Leukemia (2007) 21, 2463–2469 & 2007 Nature Publishing Group All rights reserved 0887-6924/07 $30.00 www.nature.com/leu ORIGINAL ARTICLE Further delineation of chromosomal consensus regions in primary mediastinal B-cell lymphomas: an analysis of 37 tumor samples using high-resolution genomic profiling (array-CGH) S Wessendorf1,6, TFE Barth2,6, A Viardot1, A Mueller3, HA Kestler3, H Kohlhammer1, P Lichter4, M Bentz5,HDo¨hner1,PMo¨ller2 and C Schwaenen1 1Klinik fu¨r Innere Medizin III, Zentrum fu¨r Innere Medizin der Universita¨t Ulm, Ulm, Germany; 2Institut fu¨r Pathologie, Universita¨t Ulm, Ulm, Germany; 3Forschungsdozentur Bioinformatik, Universita¨t Ulm, Ulm, Germany; 4Abt. Molekulare Genetik, Deutsches Krebsforschungszentrum, Heidelberg, Germany and 5Sta¨dtisches Klinikum Karlsruhe, Karlsruhe, Germany Primary mediastinal B-cell lymphoma (PMBL) is an aggressive the expression of BSAP, BOB1, OCT2, PAX5 and PU1 was extranodal B-cell non-Hodgkin’s lymphoma with specific clin- added to the spectrum typical of PMBL features.9 ical, histopathological and genomic features. To characterize Genetically, a pattern of highly recurrent karyotype alterations further the genotype of PMBL, we analyzed 37 tumor samples and PMBL cell lines Med-B1 and Karpas1106P using array- with the hallmark of chromosomal gains of the subtelomeric based comparative genomic hybridization (matrix- or array- region of chromosome 9 supported the concept of a unique CGH) to a 2.8k genomic microarray. Due to a higher genomic disease entity that distinguishes PMBL from other B-cell non- resolution, we identified altered chromosomal regions in much Hodgkin’s lymphomas.10,11 Together with less specific gains on higher frequencies compared with standard CGH: for example, 2p15 and frequent mutations of the SOCS1 gene, a notable þ 9p24 (68%), þ 2p15 (51%), þ 7q22 (32%), þ 9q34 (32%), genomic similarity to classical Hodgkin’s lymphoma was þ 11q23 (18%), þ 12q (30%) and þ 18q21 (24%). Moreover, 12–14 previously unknown small interstitial chromosomal low copy observed. The genetic overlap of the two entities has been number alterations (for example, À6p21, À11q13.3) and a total supported recently by the results of mRNA gene expressions 15–17 of 19 DNA amplifications were identified by array-CGH. For 17 studies. A number of critical genes, such as JAK2, REL, chromosomal localizations (10 gains and 7 losses), which were cMYC, MAL, PDL, SOCS1 and CDKN2A, have been discussed altered in more than 10% of the analyzed cases, we delineated to be involved in the pathogenesis of PMBL. However, the minimal consensus regions based on genomic base pair cascade of oncogenic events leading to PMBL is still elusive. The positions. These regions and selected immunohistochemistries detailed delineation of aberrant chromosomal regions using point to candidate genes that are discussed in the context of 18–23 NF-jB transcription activation, human leukocyte antigen class high-resolution, microarray-based genomic techniques I/II defects, impaired apoptosis and Janus kinase/signal allowing candidate gene identification provides a suitable tool transducer and activator of transcription (JAK/STAT) activation. for clarifying the genetic causes of this disease. Our data confirm the genomic uniqueness of this tumor and provide physically mapped genomic regions of interest for focused candidate gene analysis. Leukemia (2007) 21, 2463–2469; doi:10.1038/sj.leu.2404919; Materials and methods published online 30 August 2007 Keywords: mediastinal B-cell lymphoma; array-CGH; genomic Thirty-seven fresh, frozen tumor samples of PMBL were alterations included in the study. Histopathological diagnosis was based on morphologic and immunohistochemical criteria according to the WHO classification of malignant lymphomas1 by two expert hematopathologists (PM and TFEB). A total of 22 male and 15 female patients were analyzed. The median age was 36 years Introduction (for more details see Table 1). For all cases, chromosomal comparative genomic hybridization (cCGH) data along with Primary mediastinal B-cell lymphoma (PMBL) is a distinct selected fluorescent in situ hybridization and cytogenetic data clinicopathological entity in the WHO classification of non- were published earlier.11,12 However, for most cases of the 1 Hodgkin’s lymphomas (ICD-O: 9679/3). Due to the location analyzed series, sufficient material for systematic mRNA and morphologic characteristics, PMBL is a unique lymphoma expression analysis was not available any more. subset that probably derives primarily from asteroid thymic B For the construction of a 2800 targets comprising genomic 2–5 cells. PMBL is positive for CD19, CD20, CD45, and often microarray, a genome-wide distributed core set of DNA CD30, CD79a, CD11c, CD23 and negative for CD10 and fragments was selected out of the previously published, 6 CD21. Highly characteristic is a lack of immunoglobulin acknowledged ‘GoldenPath’ clone set. The targets were set at surface antigen and a missing expression of major histocompati- intervals of approximately 2 Mb across the entire human 7,8 bility complex (MHC) class I and II molecules. More recently, genome (alternating clones of the GoldenPath; in total 1500 clones). This compilation was supplemented with a set of 600 Correspondence: Dr S Wessendorf, Klinik fu¨r Innere Medizin III, DNA fragments, which focuses critical regions of B-cell Zentrum fu¨r Innere Medizin der Universita¨t Ulm, Robert-Koch-Strasse neoplasms as detected by previous cytogenetic studies, for 8, Ulm 89081, Germany. example, 2p13, 9p24 or 11q13. Finally, 699 DNA targets E-mail: [email protected] 6These authors contributed equally to this study. specific for, for example, proto-oncogenes or tumor suppressor Received 30 May 2007; revised 25 July 2007; accepted 26 July 2007; genes with discussed pathogenetic relevance in B-cell non- published online 30 August 2007 Hodgkin’s lymphoma were added. The final chip contained Array-CGH in primary mediastinal B-cell lymphoma S Wessendorf et al 2464 Table 1 Patient characteristics Clinical feature No. of patients Patients evaluable for characteristic Percentage Gender, male/female 22/15 37 59.5/40.5 Stage, XIII/IV 6 29 21 LDH elevated 20 27 74 WHO performance 4 27 15 Age, 460 years 3 37 8 Figure 1 Ideogram-based synopsis of all identified chromosomal imbalances for 37 analyzed tumor samples for chromosomes 1–22. Chromosomal gains (low copy number and amplifications) are indicated by green bars on the right of each ideogram, losses are indicated in red. Sites of known copy number polymorphisms (CNPs) are indicated as light blue dots on the right side of each ideogram. 2799 clones. A list of all clones with respective physical clone on the array was compared with the ‘Database of mapping information is given in Supplementary Table S1. Genomic Variants’ (http://www.projects.tcag.ca/variation/).26,27 DNA preparation of all bacterial artificial chromosome and P1- Clones and sites located in regions with known copy number derived artificial chromosome clones and subsequent sequential polymorphisms are marked in Figure 1 and in Supplementary degenerative oligonucleotide primer-mediated DNA amplifica- Table S1, enabling a conservative evaluation of the array data. tion was performed as described by Fiegler et al.,24,25 with three Alterations, which entirely covered CNPs, were excluded from minor modifications of the original protocol. A detailed descrip- the evaluation (for example, 5p11, 7q35, 14q32 and 15q11). For tion of the applied PCR procedures is listed in the supplement. the present study, the Y chromosome was excluded from the The PCR products were spotted onto Corning CMT-Gaps II glass analysis and the X chromosome was only evaluated with regard slides using an Omnigrid microarrayer (Gene Machines, San to DNA amplifications, since in all experiments, gender Carlos, CA, USA). DNA labeling, hybridization and data difference between tumor DNA and control DNA was used to acquisition were performed as described previously.21–23,25 monitor the integrity of each experiment. Fluorescence ratios were obtained using an Axon 4000 B dual laser scanner, computed as log2 ratios and normalized based on the linear clone set of 1500 clones (for details, see Schwaenen Results et al.21), excluding X and Y clones for normalization. There- upon, the ratios of two reverse color experiments were In 33 of the 37 (89%) tumor samples, a total of 199 averaged. The diagnostic cutoff level for each individual chromosomal imbalances were detected by array-CGH. The experiment was determined by calculating the mean and median number of aberrations was 5 (range ¼ 0–19). A total of subsequently using plus/minus three times the standard devia- 130 chromosomal gains and 69 losses were identified. In four tion of all clones from chromosomes 1–22. Additionally, those patients, no aberrations were found. Chromosomal gains in regions were scored as aberrant, whose signal ratios narrowly more than 10% of all cases (nX4) were detected on 9p24 missed the threshold criteria but were deviated unidirectionally (n ¼ 24), 2p15 (n ¼ 19), 7q22 (n ¼ 12), 12q (n ¼ 11), 11q13 in at least three adjacent targets. This was done to take into (n ¼ 7), 12p (n ¼ 6), 3q27 (n ¼ 5), 1q and 21q (both n ¼ 4). account aberrations of tumors with lower tumor cell fractions Genomic losses affected preferably 15q (n ¼ 6), 4q and 13q14 (for more details see Schwaenen et al.21). The cutoff value for (both n ¼ 5), and 8p, 6p21, 11q13 and 14q32 (all n ¼ 4). the detection of DNA amplifications was determined by Nineteen high-level DNA amplifications were identified by repetitive hybridizations of normal male vs normal female array-CGH localizing to (i) the previously described regions DNA. Due to the ratios for clones mapping to chromosome X in 2p15 (n ¼ 4); 9p24 (n ¼ 4) and X (n ¼ 3; see also annotations for these experiments, the cutoff ratio for amplifications was 1.6 (for Table 4 in which an overview of all DNA amplifications is details see Wessendorf et al.20).