[CANCER RESEARCH 60, 549 –552, February 1, 2000] Advances in Brief Genomic Imbalances Including Amplification of the Tyrosine Kinase Gene JAK2 in CD30  Hodgkin Cells 1 Stefan Joos, 2 Manfred Ku ¨ pper, Sibylle Ohl, Frederike von Bonin, Gunhild Mechtersheimer, Martin Bentz, Peter Marynen, Peter Mo ¨ller, Michael Pfreundschuh, Lorenz Tru ¨ mper, and Peter Lichter Deutsches Krebsforschungszentrum, Abteilung Organisation Komplexer Genome (H0700), D-69120 Heidelberg [S. J., M. K., S. O., P. L.]; Universita ¨tskliniken des Saarlandes, Innere Medizin I, D-66421 Homburg/Saar [F. v. B., M. P., L. T.]; Pathologisches Institut, Universita ¨t Heidelberg, D-69120 Heidelberg [G. M.]; Abteilung Innere Medizin III, D- 89081 Ulm [M. B.]; Human Genome Laboratory, B-3000 Leuven [P. Ma.]; Abteilung fu ¨r Pathologie des Universita ¨tsklinikums Ulm, D-89081 Ulm [P. Mo ¨.], Germany Abstract Comparative genomic hybridization was applied for a comprehensive screening of frequently occurring net gains and losses of chromosomal subregions in small populations of CD30  Hodgkin cells and their mor- phological variants. In 12 Hodgkin’s lymphomas, recurrent gains were detected on chromosomal arms 2p, 9p, and 12q (in six, four, and five tumors, respectively) and distinct high-level amplifications were identified on chromosomal bands 4p16, 4q23-q24, and 9p23-p24. In Hodgkin cells with 9p23-p24 amplification, fluorescence in situ hybridization revealed an increased copy number of chromosomal sequences spanning the tyro- sine kinase gene JAK2. Several of the imbalances described, in particular a gain in chromosomal arm 9p that includes JAK2 amplification, are similar to the genomic changes detected in primary mediastinal B-cell lymphoma. Introduction In Hodgkin’s disease, four well-defined histotypes of cHL 3 have been distinguished according to the recent REAL classification, i.e., cHL-LR, cHL-NS, cHL-MC, and cHL-LD, in addition to the clini- cally and immunophenotypically distinct subtype of paragranuloma, or NLPHL (1). All subtypes are characterized by the presence of only a small fraction of malignant cells, referred to hereafter as “Hodgkin cells,” which exist in several variants: the mononuclear variant, the L&H cell, and the lacunar cell variant, as well as the multinucleated Reed-Sternberg cell (2). Because of the small numbers, low mitotic index, and frequently poor chromosome morphology of these cells, chromosome banding analysis is particularly difficult and has not revealed specific chromosomal changes that would immediately indi- cate the localization of genes involved in the etiology of Hodgkin’s disease (3). Therefore, we used an alternative approach and collected pools of 30 CD30 + Hodgkin cells from a series of 11 cHLs and a single case of NLPHL. The genomic DNA of the pooled cells was subjected to universal PCR amplification and used as probe for CGH. On the basis of the results obtained, the copy number of a candidate gene, JAK2, was analyzed by FISH and Southern blot analysis in Hodgkin cells and PMBL, in which similar chromosomal imbalances have been found previously (4). Materials and Methods Tumor Material. Lymph node biopsies from seven male and five female patients were used for cytogenetic analysis of Hodgkin cells. The mean age of the patients at the time of diagnosis was 38.3 years. The 11 cHLs comprised 1 nodular variant of cHL-LR, 4 cases of cHL-MC, 5 cases of cHL-NS, 1 case of cHL-LD, and 1 case of a nodular variant of NLPHL. Tumors were classified according to the guidelines of the pathology panel of the German Hodgkin’s study, anticipating the upcoming WHO classification (5) based on routinely stained paraffin sections and immunohistology that included stains for CD3, CD15, CD20, and CD30 as well as epithelial membrane antigen. The tumor staging and immunophenotypes of CD3, CD20, and CD30 are listed in Table 1. From each tumor, fresh, unfixed tissue material was available. Isolation of Hodgkin Cells. For isolation of Hodgkin cells, lymph nodes derived from Hodgkin’s disease were prepared as described previously (6). Briefly, lymphocytes and Hodgkin cells from viable single-cell suspensions were fixed in 3% w/v paraformaldehyde and applied to glass slides by cytospin centrifugation. For immunostaining of CD30 antigen, Alkaline-phosphatase- anti-alkaline-phosphatase assay 4 was applied (6), using monoclonal antibody HRS-4. Identification of Hodgkin cells was based on positive staining for CD30 antigen and on morphological criteria. In the single case of NLPHL, which exhibited large CD20 + /CD30 + malignant cells, primarily morphologi- cal criteria were applied. Thirty Hodgkin cells were isolated and collected with glass capillaries, using a micromanipulation device (Eppendorf, Hamburg, Germany). DOP-PCR. Hodgkin cells were first digested with proteinase K (250 g/ml) in 20 l of 1 PCR buffer (2 mM MgCl 2 , 50 mM KCl, 10 mM Tris, pH 8.3, 0.1 mg/ml gelatin) for 1 h at 55°C, with subsequent inactivation of the enzyme at 95°C for 15 min. For universal amplification of the genomic DNA, degenerate oligonucleotide-primed DOP-PCR as described by Telenius et al. (7) was applied. After the PCR reaction, excessive DOP primers were sepa- rated from the amplified genomic DNA on the appropriate columns (Qiagen, Hilden, Germany). CGH. Preparation of metaphase chromosomes, probe labeling, hybridiza- tion, and image acquisition were performed as described previously (4, 8). Chromosomal imbalances were detected based on the ratio profile deviating from the balanced value of 1.0. Chromosomal regions were scored as gains or losses when the ratio profile either reached or exceeded the diagnostic thresh- olds of 1.25 or 0.75, respectively. Overrepresentations were considered high- level amplifications when the CGH ratio exceeded the value of 2.0 or when the fluorescence showed a strong distinct signal detected by visual inspection and the corresponding ratio profile was diagnostic for overrepresentation. Centro- meric regions as well as chromosomes 1p32-p36 and 19 were not scored in the results for reasons specified elsewhere (9). The quality of the CGH experi- ments was assessed using genomic DNA of male individuals as internal control probes; monosomy of the X chromosome was clearly visible in all experiments performed in this study. FISH. Hodgkin cells were detected by staining with monoclonal anti-CD30 (HRS-4), a secondary alkaline phosphatase conjugated antibody (Dako Envi- sion; DAKO Diagnostica, Hamburg, Germany) and fast red substrate. Slides were then treated with 1.5% Triton X-100. After denaturation of chromosomal Received 11/8/99; accepted 12/13/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the Tumorzentrum Heidelberg/Mannheim (Grants I./I.1. and I./I.2.) and by the Deutsche Forschungsgemeinschaft (Grants Be 1454/5-2, Li 406/4-1, and SFB 399-A8). 2 To whom requests for reprints should be addressed, at Deutsches Krebsforschung- szentrum, H0700, D-69120 Heidelberg, Germany. Phone: 49-6221-424620; Fax: 49- 6221-424639; E-mail: s.joos@dkfz-heidelberg.de. 3 The abbreviations used are: cHL, classical Hodgkin’s lymphoma; cHL-LR, lympho- cyte-rich cHL; cHL-NS, nodular sclerosis cHL; cHL-MC, mixed-cellularity cHL; cHL- LD, lymphocyte-depletion cHL; NLPHL, nodular lymphocyte predominant Hodgkin’s lymphoma; CGH, comparative genomic hybridization; PMBL, primary mediastinal B-cell lymphoma; DOP-PCR, degenerate-oligo-primed-PCR; FISH, fluorescence in situ hybrid- ization. 549 Research. on December 7, 2021. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from