Osteosarcoma (OS) is characterized by an unstable karyotype which typically has a heterogeneous pattern of complex chromosomal abnormalities. High-resolution array comparative genomic hybridization (CGH) in combination with interphase fluorescence in situ hybridization (FISH) analyses provides a complete description of genomic imbalances together with an evaluation of the contribution of cell-to-cell variation to copy number changes. There have been no analyses to date documenting genomic signatures consistent with chromosomal instability mechanisms in OS tumors using array CGH. In this study, we utilized high-resolution array CGH to identify and characterize recurrent signatures of genomic imbalances using ten OS tumors. Comparison between the genomic profiles identified tumor groups with low, intermediate and high levels of genomic imbalance. Bands 6p22→p21, 8q24 and 17p12→ p11.2 were consistently involved in high copy gain or amplification events. Since these three locations have been consistently associated with OS oncogenesis, FISH probes from each cytoband were used to derive an index of cellular heterogeneity for copy number within each region. OS with the highest degree of genomic imbalance also exhibited the most extreme cell-to-cell copy number variation. Significantly, the three OS with the most imbalance and genomic copy number heterogeneity also had the poorest response to preoperative chemotherapy. This genome wide analysis is the first utilizing oligonucleotide array CGH in combination with FISH analysis to derive genomic signatures of chromosomal instability in OS tumors by studying genomic imbalance and intercellular heterogeneity. This comprehensive genomic screening approach provides important insights concerning the mechanisms responsible for generating complex genomes. The resulting phenotypic diversity can generate tumors with a propensity for an aggressive disease course. A better understanding of the underlying mechanisms leading to OS tumor development could result in the identification of prognostic markers and therapeutic targets.

1.
Ashton KJ, Weinstein SR, Maguire DJ, Griffiths LR: Molecular cytogenetic analysis of basal cell carcinoma DNA using comparative genomic hybridization. J Invest Dermatol 117:683–686 (2001).
2.
Atiye J, Wolf M, Kaur S, Monni O, Bohling T, et al: Gene amplifications in osteosarcoma-CGH microarray analysis. Genes Chromosomes Cancer 42:158–163 (2005).
3.
Bayani J, Zielenska M, Pandita A, Al-Romaih K, Karaskova J, et al: Spectral karyotyping identifies recurrent complex rearrangements of chromosomes 8, 17, and 20 in osteosarcomas. Genes Chromosomes Cancer 36:7–16 (2003).
4.
Bayani J, Selvarajah S, Maire G, Vukovic B, Al-Romaih K, et al: Genomic mechanisms and measurement of structural and numerical instability in cancer cells. Semin Cancer Biol 17:5–18 (2007).
5.
Bech-Otschir D, Kraft R, Huang X, Henklein P, Kapelari B, et al: COP9 signalosome-specific phosphorylation targets p53 to degradation by the ubiquitin system. EMBO J 20:1630–1639 (2001).
6.
Bridge JA, Nelson M, McComb E, McGuire MH, Rosenthal H, et al: Cytogenetic findings in 73 osteosarcoma specimens and a review of the literature. Cancer Genet Cytogenet 95:74–87 (1997).
7.
Castellano MM, Sanz-Burgos AP, Gutierrez C: Initiation of DNA replication in a eukaryotic rolling-circle replicon: identification of multiple DNA-protein complexes at the geminivirus origin. J Mol Biol 290:639–652 (1999).
8.
Chen D, Gallie BL, Squire JA: Minimal regions of chromosomal imbalance in retinoblastoma detected by comparative genomic hybridization. Cancer Genet Cytogenet 129:57–63 (2001).
9.
Ciullo M, Debily MA, Rozier L, Autiero M, Billault A, et al: Initiation of the breakage-fusion-bridge mechanism through common fragile site activation in human breast cancer cells: the model of PIP gene duplication from a break at FRA7I. Hum Mol Genet 11:2887–2894 (2002).
10.
Cloud JE, Rogers C, Reza TL, Ziebold U, Stone JR, et al: Mutant mouse models reveal the relative roles of E2F1 and E2F3 in vivo. Mol Cell Biol 22:2663–2672 (2002).
11.
Fioretos T, Strombeck B, Sandberg T, Johansson B, Billstrom R, et al: Isochromosome 17q in blast crisis of chronic myeloid leukemia and in other hematologic malignancies is the result of clustered breakpoints in 17p11 and is not associated with coding TP53 mutations. Blood 94:225–232 (1999).
12.
Forus A, Weghuis DO, Smeets D, Fodstad O, Myklebost O, Geurts van Kessel A: Comparative genomic hybridization analysis of human sarcomas: II. Identification of novel amplicons at 6p and 17p in osteosarcomas. Genes Chromosomes Cancer 14:15–21 (1995).
13.
Gisselsson D, Pettersson L, Hoglund M, Heidenblad M, Gorunova L, et al: Chromosomal breakage-fusion-bridge events cause genetic intratumor heterogeneity. Proc Natl Acad Sci USA 97:5357–5362 (2000).
14.
Gudmundsson J, Sulem P, Manolescu A, Amundadottir LT, Gudbjartsson D, et al: Genome-wide association study identifies a second prostate cancer susceptibility variant at 8q24. Nat Genet 39:631–637 (2007).
15.
Hellman A, Zlotorynski E, Scherer SW, Cheung J, Vincent JB, et al: A role for common fragile site induction in amplification of human oncogenes. Cancer Cell 1:89–97 (2002).
16.
Henriksen J, Aagesen TH, Maelandsmo GM, Lothe RA, Myklebost O, Forus A: Amplification and overexpression of COPS3 in osteosarcomas potentially target TP53 for proteasome-mediated degradation. Oncogene 22:5358–5361 (2003).
17.
Huppi K, Volfovsky N, Runfola T, Jones TL, Mackiewicz M, et al: The identification of microRNAs in a genomically unstable region of human chromosome 8q24. Mol Cancer Res 6:212–221 (2008).
18.
Knuutila S, Bjorkqvist AM, Autio K, Tarkkanen M, Wolf M, et al: DNA copy number amplifications in human neoplasms: review of comparative genomic hybridization studies. Am J Pathol 152:1107–1123 (1998).
19.
Kuwahara Y, Tanabe C, Ikeuchi T, Aoyagi K, Nishigaki M, et al: Alternative mechanisms of gene amplification in human cancers. Genes Chromosomes Cancer 41:125–132 (2004).
20.
Larramendy ML, Koljonen V, Bohling T, Tukiainen E, Knuutila S: Recurrent DNA copy number changes revealed by comparative genomic hybridization in primary Merkel cell carcinomas. Mod Pathol 17:561–567 (2004).
21.
Lau CC, Harris CP, Lu XY, Perlaky L, Gogineni S, et al: Frequent amplification and rearrangement of chromosomal bands 6p12-p21 and 17p11.2 in osteosarcoma. Genes Chromosomes Cancer 39:11–21 (2004).
22.
Lee JA, Carvalho CM, Lupski JR: A DNA replication mechanism for generating nonrecurrent rearrangements associated with genomic disorders. Cell 131:1235–1247 (2007).
23.
Lengauer C, Kinzler KW, Vogelstein B: Genetic instability in colorectal cancers. Nature 386:623–627 (1997).
24.
Lim G, Karaskova J, Vukovic B, Bayani J, Beheshti B, et al: Combined spectral karyotyping, multicolor banding, and microarray comparative genomic hybridization analysis provides a detailed characterization of complex structural chromosomal rearrangements associated with gene amplification in the osteosarcoma cell line MG-63. Cancer Genet Cytogenet 153:158–164 (2004).
25.
Lim G, Karaskova J, Beheshti B, Vukovic B, Bayani J, et al: An integrated mBAND and submegabase resolution tiling set (SMRT) CGH array analysis of focal amplification, microdeletions, and ladder structures consistent with breakage-fusion-bridge cycle events in osteosarcoma. Genes Chromosomes Cancer 42:392–403 (2005).
26.
Loeb KR, Loeb LA: Significance of multiple mutations in cancer. Carcinogenesis 21:379–385 (2000).
27.
Macris MA, Krejci L, Bussen W, Shimamoto A, Sung P: Biochemical characterization of the RECQ4 protein, mutated in Rothmund-Thomson syndrome. DNA Repair 5:172–180 (2006).
28.
Man TK, Lu XY, Jaeweon K, Perlaky L, Harris CP, et al: Genome-wide array comparative genomic hybridization analysis reveals distinct amplifications in osteosarcoma. BMC Cancer 4:45 (2004).
29.
Mann MB, Hodges CA, Barnes E, Vogel H, Hassold TJ, Luo G: Defective sister-chromatid cohesion, aneuploidy and cancer predisposition in a mouse model of type II Rothmund-Thomson syndrome. Hum Mol Genet 14:813–825 (2005).
30.
Masramon L, Vendrell E, Tarafa G, Capella G, Miro R, et al: Genetic instability and divergence of clonal populations in colon cancer cells in vitro. J Cell Sci 119:1477–1482 (2006).
31.
McClintock B: The stability of broken ends of chromosomes in Zea mays. Genetics 26:234–282 (1941).
32.
Ozaki T, Schaefer KL, Wai D, Buerger H, Flege S, et al: Genetic imbalances revealed by comparative genomic hybridization in osteosarcomas. Int J Cancer 102:355–365 (2002).
33.
Ozaki T, Neumann T, Wai D, Schafer KL, van Valen F, et al: Chromosomal alterations in osteosarcoma cell lines revealed by comparative genomic hybridization and multicolor karyotyping. Cancer Genet Cytogenet 140:145–152 (2003).
34.
Picci P, Sangiorgi L, Rougraff BT, Neff JR, Casadei R, Campanacci M: Relationship of chemotherapy-induced necrosis and surgical margins to local recurrence in osteosarcoma. J Clin Oncol 12:2699–2705 (1994).
35.
Prat E, Bernues M, Caballin MR, Egozcue J, Gelabert A, Miro R: Detection of chromosomal imbalances in papillary bladder tumors by comparative genomic hybridization. Urology 57:986–992 (2001).
36.
Reams AB, Neidle EL: Selection for gene clustering by tandem duplication. Annu Rev Microbiol 58:119–142 (2004).
37.
Saglio G, Storlazzi CT, Giugliano E, Surace C, Anelli L, et al: A 76-kb duplicon maps close to the BCR gene on chromosome 22 and the ABL gene on chromosome 9: possible involvement in the genesis of the Philadelphia chromosome translocation. Proc Natl Acad Sci USA 99:9882–9887 (2002).
38.
Saha S, Bardelli A, Buckhaults P, Velculescu VE, Rago C, et al: A phosphatase associated with metastasis of colorectal cancer. Science 294:1343–1346 (2001).
39.
Sangrithi MN, Bernal JA, Madine M, Philpott A, Lee J, et al: Initiation of DNA replication requires the RECQL4 protein mutated in Rothmund-Thomson syndrome. Cell 121:887–898 (2005).
40.
Santos GC, Zielenska M, Prasad M, Squire JA: Chromosome 6p amplification and cancer progression. J Clin Pathol 60:1–7 (2007).
41.
Saunders WS, Shuster M, Huang X, Gharaibeh B, Enyenihi AH, et al: Chromosomal instability and cytoskeletal defects in oral cancer cells. Proc Natl Acad Sci USA 97:303–308 (2000).
42.
Savelyeva L, Schwab M: Amplification of oncogenes revisited: from expression profiling to clinical application. Cancer Lett 167:115–123 (2001).
43.
Selvarajah S, Yoshimoto M, Park PC, Maire G, Paderova J, et al: The breakage-fusion-bridge (BFB) cycle as a mechanism for generating genetic heterogeneity in osteosarcoma. Chromosoma 115:459–467 (2006).
44.
Selvarajah S, Yoshimoto M, Maire G, Paderova J, Bayani J, et al: Identification of cryptic microaberrations in osteosarcoma by high-definition oligonucleotide array comparative genomic hybridization. Cancer Genet Cytogenet 179:52–61 (2007).
45.
Sharma S, Stumpo DJ, Balajee AS, Bock CB, Lansdorp PM, et al: RECQL, a member of the RECQ family of DNA helicases, suppresses chromosomal instability. Mol Cell Biol 27:1784–1794 (2007).
46.
Sharma SV, Gajowniczek P, Way IP, Lee DY, Jiang J, et al: A common signaling cascade may underlie ‘addiction’ to the Src, BCR-ABL, and EGF receptor oncogenes. Cancer Cell 10:425–435 (2006).
47.
Shaw CJ, Lupski JR: Implications of human genome architecture for rearrangement-based disorders: the genomic basis of disease. Hum Mol Genet 1:R57–64 (2004).
48.
Shimizu N, Shingaki K, Kaneko-Sasaguri Y, Hashizume T, Kanda T: When, where and how the bridge breaks: anaphase bridge breakage plays a crucial role in gene amplification and HSR generation. Exp Cell Res 302:233–243 (2005).
49.
Shuster MI, Han L, Le Beau MM, Davis E, Sawicki M, et al: A consistent pattern of RIN1 rearrangements in oral squamous cell carcinoma cell lines supports a breakage-fusion-bridge cycle model for 11q13 amplification. Genes Chromosomes Cancer 28:153–163 (2000).
50.
Simons A, Janssen IM, Suijkerbuijk RF, Veth RP, Pruszczynski M, et al: Isolation of osteosarcoma-associated amplified DNA sequences using representational difference analysis. Genes Chromosomes Cancer 20:196–200 (1997).
51.
Soares LM, Zanier K, Mackereth C, Sattler M, Valcarcel J: Intron removal requires proofreading of U2Af/3′ splice site recognition by DEK. Science 312:1961–1965 (2006).
52.
Squire JA, Pei J, Marrano P, Beheshti B, Bayani J, et al: High-resolution mapping of amplifications and deletions in pediatric osteosarcoma by use of CGH analysis of cDNA microarrays. Genes Chromosomes Cancer 38:215–225 (2003).
53.
Stark G, Debatisse M, Giulotto E, Wahl GM: Recent progress in understanding mechanisms of mammalian DNA amplification. Cell 57:901–908 (1989).
54.
Stock C, Kager L, Fink FM, Gadner H, Ambros PF: Chromosomal regions involved in the pathogenesis of osteosarcomas. Genes Chromosomes Cancer 28:329–336 (2000).
55.
Tarkkanen M, Karhu R, Kallioniemi A, Elomaa I, Kivioja AH, et al: Gains and losses of DNA sequences in osteosarcomas by comparative genomic hybridization. Cancer Res 55:1334–1338 (1995).
56.
Tarkkanen M, Bohling T, Gamberi G, Ragazzini P, Benassi MS, et al: Comparative genomic hybridization of low-grade central osteosarcoma. Mod Pathol 11:421–426 (1998).
57.
Tarkkanen M, Elomaa I, Blomqvist C, Kivioja AH, Kellokumpu-Lehtinen P, et al: DNA sequence copy number increase at 8q: a potential new prognostic marker in high-grade osteosarcoma. Int J Cancer 84:114–121 (1999a).
58.
Tarkkanen M, Kiuru-Kuhlefelt S, Blomqvist C, Armengol G, Bohling T, et al: Clinical correlations of genetic changes by comparative genomic hybridization in Ewing sarcoma and related tumors. Cancer Genet Cytogenet 114:35–41 (1999b).
59.
Thomas DM, Johnson SA, Sims NA, Trivett MK, Slavin JL, et al: Terminal osteoblast differentiation, mediated by runx2 and p27KIP1, is disrupted in osteosarcoma. J Cell Biol 167:925–934 (2004).
60.
Toledo F, Smith KA, Buttin G, Debatisse M: The evolution of the amplified adenylate deaminase 2 domains in Chinese hamster cells suggests the sequential operation of different mechanisms of DNA amplification. Mutat Res 276:261–273 (1992).
61.
Tomovska S, Richter J, Suess K, Wagner U, Rozenblum E, et al: Molecular cytogenetic alterations associated with rapid tumor cell proliferation in advanced urinary bladder cancer. Int J Oncol 18:1239–1244 (2001).
62.
Trask BJ, Hamlin JL: Early dihydrofolate reductase gene amplification events in CHO cells usually occur on the same chromosome arm as the original locus. Genes Dev 3:1913–1925 (1989).
63.
van Dartel M, Cornelissen PW, Redeker S, Tarkkanen M, Knuutila S, et al: Amplification of 17p11.2 approximately p12, including PMP22, TOP3A, and MAPK7, in high-grade osteosarcoma. Cancer Genet Cytogenet 139:91–96 (2002).
64.
van Dartel M, Redeker S, Bras J, Kool M, Hulsebos TJ: Overexpression through amplification of genes in chromosome region 17p11.2 approximately p12 in high-grade osteosarcoma. Cancer Genet Cytogenet 152:8–14 (2004).
65.
Vissers LE, Stankiewicz P, Yatsenko SA, Crawford E, Creswick H, et al: Complex chromosome 17p rearrangements associated with low-copy repeats in two patients with congenital anomalies. Hum Genet 121:697–709 (2007).
66.
Weinstein IB: Cancer. Addiction to oncogenes – the Achilles heal of cancer. Science 297:63–64 (2002).
67.
Yan T, Wunder JS, Gokgoz N, Gill M, Eskandarian S, et al: COPS3 amplification and clinical outcome in osteosarcoma. Cancer 109:1870–1876 (2007).
68.
Zanke BW, Greenwood CM, Rangrej J, Kustra R, Tenesa A, et al: Genome-wide association scan identifies a colorectal cancer susceptibility locus on chromosome 8q24. Nat Genet 39:989–994 (2007).
69.
Zielenska M, Bayani J, Pandita A, Toledo S, Marrano P, et al: Comparative genomic hybridization analysis identifies gains of 1p35 approximately p36 and chromosome 19 in osteosarcoma. Cancer Genet Cytogenet 130:14–21 (2001).
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2008
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