Numerical and Structural Chromosomal Anomalies in Undifferentiated Pleomorphic Sarcoma

ANTICANCER RESEARCH 34: 7119-7128 (2014)

MUSTAFA BECERIKLI1, STEFAN WIECZOREK2, INGO STRICKER3, SANDEEP NAMBIAR3, ANDREA RITTIG1, JOERG THOMAS EPPLEN2, ANDREA TANNAPFEL3, MARCUS LEHNHARDT1, LARS STEINSTRAESSER1* and FRANK JACOBSEN1*

1Department of Plastic Surgery, BG University Hospital Bergmannsheil, Ruhr-University Bochum, Bochum, Germany; 2Department of Human Genetics, Ruhr-University Bochum, Bochum, Germany; 3Institute of Pathology, Ruhr-University Bochum, Bochum, Germany

Abstract. Background: Malignant fibrous histiocytoma include liposarcomas, leiomyosarcomas, synovial sarcomas, (MFH) or undifferentiated pleomorphic sarcoma (UPS) is the malignant peripheral nerve sheath tumors (MPNST) and most common soft-tissue sarcoma of late adult life. Further malignant fibrous histiocytomas (MFH), also referred to as advances in genetic characterization are warranted. The aim undifferentiated pleomorphic sarcomas (UPS) (3, 4). Since of this study was to search for numerical and structural STS often show highly aggressive potential, the overall five- chromosomal anomalies in UPS. Materials and Methods: We year survival rate is approximately 50% (5). Surgical investigated five sarcoma-specific chromosomal translocations, resection with free margins and adjuvant radiotherapy five oncogene amplifications as well as the numerical represents the current gold standard for STS therapy. Yet, karyotype of 19 UPS samples and one UPS/MFH cell line morbidity and mortality remain comparatively high and (U2197) using FISH probes on interphase nuclei. Results: Our severe late-effects occur quite often (6-8). results demonstrate that chromosomal translocations involving Although it is assumed that sarcomas arise due to CHOP, SYT, EWS, FUS and FKHR genes are absent. chromosomal aberrations and/or mutations in mesenchymal Furthermore, amplification of ERBB2 (10.5%) and MDM2 progenitor cells, the exact cellular origin of most of these (10.5%) was observed whereas the EGFR, C-MYC and N- tumors remains elusive (9). With regard to genetic MYC genes were not amplified. Interestingly, predominant abnormalities sarcomas are divided into two classes: The first aneuploidies were found in eight chromosomes. Conclusion: class encompasses tumors that have specific genetic mutations, The data demonstrate rarity of sarcoma-specific chromosomal simple karyotypes and translocations potentially resulting in breaks and oncogene amplifications in UPS, yet polysomic the formation of fusion genes. The second class comprises of chromosomes appear more characteristically in this condition. sarcomas without specific mutations and chromosomal aberrations, but comprising complex karyotypes with numerous Soft tissue sarcomas (STS) are malignant tumors of genetic gains and losses with no specific pattern (10). For the mesenchymal origin. With approximately 11,280 new cases sarcoma of the first class, various fusion genes, which represent diagnosed annually in the United States they comprise less a characteristic feature for the appropriate entity, have been than 1% of all malignancies (1, 2). Nevertheless, STS described over the years. Fusion genes like SS18-SSX1 and comprise a large heterogeneous group with more than 50 EWS-FLI1 are specifically linked to synovial sarcoma and diagnostic entities described, the more common entities Ewing’s sarcoma/Primitive Neuroectodermal Tumor (PNET), respectively (11, 12). Some further sarcoma-specific chromosomal break regions are present in the CHOP gene in myxoid liposarcoma (13, 14) and FKHR in alveolar *These Authors contributed equally and are named in alphabetical order. rhabdomyosarcoma (15). Recently, methods such as fluorescence in situ-hybridization (FISH) have been established Correspondence: Mustafa Becerikli, Department of Plastic and to screen for known fusion genes (16). The resulting fusion Reconstructive Surgery, BG University Hospital Bergmannsheil, proteins do not interact with the same interaction partners like Ruhr University Bochum, Germany. Tel: +49 2343024787, Fax: +49 2343024790, e-mail: [email protected] the physiological wild- type proteins (17). This can alter the regulation of key proteins or transcription factors in tumor Key Words: Chromosome aberrations, aneuploidy, gene amplification, cells. Consequently, an altered expression profile can result, sarcoma, fluorescent in situ hybridization. which may contribute to tumorigenesis. Accordingly, it has

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Table I. Clinical and histopathological data of the UPS study cohort (n=19).

Case no. # Age/Gender Grade Karyotype Histopathological type

1 41/F 3 Complex Without further differentiation 2 41/F 3 Complex Pleomorphic 3 63/M 3 Complex Storiform pleomorphic 4 67/M 3 Near diploid Without further differentiation 5 77/F 3 Complex Pleomorphic 6 52/F 3 Complex Myxoid 7 90/F 3 Complex Pleomorphic 8 85/M 3 Complex Pleomorphic 9 69/M 3 Near diploid Without further differentiation 10 50/M 3 Near diploid Pleomorphic 11 45/M 3 Complex Without further differentiation 12 82/M 2 Near diploid Without further differentiation 13 60/F 3 Near diploid Storiform pleomorphic 14 63/F 3 Complex Pleomorphic 15 74/M 3 Complex Pleomorphic 16 71/F 2 Near diploid Pleomorphic 17 56/F 3 Complex Storiform pleomorphic 18 79/F 2 Complex Storiform pleomorphic 19 78/M 2 Complex Pleomorphic

been found that the SS18-SSX1 fusion protein reduces the breaks, which have already been found in different sarcoma tumor-suppressive function of p53 by stabilizing its negative entities (as described above). Secondly, we explored the regulator MDM2 (18). This example demonstrates the amplification of five genes that harbor tumorigenic potential complexity of genetic alterations in relation to the mechanisms in diverse neoplasms. Finally, we determined the numbers of of regulation. The discovery of particular chromosomal breaks chromosomes in several cell nuclei of UPS cells. and translocations is significant - not only for the development of new therapeutic strategies, but also for the confirmation of Materials and Methods diagnosis and pathological findings. Another important molecular genetic feature of cancer cells Ethics statement. The participants gave their written informed concerns copy number gains and amplifications of proto- consent, and the study was reviewed and approved by the ethical oncogenes like the transcription factor C-MYC which binds committee of the BG University Hospital Bergmannsheil, Ruhr- University Bochum, Germany with the registration number 3974- to promoters and regulates up to 15% of all genes (19). C- 11. Experiments comply with the current laws of Germany. MYC is amplified in diverse malignancies like in breast and prostate cancer (20, 21). Amplification of further factors as N- Cell culture. The human MFH/uUPS cell line U2197 obtained from MYC and the receptor tyrosine kinases ERBB2 and EGFR German Collection of Microorganisms and Cell Cultures (DSMZ), have also been described in various malignancies like Braunschweig, Germany was authenticated via DNA (STR) neuroblastoma, breast cancer and gliomas, respectively (22- profiling by the DSMZ in November 2013. Cells were grown in 24). The oncogene MDM2 is an inhibitor of apoptosis, whose MEM supplemented with 20% FCS (Thermo Fisher Scientific Inc., Waltham, MA, USA), 0.165% sodium bicarbonate (PAA amplification has been shown in sarcomas (25). Amplification laboratories, Pasching, Austria) and 1% penicillin/streptomycin or translocation of such genes could result in altered gene (PAA laboratories). The primary human undifferentiated sarcoma expression profile and consequently favor oncogenesis. samples were harvested from patients’ tissues at the BG University Profound knowledge of the origin and pathogenesis of STS Hospital Bergmannsheil (Table 1). This study was approved by the is missing, and new insights or diagnostic characteristics are local Ethics Committee and all of the patients gave written informed necessary. For a better understanding of the molecular consent. Cell cultures were obtained from freshly received tumor mechanisms, intensive molecular genetic analysis is required, resections by separation of the cells using Collagenase type II (Worthington Biochemical Corporation, Lakewood, NJ) as which is difficult due to the rarity and the large number of previously described (26). Cultures were maintained at 37˚C in a different histological subtypes of STS. To further investigate humidified 5% CO2 atmosphere. the genetics of UPS we examined three types of chromosomal aberrations in 19 UPS samples and one UPS/malignant fibrous FISH probes. All probes were obtained from Kreatech (Kreatech, histiocytoma cell line (U2197) by the use of specific FISH Amsterdam, Netherlands). All used probes are listed in Tables V- probes: First, we tried to identify five specific chromosomal VII. 100 nuclei of each UPS sample and the U2197 cell line were

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Table II. Presence of chromosomal breaks per hundred nuclei of each Table III. Prevalence of gene amplifications in UPS. UPS sample. Gene amplification Genes affected by break Sample EGFR ERBB2 MDM2 C-MYC N-MYC Sample CHOP SYT EWS FUS FKHR UPS #1 - - - - - UPS #1 - - - - - UPS #2 - - + - - UPS #2 - - - - - UPS #3 - - - - - UPS #3 - - - - - UPS #4 - - - - - UPS #4 - - - - - UPS #5 - - - - - UPS #5 2/100 - - 1/100 - UPS #6 - - - - - UPS #6 - - - - - UPS #7 - - - - - UPS #7 - - - - - UPS #8 - - - - - UPS #8 2/100 - - - - UPS #9 - - - - - UPS #9 8/100 - - - - UPS #10 - - - - - UPS #10 - - - - - UPS #11 - + - - - UPS #11 - - - - - UPS #12 - - - - - UPS #12 - - - - - UPS #13 - - - - - UPS #13 - 1/100 - - - UPS #14 - + + - - UPS #14 - - 4/100 - - UPS #15 - - - - - UPS #15 - - - - - UPS #16 - - - - - UPS #16 4/100 - 2/100 - - UPS #17 - - - - - UPS #17 - - - - - UPS #18 - - - - - UPS #18 - 1/100 - - - UPS #19 - - - - - UPS #19 - - - - - Prevalence 0/19 2/19 2/19 0/19 0/19 Percentage 0 10.5 10.5 0 0

analyzed. For comparison and validation each probe was Table IV. Prevalence of chromosome-specific trisomy, pentasomy and additionally tested on 100 nuclei of fibroblasts from a non-sarcoma hexasomy in UPS. patient defined as "healthy control". Chromosome Trisomy Pentasomy Hexasomy Preparation of samples for FISH. Sample preparation and the following FISH analysis were performed by standard procedures. 1 2/19 2/19 - Briefly, when nearly reaching confluency, cells were trypsinized and 3 4/19 - - harvested in Carnoy’s fixative (3:1 methanol to glacial acetic acid). 4 3/19 - 1/19 The pellet was dripped onto the cold, wet slides, air-dried and pre- 7 2/19 1/19 1/19 treated in 2x SSC prior to hybridization. 8 4/19 - 1/19 11 2/19 - - 12 2/19 - 1/19 Hybridization of FISH compounds by co-denaturation. FISH probe 17 1/19 1/19 1/19 mixtures were applied according to the manufacturer’s protocols (Kreatech, Amsterdam, The Netherlands). Samples were denatured for 5 min at 75˚C in a HYBrite hybridization system (Medcompare, South San Francisco, CA, USA), followed by hybridization of 12-16 h at 37˚C. After hybridization, slides were immediately incubated in 0.4x hundred nuclei of each sample were analyzed by break-apart SSC/0.3% NP40 at 72˚C for 120 sec and afterwards in 2x SSC/0.1% NP40 for 60 sec at room temperature and air dried. After addition of FISH probes. A chromosomal break was assumed when 5-7 μl DAPI solution, slides were evaluated by fluorescence signals were separated by more than the 2-fold of the signal microscopy with the appropriate image processing software (Isis; diameter. Based on the experience from routine diagnostics, Metasystems, Altlussheim, Germany). where among other things the same probes are used, the presence of chromosomal breaks in >10% nuclei was used Results as a cut-off to categorize the sample as positive for the respective chromosomal break. The analyses revealed that all Absence of special chromosomal breaks. We investigated the 19 UPS samples, U2197 cells and normal human fibroblasts distribution of five sarcoma-specific chromosomal breaks (in were negative for chromosomal breaks in the CHOP, SYT, the CHOP, SYT, EWS, FUS and FKHR genes), in 19 UPS EWS, FUS and FKHR genes (Figure 1A, B, C, D and E). samples, U2197 cells and normal human fibroblasts. One Among the negative samples, CHOP (4/19), SYT (2/19),

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Figure 1. Absence of translocations in UPS. In the slide preparations DNA was denatured and hybridized with appropriate two-color FISH probes; signals were evaluated by fluorescence microscopy. Chromosomal breaks were absent in CHOP (A) SYT (B) EWS (C) FUS (D) FKHR (E) Negligible numbers (1-8%) of the nuclei exhibited chromosomal breaks in the CHOP (F) SYT (G) EWS (H) FUS (I) genes. The red and green signals are spatially dissociated as indicated by arrowheads for chromosomal breaks.

Figure 2. Gene amplifications in UPS. In the slide preparations DNA was denatured and hybridized with appropriate two-color FISH probes; signals were evaluated by fluorescence microscopy. Gene amplification was not demonstrable in EGFR (A), yet present in ERBB2 (B) and MDM2 (C) yet absent in C-MYC (D) and N-MYC (E). The centromere- and gene-specific probes exhibit green or red fluorescence signals, respectively.

EWS (2/19) and FUS (1/19), showed negligible nuclei (1- FISH probes, and the presence of duplication/amplification in 8%) with putative chromosomal breaks (signals more than 2 >10 % nuclei were used as a cut-off to categorize the sample as diameters apart) where as FKHR exhibited no chromosomal positive for the respective gene. ERBB2 and MDM2 break at all (Figure 1F, G, H and I). The chromosomal breaks amplifications were prevalent in 2 of the 19 samples, each in a few nuclei (1-8%) were below the cut-off value, applied (Table III). No amplification of EGFR, C-MYC and N-MYC to clinical diagnostics of tumor entities (>10% of nuclei). was observed. Representative images of amplifications are Such chromosomal breaks/translocations were neither found shown in Figure 2. EGFR gene amplification was observed in in the U2197 cell line nor in the healthy control. The results U2197 cells. Normal human fibroblasts showed no are summarized in Table II. Thus, UPS appear characterized amplification in any of the 5 genes. by an absence of chromosomal breaks on CHOP, SYT, EWS, FUS and FKHR genes. Presence of chromosome-specific aneuploidy in UPS. In order to address whether UPS are characterized by Prevalence of ERBB2 and MDM2 amplification. We aneuploidy or specific chromosomal gains, numerical investigated the prevalence of amplification of five potential karyotype of all samples were established. In normal oncogenes (EGFR, ERBB2, MDM2, C-MYC and N-MYC). One human fibroblasts, UPS and U2197 disomy and tetrasomy hundred nuclei of each sample were analyzed using dual-color were observed (Figure 2A). As normal cells are diploid and

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Figure 3. Numerical karyotyping of fibroblasts, UPS and U2197 cells. In the slide preparations DNA was denatured and hybridized with appropriate two-color FISH probes; signals were evaluated by fluorescence microscopy. The bar graphs represent percentage of various ploidies.

natural DNA replication in the S phase can exhibit tetrasomy, i.e. 2 chromatids of each chromosome (2n4c), it is indistinguishable from disomy with pathological rearrangements and tetraploidy (4n4c). Hence we ignored chromosomes with 2 and 4 copies from the analysis. In normal human fibroblasts, the presence of DNA ploidies other than disomy and tetrasomy were negligible (see below). In sharp contrast, UPS and U2197 conspicuously exhibited abnormal ploidies (Figure 3). In UPS cells and U2197 cells, the most noticeable increase in aneuploidy was trisomy in 5.1% and 20.9%, respectively, in comparison to a negligible 0.6% in normal human fibroblasts. Also, increased rates of pentasomy (p) and hexasomy (h) were observed in UPS cells (p=1%, h=1.7%) Figure 4. Percentage of the different chromosome dosages of fibroblasts, and U2197 cells (p=8%, h=4.1%) in comparison to a UPS and U2197 cells. In the slide preparations DNA was denatured and negligible percentage in normal human fibroblasts (p=0%, hybridized with appropriate two-color FISH probes; signals were h=0.1%). Furthermore, 13/19 UPS samples displayed evaluated by fluorescence microscopy. The bar graphs represent highly complex karyotypes whereby each chromosome was individual chromosome-specific aneuploidy. Table IV summarizes the present in five to ten copies in more than 4% of the nuclei. most frequent aneuploidies. In contrast to the constant karyotype of fibroblasts, in UPS, eight chromosomes exist in more than three copies In U2197 cells each chromosome was present in five to ten in excess of 10% of cell nuclei with the most frequent aneuploidy copies in almost 16% of the nuclei characteristic for a affecting the chromosome 8. U2197 cells exhibited an inhomogeneous deteriorated karyotype. number of nuclei with diverse chromosomal imbalances.

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We also investigated individual chromosome-specific activity or inactivation of transcriptional suppressors, need aneuploidy in fibroblasts, UPS and U2197 cells. The to be considered as plausible mechanisms of EGFR fibroblasts showed a constant karyotype. None of the overexpression in MFH. Interestingly, ERBB2 and MDM2 chromosomes existed in more than three copies in excess of genes demonstrate amplification in UPS. In 10.5% (2/19) 10% of cell nuclei. For each chromosome only 1-2% of UPS studied, the number of signals for ERBB2 (17q12) and nuclei showed a chromosomal copy number gain. In sharp MDM2 (12q15) was greater than the number of signals for contrast, UPS exhibited copy number changes in different centromeres of chromosome 17 and chromosome 12, chromosomes, namely 1, 3, 4, 7, 8, 11, 12, 17 (Figure 4, respectively. In addition, chromosome 12 and 17 also Table IV) in excess of 10% of cell nuclei. Chromosomes 1, exhibited conspicuous gains of copy numbers. The elevated 3, 4, 7, 8, 11, 12 and 17 were found trisomic in 5.3% to dosage of ERBB2 and MDM2 in UPS may result from 21.1% (4/19) UPS, respectively, in >10% of nuclei. complex chromosomal rearrangements or, both, individual Chromosomes 4, 7, 8, 12 and 17 exhibited hexasomy in gene amplification as well as copy number gain of 5.3% (1/19) UPS in >10% of nuclei. Chromosomes 1, 7, and chromosome 17 and 12. Such increases in gene dosage are 17 demonstrated pentasomy in 10.5% (2/19), 5.3% (1/19) consistent with the hypothesis that gene amplifications and 5.3% (1/19) UPS in >10% of nuclei. The most frequent confer growth advantages (31, 32). Momand et al. aneuploidies in UPS patients are summarized in Table IV. In investigated MDM2 amplification in 3889 samples of 28 U2197 cells, twelve different chromosomes appeared in more tumor types from previously published sources and reported than three copies with nine of these chromosomes existing a 7% overall frequency of MDM2 amplification. MDM2 in excess of 90% of nuclei. amplification was observed in 19 tumor types with the highest frequency observed in soft tissue tumors (20%) (33). Discussion Interestingly, 21% among the 163 MFH exhibited MDM2 amplification (33). Recently, a small-molecule MDM2 We investigated numerical and structural chromosome antagonist has been described as a possible therapy, and anomalies in undifferentiated pleomorphic sarcoma and MDM2 overexpressing sarcomas yielded good responses to examined five special chromosomal breaks, five potential the respective inhibitor (34, 35). Despite these advances, gene amplifications as well as performed numerical further studies on amplification mechanisms, and in interphase karyotyping in UPS and the UPS/MFH cell line particular, about the initiating processes of gene (U2197). Chromosomal breaks/translocations in the genes amplification are certainly warranted. CHOP, SYT, EWS and FKHR are characteristic for myxoid Sarcomas display multiple, complex karyotypic liposarcoma (13, 14), synovial sarcomas (11), Ewing’s abnormalities, and these genetic alterations are an important sarcoma/PNET(12) and alveolar rhabdomyosarcoma(15), adjunct to standard morphological and immunohistochemical respectively. Our study demonstrates that UPS exhibit no diagnoses (36-39). Consistent with our observation of chromosomal breaks of the CHOP, SYT, EWS, FUS and aneuploidy in UPS, independent cytogenetic studies in MFH FKHR genes detectable by standard FISH probes. Absence mention also aneuploidy (36-39). In the current analysis, we of CHOP rearrangements has been independently reported in omitted to include disomy and tetrasomy, as normal cells are MFH (27). We propose that although the above-mentioned diploid and natural DNA replication in the S phase can five different breaks/translocations may be relevant in the pretend tetraploidy, making pathological diploidy and pathogenesis of other sarcoma entities, they play no critical tetraploidy indistinguishable from normal disome and role in the pathogenesis of UPS. To date, 41 gene fusions tetrasome phases of the cell. Thereafter, the most have been described in 17 different sarcoma types (28), but conspicuous copy number gains that emerged were in no specific chromosomal translocation has been reported in specific chromosomes namely trisomy (1, 3, 4, 7, 8, 11, 12, UPS. The present study reinforces this observation (29). 17), pentasomy (1, 7, 17) and hexasomy (4, 7, 8, 12, 17). Of EGFR, ERBB2, MDM2, C-MYC and N-MYC gene these, the copy number gains in chromosomes 1, 7, 8 in amplifications have been reported in various cancers. Our MFH has been independently reported by Tarkkanen et al. study demonstrates that UPS exhibit no EGFR, C-MYC and using comparative genomic hybridizations (CGH) (40). In N-MYC amplifications. EGFR has been reported to be over- our study, on average each chromosome exhibited 3-10 copy expressed in MFH (30), and the EGFR gene amplification in gains in ~10% of UPS nuclei. Chromosomal mis-segregation UPS was analyzed to explain high EGFR expression levels. in normal diploid cells occurs generally below 1% (reviewed We observed no EGFR amplification in UPS (yet exclusively in (41)), as observed in our results from normal human in U2197 cell line). Therefore, we conclude that the reported fibroblasts. Not surprisingly, the numerical aberrations in the over-expression of EGFR in UPS/MFH cannot be UPS/MFH cell line U2197 was larger than in the UPS substantiated via gene amplification/dosage events. Hence, samples. In U2197 cells, each chromosome was on average other regulatory mechanisms, like elevated transcriptional present in 3-10 copies in almost 36.8% of the nuclei. During

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Table V. FISH probes used in the detection of chromosomal breaks. C-MYC copy number (49) and its availability to bind and regulate up to 15% of all genes (19). Although chromosome Probe Catalogue number 8 exhibited trisomy and hexasomy in UPS, the number of ON EWSR1 (22q12) Break KBI-10708 signals for 8q24 (the locus of C-MYC) was similar to the ON SYT (18q11) Break KBI-10713 number of signals for centromeres of chromosome 8 ploidy. ON CHOP (12q13) Break KBI-10714 Therefore, our data may indicate that elevation of C-MYC ON FUS (16p11) Break KBI-10715 protein in MFH may be achieved via copy number gain of ON FKHR (13q14) Break KBI-10716 the entire chromosome 8 and not through individual C-MYC amplifications (40). Other genes involved in cell growth, motility and survival located on chromosome 8q and Table VI. FISH probes used to investigate gene amplifications. reported in various cancer entities are SNAI2, PLAG1, Sulf1, CTHRC1, ENPP2 and ASAP1. Therefore their role in UPS Probe Catalogue number pathogenesis needs further evaluation. ON C-MYC (8q24)/SE 8 KBI-10106 UPS cells lack specific chromosomal breaks but are ON MYCN (2p24)/LAF (2q11) KBI-10706 characterized by ERBB2 and MDM2 amplification and ON EGFR, Her-1 (7p11)/SE 7 KBI-10702 specific polysomies. In the current study we observed no ON ERBB2, Her2/Neu (17q12)/SE 17 KBI-10701 correlation between these structural and numerical aberrations ON MDM2 (12q15)/SE 12 KBI-10717 and the histological subtype or tumor grading. This observation is consistent with the report of the chromosomes and morphology (20) study group, that a differential diagnostic sub-classification of pleomorphic sarcomas by Table VII. FISH probes used for determining chromosome copy number. means of cytogenetic analysis is implausible and that the Probe Fluorescence Label Catalogue number karyotype could not be used to predict clinical outcome (50). Metaphase analyses and array CGH may be useful tools to SE 1 (1qh) Red KBI-20001 further elucidate clonal chromosomal changes in UPS. SE 2 (D2Z2) Red KBI-20002 SE 3 (D3Z1) Red KBI-20003 SE 4 (D4Z1) Red KBI-20004 Acknowledgements SE 7 (D7Z1) Red KBI-20007 SE 8 (D8Z1) Red KBI-20008 The Authors would like to thank Viktoria Albrecht for her expert SE 9 (classical) Green KBI-20009 technical assistance. This work was supported by the Medical SE 10 (D10Z1) Green KBI-20010 Faculty of the Ruhr-University Bochum (FORUM: F667N-2010). SE 11 (D11Z1) Green KBI-20011 SE 12 (D12Z3) green KBI-20012 SE 15 (D15Z4) Green KBI-20015 References SE 16 (D16Z2) Blue KBI-20016 SE 17 (D17Z1) Blue KBI-20017 1 Siegel R, Naishadham D and Jemal A: Cancer statistics, 2012. SE 18 (D18Z1) Blue KBI-20018 CA Cancer J Clin 62: 10-29, 2012. SE 20 (D20Z1) Blue KBI-20020 2 Rubin BP, Fletcher CD, Inwards C, Montag AG, Peabody T, Qualman SJ, Rosenberg AE, Weiss S and Krausz T: Protocol for the examination of specimens from patients with soft tissue tumors of intermediate malignant potential, malignant soft tissue a long period of cell cultivation, cell lines may progressively tumors, and benign/locally aggressive and malignant bone tumors. Arch Pathol Lab Med 130: 1616-1629, 2006. “evolve” in several aspects including morphology, vitality, 3 Coindre JM, Terrier P, Guillou L, Le Doussal V, Collin F, genetics and/or epigenetics (42). Ranchere D, Sastre X, Vilain MO, Bonichon F and N’Guyen Bui Among the chromosomal gains reported in our study, gain B: Predictive value of grade for metastasis development in the of chromosome 8 has been earlier reported in prostate cancer main histologic types of adult soft tissue sarcomas: a study of (21) and gastric cancer (43), gains of chromosomes 8 and 12 1240 patients from the French Federation of Cancer Centers in Ewing’s sarcoma (44) and gains of chromosomes 8, 12 Sarcoma Group. Cancer 91: 1914-1926, 2001. and 17 in stage c colon cancer (45). Additionally, gain of the 4 Matushansky I, Charytonowicz E, Mills J, Siddiqi S, Hricik T q-arm of chromosome 8 has also been reported in a wide and Cordon-Cardo C: MFH classification: differentiating undifferentiated pleomorphic sarcoma in the 21st Century. variety of cancers like breast, head and neck, gastric and Expert Rev Anticancer Ther 9: 1135-1144, 2009. pancreatic cancer (43, 46-48). Such numerical abnormalities 5 Kotilingam D, Lev DC, Lazar AJ and Pollock RE: Staging soft of chromosome 8, on which C-MYC is located, has been tissue sarcoma: evolution and change. CA Cancer J Clin 56: suggested as an important mechanism in the increase of the 282-291; quiz 314-285, 2006.

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