Leukemia (2011) 25, 387–399 & 2011 Macmillan Publishers Limited All rights reserved 0887-6924/11 www.nature.com/leu LEADING ARTICLE Comprehensive array CGH of normal karyotype myelodysplastic syndromes reveals hidden recurrent and individual genomic copy number alterations with prognostic relevance A Thiel1, M Beier1, D Ingenhag1, K Servan1, M Hein1, V Moeller1, B Betz1, B Hildebrandt1, C Evers1,3, U Germing2 and B Royer-Pokora1 1Institute of Human Genetics and Anthropology, Medical Faculty, Heinrich Heine University, Duesseldorf, Germany and 2Department of Hematology, Oncology and Clinical Immunology, Heinrich Heine University, Duesseldorf, Germany About 40% of patients with myelodysplastic syndromes (MDSs) 40–50% of MDS cases have a normal karyotype. MDS patients present with a normal karyotype, and they are facing different with a normal karyotype and low-risk clinical parameters are courses of disease. To advance the biological understanding often assigned into the IPSS low and intermediate-1 risk groups. and to find molecular prognostic markers, we performed a high- resolution oligonucleotide array study of 107 MDS patients In the absence of genetic or biological markers, prognostic (French American British) with a normal karyotype and clinical stratification of these patients is difficult. To better prognosticate follow-up through the Duesseldorf MDS registry. Recurrent these patients, new parameters to identify patients at higher risk hidden deletions overlapping with known cytogenetic aberra- are urgently needed. With the more recently introduced modern tions or sites of known tumor-associated genes were identi- technologies of whole-genome-wide surveys of genetic aberra- fied in 4q24 (TET2, 2x), 5q31.2 (2x), 7q22.1 (3x) and 21q22.12 tions, it is hoped that more insights into the biology of disease (RUNX1, 2x). One patient with a 7q22.1 deletion had an additional 5q31.2 deletion of the acute myeloid leukemia/MDS progression might be obtained. region, the smallest deletion identified so far and including Array comparative genomic hybridization (aCGH) studies the putative tumor suppressor (ts) genes, EGR1 and CTNNA1. with bacterial artificial chromosomes (BACs) or oligonucleotides One TET2 deletion was homozygous and one heterozygous, using exclusively MDS samples are currently rare. The first with a missense mutation in the remaining allele, further BAC-based aCGH study of MDS and AML patients with trisomy supporting its role as a ts gene. Besides these recurrent 8 as sole cytogenetic aberration demonstrated that additional alterations, additional individual imbalances were found in 34 duplications and homozygous deletions can be detected, and cases; in total, 42/107 (39%) cases had genomic imbalances. 4 These patients had an inferior survival as compared with that trisomy 8 is not always the primary genetic event. Another the rest of the patients (P ¼ 0.002). This study emphasizes the BAC aCGH analysis of 38 MDS patients, 19 with and 19 without heterogeneity of MDS, but points to interesting genes that may cytogenetic abnormalities, compared with a normal age- have diagnostic and prognostic impact. matched control group revealed many cryptic aberrations, Leukemia (2011) 25, 387–399; doi:10.1038/leu.2010.293; which were confirmed by other methods.5 A BAC aCGH study published online 28 January 2011 using CD34 þ cells from 44 low-risk MDS patients (IPSS p1) Keywords: myelodysplastic syndromes; aCGH; normal karyotype; 6 prognosis included 25 cytogenetically normal cases. This aCGH analysis uncovered hidden aberrations in the cytogenetically normal cases, but confirmed only 11 of 16 cytogenetically visible Introduction aberrations. These authors divided the patients into two groups based on the presence of a total of p3 Mb genomic aberrations The myelodysplastic syndromes (MDSs) are clonal disorders of (TGA) or 43 Mb TGA. It is not surprising that the group of the hematopoietic system, with morphologic dysplasia, ineffec- 43 Mb TGA had a significant shorter overall survival, as most tive hematopoiesis and peripheral blood cytopenias. MDS is cases with large cytogenetically visible alterations fell into this currently classified according to the WHO (World Health group. A separate analysis of the 25 cytogenetically normal Organization), which is based on the morphological, cytoge- cases also showed a significant difference in overall survival in netic and hematological criteria and defines six subtypes.1 the group with 43 Mb TGA, although the numbers are still In addition, the French American British classification, which small.6 Using oligonucleotide aCGH on MDS/AML with a comprises five subtypes, is also still in use.2 The International 5q-syndrome or del(5) as the sole cytogenetic abnormality, we Prognostic Scoring System (IPSS), a risk stratification system that have described the presence of additional hidden aberrations in takes into account clinical parameters as well as cytogenetic 2/12 patients.7 With single-nucleotide polymorphism (SNP) results, assigns four risk categories for death or transformation arrays, copy number alterations as well as loss of heterozygosity to acute myeloid leukemia (AML) (low, Int-1, Int-2 and high). through uniparental disomy (UPD) can be detected. Two reports Specific cytogenetic abnormalities were also included in described the use of SNP arrays to study low-risk MDS for the updated prognostic classification system.3 Approximately hidden genomic imbalances. One SNP array study of 119 low- risk MDS patients, 77 with a normal karyotype, 36 with visible Correspondence: Dr B Royer-Pokora, Institute of Human Genetics cytogenetic alterations and 6 with unavailable cytogenetics, and Anthropology, Medical Faculty, Heinrich Heine University, identified 125 UPDs with a median size of 3.78 Mb in 46% of Moorenstr. 5, 40225 Duesseldorf, Germany. the cases studied.8 Another study showed that 82% of MDS E-mail: [email protected] patients harbor gains and losses detected by SNP arrays, 3Current address: Institute of Human Genetics, Heidelberg University, whereas conventional cytogenetics detected alterations Heidelberg, Germany. 9 Received 26 October 2010; accepted 8 November 2010; published in 50%. In addition, UPD was found in 33% of the patients. online 28 January 2011 In both studies, first correlations of genomic imbalances Prognostic relevance of hidden imbalances in MDS A Thiel et al 388 including UPD with survival indicated that the presence of these Germany) or Marrow Max Medium (Invitrogen, Gibco, may correlate with a shorter survival.8,9 This high rate of UPDs Karlsruhe, Germany) with 10% fetal calf serum (Cytogen, Sinn, in MDS was recently challenged in a study that showed, when Germany). In total, 20–25 mitoses were analyzed according to using buccal DNA samples from the same patients as controls, the ISCN (International System for Human Cytogenetic Nomen- that only a small number of the potential loss of heterozygosity clature), with a resolution of 100–300 bands. All 107 cases regions represent tumor-specific UPDs and gains and losses.10 In had a successful cytogenetic analysis. Material left over after that report only 4/33 cytogenetically normal cases had cytogenetics was stored in methanol–acetic acid (3:1) at À20 1C. additional alterations. DNA was isolated from archived bone marrow samples with the To further explore the frequency of hidden genomic QIAmp DNA Blood Midi Kit (Qiagen, Hilden, Germany) or with imbalances, specifically recurrent abnormalities, we performed a phenol chloroform extraction. DNA concentration and purity a large retrospective study on 107 karyotypically normal MDS/ were determined with the NanoDrop ND-1000 spectrophoto- AML patients (WHO) using long oligonucleotides aCGH and meter (Thermo Scientific, Wilmington, NC, USA). non-amplified DNA. Recurrent and individual imbalances were detected, which correlated with an inferior survival. Oligonucleotide aCGH Materials and methods For aCGH, 44k (n ¼ 37), 105k (n ¼ 21) or 244k (n ¼ 49) 60mer oligonucleotide microarrays were used (Human Genome CGH Patients, cytogenetic analysis and DNA isolation Microarray, Agilent Technologies, Palo Alto, CA, USA). CGH In total, 107 cytogenetically normal MDS patients at the time of analyses were performed essentially as described in the protocol diagnosis and before therapy were included in this retrospective of the manufacturer (Protocol Version 5.0, June 2007; Agilent). study. The patients had been entered into the Duesseldorf Same-gender reference DNAs were purchased as pools (Promega, MDS registry and followed for survival. Median follow-up time Mannheim, Germany). In total, 1.5 mg of cyanine 5-dUTP- was 28 months (1–150). Median survival time of the entire labeled test DNA and cyanine 3-dUTP-labeled reference DNA cohort was 32 months (1–150). The clinical details of the were hybridized in the presence of Cot-1-DNA (Invitrogen) for patients are shown in Table 1. Approval for the study was 40 h (244, 105k) and 24 h (44k) at 65 1C. Arrays were washed obtained from the local ethics committee. (Wash Procedure B, Agilent) and slides were scanned with the For conventional G-banding, bone marrow cells were Agilent array scanner G2505B. cultivated for 24, 48 or 72 h in RPMI 1640 (Biochrom, Berlin, Table 1 Clinical characteristics of the patient cohort (n ¼ 107) Data analysis Microarray images were scanned and Agilent’s Feature Extrac- No. % tion Software Version 10.1 was used for quantification. Aberrant regions were determined by automatic breakpoint detection Cytogenetics using the GLAD library11