- Email: [email protected]
Molecular basis of benign mesenchymal tumors
133
Abstracts IN SITU HYSRIDIZATION 15 TLUOREStXNCE t(2;lB) IN FOR
TEE
PRESENCE
ASSAY
OF
16
wABDoMYos~cw4. Viars, CS , Cavenee, WK, Arde", KC, Anderson MA; Ludwig Institute for Cancer Research, San Diego Branch and Department of Medicine, University of California, San Diego, CA 92093-0660 Differential diagnosis among the small round cell tumors of childhood can be particularly difficult due to their primitive or undifferentiated state. Rhabdomyosarcoma, a tumor of skeletal muscle and the most cormno" soft tissue neoplasm found in children, is a member of this group of tumors. The alveolar subtype has been shown to carry a specific and unique reciprocal chromosome translocation, t(2;13j (q35;q14j which disrupts the PAX3 gene on chromosome 2 and the FKHR gene on chromosome 13 and generates two new fusion genes. We have used two color fluorescence in situ hybridization (FISH) with genomic clones encompassing the breakpoints in the PAX3 and FKHR genes to detect the presence of the PAX3/FKHR and FKHR/PAX3 fusions. This technique was applied to metaphase spreads and interphase nuclei prepared from normal lymphocytes and 6 rhabdomyosarcoma cell lines. Both metaphase and interphase analysis detected the presence of the t(2;13) in the 5 tumor cell lines known to have the t(2:13) by karyotypic analysis. These studies demonstrate the potential use of this technique for rapid differential diagnosis by FISH particularly when DNA and RNA are not available for molecular analysis.
DELETION MAPPING AND CANDIDATE GENE ANALYSIS OF CHROMOSOME 17 IN PRIMITIVE NEIJROECTODERMAL TUMORS OF THE CNS Jaclyn Biegcl. Annette Parmiter, Ignacio Rodriguez, Krzyzstof Mazuruk. Gerald Chader and Irene Slave. Children’s Hospital of Philadelphia, Philadelphia, PA and National Eye Institute, NII-I, Bethesda, MD.
17
The most frequent chromosomal abnormality in pediatric PNETs of the central nervous system is an i(17q). Molecular studies have confirmed LOH of 17~ in PNETs and suggest that a region in 17~13. distal to ~53, contains a tumor-suppressor gene. As a rapid means of identifying tumors with deletions of 17~. and specifically an i(17q). we have utilized two-color interphase FISH on primary brain tumor specimens. Thirty-six PNETs were analyzed with a series of chromosome 17-specific cosmids. The copy number for each 17p and 17q probe was determined relative to the number of signals for the 17 centromere probe. Karyotypes were prepared from each case for comparison with the FISH results. Cytogenetic studies were successful in 21 of 36 cases. Results demonstrated deletion of 17~ in 2 cases and an i(17q) in 10 cases, which were confirmed by FISH. Two additional cases with deletion of 17~ and 5 tumors with an i(17q) were demonstrated by FISH alone. Loss of 17p was seen in a total of 19 of the 36 (53%) PNETs. FISH with chromosome 17specific cosmids is a useful adjunct to the pathologic diagnosis of PNETs. and will enable us to define a critical regton on 17p which should contain a tumor-related gene. Pigment epithelium derived factor (PEDF) is a member of the serpin gene family. It maps to 17~13 and has been shown to induce neuronal differentiation in Y79 retinoblastoma cells. We have recently initiated studies to explore the potential role of PEDF as a candidate gene for PNETs. We developed SSCP assays for each of the 8 exons of the PEDF gene, and identified Polymorphisms in exons 4 and 7. Loss of heterozygosity for exons 4 or 7 was demonstrated in 10 of 20 PNETs. Matched normal blood and tumor DNAs are being sequenced to determine if there are mutations in the coding sequence.
VISUAL ANALYSIS OF DNA REARRANGEMENT IN SMALL CELL LUNG CANCER (SCLC) BY FISH TO EXTENDED DNA
Mervi Heiskanen, Elina Hellsten, Olli-Pekka Kalliomemil, Tomi MakeIll, Kari Alitalo3 and Aamo Palotie Department of Clinical Chemistry, University of Helsinki, Helsinki, Finland and ILaboratory of Cancer Genetics, Tampere University Hospital, Tampere, Finland and 2Whitehead Institure for Biomedical Research, Cambridge, USA and 3Laboratory of Cancer Biology, Departments of Virology and Pathology, University of Helsinki, Finland. Several SCLC cell lines and also primary carcinomas are characterized by extensive amplification of L-myc protooncogene on 1~32. MLkelH et al described a chimeric protein where the aminoterminal sequences from a novel rlfgene were loined to the L-myc protein. Further characterization of this rearrangement revealed that the regulatory region and at least the first exon of rlf was placed upstream L-myc and this fusion gene had also been involved in DNA amplification (Mlkela et al 1992). We have further analyzed this rearrangement using new high resolution FISH technique, which allows visual analysis of gene loci on DNA fibers (Heiskanen et al 1994). By fiber FISH we were able to determine an inverted orientation of the rearranged rlf-gene in two SCLC cell hnes. Our results show the usefullness of fiber FISH in the structural characterization of amplicons. Make]& T.P., Shiraishi, M., Borrello, M., Sekiya, T. and Alitalo, K. (1992) Oncogene 7~405-409. Heiskanen, M., Karhu, R., Hellsten, E., Peltonen. L., Kallioniemi, O-P and Palotie, A. (1994) BioTechniques 17:928-934
18
HOLECULAR BASIS OF BENIGN MESENCXYNAL
1: “~::,anlL,“.
TUMOR9
yr&llJy;~s’~-f~“~~
Bartnitzke. H. Van den Berghe, and J. Bullerdick. Center For Human Genetics (WDV. HS. PK. JG. lf.'DB), University of Leuven. Belgium; and Center for Human Genetics (SW. BK. SB, JB), University of Bremen. GerUlany. Chromosome translocations involving region q13-q15 of chromosome 12 have been observed in a wide variety of solid tumors. In cytogenetically abnormal uterine leiomyomas. pleomorphic salivary gland adenomas. and benign adipose tissue tumors, 12ql3-q15 aberrations are frequently observed. In a recent study, we have identified and molecularly characterized ULCR12. the uterine leiomyoma cluster region of chromosome 12 breakpoints (Schoenmakers et al., Genes Chromosomes 6 Cancer 11, 106-118, 1994j. Here, we report results of further molecular cytogenetic studies of these three distinct benign tumor types. FISH analysis of these tumors using probes derived from a 2.8 Mb genomic DNA region of chromosome segment 12ql3-q15 encompassing ULCR12 revealed that the 12q breakpoints of all three tumor types are clustering in the same DNA region, which we therefore have designated HAi7 (nultiple Aberration Region) (Van de Ven et al.. Genes Chromosomes h Cancer, In press). Similar FISH analysis revealed that the MAR region is also involved in other mesenchymal tumors. Using various approaches, genes residing in the HAR region were identified and The molecularly characterized. possible involvement of MAR genes in benign mesenchymal fumors will be discussed.
Abstracts IN SITU HYSRIDIZATION 15 TLUOREStXNCE t(2;lB) IN FOR
TEE
PRESENCE
ASSAY
OF
16
wABDoMYos~cw4. Viars, CS , Cavenee, WK, Arde", KC, Anderson MA; Ludwig Institute for Cancer Research, San Diego Branch and Department of Medicine, University of California, San Diego, CA 92093-0660 Differential diagnosis among the small round cell tumors of childhood can be particularly difficult due to their primitive or undifferentiated state. Rhabdomyosarcoma, a tumor of skeletal muscle and the most cormno" soft tissue neoplasm found in children, is a member of this group of tumors. The alveolar subtype has been shown to carry a specific and unique reciprocal chromosome translocation, t(2;13j (q35;q14j which disrupts the PAX3 gene on chromosome 2 and the FKHR gene on chromosome 13 and generates two new fusion genes. We have used two color fluorescence in situ hybridization (FISH) with genomic clones encompassing the breakpoints in the PAX3 and FKHR genes to detect the presence of the PAX3/FKHR and FKHR/PAX3 fusions. This technique was applied to metaphase spreads and interphase nuclei prepared from normal lymphocytes and 6 rhabdomyosarcoma cell lines. Both metaphase and interphase analysis detected the presence of the t(2;13) in the 5 tumor cell lines known to have the t(2:13) by karyotypic analysis. These studies demonstrate the potential use of this technique for rapid differential diagnosis by FISH particularly when DNA and RNA are not available for molecular analysis.
DELETION MAPPING AND CANDIDATE GENE ANALYSIS OF CHROMOSOME 17 IN PRIMITIVE NEIJROECTODERMAL TUMORS OF THE CNS Jaclyn Biegcl. Annette Parmiter, Ignacio Rodriguez, Krzyzstof Mazuruk. Gerald Chader and Irene Slave. Children’s Hospital of Philadelphia, Philadelphia, PA and National Eye Institute, NII-I, Bethesda, MD.
17
The most frequent chromosomal abnormality in pediatric PNETs of the central nervous system is an i(17q). Molecular studies have confirmed LOH of 17~ in PNETs and suggest that a region in 17~13. distal to ~53, contains a tumor-suppressor gene. As a rapid means of identifying tumors with deletions of 17~. and specifically an i(17q). we have utilized two-color interphase FISH on primary brain tumor specimens. Thirty-six PNETs were analyzed with a series of chromosome 17-specific cosmids. The copy number for each 17p and 17q probe was determined relative to the number of signals for the 17 centromere probe. Karyotypes were prepared from each case for comparison with the FISH results. Cytogenetic studies were successful in 21 of 36 cases. Results demonstrated deletion of 17~ in 2 cases and an i(17q) in 10 cases, which were confirmed by FISH. Two additional cases with deletion of 17~ and 5 tumors with an i(17q) were demonstrated by FISH alone. Loss of 17p was seen in a total of 19 of the 36 (53%) PNETs. FISH with chromosome 17specific cosmids is a useful adjunct to the pathologic diagnosis of PNETs. and will enable us to define a critical regton on 17p which should contain a tumor-related gene. Pigment epithelium derived factor (PEDF) is a member of the serpin gene family. It maps to 17~13 and has been shown to induce neuronal differentiation in Y79 retinoblastoma cells. We have recently initiated studies to explore the potential role of PEDF as a candidate gene for PNETs. We developed SSCP assays for each of the 8 exons of the PEDF gene, and identified Polymorphisms in exons 4 and 7. Loss of heterozygosity for exons 4 or 7 was demonstrated in 10 of 20 PNETs. Matched normal blood and tumor DNAs are being sequenced to determine if there are mutations in the coding sequence.
VISUAL ANALYSIS OF DNA REARRANGEMENT IN SMALL CELL LUNG CANCER (SCLC) BY FISH TO EXTENDED DNA
Mervi Heiskanen, Elina Hellsten, Olli-Pekka Kalliomemil, Tomi MakeIll, Kari Alitalo3 and Aamo Palotie Department of Clinical Chemistry, University of Helsinki, Helsinki, Finland and ILaboratory of Cancer Genetics, Tampere University Hospital, Tampere, Finland and 2Whitehead Institure for Biomedical Research, Cambridge, USA and 3Laboratory of Cancer Biology, Departments of Virology and Pathology, University of Helsinki, Finland. Several SCLC cell lines and also primary carcinomas are characterized by extensive amplification of L-myc protooncogene on 1~32. MLkelH et al described a chimeric protein where the aminoterminal sequences from a novel rlfgene were loined to the L-myc protein. Further characterization of this rearrangement revealed that the regulatory region and at least the first exon of rlf was placed upstream L-myc and this fusion gene had also been involved in DNA amplification (Mlkela et al 1992). We have further analyzed this rearrangement using new high resolution FISH technique, which allows visual analysis of gene loci on DNA fibers (Heiskanen et al 1994). By fiber FISH we were able to determine an inverted orientation of the rearranged rlf-gene in two SCLC cell hnes. Our results show the usefullness of fiber FISH in the structural characterization of amplicons. Make]& T.P., Shiraishi, M., Borrello, M., Sekiya, T. and Alitalo, K. (1992) Oncogene 7~405-409. Heiskanen, M., Karhu, R., Hellsten, E., Peltonen. L., Kallioniemi, O-P and Palotie, A. (1994) BioTechniques 17:928-934
18
HOLECULAR BASIS OF BENIGN MESENCXYNAL
1: “~::,anlL,“.
TUMOR9
yr&llJy;~s’~-f~“~~
Bartnitzke. H. Van den Berghe, and J. Bullerdick. Center For Human Genetics (WDV. HS. PK. JG. lf.'DB), University of Leuven. Belgium; and Center for Human Genetics (SW. BK. SB, JB), University of Bremen. GerUlany. Chromosome translocations involving region q13-q15 of chromosome 12 have been observed in a wide variety of solid tumors. In cytogenetically abnormal uterine leiomyomas. pleomorphic salivary gland adenomas. and benign adipose tissue tumors, 12ql3-q15 aberrations are frequently observed. In a recent study, we have identified and molecularly characterized ULCR12. the uterine leiomyoma cluster region of chromosome 12 breakpoints (Schoenmakers et al., Genes Chromosomes 6 Cancer 11, 106-118, 1994j. Here, we report results of further molecular cytogenetic studies of these three distinct benign tumor types. FISH analysis of these tumors using probes derived from a 2.8 Mb genomic DNA region of chromosome segment 12ql3-q15 encompassing ULCR12 revealed that the 12q breakpoints of all three tumor types are clustering in the same DNA region, which we therefore have designated HAi7 (nultiple Aberration Region) (Van de Ven et al.. Genes Chromosomes h Cancer, In press). Similar FISH analysis revealed that the MAR region is also involved in other mesenchymal tumors. Using various approaches, genes residing in the HAR region were identified and The molecularly characterized. possible involvement of MAR genes in benign mesenchymal fumors will be discussed.