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Molecular cytogenetic analyses of 7Q31 in prostate cancer
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MULTIFACETED ASSESSMENT OF GENETIC ALTERATIONS IN ORALCANCER. SM Qollin, CM Lese, M Shuster, IC Law, CS Ishwad, KM Rossie, RE Ferrell, CM Croce, K Huebner, T Ried, EN Myers, and JT Johnson. University of Pittsburgh and the University of Pittsburgh Cancer Institute, Pittsburgh, PA; Kimmel Cancer Center and Thomas Jefferson Univ., Philadelphia, PA; National Center for Human Genome Research, Bethesda, MD. We are carrying out a hierarchical genetic analysis of oral squamous cell carcinoma and derived cell lines to identify frequent genetic alterations. Classical cytogenetic analyses of 30 cell lines identified common abnormalities involvingthree genomic regions: 3p, 9p, and 1lq. Karyotyping of 30 cell lines revealed numerical and/or structural abnormalities of chromosome 3 in 87 percent, loss of 9p in 47 percent, and amplification of llq13 in 53pereant. These regions were then examined by FISH, comparative genomic hybridization (CGH), and/or molecular genetic analysis, and specific protein products were assessed. By FISH, loss of exons 5 and/or 9-10 from FHIT, a putative tumor suppressor gene at 3p14.2, was observed in 21 of 27 cell lines (78 percent). Aberrant FI-I1Ttranscripts were detected by RT-PCR in 16 of 26 (62 percent) cell lines examined. 9p21 is the site of the pl6 or MTS1 or cyclin dependent kinase inhibitor 2 (CDKN2) gene. 9p21 loss, assessed by enumeration of Cos66 (a gifc from Dr. E Olopade) compared to ABL as a control, was seen in 12 of 15 cell lines (80 percent), 6 of 7 primary tumors, and 4 of 7 adjacent mucosal biopsies. Homozygous CDKN2 loss was confirmed by FISH in three selected matched primary tumors and in others by microsatellite analysis. CDKN2 mutations were identified in frozen tumors by SSCP and DNA sequencing. Amplification of llq13 (INT2) was observed by FISH analysis in 25 of 49 (51 percent) freshly harvested tumors and 19 of 34 cell lines (56 percent) and by CGH in 5 of 11 cell lines examined (45 percent). Cyclin DI overexpression was evident in tumor sections by immanocytochemistry and in amplified cell lines by Western blotting. FHIT, CDKN2, and one or more genes at llq13 appear to play key roles in the development and/or progression of oral carcinoma. The clinical and biologic significance of these findings and the utility of these markers for risk assessment and prognosis will be defined by continued studies of our patients and cell lines. [Supported by R01 DEI0513]. 26
CLONING OF A NOVEL TRANSCRIPTION FACTOR LIKE GENE AMPLIFIED IN HUMAN LOW AND HIGH GRADE GLIOMA U FischerI, D HeckelI, A Michel1, M Janka2, T Hulsebos3, and E Meese 1 IDepartment of Human Genetics, Medical School, Universityof Saar, D-66421 Homburg, Germany; 2Departmem of Neurosurgery, Medical School, 97080 Wiirzburg, Germany; 3Department of Human Genetics, AMC, 1105 AZ Amsterdam, The Netherlands Although gene amplification is a common feature of high grade glioma there are no data on amplificationin pilocytic astrocytoma but one case of Met amplification in an astrocytoma grade I1. In this study, we report cloning and sequencing of a full length eDNA termed glioma amplified sequence (GAS41) which was recently identified in a glioblastomacell line by microdissection mediated cDNA capture. GAS41 was found to be amplified not only in glioblastomamultiforme and astrocytoma III but also in astrocytoma grade I and II. Sequence comparison indicates a high homology between the GAS41 protein, yeast and human AF-9 protein and human ENL protein. Both AF-9 and ENL represent a new class of transcription factors. Our results strongly indicate that GAS 41 is a new transcription factor which is the In'st gene reported to be amplified early in the developmentof human glioma.
Abstracts
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MOLECULAR CYTOGENETIC ANALYSES OF 7Q31 IN PROSTATE CANCER. R Jenkins J Qian, H Lee, K Hirasawa, and M Lieber. Mayo Clinic, Rochester, MN Gains of chromosome 7 and alterations of the 7q-arm have been frequently observed in multiple cancers using various cytogeuetic and molecular genetic techniques. Using PCR analysis of microsatellite markers, we have previously reported that allelic imbalance of 7q31 is common in prostate cancer and is associated with tumors of high grade and stage (Takahashi et al Cancer Res 55:4114,1995). In an effort to better understand the chromosome 7 alterations and to map the minimal region of alteration, we have begun a molecular cytogenetic analysis of prostate cancer specimens with FISH probes mapped to 7q31 (D7S486 and D7S522) and to the chromosome 7 centromere. We have completed analysis of 25 tumors. Six had no apparent anomaly of chromosome 7. Among the remaining tumors, we observed 8 and 1 with simple gain and loss of chromosome 7, respectively. Two tumors had gain of the chromosome 7 cantromere and relative loss of 7q31. Five tumors had gain of the chromosome 7 centromere and additional overrepresentation of 7q31. Two tumors had overrepresentation of 7q31 without any apparent anomaly of the chromosome 7 centromere. We conclude that alterations of chromosome 7, and particularly 7q31, are common in prostate cancer. Unexpectedly, overrepresentation (gain) of 7q31 was much more common than deletions of 7q31. Careful multiplex PCR studies of some of these specimens support these in situ hybridization observations. Additional studies will be necessary to further map the extent of the 7q31 alterations and to correlate these alterations with clinical and pathologic parameters. (Supported by CA58225) 28
DISCRIMINATION BETWEEN MULTICENTRIC AND MULTIFOCAL BREAST CARCINOMA BY CYTOGENETIC INVESTIGATION OF MACROSCOPICALLY DISTINCT IPSILATERAL LESIONS MR Teixeira. N Pandis, and S Heim Department of Genetics, The Norwegian Radium Hospital and Institute for Cancer Research, Oslo, Norway; Department of Genetics, Saint Savas Hospital,Athens, Greece Whether macroscopically distinct carcinomas in the same breast are clonally related (multifocal breast carcinoma) or unrelated (multicentricbreast carcinoma) is no longer only a scientificpathological issue but, because differenttherapeutic strategies may be preferable for cases with intramammary metastatic disease compared with cases of multiple primary breast carcinomas, one that may have profound clinical implications.We studied the evolutionaryrelationship among macroscopically distinct, ipsilateralbreast carcinomas by cytogenetic analysis of 37 tumorous lesions from 17 patients. Twenty-seven of the 37 foci (73 percent) were found to contain chinal chromosome abnormalities.At least two carcinoma loci were karyotypicallyabnormal in each of twelve patients. Nine of these cases (75 percent) had an evohitionarilyrelated, cytogenetically abnormal clone in the different lesions from the same breast, whereas the remaining three cases (25 percent) had completelydifferentclonal karyotypic aberrations in the separate foci. These results show that multiple, synchronous breast tumors sometimesarise through intramammary spreading of a single primary carcinoma, whereas on other occasions they are the result of the simultaneous emergence of pathogeneticallyindependent carcinomas within the breast. An association was seen between the proximity of the foci and the likelihood of them being karyotypically related.
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MULTIFACETED ASSESSMENT OF GENETIC ALTERATIONS IN ORALCANCER. SM Qollin, CM Lese, M Shuster, IC Law, CS Ishwad, KM Rossie, RE Ferrell, CM Croce, K Huebner, T Ried, EN Myers, and JT Johnson. University of Pittsburgh and the University of Pittsburgh Cancer Institute, Pittsburgh, PA; Kimmel Cancer Center and Thomas Jefferson Univ., Philadelphia, PA; National Center for Human Genome Research, Bethesda, MD. We are carrying out a hierarchical genetic analysis of oral squamous cell carcinoma and derived cell lines to identify frequent genetic alterations. Classical cytogenetic analyses of 30 cell lines identified common abnormalities involvingthree genomic regions: 3p, 9p, and 1lq. Karyotyping of 30 cell lines revealed numerical and/or structural abnormalities of chromosome 3 in 87 percent, loss of 9p in 47 percent, and amplification of llq13 in 53pereant. These regions were then examined by FISH, comparative genomic hybridization (CGH), and/or molecular genetic analysis, and specific protein products were assessed. By FISH, loss of exons 5 and/or 9-10 from FHIT, a putative tumor suppressor gene at 3p14.2, was observed in 21 of 27 cell lines (78 percent). Aberrant FI-I1Ttranscripts were detected by RT-PCR in 16 of 26 (62 percent) cell lines examined. 9p21 is the site of the pl6 or MTS1 or cyclin dependent kinase inhibitor 2 (CDKN2) gene. 9p21 loss, assessed by enumeration of Cos66 (a gifc from Dr. E Olopade) compared to ABL as a control, was seen in 12 of 15 cell lines (80 percent), 6 of 7 primary tumors, and 4 of 7 adjacent mucosal biopsies. Homozygous CDKN2 loss was confirmed by FISH in three selected matched primary tumors and in others by microsatellite analysis. CDKN2 mutations were identified in frozen tumors by SSCP and DNA sequencing. Amplification of llq13 (INT2) was observed by FISH analysis in 25 of 49 (51 percent) freshly harvested tumors and 19 of 34 cell lines (56 percent) and by CGH in 5 of 11 cell lines examined (45 percent). Cyclin DI overexpression was evident in tumor sections by immanocytochemistry and in amplified cell lines by Western blotting. FHIT, CDKN2, and one or more genes at llq13 appear to play key roles in the development and/or progression of oral carcinoma. The clinical and biologic significance of these findings and the utility of these markers for risk assessment and prognosis will be defined by continued studies of our patients and cell lines. [Supported by R01 DEI0513]. 26
CLONING OF A NOVEL TRANSCRIPTION FACTOR LIKE GENE AMPLIFIED IN HUMAN LOW AND HIGH GRADE GLIOMA U FischerI, D HeckelI, A Michel1, M Janka2, T Hulsebos3, and E Meese 1 IDepartment of Human Genetics, Medical School, Universityof Saar, D-66421 Homburg, Germany; 2Departmem of Neurosurgery, Medical School, 97080 Wiirzburg, Germany; 3Department of Human Genetics, AMC, 1105 AZ Amsterdam, The Netherlands Although gene amplification is a common feature of high grade glioma there are no data on amplificationin pilocytic astrocytoma but one case of Met amplification in an astrocytoma grade I1. In this study, we report cloning and sequencing of a full length eDNA termed glioma amplified sequence (GAS41) which was recently identified in a glioblastomacell line by microdissection mediated cDNA capture. GAS41 was found to be amplified not only in glioblastomamultiforme and astrocytoma III but also in astrocytoma grade I and II. Sequence comparison indicates a high homology between the GAS41 protein, yeast and human AF-9 protein and human ENL protein. Both AF-9 and ENL represent a new class of transcription factors. Our results strongly indicate that GAS 41 is a new transcription factor which is the In'st gene reported to be amplified early in the developmentof human glioma.
Abstracts
27
MOLECULAR CYTOGENETIC ANALYSES OF 7Q31 IN PROSTATE CANCER. R Jenkins J Qian, H Lee, K Hirasawa, and M Lieber. Mayo Clinic, Rochester, MN Gains of chromosome 7 and alterations of the 7q-arm have been frequently observed in multiple cancers using various cytogeuetic and molecular genetic techniques. Using PCR analysis of microsatellite markers, we have previously reported that allelic imbalance of 7q31 is common in prostate cancer and is associated with tumors of high grade and stage (Takahashi et al Cancer Res 55:4114,1995). In an effort to better understand the chromosome 7 alterations and to map the minimal region of alteration, we have begun a molecular cytogenetic analysis of prostate cancer specimens with FISH probes mapped to 7q31 (D7S486 and D7S522) and to the chromosome 7 centromere. We have completed analysis of 25 tumors. Six had no apparent anomaly of chromosome 7. Among the remaining tumors, we observed 8 and 1 with simple gain and loss of chromosome 7, respectively. Two tumors had gain of the chromosome 7 cantromere and relative loss of 7q31. Five tumors had gain of the chromosome 7 centromere and additional overrepresentation of 7q31. Two tumors had overrepresentation of 7q31 without any apparent anomaly of the chromosome 7 centromere. We conclude that alterations of chromosome 7, and particularly 7q31, are common in prostate cancer. Unexpectedly, overrepresentation (gain) of 7q31 was much more common than deletions of 7q31. Careful multiplex PCR studies of some of these specimens support these in situ hybridization observations. Additional studies will be necessary to further map the extent of the 7q31 alterations and to correlate these alterations with clinical and pathologic parameters. (Supported by CA58225) 28
DISCRIMINATION BETWEEN MULTICENTRIC AND MULTIFOCAL BREAST CARCINOMA BY CYTOGENETIC INVESTIGATION OF MACROSCOPICALLY DISTINCT IPSILATERAL LESIONS MR Teixeira. N Pandis, and S Heim Department of Genetics, The Norwegian Radium Hospital and Institute for Cancer Research, Oslo, Norway; Department of Genetics, Saint Savas Hospital,Athens, Greece Whether macroscopically distinct carcinomas in the same breast are clonally related (multifocal breast carcinoma) or unrelated (multicentricbreast carcinoma) is no longer only a scientificpathological issue but, because differenttherapeutic strategies may be preferable for cases with intramammary metastatic disease compared with cases of multiple primary breast carcinomas, one that may have profound clinical implications.We studied the evolutionaryrelationship among macroscopically distinct, ipsilateralbreast carcinomas by cytogenetic analysis of 37 tumorous lesions from 17 patients. Twenty-seven of the 37 foci (73 percent) were found to contain chinal chromosome abnormalities.At least two carcinoma loci were karyotypicallyabnormal in each of twelve patients. Nine of these cases (75 percent) had an evohitionarilyrelated, cytogenetically abnormal clone in the different lesions from the same breast, whereas the remaining three cases (25 percent) had completelydifferentclonal karyotypic aberrations in the separate foci. These results show that multiple, synchronous breast tumors sometimesarise through intramammary spreading of a single primary carcinoma, whereas on other occasions they are the result of the simultaneous emergence of pathogeneticallyindependent carcinomas within the breast. An association was seen between the proximity of the foci and the likelihood of them being karyotypically related.