 CpGs residues) for calculation of methylated CpG percentage. The p15/INK4b was highly methylated in all 3 cell lines (methylated CpGs Kasumi-1 95.2 %; KG-1 89.6 %; KG-1a 98.4 % ). In Kasumi-1 cells, treatment with DAC resulted in a striking, dose-dependent regional demethylation of the p15/INK4b promoter (demethylated CpGs at 200 nM of DAC: Kasumi-1 63.8 %, KG-1 48.9 %, KG-1a 9.3 %). No demethylating effect was achieved with equitoxic doses of cytarabine or melphalan. Effective demethylation of the p15/INK4b promoter was associated with p15/INK4b protein induction as determined by Western Blot. Simultaneous treatment with all-trans retinoic acid (ATRA) enhanced the effects of DAC treatment upon growth inhibition, but not upon p15/INK4b induction. U937 cells with ecdysone inducible AML1/ETO expression (Fliegauf et al, Oncogene 2004) were also treated with different doses of DAC. When AML1/ETO was induced, U937 cells showed a higher growth inhibition (U937 + AML1/ETO 38.6 %, U937 - AML1/ETO 18 % at 25 nM) and increase in Sub-G1 (U937 + AML1/ETO 18 %, U937 - AML1/ETO 10.55 % at 100 nM) after treatment with DAC. Our results imply that the growth-inhibitory and proapoptotic effect of DAC on leukemia cells is modulated by AML1/ETO protein (or its target genes). The greater accessibility of the p15/INK4b promoter to the demethylating effect of DAC in AML1/ETO expressing Kasumi-1 cells may also be due to differences in regional chromatin structure. With their differential sensitivity to DAC, the cell lines Kasumi-1 and KG-1a provide a model for the different responses of leukemic blasts to DAC.