Identification of Gene Expression–Based Prognostic Markers in the Hematopoietic Stem Cells of Patients With Myelodysplastic Syndromes Andrea Pellagatti, Axel Benner, Ken I. Mills, Mario Cazzola, Aristoteles Giagounidis, Janet Perry, Luca Malcovati, Matteo G. Della Porta, Martin Ja ¨dersten, Amit Verma, Emma-Jane McDonald, Sally Killick, Eva Hellstro ¨m-Lindberg, Lars Bullinger, James S. Wainscoat, and Jacqueline Boultwood Author affiliations appear at the end of this article. Published online ahead of print at www.jco.org on September 3, 2013. Supported by Leukaemia and Lymphoma Research of the United Kingdom; by the National Institute for Health Research Oxford Biomedical Research Centre Programme (financial support for patient sample collection); by grants from Associazione Italiana per la Ricerca sul Cancro (Special Program Molecular Clinical Oncology 5x1000, Project No. 1005) and Fondazione Cariplo to M.C., and from Fondazione Berlucchi to M.G.D.P. (for studies performed at Department of Hematol- ogy Oncology, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo, and Department of Molecular Medicine, University of Pavia, Pavia, Italy); in part by European Program for Cooperation in Science and Technology (COST) Action No. BM0801 WG1 (work is based on joint research activities under COST framework); and in part by German Research Founda- tion (Heisenbergstipendium BU 1339/ 3-1; L.B.). J.S.W. and J.B. contributed equally to this work. Authors’ disclosures of potential con- flicts of interest and author contribu- tions are found at the end of this article. Corresponding author: Jacqueline Boultwood, PhD, Professor of Molecu- lar Haematology, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom; e-mail: jacqueline.boultwood@ ndcls.ox.ac.uk. © 2013 by American Society of Clinical Oncology 0732-183X/13/3128w-3557w/$20.00 DOI: 10.1200/JCO.2012.45.5626 A B S T R A C T Purpose The diagnosis of patients with myelodysplastic syndromes (MDS) is largely dependent on morphologic examination of bone marrow aspirates. Several criteria that form the basis of the classifications and scoring systems most commonly used in clinical practice are affected by operator-dependent variation. To identify standardized molecular markers that would allow prediction of prognosis, we have used gene expression profiling (GEP) data on CD34+ cells from patients with MDS to determine the relationship between gene expression levels and prognosis. Patients and Methods GEP data on CD34+ cells from 125 patients with MDS with a minimum 12-month follow-up since date of bone marrow sample collection were included in this study. Supervised principal components and lasso penalized Cox proportional hazards regression (Coxnet) were used for the analysis. Results We identified several genes, the expression of which was significantly associated with survival of patients with MDS, including LEF1, CDH1, WT1, and MN1. The Coxnet predictor, based on expression data on 20 genes, outperformed other predictors, including one that additionally used clinical information. Our Coxnet gene signature based on CD34+ cells significantly identified a separation of patients with good or bad prognosis in an independent GEP data set based on unsorted bone marrow mononuclear cells, demonstrating that our signature is robust and may be applicable to bone marrow cells without the need to isolate CD34+ cells. Conclusion We present a new, valuable GEP-based signature for assessing prognosis in MDS. GEP-based signatures correlating with clinical outcome may significantly contribute to a refined risk classifi- cation of MDS. J Clin Oncol 31:3557-3564. © 2013 by American Society of Clinical Oncology INTRODUCTION Myelodysplastic syndromes (MDS) are clonal hematopoietic stem-cell (HSC) malignancies char- acterized by blood cytopenias, ineffective hemato- poiesis, and hypercellular bone marrow. 1,2 Disease course and prognosis are highly variable in MDS, and approximately 30% to 40% of patients with MDS will develop acute myeloid leukemia (AML). 1,2 It is important to establish prognosis of patients with MDS, because treatment options vary from sup- portive care to bone marrow transplantation. Diagnosis of MDS is largely dependent on morphologic examination of bone marrow aspi- rates. The classifications of MDS proposed by the French-American-British Study Group 3 and more recently by WHO 4 are still widely used in clinical practice. The International Prognostic Scoring Sys- tem (IPSS) 5 and WHO classification– based prog- nostic scoring system 6 are able to classify patients into risk groups with different survival rates. A re- vised IPSS was recently reported that was shown to have improved prognostic ability for survival and AML evolution in patients with MDS. 7 Several cri- teria that form the basis of these classifications and scoring systems are subjective, although a high con- cordance rate among experts has been reported. 6-8 The identification of standardized molecular markers would allow prediction of disease pro- gression and prognosis in patients with MDS. 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