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Complex Rearrangement of Chromosomes 19, 21, and 22 In Cancer Genetics and Cytogenetics 181 (2008) 81e92 Complex rearrangement of chromosomes 19, 21, and 22 in Ewing sarcoma involving a novel reciprocal inversioneinsertion mechanism of EWSeERG fusion gene formation: a case analysis and literature review Georges Mairea, Christopher W. Brownb,c,d, Jane Bayania, Carlos Pereirae, Denis H. Gravelf, John C. Bellc, Maria Zielenskae,g,h, Jeremy A. Squirea,h* aDivision of Applied Molecular Oncology, Ontario Cancer Institute, Princess Margaret Hospital, University Health Network, 610 University Avenue, Room 9-717, Toronto, Ontario M5G 2M9, Canada bOttawa Health Research Institute, Centre for Cancer Therapeutics, Ottawa, Ontario, Canada cDepartment of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada dDepartment of Orthopaedic Surgery, Ottawa Hospital and University of Ottawa, Ottawa, Ontario, Canada eDepartment of Pediatric Laboratory Medicine and Pathology, The Hospital for Sick Children, Toronto, Ontario, Canada fDepartment of Pathology and Laboratory Medicine, Ottawa Hospital and University of Ottawa, Ottawa, Ontario, Canada gGenetics and Genome Biology, Hospital for Sick Children, Toronto, Ontario, Canada hDepartment of Laboratory Medicine and Pathology, University of Toronto, Toronto, Ontario, Canada Received 9 August 2007; received in revised form 5 November 2007; accepted 7 November 2007 Abstract EWSeERG Ewing sarcoma (ES) gene fusions often result from complex chromosomal rearrange- ments. We report an unusually aggressive case of ES with an EWSeERG fusion gene that appeared to be a result of a simple balanced and reciprocal translocation, t(19;22)(q13.2;q12.2). Subsequent molecular investigation of the primary tumor, the metastasis, and a cell line generated from this ES permitted reconstruction of each genomic step in the evolution of this complex EWSeERG fusion. We elucidated a new mechanism of reciprocal insertion inversion between chromosome 21 and 22, involving cryptic alterations to both the ERG and EWS genes. Molecular cytogenetic investigation, using systematic analysis with locus-specific probes, identified the cognate genomic breakpoints within chromosome 21 and 22, mandatory for the excision and exchange of both 30ERG and 30EWS, resulting in the formation of the EWSeERG fusion gene present on the der(22). Array comparative genomic hybridization and fluorescence in situ hybridization studies of the ES cell line derived from this tumor identified additional acquired chromosomal and genomic abnormalities, likely as- sociated with establishment and adaptation to in vitro growth. Notably, the cell line had lost one copy of the RB1 gene within the 13q13.1~q14.2 region, and also had a near-tetraploid karyotype. The significance of these findings and their relationship to other reports of variant and complex ES translocations involving the ERG gene are reviewed. Ó 2008 Elsevier Inc. All rights reserved. 1. Introduction and various genes of the ETS family of transcription fac- tors. In 90% of the cases, FLI1 on chromosome 11 is the Ewing sarcoma (ES) is the primary aggressive tumor of 30 partner of the EWSeFLI1 fusion gene. Alternatively, bone occurring commonly during childhood. Some ES ERG on chromosome 22 is found as the EWS partner in tumors may show the presence of small round blue cells or- 10% of the cases [3]. ganized in Homer Wright rosettes, which express the sur- Many other ETS family gene members (FEV, ETV1, face marker CD99 and the neuron-specific enolase ETV4, .) have been described fused to the 50 end of antigen [1,2]. The common genetic alteration in ES is EWS in ES, as well as in other malignancies, including a translocation between the gene on chromosome 22 EWS acute leukemia, clear cell sarcoma, and myxoid liposarco- ma [4e6]. To date, 13 variants of the EWS fusion gene have * Corresponding author. Tel.: (416) 946-4509; fax: (416) 946-2840. been described, involving nearly as many different cyto- E-mail address: [email protected] (J.A. Squire). bands: 2q31.1, 2q33.3, 2q36, 6p21.33, 7p21.2, 9q31.1, 0165-4608/08/$ e see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2007.11.002 82 G. Maire et al. / Cancer Genetics and Cytogenetics 181 (2008) 81e92 11p13, 12p12, 12q13.13, 12q13.3, 17q12, and 22q12. They for a total of four cycles. Two months after diagnosis, he are extremely rare, representing altogether !1% of the was treated with palliative radiation to the right arm with cases [3,4,7e11]. a dose of 2800 cGy in 14 fractions. Five months after the di- Secondary chromosomal abnormalities have also been agnosis, the patient presented with perianal numbness and described, over many years, but their significance in terms mildly decreased rectal tone. Magnetic resonance imaging of prognosis seems to be related to the overall complexity (MRI) did not show compression of the cauda equina or the of the karyotype rather than to any specific abnormality spinal cord; however, there was involvement of the sacral fo- [3,12]. So far, these secondary abnormalities have not been ramina and nerves. Radiotherapy was applied to the diseased correlated with any specific subgroups of ES. The most pelvis, 2,000 cGy in five fractions, and the patient’s symp- common aberrations consist in gains of chromosomes or toms responded almost immediately. Six months after diag- chromosome arms 1q, 8, and 12; deletion of 1p; and an nosis, the patient was readmitted, for neck pain, and an unbalanced translocation t(1;16) [13e16]. MRI scan detected an epidural tumor causing spinal cord Although more than 100 ES karyotypes have been pub- compression. A C2 vertebral laminectomy and decompres- lished to date, very few cases with the EWSeERG fusion sion were performed. Shortly thereafter, the patient devel- gene have been analyzed using locus-specific fluorescence oped bilateral pleural effusions; he died 6 months after in situ hybridization (FISH) probes. Some studies report being diagnosed with ES. the presence of complex and often cryptic rearrangement of EWSeERG, but the step-by-step events that lead to such 2.2. Tumor sample, histology, immunohistochemistry structural rearrangement are still poorly understood [17e29]. We report here a new three-chromosome rearrange- Approval for the study was obtained from the Ottawa ment involving chromosomes 19, 21, and 22 that carries Hospital Research Ethics Board. A biopsy sample of the a cytogenetically cryptic EWSeERG fusion gene. Molecular proximal humerus lesion was divided for cell culture, fro- cytogenetic analysis of the primary tumor, the metastasis, zen tissue bank, and formalin fixation and paraffin embed- and the derived cell line provided a unique opportunity to ding. Immunostaining was performed according to the e e detail the steps in the generation of a EWSeERG fusion gene streptavidin biotin peroxidase method of Hsu et al. by a new conservative reciprocal insertioneinversion mech- [30]. The following monoclonal antibodies were used: anism; followed by a secondary balanced and reciprocal CD99, vimentin, desmin, CD20, CD45, CD45RO, synapto- translocation with chromosome 19. In this case analysis, physin, leukocyte common antigen (LCA) (DAKO, Glostr- we show that a clinically aggressive ES was associated with up, Denmark; Mississauga, Ontario, Canada); keratin the acquisition of complex chromosomal abnormalities, and CAM5.2 (Becton Dickinson, Franklin Lakes, NJ; Oakville, that in vitro spontaneous transformation of cultured cells Ontario, Canada); keratin AE1:AE3 (Millipore, Billerica, from the primary biopsy was accompanied by RB1 loss and MA; Toronto, Ontario, Canada); and S-100 protein (ESBE pseudo-tetraploidization. Scientific, Markham, Ontario, Canada) and neuron-specific enolase (Intermedico, Markham, Ontario, Canada). 2.3. Cell culture 2. Materials and methods Biopsy material isolated from the proximal humerus le- 2.1. Case history and treatment sion was mechanically disaggregated and cultured in Dul- becco’s modified Eagle’s mediumehigh glucose medium A 25-year-old man presented with a 9-month history of (HyQ; Hyclone, Logan, UT) without antibiotics and con- right shoulder pain, but otherwise had no abnormal clinical taining 10% heat-inactivated bovine serum (3:1 of donor findings. Radiographic investigations revealed a pathologic calf serum [PAA Laboratories, Etobicoke, Ontario, Canada] fracture of the right proximal humerus, as well as multiple and qualified fetal bovine serum [Invitrogen, Carlsbad, CA; pulmonary nodules. An open biopsy of the proximal hu- Burlington, Ontario, Canada]). After dispersion, cells dis- meral lesion was performed, revealing a small blue cell played a semiadherent phenotype and were subcultured tumor. Further histological and immunohistochemical for 4 months (1/3 dilution twice a week) in RPMI-1640 me- investigations on the biopsy material confirmed the diagno- dium and antibiotics (streptomycin, 100 mg/mL, penicillin, sis of a ES. A computed tomography scan demonstrated the 100 mg/mL) supplemented with 10% heat-inactivated fetal presence of multiple bilateral pulmonary nodules (the larg- calf serum (Invitrogen). est measuring 14 mm by 23 mm), as well as lesions in the vertebrae (T1, T11, L3, and L5) and in the ischial tuberos- 2.4. Cytogenetic and FISH analyses ity. A bone scan confirmed the presence of multiple bone lesions. Ultrasonography of testes and thyroid yielded Metaphase spreads for cytogenetic analysis were prepared normal findings. from the cultured cell line using conventional methods [31]. The patient was treated with a monthly
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