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The use of fluorescence in situ hybridization (FISH) to predict radiosensitivity of human tumor cell lines
166
Abstracts
EVALUATION OF [NTERPHASE FLUORESCENCE IN SITU HYBRIDIZATION (FISH) IN DNA MALIGNANCY GRADING AND COMPARISON TO STATIC DNA CYTOMETRY IN TUMOR CELLS Barbara Roitzheim ~, Dietmar Kindermann 2, Wulf-Dietrich Miersch 3, J/irgen Vogel 2, Peter Briihl3; tlllstitute of Human Genetics, 21nstitut of Pathology, ~Department of Urology; University of Bonn, Germany Investigation of interphase ceils offers a valuable tool in tumor cytogenetics if metaphase spreads are not available in sufficient number and quality. The aim of our study was to test the reliability of the determination of the DNA content in cancer cells (testicular and kidney tumors) by means of FISH and static DNA cytometry. FISH is a powerful method to detect numerical anomalies in interphase cells when using repetitive centromeric DNA probes. Static DNA cytometry ascertains quantitatively the DNA content by measuring the specific monochromatic light extintion of Feulgen-stained nuclei and is widely used for DNA malignancy grading. Three types of cell populations of 4 testicular and 2 kidney tumors were analysed: histologic slides from tumor tissues, direct cell preparations of tumor biopsies after short term incubation and long-term cultures of the transformed cells. For each tumor, a control sample was set up from normal tissue adjacent to the tumor. To analyse the polyploidy-rate of the tumor cells we applied centromere 6 probes in tnsticutar cancer cells and centromere 2 probes in kidney cancer cells (both were proved not to be involved in the tumor progression). According to static DNA cytometry the DNA content of the tumor stemline and the amount of polyploid cells were determined. The cells in G2-phase of the ceil cycle with their doubled DNA content gave false-positive results in all measurements, because they could not be distinguished from polyploid G0-phaso tumor cells. In each preparation type of every tumor 100 inte~hase nuclei were analysed. After performing FISH, the number of the centromere signals in 100 interphase cells of each tumor was counted. Both, the DNA cylometric results and the FISH-analysis showed that 3 / 6 tumors were polyploid. The DNA values of all cases determined by cytometry ranged from 2.00c to 4.59c. In addition to the DNA content of the whole cell population, FISH could demonstrate the distribution of the polyploidy-grades: for example, one seminoma with a cytometric measured stemlme of 4.15c had the polyploidy-grades In : 2n : 3n : 4n : 5n in the ratio 1.00 : 10.65 : 1.33 : 11.99 : 1.00. Therefore, cells after S-phase are not giving false-positive results. On the other hand, a probe-specific discrepancy between expected and observed results has to be taken into coasideration.
THE USE OF FLUORESCENCE IN SITU HYBRIDIZATION (FISH) TO MEASURE INITIAL AND RADIATION INDUCED CHROMOSOME ABERRATIONS IN SOLID TUMORS. Jose M Coco-Martin, Cecile Ottenheim Ingrid Hofland, Fons J.M Balm, Adrian C. Begg.The Netherlands Cancer Institute (N.K.I), Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
Chromosome aberrations as determined with FISH could be predictive for human tumor radiosensitivity. In our studies wholechromosome specific probes were used to screen initial aberrations in fresh solid tumors from head and neck. The expression of different cytokeratins in the tumor cells was also determined using monoclonal antibodies, as discriminators between normal and tumor cells. Cytokeratin expression will be determined by flow cytometry. After preparing a celt suspension, cells were cultured for a short period to obtain mitotic cells. Many tumors showed numerous initial chromosome aberrations, numerical as well as structural. A high number of initial aberrations could complicate the determination of radiation induced chromosome aberrations, necessiteting the testing of several probes.
DNA ANALYSIS BY IMAGE CYTOMETRY AND CORRESPONDING CYTOGENETIC INVESTIGATIONS IN UROGENITAL CARCINOMAS Eva Sleeger~(I), Dietmar Kindermann(2) (1)Institute of Human Genetics, University of Bonn, Germany (2)Department of Pathology, University of Bonn, Germany DNA image cytometry is widely used to determine the DNA content of tumor cells. This technique offers the possibility to record a high number of cells in interphase. Thus, even if no mitoses are available it is possible to assess the malignancy of the tumor material. The simultaneous analysis of karyotype and DNA content was performed on two testicular germ cell tumors and two renal cell carcinomas and their adjacent normal tissues. 8 direct preparations and 8 long term cell cultures were analyzed. The influence of different cultivation mediums on cell growth was tested. In all cases the histological diagnosis of malignancy was confirmed by DNA image cytometry. Altogether 2976 tumor and nonmalignant cells, spermatids as well as different reference cell populations (lymphocytes, fibroblasts) were investigated to determine the reliability of DNA measurement compared to the known karyotype. Two cases showed correspondence between karyotypo and DNA content. We could demonstrate that the two testicular germ cell tumors (mixed tumor and seminoma) had a hypotriploid DNA content. Due to fibroblasts overgrowing the tumor cells in the seminoma cell culture the chromosomes that were analyzed showed a false normal karyotype. This investigation of the wrong cell population could be refuted by the DNA cytometry results because of the hypotriploid stemline. In both cases of direct preparations of the normal adjacent tissues the malignancy of existing dysplastic germ cells was proved. The two renal cell carcinomas showed a hyperdiploid DNA content. In one case a trisomy of the chromosomes l, 11 and X was found m several metaphases. In the second case no mitotic stages were available, so that the malignancy of the tissue was only proved by cytometry, but the given type ofaneuploidy could not be ascertained. In our cases measurement of tumor cells with a divergent DNA content to 2c did not allow exact conclusions on the overrepresented chromosomes. The study of the DNA content of 7 different numerical aberrations on G0,Gl-phase fibroblasts (proliferating cells immunophenotyped by Ki-67) demonstrated that the minimal analyzable deviation of DNA content to 2c is 2%. Therefore, additional karyotyping is necessary to characterize the different tumor types. Especially marker chromosomes of prognostic importance like isochromosome i(12p) can be registrated only by analyzing banded metaphases.
THE USE OF FLUORESCENCE IN SITU HYBRIDIZATION (FISH) TO PREDICT RADIOSENSITIVlTY OF HUMAN TUMOR CELL LINES. Jose M. Coco-Martin Monique FM.A. Smeets, Marilyn Poggensee, EIs Mooren, Cecile Ottenheim, Adrian.C. Begg. The Netherlands Cancer Institute (N.K.I), Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Chromosome damage as measured with FISH can be a potential method to predict intrinsic radiosensitivity of human tumor cell lines in both mitotic and interphase cells. Whole-chromosome specific probes were used to determine the radiosensitivity of four human tumor cell lines, including a human lung adenocarcinoma (A549), a human ovarian carcinoma cell line (A1847) and two squamous cell carcinomas (SCC61, SQ20B). The difference in radiosensitivity of these cell lines cover most of the range in radiosensitivity seen for mammalian cell lines. Using the appropriate probe for each cell line, dose response curves for chromosome aberration induction were obtained 24h post-irradiation. Three cell lines (A549, A1847, SCC61) showed the same correlation with cell survival, although the fourth (SQ20B) showed more aberrations for a given survival. Dose response curves were also obtained 14 days post-irradiation to determine the stability of the induced aberrations. Differences between number of aberrations at day 1 and day 14 showed the same correlation for all four cell lines with cell survival, indicating that this parameter is a better predictor of survival. In conclusion, FISH using whole-chromosome specific probes can be a rapid and quantitative assay for cellular radiosensitivity, but remaining aberrations (stable) should be taken into consideration.
Abstracts
EVALUATION OF [NTERPHASE FLUORESCENCE IN SITU HYBRIDIZATION (FISH) IN DNA MALIGNANCY GRADING AND COMPARISON TO STATIC DNA CYTOMETRY IN TUMOR CELLS Barbara Roitzheim ~, Dietmar Kindermann 2, Wulf-Dietrich Miersch 3, J/irgen Vogel 2, Peter Briihl3; tlllstitute of Human Genetics, 21nstitut of Pathology, ~Department of Urology; University of Bonn, Germany Investigation of interphase ceils offers a valuable tool in tumor cytogenetics if metaphase spreads are not available in sufficient number and quality. The aim of our study was to test the reliability of the determination of the DNA content in cancer cells (testicular and kidney tumors) by means of FISH and static DNA cytometry. FISH is a powerful method to detect numerical anomalies in interphase cells when using repetitive centromeric DNA probes. Static DNA cytometry ascertains quantitatively the DNA content by measuring the specific monochromatic light extintion of Feulgen-stained nuclei and is widely used for DNA malignancy grading. Three types of cell populations of 4 testicular and 2 kidney tumors were analysed: histologic slides from tumor tissues, direct cell preparations of tumor biopsies after short term incubation and long-term cultures of the transformed cells. For each tumor, a control sample was set up from normal tissue adjacent to the tumor. To analyse the polyploidy-rate of the tumor cells we applied centromere 6 probes in tnsticutar cancer cells and centromere 2 probes in kidney cancer cells (both were proved not to be involved in the tumor progression). According to static DNA cytometry the DNA content of the tumor stemline and the amount of polyploid cells were determined. The cells in G2-phase of the ceil cycle with their doubled DNA content gave false-positive results in all measurements, because they could not be distinguished from polyploid G0-phaso tumor cells. In each preparation type of every tumor 100 inte~hase nuclei were analysed. After performing FISH, the number of the centromere signals in 100 interphase cells of each tumor was counted. Both, the DNA cylometric results and the FISH-analysis showed that 3 / 6 tumors were polyploid. The DNA values of all cases determined by cytometry ranged from 2.00c to 4.59c. In addition to the DNA content of the whole cell population, FISH could demonstrate the distribution of the polyploidy-grades: for example, one seminoma with a cytometric measured stemlme of 4.15c had the polyploidy-grades In : 2n : 3n : 4n : 5n in the ratio 1.00 : 10.65 : 1.33 : 11.99 : 1.00. Therefore, cells after S-phase are not giving false-positive results. On the other hand, a probe-specific discrepancy between expected and observed results has to be taken into coasideration.
THE USE OF FLUORESCENCE IN SITU HYBRIDIZATION (FISH) TO MEASURE INITIAL AND RADIATION INDUCED CHROMOSOME ABERRATIONS IN SOLID TUMORS. Jose M Coco-Martin, Cecile Ottenheim Ingrid Hofland, Fons J.M Balm, Adrian C. Begg.The Netherlands Cancer Institute (N.K.I), Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
Chromosome aberrations as determined with FISH could be predictive for human tumor radiosensitivity. In our studies wholechromosome specific probes were used to screen initial aberrations in fresh solid tumors from head and neck. The expression of different cytokeratins in the tumor cells was also determined using monoclonal antibodies, as discriminators between normal and tumor cells. Cytokeratin expression will be determined by flow cytometry. After preparing a celt suspension, cells were cultured for a short period to obtain mitotic cells. Many tumors showed numerous initial chromosome aberrations, numerical as well as structural. A high number of initial aberrations could complicate the determination of radiation induced chromosome aberrations, necessiteting the testing of several probes.
DNA ANALYSIS BY IMAGE CYTOMETRY AND CORRESPONDING CYTOGENETIC INVESTIGATIONS IN UROGENITAL CARCINOMAS Eva Sleeger~(I), Dietmar Kindermann(2) (1)Institute of Human Genetics, University of Bonn, Germany (2)Department of Pathology, University of Bonn, Germany DNA image cytometry is widely used to determine the DNA content of tumor cells. This technique offers the possibility to record a high number of cells in interphase. Thus, even if no mitoses are available it is possible to assess the malignancy of the tumor material. The simultaneous analysis of karyotype and DNA content was performed on two testicular germ cell tumors and two renal cell carcinomas and their adjacent normal tissues. 8 direct preparations and 8 long term cell cultures were analyzed. The influence of different cultivation mediums on cell growth was tested. In all cases the histological diagnosis of malignancy was confirmed by DNA image cytometry. Altogether 2976 tumor and nonmalignant cells, spermatids as well as different reference cell populations (lymphocytes, fibroblasts) were investigated to determine the reliability of DNA measurement compared to the known karyotype. Two cases showed correspondence between karyotypo and DNA content. We could demonstrate that the two testicular germ cell tumors (mixed tumor and seminoma) had a hypotriploid DNA content. Due to fibroblasts overgrowing the tumor cells in the seminoma cell culture the chromosomes that were analyzed showed a false normal karyotype. This investigation of the wrong cell population could be refuted by the DNA cytometry results because of the hypotriploid stemline. In both cases of direct preparations of the normal adjacent tissues the malignancy of existing dysplastic germ cells was proved. The two renal cell carcinomas showed a hyperdiploid DNA content. In one case a trisomy of the chromosomes l, 11 and X was found m several metaphases. In the second case no mitotic stages were available, so that the malignancy of the tissue was only proved by cytometry, but the given type ofaneuploidy could not be ascertained. In our cases measurement of tumor cells with a divergent DNA content to 2c did not allow exact conclusions on the overrepresented chromosomes. The study of the DNA content of 7 different numerical aberrations on G0,Gl-phase fibroblasts (proliferating cells immunophenotyped by Ki-67) demonstrated that the minimal analyzable deviation of DNA content to 2c is 2%. Therefore, additional karyotyping is necessary to characterize the different tumor types. Especially marker chromosomes of prognostic importance like isochromosome i(12p) can be registrated only by analyzing banded metaphases.
THE USE OF FLUORESCENCE IN SITU HYBRIDIZATION (FISH) TO PREDICT RADIOSENSITIVlTY OF HUMAN TUMOR CELL LINES. Jose M. Coco-Martin Monique FM.A. Smeets, Marilyn Poggensee, EIs Mooren, Cecile Ottenheim, Adrian.C. Begg. The Netherlands Cancer Institute (N.K.I), Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Chromosome damage as measured with FISH can be a potential method to predict intrinsic radiosensitivity of human tumor cell lines in both mitotic and interphase cells. Whole-chromosome specific probes were used to determine the radiosensitivity of four human tumor cell lines, including a human lung adenocarcinoma (A549), a human ovarian carcinoma cell line (A1847) and two squamous cell carcinomas (SCC61, SQ20B). The difference in radiosensitivity of these cell lines cover most of the range in radiosensitivity seen for mammalian cell lines. Using the appropriate probe for each cell line, dose response curves for chromosome aberration induction were obtained 24h post-irradiation. Three cell lines (A549, A1847, SCC61) showed the same correlation with cell survival, although the fourth (SQ20B) showed more aberrations for a given survival. Dose response curves were also obtained 14 days post-irradiation to determine the stability of the induced aberrations. Differences between number of aberrations at day 1 and day 14 showed the same correlation for all four cell lines with cell survival, indicating that this parameter is a better predictor of survival. In conclusion, FISH using whole-chromosome specific probes can be a rapid and quantitative assay for cellular radiosensitivity, but remaining aberrations (stable) should be taken into consideration.