ALBERT

Description: Introduction Gene expression profiling studies in the Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) have unraveled significant deregulation of several immune and inflammation genes of potential importance for clonal evolution (Skov V et al., Eur J Haematol 2011; Leuk Res 2012; Exp Hematol 2012). Other mechanisms might be downregulation of major histocompatibility (MHC) class I and II genes, which are used by tumor cells to escape antitumor T-cell-mediated immune responses. In a previous study, several genes encoding human leukocyte antigen (HLA) class I and II molecules, beta2microglobulin and members of the antigen processing machinery of HLA class I molecules (LMP2, LMP7, TAP1, TAP2 and tapasin) have been shown to be significantly downregulated (Skov V, Leuk Lymphoma 2012). Upregulation of HLA-genes is considered one of the mechanisms of action of interferon-alpha2 (IFN-alpha2) but regulation of these genes during IFN-alpha2 treatment of patients with MPNs has never been studied. The purpose of this study was to assess the regulation of several HLA genes before and during treatment with IFN-alpha2, the hypothesis being that IFN-alpha2 - as in other cancers - may restore downregulation of HLA genes in MPNs as well. Patients and Methods Using Affymetrix HG-U133 2.0 Plus microarrays, gene expression profiling have been performed on whole blood from patients with ET (n =8), PV (n = 21), and PMF (n = 4) before and after 3 months of treatment with IFN-alpha2. Background correction, normalization, and gene expression index calculation were performed with the robust multi-array (rma) method. The regularized t-test limma for pairwise data was used to calculate differences in gene expression between patients before and after treatment with IFN-alpha2. A p-value 〈 0.05 was considered significant. Results Statistical analysis of all 54,675 probe sets on the microarray revealed 6261, 10,008, 2828, and 12,390 probe sets to be significantly differentially expressed in ET, PV, PMF, and MPNs as a whole, respectively, in response to treatment with IFN-alpha2 (P-value

Description: Abstract 1477 Previous studies have documented the underrepresentation of women and elderly patients in American clinical trials of leukemia. If, characteristics of patients included in clinical protocols differ markedly from the characteristics of the majority of patients treated outside protocols the external validity of clinical trials may be threatened. Methods: The Danish National Acute Leukaemia Database (ALDB) includes detailed data on a large well-defined non-selected population of 2729 AML patients (covering 〉95% of AML patients diagnosed since Jan 2000). Since 2000 Danish AML patients have been included in 3 different British protocols (AML15, 16 and 17). We analysed a cohort of 2624 patients diagnosed with AML in Denmark since Jan. 2000 (105 APL-patients were excluded). We compared patients treated with curative intent according to the British protocols with patients treated with curative intent off-protocol with regard to characteristics, possible prognostic factors, CR-rate, and survival. For comparable groups we divided patients into 2 age groups (

Description: Introduction: Primary myelofibrosis (PMF) belongs to the heterogeneous group of chronic myeloproliferative neoplasms (MPN) together with essential thrombocytosis (ET) and polycythemia vera (PV). It has been suggested that these neoplasms represent a biological continuum from early cancer stage (ET,PV) to advanced MF. Multiple studies report frequent mutations in epigenetic regulators. However, the association to epigenetic changes and the role of epigenetic aberrations in different cell populations is still unknown. We therefore performed DNA methylation profiling of sorted cells from MF patients to unravel pathways contributing to disease phenotype and gain insight into MF pathogenesis. As an aberrant DNA methylation pattern may be an early event in tumorigenesis and may be crucial for progression of the malignant clone towards the more aggressive forms of MPN, we further aimed to identify methylated candidate driver genes. Material and methods: Peripheral blood samples from 16 MF patients were together with BM (bone marrow) and peripheral blood from 3 healthy age matched controls sorted in CD34+ cells, granulocytes and mononuclear cells, and analysed for differential methylated regions using Illumina Infinium HumanMethylation 450K BeadChip. Candidate genes were validated by pyrosequencing in a second cohort of 30 MF patients. To identify potential driver genes the DNA methylation status of candidate genes were likewise analyzed in a larger cohort consisting of 60 ET and PV patients. Results: The number of differential methylated CpG sites between MF cells and the healthy counterparts differed extensively among the three cell populations analyzed. In MF CD34+ cells 1628 CpG sites were differential methylated compared to normal CD34+ cells, and 519 and 213 differential methylated CpG sites were observed in MF granulocytes and MF mononuclear cells, respectively (Δβ was set to 0.2 with an adjusted p-value 〈 0.05, T-test). Differentially methylated genes were mainly involved in cancer and embryogenic pathways in both the MF CD34+ and mononuclear cells, while mononuclear cells also showed aberrant methylation of genes involved in the inflammatory disease pathways. MF granulocytes showed significant aberrations in pathways involving immunological diseases, cell death and survival. Candidate genes have been identified and validation is ongoing. Interestingly, a gradual increase of the DNA methylation level of TRIM59 was observed from the healthy controls (31%) over ET (53%) to PV (64%) and MF (65%). ET patients could be distinguished from both healthy controls (P= 0.0004, Mann-Whitney test) and from the more progressed stages PV and MF (P=0.0132, Mann-Whitney test) based on the TRIM59 DNA methylation level. TRIM59 promoter methylation could, however, not discriminate between PV and MF (P=0.4721, Mann-Whitney test). Conclusion: Genome-wide DNA methylation profiling of sorted MF blood cells provided an exclusive insight into which pathways that are contributing to MF disease phenotype at a cell specific level. The MF CD34+ cells had the highest number of differential methylated CpG sites (n=1628) when comparing to granulocytes (n=519) and mononuclear cells (n=213) and should be cells of choice when exploring new treatment strategies. Interestingly, the mononuclear compartment show aberrant methylation of inflammatory genes supporting a role of aberrant immune regulation in the pathogenesis of MPN. Earlier studies have failed to identify aberrant methylation in early ET and PV, however, the preliminary data on the methylation of individual genes (TRIM59 promoter methylation) shows that it might be possible to identify early driver genes, and that it may be possible to select a panel of genes that can discriminate early MPN from the late MF stage. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3542 The prognosis of acute promyelocytic leukemia (APL) has improved markedly over the last two decades. Clinical trials have demonstrated excellent survival of patients receiving anthracycline-based chemotherapy in combination with all-trans-retinoid acid (ATRA). It is not clear whether the excellent survival results demonstrated in clinical trials during recent years, also do apply to unselected patients including elderly and frail patients for whom a clinical trial may not be available. In order to investigate survival and death rates in APL over the course of treatment, we conducted a retrospective analysis of survival in 105 consecutive APL-patients diagnosed in Denmark January 2000 through June 2012. Data were retrieved from the Danish National Acute Leukemia Registry which covers 95 – 100 % of all acute leukemia cases diagnosed in Denmark since January 2000 (not including children). A diagnosis of APL was confirmed in 105 (3.9 %) of 2726 adult patients (age ≥ 15 years) with acute myeloid leukemia (AML) diagnosed in Denmark during the 12½ year period. This corresponds to an incidence rate of 1.53 APL cases per million inhabitants per year. The diagnosis of APL was based upon cytogenetic analysis (performed in 96 (91 %) of cases), and/or interphase FISH (iFISH), and/or RT-PCR in a total of 100 cases. In 5 cases clinical signs and morphological changes in bone marrow were highly suggestive of APL and these cases were, thus, deemed to have APL. Median age at diagnosis of APL was 50 years (range 15 to 83 years) and median leukocyte count was 2.65 (range 0.2 to 99.0 × 109/L). In 104 patients with data available for analysis, very early death (VED, within the first week from the date of diagnostic bone marrow) occurred in 10 cases (9.6 %), and early death (ED, death within 30 day from diagnosis) occurred in 22 (21 %) cases. Death between day 30 and 5 years from diagnosis occurred in 11 cases. No deaths were seen after 5 years from diagnosis. For all patients, estimated overall survival at 10 years was 65 % (Figure 1). Patients surviving beyond day 30 from day of diagnosis (82 cases) had an excellent overall survival of greater than 80 % at 10 years (Figure 2). Survival of patients were strongly correlated with WHO-performance status whereas age over 50 years and presenting leukocyte count greater than 10 × 109/L could not be definitely associated with inferior survival (Table 1 and Figure 3). Table 1. Factors of importance to survival in unselected APL-patients Probability of overall survival (Univariate Cox Regression, nevaluable= 104) Probability of overall survival (multivariate Cox Regression, nevaluable= 101) Variable Hazard ratio 95% CI of HR P value Hazard ratio 95% CI of HR P value Age (≤50 vs. 〉50 years) 2.17 1.07–4.42 0.03 1.44 0.67–3.09 NS WHO performance status (0–1 vs. ≥2) 5.20 2.57–10.5 〈 10−4 4.06 1.88–8.78 〈 10−4 WBC (≤10 × vs.〉10 × 109/L) 1.76 0.86–3.61 NS 1.62 0.79–3.33 NS From this population-based analysis we conclude that early death continues to be the predominant hazard to patients with APL. Two thirds of deaths in APL-patients do occur during the first month from diagnosis. Patients with a poor performance status at disease presentation carry a particularly dismal prognosis. For all APL-patients, every possible effort should be made to facilitate prompt diagnosis and speedy initiation of treatment with ATRA and anthracycline-based chemotherapy. When timely applied, currently available treatments are highly effective and result in cure in a high proportion of patients including elderly patients and patients with high leukocyte count at time of disease presentation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 803 Background: Recent findings lend strong support to the contention that histone deacetylase inhibition may be an important epigenetic therapy in the treatment of patients with myeloproliferative neoplasms and emphasize the need to characterize the efficacy and safety of this novel class of cytoreductive agents. Aims: This study was a non-randomized, open-label phase II multicenter study with sixty-three patients (21 essential thrombocythaemia (ET), 42 polycythemia vera (PV)) included from 15 centers. The primary objective was to investigate, if vorinostat as monotherapy in patients with PV and ET was followed by a decline in clonal myeloproliferation as assessed by conventional disease activity parameters. Secondary objectives included assessment of adverse effects during treatment; changes in bone marrow morphology before and after treatment with vorinostat and to investigate whether treatment with vorinostat influences the JAK2 mutant allele burden as assessed by quantitative PCR. (qPCR) Results: Thirty-one patients (49%) were followed to the end of the intervention period. Eighty-one percent of these had a partial (n=20) – or complete (n=5) hematological response according to ELN criteria. Response rates were found to be independent of JAK-status in ET patients. (P=0.83). A tendency towards less favorable responses was observed among patients with 〉 1 previous therapies albeit statistically insignificant (P=0.11). For all but two patients, a clinicohematological response was not followed by a histological remission. The prevalence of splenomegaly was lowered from 48% to 24% (P=0.02). A statistically significant reduction of JAK2 mutant allele burden was observed (P=0.006). No JAK2 positive patients experienced a complete molecular response defined as undetectable JAK2V617F by qPCR. No significant correlation between severity of tumor allele burden at inclusion and a more pronounced molecular response to vorinostat was found using a Spearman correlation analysis (rho = 0.16, p = 0.3). The most commonly reported adverse effects (AEs) during the intervention period among patients who completed the protocol were fatigue and gastrointestinal (anorexia, nausea, vomiting, diarrhea, dryness of the mouth). Gastrointestinal symptoms were generally manageable. Seventy percent of included patients experienced hair loss. Seventeen percent experienced renal toxicity (3 pts. grade I, 1 pt. grade II) and 1 pt. (4%) liver toxicity of unknown grade, which resolved after withdrawal of vorinostat. Forty-three percent of included patients required at least one dose reduction due to AE′s. Forty patients (63 % of patients) discontinued study drug before end of study period due to adverse events (65%), unknown (17.5%), withdrawal of consent (7.5%), no response (2.5%), or progression to acute leukemia (7.5%). Response rates for patients who discontinued therapy, however, showed that approximately 50% of patients who discontinued experienced a clinicohematological response providing evidence that toxicity issues were of concern rather than lack of clinical effect. Conclusions: Vorinostat is effective in patients with ET and PV normalizing elevated leukocyte and platelet counts as well as providing a statistically significant reduction of the JAK2 mutant allele burden and splenomegaly. However, vorinostat was associated with a high dropout rate. Considering the heterogeneity and complexity of oncogenic events, involving both deregulated tyrosine kinase activity as well as epigenetic deregulation in MPN-pathogenesis combination therapy (eg. JAK1-2 inhibitors, DNA-hypomethylating agents, interferon-alpha2) may be more efficacious than single agent therapy. This strategy might also allow for dose reduction of single agents and accordingly a decrease in toxicity, which was the major limiting factor for patients adhering to treatment in the present study. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2003 The prognosis of patients suffering from AML with manifestations of accompanying extramedullary leukemia (EML) including myeloid sarcoma (MS) compared to that of AML patients not exhibiting EML manifestations is still an open question as results from previous studies have been contradictory most likely due to selection bias. Here we present an analysis performed in a cohort of 2261 patients representing 〉90% of all AML patients diagnosed and treated in Denmark during the eleven-year period January 2000 through December 2010. The goal was to investigate the prognostic impact of presence of EML at time of AML diagnosis by a retrospective population- and registry-based analysis Of these patients, 219 (9.7%) showed signs of EML at time of AML presentation. Anatomic sites of EML were: lymph nodes (3.0%), skin (2.7%), spleen (1.7%), oral (1.3%), CNS (0.4%), testes (0.2%), other sites (1.1%), and two or more anatomical sites (0.5%). In 27 cases myeloid sarcoma was not accompanied by AML in the bone marrow and, thus, presented as isolated MS. In total, 1168 of the 2261 (52 %) patients were treated with curative intention. Allogeneic stem cell transplantation (Standard allo in 105 cases, and reduced intensity conditioning (RIC) transplant in 90 cases) was conducted in a total of 195 patients (118 in CR1, 65 in CR2, and 12 during other disease stages). Overall the frequencies of allogeneic transplantations in curatively treated patients were 13.7% in patients with EML and 8.5% in patients without EML. The presence of EML at time of leukemia diagnosis had no statistical significance to probability of obtaining complete remission (CR), nor to duration of overall survival (OS) (Table 1. and Fig. 1). By contrast, well-established prognostic parameters such as presenting cytogenetic abnormalities (categorized according to revised MRC-criteria, D. Grimwade et al. Blood, 2010), age, leukocyte count, and type of leukemia (secondary vs de novo) were all found to be statistically significant to probability of attainment of (CR) and to duration of OS in uni- as well as multivariate analyses. Gender was of borderline statistical significance with respect to probability of attainment of CR and to OS (Table 1).Figure 1Years from AML diagnosisPatients with EML(n = 132)Patients without EML(n = 1007)p-value (log-rank test) = 0.51Figure 1. Years from AML diagnosis. / Patients with EML. / (n = 132). / Patients without EML. / (n = 1007). / p-value (log-rank test) = 0.51Table 1.Factors of significance to probability of attainment of CR and to overall survival (OS)Probability of CR (Logistic regression, nevaluable = 927)Probability of overall survival (Cox regression, nevaluable = 958)VariableOdds ratio (OR)95% CI of ORP valueHazard ratio95% CI of HRP valueEML––0.82––0.54Age1.061.04–1.08