[Show abstract][Hide abstract] ABSTRACT: Radiotherapists are highly interested in optimizing doses especially for patients who tend to suffer from side effects of radiotherapy (RT). It seems to be helpful to identify radiosensitive individuals before RT. Thus we examined aberrations in FISH painted chromosomes in in vitro irradiated blood samples of a group of patients suffering from breast cancer. In parallel, a follow-up of side effects in these patients was registered and compared to detected chromosome aberrations.
Blood samples (taken before radiotherapy) were irradiated in vitro with 3 Gy X-rays and analysed by FISH-painting to obtain aberration frequencies of first cycle metaphases for each patient. Aberration frequencies were analysed statistically to identify individuals with an elevated or reduced radiation response. Clinical data of patients have been recorded in parallel to gain knowledge on acute side effects of radiotherapy.
Eight patients with a significantly elevated or reduced aberration yield were identified by use of a t-test criterion. A comparison with clinical side effects revealed that among patients with elevated aberration yields one exhibited a higher degree of acute toxicity and two patients a premature onset of skin reaction already after a cumulative dose of only 10 Gy. A significant relationship existed between translocations in vitro and the time dependent occurrence of side effects of the skin during the therapy period.
The results suggest that translocations can be used as a test to identify individuals with a potentially elevated radiosensitivity.
[Show abstract][Hide abstract] ABSTRACT: Structural genomic rearrangements are frequent findings in human cancers. Therefore, papillary thyroid carcinomas (PTCs) were investigated for chromosomal aberrations and rearrangements of the RET proto-oncogene. For this purpose, primary cultures from 23 PTC have been established and metaphase preparations were analysed by spectral karyotyping (SKY). In addition, interphase cell preparations of the same cases were investigated by fluorescence in situ hybridisation (FISH) for the presence of RET/PTC rearrangements using RET-specific DNA probes. SKY analysis of PTC revealed structural aberrations of chromosome 11 and several numerical aberrations with frequent loss of chromosomes 20, 21, and 22. FISH analysis for RET/PTC rearrangements showed prevalence of this rearrangement in 72% (16 out of 22) of cases. However, only subpopulations of tumour cells exhibited this rearrangement indicating genetic heterogeneity. The comparison of visual and automated scoring of FISH signals revealed concordant results in 19 out of 22 cases (87%) indicating reliable scoring results using the optimised scoring parameter for RET/PTC with the automated Metafer4 system. It can be concluded from this study that genomic rearrangements are frequent in PTC and therefore important events in thyroid carcinogenesis.
BioMed Research International 03/2011; 2011(2):693691. DOI:10.1155/2011/693691 · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytogenetic analysis of head and neck squamous cell carcinoma (HNSCC) established several biomarkers that have been correlated to clinical parameters during the past years. Adequate cell culture model systems are required for functional studies investigating those potential prognostic markers in HNSCC. We have used a cell line, CAL 33, for the establishment of a cell culture model in order to perform functional analyses of interesting candidate genes and proteins. The cell line was cytogenetically characterized using array CGH, spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH). As a starting point for the investigation of genetic markers predicting radiosensitivity in tumor cells, irradiation experiments were carried out and radiation responses of CAL 33 have been determined. Radiosensitivity of CAL 33 cells was intermediate when compared to published data on tumor cell lines.
[Show abstract][Hide abstract] ABSTRACT: It is well established that genetic alterations may be associated to prognosis in tumor patients. This study investigates chromosomal changes that predict the clinical outcome of head and neck squamous cell carcinoma (HNSCC) and correlate to characteristic clinicopathological parameters. We applied comparative genomic hybridization (CGH) to tissue samples from 117 HNSCC patients scheduled for radiotherapy. Genomic aberrations occurring in more than five patients were studied for impact on locoregional progression (LRP)-free survival. p values were adjusted by the Hochberg-Benjamini procedure and significant aberrations and clinical variables subjected to a stepwise backwards Cox proportional model. Significant alterations were further analyzed by array-CGH and fluorescence in situ hybridization (FISH). In multivariate survival analysis gains on 1q and 16q predict reduced LRP-free survival independently from known prognostic factors. Cluster analysis separated the HNSCC cases into two groups (cluster 1 and 2) that are characterized by significant differences for imbalances in 13 chromosomal regions. Moreover, it became apparent that cluster 1 correlates to nonanemic patients, while cluster 2 represents predominantly anemic cases. Array-CGH pinpoints 16q24.3 to be the region of interest on chromosome 16 which was further verified by FISH analysis where an increased copy number of FANCA, a member of the Fanconi anemia/breast cancer pathway, could be identified. This study demonstrates that chromosomal gains on 1q and 16q as well as chromosomal loss on 18q represent prognostic markers in HNSCC and that these alterations may explain to some extent the dismal course of a subgroup of patients.
Journal of Molecular Medicine 10/2008; 86(12):1353-65. DOI:10.1007/s00109-008-0397-0 · 5.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the DNA-proportional distribution of radiation-induced chromosome aberrations for all chromosomes of a male and a female human karyotype.
Metaphases were prepared from whole blood cultures obtained from two healthy donors and set up after irradiation with 3 Gy 220 kV X-rays. Single whole-chromosome FISH painting simultaneously with pancentromeric DNA painting were performed separately for each chromosome of the human karyotype. One thousand exclusively first-division metaphases were analysed per chromosome and donor. After statistical analysis, the data obtained were compared with theoretically expected values.
All aberration types (translocations, dicentrics) showed deviations from a DNA-proportional distribution. For both donors, chromosomes 2 and 3 exhibited significantly less and chromosome 4 more symmetrical translocations than expected. Chromosomes 15 and 22 showed more symmetrical translocations than predicted for one of the two donors. Less dicentrics than expected became apparent for chromosomes 2, 3 and 18, while more dicentrics were seen for chromosomes 15, 16 and 17. Moreover, chromosomes 4, 14 and 22 showed a significant deviation from the theoretically expected 1:1 ratio of the yields of symmetrical translocations to the yields of dicentrics.
The results from the whole-chromosome complement in two different donors confirmed published data from the analysis of single chromosomes that some human chromosomes were not involved in radiation-induced dicentrics and symmetrical translocations proportional to their DNA content. This must be taken into account if chromosome subsets for dose reconstruction are selected or if whole genomic frequencies have to be calculated from partial genome analysis.
International Journal of Radiation Biology 07/2003; 79(6):393-403. DOI:10.1080/0955300031000137805 · 1.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Metafer2 fluorescence scanning system was used for routine analysis of radiation-induced exchange aberrations measured by fluorescence in situ hybridisation (FISH) chromosome painting in human peripheral lymphocytes. The system enables a rapid and unbiased fully-automated finding and image acquisition of fluorescently stained metaphase spreads. The chromosome aberration analysis is performed interactively from stored digitised processed gallery images, presented on a screen. Appropriate software image filters are available to further improve these pictures by background correction, noise reduction and fluorescence signal enhancement. Data sets generated by computer-assisted and manual scoring of radiation-induced reciprocal translocations (2B) and total 2B (2B+related 'one-way' types) or complete dicentrics (2A) and total 2A (2A+related 'one-ways') involving painted target chromosomes 2, 3 or 4 were compared and no significant differences were found.A linear-quadratic dose-response curve for total translocations (2B+'one-ways'+complex-derived types) based on computer-assisted analysis of 27,741 metaphases with chromosome 4 painting was compared to a curve obtained earlier for manually scored translocations in a set of target chromosomes 1, 4 and 12. After extrapolation to the whole genome, no significant difference between both curves was found. From our results it can be derived that computer-assisted aberration analysis using the Metafer2 system is a reliable alternative to manual analysis. Since time saving for computer-assisted translocation analysis is about 50% compared to manual scoring, this system is highly promising for a practical application in retrospective biodosimetry of human radiation exposure.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 06/2001; 492(1-2):51-7. DOI:10.1016/S1383-5718(01)00151-6 · 3.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Follow-up translocation and dicentric measurements in blood lymphocytes of five breast cancer patients were performed by FISH using painting probes for chromosomes 1, 4, and 12 simultaneously with a pancentromeric DNA probe, during 14 months after fractionated photon therapy affecting only small areas of the bone marrow (about 5%). The analysis of individual time-courses for translocations and dicentrics revealed a significant temporal decline of the yields with comparable half-times, both for these stable and unstable aberration types in two patients. In three patients, the aberration yields remained fairly unchanged during the observation period. Regarding retrospective biodosimetry for cases with partial-body exposures or large dose inhomogeneity, it follows that even FISH chromosome painting is limited in assessing initial doses correctly in terms of stable translocations.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 10/1999; 446(1):103-9. DOI:10.1016/S1383-5718(99)00153-9 · 3.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The quantification of radiation-induced chromosome aberrations identified by multicoloured FISH painting and classified according to different nomenclature systems (PAINT, S&S and a conventional method).
Blood samples were irradiated with five different doses of 220 kV X-rays or fission neutrons respectively. Cell cycle-controlled, multicoloured FISH painting was performed with a cocktail of chromosomes 1, 4, 12 and a pancentromeric probe.
Ten aberration parameters or categories were selected according to the three nomenclature systems and dose-response curves were constructed for the observed yields. Fitted coefficients of the linear-quadratic dose-response function show the relative importance of the quadratic term for aberration parameters of the low-LET radiation (X-rays) data set and of the linear term for those of the high-LET radiation (fission neutrons) data set. The relative proportion of complex aberrations observed was larger for the densely ionizing fission neutrons than for sparsely ionizing X-rays.
Compared with single-colour FISH painting, a multicolour approach provides extended information for a mechanistic and quantitative interpretation of radiation-induced chromosome aberrations. The choice of a nomenclature system and the selection of an appropriate aberration parameter or category depend on these specific aspects. Practical application requires a rapid and reproducible description of the observed painting patterns and should also throw light on the origin of aberrations.
International Journal of Radiation Biology 04/1999; 75(4):407-18. · 1.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Based on the technical configuration of the Metafer2 metaphase finder (Metasystems, Altlussheim, Germany), an automated system was developed that is able to discriminate first/M1 or second cycle/M2 metaphases. For an evaluation of the system in fluorescence plus Giemsa stained preparations of human lymphocytes, a learning sample and an independent test sample was used. Between 8 and 14 separated chromosomes per metaphase were sufficient to achieve correct classification rates between 95 and 100%. For practical application the system can be most efficiently used for biodosimetry of human radiation exposure which requires scoring of thousands of metaphases exclusively in M1. An application for selection of M2 for SCE scoring in mutagenicity testing was possible, but does not provide a comparable advantage.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 12/1998; 419(1-3):27-32. DOI:10.1016/S1383-5718(98)00121-1 · 3.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: For evaluation of the risk borne by hospital pharmacy personnel exposed to antineoplastic agents, the incorporation of cyclophosphamide,
ifosfamide, and platinum-containing drugs was quantified by the determination of urinary concentrations. In addition, the
induction of micronuclei (MN) and sister-chromatid-exchange (SCE) rates in peripheral blood lymphocytes were studied for correlation
with the urinary excretion of cytostatic drugs. Cyclophosphamide and ifosfamide were determined in 24-h urine samples using
gas chromatography with electron capture (detection limit 2.5 μg/l). Voltammetric analysis enabled the determination of platinum
concentrations of 4 ng/l. Heparinized blood (20 ml) was drawn and lymphocytes were cultured for MN and SCE studies. In all,
13 hospital pharmacists and pharmacy technicians regularly involved in the preparation of cytostatic drugs participated in
this investigation (7 persons represent a follow-up group). All subjects applied standard safety precautions, including the
use of a vertical laminar air-flow hood, protective gowns, and latex gloves. On the day of urine sampling an average of 4,870
mg cyclophosphamide, 5,580 mg ifosfamide, and 504 mg platinum-containing drugs were handled. The excretion of 5 and 9 μg cyclophosphamide/1
urine was measured in two samples, respectively. An elevated level of urinary platinum was found in one pharmacist (22.3 ng/g
creatinine) in comparison with a nonexposed control group. Mean frequencies of MN and SCE did not differ significantly between
the drug exposed group and control group. The employees who had incorporated chemotherapeutic agents were part of the follow-up
group and, thus, particularly cautious and sensitive to a possible hazard. The results emphasize the necessity of improving
personal protection of hospital pharmacy personnel occupationally exposed to cytostatic drugs and support the importance of
biological monitoring. In an ongoing project in our department the sources of contamination are being investigated parallel
to biological monitoring so as to determine critical situations and improve personal protection.
International Archives of Occupational and Environmental Health 07/1997; 70(3):205-208. DOI:10.1007/s004200050208 · 2.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chromosome painting (target chromosomes 1, 4, 12) was performed in peripheral lymphocytes from 25 occupants of nine houses with indoor radon concentrations of 210-3000 Bqm-3. Compared to a control group, the mean frequency of symmetrical translocations of the radon group was slightly but not significantly (p < 0.10) increased. A similar tendency became apparent for a comparison of two groups of subjects with cumulative radon exposures above and below 2800 Bqm-3 y. It is concluded that FISH-based measurements of stable symmetrical translocations should reflect the cumulative radon exposure to haematopoietic compartments such as the red bone marrow rather than to mature blood lymphocytes. Since, however, radon-derived bone marrow doses are low and control frequencies of translocations are very high (about 10-fold higher than the value for conventionally scored dicentrics), the observed relative increase (1.5-fold) of the translocation frequency in blood lymphocytes is too small to discriminate chronic radon exposure from background.
International Journal of Radiation Biology 12/1996; 70(6):657-63. · 1.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fluorescence in situ hybridisation (FISH) with a human alphoid satellite pancentromeric DNA probe was used to detect centromeres in micronuclei of human lymphocytes induced by gamma irradiation and by Vinblastine sulfate. In a cytokinesis-block micro-nucleus assay a dose-dependent increase of micronuclei was detected for both agents. 72-89% of vinblastine-induced micronuclei, but only 7-48% of radiation-induced micronuclei showed centromere-positive fluorescence signals. Vinblastine treatment frequencies of centromere-negative micronuclei did not increase compared to control values, nor did frequencies of centromere-positive micronuclei in irradiated lymphocytes. Since FISH with an alpha satellite DNA probe allows the direct detection of centromeric DNA sequences the spindle damaging or clastogenic effectiveness of a compound can be easily and reliably examined in a cytokinesis-block micronucleus assay in human lymphocytes.
[Show abstract][Hide abstract] ABSTRACT: We present a simple method that allows scoring of FISH-painted chromosomes exclusively in the first division human lymphocytes. It consists of a combination of FISH with chromosome-specific libraries and a differential sister chromatid staining after BrdU treatment of lymphocyte cultures. The method allows a precise quantification of induced chromosome damage for human biodosimetry.
International Journal of Radiation Biology 08/1995; 68(1):25-7. DOI:10.1080/09553009514550881 · 1.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The efficiency of the automated metaphase finding system METAFER2 is assessed in a routine mutagenicity assay using an aneuploid rat liver cell line treated with various promutagens. Data sets generated by automated and manual selection of metaphases are compared. It is demonstrated that METAFER2 routinely allows an efficient automatic identification of metaphases not only in lymphocyte preparations, but also in preparations from mammalian cell lines with varying chromosome numbers. Although larger slide areas are required for automated compared to manual metaphase scanning, the automatic system is faster by a factor of about 5. The interactive visual elimination of metaphases of insufficient quality is an easy and fast procedure. METAFER2 allows an unbiased selection of metaphases irrespective of their appearance as homogeneously stained first or harlequin-stained second division cells. Random selection of metaphases is neither influenced by various structural chromosome changes nor by increased frequencies of sister-chromatid exchanges.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 03/1995; 334(1):97-102. DOI:10.1016/0165-1161(95)90035-7 · 3.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The induction of micronuclei in cytokinesis-blocked human lymphocytes of different donors has been measured after G0 irradiation in vitro with a mixed fission neutron-gamma-ray beam, 220 kV X-rays and 60Co gamma-rays. The dose-response relationships for the micronucleus yields were linear-quadratic for both types of low LET radiations. The linear model applied for neutron-induced micronuclei over the dose range 0-0.66 Gy. At higher doses a saturation effect became apparent. For neutron-induced micronucleus yields a limiting RBE value of 12.2 was derived from the ratio of the alpha coefficients of neutrons and 60Co gamma-rays.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 05/1994; 306(2):135-41. DOI:10.1016/0027-5107(94)90024-8 · 3.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Serial blood samples were taken from four healthy individuals (three males, one female, aged between 26 and 51 years) in 3-monthly intervals during 1 year. Leucocyte suspensions were prepared and exposed to 3 Gy of 137Cs gamma-rays or left unirradiated as controls. In a cytokinesis-blocked (CB) micronucleus (MN) assay significant inter- and intra-donor variations of background and radiation-induced MN incidences became apparent. The two sources of variation lead to an extra variance sigma I2, in addition to the sample variance sigma e2 of MN incidences. The contributions of the different components to the total variance were estimated by means of a variance component model. The deviation sigma I for the mean background MN level of 1.53 x 10(-2) MN/CB cell was +/- 0.67 x 10(-2) and for the mean radiation-induced MN level of 0.53 MN/CB cell it was +/- 0.10. The contribution of the intra-individual variance to sigma I2 was about 50% for background MN levels and 75% for radiation-induced MN frequencies. With respect to the application of the CB-MN assay as a biological dosimetry system, the consequences of the present findings for calibration purposes and low-dose estimation are discussed. The calculation of the variance components is explained in an appendix, which serves also as an example for the adaptation of analysis of variance techniques to the evaluation of data derived from scoring of MN, as well as from scoring of metaphase chromosomal aberrations.
International Journal of Radiation Biology 06/1992; 61(5):655-61. DOI:10.1080/09553009214551461 · 1.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Non-isotopic in situ hybridization using a mouse gamma (major) satellite probe DNA was applied to detect centromeres in micronuclei, which were induced in vitro in mouse liver cells by ionizing radiation and by vinblastine sulfate. In a cytokinesis-blocked micronucleus assay a dose-dependent induction of micronuclei was found for both agents. After vinblastine exposure the observed micronuclei showed centromere-positive hybridization signals in an order of magnitude of 70-90%, but after radiation exposure the magnitude was only 10-20%. Since the in situ hybridization technique detects centromeric DNA directly, it can be used in a cytokinesis-blocked micronucleus assay for a rapid and reliable discrimination between aneuploidy-inducing and clastogenic agents.
[Show abstract][Hide abstract] ABSTRACT: The response of unstimulated peripheral lymphocytes to a single dose of 3 Gy of 137Cs gamma rays was analysed in blood samples from 30 donors by a conventional micronucleus assay and from 14 donors by the cytokinesis-block (CB) method. Significant interindividual variations could be detected for the baseline levels and for induced levels of micronuclei. An age effect could be demonstrated with the conventional method for the number of spontaneous MN, but not with the CB method. The corresponding numerical estimate was 3.4 +/- 1.3% increase per year. No such increase was apparent for induced frequencies. Provided that cell proliferation kinetics is reliably taken into account the micronucleus assay could be helpful for diagnosing potential radiosensitive individuals.