ALBERT

Description: Discriminating between cytopenia(s) due to myelodysplastic syndromes (MDS) and due to other (non-clonal) causes can be challenging, especially when dysplasia as assessed by cytomorphology is minimal, and when other MDS-specific features (such as ring sideroblasts or cytogenetic aberrations) are absent. Current recommendations for diagnosing MDS endorse flow cytometry (FC) as a valuable and informative diagnostic tool. Most FC protocols focus on analyzing the progenitor cells and the maturing myelomonocytic lineage. However, one of the most frequently observed symptoms in MDS is anemia, which is often associated with erythrodysplasia. Therefore, flow cytometric features of nucleated erythroid cells may complement current validated FC tools. The international, multicenter study within the European LeukemiaNet MDS-FC working group (ELNet-IMDS-Flow) reported herein focused on defining those erythroid parameters that enable discrimination of dyserythropoiesis associated with MDS from erythropoiesis in non-clonal cytopenias. This analysis was based on ELNet iMDS-flow guidelines for studying nucleated erythroid cells and their expression of CD117, CD71, CD36, CD235a and CD105. [Westers et al., Leukemia 2012] Nineteen centers (members of the ELNet-iMDS-flow) collected FC data on the erythroid lineage in mainly low grade MDS cases and pathological and normal controls. Bone marrow aspirates were taken after informed consent in accordance with the Declaration of Helsinki and local ethics committee approval. Data from a learning cohort were compared among MDS patients and controls; the results were validated in a separate cohort. The learning cohort comprised 685 cases and the validation cohort 352 cases; in total 191 normal controls, 443 pathological controls, and 403 MDS cases were included. The data revealed that the analysis of the expression pattern of CD71 and that of CD36 on erythroid cells in combination with the percentage of CD117+ erythroid progenitors provides the best discrimination between MDS and non-clonal cytopenia. The selected markers were used to build an FC erythroid dysplasia score which displayed a sensitivity of 59% (95% CI: 49-68%) and a specificity of 84% (95% CI: 77-89%). Of note, not every MDS case shows signs of erythrodysplasia by cytomorphology whereas some non-clonal conditions do. Evaluation of the results in the validation cohort displayed a specificity of 77% (95% CI: 29-50%) and a sensitivity of 39% (95% CI: 66-85%) for separating pathologic controls and MDS cases based on FC erythroid dysplasia. Most “FC-dysplastic” cases in the pathological control group involved reactive conditions and cytopenia associated with infections. The majority of the “FC-dysplastic” controls demonstrated abnormal CD71 expression, which argues against the application of single aberrancies to indicate dysplasia. Considering only the presence of multiple erythroid aberrancies as erythroid dysplasia by FC increased the specificity to 96% and 95% in the learning and validation cohorts, respectively; however, at the cost of a markedly reduced sensitivity (37% and 21%, respectively). Ultimately, analysis of the erythroid and myeloid lineages should be combined to increase both sensitivity and specificity. In summary, the defined erythroid marker combination may aid the diagnostic work-up of cytopenic cases with suspected MDS, particularly in combination with flow cytometric evaluation of the progenitor cells and maturing myelomonocytic lineage. This will be implemented in an upcoming multicenter data collection exercise within ELNet iMDS-flow. Disclosures Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Loosdrecht:Celgene: Consultancy.

Description: Myelodysplasia is a diagnostic feature of myelodysplastic syndromes (MDSs) but is also found in other myeloid neoplasms. Its molecular basis has been recently elucidated by means of massive parallel sequencing studies. About 90% of MDS patients carry ≥1 oncogenic mutations, and two thirds of them are found in individuals with a normal karyotype. Driver mutant genes include those of RNA splicing (SF3B1, SRSF2, U2AF1, and ZRSR2), DNA methylation (TET2, DNMT3A, and IDH1/2), chromatin modification (ASXL1 and EZH2), transcription regulation (RUNX1), DNA repair (TP53), signal transduction (CBL, NRAS, and KRAS), and cohesin complex (STAG2). Only 4 to 6 genes are consistently mutated in ≥10% MDS patients, whereas a long tail of ∼50 genes are mutated less frequently. At presentation, most patients typically have 2 or 3 driver oncogenic mutations and hundreds of background mutations. MDS driver genes are also frequently mutated in other myeloid neoplasms. Reliable genotype/phenotype relationships include the association of the SF3B1 mutation with refractory anemia with ring sideroblasts, TET2/SRSF2 comutation with chronic myelomonocytic leukemia, and activating CSF3R mutation with chronic neutrophilic leukemia. Although both founding and subclonal driver mutations have been shown to have prognostic significance, prospective clinical trials that include the molecular characterization of the patient’s genome are now needed.

Description: The WHO classification of myelodysplastic syndromes (MDS) is based on uni- or multi-lineage hematopoietic involvement, blast count and cytogenetic features. We recently confirmed the prognostic value of this classification and demonstrated that cytogenetics and transfusion requirement are the main prognostic factors affecting survival of WHO subgroups (JCO 2005, in the press). The aim of the present study was to define and validate a scoring system for evaluating prognosis in MDS classified according to WHO criteria. The patients comprised a learning cohort, in whom investigations were aimed at defining the set of variables to be included in the prognostic model and their weighted scores, and a validation cohort, in whom we evaluated whether the prognostic value of the scoring system was confirmed. The learning cohort was formed of 467 consecutive patients with a diagnosis of de novo MDS made at the IRCCS Policlinico S. Matteo, Italy, between 1992 and 2002, while the validation cohort consisted of 620 consecutive patients diagnosed at the Heinrich-Heine-University Hospital between 1982 and 2003. All cases were reclassified by independent cytologists according to the WHO criteria. Those patients who were treated with allogeneic stem cell transplantation or chemotherapy, were censored at the time of this therapeutic intervention. Uni- and multivariate analyses were performed by means of Cox proportional hazards regression. The actuarial probabilities of overall survival (OS) and leukemia-free survival (LFS) were estimated using the Kaplan-Meier product limit method. Based on the results of the uni- and multivariate analyses, the most significant variables selected for the prognostic model were WHO subgroups, cytogenetic abnormalities scored according to the IPSS and transfusion requirement. Risk scores for each variable were estimated based on coefficients from the proportional hazards regression (Table 1). Table 1 - WHO-Classification Based Prognostic Scoring System (WPSS) for MDS Prognostic variable Score value 0 1 2 3 WHO category RA, RARS, 5q- RCMD, RCMD-RS RAEB-1 RAEB-2 Karyotype Good Intermediate Poor - Transfusion requirement No Regular - - By summing the score values for the three variables, patients were stratified into five distinct risk groups [very low (score 0), low (1), intermediate (2), high (3–4), very high (5–6)], showing significantly different OS and probability of leukemia evolution (P

Description: In the last few years, several studies have evaluated the clinical utility of flow cytometry immunophenotyping in myelodysplastic syndromes (MDS). We developed a flow cytometry approach for the evaluation of marrow dysplasia in MDS using discriminant analysis to generate erythroid and myeloid classification functions (Hematol J2004; vol 5 S2: pag S91). However, a major problem in the immunophenotypic analysis of MDS is evaluation of erythroid dysplasia because of the limited availability of specific antibodies (Ab). In order to define new tools, we prospectively analyzed the expression of mitochondrial ferritin (MtF) and H ferritin subunit (HF) in bone marrow cells from 69 consecutive patients with MDS and 40 pathologic controls. Six-parameter, 4-color flow cytometry was performed with an EPIX XL flow cytometer (Beckmann Coulter, Fullerton, CA). Erythroid bone marrow cells were analyzed for the expression of HF using a monoclonal mouse Ab (rH02), expression of MtF using a polyclonal mouse Ab, and expression of Glycophorin A, CD71 and CD105 using monoclonal Abs (Instrumentation Laboratory, Milan, Italy). Data on HF and MtF expression obtained by flow cytometry were compared with those obtained on bone marrow smears by an immunocytochemical method (LSAB kit, Dakopatts, Glostrup, Denmark). Discriminant function analysis was performed to identify erythroid dysplasia, with the aim of distinguishing MDS without ringed sideroblasts (RS), MDS with RS, and controls. A significant correlation was found between immunocytochemically and cytometrically detected HF expression (r=.46, P=.016). Overall, the expression of HF was higher in MDS than in controls (P

Description: The WHO recently proposed a new classification of myelodysplastic syndromes (MDS) based on uni- or multi-lineage hematopoietic involvement, blast count and cytogenetic features. The aims of this study were to evaluate the prognostic value of the WHO classification, to assess the role of known prognostic factors in MDS within these WHO subgroups, and to estimate mortality rates and life expectancy of the patients in the subgroups. Four hundred and ninety-one consecutive patients with a diagnosis of MDS made at the IRCCS Policlinico San Matteo, University of Pavia Medical School, Italy, between 1992 and 2002 were retrospectively evaluated and reclassified according to the WHO criteria. Cox proportional hazards regression was used to identify the most significant prognostic factors. A standardized mortality ratio (SMR) was calculated to compare the mortality of MDS patients with that of the general population. Overall survival (OS) and leukemia-free survival (LFS) differed significantly between RA and RCMD (P

Description: According to evidence- and consensus-based practice guidelines (Haematologica2002;87:1286–306), red cell transfusion is the therapy of choice for the majority of patients with myelodysplastic syndrome (MDS) and symptomatic anemia. Previous studies have shown that widespread organ dysfunction can result from transfusion iron overload developing in non-thalassemic adults (Schafer et al, N Engl J Med1981;304:319–24; Cazzola et al, Blood1988;71:305–12). In this study, we evaluated the effect of transfusion dependency and secondary iron overload on survival of MDS patients classified according to WHO criteria. Four hundred and sixty-seven consecutive patients with a diagnosis of de novo MDS made at the IRCCS Policlinico San Matteo, University of Pavia Medical School, Pavia, Italy, between 1992 and 2002 were retrospectively evaluated and reclassified according to the WHO criteria. The effects that developing transfusion dependency or secondary iron overload had on survival were evaluated by applying Cox proportional hazards regression with time-dependent covariates. Transfusion-dependent patients had a significantly shorter overall survival (OS) and leukemia-free survival (LFS) than did patients who did not become transfusion-dependent (HR=2.16, P

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: Ring sideroblasts (RS) characterize a group of myelodysplastic syndromes (MDS) categorized in the WHO classification as refractory anemia with RS (RARS) or refractory cytopenia with multilineage dysplasia and RS (RCMD-RS), according to the presence of 15% or more bone marrow RS and dysplasia in one or more myeloid lineages. A high prevalence of somatic mutations in SF3B1 was reported in MDS with RS [N Engl J Med 2011;365:1384-95], and recent unsupervised analyses suggested that MDS with SF3B1 mutation represent a homogeneous subset [Blood 2014 Jun 26]. In this study, we performed a comprehensive mutation analysis of genes implicated in myeloid disorders in a large and well-characterized cohort of myeloid neoplasms with 1% or more RS with the aim to identify mutation patterns that affect disease phenotype and clinical outcome. The study population consisted of 309 patients (pts), including: a) 244 with MDS, of whom 160 assigned to sideroblastic categories (RARS, RCMD-RS) and 84 to other WHO categories [34 RA or RCMD, 7 MDS with isolated del(5q), 20 RAEB-1, 23 RAEB-2]; b) 51 with myelodysplastic/myeloproliferative neoplasms (MDS/MPN: 9 CMML, 42 RARS-T); c) 14 with AML-MDS. SF3B1 mutations were observed in 151/244 pts with MDS and RS (62%). Within sideroblastic categories, SF3B1 mutation was found in 81/91 cases of RARS (89%), and 48/69 RCMD-RS (70%). Among pts classified in other MDS categories, significantly lower rate of SF3B1 mutations (22/84, P

Description: Chronic myelomonocytic leukemia (CMML) is a myelodysplastic/myeloproliferative neoplasm characterized by a highly variable clinical course. Based on clinical, hematologic and cytogenetic parameters, we previously developed a CMML-specific Prognostic Scoring System (CPSS) that stratifies patients into 4 different risk groups [Blood 2013;121:3005-15]. Recently, recurrent somatic mutations have been identified in CMML, and preliminary evidence suggests that selected mutated genes may provide useful prognostic information. In this study, we performed a comprehensive mutation analysis of genes implicated in myeloid malignancies in a large and well characterized cohort of CMML patients with the aim to dissect relationships between genotype and disease phenotype and to integrate somatic mutations into a clinical/molecular prognostic model. Thirty-eight genes were analyzed by high throughput sequencing (Illumina MiSeq, San Diego, CA) in a cohort of 199 CMML patients (pts) diagnosed according to WHO classification, and in 12 pts with monocytosis not fulfilling WHO diagnostic criteria. Myelodysplastic and myeloproliferative subtypes (CMML-MD and CMML-MP, respectively) were defined according to FAB criteria. Least absolute shrinkage and selection operator (Lasso) and Cox proportional hazards methods were adopted to select and weight variables for prognostic scoring. Ninety-three percent of pts showed at least one somatic mutation (median number per patient: 2, range 0-6). The most frequently mutated genes were TET2 (44%), SRSF2 (43%), ASXL1 (34%), KRAS (11%), NRAS (10%), CUX1 (10%), CBL (9%), RUNX1 (7%), SETBP1 (7%), JAK2 (6%), SF3B1 (6%), and U2AF1 (5%). A significant association was found between mutations in TET2 and RNA splicing factors (P=.037), 42 of 199 CMML pts (21%) showing co-occurrence of TET2 and SRSF2 mutations. Mutations in genes involved in signaling were significantly associated with CMML-MP (P=.002), whereas SF3B1 mutations were associated with CMML-MD (P=.024). The number of mutations per patient inversely correlated with overall survival (OS) (HR=1.32, P=.021). In univariate analysis, mutations in ASXL1 (HR=2.31, P=.026) , RUNX1 (HR=3.53, P=.02) and SETBP1 (HR=3.85, P=.005) significantly affected OS. Focusing the analysis on disease subtype, ASXL1 mutations significantly affected survival in CMML-MD (HR=3.45, P=.025), whereas CUX1 and SETBP1 had a significant prognostic value in CMML-MP (HR=4.33, P=.013 and HR=4.4, P=.025, respectively). In order to investigate the additive value of somatic mutations to current prognostic assessment, we first fitted a Lasso Cox regression model for genetic variable selection. The selected variables were then included in an unpenalized Cox regression in order to obtain unbiased coefficients. The statistically significant variables were CPSS-specific cytogenetic risk groups (HR=2.49, P=.001), mutations in ASXL1 (HR=2.77, P=.018), RUNX1 (HR=5.39, P=.009) and SETBP1 (HR=3.96, P=.013). Based on regression coefficients, we defined a CMML-specific genetic risk score that was able to identify three different groups (Low risk: normal karyotype and –Y; Intermediate: other abnormalities, mutations in ASXL1; High: trisomy 8, complex karyotype, mutations in RUNX1 or SETBP1), with significantly different OS (HR=2.24, P