Cancer Genetics and Cytogenetics 181 (2008) 81e92

Complex rearrangement of 19, 21, and 22 in Ewing sarcoma involving a novel reciprocal inversioneinsertion mechanism of EWSeERG fusion 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 21 and 22, involving cryptic alterations to both the ERG and EWS . 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 . 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 (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 chemotherapy reg- Spectral karyotyping was performed on metaphases accord- imen of vincristine, actinomycin-D, and cyclophosphamide ing to the manufacturer’s instructions (ASI, Carlsbad, CA) G. Maire et al. / Cancer Genetics and Cytogenetics 181 (2008) 81e92 83 and as previously published [31,32]. The probes used are For FISH on the frozen humerus biopsy, tissue sections 5 listed in Table 1. Commercial probes were used according mm thick were put on polylysine coated glass slides and to the manufacturer’s instructions. Bacterial artificial chro- were processed using a frozen-tissue prep kit (Abbott Mo- mosome (BAC) probes from the RP11 library were obtained leculareVysis). The formalin-fixed and paraffin-embedded from the Centre for Applied Genomics (Toronto, Ontario, tissue from the fine-needle aspiration of the cervical Canada) and were labeled by means of a nick-translation metastasis was processed for FISH analysis as described kit (Abbott MoleculareVysis, Des Plaines, IL) using Spec- previously [34]. Slides were observed using an epifluores- trumOrange, SpectrumGreen (both Vysis), Cy5-dUTP, or cence Zeiss Imager Z1 microscope equipped with a digital DEAC-dUTP (both PerkinElmer, Waltham, MA). Standard camera Axio Cam MRm and AxioVision 4.3 capturing FISH procedures were followed [33]. software.

Table 1 Description and origin of the FISH probes used for cell line characterization Gene or chromosomal Result Probe Cytoband location Source Whole chromosome painting probes der(19) and der(22) WCP 19 chromosome 19 Q-Biogene (Gaithersburg, MD) der(21) and der(22) WCP 21 chromosome 21 Q-Biogene der(19) and der(22) WCP 22 chromosome 22 Abbott MoleculareVysis (Des Plaines, IL) Gene- and locus-specific probes Chromosome 13 2 copies LSI 13 (RB1) 13q14.2 RB1 Abbott MoleculareVysis SpectrumOrange 4 copies RP11-172A19 13q14.3 RMC 4 copies RP11-475F14 13q21.33 RMC 4 copies RP11-165M6 13q33.2 RMC Chromosome 19 der(19) RP11-434E22 19q13.11 UCSC der(19) RP11-615P15 19q13.11 UCSC der(19) RP11-316I4 19q13.11 UCSC der(19) RP11-3L5 19q13.11 UCSC der(19) RP11-314G8 19q13.11 UCSC der(19) RP11-737K14 19q13.11 UCSC der(19) RP11-60A22 19q13.11 HPN, FXYD5 UCSC Split der(19)/der(22) RP11-44C9 19q13.12 30HPN, FXYD5, UCSC TMEM162 der(22) RP11-412D22 19q13.12 TMEM162 UCSC der(22) RP11-111F21 19q13.12 UCSC der(22) RP11-808P14 19q13.12 UCSC der(22) RP11-430N3 19q13.12 UCSC der(22) RP11-45P2 19q13.12 UCSC der(22) RP11-150D11 19q13.12 UCSC der(22) RP11-569M1 19q13.2 Kawamura-Saito et al., 2006 [48] der(22) RP11-208I3 19q13.2 [48] der(22) RP11-10I11 19q13.33 UCSC der(22) RP11-667O16 19q13.43 UCSC Chromosome 21 Split der(22)/der(21) RP11-476D17 21q22.2 30ERG Tomlins et al., 2005 [49]; Yoshimoto et al., 2006 [50]; UCSC der(21) RP11-95I21 21q22.2 50ERG Tomlins et al., 2005 [49]; Yoshimoto et al., 2006 [50]; UCSC der(21) RP11-535H11 21q22.3 TMPRSS2 UCSC der(21) RP11-35C4 21q22.3 UCSC der(21) RP11-891L10 21q22.3 UCSC der(21) RP11-260O11 21q22.3 UCSC Chromosome 22 50der(22) 30der(21) LSI EWSR1 dual-color, 22q12.2 50EWS (SpectrumOrange)/ Abbott MoleculareVysis break-apart 30EWS (SpectrumGreen) der(21) RP11-91J21 22q12.2 57 kb telomeric to 30 end UCSC of EWS BAC probes (prefixed RP11) from the BAC library of the Roswell Park Cancer Institute (Buffalo, NY) were identified using either the University of California Santa Cruz (UCSC) Genome Browser or Resources for Molecular Cytogenetics (RMC) (Bari, Italy). Abbreviations: FISH, fluorescence in situ hybridization; WCP, whole chromosome painting. 84 G. Maire et al. / Cancer Genetics and Cytogenetics 181 (2008) 81e92 2.5. Reverse transcriptaseepolymerase chain reaction Total RNAs were extracted from the cell line during the same culture period as when metaphase preparations were made for molecular cytogenetic analyses, using Trizol reagent (Invitrogen), and were submitted to reverse transcriptasee polymerase chain reaction (RT-PCR)for the detection of the EWSeERG fusion gene [35]. Duplicate experiments were done with positive (TTC-466 cell line) and negative (tonsil) control RNA.

2.6. Array comparative genomic hybridization Normal female 46,XX genomic DNA was obtained from Promega (Madison, WI). Genomic DNA was extracted from the cell line during the same culture period that meta- phase preparations were made for molecular cytogenetic analyses using Puregene DNA purification tissue kits (Gen- tra Systems, Minneapolis, MN). Human microarray con- taining unique 60-mer oligonucleotides representing 244,000 features was used (Agilent Technologies, Santa Clara, CA). Labeling reactions were performed as de- scribed previously, with 2 mg of purified DNA and either Cy5-dUTP for the experimental sample or Cy3-dUTP for the female reference (PerkinElmer Life and Analytical Sci- ences, Waltham, MA; Woodbridge, Ontario, Canada) [36]. A dye-swap experiment was performed in parallel. Hybrid- ization, washes, scanning, and analysis were performed as described by Selvarajah et al. [36] with minor modifica- Fig. 1. Histologic and immunohistochemical analysis of the biopsy of the tions. To analyze the array comparative genomic hybridiza- humerus lesion. Immunohistochemistry analysis shows a very weak stain- tion (CGH) data, Agilent Technologies software (CGH ing of the neuron-specific enolase (A) and a strong expression of CD99 (B). Hematoxylineeosin staining shows the presence of high-density small Analytics version 3.4) was used. round blue cells associated with bone trabeculae (arrow) (C), and rare Homer Wright rosettes are observed (arrow) (D). All images are taken at 40 magnification. 3. Results demonstrated a mixed interval response after the first two 3.1. Clinical course and pathology rounds of chemotherapy, but within 5 months of diagnosis signs of disease progression were evident both radiologi- The present ES case was unusual by clinical, histologi- cally and clinically. An unusually aggressive disease course cal, and immunohistological standards. First, the patient was apparent, and the patient died of his disease ~6 months was diagnosed at 25, a relatively old age, compared with after his first presentation. the usual onset for ES (the incidence peak is between 15 and 19 years old). Second, the anatomical lesion of the pri- mary tumor was in the upper rather than the lower extrem- 3.2. Molecular analysis ity. Third, the neuron-specific enolase immunophenotype was atypical, in that very few cells were positive (Fig. 1A). 3.2.1. Identification of a t(19;22), and detection Immunohistochemical analysis of both the initial hu- of EWSeERG fusion gene meral biopsy sample and the metastatic cervical spine le- A total of 10 metaphases derived from the ES culture sion demonstrated staining for CD99 (Fig. 1B) and were analyzed by 24-color spectral karyotyping (SKY). vimentin, and slight positivity in the PAS stain (data not Most cells exhibited a tetraploid karyotype. The sole de- shown); staining was negative for synaptophysin, keratin tectable structural abnormality was an apparent reciprocal CAM5.2, keratin AE1:AE3, S-100 protein, desmin, and balanced translocation, t(19;22)(q13;q12) (Fig. 2A). CD20, CD45 and CD45RO, and LCA. At the morphologi- No normal cell was detected. The composite karyotype, de- cal level, the biopsy specimen exhibited the classical high scribed according to ISCN 1995 guidelines [37], was density of small round blue cells (Fig. 1C), as well as the 75~80,XXYY,2,4,5,5,6,6,7,7,10,10,14, rare Homer Wright rosettes (Fig. 1D), suggesting neuroec- 15,16,16,t(19;22)(q13;q12)2[cp10]. FISH analysis todermal differentiation. Radiologically, the patient initially with whole chromosome painting probes (WCP) for G. Maire et al. / Cancer Genetics and Cytogenetics 181 (2008) 81e92 85

Fig. 2. Molecular cytogenetic analyses. (A) Representative spectral karyotyping (SKY) karyogram. (BeD,F,GeI) Fluorescence in situ hybridization (FISH) and (E) array comparative genomic hybridization (CGH) analyses. In (A), an apparent balanced and reciprocal translocation between chromosome 19 and22 is observed as the sole structural rearrangement. (B through I) Legend: Derivative chromosomes are identified as follows: cross, der(13); arrowhead, der(19); 86 G. Maire et al. / Cancer Genetics and Cytogenetics 181 (2008) 81e92 chromosome 19 and 22 confirmed the t(19;22)(q13;q12) the der(22) was identified by the translocated BAC clone rearrangement (Table 1). The chromosome 22 breakpoint RP11-667O16, which is normally located on 19q13.43. was located on the 22q12.2 cytoband within the EWS The der(21), identified by the RP11-91J21 BAC clone, gene; hybridization with the EWS break-apart FISH was uniformly stained with the WCP probe for chromo- probes showed two separate signals (Fig. 2B). The RT- some 21. No chromosome 21 material was observed on PCR analysis of RNA extracted from the same sample the der(19), identified by the RP11-44C9 BAC clone, span- demonstrated that the fusion partner of EWS was the ning the 19q13.12 breakpoint. ERG gene, located on 21q22.2 (Fig. 3). The distinction between the normal chromosome 19 and its derivative was assessed with the BAC clone RP11- 667O16 (19q13.43) (Fig. 2C). As expected, based on the 3.2.2. Complex rearrangement of chromosomes RT-PCR results (Fig. 3), the cohybridization of the EWS 19, 21, and 22 and ERG break-apart probes (Table 1) showed a colocaliza- Positional cloning of the 19q13 breakpoint by FISH (Ta- tion of the 50 end of EWS with the 30 end of ERG on the ble 1) was narrowed down to an interval of 44 kb at der(22) (Fig. 2D, arrow). The observation of signals from 19q13.12, between the RP11-60A22 (proximal) and the BAC clone RP11-476D17 (containing the 30 end of RP11-412D22 (distal) BAC clones (Fig. 2B). As expected the ERG gene) present on both the der(21) and the from the UCSC genome browser (March 2006 release: der(22) suggested another breakpoint within the 21q22.2 hg18, NCBI Build 36.1; http://genome.ucsc.edu), the region, proximal to the 30 end of the ERG gene (Fig. 2D). BAC clone RP11-44C9, spanning the 19q13.12 breakpoint We therefore concluded that a fragment of ~350 kb, con- showed a split signal on both der(19) and der(22) (Table 1). taining the ERG 30 end has been excised, inverted and in- Moreover, as shown in Figure 2B, the 30EWS probe did not serted on 22q12.2 to generate the EWSeERG in-frame colocalize with the RP11-412D22 probe (distal side of the fusion gene. 19q13.12 breakpoint), and was observed on the der(21). The fragment of chromosome 22 that was translocated to the der(21) was estimated to be 250 kb, because both the 3.2.3. Genomic imbalances: array CGH 30EWS probe (Fig. 2B) and the RP11-91J21 BAC clone, Array CGH analysis was performed to determine the normally located 57 kb apart from the telomeric side of overall pattern of genomic imbalance associated with this the 30 end of the EWS gene, were observed on der(21) (Ta- complex EWSeERG fusion at higher resolution. The pro- ble 1)(Fig. 2BeC). files indicated a relatively small proportion of imbalance Hybridization with chromosome 21 WCP probe showed present using DNA derived from the ES culture. Twenty the presence of a very small insertion of chromosome 21 chromosomes exhibited flat profiles from the pter to the material within the 22q12 cytoband (Fig. 2C). Notably, qter extremities: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14,

= star, der(21); arrow, der(22). Normal chromosomes are indicated by their number. (BeD) Metaphase FISH. Complex tri-chromosome rearrangement revealed by overlapped signals from the 50EWS and the 30ERG probes. (B) Mapping of 19q13.12 and 22p12.2 breakpoints. Probes RP11-60A22 (violet) and RP11- 412D22 (pale blue) located on 19q13.12 are observed on der(19) and der(22), respectively. The 22q12.2 probes (50 and 30 extremities of EWS gene) are observed on der(22) for the 50EWS probe (red) and on the der(21) for the 30EWS probe (green). Although the t(19;22) seemed to be balanced and reciprocal by SKY, a third chromosome is here seen to be involved in the rearrangement. Insets: Magnified view of the three derivative chromosomes. (C) Chromosome 21 rearrangements. Whole chromosome painting probe (WCP) for chromosome 21 (green) shows chromosome 21 material on the der(22), which is identified by the bacterial artificial chromosome (BAC) clone RP11-44C9 (violet) (spanning the 19q13.12 breakpoint) and RP11-667O16 (pale-blue) (19q13.43 trans- located to the der(22) [arrow]). The der(21) is identified by the colocalization of the BAC clone RP11-91J21 (red) (57 kb from the 30 end of EWS ) and the WCP probe for chromosome 21 (green) as a yellow signal (star). Insets: Magnified view of derivative chromosomes 21 (star) and 22 (arrow). (D) Mapping of 21q22.2 and 22q12.2 breakpoints. The 30 extremity of ERG (RP11-476D17, pale-blue probe) colocalizes with the 50EWS (red probe) on the der(22) (arrow). The 30EWS probe (green) is observed on the der(21) (star), very close to the 50ERG probe (violet), with an additional pale-blue signal (RP11-476D17) as well. This observation of split signals of the pale-blue RP11-476D17 probe on both der(21) and der(22) maps another breakpoint within 21q22.2, centromeric to the 30ERG extremity. (E,F) Hemizygotic loss of the RB1 gene identified by array CGH and validated by FISH. (E) Chromosome 13 profile from array CGH experiment shows the presence of a 16-Mb loss from RH80830 to RH103912 loci (13q13.3~q14.3) (red shaded box), which encompasses the RB1 locus (13q14.2). Each feature of the array is indicated by a dot positioned according to its localization on chromosome 13 (on the y-axis); the G-banding ideogram

at 850-band resolution is shown on the left. The x-axis represents the log2 ratio value for a feature. The solid vertical line represents a log2 ratio of zero; the dashed line, a log2 ratio of 1. (F) Validation of the hemizygotic loss of the RB1 locus. Metaphase from the cell line was cohybridized with a probe con- taining the RB1 gene (red), with a control probe (BAC clone RP11-156M6) (green) located on 13q33.2. The observation of two chromosomes 13, and two additional chromosome 13 derivatives (cross), lacking the RB1 (red) signal, was interpreted as a hemizygotic loss of the RB1 gene. (GeI) Interphase FISH analysis of the humerus lesion (biopsy). Hybridization with the break-apart probes for the 22q12.2 and 19q13.12 breakpoints as well as the probes for 30ERG and RB1 shows a diploid pattern. The 19q13.2 and 22q12.2 breakpoints are observed on the humerus lesion (G). The 50EWS (red) and the 30EWS (green) signals are separated, as well as the RP11-60A22 (violet) and RP11-412D122 (pale-blue) signals for the 19q13.12 breakpoint. The der(19) is identified as a single violet signal (arrowhead), the der(21) as a single green signal (star), and the der(22) by the colocalization of red and pale-blue signals (arrow). (H) The rearrangement of EWS and ERG is revealed, with the EWSeERG fusion on the der(22) (arrow) is identified by the colocalization of the 50EWS (red) with the 30ERG (RP11-476D17 pale-blue). The breakage within the RP11-476D17 BAC is also observed, with an additional pale-blue signal colocalized with the 30EWS probe (green) and the 50ERG probe (RP11-95I21 violet) on the der(21) (star). (I) Hybridization with the RB1 probe (red) and 13q33.2 probe (green) shows a normal diploid pattern of signals. G. Maire et al. / Cancer Genetics and Cytogenetics 181 (2008) 81e92 87 12 3 4 tumor biopsy. Both the 19q13.12 break-apart probes and the EWS break-apart probes exhibited separated signals 700 bp (Fig. 2G). The EWSeERG rearrangement was confirmed 500 bp EWS-ERG by the colocalization of the 50EWS and the 30ERG signals 300 bp (Fig. 2H); however, the RB1 deletion was not observed 200 bp (Fig. 2I), nor was the polyploidy. All the probes tested by interphase FISH showed a diploid pattern of signals (Fig. 2- GeI). Similarly, the interphase FISH analysis of the cervi- 50 bp cal metastasis fine-needle aspiration also exhibited the 19q13.12, ERG, and EWS rearrangements, but not the Fig. 3. EWSeERG fusion gene detection by reverse transcriptaseepoly- RB1 hemizygotic loss and the polyploidy (data not shown). merase chain reaction (RT-PCR). Total RNA was extracted from cell lines (lane 1, TTC-466, positive control; lane 4,l cell line of interest) or tissue (lane 2, tonsil, negative control) and subjected to RT-PCR using primers to detect the EWSeERG fusion gene. Water was used as a template (lane 4. Discussion 3). A negative picture of the ethidium-bromide-stained agarose electropho- resis gel is shown. RT-PCR product is observed at 465 bp, as expected for In this study, we characterized a primary ES tumor, the the TCC-466 cell line, and also for the cell line of interest, indicating that metastasis, and the cell line derived from the primary tu- they harbor the EWSeERG fusion gene (arrow). mor. The combination of SKY, FISH, and array CGH anal- yses allow us to establish a possible sequence of 15, 16, 17, 18, 20, X, and Y. Among these, eight chromo- chromosomal alterations leading to the complex rearranged somes showed no copy changes (1, 3, 6, 8, 12, 17, 18, chromosomes (Fig. 4). We propose that the two first steps and 20), with a log2 ratio equal to zero. Six chromosomes occurred in vivo within the tumor, and the later steps were (2, 4, 9, 11, 14, and 15) presented a log2 ratio close to related to the spontaneous in vitro transformation of the 0.25. A third group of chromosomes was characterized cultured primary cells derived from the humerus biopsy. by a log2 ratio close to 0.75 (chromosomes 5, 7, 10, Evidence in support of this model was provided by the and 16) (data not shown). These observations were in ac- systematic FISH analyses of the rearranged cytobands asso- cordance with the SKY data. ciated with the complex EWSeERG fusion. For the inverted The three chromosomes (19, 21, and 22) involved in the insertion of the 30 end of the ERG gene on the chromosome rearrangements characterized by FISH, showed a variation 22, the two mandatory breakpoints have been identified on of the log2 ratio value at the proximity of the identified the 21q22.2 cytoband. The proximal breakpoint is within the breakpoints: 19q13.12 (within the 44-kb interval identified RP11-476D17 BAC clone, and the distal one is between the by FISH analysis, Fig. 2B and Table 1), 21q22.2 (39 Mb RP11-476D17 and RP11-95I21. The excised segment, ~350 telomeric to the ERG gene), and 22q12.2 (1 Mb telomeric kb in length and containing the 30 end of the ERG gene, was to the EWS gene). Regions from chromosome 19 observed on the der(22) and colocalized with the 50EWS (19q13.12~q13.43) and 22 (22q11.21~q12.1) that com- probe. The inversion of that inserted segment was verified posed the der(22) showed a log2 ratio of zero, while the rest by the positive detection of the EWSeERG fusion gene of the chromosomes (19q12~q13.12 and 22q12.2~13.33) by RT-PCR. Indeed, according the to the UCSC Genome were characterized by a log2 ratio value close to 0.25 Browser (March 2006 release; hg18, NCBI Build 36.1; (data not shown). http://genome.ucsc.edu), the ERG gene is oriented 30 to 50 A 16-Mb region spanning the 13q13.3~q14.2 region, on the long arm of chromosome 21, from cen(21) to 21qter, from RH80830 to RH103912, was identified with a log2 and the EWS gene is oriented on the opposite: 50 to 30 on the ratio of 1(Fig. 2E). This region, which includes the RB1 long arm of chromosome 22, from cen(22) to 22qter. To gene within the 13q14.2 cytoband, was therefore hemizy- avoid the tail-to-tail or head-to-head fusion of the genes, gously deleted in these cells. This conclusion was con- an inversion of one the genes (in this instance, ERG) is nec- firmed by FISH, using a RB1 probe with control from essary for the formation of the in-frame fusion gene EW- chromosome 13 (Table 1). Two normal chromosomes 13 SeERG. The insertion of the chromosome 22 material were observed, with both signals from the RB1 and the into chromosome 21 extends towards the 30 end of the RP11-165M6 BAC clone (located on 13q33.2), whereas EWS gene, for up to ~200 kb on the telomeric side. The ori- the two der(13) were characterized by the lack of the entation of that chromosome 22 fragment, of ~250 kb in RB1 signal (Fig. 2F). The FISH results validate the array length, has not been investigated. CGH observation of a hemizygotic loss of the RB1 gene. Within the ISCN 1995 guidelines [37], the karyotype can be described as inv ins(21;22)(21pter/21q22.2::22q12.2::21q 3.2.4. Interphase FISH analysis of the primary tumor 22.2/21qter;22pter/22q12.2::21q22.2::22q12.2/22qter). and the metastasis A secondary translocation between the derivative 22 and We found that the 19q13.12, 21q22.2, and 22q12.2 rear- the chromosome 19 occurred, with a breakpoint on rangements were also present on the humerus primary 19q13.12 precisely mapped by FISH (Fig. 2B and Table 88 G. Maire et al. / Cancer Genetics and Cytogenetics 181 (2008) 81e92

19 21 22

13 ABCD

WCP1 9 60 A22 60 A22 RB1 412D21 WCP2 2

172 A19 172 A1 9 667O19

165M6 165M6

476D17 WCP22 5’ EW S WCP2 1 5’EW S 3’ER G, 476D17 3’ER G, 476D17 91J21 WCP21 3’EW S 412D22 5’ER G 3’EW S WCP2 1 91J21 WCP1 9 5’ER G

260O16 667O16 260O11 E

Fig. 4. Model of the progression of chromosomal abnormalities as a sequence of events (AeE). Legend: Derivative chromosomes are identified as follows: cross, der(13); arrowhead, der(19); star, der(21); arrow, der(22). Chromosomes 13 (gray), 19 (blue), 21 (green), and 22 (red) are represented according to combined findings from RT-PCR, SKY, FISH, and array CGH. From observations of cell line vs. biopsy, breakpoints identified by black double-ended arrow occurred in the tumor (A,B), and the others (C) (white double-ended arrow) occurred during the line establishment; panel D is showing the structural ab- normalities characterized. The detailed breakpoint mapping is summarized in panel E. All BAC clones used were from the RP11 library. As a first step, an inverted insertion occurred between chromosomes 21 and 22 (A); the proximal breakpoint on 21q22.2 is located within the BAC clone RP11- 476D17, and the distal breakpoint is within the ERG gene. As a result, a fragment of ~350 kb has been inserted, and inverted at 22q12.2 within the EWS gene, leading to the formation of the EWSeERG fusion gene (B). This insertion is reciprocal, in that a 22q12.2 fragment of ~250 kb has been observed on der(21). The proximal breakpoint on 22q12.2 is located within the EWS gene, and the distal breakpoint should be close to the distal end of the BAC clone RP11-91J21 (A). The second step in the progression consists of a reciprocal translocation between a normal chromosome 19 and the derivative 22. The break- point on 19q13.12 is located between BAC clones RP11-60A22 (proximal border) and RP11-412D21 (distal border). The breakpoint on der(22) has not been characterized. The third step was not observed on the biopsy, nor on the cervical metastasis, and therefore must have occurred during cell line establishment. Array CGH identified a 13q13.1~q14.2 deletion of 16 Mb, including the RB1 gene (chr13:21,131,883e37,971,206, according to the UCSC Browser, release hg18, March 2006; NCBI build 36; http://www.genome.ucsc.edu). The final step in the progression is the tetraploidization process. Indeed, every normal and derivative chromosomes was observed in duplicate, whereas a diploid pattern was observed on the biopsy and the metastasis.

1), and a breakpoint on der(22), distal to the RP11-91J21 and 22. Polyploidization likely took place subsequent to BAC clone location. This is supported by the absence of acquisition of the structural rearrangements, because SKY chromosome 21 material identified on der(19) by the analyses showed that some chromosomes were present as WCP probe for chromosome 21 (Fig. 2C). two or three copies, whereas the majority exhibited a tetra- The last two steps in the generation of this complex kar- ploid pattern. Notably, the fragments of chromosomes 19, yotype occurred in vitro. We observed an in vitro hemizy- 21, and 22 composing the der(22) are consistently present gotic loss of the 13q13.1~q14.2 region, which includes in four copies. the RB1 gene. Because this loss is observed neither in the ES tumors with the EWSeERG fusion gene are rare, and primary tumor (humerus biopsy) nor in the cervical metas- most of the cases reported to date have been simple obser- tasis, it is thought to be part of the selection for enhanced in vational studies using RT-PCR or other DNA-based vitro growth. We also observed that all derivatives in the methods (Table 2). Although such approaches are useful cell linedder(13), der(19), der(21) and der(22)d are pres- in the context of diagnosis and clinical studies, neither ent in duplicate, as are the normal chromosomes 13, 19, 21, the mechanism of the fusion gene formation nor the Table 2 Cytogenetics and molecular abnormalities in Ewing sarcoma with the EWSeERG fusion gene Abnormalities of Molecular Location of Sample type (name) Chr 21 and 22 FISH data biology data EWSeERG Mechanism of EWSeERG formation References Tumor (T91-124) der(21)t(21;?)(q22;?) ND RT-PCR sequence NE Complex chromosomal rearrangement Giovannini et al., 1994 [17] ?der(22)t(22;?)(q12;?) (21q22 YACs map) Tumor (T89-135) no obvious rearrangement Rearrangement of ERG RT-PCR sequence NE Complex chromosomal rearrangement [17] of Chr 21, one 22 missing (break-apart probes) (21q22 YACs map) Tumor (T92-60) der(22)t(11;22)(q24;q12) ND RT-PCR sequence NE Complex chromosomal rearrangement [17] (21q22 YACs map) Cell line (SK-PN-LI) t(21;22)(q22;q12) ND RT-PCR sequence NE Complex chromosomal rearrangement [17] (21q22 YACs map) Tumor (T93-113) t(12;22;22)(q14;p1;q12) ND RT-PCR sequence NE Complex chromosomal rearrangement [17] (21q22 YACs map) Cell line (5838) t(11;22)(q24;q12) WCP 11 and WCP 22, no RT-PCR sequence NE NI Dunn et al., 1994 [18] .Miee l acrGntc n yoeeis11(08 81 (2008) 181 Cytogenetics and Genetics Cancer / al. et Maire G. rearrangement with WCP 21 Tumor t(21;22)(q22;q12) Rearrangement of 21 and EWS and ERG NE NI Mugneret et al., 1996 [19] 22 by WCP 21 and WCP 22 rearrangement Tumor, I and met (MON) t(21;22)(q22;q12) ND PCR sequence NE NI Peter et al., 1996 [22] Tumor t(21;22)(q22;q12) Juxtaposition of WCP 21 ND NE NI Massey et al., 1996 [23] and WCP 22 Cell line (IARC-EW18) Complex karyotype with no WCP 22 and ERG cosmid RT-PCR sequence on der(22) Complex chromosomal rearrangement Desmaze et al., 1997 [20] clear evidence of rearranged (opposite orientation of each gene) Chr 21 or 22 Tumor No evidence of rearranged Chr ND RT-PCR sequence NE Complex chromosomal rearrangement Stark et al., 1996 [21] 21 or 22 (opposite orientation of each gene) Tumor (1296) No obvious rearrangement of EWS and ERG rearrangement RT-PCR sequence on der(21) Inversion of 22q12.2 that brakes EWS, Kaneko et al., 1997 [24] Chr 21 and 22 reciprocal translocation with 21q22.2 and fusion with ERG Tumor t(15;22)(p11;q12) EWS rearrangement RT-PCR sequence NE Complex chromosomal rearrangement Minoletti et al., 1998 [25] (opposite orientation of each gene) Tumor der(20)?t(20;21)(q1;q11)? WCP 21 and WCP 22. 21 and RT-PCR sequence NE NI Tan et al., 2001 [27] t(21;22)(q22;q12),add 22 are rearranged, but (21)(q11),add(22)(q12) juxtaposition could not be established, as no co-hybridization, with WCP, was done.

Tumor (no. 1) Apparently normal Chr 21 Rearrangement of EWS and RT-PCR sequence on der(22) Insertion of ERG within EWS,no Hattinger et al., 2001 [26] e and 22 ERG (fiber FISH) insertion of EWS within Chr 21 92 Tumor (no. 2) Apparently normal Chr 21 Rearrangement of EWS and ERG RT-PCR sequence on der(22) Insertion of ERG within EWS,no [26] and 22 (fiber FISH) insertion of EWS within Chr 22 Tumor (no. 3) t(2;22)(p25;q12) Rearrangement of EWS and ERG RT-PCR sequence on der(22) Insertion of ERG and distal sequences [26] (fiber FISH) within EWS,30EWS translocated to 2p25 Tumor der(22)ins(21)(q21~q22) WCP 21 and WCP 22 juxtaposed ND NE NI Lee et al., 2005 [28] possibly and EWS rearranged Cell line (TTC-466) t(21;22)(q22;q12),þder(22)t EWS rearrangement, WCP 21 RT-PCR sequence on der(21) Insertion, duplication and inversion of Sorensen et al., 1994 [35]; (21;22)(q22;q12) and WCP 22 juxtaposed the EWS gene within the der(21) Szuhai et al., 2006 [29] Cell line (EW3) der(15)t(15;22)(q21;q12), EWS rearrangement (break-apart ND on der(21) Insertion of 50EWS within der(21), Szuhai et al., 2006 [29] der(21)(21pter/q22:: probes), WCP 21 and WCP 22 translocation of 30EWS to Chr 15 22q12::21q22/qter),der and WCP 15 (the rearrangements (22)t(15;22)(q21;q12) observed are described in the previous column) 89 Abbreviations: Chr, chromosome; FISH, fluorescence in situ hybridization; I, primary tumor; met, metastasis; ND, no data; NE, not elucidated; NI, not investigated; PCR, polymerase chain reaction; RT-PCR, reverse transcriptaseepolymerase chain reaction; WCP, whole chromosome painting probe; YAC, yeast artificial chromosome. 90 G. Maire et al. / Cancer Genetics and Cytogenetics 181 (2008) 81e92 evolution of chromosomal abnormalities during progres- translocation between a normal chromosome 19 and the sion can be assessed. Giovannini et al. [17] were the first to der(22) was identified. The involvement of a third chromo- hypothesize that the EWSeERG rearrangement was likely some associated with rearrangement of EWS has previously to be more complex than the simple reciprocal t(21;22) been described in ES, either with EWSeFLI1 or EWSe translocation proposed in the initial description [35], be- ERG, but none of those cases exhibited a chromosome 19 cause of the orientation requirements for an in-frame fu- rearrangement [26,29,35,40e44]. In fact, chromosome 19 sion. Later more detailed analyses, with locus-specific is rarely involved in ES tumors [3]. Only two cases showed FISH probes or fiber FISH experiments, demonstrated the rearrangement within the 19q13 cytoband, one involving complexity of the EWSeERG rearrangement [24,26,29]. 1p13 and one involving 1q10 [20,45]. The 19q13 cytoband Indeed, at least five cases (three tumors and two cell lines) has been lost or gained in six tumors [13,15,16,40,46,47]. were positive for RT-PCR detection of EWSeERG fusion In the present study, identical der(19), der(21), and transcripts, but companion R- or G-banding analyses failed der(22) abnormalities were found in the primary tumor, to show any evidence of rearrangement of chromosome 21 the cervical node metastasis, and the derived cell line, sug- [17,18,20,21]. gesting that secondary rearrangements were not acquired The description of the mechanisms leading to the forma- during either the tumor dissemination or the in vitro trans- tion of the EWSeERG fusion gene showed that the EWS formation. Therefore, the physiological consequences of gene can be broken by a paracentric inversion of 22q12.2 the characterized rearrangements are most likely due to [24], or by a translocation with a chromosome other than the presence of the oncogenic fusion gene EWSeERG chromosome 21 [26,29]. The fusion with the 30ERG has [3]. The absence of any known gene in the 19q13.12 break- been described as the result of an inverted insertion on point interval supports the idea of a secondary, or passen- 22q12.2 [20,26], or the insertion of either the EWS full ger, role of the der(19) abnormality. length (associated with an inverted duplication) or the Finally, during the in vitro transformation process, char- 50EWS end insertion within chromosome 21 [24,29].As acterized by the loss of RB1 and polyploidization, the a result, the EWSeERG fusion gene has been observed der(22) carrying the EWSeERG fusion gene was preserved on the der(21) or alternatively on the der(22). The fusion from loss in the context of a pseudo-tetraploid composite gene was first identified by an apparent t(21;22) reciprocal karyotype. This demonstrates that even after the in vitro translocation, but no study has yet demonstrated the con- transformation process, the EWSeERG fusion gene is still comitant exchange of material between chromosome 21 the major determinant for unlimited growth of these cells. and 22. Either EWS or ERG translocates to the other chro- mosome by an inversioneinsertion mechanism. With the present study, we have observed for the first Acknowledgments time a reciprocal insertioneinversion mechanism that is a true equivalent of a balanced reciprocal translocation G.M. is the recipient of the Helena Lam award. This (Fig. 4). Having characterized the breakpoints within chro- study was supported by the National Cancer Institute of mosome 21 and 22, we have proposed a step-by-step recip- Canada with funds from the Canadian Cancer Society. rocal mechanism that generated the EWSeERG fusion gene. During our analyses, we precisely identified the two breakpoints necessary for the excision of the 30ERG end References from chromosome 21, and the proximal breakpoint within [1] Enzinger FM, Weiss SW. Soft tissue tumors. 3rd ed. St. Louis: Mos- 0 chromosome 22 for the excision of the 3 EWS. The distal by, 1995. breakpoint on chromosome 22 is thought to be located [2] Bernstein M, Kovar H, Paulussen M, Randall RL, Schuck A, ~200 kb away from the telomeric side of the EWS gene. Teot LA, Juergens H. Ewing’s sarcoma family of tumors: current e This mechanism bears some similarity to homologous management. Oncologist 2006;11:503 19. [3] Sandberg AA, Bridge JA. 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