134 Abstracts

19 Correlation of phenotype and genotype .reveals a sequence of genetic events in the progression of breast and cervix tumors

Thomas Ried, Evelin Schrocl. Kersrln Heselme),er’. Cert Auer’. Kelly Just, Stanislas du blanoir

National Center for Human Genome ResearchINIH, Bethesda, Maryland, *Karolinska Institute. Stockholm. Sweden

Comparative genomic h!,bridlzatlon (CGH) was used to analyze 15 breast fibroadenomas and 25 adeno- carcinomas of the mammary gland and metastates. We observed different patterns of chromosomal gains and losses in diploid and aneuploid breast carcinomas \vith a significantly higher number of genetic imbalances in aneuploid tumors. Fibroadenomas of the breast did not reveal DNA copy number changes. In order to define a sequence of genetic events during tumor progression we analyzed histologically defined sections of different stages during the progression of cervix tumors (normal epithelium. mild dysplasia, moderate dysplasia, severe d! splasia, and carcinoma) using a combination of microdissection, sequence independent DNA amplification and CCH. The results for all cases were compared to DNA ploidy measurements, mutant p53-expression. immunohistochemical detectlon of proliferation markers and oncogene amplification.

21 Genetic evolution of breast cancer from primae tumors to distant metastases evaluated b>

comparative genomic hybridization. 0 -P. Kallioniemil. T Kuukasi%ti E. Hwtinen. R. K&u’. M Tanne?. J. Isola. Laboratory of Cancer Genetics. Tampere University Hospital. Taxnpere. Fiid.

According lo the hypothesis on the multi-step progression of cancer. successive genetic changes targeting specific genes gradually lead to an increasingly malignant cell clone. An important step of tumor pro@ession is metastasis as this ultimately determines survival of the cancer patient. We are developing strategies for comprehensive analyses of genetic differences between a primary tumor and it’s metastasis in order to determine the clonal relationships between these two samples. as well as to highlight ctuomosomal regions and genes that might be implicated in the actual metastatic process Comparative genomic hybridization (CGH) is used to analyze sample pans 6om archivaL formal&fixed rumor tissues from the same patients, up fo decades apart. and DNA sequence gains and losses along all chromosome arms are evaluated.

“Copy number karyotypes” of primary breast tumors and distant metastases appearing 1-3 years after operation shared several characteristic losses and gains with identical breakpoints. Thus, identical clonal origin of the two samples could usually be established. However. up to 30% of the genetic changes were different between the two sample pairs. and unexpectedly, the metastases often had fewer genetic aberrations than the primary tumor. Several deletions present in the primary tumors were absent from the metastases, although occasionally gains, such as 8q+ were only seeo in the metastases. Absence of certain changes io the metastasis suggests that the metsstatic clone diverged early on during hunor evohrtion. Analyses of large numbers of specimen pairs are now in progess to allow studies of the involvement of individual chromosomal regions.

20 Detection of genetic imbalances in human brain tumors using keryotyping, interphase cytogenetics, and comparative genomic hybridization

Evelin Schr6ck. Stanislas du Mano~r. ‘Gundula Thiel. Thomas Red Nafional Center for Human Genome ResearcMVlH, Bethesda, Maryland, ‘Institute of Medical Generw HumboldtUn~versily. Berlin

Astrocytic tumors are the most common bram tumors both in children and adults. Cylogenetlc analysis revealed a number of recurrent genebc changes for high grade tumors. however, lrltle IS known on chromosomal aberrations I” low grade and recurrent astrocytomas. CGH was used to investtgate bentgn astrocytomas, recurrent tumors and malign gltomas. Low grade astrocylomas revealed a gain of the long arm of chromosome 7. which has not been known from cytogenebc analysis. CGH with DNA extracted from normal braln tissue dtd not show copy number increases for chromosome 7. CGH studies of recurrent astrocylomas with the same or increasing stage of malignancy demonstrated that the primary and the recurrent tumor have a common clonal origin. Multiple amplification sites and nonrandom chromosomal gains and losses were ldentlfled for anaplastlc astrocytomas and glloblastomas. with overrepresentation of chromosome 7 and underrepresentatlon of chromosome 10 as the most frequent events. The validity of the CGH data was confirmed usmg Interphase cytogenetlcs with YAC clones on uncultured tumor nuclei and chromosome paintlng In tumor metaphase spreads. Ependymomas represent a subset of braln tumors that are derived from ependymal cells covenng ventrtcles and the spmal cord. Karyotype infonabon tn these tumors was restricted lo few cases. CGH and interphase cyiogenetlcs revealed recurrent gams of chromosomes 7 and 12 and losses of chromosome 17 and 22. A umque pattern of high copy number Increases was mapped to chromosome 12. No evidence of gene amplificabon was reported lo date.

22 GENE AMPLIFICATION IN THYROID CANCER: A NEW MECHANISM DEFINED BY CGH and A BAC MAP OF THE HUMAN GENOME. lx-N Chen,

‘R. Gonsky, 2M. Wang, 2E. Lai. ‘1. A. Fags, ‘1 R. Korenkrg Cedars- Sinai Medical Ctr, UCLA, CA; Dept. of Pharmacology, UNC-CH, NC

More than 12,000 new cases of thyroid cancer develop each year m the USA for which scant information is available on cytogenetic abnormalities. Comparative genomic hybridization (CGH) now permits the detection and mapping of amplified regions in DNAs without the need for metaphase tumor preparations. We have now combined two advances to increase the resolution and speed of gene identification using CGH. These include increasing the resolution of CGH by combining it with a high resolution chromomycin and dlstamycin reverse- banding technique, and elaborating a dense, genome-wide array of molecular cytogenetic agents (bacterial artificial chromosomes (BACs) with which to identify and isolate genes from amplified regions. Using CGH, we have analyzed the DNA copy number changes in 15 human thyroid tumors. The results revealed amplifications of chromosome bands 1~36, lq42, 2~13, 2~21, 19q13.1 and losses of chromosome bands 16q12-13 and 16q22-23. One of the 140 chromosome 2 BACs mapping at the 2~21-22 border revealed amplification of 40-70 copies by metaphase and interphase analyses m WRO, a thyroid follicular cell line containing double mmutes(dm). The variable signals suggest the existence of mmor populations with higher number and smaller amplicon size suitable for microdissection and- more rapid gene cloning. In summary: High resolution CGH has been combined with whole genome BAC Map construction to reveal a very specific region amplified on chromosome 2~21 likely to contain new genes involved In thyroid cancer tumorigenesis.