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Detection and mapping of amplifications and deletions in ovarian cancer by comparative genomic hybridization (CGH)
Abstracts DBTECTION AND MAPPING OF A M P L I F I C A T I O N S A N D D E L E T I O N S IN O V A R I A N C A N C E R BY C O M P A g A T I V E GEIqOMIC HYBKIDIZATION (CGED M. Sakamoto 1, 3,,, H. S a k u z ~ t I, T.L. Yang-Femg2, S.B. IA 2,, A. g a l l i o n k a m l , O. KaRionlemil, D. Su~hurl, D. Piaka11, a n d J.W. G r a y 1. 1: Div. Molecular C y l o m e t ~ , U.C. San Francisco, CA. 2: Dept. H a m a n Genetics, Yale Univ., N e w H a w m , CT. 3: Sanaki Inst. Kyoudo Hosp,, Tokyo, Japan. C G H is a n e w p o w e r f u l m e t h o d for detecting a n d m a p p i n g c h a n g e s in D N A a e q u e n e e e n p y n u m b ~ a n y w h e r e in t h e t u m o r g e n o m e in a single expertme~t. T h e m e t h o d is b a s e d on a differential detection of the h y b r i d i z a t i o n of tt,mmr a n d n o r m a l D N A to n o r m a l m e t a p h a . ~ c h r o m o s o m e s . Variations ot D N A ~ . q u e a ~ copy n u m b e r in the t u m o r are detected u varlati0ms in the ratios ~ t h e intensities of t u m o r a n d n o r m a l D N A hybridization •lmag each c h r o m m e m e . We spplied C G H artalysiA to 27 ~ a r ~ of ovarian cancer. Several r e g t m ~ of increased ~opy n u m l m " were ~ . Soa~, e.g., lp~2p34 (most likely, MYCL), 8o,24 (MYC), 12p12 (KRA$2), were previoualy k n o w n to be amplhqed i a aolld t u m m y , while others (e.g., 3q26, 6p22, 9q31-q33, anti 17q2~ have n o t been reported. Regions m o s t c o m m o n l y associated w i t h i n . e a s e d c o p y n m n l ~ r in this s t u d y were 3p26 (41%), 8q24 (37%), a n d 12p12 (22%). Regions s h o w i n g r e d u c e d copy n u m b e r also w~,a kl~mti/iad b y the C G H analyld& These W¢~'e highly correlated with , I t . l i,: detected by RFLP analysis om the s a m e D N A (p=O.O001). C h r o m o s o m a l r e g i o n s s l m w i n g frequent reduced copy n u m b e r wene l p , 3p. gp, 13q, 17, 19 a n d Xp. The r t ~ o a of c o m m o n reduced copy n u m b e r on e.J,a-. 1 was narrowed to lp35-p36, s u g g e s t i n g the presence of • t u m o r suppressor geme associated w i t h ovarian c a n c ~ at that site. Sui:pta.~ by Nn'H ip.a.nt C.A45919, DOg ~ata'a~ D~-AC-O~76SFI~0~, amd
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USE OF FISH FOR ANALYSIS OF STRUCTURAL AND NUMERICAL CHROMOSOME ABERRATIONS IN TUMOR CELLS FROM PARAFFIN BLOCKS. W. Lee, K. Han, C. Hams and L. Meisner University of Wisconsin, State Lab of Hygiene. Madison, WI U,S.A.
We have developed techniques for processing cells from paraffin blocks for in situ hybridization with centromenc. cosmid, or painting probes. Use of these techniques has demonstrated unexpected chromosome changes in diverse tumor types. For example, although the interphase chromosome findings in ependymoma confu'med those obtained in metaphase cytogenetic studies, breast carcinoma, rhalxtomyosarcoma and other malignancies demonstrated chromosome changes not previously reported in metaphase studies. Use of paraffin blocks for retrospective studies permits selection of cases based on known patient outcomes to determine which chromosome changes may have prognostic value. It also eliminates the potential bias associated with selection of minor tumor clones which are better adapted to in vitro culture than are the more representative and presumably more diagnostic tumor cell populations. Techniques for probing cells from paraffin blocks will be described with examples presented of chromosome changes in tumor cells from paraffin blocks compared with metaphase findings.
15 7
A~0
GENERATION OF REGION-SPECIFIC LIBRARY AND PAINTING PROBES ON CHROMOSOME 6 B Y MICRO-DISSECTION
X-Y. Guan I, P. S. Meltzerl,2, A. Burgess I, J. Cao 1, and J. M. Trent L3. Deparm~nts of Radiation Oncology t , l~ia__n'ics 2, ~ld Human Genetics 3, The University of Michigan, 1150 W. Medical Center Drive, MSRBII Room C560, Ann Arbor, Michigan 481094)668. USA Chromosome mic~dissection has been applied to generate both chromosomal band-specific microclone libraries for physical mapping, and band-specific DNA painting probes for fluon~ent in sire hybridization (FISH). Chromosome 6 h u been divided by microdissecdon into a series of I 1 different regions, and a micrediss~don of the whole 6 has also been performed. Direct PCR amplification of dissected DNA was first used to generate painting probes (Micro-FISH probes). Chromosomal in sire hybridization of normal metaphases were then performed using these band-specific Micro-FISH probes to conf'urm their reciprocal specificity. The FISH results demonstrated that all of these painting probes gave strong and specific signals on the dissected region. Micro-FISH probes have also been used successfully to determine c h r o m o s o m e rearrangements unidentifiable by standard chromosome banding analysis. Malignant melanoma is frequently characte=ized by the deletion of the long arm of chromosome 6 (usually encompassing 6¢116q21). In an effort to satmatc this region with DNA markers, a genomic library of 20,000 clones was onnstrueted from 6q16-21 dissection (Guan et at., Oenomics, In press). Clones from this library were mapped against a human-rodent somatic cell hybrid mapping panel that divides chromosome 6 into 7 regions, confirming their localization within the target region. The generation of these Micro-FISH probes and DNA mierocione libraries from chromosome 6 should assist in establishing a physical map of this chromosome'and may assist in identifying the melanoma dei&ion region.
FLUORESCENT IN SITU HYBRIDIZATION(FISH) AS A TOOL To IDENTIFY CHROMOSOMAL ABERRATIONSIN HUMAN TUMOR CELLS IRae.heiA. Jema~uma.2M. Rezanr Rahnum.2K.L..Ying. IC. Patrick Reynolds and IEri S. Srivauam IDiviJio~of HematologyOaoology, 2Divisiooof Medical Ge.a~ics, Childreus Hospitalof I..o6 Angelea, l ~ t of Pediatrics, USC School of Medicine, Los/mgeles. California 90027.
A32
Molecular geneac studies in our hbtmm~ry have identified deletion of cinomagomc I lq seqaeacus in cervical ¢.ucmon~s and aearoblLstomas.We are daetefoaeinterestedin de~tmininS wheXbetfloor.cent in siVahybridizatitm (FISH) ~.baiquus could be used W glcmify chromosome I I deletions ~ g e m e a ~ . in tbe present study we have used chrorat~0m¢ I 1 specific cenuomm'~ and lmin~agprobe=to detect ¢.bn)musome11 aberrationsin Sills, • cervical carcinoma cell line and in LA-N-6, a ae,utoblastoma cell line. C ~ emalysiaby G-II l~lad~g showed 3 normal appearing II's and few mark~ ~ m u s in the SiHa cells. FISH amdysis using both ceatmmmac and painting probes ¢oatirmed the ptmmce of the 3 normal appearing 1i's. FISH also idemified the premmce of • a~mrketchromosome whichctmvaiusonly the I i omtmmetic seq~t,,m,5~__.FISH using I l ceatromeri¢ and painting pmabe¢ oa the ~ cell line. LA-N-6, identified • normal appearing 11 and a dmivative it wheNmfl~edimudIlq is rcplaoed by • aegmeat of an unknown dmmaoanme. Thus by using FISH we have idantifiedchrommomal ~ in the two differant tom~ cell linen.We are • , preamt tsyia$ to iocaiizz precisely deintim/tmmdocationbruskpointsusing singiz copy sequences.
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USE OF FISH FOR ANALYSIS OF STRUCTURAL AND NUMERICAL CHROMOSOME ABERRATIONS IN TUMOR CELLS FROM PARAFFIN BLOCKS. W. Lee, K. Han, C. Hams and L. Meisner University of Wisconsin, State Lab of Hygiene. Madison, WI U,S.A.
We have developed techniques for processing cells from paraffin blocks for in situ hybridization with centromenc. cosmid, or painting probes. Use of these techniques has demonstrated unexpected chromosome changes in diverse tumor types. For example, although the interphase chromosome findings in ependymoma confu'med those obtained in metaphase cytogenetic studies, breast carcinoma, rhalxtomyosarcoma and other malignancies demonstrated chromosome changes not previously reported in metaphase studies. Use of paraffin blocks for retrospective studies permits selection of cases based on known patient outcomes to determine which chromosome changes may have prognostic value. It also eliminates the potential bias associated with selection of minor tumor clones which are better adapted to in vitro culture than are the more representative and presumably more diagnostic tumor cell populations. Techniques for probing cells from paraffin blocks will be described with examples presented of chromosome changes in tumor cells from paraffin blocks compared with metaphase findings.
15 7
A~0
GENERATION OF REGION-SPECIFIC LIBRARY AND PAINTING PROBES ON CHROMOSOME 6 B Y MICRO-DISSECTION
X-Y. Guan I, P. S. Meltzerl,2, A. Burgess I, J. Cao 1, and J. M. Trent L3. Deparm~nts of Radiation Oncology t , l~ia__n'ics 2, ~ld Human Genetics 3, The University of Michigan, 1150 W. Medical Center Drive, MSRBII Room C560, Ann Arbor, Michigan 481094)668. USA Chromosome mic~dissection has been applied to generate both chromosomal band-specific microclone libraries for physical mapping, and band-specific DNA painting probes for fluon~ent in sire hybridization (FISH). Chromosome 6 h u been divided by microdissecdon into a series of I 1 different regions, and a micrediss~don of the whole 6 has also been performed. Direct PCR amplification of dissected DNA was first used to generate painting probes (Micro-FISH probes). Chromosomal in sire hybridization of normal metaphases were then performed using these band-specific Micro-FISH probes to conf'urm their reciprocal specificity. The FISH results demonstrated that all of these painting probes gave strong and specific signals on the dissected region. Micro-FISH probes have also been used successfully to determine c h r o m o s o m e rearrangements unidentifiable by standard chromosome banding analysis. Malignant melanoma is frequently characte=ized by the deletion of the long arm of chromosome 6 (usually encompassing 6¢116q21). In an effort to satmatc this region with DNA markers, a genomic library of 20,000 clones was onnstrueted from 6q16-21 dissection (Guan et at., Oenomics, In press). Clones from this library were mapped against a human-rodent somatic cell hybrid mapping panel that divides chromosome 6 into 7 regions, confirming their localization within the target region. The generation of these Micro-FISH probes and DNA mierocione libraries from chromosome 6 should assist in establishing a physical map of this chromosome'and may assist in identifying the melanoma dei&ion region.
FLUORESCENT IN SITU HYBRIDIZATION(FISH) AS A TOOL To IDENTIFY CHROMOSOMAL ABERRATIONSIN HUMAN TUMOR CELLS IRae.heiA. Jema~uma.2M. Rezanr Rahnum.2K.L..Ying. IC. Patrick Reynolds and IEri S. Srivauam IDiviJio~of HematologyOaoology, 2Divisiooof Medical Ge.a~ics, Childreus Hospitalof I..o6 Angelea, l ~ t of Pediatrics, USC School of Medicine, Los/mgeles. California 90027.
A32
Molecular geneac studies in our hbtmm~ry have identified deletion of cinomagomc I lq seqaeacus in cervical ¢.ucmon~s and aearoblLstomas.We are daetefoaeinterestedin de~tmininS wheXbetfloor.cent in siVahybridizatitm (FISH) ~.baiquus could be used W glcmify chromosome I I deletions ~ g e m e a ~ . in tbe present study we have used chrorat~0m¢ I 1 specific cenuomm'~ and lmin~agprobe=to detect ¢.bn)musome11 aberrationsin Sills, • cervical carcinoma cell line and in LA-N-6, a ae,utoblastoma cell line. C ~ emalysiaby G-II l~lad~g showed 3 normal appearing II's and few mark~ ~ m u s in the SiHa cells. FISH amdysis using both ceatmmmac and painting probes ¢oatirmed the ptmmce of the 3 normal appearing 1i's. FISH also idemified the premmce of • a~mrketchromosome whichctmvaiusonly the I i omtmmetic seq~t,,m,5~__.FISH using I l ceatromeri¢ and painting pmabe¢ oa the ~ cell line. LA-N-6, identified • normal appearing 11 and a dmivative it wheNmfl~edimudIlq is rcplaoed by • aegmeat of an unknown dmmaoanme. Thus by using FISH we have idantifiedchrommomal ~ in the two differant tom~ cell linen.We are • , preamt tsyia$ to iocaiizz precisely deintim/tmmdocationbruskpointsusing singiz copy sequences.