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Deletion map of chromosome 16q in ductal carcinoma in situ of the breast: refining a putative tumor suppressor gene region
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CLONING OF GENES INVOLVED IN CANCER PROGRESSION BY A COMBINATION OF FUNCTIONAL AND POSITIONAL CLONING JC B0rrett, N a t i o n a l Institute o f E n v i r o n m e n t a l H e a l t h Sciences, N a t i o n a l Institutes of Health, R e s e a r c h T r i a n g l e Park, N C L i n k a g e o f disease waits in f a m i l i e s to genetic m a r k e r s has led to the isolation of many important genes t h r o u g h positional cloning. H o w e v e r , at-risk f a m i l i e s are not available for all diseases a n d certain disease g e n e s m a y not c o n f e r a genetic predisposition and be altered only in somatic tissues. Identification o f genes by their function can be studied in somatic cells in culture i f a c e l l u l a r phenotype can be associated w i t h the gene. M i c r o c e l l - m e d i a t e d c h r o m o s o m e transfer can be used to map g e n e s i n v o l v e d in a n u m b e r od diseases and c e l l u l a r processes. O n c e the gene has b e e n m a p p e d to a s p e c i f i c c h r o m o s o m e , then a variety o f methods can be used to further define the p o s i t i o n o f the g e n e to a l l o w its isolation. G e n e s involved in the escape from c e l l u l a r s e n e s c e n c e ( i m m o r t a l i z a t i o n ) and suppression o f metastasis have been i d e n t i f i e d by c o m b i n e d functional/positional cloning. N e w approaches to i m p r o v e this method o f functional/positional c l o n i n g w i l l be discussed.
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MICRODISSECTING THE MOLECULAR STAGES OF CANCER PROGRESSION, INVASION AND METASTASIS M Emmert Buck, Z Zhuang, J Trent, R Bonner, R Chuaqui, D Krizman, and LA Liotta. National Institutes of Health, Bethesda, Md. USA. The major obstacle to PCR-hased molecular analysis of tissue is the need to isolate the microscopic cellular populations of interest away from the vast majority of contaminating ceils. Under direct microscopic visualization, Laser Capture Microdissection (LCM) permits rapid one step procurement of selected cell populations directly from a section of complex heterogeneous tissue. A transparent ethylene vinyl acetate (e.v.a.) thermoplastic film is applied to the surface of the tissue section on a glass slide held in a standard microscope stage. The operator moves the slide under a fixed pulsed laser which irradiates the e.v.a, film above the chosen cells. The laser pulse energy (100-200 mJ; 10.6 ,urn) is absorbed (>99 percent) by the film which adheres tightly the under/ying selected cells, which are selectively procured when the film is removed directly into PCR anaplification buffer. LCM can be applied to fixed stained tissue sections with excellent preservation of protein, DNA, and RNA, and has general utility for chip based genetic panel testing. From a single patient's tissue section RNA or DNA can be procured from normal epithelium, hyperplasia, in situ carcinoma, and invasive carcinoma, The timing and persistence during progression of somatic genetic alterations or gene expression fluctuations can be analyzed. Microdissection based LOH analysis has revealed genetic loci on llq13 and 8p21 which are first detected in cancer precursor stages. Microdissection has now permitted the development of eDNA libraries representing normal epithelium, atypical hyperplasia, in situ, and invasive lesions all from the same patient. Microdissection derived eDNA libraries have been developed for prostate. Snap shot eDNA libraries of microdissected human tumor premalignant stages can impact on the search for specific patterns of gene expression which correlate with progression, invasion, or response to therapy.
Abstracts
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D E L E T I O N M A P O F C H R O M O S O M E 16q IN D U C T A L C A R C I N O M A IN SITU OF THE BREAST: REFINING A PUTATIVE TUMOR SUPPRESSOR GENE REGION TChen, A Sahin, and CM Aldaz Department of Carcinogenesis, The University of Texas M.D. Anderson Cancer Center, Science Park, Research Division, Smithville, T X 78957 Allelic losses or imbalances affecting chromosome arm 16q appear to be early genomic abnormalities in breast carcinogenesis since they were observed in a significant number of breast ductal carcinoma in situ (DCIS) lesions in our previous study. To define the minimum region of loss of heterozygosity (LOH), we generated a high-resolution allelotype of 35 DCIS cases and completed a deletion map of chromosome 16q by means of paraffinembedded tissue microdissection and PCR microsa~llite analysis of 22 markers. We observed a strikingly high frequency of LOH in 16q, with 31 of 35 tumors (89 percent) affected. We identified three distinctive areas with high LOH. Two areas were previously described and correspond to 16q21 and 16q24.2-qter. The third and most commonly affected area spanned the region from marker D16S515 to D16S504. The most affected locus was at D16S518, in which LOH was observed in 20 of 26 informative cases (77 percent), and we estimate that it lies in sub-region q23.3-q24.1. The region of highest LOH spanned approximately 2 Mb, as determined by a YAC contig that covers this region. A human BAC contig has also been constructed spanning the region of interest. Microsatellite and STS makers were also used to screen human breast cancer lines in order to identify areas of homozygous loss. The high frequency of LOH observed at a preinvasive stage of breast cancer suggests that a candidate tumor suppressor gene or genes at this location may play an important role in breast carcinogenesis. 36
GENETIC BASIS OF A HISTOGENETIC M O D E L OF TUMORIGENESIS OF EPITHELIAL RENAL TUMORS HJ Decker, R Schwerdtle, J Brieger, E Weidt, F Grian, C Neuhaus§, C Puranakanitstha, S St6rkel °, W Brenner*, C Huber. II1. Med., Dep. Hematology, * Urological Clinic, University of Malnz, § new position: Aristogen GmbH, Ingelheim, ° Inst. Pathology, Wuppertal/University of Diisseldorf, GERMANY. The WHO classification system does not reflect the high extent of differences in tumorhiology and in clinical outcome of renal tumors. It is insufficient for predicting prognosis and for adequate treatment stratification. A new system has been proposed by Thoenes et al. based on immunohistochemistry. Here, we present a classification system based on genetic characterization perfectly corresponding to the 'Malnzer System' of Thoenes et al. We have studied more than 180 renal tumors including all four major subtypes applying various genetic methods such as classical tumor Cytogenetics, fluorescence in situ hybridization (FISH), restriction fragment lengths polymorphism (RFLP) and microsatallite analysis of more than 10 chromosomal regions, mutational analysis of the yon HippeI Lindau (VHL) tumor suppressor gene by PCPdSSCP/direct sequencing, RT-PCR and Northern analysis of the VHL gene. According to our findings there are genetic equivalents for the immunohistogenetic classification system of renal tumors, defining at least four genetic subgroups, such as clear cell, ehromophilic, chromophobe subtypes and oncocytomas. There is also a prognostic significance of this classification system as the latter two subtypes clearly show a more favorable outcome compared to the poor prognosis of the clear cell and the chromophilic subtypes. We think that defining the genetic changes in these clinically relevant subgroups will be an extremely helpful tool for understanding tumorigenesis and developing new strategies for the management of renal tumors.
33
CLONING OF GENES INVOLVED IN CANCER PROGRESSION BY A COMBINATION OF FUNCTIONAL AND POSITIONAL CLONING JC B0rrett, N a t i o n a l Institute o f E n v i r o n m e n t a l H e a l t h Sciences, N a t i o n a l Institutes of Health, R e s e a r c h T r i a n g l e Park, N C L i n k a g e o f disease waits in f a m i l i e s to genetic m a r k e r s has led to the isolation of many important genes t h r o u g h positional cloning. H o w e v e r , at-risk f a m i l i e s are not available for all diseases a n d certain disease g e n e s m a y not c o n f e r a genetic predisposition and be altered only in somatic tissues. Identification o f genes by their function can be studied in somatic cells in culture i f a c e l l u l a r phenotype can be associated w i t h the gene. M i c r o c e l l - m e d i a t e d c h r o m o s o m e transfer can be used to map g e n e s i n v o l v e d in a n u m b e r od diseases and c e l l u l a r processes. O n c e the gene has b e e n m a p p e d to a s p e c i f i c c h r o m o s o m e , then a variety o f methods can be used to further define the p o s i t i o n o f the g e n e to a l l o w its isolation. G e n e s involved in the escape from c e l l u l a r s e n e s c e n c e ( i m m o r t a l i z a t i o n ) and suppression o f metastasis have been i d e n t i f i e d by c o m b i n e d functional/positional cloning. N e w approaches to i m p r o v e this method o f functional/positional c l o n i n g w i l l be discussed.
34
MICRODISSECTING THE MOLECULAR STAGES OF CANCER PROGRESSION, INVASION AND METASTASIS M Emmert Buck, Z Zhuang, J Trent, R Bonner, R Chuaqui, D Krizman, and LA Liotta. National Institutes of Health, Bethesda, Md. USA. The major obstacle to PCR-hased molecular analysis of tissue is the need to isolate the microscopic cellular populations of interest away from the vast majority of contaminating ceils. Under direct microscopic visualization, Laser Capture Microdissection (LCM) permits rapid one step procurement of selected cell populations directly from a section of complex heterogeneous tissue. A transparent ethylene vinyl acetate (e.v.a.) thermoplastic film is applied to the surface of the tissue section on a glass slide held in a standard microscope stage. The operator moves the slide under a fixed pulsed laser which irradiates the e.v.a, film above the chosen cells. The laser pulse energy (100-200 mJ; 10.6 ,urn) is absorbed (>99 percent) by the film which adheres tightly the under/ying selected cells, which are selectively procured when the film is removed directly into PCR anaplification buffer. LCM can be applied to fixed stained tissue sections with excellent preservation of protein, DNA, and RNA, and has general utility for chip based genetic panel testing. From a single patient's tissue section RNA or DNA can be procured from normal epithelium, hyperplasia, in situ carcinoma, and invasive carcinoma, The timing and persistence during progression of somatic genetic alterations or gene expression fluctuations can be analyzed. Microdissection based LOH analysis has revealed genetic loci on llq13 and 8p21 which are first detected in cancer precursor stages. Microdissection has now permitted the development of eDNA libraries representing normal epithelium, atypical hyperplasia, in situ, and invasive lesions all from the same patient. Microdissection derived eDNA libraries have been developed for prostate. Snap shot eDNA libraries of microdissected human tumor premalignant stages can impact on the search for specific patterns of gene expression which correlate with progression, invasion, or response to therapy.
Abstracts
35
D E L E T I O N M A P O F C H R O M O S O M E 16q IN D U C T A L C A R C I N O M A IN SITU OF THE BREAST: REFINING A PUTATIVE TUMOR SUPPRESSOR GENE REGION TChen, A Sahin, and CM Aldaz Department of Carcinogenesis, The University of Texas M.D. Anderson Cancer Center, Science Park, Research Division, Smithville, T X 78957 Allelic losses or imbalances affecting chromosome arm 16q appear to be early genomic abnormalities in breast carcinogenesis since they were observed in a significant number of breast ductal carcinoma in situ (DCIS) lesions in our previous study. To define the minimum region of loss of heterozygosity (LOH), we generated a high-resolution allelotype of 35 DCIS cases and completed a deletion map of chromosome 16q by means of paraffinembedded tissue microdissection and PCR microsa~llite analysis of 22 markers. We observed a strikingly high frequency of LOH in 16q, with 31 of 35 tumors (89 percent) affected. We identified three distinctive areas with high LOH. Two areas were previously described and correspond to 16q21 and 16q24.2-qter. The third and most commonly affected area spanned the region from marker D16S515 to D16S504. The most affected locus was at D16S518, in which LOH was observed in 20 of 26 informative cases (77 percent), and we estimate that it lies in sub-region q23.3-q24.1. The region of highest LOH spanned approximately 2 Mb, as determined by a YAC contig that covers this region. A human BAC contig has also been constructed spanning the region of interest. Microsatellite and STS makers were also used to screen human breast cancer lines in order to identify areas of homozygous loss. The high frequency of LOH observed at a preinvasive stage of breast cancer suggests that a candidate tumor suppressor gene or genes at this location may play an important role in breast carcinogenesis. 36
GENETIC BASIS OF A HISTOGENETIC M O D E L OF TUMORIGENESIS OF EPITHELIAL RENAL TUMORS HJ Decker, R Schwerdtle, J Brieger, E Weidt, F Grian, C Neuhaus§, C Puranakanitstha, S St6rkel °, W Brenner*, C Huber. II1. Med., Dep. Hematology, * Urological Clinic, University of Malnz, § new position: Aristogen GmbH, Ingelheim, ° Inst. Pathology, Wuppertal/University of Diisseldorf, GERMANY. The WHO classification system does not reflect the high extent of differences in tumorhiology and in clinical outcome of renal tumors. It is insufficient for predicting prognosis and for adequate treatment stratification. A new system has been proposed by Thoenes et al. based on immunohistochemistry. Here, we present a classification system based on genetic characterization perfectly corresponding to the 'Malnzer System' of Thoenes et al. We have studied more than 180 renal tumors including all four major subtypes applying various genetic methods such as classical tumor Cytogenetics, fluorescence in situ hybridization (FISH), restriction fragment lengths polymorphism (RFLP) and microsatallite analysis of more than 10 chromosomal regions, mutational analysis of the yon HippeI Lindau (VHL) tumor suppressor gene by PCPdSSCP/direct sequencing, RT-PCR and Northern analysis of the VHL gene. According to our findings there are genetic equivalents for the immunohistogenetic classification system of renal tumors, defining at least four genetic subgroups, such as clear cell, ehromophilic, chromophobe subtypes and oncocytomas. There is also a prognostic significance of this classification system as the latter two subtypes clearly show a more favorable outcome compared to the poor prognosis of the clear cell and the chromophilic subtypes. We think that defining the genetic changes in these clinically relevant subgroups will be an extremely helpful tool for understanding tumorigenesis and developing new strategies for the management of renal tumors.