ALBERT

Description: Myelodysplastic syndrome (MDS) and chronic myelomonocytic leukaemia (CMML) are haematological disorders that develop in haematopoietic stem or progenitor cells (HSPCs) and are characterised by ineffective haematopoiesis. 5'-Azacitidine (AZA) is a DNA demethylating agent that is effective in treating MDS and CMML. However, response rates are less than 50% and the basis for poor response is currently unknown. A patient's potential to respond cannot be currently determined until after multiple cycles of AZA treatment and alternative treatment options for poor responders are limited. To address these fundamental questions, we enrolled patients on a compassionate access program prior to the listing of AZA on the pharmaceuticals benefit scheme in Australia. We have collected bone marrow from 18 patients (10 MDS, 8 CMML) at seven different stages of treatment, starting from before treatment until after six cycles of AZA treatment, and isolated high-purity CD34+ HSPCs at each stage. 10 of these patients (5 MDS and 5 CMML) responded completely to AZA while 8 did not achieve complete response. We performed next-generation sequencing (RNA-seq) of these HSPCs to identify the basis of poor response to AZA therapy. Analysis of the RNA-seq data from pre-treatment HSPCs has revealed a striking differential expression of 1148 genes between patients who were subsequently complete (CR) or non-complete responders (non-CR) to AZA therapy (Figure 1A). Using a Fluidigm nanofluidic system, we have validated the differential expression of a subset of these genes between CR and non-CR patients in two independent cohorts, totalling 67 patients, from the U.K. and Sweden. We have additionally confirmed that our gene signature does not simply segregate patients based on disease severity or poor overall survival, but rather uniquely prognosticates best AZA response. Pathway analyses of the differentially expressed genes indicates that the HSPCs of non-CR patients have decreased cell cycle progression and DNA damage pathways, while concomitantly possessing increased signalling through integrin and mTOR/AKT pathways. Using computational methods, we have determined that the expression of 15 genes (within the 1148 gene set) is sufficient to separate CRs from non-CRs across independent cohorts (Figure 1B). We have also developed a predictive AZA response algorithm that utilises the expression of these genes to identify potential complete and non-complete responders to AZA with high specificity and sensitivity (Figure 1C). Furthermore, we have identified statistically significant correlations between recurrent DNA mutations in MDS and our prognostic gene signature (SF3B1 & TET2 with CR, STAG2 and NUP98 with non-CR, p

Description: Key Points We observed that SMAD7, a negative regulator of TGF-β receptor-I kinase, is markedly reduced in MDS, and leads to ineffective hematopoiesis. Increased levels of microRNA-21 are seen in MDS and reduce SMAD7 levels, thus overactivating TGF-β signaling.

Description: The del(5q) is the most commonly reported deletion in de novo MDS and is found in 10–15% of all patients. Our group demonstrated haploinsufficiency for the ribosomal gene RPS14, which is required for the maturation of 40S ribosomal subunits and maps to the commonly deleted region in patients with the 5q- syndrome (Boultwood et al, Br J Haematol2007, 139:578–89). Haploinsufficiency of RPS14 has been shown to be the mechanism underlying the erythroid defect in this disorder (Ebert et al, Nature2008, 451:335–9). We have recently shown that haploinsufficiency of RPS14 in patients with the 5q- syndrome is associated with deregulated expression of ribosomal- and translation-related genes, suggesting that the 5q- syndrome represents a disorder of aberrant ribosome biogenesis (Pellagatti et al, Br J Haematol2008, 142:57–64). The del(5q) in the 5q-syndrome is cytogenetically indistinguishable from the del(5q) found in other MDS and in the vast majority of these patients the CDR of the 5q- syndrome will be deleted (and therefore one allele of RPS14 will be lost). We are investigating the hypothesis that haploinsufficiency of RPS14 and consequent deregulated ribosome biogenesis may also play a role in the pathogenesis of non-5q- syndrome MDS patients with del(5q). Using Affymetrix U133 Plus2.0 arrays, we have studied the expression profiles of a group of 579 ribosomal- and translation-related genes in the CD34+ cells of 21 non-5q- syndrome MDS patients with del(5q) and 95 MDS patients without del(5q). 168 of 579 ribosomal-and translation-related probe sets were found to be significantly differentially expressed between these two groups, with approximately 90% of these showing lower expression levels in patients with del(5q). Hierarchical clustering using this set of 168 genes gave a good separation between patients with and without the del(5q). RPS14 was one of the most significant differentially expressed genes, with lower expression levels in patients with del(5q) confirming its haploinsufficient status in these patients. Other significant differentially expressed genes include the ribosomal protein RPL22L1, and the translation initiation factors EIF4EBP3 and EIF4B. Interestingly, when samples from 16 patients with 5q- syndrome were included in the analysis, hierarchical clustering using significantly differentially expressed ribosomal- and translation-related genes showed that most patients with 5q- syndrome and most patients with del(5q) clustered together. We are currently using polysome profile analysis on bone marrow cells to examine the levels of the 40S ribosomal subunit in patients with del(5q) and without del(5q). Our results support the hypothesis that haploinsufficiency of RPS14 and deregulation of ribosomal- and translation-related genes contribute to disease pathogenesis in MDS patients with del(5q). An exciting possibility is that other MDS with the del(5q) and the 5q- syndrome share a related molecular basis in that they are all disorders of defective ribosomal biogenesis.

Description: Refractory Anemia with Ringed Sideroblasts (RARS) is characterized by severe ineffective erythropoesis, cytochrome c release, and mitochondrial iron overload. (Tehranchi, 2003). Granulocyte-CSF inhibit erythroid apoptosis in vitro as well as in vivo (Tehranchi 2005) The molecular mechanisms underlying the erythroid apoptosis in RARS and the effects of G-CSF were studied by gene expression profiling of erythroblasts from 8 healthy controls and 6 RARS patients. CD34+ selected marrow cells were cultured for 7 days in Iscove’s medium with 15% BIT9500. At day 7 an aliquot of RARS cells were treated with G-CSF (100ng/ml) for 4 hours. The gene expression profiles were determined using Affymetrix, U133 Plus2.0 chips (Pellagatti, 2006). Statistical analysis showed that 1426 probe-sets were significantly differentially expressed (P

Description: Abstract 298 The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell malignancies that are characterized by ineffective hematopoiesis resulting in peripheral cytopenias and a hypercellular bone marrow. Approximately 40% of patients with MDS will develop an acute myeloid leukemia. It is important to establish prognosis of MDS patients since the treatment options vary from supportive care to bone marrow transplantation. In order to determine the relationship of gene expression levels to prognosis and so identify new molecular markers, we have used gene expression profiling to study the transcriptome of the hematopoietic stem cells of 125 MDS patients with a minimum 12 month follow up. The CD34+ cells obtained from MDS patients and healthy individuals were analyzed using Affymetrix U133 Plus2.0 arrays. The patients were split randomly in a training set (n=84) and a test set (n=41). Supervised principal components analysis was used to identify genes correlated with survival. Using the 84 patients in the training set, the Cox scores were computed for each gene, and the principal components calculated on the genes with the highest Cox scores. The first of the principal components was then used to generate a regression model to predict the survival in the test set. Finally, for each probe set an importance score was calculated equal to its correlation with the supervised principal component predictor. This approach returned a list of 150 top ranked probe sets correlated with survival. Patients in the training set were split into tertiles based on the predictor (low, medium and high score) and patients in the test set were assigned to their predicted class, and Kaplan-Meier plots were generated for both training and test set. The differences in survival for both training and test set were statistically significant (Figure 1). Top ranked genes showing lower expression levels in patients with shorter survival include CDH1, LEF1 and AKAP12/Gravin. Top ranked genes showing higher expression levels in patients with shorter survival include IL23A, WT1 and PTHR2. Figure 2 shows survival of patients divided into tertiles of expression for the individual genes CDH1, LEF1 and WT1. It is probable that the genes identified in this study will become the first validated molecular markers for MDS prognosis. Multivariate analysis is currently being performed. Figure 1 Figure 1. Figure 2 Figure 2. Disclosure: No relevant conflicts of interest to declare.

Description: Abstract 1862 Refractory anemia with ring sideroblasts (RARS) is a myelodysplastic syndrome (MDS) characterized by isolated anemia, erythroid dysplasia only, less than 5% blasts and 15% or more ring sideroblasts in the bone marrow (2008 WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues). The natural history of RARS is characterized by an initial phase of erythroid hyperplasia and ineffective erythropoiesis, which is usually stable for many years but in a proportion of patients may be followed by a phase of marrow failure, with or without the later emergence of leukemic blasts. Overall, RARS is a benign condition with a median survival of about 9 years (J Clin Oncol. 2005;23:7594-603). Since the vast majority of these patients have no cytogenetic abnormalities, the clonal nature of RARS has been questioned. However, a few studies of X-chromosome inactivation patterns performed in female patients have suggested that RARS derives from clonal proliferation of a multipotent hematopoietic stem cell with the potential for myeloid and lymphoid differentiation. Somatic mutations of TET2 have been recently found in myeloid neoplasms including MDS, where they appear occur early during disease evolution (Nat Genet. 2009;41:838-42), and are currently considered as a reliable clonal marker of these disorders. In this study, we therefore performed a mutation analysis of TET2 in patients with myeloid neoplasms associated with ring sideroblasts. Using direct sequencing, we studied 33 patients with RARS and 28 patients with refractory cytopenia with multilineage dysplasia (RCMD) having 15% or more ring sideroblasts in the bone marrow. Somatic mutations of TET2 were detected in circulating granulocytes from 10 out of 33 (30%) patients with RARS and 10 out of 28 (36%) patients with RCMD and ring sideroblasts. Most of these mutations were novel at the time of this writing. Fourteen patients had a single somatic mutation, and the mutation burden ranged from 10 to 80%. In 9 of these 14 cases, the mutation burden was approximately 50%, consistent with a fully clonal hematopoiesis characterized a single dominant clone that was heterozygous for the mutation. In a female patient with 10% mutant alleles, however, granulocytes carrying mutant TET2 represented only one tenth of clonal granulocytes as determined by X-chromosome inactivation patterns, suggesting the existence of alternative genetic events preceding the TET2 mutation and sustaining clonal dominance. Six patients had multiple somatic mutations of TET2: two mutations in 3 cases, three mutations in 2 cases, and four mutations in the last case. Quantitative evaluation of mutation burden showed concordant values (about 50%) for the multiple mutations in two patients (one with 4 and the other one with 3 somatic mutations of TET2), indicating the existence of a single dominant clone with multiple mutations. In the remaining 4 patients, discordant mutation loads were detected: the dominant mutation was present in about 50% alleles, while the remaining one(s) involved a lower proportion (10-35%) of alleles. These findings are consistent with the initial emergence of a clone of hematopoietic cells carrying a single mutation of TET2 and the subsequent development of subclones that carry additional TET2 mutations and become dominant with time. We also compared gene expression profiles of CD34-positive cells from patients with and without somatic mutations of TET2. While these 2 patient groups both had up-regulation of ALAS2 and down-regulation of ABCB7, distinctive “sideroblastic” features at the molecular level (Blood. 2006;108:337-45), no differentially expressed gene was identified between the 2 groups. These data indicate that somatic mutations of TET2 are unlikely to have a major impact on metabolic pathways at the CD34-positive cell level, and are more consistent with an epigenetic regulation function of TET2. In summary, this study shows that about one third of patients with RARS carry somatic mutations of TET2 in circulating granulocytes, clearly indicating that RARS is a true clonal disorder of hematopoiesis despite it presents as a benign erythroid disorder. In most cases, TET2 mutations appear to cause clonal dominance of hematopoietic stem cells, thus initiating the myelodysplastic process. During the clinical course of the disease subclonal evolution may occur through the acquisition of additional somatic mutations of TET2. Disclosures: No relevant conflicts of interest to declare.

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.