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E Gebhart

Publications and source records attributed to E Gebhart.

At least 37 records · Page 2Linked to original sources
PubMed2000

Delimiting the use of comparative genomic hybridization in human myeloid neoplastic disorders.

Hematopoietic disorders can be used as a suitable tool of additional information on the actual resolving power of comparative genomic hybridization (CGH). Therefore, CGH examination was performed of DNA extracted from 23 acute and 15 chronic myeloproliferative disorders which had just been analyzed using classical cytogenetic techniques. In nearly all cases CGH analysis was repeated with reversely labeled probes. A Zeiss axioplan microscope was equipped with the ISIS 3 system for photometric evaluation of the CGH data. A main group was selected of 34 cases showing karyotypic mosaics when routinely diagnosed by classical cytogenetics. The grade of mosaicism was basically determined from the classical cytogenetic analysis and was additionally defined examining target anomalies by I-FISH analysis in 28 of the cases. The second group included 23 cases with deletions, and in 1 case another informative genomic imbalance could be analyzed. Every target anomaly irrespective of its type could be detected in all cases with an affected cell population equalling or exceeding about 25%, but in none was it below 23%. This value was the lowest and was found in a case, with CGH-detected 20q deletion. The smallest deletions of two bands on 20q which could visually be detected by CGH were estimated in the range of 5-7 Mb. CGH was also suitable to detect imbalances which were not clearly detected by routine cytogenetics. Reverse labelling, performed in nearly all cases, confirmed the result of the original CGH analysis. These data not only document the readiness and reliability of CGH studies on human leukemia, but also further contribute to a clearer definition of the limits of the resolving power of this technique.

Adult↗
PubMed1999

Deletion monitoring in skin tumors by interphase-FISH using band-specific DNA probes.

Thin sections from archival paraffin blocks of various skin tumors (26 melanomas, 15 squamous cell carcinomas, 5 keratoacanthomas, 5 basal cell carcinomas) were subjected to interphase-FISH (I-FISH) with DNA probes which are specific for chromosomal regions often involved in deletions in human cancer. These were probes for chromosome 3p21, the p53 gene on chromosome 17p13, and, in a few selected cases, a probe for chromosome 9p21. It was demonstrated that deletions of these regions could be reliably detected and related to tumor type and histology, i.e. grading. The most common deletion was that of 3p21 which was found in all studied squamous cell carcinomas (SCC) of low differentiation, in 60% of the Bowen carcinomas, in 70% of the metastatic melanomas less than 1.5 mm thick, and in over 55% of those which thickness over 2 mm. In contrast, FISH-detected p53 deletion was a rare finding in the investigated tumors. However, this gene was even found in an increased copy number in 60% of the poorly differentiated SCCs (grade 4) and in 50% of the non-metastatic melanomas less than 1.5 mm thick. Deletion of 9p21 was detected in 13 of the 14 tumors on which pertinent examinations could be performed. I-FISH was shown to be a reliable technique for the rapid detection of chromosome band specific deletions in archival material of human skin tumors.

Adult↗
PubMed1999

Differentiation between Spitz nevi and malignant melanomas by interphase fluorescence in situ hybridization.

Spitz nevi are benign melanocytic neoplasias which have distinct pathological features that make the pathological differential diagnosis from malignant melanomas extremely difficult. The Spitz nevi may be misdiagnosed as malignant melanoma and vice versa. Therefore, interphase fluorescence in situ hybridization (I-FISH) was used for a possible discrimination between Spitz nevi and malignant melanomas on the basis of numerical aberrations of the chromosome complement in interphase nuclei of thin sections. Previous studies had shown changes in malignant melanomas which were not found at the same level in normal tissue or benign tumors. Thin sections of archival paraffin material from 42 Spitz nevi with different histological type and grade of anomaly were subjected to FISH-analyses using commercially available biotinylated and/or digoxigenated alphoid DNA probes of chromosomes 1, 6, 7, 9, 17 and 18, which were applied in combinations in a two- or three-color-FISH. Unaffected epithelial areas from the same sections served as. The obtained data were compared with those collected previously from thin sections of malignant melanomas prepared in the same way. Due to the sometimes limited nevus area investigated, the number of evaluable nuclei was lower than expected from previous experiences with malignant melanomas. Therefore, only 20 nevi could be reliably evaluated. The comparison of the group of Spitz nevi with the group of controls did not show any significant difference regarding chromosomes 1, 6, 7, 9 and 17 (Wilcoxon test). The method used to detect chromosomal loss or gain in the individual Spitz nevi demonstrated only two nevi (one of the spindle cell type with a low to middle grade of anomaly, the other of the epitheloid cell type with a middle grade of anomaly) with a gain of chromosome 7 and chromosome 17, respectively. So, with respect to the histological type and grade of anomaly, no numerical aberrations could be detected in Spitz nevi. The comparison of the group of Spitz nevi with subgroups of malignant melanomas (metastatic, non-metastatic, melanomas with a thickness <1.5 mm and melanomas with a thickness >2. 0 mm) and with the whole group of malignant melanomas showed significant differences concerning chromosome 9 (Mann-Whitney U test), signal indices, which were higher in the melanomas than in the Spitz nevi. Regarding chromosomes 6, 7 and 17 no significant differences could be shown, although a trend of gain in melanomas and of loss in Spitz nevi was observed of these chromosomes.

Adolescent↗
PubMed1998

New eukaryotic semaphorins with close homology to semaphorins of DNA viruses.

Semaphorins were initially described as a family of repulsive guidance molecules in embryonal development. Their basic structure consists of an N-terminal signal sequence, the defining semaphorin domain ofapproximately 500 amino acids, an Ig-like domain,and a variable carboxy-terminus. We recently described a viral semaphorin homologue encoded by the alcelaphine herpesvirus type 1. Less conserved, truncated homologues were also identified in poxviruses. Here we describe new human and murine semaphorin homologues. The respective genes were cloned and sequenced, and they were termed H-Sema-L and M-Sema-L (HGMW-approved symbols SEMAL and Semal, respectively). A multiply spliced mRNA of 3.2 kb is expressed in human placenta, spleen, thymus, and gonadal tissue. H-Sema-L maps to chromosome 15q22.3-q23 and M-Sema-L to the homologous locus 9A3.3-B in the mouse genome. The expression patterns and the presence of related genes in large DNA viruses suggest that this new semaphorin has a relevant function in the immune system.

Adjuvants, Immunologic↗
PubMed1998

Chromosomal in-vitro radiosensitivity of lymphocytes in radiotherapy patients and AT-homozygotes.

BACKGROUND: We investigated the in-vitro radiosensitivity of peripheral blood lymphocytes with a special FISH/CISS-technique. PATIENTS AND METHODS: From October 1993 through April 1996, a total number of 52 cancer patients was enrolled in the study. The tumor sites in these patients were: breast (n = 41), lung (n = 4), head and neck (n = 3) as well as prostate, bladder, rectal cancer and Hodgkin's disease (each n = 1). Twenty-six of them were examined prior to planned radiotherapy (prospective group) and 26 after radiotherapy (retrospective group). Three additional individuals (without cancer or radiotherapy) with proven ataxia telangiectasia (Louis-Bar syndrome, AT-homozygotes) were also investigated and their blood samples served as positive control for radiosensitivity. The clinical radiation response of normal tissue in radiotherapy patients was scored according to the WHO grading system for acute and according to the RTOG grading system for late effects. For to estimate the intrinsic radiosensitivity, blood samples were taken and irradiated in vitro with 0 (control) or 0.7 or 2 Gy with a 6 MV-linear accelerator, standard 48-hour lymphocyte cultures were prepared, chromosomes #1, #2 and #4 were simultaneously labeled with a FISH/CISS-technique and 200 to 1,000 metaphase spreads were scored for chromosomal aberrations. The radiation sensitivity of lymphocytes was expressed as the number of radiation-induced chromosomal breaks per mitosis after 0.7 Gy or 2 Gy corrected for the 0-Gy control value. RESULTS: The frequency of chromosomal breaks/mitosis in the unirradiated control lymphocytes was 0.020 +/- 0.015 in prospective patients who had not yet received radiotherapy. It was significantly higher in retrospective patients (0.264 +/- 0.164 breaks/mitosis) as a result of the previous radiation exposure. The 3 AT-homozygotes showed also an increased number of spontaneous chromosomal breaks (0.084 +/- 0.016 breaks/mitosis), probably resulting from the chromosomal instability in this disease. This figure, however, was significantly lower than in retrospective patients. The number of radiation-induced breaks after in-vitro irradiation was comparable in lymphocytes of patients who showed no normal tissue reaction (n = 11) as compared to those with mild to moderate radiation reaction (n = 32, acute reactions Grade 1 to 2, late reactions Grade 0 to 2). In 9 patients with unexpected severe plus late Grade 3 to 4 reactions, however, a significantly higher number of radiation-induced chromosomal breaks was measured; the highest number was observed in a patient with a radiation myelitis. The 3 AT-homozygotes showed, as expected, an extreme radiosensitivity of their lymphocytes. The number of breaks/mitosis after 0.7 Gy in vitro irradiation of lymphocytes was 0.103 +/- 0.059 in patients with no normal tissue radiation reaction (n = 11), 0.122 +/- 0.146 in the group with mild to moderate radiation reactions Grade 1 to 2 (n = 32), 0.359 + 0.226 in patients with unexpected Grade 3 to 4 normal tissue reactions (n = 9) and 0.550 +/- 0.243 in the 3 AT-homozygotes (p < 0.01, t-test). The difference in lymphocyte radiosensitivity between these 4 groups was also detected after in-vitro irradiation with 2 Gy (0.484 +/- 0.132 vs. 0.535 +/- 0.228 vs. 0.926 +/- 0.349 vs. 1.423 +/- 0.072). CONCLUSIONS: We found a significantly higher number of chromosomal breaks in lymphocytes of patients with severe or extreme radiation reaction of normal tissues as compared to patients with no or only mild to moderate radiation reactions. The radiosensitivity of lymphocytes in these radiosensitive patients was in the range between normal radiosensitivity and the radiosensitivity of AT-homozygotes. Detection of patients with severely enhanced intrinsic radiosensitivity might be possible with this method.

Adult↗
PubMed1998

Estimation of DNA single-strand breaks by single cells gel electrophoresis in tumor cells.

To estimate the frequency of single-strand breaks on a single cell level the so-called comet-assay (single cell gel electrophoresis) is a well-established technique. We present a modified protocol suitable for testing primary tumors, a kind of tissue very uneasy to be analysed in former single cell gel electrophoresis assays. Tumor cells of 12 studied cases showed a typical dose-rate effect on in vitro irradiation with different X-ray doses, as observed in peripheral blood leukocytes. Interestingly, the repair capability of primary tumor cells was lower than that of peripheral blood leukocytes of the same patients.

Adenocarcinoma↗
PubMed1998

Oral squamous cell carcinomas are characterized by a rather uniform pattern of genomic imbalances detected by comparative genomic hybridisation.

Total genomic DNA sampled from 20 oral squamous cell carcinomas (SCCs) and from four SCC cell lines, was examined for genomic imbalances using comparative genomic hybridisation (CGH). Gains and losses of DNA copy number aberrations (CNAs) were found in the primary tumours, but also in the cell lines at a varying number. The patterns of CNAs proved to be rather peculiar in oral SCCs, gains of genetic material clearly dominating compared with losses, and a rather high uniformity of these patterns was an impressive finding. Hypersomies of whole chromosomes, e.g. numbers 17 and 19 or of whole chromosome arms, e.g. 20q, were particularly evident. The segments most frequently gained in oral SCCs were 3q26-q27, 5p15 and 9q34 (16 of 20 tumours each), as well as 1p36.3, 8q24, 10q26, 19 and 20q (15/20 each). Among the 15 tumours with more than 10 CNAs, all showed these imbalances. 11q13 was a band often involved in increases (14/20 tumours), but in several tumours was involved in amplification of DNA copy number. Several other chromosomal segments over represented in more than 60% of the tumours, as, for example, 12q24, 15q22-q24, 16p13.2 and 17q (14/20 tumours each), 6q26-qter, 7p22, 12p12.2-p13, 14q31-q32.2 (13/20) and 1q32-q41, 2q37, 16q23-q24 (12/20 each). In contrast, loss of material affected only a few chromosomal segments, as, for example, 3p12 (12 of the 20 tumours), 5q21 (10/20), 6q13 (8/20). The peculiarities of these findings, in some respect, differ from those found in other epithelial tumours, suggesting a high impact of environmental factors in the generation and progression of these tumours.

Adult↗
PubMed1998

Gain of DNA copy number on chromosomes 3q26-qter and 5p14-pter is a frequent finding in head and neck squamous cell carcinomas.

Comparative genomic hybridization (CGH) was used to study genomic imbalances in 77 squamous cell carcinomas of the head and neck (HNSCC) and in four cell lines derived from oral carcinomas. Particular attention was paid to all tumors characterized by a gain of two specific chromosomal segments, i.e. 3q26-qter and 5p14-p15. The 57 tumors containing both or one of the two imbalances were compared with 20 tumors lacking both with regard to genomic complexity, as well as to associated genomic, histopathologic and clinical peculiarities. 60% of the oral, and 66% of the non-oral cancers exhibited a gain of 3q26-qter, while a gain of 5p14-p15 was found in 66% of the oral, but only in 48% of the non-oral tumors. 48% of all tumors were characterized by both gains together, 26% exhibited only one of the two alterations. It could be shown that presence of both, gain of 3q26-qter and 5p14-p15, was clearly associated with a significantly higher complexity of genomic changes which was not only expressed as a high frequency of DNA copy number alterations (CNAs) but was also connected with a considerable number of additional amplifications of various chromosomal segments and a high conformity of the patterns of genomic imbalances in these tumors. Clear differences of the extent and of the patterns of genomic imbalance could be observed between oral and non-oral tumors. With respect to histopathological parameters, no clear association could be found for specific imbalances to the grade of differentiation, nor the invasiveness or metastatic status of the tumors. However, a higher number of patients with tumors characterized by gain of 3q26-qter plus 5p14-p15 died within a short period (i.e. less than 15 months) after excision of the tumor compared to the group without these imbalances. The implications of these findings are discussed from the oncogenetic and clinical aspects and in comparison with other reports.

Journal Article↗
PubMed1998

CGH-detected DNA sequence copy number amplifications can be confirmed by interphase-FISH: new possiblities for prognostic approaches in oral squamous cell carcinomas.

In order to control the data obtained by comparative genomic hybridization (CGH) on DNA sequence copy number amplifications, 20 oral squamous cell carcinomas (SCC) were subjected to interphase fluorescence in situ hybridization (I-FISH) examination using specific DNA probes for the oncogenes int2 and erbB-2, and the corresponding centromeric probes of chromosomes 11 and 17. In all cases characterized by distinct peaks of the CGH profile on the critical chromosomal segment 11q13, these data could be clearly substantiated by the I-FISH analyses using the int2 probe and estimating the signal index, the int2/centromer 11 relation, and the fraction of nuclei with high int2 signal numbers. In addition, I-FISH detected smaller cell fractions with high signal numbers (and/or signal clusters) in some tumors which were not definitely conspicuous in CGH. In contrast to int2, erbB-2 amplification apparently does not play a major role in oral SCCs, as the blurred peaks of CGH profiles on chromosome 17ql 1.2-q12 corresponded well with the findings of I-FISH using the erbB-2 probe. Gain of a whole chromosome 17 is apparently a rather common feature of these tumors. In conclusion, the combination of interphase FISH with oncogene-specific probes and CGH is regarded as a valuable means of practical molecular cytogenetic analysis of oral SCCs which could eventually achieve high practical importance in the pathologic analysis of these tumors and in prognosis of their development.

Adult↗
PubMed1998

Pattern of genomic imbalances in oral squamous cell carcinomas with and without an increased copy number of 11q13.

Among 23 squamous cell carcinomas (SCC) of the oral cavity which were screened for DNA copy number alterations (CNAs) using comparative genomic hybridization, 14 showed a gain of, and 5 of these 14 even an amplification of band 11q13. Amplification of 11q13 was also detected in three of the four studied SCC cell lines and was confirmed by interphase FISH. The number of CNAs in addition to 11q13 varied from 14 to 47 in these carcinomas. All these tumors had seven other specific CNAs in common, i.e. gain on 1p36.3-36.6, 5p15, 9q34, 12p12-13, 14q32, 19 and 20q, all but one showed also an increase of copy number in 7p22, 8q24, 10q26, 12q26, 15q24-25, 16p, 16q23-24, 17q and 22q12-qter. These imbalances were distinctly rarer in the tumors without CNA in 11q13. Loss of material apparently played a minor role in these tumors with gain of 11q13, the most frequent losses (3p12-14 and 5q21) being present in 10 of the 14 cases and loss of 9p13-21 in 5/14 tumors. The three tumors with the highest number of CNAs in addition to 11q13, were histologically classified as pT4, three of the five tumors with 11q13 amplification were highly node-positive (pN 2b-2c). Two of the pT4 tumors shared as many as 23 specific chromosomal segments affected by CNA. Thus, gain of 11q13, though being found at different stages of karyotypic evolution, is apparently associated with a rather specific pattern of other CNAs and involved in progressed stages of malignancy in oral squamous cell carcinoma. In addition, the proportion of patients deceased within one year after diagnosis was clearly higher in the group whose tumors showed an increased 11q13 copy number as compared to the group without this increase. This could point to an association of gain in 11q13 and aggressiveness of the respective tumor.

Aged↗
PubMed1997

The role of N-acetylcysteine as a putative radioprotective agent on X-ray-induced DNA damage as evaluated by alkaline single-cell gel electrophoresis.

Samples of human whole blood from 8 different donors were incubated with physiological saline or N-acetyl-L-cysteine (NAC, 1 x 10(-3) M) before being irradiated in vitro with high-energy X-rays (0.7 or 2.0 Gy). Primary DNA damage was evaluated in isolated lymphocytes using alkaline single-cell gel electrophoresis. Whereas the lymphocytes from non-irradiated blood samples showed a similar 'background level' of damage, there was a difference in sensitivity towards the radiation-induced DNA damage, especially at 2.0 Gy. When the data were pooled there was a clear and dose-related increase (p < 0.001) in damage, both in the absence and presence of NAC. Using the two most sensitive 'comet parameters' for DNA damage, i.e., the tail inertia and tail moment, the radiation-induced damage was found to be significantly increased already at 0.7 Gy in the samples that had been irradiated without NAC. Overall, NAC was found to be without radioprotective effects. Instead, the incubation with NAC itself was found to be associated with a slightly increased level of DNA damage. If the present findings are relevant also in an in vivo situation using peripheral lymphocytes as a surrogate for non-malignant cells in the body, NAC seems to be of limited value as a radioprotective agent in the clinic, at least when it comes to the acute DNA-damaging effects of therapeutic doses of high-energy X-rays.

Acetylcysteine↗
PubMed1997

The impact of complex chromosomal rearrangements on the detection of radiosensitivity in cancer patients.

BACKGROUND AND PURPOSE: Lymphocytes of a small fraction of cancer patients responded to in vitro irradiation with an extreme chromosomal reaction. A large portion of the observed chromosome aberrations were complex chromosomal rearrangements (CCR). The present study is an attempt to define the impact of CCR on the predictive detection of an intrinsic clinical radiosensitivity in cancer patients in more detail. MATERIALS AND METHODS: A three-colour 'FISH-painting' technique (chromosome in situ suppression (CISS) hybridization) was used for the detection of chromosomal rearrangements, induced by in vitro irradiation, in 81 samples of peripheral blood lymphocytes from 66 cancer patients. Thirty-three of those were assigned for radiation therapy, the others having just undergone radiation therapy. Seven healthy individuals served as controls. RESULTS: CCRs are a very rare event in non-irradiated cells. Lymphocytes of patients who had just undergone therapeutic irradiation, however, not only exhibited high basic frequencies of CCR but also responded to in vitro irradiation with a more drastic increase of CCR than did the lymphocytes of non-exposed patients. A high inter-individual variability of the reaction to in vitro irradiation could be generally stated. The lymphocytes of patients with clinical signs of an outstanding radiosensitivity responded with an unusually high frequency of CCR. The total number of CCRs detected by CISS was found to be dependent on the interval from a previous radiation therapy and was slightly influenced by previous cytostatic therapy. Irrespective of these influences, patients with clinically defined radiation hypersensitivity were those with the highest radiosensitivity also in cytogenetic terms (including CCR). CONCLUSION: The successful use of FISH-painting for the detection of CCR, in addition to the general breakage frequency, highlights its suitability in the identification of individual hypersensitivity to ionizing radiation. The time-consuming cytogenetic examination can be considerably reduced by its use.

Chromosome Aberrations↗
PubMed1996

LW/SO cell line: a tool for studying the phenotypical characterization and commitment of hematopoietic stem cells.

We report our observations with the cell line LW/SO, which was recently derived from the bone marrow of a patient with acute myeloid leukemia. Based on the morphological and histochemical examination, the leukemic cells were classified primarily as FAB type M4. However, 2 years later, in relapse, the cells changed their morphology and were hence specified as FAB type M2 (slightly positive for acid phosphatase and Sudan black). The cells established have now been in culture for approximately 11 months and display nearly 100% CD4/5/7/15/25/71/120a,b at varying densities. Some of them spontaneously and reversibly become either CD34 + /38- or CD34 - /38+, yet the majority of the cells remain negative for both. All attempts to separate the cells with a distinct phenotype by limiting dilution or sorting through a flow cytometer failed repeatedly. The subsets, enriched up to 98% (regardless of their primary immunophenotype CD34 - / 38-, CD34 + /38-, or CD34 - /38+), soon displayed a phenotypical constellation similar to that before sorting. The ratio of CD34- to CD34+ seems to be influenced by the cell density: The greater the cell-to-cell contact, the lower the percentage of CD34-expressing cells. Some of the cells apparently differentiate into T-cell phenotype and acquire CD3 and T-cell receptor (TCR) alpha/beta molecules. While the quantity of CD34-expressing cells significantly increased in the presence of dexamethasone (10(-7) M), and some of them additionally acquired CD33 antigen, the percentage of CD3-positive cells was enhanced by adding 1% DMSO in medium. In contrast, cytokines such as IL-1, IL-2, IL-3, IL-4, IL-6, G-CSF, GM-CSF, or SCF (c-kit ligand) altered neither the proliferation capacity nor the phenotypical constellation of LW/SO cells (each tested alone). Although normal karyotype was obtained from the bone marrow cells, the LW/SO cells revealed a homogeneous chromosomal composition of 45, X, -X, der(9) inv(9) (p12q13) del(9) (p22?). These data suggested that LW/SO cells might be the leukemic counterpart of putative pre-CD34-positive progenitors. In order to substantiate this assumption, we analyzed the expression of other so-called T-cell markers on CD34+ cells from peripheral blood stem cell aphereses of five patients who later underwent high-dose chemotherapy and subsequent stem cell retransfusion. These data clearly revealed that a considerable amount of CD34+ hematopoietic progenitors co-express CD2/4/(5)/(7)/25 at an early stage of differentiation, and support the notion that CD34-negative LW/SO cells with the surface markers CD4/5/7/25 are probably phenotypical representatives of pluripotent stem cell. Hence, not all CD34-negative populations with so-called T-cell surface markers should be considered T-cells; some may constitute the ancestor of CD34 antigen-expressing progenitors.

ADP-ribosyl Cyclase↗
PubMed1996

Use of a three-color chromosome in situ suppression technique for the detection of past radiation exposure.

A three-color chromosome in situ suppression technique and classical cytogenetic analysis were compared for the detection of chromosomal aberrations in blood lymphocytes of 27 patients who had undergone radiation therapies from 1 month to 9 years ago. Depending on the respective regimens of therapy, a high variability was found in the aberration data. Aberration rates depended on the interval between exposure and scoring rather than on the locally applied radiation doses, which were rather uniform among most patients. Chromosome in situ suppression was found to be superior to classical cytogenetics with respect not only to the spectrum of detectable aberrations but also to the uncovering of long-term effects of irradiation. Of particular interest were the relative stability of the frequency of radiation-induced reciprocal translocations and the utility of chromosome in situ suppression to uncover complex rearrangements.

Chromosome Aberrations↗