Abstracts 171

B 2 9 COMPARATIVE GENOMIC HYBRIDIZATION AND REPRESENTATIONAL DIFFERENCE ANALYSIS AS TOOLS TO

IDENTIFY AND MOLECULARLY CLONE TUMOR-SPECIFIC C H R O M O S O M A L A N O M A L I E S A Simons, I Janssen, RF Sui_ikerbu~k, A Forus*, A Geurts van Kessel. Dept. of Human Genetics, university Hospital Nijmegen, The Netherlands; *Dept. of Tumor Biology, Norwegian Radium Hospital, Oslo, Norway

Gains and losses of chromosomal sequences within a tumor genome can be detected in a singie step in situ hybridization procedure following the comparative genomic hybridization (CGH) strategy. Previously, we have used this technique to define novel genomic imbalances (i.e., amplifications) in human osteosarcomas and soft tissue sarcomas of various histologic (sub)-types. In order to isolate these amplified sequences we have now extended these CGH analyses by employing the recently developed representational difference analysis (RDA) technique. RDA allows for the direct isolation of differences between tumor DNA and normal DNA through the combination of subtractive hybridization and PCR amplification processes. An nsteosarcoma showing clear amplifications of genomic regions on chromosomes 17 (pll.2) and 19 (q13.1) by CGH was subjected to RDA. Using tumor DNA as tester and a mixture of DNAs from 5 normal individuals as driver several DNA fragments were obtained after three rounds of subtraction and amplification. Southern blotting revealed the amplification of some of these RDA fragments in the tumor genuine. Subsequent somatic cell hybrid analysis disclosed their chromosome 19 origin. FISH analysis using a YAC isolated with one of the chromosome 19-specific RDA fragments on normal human chromosomes and on tumor nuclei confirmed its chromosomal localization (i.e., 19q13.1) and its amplification in the tumor, respectively. These results demonstrate that CGH and RDA appear to be useful tools to identify and molecularly clone tumor- amplified sequences.

B 3 1 CHARACTERIZATION OF A NEW HUMAN NEUROBLASTOMA CELL LINE

YS Kao and H Correa. Louisiana State University Medical Center, New Orleans, LA 70112-1393

Background: ncuroblastoma (NB) is a common neoplasia in children. Yet, very few human cell lines have been established. We report a NB cell line.

Design: A retropetitoneai NB from a 4-year-old girl was cultured in F-10 media supplemented with fetal calf serum (FCS). The monolayer culture was karyo-typed on day 8. The culture was maintained in RPMI 1640 supplemented with FCS. On 88th day the cells (KSC) started to grow in suspension. The KSC was then infected with Herpes Simplex Virus type 1 (HSV-1) and was observed using anti-HSV-1 monoclonal antibody up to 15 months post infection.

Results: The initial karyotype had both normal (13 cells) and abnormal (18 cells) karyotypes. The abnormal cells had 45 to 47 chromosomes per cell with del (1) (p13), del (9) (p21) and double minutes (drain). The KSC, which has been growing in suspension culture for more than 17 months, is hyperdiploid with 47 to 71 chromosomes per cell. The drain has disappeared, but, the del (1) (p13) persists. The HSV-I is still growing in KSC 15 months pest infection.

Conclusion: A newly established human NB cell line, KSC, is described. The cells are persistently infected with HSV-1 15 months pest infection.

B30 COMPARATIVE G E N O M I C HYBRIDIZATION ANALYSIS OF MENINGIOMAS.

Khan 11 , Parsa NZ 1, Harada T 2, Meltzer PSI , and Carter NP 3

1Laboratory of Cancer Genetics, NCHGR/NIH, Bethesda, MD; 2Molecular Genetics, Cambridge University, England; and 3Sanger Center, Cambridge, England

The gene for the dominantly inherited familial cancer syndrom neumfibramatosis type 2 (NF2) maps to 22q12. There is strong evidence that NF2 gene acts as a tumor suppressor gene involved in the etiology of central nervous system tumors including meningiomas. Combined molecular and cytogenefic studies have demonstrated loss of heterozygosity and partial or complete loss of chromosome 22 in 50-70% cases. The aim of this CGH study was to confirm the LOH findings on chromosome 22 and to detect additional areas of gain or loss in meningiomas, which may implicate novel tumor suppressors or oncogenes in this disease. CGH was applied to investigate 18 meningioma tumors. The patient's own lymphocyte DNA was used as control. The results were combined for CGH analysis. Labelling biases were seen with increased Rhodamine (red) signal compared to FITC (grce) in GC rich areas of chromosomes using our experimental conditions. These biases were compensated by labelling tumor green and normal red in one set of experiments, reversing the labels in another set and combining the results, partial or complete losses of chromosome 22 were seen in only 8/1g cases by CGH. This compares with 12/18 losses of a single or more locus on chromosome 22 by LOH studies. The differences in these results could be due to the increased sensitivity of LOH using micro-satellite for detection of small losses. Other regions of loss observed in two or more cases included lp, 8, 19p, and 19q. Gains were infrequent and in single cases included 3q, 5p,q, 11 ql 3, and 12p. In one tumor in which LOH and CGH studies failed to demonstrate loss on chromosome 22, there was an amplification of 17q23 which may be the site of a novel oncogene.

B 3 2 C H R O M O S O M E INSTABILITY IN 75 UNTREATED OVARIAN CANCER PATIENTS

*Parza NZ l , *Taede R 2, Nelson M 2, Adair T 2, Albelts D 2, Leong S 2, Salmon S 2, Krizman D 1 , Thompson FG 2, Trent j l lNational Center for Human Genome Researcl~NIH, Bethesda, MD; 2Arizona Cancer Center, Tucson, AZ

*These authors participated equally in this work

The etiology of ovarian cancer is poorly understood. However, chromosome rearrangements associated with ovarian cancer have been defined previously (for review see Thompson et al., Cancer Gene & Cytogene 73:33, 1994). In this study, we have completed a detailed eytogenetic review of 75 untreated ovarian cancer patients documented to contain chromosomal instability (breaks, gaps, complex chromatid exchange, etc.). For each patient, 1 to 5 mitotic cells could be completely examined cytogenetically The average chromosome number ranged from 17 to 250 per cell, with the majority of cases examined being hyperdiphiid. These abewant karyotypes were extremely complex and all exhibited a significant degree of chromosome and chromatid breakage resulting from a variety of types of chromosome aberrations, out of 75 cases, 65 bad chromosome breaks, 71 had chromosome or chromatid gaps, 13 had rings, 9 had isochromosomes, 73 had deletions, 72 had translocations, 10 had double minutes, 8 had homogeneously staining regions, 71 had fragments, 50 had diradials, 48 had ttiradials, 33 had quadriradials, and all 75 had unidentifiable marker chromosomes. The highly rearranged chromosomes in these 75 patients were associated with a less well differentiated and more aggressive clinical course, when compared to age and stage matched patients in our study without evidence of chromosome breakage. The mechanism underlying this plethora of breakage events in a series of untreated patients is currently indeterminate. Evidence of an PER + phenotype was not observed in DNA isolated from microexcised tumors from 5 representative patients with high levels of chromosome Instability. Further studies are underway to attempt to clarify the biological explanation for this phenomen.