ALBERT

Beschreibung: Abstract 223 A coding variant form of GFI1 (GFI136N) increases the risk to develop AML by 60% and is present in about 10–15 % of all Caucasian AML patients. To determine the underlying molecular mechanism and potentially develop new therapeutic approaches, we generated “knockin” mouse strains wherein the endogenous murine Gfi1 gene was replaced either by the human GFI1 variant (GFI136N, the form predisposing to AML) or by the more common form of GFI1 (GFI136S). In most hematopoietic compartments no difference was observable between GFI136N and GFI136S expressing mice; however, there was a 3–5 fold increase in the number of granulocytic monocytic progenitors (GMPs) and common myeloid progenitors (CMPs) in Gfi136N expressing (either homozygous or heterozygous) mice compared to wild-type or Gfi136S expressing mice(p≤0.01). Interestingly, both human and murine AML leukemic cells are thought to originate from GMPs and CMPs. To assess functional differences, we seeded GMPs from GFI136N or GFI136S knockin mice on methylcelluose or transplanted them into into syngenic animals. We found that GFI136N expressing GMPs proliferate faster and have an increased self-renewal capacity both in-vitro and in-vivo compared to GMPs carrying Gfi136S alleles. A gene expression array analysis showed that GFI136N GMPs have a stem cell-like gene signature with elevated levels of Hoxa9 expression and a deregulation of a number of oncogenes involved in the development of human AML such as Trib2, Tet2 or Idh2. It is of particular interest that Hoxa9, a known GFI1 target gene, was up-regulated 3–4 fold in GFI136N GMPs compared to in GFI136S GMPs (p≤0.01). It is known that high levels of Hoxa9 accelerate AML development in mice and are associated with a poor prognosis in AML patients. GFI1 is a transcriptional repressor and exerts its function by recruiting different histone modifying enzymes, in particular LSD1, which de-methylates histone 3 (H3) at lysine 4 (K4), or histone deacetylases (HDACs), which remove acetyl groups from H3K9 residues and G9a, which initiates dimethylation of H3K9. Both H3K4 methylation and H3K9 acetylation correlate with actived gene expression, whereas H3K9dimethyl correlates with repession. Chromatin-immuno-precipitation (ChIP) of Gfi1-bound chromatin from Lin−Sca1−c-Kit+ cells, which contains the GMP population, showed that GFI136N binds to a lesser degree to the Hoxa9 locus than GFI136S. This diminished binding of Gfi136N correlated with an increased H3K4 dimethylation and H3K9 acetylation as well as diminished H3K9 dimethylation across the Hoxa9 locus in GFI136N cells. It is likely that these epigenetic changes lead to the increased Hoxa9 expression observed in GFI136N GMPs. A more exhaustive ChIP-Seq analysis with antibodies recognizing H3K4dimethyl in Lin−Sca1−c-Kit+ cells from Gfi136N or Gfi136S mice showed significant epigenetic alterations throughout the Hoxa9 locus genome and at other GFI1 target genes. It is conceivable that these epigenetic alterations explain, at least in part, the changed gene expression signatures in GFI136N GMPs. To investigate the role of GFI136N in myeloid leukemogenesis, we induced the expression of a mutated form of KRAS (K12D) in both GFI136N and GFI136S mice. All mice developed a deadly myelo-proliferative disorder, but animals carrying the GFI136N allele succumbed to the disease within a significantly shorter latency period (17 against 31 days, p≤0.01) than GFI136S mice. We also transduced GFI136N and GFI136S GMPs with retroviral vectors directing the expression of either the AML1-Eto9a or the MLL-AF9 onco-fusion proteins typically found in human AML. We observed that GFI136N GMPs expressing MLL-AF9 or AML1-Eto9a generated 5–10 fold more colonies (p≤0.01) on methylcellulose and exhibited a higher replating efficiency than the respective GFI136S GMPs. Finally, AML blast cells from GFI136N heterozygous patients expressed higher levels of HOXA9 compared to AML blasts from GFI136S homozygous patients, suggesting that our mouse model reflects the disease predisposition in human patients. Our knockin mice are, to our knowledge, the first animal model for a human genetic variation that predisposes to leukemia. Based on the findings with this model, we propose that the human GFI136N variant predisposes to AML by inducing epigenetic changes affecting the expression of important regulators with oncogenic potential such as Hoxa9. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: While allogeneic hematopoietic stem cell transplantation (alloHSCT) has extensively been studied in patients with AML

Beschreibung: The GFI1 gene encodes a transcriptional repressor, which regulates myeloid differentiation. In the mouse, Gfi1 deficiency causes neutropenia and an accumulation of granulomonocytic precursor cells that is reminiscent of a myelodysplastic syndrome. We report here that a variant allele of GFI1 (GFI136N) is associated with acute myeloid leukemia (AML) in white subjects with an odds ratio of 1.6 (P 〈 8 × 10−5). The GFI136N variant occurred in 1806 AML patients with an allele frequency of 0.055 compared with 0.035 in 1691 healthy control patients in 2 independent cohorts. We observed that both GFI1 variants maintain the same activity as transcriptional repressors but differ in their regulation by the AML1/ETO (RUNX1/RUNX1T1) fusion protein produced in AML patients with a t(8;21) translocation. AML1/ETO interacts and colocalizes with the more common GFI136S form in the nucleus and inhibits its repressor activity. However, the variant GFI136N protein has a different subnuclear localization than GFI136S. As a consequence, AML1/ETO does not colocalize with GFI136N and is unable to inhibit its repressor activity. We conclude that both variants of GFI1 differ in their ability to be regulated by interacting proteins and that the GFI136N variant form exhibits distinct biochemical features that may confer a predisposition to AML.

Beschreibung: Increased expression levels of miR-181 family members have been shown to be associated with favorable outcome in patients with cytogenetically normal acute myeloid leukemia. Here we show that increased expression of miR-181a and miR-181b is also significantly (P 〈 .05; Cox regression) associated with favorable overall survival in cytogenetically abnormal AML (CA-AML) patients. We further show that up-regulation of a gene signature composed of 4 potential miR-181 targets (including HOXA7, HOXA9, HOXA11, and PBX3), associated with down-regulation of miR-181 family members, is an independent predictor of adverse overall survival on multivariable testing in analysis of 183 CA-AML patients. The independent prognostic impact of this 4-homeobox-gene signature was confirmed in a validation set of 271 CA-AML patients. Furthermore, our in vitro and in vivo studies indicated that ectopic expression of miR-181b significantly promoted apoptosis and inhibited viability/proliferation of leukemic cells and delayed leukemogenesis; such effects could be reversed by forced expression of PBX3. Thus, the up-regulation of the 4 homeobox genes resulting from the down-regulation of miR-181 family members probably contribute to the poor prognosis of patients with nonfavorable CA-AML. Restoring expression of miR-181b and/or targeting the HOXA/PBX3 pathways may provide new strategies to improve survival substantially.

Beschreibung: Abstract 6 Acute promyelocytic leukemia (APL) is a curable disease, and contemporary treatment based on the combination of all-trans retinoic acid (ATRA) with anthracyclines results in overall survival (OS) rates of around 90% at five years. Unfortunately, the treatment outcome of patients with APL in developing countries is significantly less. A recent Brazilian study had reported an OS of 53% with a first 5-days mortality of 13.4%. The International Consortium on Acute Promyelocytic Leukemia (IC-APL) is an initiative of the International Members Committee of the ASH and the project aims to reduce this gap through the establishment of international network, which was launched in Brazil, México and Uruguay. All patients with a suspected diagnosis of APL were immediately started on ATRA, while bone marrow samples were shipped to a national central lab where genetic verification of the diagnosis was performed. Results of the immunofluorescence for PML was obtained within hours and upon confirmation of the diagnosis, patients were enrolled in a protocol identical to the PETHEMA-LPA 2005, except for the replacement of idarubicin by daunorubicin. Supportive care aimed at maintaining platelet counts above 30,000/μl and fibrinogen levels above 150 mg/dl. In each country, cases were discussed every other week through internet and whenever needed international experts were involved. As of June 2009, 102 (70 Brazil, 25 Mexico, 7 Uruguay) APL patients were enrolled. The median age was 34 y (range: 9–72y) with 55 males (54%).The median white blood cell counts (WBC) at baseline was 3.6×109 /L(range: 0.2–149.7). The distribution of the relapse risk score at diagnosis according to PETHEMA-GIMEMA criteria was 14 low (14%), 54 intermediate (53%) and 34 high risk(33%) respectively. The incidence of low risk APL appeared lower than the values reported in developed countries. Of 102, 97 patients have toxicity and response data available. Of these 97, 12 (12.3%)experienced at least three symptoms/signs of differentiation syndrome (DS) and 77 (79%) patients achieved a complete remission (CR). Twenty-three deaths occurred and the cause of deaths included 9 hemorrhage, 8 infection, 2 DS . The 7 and 30 day mortality rates were 8% and 19.6%, respectively, and the one- year overall survival was 75% (95%CI:68%–84%). The median follow-up time among survivors was 14 months (range: 1.3–35). Among 77 patients who achieved CR, the 1-year OS and disease-free survival from the date of CR was 95% (95% CI: 89%–100%). Only one patient relapsed. For patients surviving a minimum of 30 days the outcome was similar to that reported by the twin PETHEMA-LPA 2005 protocol in European patients. Prognostic factors for overall survival were examined using log-rank test as well as multivariate Cox models. Factors predicting OS were a high relapse risk score at baseline (1-year OS: 59% for high, 87% for intermediate, 91% for low, p=0.0007) and age. The 1-year OS was 85% for age

Beschreibung: Background. CHR-2797 is a novel, orally bioavailable agent which displays potent, tumor cell-selective, anti-proliferative properties. It is an inhibitor of Zn++-dependent aminopeptidases and generates signs of amino acid deprivation in sensitive cells, decreased protein synthesis and an increase in the level of the pro-apoptotic protein, NOXA. CHR-79888 is an active metabolite of CHR-2797. Methods. This was an open label, single agent, dose escalating phase I salvage study to assess tolerance, MTD/DLT, activity, and pharmacokinetics of CHR-2797 in patients with hematological malignancies. Elderly patients and/or relapsed patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and multiple myeloma (MM) were eligible. Patients were treated with escalating once daily doses (60–180 mg) for up to 84 days or until progressive disease (PD). Clinical responses were assessed by monthly bone marrow aspirates in AML/MDS patients and by M-protein levels in MM patients. Results. Sixteen adults (4 women, 12 men) of median age 70 yrs, (range 45–84 yrs) were accrued between May 2006 and Jan 2007: 13 patients with AML, 1 with MDS, and 2 with MM. Thirteen patients finished the dose finding phase of 28 days and 6 patients continued for at least 84 days. CHR-2797 was well tolerated and, except for one patient with grade III ALT elevation, no grade III/IV drug related non-hematological toxicity was observed during the first 28 days of treatment. Two patients on 180 mg developed DLT that was considered drug related: 〉75 percent reduction in platelet count. CHR-2797 had no influence on hemoglobin or neutrophils in this trial. Overall the most frequently reported adverse events were thrombocytopenia (6.7%), diarrhea (4.5%), dizziness (3.9%), and fatigue (3.9%). Five AML patients died in the first 3 months of the trial or within 4 weeks of discontinuing CHR-2797: 3 due to disease progression and 2 following a MI (not related to drug). Bone marrow studies revealed complete responses (〈 5% blasts in bone marrow) in 3/12 AML patients after 1–3 months of therapy (60 and 130mg), one of which was also a cytogenetic response. One of the 2 responding patients on 130 mg was evaluated as a CRp at 3 months; this patient was in remission for 3 months following platelet recovery after the drug was stopped. One further AML patient (60 mg) became completely transfusion independent and remained so for 6 weeks. Good exposure to CHR-2797, including levels of the active metabolite CHR-79888 has been observed on days 1 and 28 with a terminal half life (for 79888) of 8– 11 hours. Conclusions. Oral once daily CHR-2797 in AML/MDS/MM patients with adverse prognostic risk was well tolerated. MTD for maintenance therapy was reached at 180 mg. Single agent CHR-2797 therapy showed encouraging clinical activity (incl. 3/12 CRs) in these elderly and poor risk AML patients who were able to continue therapy for at least 28 days. Because of the favorable results a phase II study with CHR-2797 in advanced AML is currently in progress.

Beschreibung: Acute myeloid leukemia (AML) is a heterogeneous disease with variable responses to treatment, characterized by specific chromosomal breakpoint products such as AML-ETO, PML-RAR or mutations in e.g., c/EBPα or FLT3 genes. These abnormalities are not sufficient to cause AML and approximately 40% of AML cases present without these defects or have more complex abnormalities. To find genes involved in AML, we recently isolated common integration sites (CIS) from Graffi-1.4 MuLV (Gr1.4)-induced mouse myeloid leukemia and identified the genes affected by these integrations (Erkeland S.J., et al, J. Virol., 2004). To assess the significance of these genes for human AML, we determined their contribution to 16 distinct classes of patients recently defined based on gene expression profiling of 285 AML samples using Affymetrix U133A microarrays (Valk P.J., et al, NEJM, 2004) plus 11 additional groups based on known chromosomal aberrations or gene mutations. Using the significance analysis of microarrays (SAM) methods we could link specific gene sets to these 27 patient classes. Genes were considered to contribute to the signature of the class when the following criteria were met: an RNA expression Fold Change over 1.5 or under 0.67, a score over 4 or less than −4 and a q-value less then 5%. Three gene lists derived from the Gr1.4-induced leukemia samples were generated: (I) genes (n=51, represented by 116 probe sets), within or most proximal to CIS; (II) genes (n=53, 81 probe sets) located next to the CIS flanking genes; (III) genes (n=279, 468 probe sets) located more distantly from the CIS (up to 1 megabases), with a maximum of 5 genes in each direction. Each of these gene lists was compared to the 27 outcomes of the SAM analyses to count the number of cases where a probe set of one of the three lists was significantly deregulated. To establish whether the fraction of deregulated genes in one of the three lists was significantly higher than the expected fraction of the total list, a chi-square test was performed. The list of CIS shows a significant overrepresentation of deregulated genes (Ψ2 =13.4 and p=0.0002). In contrast, the list of two closest flanking genes does not show overrepresentation (Ψ2= 0.4 and p=0.54), and neither does the list of 10 neighboring genes (Ψ2= 1.7 and p= 0.19). We repeated the analysis on a profiling dataset from 130 childhood AML samples (Ross ME, et al, Blood 2004). Using this dataset, we performed 5 SAM analyses based on known chromosomal aberrations or specific FAB-type. Again, this showed an overrepresentation of deregulated genes in case of the CIS (Ψ2= 6.3, p=0.012) but not for the more distant genes (2 closest genes: Ψ2= 0.006, p=0.936, 10 neighboring genes: Ψ2= 1.67, p=0.196). We conclude from these analyses that the closest CIS flanking genes have the highest probability to be involved in human AML. We next performed a similar analysis using the list of virus integrations from the BXH2 myeloid leukemia model (http://genome2.ncifcrf.gov/RTCGD/). This list, consisting of 53 genes represented by 111 probe sets also showed a higher representation of deregulated genes of adult AML (Ψ2= 112.2, p=0.000) as well as of pediatric AML samples (Ψ2= 14.0 and p=0.0001). Examples of CIS genes that are found associated with specific subgroups of clinical AML are CTNNA1, IRS-2, VDUP1, DUSP10 and PRDXII. These results demonstrate the power of combining retroviral tagging of cancer genes with expression array analysis of clinical leukemia samples for the identification of pathogenetic mechanisms in human AML.

Beschreibung: Acute Myeloid Leukemia (AML) is a heterogeneous disease from the molecular and biological standpoints. In order to resolve some of this complexity, a recent microarray-based expression profiling study segregated cohorts of patients with common gene signatures. One of these signatures was associated with alterations of the CCAAT/enhancer-binding protein alpha (CEBPA) gene. Among these patients, a subset harbored CEBPA mutations, while the remainder failed to express CEBPA, which in a number of cases correlated with hypermethylation of its promoter. This latter subgroup of leukemias with silenced CEBPA presented with significant biological differences compared to CEBPA mutant patients, including expression of T-cell markers and activating mutations of NOTCH1 (Wouters BJ et. al., PMID:17671232). Since our preliminary data show that DNA methylation profiling is extremely accurate in identifying distinct biological phenotypes in AML and other tumors, we wondered whether genome-wide epigenetic analysis would identify the biological difference between these patients. In order to determine the DNA methylation profiles of these patients we performed HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR), a quantitative genome-wide DNA methylation method, using a 400,000 feature cutom-desinged microarray, representing 24,000 gene promoters with 50-mer oligonucleotides. We studied the complete previously identified cluster of AML cases presenting with a CEBPA expression signature, and compared and contrasted the DNA methylation profiles of cases carrying the CEBPA mutation and those presenting with CEBPA silencing. Remarkably, unsupervised (unbiased) clustering of DNA methylation profiles revealed that samples were readily segregated into two groups that overlapped perfectly with the presence or absence of the CEBPA mutation, indicating the presence of underlying genome-wide DNA methylation differences between these two groups. We next performed supervised analysis of the samples to compare the DNA methylation profiles of CEBPA mutated vs. CEBPA non-mutated samples. The analysis was performed using a moderated T test, and 291 genes promoters were identified as differentially methylated between the two groups at a significance level of p

Beschreibung: AML is heterogeneous group of diseases with variable clinical outcomes. While cytogenetics, molecular markers and gene expression profiling can help to classify these patients, they still cannot fully explain the biology and clinical outcomes of the disease. Epigenetic gene deregulation is a hallmark of cancer and our preliminary data suggest that epigenetic signatures are critical determinants of cellular phenotype in AML. Therefore, we hypothesized that aberrant epigenetic regulation of genes would provide critical insight into the biological complexity of AML and identify new and clinically relevant disease subtypes. We studied genome wide DNA methylation in a cohort of 295 patients from HOVON multicenter clinical trials using the HELP assay, which measures with 〉95% accuracy the abundance of DNA methylation at ~50,000 CpG sites covering ~13,000 promoter regions. Median follow-up was 18.2 months (range=0.1–214.5); median age: 48.1 years (range=15.8–75). Unsupervised analysis using hierarchical clustering (Pearson correlation distance with Ward’s clustering method) segregated the AMLs into 16 well-defined epigenetic clusters. Cluster 1 consisted 100% of patients with acute promyelocytic leukemia (n=6); 100% of cluster 4 harbored CEBPA mutations (n=14); 21/23 patients in cluster 6 carried an inv(16); clusters 7 and 9 were enriched for cases carrying the NPM1 mutation (#7: 80% NPM1+ and #9: 96%) and cluster 12 was enriched for t(8;21) AMLs (18/23). Most of the clusters however define previously unknown biological entities. Next we used a supervised analysis and identified the differentially methylated genes and gene networks that define each cluster, which revealed previously unknown biological differences among these patients. Moreover, Kaplan-Meier survival analysis revealed significant differences in event-free survival (EFS) and overall survival (OS) for the 8 clusters that consisted of 〉20 patients (clusters 5, 6, 7, 8, 9, 11, 12, and 14), which includes clusters that represent previously unidentified AML subtypes. The inv(16) and t(8;21) containing clusters (i.e. #6 and #12) demonstrated a 2-year EFS of 48% and 58%, respectively, compared to 2-year EFS ranging from 19%–44% for all other clusters (p=0.002 by log-rank test) and a 2-year OS of 70% and 61%, respectively, compared to 2-year OS ranging from 25%–50% for all other clusters (p=0.008 by log-rank test). After adjustment for age, cytogenetic risk, NPM1 mutation, and FLT3-itd status in a multivariate cox proportional hazards regression model, differences in EFS and OS remained between clusters i.e. multivariate analysis (utilizing cluster 12 as reference) showed that clusters 9, 5, 8 and 11 demonstrated hazard ratios for poor events of 3.2 (95% CI=1.0, 10.6; p=0.06), 3.2 (95% CI=1.1, 9.1; p=0.03), 3.4 (95% CI=1.2, 9.8; p=0.03) and 3.6 (95% CI=1.2, 10.3; p=0.02), respectively. Similarly, clusters 9, 8 and 11 demonstrated hazard ratios for mortality of 4.7 (95% CI=1.1, 19.8; p=0.03), 4.1 (95% CI=1.1, 15.2; p=0.03) and 3.7 (95% CI=1.0, 13.6; p=0.05), respectively. Interestingly none of these clusters could be entirely explained by any of the known molecular or cytogenetic markers. Clusters 9 and 5 consisted mainly of cases with normal karyotypes, while #8 and #11 grouped cases with a variety of karyotypes. Furthermore, cluster 9 was associated with a worse outcome despite the fact that 24/25 cases were NPM1+, only 11 of which also presented the poor risk association with FLT3-itd. An analysis restricted to the 125 cases with normal karyotype (NK-AML) segregated them into 2 main clusters, one enriched for NPM1+ cases (81.9%) and the other not (29.6% NPM1+) (Fisher exact test: p-value