GENES, CHROMOSOMES & CANCER 49:885–900 (2010) Clonal Heterogeneity in Childhood Myelodysplastic Syndromes—Challenge for the Detection of Chromosomal Imbalances by Array-CGH Inka Praulich, 1 Marcel Tauscher, 1 Gudrun Go ¨ hring, 1 Stefanie Glaser, 1 Winfried Hofmann, 1 Simone Feurstein, 1 Christian Flotho, 2 Peter Lichter, 3 Charlotte M. Niemeyer, 2 Brigitte Schlegelberger, 1 and Doris Steinemann 1 * 1 Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover,Germany 2 Division of Hematology and Oncology, Department of Pediatrics and Adolescent Medicine,University of Freiburg, Freiburg,Germany 3 Department of Molecular Genetics, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg,Germany To evaluate whether copy number alterations (CNAs) are present that may contribute to disease development and/or pro- gression of childhood myelodysplastic syndromes (MDS), 36 pediatric MDS patients were analyzed using array-based com- parative genome hybridization (aCGH). In addition to monosomy 7, the most frequent chromosome aberration in childhood MDS, novel recurrent CNAs were detected. They included a loss of 3p14.3–p12.3, which contains the putative tumor suppressor gene FHIT , a loss of 7p21.3–p15.3, a loss of 9q33.3–q34.3 (D184) and microdeletions in 17p11.2, 6q23 containing MYB, and 17p13 containing TP53. In this small patient cohort, patients without CNA, patients with monosomy 7 only and patients with one CNA in addition to monosomy 7 did not differ in their survival. As expected, all patients with complex karyotypes, including two patients with deletions of TP53, died. A challenge inherent to aCGH analysis of MDS is the low percentage of tumor cells. We evaluated several approaches to overcome this limitation. Genomic profiles from isolated granulocytes were of higher quality than those from bone marrow mononuclear cells. Decreased breakpoint calling stringency increased recognition of CNAs present in small clonal populations. However, further analysis using a cus- tom-designed array showed that these CNAs often did not confirm the findings from 244k arrays. In contrast, constitu- tional CNVs were reliably detected on both arrays. Moreover, aCGH on amplified DNA from distinct myeloid clusters is a new approach to determine CNAs in small subpopulations. Our results clearly emphasize the need to verify array-CGH results by independent methods like FISH or quantitative PCR. V V C 2010 Wiley-Liss, Inc. INTRODUCTION Myelodysplastic syndromes (MDS) are a heter- ogeneous group of myeloid disorders with cytope- nias affecting one or more hematopoietic lineages. In the elderly, MDS constitute one of the most frequent hematologic malignancies, but MDS is a rare disease in childhood. Cytogenetic abnormalities are found in approximately 50% of patients, with monosomy 7 (7) being the most frequent alteration (Niemeyer et al., 1997; Hasle, 2007). However, it seems clear that monosomy 7 is not the initiating event. Gross karyotypic changes may represent clonal evolution in a pop- ulation with karyotypically undetectable altera- tions. In familial MDS associated with monosomy 7, the predisposing locus is not physically linked to chromosome 7 (Mufti, 1992; Hasle et al., 2003). This has led to the conclusion that addi- tional genetic or epigenetic lesions are necessary for the development of MDS with 7. To inves- tigate small copy number changes in a genome- wide manner array-based comparative genome hybridization (aCGH) can be applied (Solinas- Toldo et al., 1997; Albertson and Pinkel, 2003; Fiegler et al., 2003). In childhood acute lympho- blastic leukemia, this approach has led to the identification of alterations in genes like PAX5 or IKAROS, previously not implicated in the patho- genesis of the disease (Mullighan, 2009). One major advantage of applying aCGH to the analy- sis of MDS, a disease characterized by enhanced levels of apoptosis and often by a hypocellular marrow (Clark and Lampert, 1990; Niemeyer et al., 1992; Kerbauy and Deeg, 2007), is that no Additional Supporting Information may be found in the online version of this article. Supported by: Deutsche Jose ´ Carreras Leuka ¨ mie-Stiftung e.V, Grant number: DJCLS R05/03; EU COST Initiative EUGESMA. *Correspondence to: Doris Steinemann, Institute of Cell and Molecular Pathology, Hannover Medical School, Carl-Neu- berg-Str. 1, Hannover 30625, Germany. E-mail: steinemann.doris@mh-hannover.de Received 2 December 2009; Accepted 18 May 2010 DOI 10.1002/gcc.20797 Published online 29 June 2010 in Wiley Online Library (wileyonlinelibrary.com). V V C 2010 Wiley-Liss, Inc.