Amplification of MYCL in Atypical Ewing Tumor. Analysis of Metaphase and Microarray Comparative Genomic Hybridization
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CANCER GENOMICS & PROTEOMICS 1: 275-282 (2004) Amplification of MYCL in Atypical Ewing Tumor. Analysis of Metaphase and Microarray Comparative Genomic Hybridization TOSHIFUMI OZAKI1, YASUKO NAKAGAWA1, AKI YOSHIDA1, KUNIHIKO NUMOTO1, SHINNSUKE SUGIHARA1, TOSHIYUKI KUNISADA1, SHUJI HAMAZAKI2 and HAJIME INOUE1 1Science of Functional Recovery and Reconstruction, Okayama University Graduate School of Medicine and Dentistry, Okayama; 2Department of Pathology, Okayama University Hospital, Okayama 700-8558, Japan Abstract. A 20-year-old man developed a soft tissue mass in his rarely fuses with other ETS members (6, 8-10). These fusion right upper arm and, 3 months later, was referred to our hospital. products encode chimeric proteins, which function as The tumor cells showed brisk mitotic activity and a large amount aberrant oncogenic transcription factors. of cytoplasmic glycogen was demonstrated with periodic acid The existence of a large-cell variation of Ewing’s sarcoma Schiff stain. A diagnosis of atypical Ewing sarcoma was made. of bone has been accepted by some authors (11-13). Chemotherapy according to the VACA protocol, comprising Nasciment et al. (14) reported that 20 out of 347 ET were vincristine, actinomycin D, cyclophosphamide and doxorubicin large cell Ewing’s sarcoma. Soule et al. (15), on reviewing was started. The chemotherapy was effective and a limb salvage 26 extraskeletal Ewing’s sarcoma, classified 11 tumors as procedure was performed by implantation of an autoclaved bone large-cell or atypical tumors, yet no significant difference in after wide tumor excision. During the postoperative clinical course was seen between the two tumor types. chemotherapy, a local recurrence and multiple metastases In recent years, chromosomal aberrations in Ewing’s developed, and the patient died due to disease progression. sarcomas revealed by comparative genomic hybridization Fourteen years later, this tumor sample, preserved in a deep- (CGH), which can detect whole chromosomal imbalances in freeze, was analyzed by reverse-transcriptase polymerase chain a single analysis, have been reported. The common changes reaction (RT-PCR) to detect the fusion gene. This tumor had an are gains of chromosome 8, 12 and 1q, and losses of 16q and EWS exon 7 to FLI1 exon 6 fusion transcript. Moreover, chromosome 11(16, 17). However, oncogene amplification metaphase and microarray comparative genomic hybridization was not detected in these analyses. (CGH) was done to detect chromosomal instabilities. Many Thirteen years ago, we treated a patient compatible with gains and losses were noted on metaphase CGH, and MYCL atypical ET who showed a rapid clinical course. We amplification was identified on microarray CGH. preserved the tumor sample in a deep-freeze at -80ÆC. In this study, mRNA was extracted from this tumor sample for Ewing tumor (ET) is the second or third most frequent evaluation of the expression of chimeric transcripts of EWS primary malignant bone tumor in children and adolescents fusions with reverse-transcriptase polymerase chain reaction (1). ET is composed of small round cells and more than (RT-PCR). Moreover, DNA was extracted and the 80% of ET express EWS-FLI1 transcripts, which result from chromosomal instabilities were analyzed by metaphase fusions between EWS (22q12) and FLI1 (11q24) (2-4). In CGH and microarray CGH. This is the first report of 5% to 10% of ET, a variant EWS-ERG fusion formed by a microarray CGH analysis in an atypical Ewing tumor. t(21;22)(q22;q12) translocation (5-7) is expressed. EWS Case Report A tumor of the flexion side of the right elbow was discovered Correspondence to: T. Ozaki, MD, Science of Functional Recovery in a 20-year old man in January 1990. The tumor was palpable and Reconstruction, Okayama University Graduate School of with elastic texture, and was about 5 cm in diameter. Skin Medicine and Dentistry, Okayama 700-8558, Japan. Tel: +81-86- adhesion was not noted; however, the tumor showed deep 235-7273, Fax: +81-86-223-9727, e-mail: [email protected] layer adhesion, tenderness and local heat. Plain X-ray revealed u.ac.jp a radiolucent area from the distal metaphysis to the diaphysis Key Words: Ewing tumor, comparative genomic hybidization, of the radial side of the right humerus (Figure 1a). T1- microarray. weighted MR images showed an iso signal tumor (Figure 1b) 1109-6535/2004 $2.00+.40 275 CANCER GENOMICS & PROTEOMICS 1: 275-282 (2004) Figure 1. a) X-ray shows a soft tissue mass in the distal right upper arm and a radiolucent area in the radial side of the right humerus, b) an iso-signal intensity tumor in the distal upper arm on the T1-weighted MR, and c) a high signal intensity tumor on T2-weighted MR image. while T2-weighted images showed a high signal tumor (Figure multiple metastases developed and this patient died in 1c) in the soft tissue from the right upper arm to the elbow. December 1991 (21 months after the diagnosis). Intramedullary infiltration was not noted on MR images. Open biopsy revealed the diagnosis of this tumor as an extraskeletal Histology. Histologically, both the primary and the recurrent Ewing’s sarcoma, which was classified as an atypical Ewing’s lesions were composed of a solid proliferation of polygonal sarcoma (Figure 2). This patient received 4 cycles of cells separated by thin fibrous septa. The tumor cells had preoperative chemotherapy (VACA; composed of vincristine, irregular oval nuclei with prominent nucleoli. The cytoplasm actinomycin-D, cyclophosphamide and doxorubicin) (18). The was clear to eosinophilic with indistinct cell boundaries. The preoperative chemotherapy was effective and a decrease of the mitotic activity was high and a large amount of cytoplasmic tumor size was noted on MR images. In July 1990, wide glycogen was demonstrated with periodic Schiff stain. excision (19) of the tumor, including the distal humerus, was Immunohistochemically, the tumor cells were positive for performed. The resected distal humerus was autoclaved at neuron specific enolase (NSE), negative for S-100 and 120ÆC at 0.26 MPa for 20 min. Then, the bone was implanted myoglobin. Electron scanning microscopy disclosed intercellular at the previous site with plate and screw fixation. During the junctions and rare neurosecretory granules. Based on these postoperative chemotherapy, in November 1990, local findings, a diagnosis of atypical Ewing tumor was made. recurrence of the tumor developed in the soft tissue around the autoclaved bone. The locally relapsed tumor was obtained Materials and Methods by surgical excision and promptly put in the deep freezer at - 80ÆC. This patient underwent disarticulation of the shoulder RT-PCR for detection of fusion transcripts. Total RNA from frozen joint. As an axillary lymph node metastasis of the tumor was material was isolated using the acid-guanidium -phenol/chloroform method. RT-PCR from EWS-FLI1 and EWS-ERG transcripts was detected by computed tomography in December 1990, performed using 1 Ìg total RNA, as published previously (21-24). radiotherapy was started. The axillary lymph node was resected following radiotherapy and intensive chemotherapy, including Metaphase CGH. Reference DNA from healthy blood donors (male) ifosfamide (IFO), etoposide (VP-16), pirarubicin (THP-ADR) and tumor DNA were labelled by the nick translation method with and cyclophosphamide (CPA), was started (20); however, SpectrumRed-dUTP (Vysis, IL, USA) and SpectrumGreen-dUTP 276 Ozaki et al: MYCL Amplification, Metaphase CGH in Atypical Ewing Tumor Figure 2. Histological findings. Polygonal cells with oval nuclei, prominent nucleoli, and ill-defined cytoplasm were evident. Mitotic activity was high. (Vysis), respectively. Nick translation and CGH hybridization were Microarray CGH. Reference DNA and test tumor DNA samples performed according to the manufacturer’s protocol. Normal were labelled using the Random Priming Reagent kit (Vysis). lymphocyte metaphase preparations were denatured at 73ÆC for 5 Sample DNA was labelled with 1 mM Cyì 3-dCTP (Perkin Elmer, min in 70% formamide/2xSSC (pH 7) and dehydrated. The probe Boston, USA). This was mixed with whole genomic reference DNA mixture, after ethanol precipitation and re-suspension in 10 Ìl CGH labelled with Cyì 5-dCTP (Perkin Elmer). The double-labelled hybridization buffer (Vysis), was denatured at 75ÆC for 5 min, DNA was co-hybridized to a microarray in the presence of human applied to the slides and hybridized for 3 days at 37ÆC. Cot 1 DNA to suppress hybridization of the labelled probe to The hybridization was analyzed using a Leica microscope repeat sequences. The microarray (GenoSensor® Array 300, Vysis) (DMRA2) (Leica Microsystems Wetzlar GmbH, Wetzlar, contained 287 kinds of target clone DNA (P1, PAC or BAC clones) Germany) and the Leica Cytogenetic Workstation (CW4000) representing regions important in cytogenetics and oncology (Leica Microsystems Imaging Ltd., Cambridge, UK) based on a including endogens, tumor suppressor genes, subtelomere, etc. high-sensitivity interrating CCD camera (SenSys0401E, Roper (Table I). The target spots were approximately 75-125 Ìm in Scientific, Ottobrunn, Germany). Ratio profiles were averaged diameter and each clone was represented by 3 target spots. from 10 metaphases per sample. Gains of DNA sequences were Following hybridization and removal of the unhybridized probe, defined as chromosomal regions with a fluorescence ratio above the arrays were air-dried and target spots were counter-stained with 1.25 and losses as regions with a ratio below 0.75. A positive a blue fluorophore included in the 4’, 6-diamidino-2-phenylindole control with known aberrations and a negative control were IV (DAPI IV, Vysis). The array was imaged using the Genosensor included in each CGH experiment as quality controls. and the Genosensor Reader Software. The GenoSensor® Reader Regional shifts of the fluorescence ratio profile exceeding the System is a large-field multicolor fluorescence imaging system 1.5 threshold were rated as amplification (25). Heterochromatic which captures an image of the hybridized chip in 3 color planes: regions near the centromeres and the entire X and Y chromosomes Cy3, Cy5 and DAPI blue.