152 Abstracts

B 3 Rapid physical mapping at the single molecule level by hybridization to linearized high molecular weight DNA.

Heinz-Ulrich Weler’. Mei Wang’, James Mulllkin’

Aaron BensImon’, Karen Creulich’, Colin Collins’

and Joe W. Gray’

‘Center ior Molecular Cytogenetrcs, Life Sciences Div., MS 74-l 57. Lawrence Berkeley Laboratory, Berkeley, CA 94720, USA; rNeuroblologle Mol&ulalre, lnstltut Pasteur, 75015 Pans, France

We developed a novel procedure for raprd physical mapprng oi cloned DNA sequences by iluorescence In sttu hvbrtdizatlon (FISH) to iacilftate contlg assembly and locallzatlon oi expressed sequences.

Purified htgh molecular DNA is deposited on glass slides, anchored with one end to the substrate and linearized by the actton oi a receding mentcus. Nucleic acid probes can then be hybrldlzed to the straightened molecules and physicallv mapped by fluorescence microscopy. Mapplng posItIons at a resolution oi l-2 kb are calculated trom the analvsls OT a limited number oi recorded images itypIcally lo-40 molecules). A detection sensitlvlty oi under 1 kb allows accurate mapping of relatively short sequences on targets OI several hundred kb.

We applied this technique to generate physical maps oi overlapping cosmld and Pl clones, and began to map Pl clones onto YAC’s. The method does not require use oi blocklng DNA or slmllar competitive hybridlzatlon schemes, hence enabling us to directly map repetitive elements in large Insert clones.

This work was iunded by the Office of Health and EnvIronmental Research, Department of Energy, under contract DE-AC-03-76SF00098.

B5 CHARACTERIZATION OF CHROMOSOMAk CHANGES IN A BLADDER CANCER CELL LINE BY CLASSICAL CYTOGENETICS, FISH AND CGH

M. Giollant. B. Perissel, M. R. Wang, A. Tchirkov. E. DOS Santos. B. Hemery and P. Malet. Laboratoire de CytogCnttique Mtdicale. CHU de Clermont-Fd. France.

Chromosome studies of bladder tumors usuallv show complex structural and numerical chromosome aberrahons. A precise analysis of genomic imbalances is therefore difficult This work is an attempt to demonstrate the usefulness of a combination of classical and molecular techniques m order to improve the study of genomic imbalances in the RTI IV84 bladder

cancer cell line (Hasting and Franks. 198 1). The classical cytogenetic study suggested the following aberrations: t(3;ll). del(3)(p21). i(4p). i(8q). I( 17;?). t( 18;?) and a loss of one 21. FISH analysis with chromosome 1 I centromere specific and chromosome 3 specific “painting” probes allowed to confirm the t(3;ll) and del(3). The t( 17;?) translocation was identified as t(17:21) by simultaneous hybridization of a centromere specific and 21 “painting” probes. By using chromosome 4 and chromosome 8 specific libraries the i(4p) and i(8q) were confirmed. Comparative genomic hybridization was performed according Du Manoir et al.. 1993 with new procedure of image acquisition, display and treatment with a SCIL image software. CGH allowed to con!irm previous results and to identify the following aberrations: i(4p). i(8q) and amplifications sites on lp. 3q and 20q. Classical cytogenetics remains a good method for determination of the modal number and identification of major chromosome aberrations. FISH allows to detect aneuploidies on metaphases or interphase nuclei and to anaiyse more precisely structural aberrations. CGH permits to identify and to quantify genetic imbalances in a global way.

B4 EVALUATION OF A SERIES OF CASES OF LANGERHANS CELL HISTIOCYTOSIS BY COMPARATIVE GENOMIC HYBRIDIZATION

MM. Heeg’-: H. Aver-Loisa?, 6. E. Fevers’, A. Steele’, J. Smith’, M. G. P~llevicin?, ‘All Children f Hospital, Department of Pathology, St. Petersburg, FL. ‘University of South Florida, Tampa, FL. ‘Molecular Cytometw, Univerwty of California, San Frencisco.

Langerhans Cell Histlocvtosis (LCHI is a rare enigmatic disease of unknown etfology and pathogenesis. The pathologic lesion features proliferation of hlstiocytes with a phenotype of the Langerhans cell. The climcal spectrum of the disease is diverse and includes significant morbidity and mortality. however the pathologic characteristics of the disease are uniform. Monoclonality of the proliferating histiocytes has been recently demonstrated IWillman. et al., 1994, NEJM.331: 1541. This findmg suggests that somatic mutation may play a role in the pathogenesis of the disease. Flow cytometry has demonstrated dlploidy in all but the rarest case, however standard cvtogenetlc analysis has been largely “ns”ccesshJl.

We have analyzed 14 cases of LCH by the technique of comparative genomz hybridization (CGHI in an attempt to measure numerical chromosome abnormalities such as gain or loss of specific chromosomes or chromosome regions. Specimens were analyzed by flow cvtometry and Image analysis (IA) for ploidy. Frozen

specimens were then selectively dlssected to include at least 75% of lesional cells for DNA isolation for CGH analysis. CGH profiles of these 14 LCH specimens appeared normal, with no visible regions of DNA loss or gain. One very homogeneous LCH sample. which contained 26% hyperdiploid cells by flow and IA analysis. showed a normal genome profile by CGH. The discrepancy between the hyperdiploid flowllA analyses and CGH analysis of this specimen may result from a subpopulatlon selection during specimen processmg or from the dilutton of the hyperdlploid tumor DNA by diploid cells which decreases sensmwty of CGH analysis. The lack of an abnormal genome copy number in this series of LCH by CGH does not preclude somatic mutation below the limit of detection of the technique. However, this study does reduce the likelihood of a gross emplification or deletion in the etiology of LCH.

B6 Detection of Malignant Hematological

Disorders by Comparative Genomic Hybridization

<;uanfpinf Shl*. Angela (ioodcrc, Stephan Stilgcnbaucr. Mlchacl Andrecfl

Experimental Hematology. I>epartmenl of Hematology, The University of Texas. M. 11. Anderson Cancer Center, Houston, Texas 77030. USA

The comparatlvc gcnomic hybridl7atlon(CGH) is a new dcvclopcd method lor screcnlng entire tumor genomc for &cnelic imbalances as well as map the amplification of gents in the tumor genomc. In the comparison with the convenllonal cytogenctic analysis. the CGH isn’l influcnccd by low milosis index of the tumor cells and low-quality metaphasc chromosome spreads, and it don’[ need to make the IISSUC culture. We have performed this technique for the detection of the chromosomal abnormalities In 19 cases of malignant hematological disorders and a lymphoma cell line. In case with the diploid acute lymphoblastic leukemia(ALL) the genetic changes found by CGH were dup(lq). del(l4)(pter-ql I), del(5q). In diploid blast crisis of chronic myeloid leukemia(CML) the CGH didn’t reveal any abnormality. In diploid acute nonlymphoblastic leukemia(ANLL) the chromosomal abnormalities identified by CGH were monosomies 5 and 7. The results indicated that the CGH can detect the masked cytogenetic aberrations which aren’t identified by the conventional chromosomal analysis and further demonstrated that the diploid blasl crisis of CML is a characteristic entity of CML.

*the present address : Departmen of Pathology. Albert Einstein College of Medicme Bronx, New York 10461, USA.