- Email: [email protected]
Detection of new amplified regions in breast cancer by comparative genomic hybridization
Abstracts
142 THE USE OF FLUORESCENT IN $1TU HYBRIDIZATION (FISH) FOR PLOIDY ANALYSIS IN PROffrATIC ADENOCARCINOMA. R.B. Jenkins, K. Takai, DJ. Gibney, J.A. Katzmann, M.M. Lieber, and D . L Persons, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905 USA. Nuclear DNA ploidy has been shown to have an important prognostic association for patients with adenocarcinoma of the prostate. Flow cytometry has been the standard method in pinidy analysis from prostate carcinoma. More recently, static image analysis has also been utilized for DNA ploidy analysis in these tumors. FISH is a new technique which can be used for pioidy determination by enumerating specific chromosomes with in interphase nuclei. In a series of studies we have compared FISH with flow cytometry (FCM) and static image analysis (tA) in the da~mination of ploidy within touch preps and in paraffin ~ tissue from prostatic adenocarcinomas. Ploidy status using FISH was determined by enumerating the centrom~res of two randomly d m u a chromosomes 8 and 12 by the use of directly4abeled a-smadlit¢ DNA probes. These probes are hybridized to touch preps of prostm~c tumor specimens as well as isolated nuclei obtained by the Hedleytechniqoe by paraffin embedded material. Although both FISH and FCM could distinguish diploid, v*treploid and aneupinid tumors, IA classification was only able to distinguish diploid versus nondiploid tumors. Our data suggest that FISH analysis, using just two probes, is a sensitive method of deteeting ploidy anomalias in prostate ranter. Furthermore with the use of additional probes, FISH has the potentisi of superior sensitivity for the detection of aneupioidy ¢ompased to currently available methods and should be applicable to small biopsy specimens. 5
DETECTION OF NEW AMPLIFIED REGIONS IN BREAST CANCER BY COMPARATIVE GENOMIC HYBRIDIZATION Kallioniemi A, Kallioniemi O-P, Rutovitz D, Sudar D, Gray JW, Pinkel D, Waldman F. Div. of Molecular Cytometry, Dept. Lab. Medicine, Univ. California, San Francisco, CA 94143; MRC Genetics Unit, Edinburgh, UK and Geraldine Brush Cancer Research Center, 2330 Clay St., San Francisco, CA 94115. Cytogenetic analysis of solid minors is often difficult because of the scarcity or poor quality of metaphase spreads, or the complexity of marker chromosomes. When cytogenetics is su_ro~s_sful,double minums (DMs) and homogeneously staining regions (HSRs) are ohen found. The..s¢ indicate DNA amplification, but do not give any clue to the identity of the sequence. We have developed a new technique, comparative gcnomic hybridization (CGH), for detection and localization of DNA sequence copy number variation anywhere in the tumor gcmom¢. CGH is based on a simultaneous in situ hybridization of differentially labeled tumor and normal refexenc¢ DNA to normal m ~ chromosomes. The labeled DNAs are detected us/rig two different fluorochromes. The relative DNA seqmmce copy numbers of all regions in the tumor genome can then be quantitated by measuring the intensity ratios of the two fluorochromcs along each human chromosome. We studied 16 breast cancer o¢11 fines and 32 primary breast tumors using CGH and found several repeatedly amplified regions. Some of these regions cofreapond to the location of known oncogenes, such as MYC and ERBB2, but most of them where at loci where DNA amplification has not been previously d ~ b e d (e.g. 6q12, 7p21, 8q21-22, l l p l S , 15q23-25, 17q22-24, and 20o.). Suppoaed by NIH iffamsCA 45919. CA 44"/68and CA 47537, DOE contract DE-AC03-76SF00098, ami ImageaettcsInc. Napervil|e. IL.
7
THE USE OF IN SITU HYBRIDIZATION FOR M A K I N G IN VIVQ GENOTYPE/PHENOTYPE CORRELATES DURING H E A D A N D N E C K TUMORIGENESIS
Walter N. Hittelman, Narin Voravud, Dong M. Shin, Jae Y. Ro, Jin Soo Lee and Waun Ki Hong. The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA While cytogenetic and molecular abnormalities are now being described with increased detail for various solid tumors, there is still little information available regarding the pathophysioiogical role of these genetic changes during tumorigenesis. To address this issue, genetic and phenotypic analyses need to be carried out on intact tissue specimens. We have recently utilized chromosomespecific probes and ia sau hyhridization on formalin-fixed, paraffinembedded head and neck tumor tissue sections containing adjacent premalignant lesions m examine genetic changes occurring during head and neck tumorigenesis. Adjacent sections underwent immunohistochemical analyses for phenotypic changes associated with dysreguiated proliferation, differentiation, and/or cell loss. Genetic changes were detected in regions of normal histology adjacent to the tumors, suggesting a field cancerization process was ongoing in the head and neck region, increased genomic changes were observed as tissue histology passed through hyperplastic and dysplastic stages. These genetic changes were accompanied by phenotypic evidence of dysrogulation, providing direct evidence for a muitistep process. This approach will now allow a direct correlation to be made between specific genetic changes and their phenotypic consequences in vivo during tumorigenesis. Supported in part by NIH grants PO1 CA-52051, CA48364, and CA-45746.
MOLECULAR CELL GENETICS BY POLYMERASE 8 CATALYSED AMPLIFICATION OR IN SITU LABELLING OF SPECIFIC NUCLEIC ACID SEOUENCES. L. Bolund. C. Brandt. J. Hindklmr, J. Koch. S. K61vraa & S. Pedersen. Institute of Human Genetics, University of Aarhus, Denmark. The Polymerase Chain R e a c t i o n (PCR) can he performed on isolated cells or chromosomes, resulting in analysable product. The analysis can be performed by traditional DNA technology or by FISH to test metaphases. We have good experience with the analysis of aberrant chromosomes by FACS sorting, PCR amplification with degenerated primers and painting of test metsphases with the PCR product. We have also utilized polymerases for PRimed IN Situ labelling (PRINS) of specific nucleic acid s e ~ s in individual cells or chromoscaes. The ~ t h o d is based on the hybridisatlon of u n l a ~ l l e d synthetic o l i g o n u c l e o t i d e s to their c o m p l e m e n t a r y sequences ~n e i t u followed by incorporation of labelled nucleotides at the site of h y b r l d i s a t i o n w i t h the ollgonucleotide as primer. DNA or RNA leguencea are visualized by choice of p o l ~ r e s e . We use FRINS for differential atalnlng of closely r e l a t ~ DNA tandem repeat families, e. g. for chrcxaosome specific cen~E~re labelling in aneuploldy studlea o n ~ a ~ and Interphese cells. Moreover, u n i ~ DNA sequenc4s can be visualized w i t h ~ a £ 1 a of primers, whereas cells can be e t a £ ~ for specific mRNA species with a slr~le primer. We are trying to further increase the selectivity and sensitivity of the m e t h ~ to be able to analyse single gene targets a n d to detect selective allellc expression and somatic mutations ( by m R N A lahelling) in individual cells.
142 THE USE OF FLUORESCENT IN $1TU HYBRIDIZATION (FISH) FOR PLOIDY ANALYSIS IN PROffrATIC ADENOCARCINOMA. R.B. Jenkins, K. Takai, DJ. Gibney, J.A. Katzmann, M.M. Lieber, and D . L Persons, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905 USA. Nuclear DNA ploidy has been shown to have an important prognostic association for patients with adenocarcinoma of the prostate. Flow cytometry has been the standard method in pinidy analysis from prostate carcinoma. More recently, static image analysis has also been utilized for DNA ploidy analysis in these tumors. FISH is a new technique which can be used for pioidy determination by enumerating specific chromosomes with in interphase nuclei. In a series of studies we have compared FISH with flow cytometry (FCM) and static image analysis (tA) in the da~mination of ploidy within touch preps and in paraffin ~ tissue from prostatic adenocarcinomas. Ploidy status using FISH was determined by enumerating the centrom~res of two randomly d m u a chromosomes 8 and 12 by the use of directly4abeled a-smadlit¢ DNA probes. These probes are hybridized to touch preps of prostm~c tumor specimens as well as isolated nuclei obtained by the Hedleytechniqoe by paraffin embedded material. Although both FISH and FCM could distinguish diploid, v*treploid and aneupinid tumors, IA classification was only able to distinguish diploid versus nondiploid tumors. Our data suggest that FISH analysis, using just two probes, is a sensitive method of deteeting ploidy anomalias in prostate ranter. Furthermore with the use of additional probes, FISH has the potentisi of superior sensitivity for the detection of aneupioidy ¢ompased to currently available methods and should be applicable to small biopsy specimens. 5
DETECTION OF NEW AMPLIFIED REGIONS IN BREAST CANCER BY COMPARATIVE GENOMIC HYBRIDIZATION Kallioniemi A, Kallioniemi O-P, Rutovitz D, Sudar D, Gray JW, Pinkel D, Waldman F. Div. of Molecular Cytometry, Dept. Lab. Medicine, Univ. California, San Francisco, CA 94143; MRC Genetics Unit, Edinburgh, UK and Geraldine Brush Cancer Research Center, 2330 Clay St., San Francisco, CA 94115. Cytogenetic analysis of solid minors is often difficult because of the scarcity or poor quality of metaphase spreads, or the complexity of marker chromosomes. When cytogenetics is su_ro~s_sful,double minums (DMs) and homogeneously staining regions (HSRs) are ohen found. The..s¢ indicate DNA amplification, but do not give any clue to the identity of the sequence. We have developed a new technique, comparative gcnomic hybridization (CGH), for detection and localization of DNA sequence copy number variation anywhere in the tumor gcmom¢. CGH is based on a simultaneous in situ hybridization of differentially labeled tumor and normal refexenc¢ DNA to normal m ~ chromosomes. The labeled DNAs are detected us/rig two different fluorochromes. The relative DNA seqmmce copy numbers of all regions in the tumor genome can then be quantitated by measuring the intensity ratios of the two fluorochromcs along each human chromosome. We studied 16 breast cancer o¢11 fines and 32 primary breast tumors using CGH and found several repeatedly amplified regions. Some of these regions cofreapond to the location of known oncogenes, such as MYC and ERBB2, but most of them where at loci where DNA amplification has not been previously d ~ b e d (e.g. 6q12, 7p21, 8q21-22, l l p l S , 15q23-25, 17q22-24, and 20o.). Suppoaed by NIH iffamsCA 45919. CA 44"/68and CA 47537, DOE contract DE-AC03-76SF00098, ami ImageaettcsInc. Napervil|e. IL.
7
THE USE OF IN SITU HYBRIDIZATION FOR M A K I N G IN VIVQ GENOTYPE/PHENOTYPE CORRELATES DURING H E A D A N D N E C K TUMORIGENESIS
Walter N. Hittelman, Narin Voravud, Dong M. Shin, Jae Y. Ro, Jin Soo Lee and Waun Ki Hong. The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA While cytogenetic and molecular abnormalities are now being described with increased detail for various solid tumors, there is still little information available regarding the pathophysioiogical role of these genetic changes during tumorigenesis. To address this issue, genetic and phenotypic analyses need to be carried out on intact tissue specimens. We have recently utilized chromosomespecific probes and ia sau hyhridization on formalin-fixed, paraffinembedded head and neck tumor tissue sections containing adjacent premalignant lesions m examine genetic changes occurring during head and neck tumorigenesis. Adjacent sections underwent immunohistochemical analyses for phenotypic changes associated with dysreguiated proliferation, differentiation, and/or cell loss. Genetic changes were detected in regions of normal histology adjacent to the tumors, suggesting a field cancerization process was ongoing in the head and neck region, increased genomic changes were observed as tissue histology passed through hyperplastic and dysplastic stages. These genetic changes were accompanied by phenotypic evidence of dysrogulation, providing direct evidence for a muitistep process. This approach will now allow a direct correlation to be made between specific genetic changes and their phenotypic consequences in vivo during tumorigenesis. Supported in part by NIH grants PO1 CA-52051, CA48364, and CA-45746.
MOLECULAR CELL GENETICS BY POLYMERASE 8 CATALYSED AMPLIFICATION OR IN SITU LABELLING OF SPECIFIC NUCLEIC ACID SEOUENCES. L. Bolund. C. Brandt. J. Hindklmr, J. Koch. S. K61vraa & S. Pedersen. Institute of Human Genetics, University of Aarhus, Denmark. The Polymerase Chain R e a c t i o n (PCR) can he performed on isolated cells or chromosomes, resulting in analysable product. The analysis can be performed by traditional DNA technology or by FISH to test metaphases. We have good experience with the analysis of aberrant chromosomes by FACS sorting, PCR amplification with degenerated primers and painting of test metsphases with the PCR product. We have also utilized polymerases for PRimed IN Situ labelling (PRINS) of specific nucleic acid s e ~ s in individual cells or chromoscaes. The ~ t h o d is based on the hybridisatlon of u n l a ~ l l e d synthetic o l i g o n u c l e o t i d e s to their c o m p l e m e n t a r y sequences ~n e i t u followed by incorporation of labelled nucleotides at the site of h y b r l d i s a t i o n w i t h the ollgonucleotide as primer. DNA or RNA leguencea are visualized by choice of p o l ~ r e s e . We use FRINS for differential atalnlng of closely r e l a t ~ DNA tandem repeat families, e. g. for chrcxaosome specific cen~E~re labelling in aneuploldy studlea o n ~ a ~ and Interphese cells. Moreover, u n i ~ DNA sequenc4s can be visualized w i t h ~ a £ 1 a of primers, whereas cells can be e t a £ ~ for specific mRNA species with a slr~le primer. We are trying to further increase the selectivity and sensitivity of the m e t h ~ to be able to analyse single gene targets a n d to detect selective allellc expression and somatic mutations ( by m R N A lahelling) in individual cells.