148 Abstracts

9 GENOMIC ANALYSES OF THREE REGIONS OF INCREASED COPY NUMBER DISCOVERED USING CGH.

JGray 1,2, L Daneshvar 1, M Schoenberg-Fejzo 1 , S Hwang 2, T Godfrey 1, M Palazzolo 2, C Martin 2, D Kowbel 2, M Tanner 3, O-P Kallioniemi 4, J Rommens 5, D Pinkell, 2, C Collins 2

IUCSF Cancer Center, University of California, San Francisco, CA. 2Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA. 3Tampere University Hospital, Tampere, Finland. 4National Center for Human Genome Research, NIH, Bethesda, MD. 5University of Toronto, Toronto, Canada.

Panganoimc surveys using comparative genomic hybridization (CGH) have revealed numerous regions of recurrent abnormal relative DNA sequence copy number in human solid tumors. These regions may encode genes that contribute to cancer progression when differentially expressed. We have analyzed three regions of increased copy number in detail to deterrmne their genomic organizations, precisely define regions of highest copy number increase and identify candidate oncogenes that might contribute to cancer progression when overexpressed.

3q26: Ovarian cancers and cell lines have frequent 2 to 4-fold increases in copy number (relative to 3p) in an ~ 2 Mb wide region at 3q26.3 defined by the CEPH megaYACs 806I)8 and 945H6. Glucose transporter 2 (GLUT2) and catalytic alpha subunit of phospha- tidylinositul 3-kinase (PIK3CA) are genes that map to this region whose functions make them candidate oncogenes.

8q22: Breast tumors and cell lines have recurrent high level amplification and/or structural rearrangements in a region at 8q22 (proximal to CMYC) defined by the CEPH YACs 890C4 and 936B3. These results suggest the presence of an unknown oncogene in this region that can be activated either by amplification or structural rearrangement.

20q13. Increased copy number at chromosome 20q13.2 has been shown to correlate with poor prognosis in node-negative breast cancer (Tanner ct al., 1994). We have narrowed the region of highest copy number increase to an -500 kb region defined by CEPH YACs 84513 and 847g7. No known oncogeaes map to this region so we cloned the region as a PI/BAC eontig and analyzed the contig in detail using exon trapping, eDNA selection and genomic sequencing. Three new genes were identified in the region of highest recurrent amplification. One, designated ZABC-1, encodes a protein having eight predicted C2H2 zinc fingers and a leucine zipper suggesting it functions as a transcription factor. Expression studies showed the gene to be weakly expressed in most breast cancer lines that are not amplified in this region. In contrast, the gene is expressed at high levels in most cell lines that are amplified and in some primary breast tumors. Thus, ZABC-1 is a strong candidate oncogene.

Work supported by USPHS grants CA 58207, CA 64602 and DOE contract DE-AC-03-76SF00098.

10 DETECTION OF AMPLIFIED GENES IN HUMAN CANCER BY COMPARATIVE GENOMIC HYBRIDIZATION

A Kallioniemi, Laboratory of Cancer Genetics, National Center for Human Genome Research, NIH, Bethesda, MD 20892

Comparative genomic hybridization (CGH) is an effective technique in locating regions of the ganome that are amplified in cancer. These sites are likely to harbor cancer genes whose increased copy number and consequent overexpression favors cancer progression. Although CGH is also able to reveal new regions of loss, there are only a few examples where CGH has directly lead to the discovery of a new tumor suppressor gene.

Many of the amplification sites discovered by CGH in various tumors coincide with the locations of previously known oncogenes that are activated by amplification in human cancer. For example, amplifications found by CGH at 2p24, 8(124, llql3, 12q12, and 17q12 are likely to involve NMYC, MYC, Cyclin-D, CDK4, and ERBB2 oncogenes, respectively. Furthermore, CGH has lead to the discovery of amplification of a number of previously known genes that were not anticipated as being amplified in cancer. Examples of these include the androgen receptor gene amplification in hormone-refractory prostate cancer (Visakorpi et al. 1994), REL gene in NHL (Houldsworth ct al. 1996, Joos et al. 1996) and BCL-2 in lymphoma (Monni ct al. 1996). Finally, if the candidate gene approach is not successful, positional cloning is the only strategy available for gene discovery. Amplification of the 17q23 region discovered by CGH is common in breast cancer and other tumors, but does not involve a known oncogene. This chromosomal region may thus contain gene(s) whose overexpression is critical for cancer progression. Current progress towards the identification of amplification target gene(s) at 17q23 will be reported: 1) Identification of a -5/r ib minimal common amplified region in breast cancer cell lines using randomly selected cosmid clones and fluorescence in situ hybridization, 2) Establishment of a non-chimeric YAC contig for this region, 3) Further refinement of the ampficon core with the non-chimeric YAC clones, 4) Localization of candidate genes and ESTs within the YAC contig, and 5) Exclusion of positional candidate genes.

11 GENETIC PROGRESSION IN ASTROCYTOMA G Mohavatra. A Bollen, and BG. Feuerstein; Department of Laboratory Medicine and

Brain Tumor Research Center, University of California, San Francisco, CA 94143-0808

Cancer is the results from the accumulation of multiple genetic events. Each may be mediated through actions at the gross chromosomal level resulting in deletion, duplication, inversion or transhication. These have the potential to be cytogenetically visible. Molecular characterization of these rearrangements may identify cancer causing genes and illuminate mechanisms that underlie initiation and progression. We have analyzed 97 grade IV and 62 grade III astrocytomas by comparative genomic hybridization (CGH) for DNA sequence copy number abnormalities (CNAs). Several CNAs common to both tumor grades occurred in greater than 20% of samples. Increase in DNA copy number on 7q (+7q) and decrease in copy number on 9p (-9p) were the most frequent aberrations occurring in both grades, suggesting these as early events. -6p, -13q, -14q, +17q, and +19p/q were also present at equal but lower overall frequency in both grades. -10, +7p, +20q, -22q, and amplifications were more frequently observed in grade IV than in grade II1 tumors. These CNAs may mark progression and/or increased genetic instability. -4q, +8q, +lOp and -1 lp were more frequendy observed in grade III than in grade IV tumors. This results is suggest that there are multiple genetic subtypes within histologic grades of astrocytoma and that progression from grade III to grade IV is not linear. Supported by CA13525, CA61147, CA64898, and Vysis Inc.

12 DNA SEQUENCE COPY NUMBER CHANGES IN LIPOMATOUS TUMORS S Jadwiga 1 , Wolf M 1 , Tarkkanen M 1 , Aaltonen LA 1 , Virolalnan M 2, Eloman 12,

Knuutila S 1

IDepartment of Medical Genetics, Haartman Institute, EO.Box 21 (Haartmaninkatu 3), 00014-University of Helsinki; 2Department of Oncology, Helsinki University Central Hospital, Haartmaninkatu 4, FIN-00290 Helsinki, Finland.

Thirty four lipomatons tumors including eight lipoma-like liposarcomas (LLLPS), 14 liposarcomas (LPS) of other histological subtypes, and 12 lipomas (LP) were studied by comparative genomlc hybridization (CGH). The most common gain detected in 50% of LPS and in 63% of LLLPS was seen at 12q (12q14-21 and 12q13-21, respectively). Three LPS showed high-level amplification at 12q15 and two LLLPS high-level amplification at 12q14-21 as the minimal common region. Another frequent gain observed in LPS and LLLPS involved lq sequences; lq21-24 and 1q21-23, respectively. In both LPS and LLLPS gain at 12q was frequently seen with a simultaneous gain at lq. Other recurrent changes in LPS include gains of 8cen-q21, 19q and 20q (each in four of 14 tumors), and losses of 9p21-pter and 13q21-qter (each in three of 14 tumors). No copy number changes were found in benign LE

In conclusion, the CGH analysis revealed recurrent overrepresentation of lq21-23 and 12q14-21 in LPS and LLLSE However, none of these regions were involved in LE Our findings suggest that genes located in both lq and 12q could be important in the development of liposarcoma. The results also showed that CGH may help in the differential diagnosis of low-grade or borderline adipose neoplasms.

Four liposarcoma specimens with an amplicon at 12q13-22 were selected for the microsatellite assay. The amplicon was studied analyzing 55 microsatellite markers by PCR. All four specimens were informative in at least 34 loci; an amplification or allelic imbalance was identified with four to 17 markers. The study revealed the complexity of the 12q13-22 amplicons; am#icons were discontinuous, indicating the presence of separate amplicons in the 12q13-22 region.