Comparative Genomic Hybridization Reveals Novel Chromosome Deletions in 90 Primary Soft Tissue Tumors

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Comparative Genomic Hybridization Reveals Novel Chromosome Deletions in 90 Primary Soft Tissue Tumors Fabienne Parente, Josiane Grosgeorge, Jean-Michel Coindre, Philippe Terrier, Odile Vilain, and Claude Turc-Carel

ABSTRACT: Comparative genomic hybridization (CGH) was used to detect chromosomal gains and losses in a series of 90 frozen soft tissue primary tumors (STTs), all untreated. The material consisted of 69 malignant sarcomas, including 20 malignant fibrous histiocytomas (MFH), 23 liposarcomas (LPS), 6 leiomyosarcomas (LMS), 4 synovial sarcomas, 4 primitive neuroectodermal tumors (PNETs), and various others subtypes, in addition to 21 benign tumors. Within the benign tumors, only 2 of the 3 schwannomas showed genetic changes. In malignant sarcomas, genetic changes were detected in 64 of the 69 samples analyzed (92%), with a mean of 4.5 per sample (range 0–10). Gains and losses on chromosome 13 were observed in 32% of the sarcomas with genomic imbalance. Recurring low-level copy number increases were found at new sites on chromosomes 7 (6 MFH samples, 30%) and 8 (10 LPS samples, 43%), the minimal common regions being 7p15-pter and 8q24. No new recurring high-level amplifications were found. Surprisingly, losses of DNA sequences were more frequent than gains; particularly, losses were the main feature in LMS, with highly recurrent common minimal losses at 11q14-qter and 13q21–q22 (4 samples, 66%, and 5 samples, 83%, respectively). Losses of chromosome 2 sequences (minimal common regions at 2p24-pter and 2q32-qter ) were observed in 50% of the MFH analyzed. New recurrent losses of whole or part of chromosome 14 were found in 57% of the pleomorphic LPS (PLPS) analyzed. This study uncovers new clues for the diagnosis of malignant STTs and shows the importance of deletions as events in the early steps involved in the tumorigenesis of STTs. © Elsevier Science Inc., 1999. All rights reserved.

INTRODUCTION Soft tissue tumors (STTs) are a heterogeneous group of benign and malignant tumors of various supportive tissues. Soft tissue sarcomas account for 1% of all human malignancies. Liposarcoma (LPS) and malignant fibrous histiocytoma (MFH) are the most common types. Clonal consistent (nonrandom, recurrent) primary chromosome abnormalities associated with specific tumor types are potential markers of diagnostic value to distinguish between these different tumor subtypes. In this context, cytogenetic data

From the Faculté de Médecine, (F. P., J. G., C. T.-C.), Nice, France; the Institut Bergonié (J.-M. C.), Bordeaux, France; the Institut Gustave Roussy (P. T.), Villejuif, France; and the Centre Oscar Lambret (O. V.), Lille, France. Address reprint requests to: C. Turc-Carel, UMR 6549 CNRS/ UNSA, Faculté de Médecine, Avenue de Valombrose 06107 Nice Cedex 02, France. Received February 12, 1999; accepted April 20, 1999. Cancer Genet Cytogenet 115:89–95 (1999)  Elsevier Science Inc., 1999. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

have already provided support for histological grouping within some soft tissue tumor types [1, 2]. A few sarcoma types have been shown to be characterized by recurrent specific chromosomal translocations [3] or the occurrence of double minutes, ring chromosomes, and large markers generally associated with gene amplifications [4]. Amplifications leading to overexpression of cellular oncogenes may play an important role in the development of malignant tumors [5]. The 12q13–15 region has been shown to be very complex [6], and several genes known to be amplified in human STTs, for example, MDM2, SAS, GLI, CHOP, and CDK4 [7–10] have been mapped to this area. Tumor suppressor genes produce proteins which are normally involved in the negative regulation of proliferation [11]. Deletions of these genes may contribute to oncogenesis. Allelic losses of tumor suppressor genes have been reported as frequent events in some STTs, such as loss of WT1 in embryonal RMS [12]. In addition, few segmental chromosomal deletions have been consistently as-

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Table 1 Series of 90 soft tissue tumors analyzed by CGH Tumor samples

No. of samples

Malignant tumors (n 5 69) Angiosarcoma Fibrosarcoma Hemangiopericytoma Leiomyosarcoma Liposarcoma (LPS) Well-differentiated liposarcoma (WDLPS) Myxoid liposarcoma (MLPS) Pleomorphic liposarcoma (PLPS) Malignant fibrous histiocytoma (MFH) Peripheral neuroectodermal tumor (PNET) Ewing sarcoma Neuroepithelioma Synovial sarcoma Rhabdomyosarcoma Other sarcoma Chordoid sarcoma Malignant melanoma of soft part Undifferentiated sarcoma Unclassified sarcoma Benign tumors (n 5 21) Angioleiomyoma Desmoid tumor Fasciitis Fibromatosis Hemangioma Myofibroblastoma Myositis Myxoid fibroma Neurofibroma Schwannoma

sociated with some subtypes of STTs, using conventional cytogenetics, for example, deletions of 17q11–12 and 22q11–12 in neurofibromas, 13q in a subset of lipomas, and 5q21–22 in desmoid tumors [13]. Conventional cytogenetic analysis is often difficult in STTs, owing to the paucity of mitotic cells, poor chromosome morphology and banding, in addition to the complex nature of chromosomal changes. Comparative genomic hybridization (CGH) allows the detection of DNA sequence copy number variations in tumor cells when DNA is the only material available [14]. In addition, CGH is complementary to basic fluorescence in situ hybridization (FISH), because it overcomes the need for specific probes or previous knowledge of the presence and nature of chromosomal aberrations [15]. Using CGH, Forus et al.[16] detected a novel recurring amplicon at 1q21–22 in several sarcoma types, such as LMS, MFH, and LPS. The CGH technique is particularly useful for large retrospective studies. We used it to detect genomic gains and losses in a series of 90 frozen STT specimens, all untreated primary tumors. Losses of DNA were more frequent than gains; particularly, losses of chromosome 13 sequences were observed in most of the malignant STTs analyzed, as well as chromosome 2- and 14-specific deletions for MFH and pleomorphic LPS (PLPS), respectively .

No. of samples with genomic imbalances

2 1 1 6

1 1 1 6

5 11 7 20

5 11 7 19

3 1 4 1

3 0 4 1

1 1 1 4

1 1 1 2

2 1 1 4 1 1 1 1 6 3

0 0 0 0 0 0 0 0 0 2

MATERIALS AND METHODS Specimens The material consisted of 90 frozen samples, including 69 primary untreated malignant sarcomas and 21 untreated primary benign tumors of various subtypes (Table 1) collected from three different institutes (Bergonié Institute, Bordeaux; Gustave Roussy Institute, Villejuif; and Oscar Lambret Center, Lille). The IARC-20304 lymphoblastoid cell line, which had a normal 46,XX karyotype, was used as a reference in all of the experiments. The MDA-MB-134 breast cancer cell line, with known amplification at 11q13 [17], was used as a “positive” control for increased genomic imbalance.

DNA Preparation Genomic DNA from tumor tissues and the IARC-20304 cell line was isolated by standard procedures using proteinase K digestion and phenol-chloroform extraction [18]. DNA concentrations were precisely measured by fluorometry, using the DNA-binding fluorochrome Hoechst 33258 (Sigma). These measurements were performed with a scanning fluorescence spectrometer PerkinElmer LS-50B.

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Chromosome Deletions in Soft Tissue Tumors Comparative Genomic Hybridization Comparative genomic hybridization was performed according to the protocol of du Manoir et al. [19]. Briefly, tumor DNA and reference DNA were differentially labeled with digoxigenin-11-dUTP and biotin-14-dATP (Boehringer Mannheim, catalog no. 1558.706), respectively, using commercially available nick translation kits (GIBCO, Life Technologies). A total of 400 ng of both DNAs was coprecipitated in the presence of 20 mg human CotI DNA (GIBCO), dissolved in hybridization mixture (50% formamide, 10% dextran sulfate, 2 3 SSC), denatured at 708C for 10 minutes, and preannealed at 378C for 1 hour. Hybridization was carried out on 46,XY reference metaphase spreads (Vysis, catalog no. 30-806010) for 2 days, at 378C, in a moist chamber. Posthybridization washes were done according to the regular FISH protocol described by Pinkel et al. [20]. For the detection of biotin molecules, we used Cy3-conjugated avidin (Biowhittaker, France, catalog no. PA.43000). Digoxigenin-labeled DNA was visualized with an antidigoxigenin-FITC antibody (Boehringer Mannheim, catalog no. 1207.741). Preparations were counterstained with an antifading medium (Vectashield™, Vector Laboratories Inc., Burlingame, CA, USA) supplemented with 0.5 mg/mL 49, 6-diamidino-2-phenylindole-dihydrochloride (DAPI). In each CGH experiment, a “negative”control with the IARC-20304 cell line DNA, and a positive control with MDA-MB134 cell line DNA were included.

Digital Image Analysis A Zeiss Axiophot microscope equiped with a Ludl 6-position automated filter wheel (Chroma Technology 83000 filter set) and a high-sensitivity Cooled CCD camera (SenSys™1400) was connected to a Macintosh PowerPC 8500 with a Datacell SCSI interface. All digital image acquisition, processing, and analysis functions (including the automated generation of chromosome fluorescence intensity profiles for the FITC vs. Cy3 fluorescence ratio) were generated by the Vysis software package (IPLAB Spectrum™, Quips CGH). Three-color images (red for reference DNA, green for tumor DNA, and blue for counterstaining) were acquired from 5 to 10 metaphases. Only metaphases of good quality, with strong uniform hybridization, were included in the analysis (i.e., chromosomes heavily bent, overlapping, or with overlying artifacts were eliminated). Chromosomal regions were interpreted as overrepresented when the corresponding green/red ratio exceeded 1.2 (gains) or 1.5 (high-level amplification), and underrepresented (losses) when the ratio was less than 0.8. RESULTS Comparative genomic hybridization analysis of 90 soft tissue tumors revealed DNA sequence copy number changes in 73% of the specimens (Table 1). In malignant sarcomas, genetic changes with a mean of 4.5 per sample (range 0–

Figure 1 Summary of the most frequent chromosomal imbalances observed in the 90 STTs investigated.

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Figure 2 Summary of losses (left bars) of DNA sequences detected by CGH in 6 leiomyosarcomas (LMS). Because high-resolution sub-band localization was not possible, the underrepresentated regions may be smaller than indicated.

10) were detected in 64 of the 69 samples (92%). In benign tumors, only 2 of the 3 schwannomas showed gains in 1q12–32, 8q21–22, and 12q14–15. The main chromosomal imbalances were observed on chromosomes 1, 2, 7, 8, 11, 12, 13, 14, and 16 (Fig. 1). High-level amplifications were frequently found on chromosomes 1 and 12, whereas lowlevel copy number increases were observed on chromosomes 7 and 8. Surprisingly, losses of DNA sequences were more frequent than gains, specifically on chromosomes 2, 11, 13, 14, and 16. Genetic changes (gains and losses) on chromosome 13 were found in 21 soft tissue sarcomas (32%) of various subtypes. No recurrent unbalanced genetic changes were found in the four synovial sarcomas and the four PNETs analyzed. Losses were the main feature in LMS (Fig. 2), where highly recurrent common minimal losses at 11q14-qter (four samples, 66%) and 13q21–22 (five samples, 83%) were observed. Chromosomes 2 and 7 were also underrepresented. In MFH (Fig. 3), underrepresentation of sequences on chromosomes 2 (10 samples, 50%), 13 (6 samples, 30%), 11 (5 samples, 25%), and 16 (4 samples, 20%) were found, as well as low-level copy number increases on chromosome 7 (6 samples, 30%) with minimal common regions at 7p15-pter, 7q21–22 and 7q32-qter. High-level amplifications were observed in 1p and 12q (minimal common regions at 1p31 and 12q15–22, respectively). Gains and losses were both seen on 13q22-qter. Figure 4 summarizes the main genetic changes observed in the 23 LPS we analyzed. Gain and loss of chromosome 13 sequences were observed in 6 LPS samples (26%). High-level amplifications on 12q were observed in

100% of WDLPS analyzed. The different amplicons varied considerably in size, and some cases included, in addition to 12q14, parts of band 12q13 or 12q15. Amplicons covering 12q21–22 and 12q24 were also observed. Amplifications on 1q22–q31 were found in 60% of the WDLPS. Four cases of MLPS (36%) exhibit an overrepresentation of the chromosome 8 sequences. Four of the 7 PLPS analyzed (57%) showed deletion of the 14q sequences with a minimal common region at 14q23–24. DISCUSSION Previous studies have convincingly demonstrated the usefulness of the CGH technique in the detection and mapping of DNA-sequence copy number changes in solid tumors [14]. Comparative genomic hybridization provides information not only on the overall occurrence of deletions or amplifications in a given tumor, but also on the size of the implicated region [14, 15]. We used the CGH technique to detect chromosomal gains and losses in 90 untreated primary STTs, free of secondary changes induced by therapeutic regimen. Although most of the malignant tumors (92%) showed genomic imbalances involving many different regions of the genome (Fig. 1) with patterns of recurrent imbalances, chromosome 13 was the most often involved. Gains and losses on chromosome 13 were detected in 32% of the sarcomas with genomic imbalances. Losses of chromosome 13 sequences were more frequent than gains (83% in LMS, 30% in MFH, and 17% in LPS), with a minimal common deleted region at 13q22–31. To our knowledge, deletions

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Figure 3 Summary of genomic overrepresentations (right thin bars), high-level amplifications (right thick bars), and losses (left bars) of DNA sequences detected by CGH 20 malignant fibrous histiocytoma (MFH). Because highresolution sub-band localization was not possible, the over- and underrepresentated regions may be smaller than indicated.

and amplifications in this chromosome region were previously described in MFH by Larramendy et al. [21], but not in other STT types. Alterations of the tumor suppressor gene RB1 (13q14) are frequent findings in cancers in general, and also in soft tissue sarcomas [22]. On the other hand, only 14% of the malignant sarcomas analyzed by CGH (25% of MFH and 100% of WDLPS) showed highlevel amplifications of the 12q13-qter sequences widely described in STTs [7, 8, 16, 23]. The novel 1q21–22 amplicon described by Forus et al. [16] was detected in three WDLPS cases (60%). Genetic changes specific for a given tumor subtype were also detected. Losses were the main feature in the 6 LMS analyzed in which highly recurrent common minimal losses in 11q14-qter and 13q21–22 were found (Fig. 2) . Numerous amplified regions, including recurrent amplification in 1q21–23 were reported in LMS, but not in primary tumors [16, 24]. In our series of untreated primary LMS, no recurrent gains were detected. Although based on a small series, it is tempting to suggest that deletions could be related to early events in the tumorigenesis of LMS. Amplification events would occur during tumor progression. We showed, for the first time, losses of chromosome 2 sequences (minimal common regions at 2p24-pter and 2q32-qter), as well as chromosome 11 (25%) and 16 (20%) losses in MFH (Fig. 3). Numerous tumor suppressor genes are located on chromosomes 11 and 16, for example, WT1 (11p13), EXT1 (11p11–13), MEN1 (11q13), RB2, and cyld1 (16q12–13) [25–29]. On chromosome 2, the tumor sup-

pressor genes MAD (2p13) and EXTL2 (2q24–31) were described [30, 31]. The loss and/or the inactivation of one or more of these genes could be required for the tumor development of MFH. Gains in 7p15-pter, 7q32, and 1p31 have already been described in MFH by Larramendy et al. [21] as a prognostic value for the rate of overall survival. The mean value per case of imbalanced aberrations was less than 3.0 in the MFHs of this small series (Fig. 3). This is an unexpectedly low value with respect to the very complex karyotypes with numerous clonal aberrrations usually detected in MFHs by conventional banding techniques. The discrepancy in results obtained by CGH versus conventional banding techniques may be of various origins (in combination or not): (a) CGH failing to detect low-level copy number changes involving small chromosomal regions, the total length of amplified DNA being at least 15 Mb for detection by CGH [32]; (b) the presence of stromal reactive cells diluting the abnormal tumor cells; and (c) the low likelihood of detecting even large-scale deletions in hyperdiploid specimens by CGH. Specific genetic changes were found for each subgroup of LPS (Fig. 4). Some of them have been already described in CGH studies, such as high-level amplifications in 1q21–22 and 12q21–22 in WDLPS [16, 23] and overrepresentation of chromosome 8 in MLPS by the mean of conventional cytogenetics [3]. New recurrent losses of whole or part of chromosome 14 were found in 57% of the PLPS analyzed. Losses at 14q23–24 have been previously described in renal oncocytomas and nonpapillary renal cell carcinomas [33, 34].

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Figure 4 Summary of genomic overrepresentations (right thin bars), high-level amplifications (right thick bars), and losses (left bars) of DNA sequences detected by CGH in 23 liposarcomas (LPS). Genetic changes specific for well-differentiated LPS (WDLPS), pleomorphic LPS (PLPS), and and myxoid LPD (MLPS) are bracketed. Because high-resolution sub-band localization was not possible, the over- and underrepresentated regions may be smaller than indicated.

Benign soft tissue tumors in the present study did not show relevant aberrations by CGH. This is in concordance with cytogenetic data; normal karyotype or balanced rearrangements are often found in these types of tumors, for example, t(12;var) in lipoma [3]. Comparative genomic hybridization can reveal only aberrations which result from loss or gain of DNA sequences, whereas balanced genetic changes remain undetected [35]. In summary, in our series, primary malignant STTs contain more genetic alterations than benign STTs. The presence of amplifications in 12q13-qter and 1q21–22 previously described in STTs was confirmed; however, they were found only in a few samples. Comparative genomic hybridization revealed new gains and losses on chromosome 13 in many subtypes of malignant STTs, as well as chromosome 2 deletions recurrently associated with MFH, and chromosome 14 deletions associated with PLPS. This study thus suggests the importance of deletions in the early steps of the tumorigenesis of malignant STTs. However, further studies are needed to confirm the diagnosis value of these findings. Supported by CNRS, the Association pour la Recherche sur le Cancer (ARC), the Ligue Nationale contre le Cancer (France), the Fédération Nationale des Centres de Lutte Contre le Cancer (France), the Fondation de France, and the Fédération Nationale des GEFLUC. Fabienne Parente is the recipient of a fellowship from the Ministère de l’enseignement supérieur de la recherche et de la technologie.

REFERENCES 1. Sandberg AA, Turc-Carel C (1987): The cytogenetics of solid tumors. Relation to diagnosis, classification and pathology. Cancer 59:387–395. 2. Molenaar WM, De Jong B, Buist J, Idenburg VJ, Seruca R, Vos AM, Hoekstra HJ (1989): Chromosomal analysis and classification of soft tissue sarcomas. Lab Invest 60:266–274. 3. Turc-Carel C, Pedeutour F, Durieux E (1995): Characteristic chromosome abnormalities and karyotype profiles in soft tissue tumors. Curr Top Pathol 89:73–94. 4. Pedeutour F, Suijkerbuijk RF, Forus A, Van Gaal J, Van de Klundert W, Coindre JM, Nicolo G, Collin F, Van Haelst U, Huffermann K, Turc-Carel C (1994): Complex composition and co-amplification of SAS and MDM2 in ring and giant rod marker chromosomes in well-differentiated liposarcoma. Genes Chromosom Cancer 10:85–94. 5. Dati C, Muraca R, Tazartes O, Antoniotti S, Perroteau I, Giai M, Cortese P, Sismondi P, Saglio G, De Bortoli M (1991): c-erB-2 and ras expression levels in breast cancer are correlated and show co-operative association with unfavorable clinical outcome. Int J Cancer 47:833–838. 6. Wolf M, Aaltonen LA, Szymanska J, Tarkkanen M, Blomqvist C, Berner JM, Myklebost O, Knuutila S (1997): Complexity of 12q13–22 amplicon in liposarcoma: microsatellite repeat analysis. Genes Chromosom Cancer 18:66–70. 7. Forus A, Florenes VA, Maelandsmo GM, Meltzer PS, Fodstad O, Myklebost O (1993): Mapping of amplification units in the q13–q14 region of chromosome 12 in human sarcomas: some amplica do not include MDM2. Cell Growth Differ 4:1065–1070. 8. Forus A, Florenes VA, Maelandsmo GM, Fodstad O, Myklebost O (1994): The protooncogene CHOP/GADD153, involved in

95

Chromosome Deletions in Soft Tissue Tumors growth arrest and DNA damage response, is amplified in a subset of human sarcomas. Cancer Genet Cytogenet 78:165–171. 9. Khatib ZA, Matsushime H, Valentine M, Shapiro DN, Sherr CJ, Look AT (1993): Coamplification of the CDK4 gene with MDM2 and GLI in human sarcomas. Cancer Res 53:5535–5541. 10. Smith SH, Weiss SW, Jankowski SA, Coccia MA, Meltzer PS (1992): SAS amplification in soft tissue sarcomas. Cancer Res 52:3746–3749. 11. Levine AJ, Momand J (1990): Tumor suppressor genes: the p53 and retinoblastoma sensitivity genes and gene products. Biochim Biophys Acta 1032:119–136. 12. Scrable H, Witte D, Shimada H, Seemayer T, Sheng WW, Soukup S, Koufos A, Houghton P, Lampkin B, Cavenee W (1989): Molecular differential pathology of rhabdomyosarcoma. Genes Chromosom Cancer 1:23–35. 13. Sreekantaiah C (1998): The cytogenetic and molecular characterization of benign and malignant soft tissue tumors. Cytogenet Cell Genet 82:13–29. 14. Kallioniemi A, Kallioniemi OP, Sudar D, Rutovitz D, Gray JW, Waldman F, Pinkel D (1992): Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 258:818–821. 15. Kallioniemi A, Kallioniemi OP, Piper J, Tanner M, Stokke T, Chen L, Smith HS, Pinkel D, Gray JW, Waldman FM (1994): Detection and mapping of amplified DNA sequences in breast cancer by comparative genomic hybridization. Proc Natl Acad Sci USA 91:2156–2160. 16. Forus A, Weghuis DO, Smeets D, Fodstad O, Myklebost O, van Kessel AG (1995): Comparative genomic hybridization analysis of human sarcomas: I. Occurrence of genomic imbalances and identification of a novel major amplicon at 1q21-q22 in soft tissue sarcomas. Genes Chromosom Cancer 14:8–14. 17. Lafage M, Pedeutour F, Marchetto S, Simonetti J, Prosperi MT, Gaudray P, Birnbaum D (1992): Fusion and amplification of two originally non-syntenic chromosomal regions in a mammary carcinoma cell line. Genes Chromosom Cancer 5:40–49. 18. Marmur J (1961): A procedure for isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3:208–218. 19. du Manoir S, Speicher MR, Joos S, Schröck E, Popp S, Döhner H, Kovacs G, Robert-Nicoud M, Lichter P, Cremer T (1993): Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization. Hum Genet 90:590–610. 20. Pinkel D, Straume T, Gray JW (1986): Cytogenetic analysis using quantitative, high sensitivity fluorescence in situ hybridization. Proc Natl Acad Sci USA 83:2934–2938. 21. Larramendy ML, Tarkkanen M, Blomqvist C, Virolainen M, Wiklund T, Asko-Seljavaara S, Elomaa I, Knuutila S (1997): Comparative genomic hybridization of malignant fibrous histiocytoma reveals a novel prognostic marker. Am J Pathol 151:1153–1161. 22. Stratton MR, Moss S, Warren W, Patterson H, Clark J, Fisher C, Fletcher CD, Ball A, Thomas M, Gusterson BA (1990): Mutation of the p53 gene in human soft tissue sarcomas: association with abnormalities of the RB1 gene. Oncogene 5:1297–1301. 23. Suijkerbuijk RF, Olde Weghuis D, Van Den Berg M, Pedeutour F, Forus A, Myklebost O, Glier C, Turc-Carel C, van Kessel AG (1994): Comparative genomic hybridization as a tool to define two distinct chromosome 12-derived amplification

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

units in well-differentiated liposarcomas. Genes Chromosom Cancer 9:292–295. Packenham JP, du Manoir S, Schrock E, Risinger JI, Dixon D, Denz DN, Evans JA, Berchuck A, Barret JC, Devereux TR, Ried T (1997): Analysis of genetic alterations in uterine leiomyomas and leiomyosarcomas by comparative genomic hybridization. Mol Carcinog 19:273–279. The European Consortium on MEN1: Lemmens I, Van de Ven WJ, Kas K, Zhang CX, Giraud S, Wautot V, Buisson N, De Witte K, Salandre J, Lenoir G, Pugeat M, Calender A, Parente F, Quincey D, Gaudray P, De Wit MJ, Lips CJ, Hoppener JW, Khodaei S, Grant AL, Weber G, Kytola S, Teh BT, Farnebo F, Grimmond S, Phelan C, Larsson C, Pannett AAJ, Forbes SA, Bassett JHD, Thakker RV (1997): Identification of the multiple endocrine neoplasia type 1 (MEN1) gene. Hum Mol Genet 6:1177–1183. Karnik P, Chen P, Paris M, Yeger H, Williams BR (1998): Loss of heterozygosity at chromosome 11p15 in Wilms tumors: identification of two independent regions. Oncogene 17:237– 240. McCormick C, Leduc Y, Martindale D, Mattison K, Esford LE, Dyer AP, Tufaro F (1998): The putative tumour suppressor EXT1 alters the expression of cell-surface heparan sulfate. Nature Genet 19:158–161. Yeung RS, Bell DW, Testa JR, Mayol X, Baldi A, Grana X, Klinga-Levan K, Knudson AG, Giordano A (1993): The retinoblastoma-related gene, RB2, maps to human chromosome 16q12 and rat chromosome 19. Oncogene 8:3465–3468. Biggs PJ, Chapman P, Lakhani SR, Burn J, Stratton MR (1996): The cylindromatosis gene (cyld1) on chromosome 16q may be the only tumour suppressor gene involved in the development of cylindromas. Oncogene 12:1375–1377. Edelhoff S, Ayer DE, Zervos AS, Steingrimsson E, Jenkins NA, Copeland NG, Eisenman RN, Brent R, Disteche CM (1994): Mapping of two genes encoding members of a distinct subfamily of MAX interacting proteins: MAD to human chromosome 2 and mouse chromosome 6, and MXI1 to human chromosome 10 and mouse chromosome 19. Oncogene 9:665–668. Wuyts W, Van Hul W, Hendrickx J, Speleman F, Wauters J, De Boulle K, Van Roy N, Van Agtmael T, Bossuyt P, Willems PJ (1997): Identification and characterization of a novel member of the EXT gene family, EXTL2. Eur J Hum Genet 5:382– 389. Parente F, Gaudray P, Carle GF, Turc-Carel C (1997): Experimental assessment of the detection limit of genomic amplification by comparative genomic hybridization. Cytogenet Cell Genet 78:65–68. Herbers J, Schullerus D, Muller H, Kenck C, Chudek J, Weimer J, Bugert P, Kovacs G (1997): Significance of chromosome arm 14q loss in nonpapillary renal cell carcinomas. Genes Chromosom Cancer 19:29–35. Schwerdtle RF, Winterpacht A, Storkel S, Brenner W, Hohenfellner R, Zabel B, Huber C, Decker HJ (1997): Loss of heterozygosity studies and deletion mapping identify two putative chromosome 14q tumor suppressor loci in renal oncocytomas. Cancer Res 57:5009–5012. Kallioniemi OP, Kallioniemi A, Piper J, Isola J, Waldman FM, Gray JW, Pinkel D (1994): Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in solid tumors. Genes Chromosom Cancer 10:231– 243.