Chromosomal Rearrangements in Post-Chernobyl Papillary Thyroid Carcinomas: Evaluation by Spectral Karyotyping and Automated Interphase FISH

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

Chromosomal radiosensitivity and acute radiation side effects after radiotherapy in tumour patients - a follow-up study

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. METHODS: 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. RESULTS: 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. CONCLUSIONS: The results suggest that translocations can be used as a test to identify individuals with a potentially elevated radiosensitivity

Positive Cofactor 4 (PC4) is critical for DNA repair pathway re-routing in DT40 cells

PC4 is an abundant single-strand DNA binding protein that has been implicated in transcription and DNA repair. Here, we show that PC4 is involved in the cellular DNA damage response. To elucidate the role, we used the DT40 chicken B cell model, which produces clustered DNA lesions at Ig loci via the action of activation-induced deaminase. Our results help resolve key aspects of immunoglobulin diversification and suggest an essential role of PC4 in repair pathway choice. We show that PC4 ablation in gene conversion (GC)-active cells significantly disrupts GC but has little to no effect on targeted homologous recombination. In agreement, the global double-strand break repair response, as measured by gamma H2AX foci analysis, is unperturbed 16 hours post irradiation. In cells with the pseudo-genes removed (GC inactive), PC4 ablation reduced the overall mutation rate while simultaneously increasing the transversion mutation ratio. By tagging the N-terminus of PC4, gene conversion and somatic hypermutation are all but abolished even when native non-tagged PC4 is present, indicating a dominant negative effect. Our data point to a very early and deterministic role for PC4 in DNA repair pathway re-routing

Chromosomal radiosensitivity and acute radiation side effects after radiotherapy in tumour patients - a follow-up study

Abstract Background 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. Methods 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. Results 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. Conclusions The results suggest that translocations can be used as a test to identify individuals with a potentially elevated radiosensitivity.</p

Chromosomal imbalances are associated with metastasis-free survival in breast cancer patients,

Abstract. Multiple chromosomal imbalances have been identified in breast cancer using comparative genomic hybridization (CGH). Their association with the primary tumors&apos; potential for building distant metastases is unknown. In this study we have investigated 39 invasive breast carcinomas with a mean follow-up period of 99 months (max. 193 months) by CGH to determine the prognostic value of chromosomal gains and losses. The mean number of chromosomal imbalances per tumor was 6.5±0.7 (range 2 to 18). The most frequent alterations identified in more than 1/3 of cases were gains on chromosomes 11q13, 12q24, 16, 17, and 20q, and losses on 2q and 13q. A significantly different frequency of chromosomal aberrations (p 0.05) was found between DNA-diploid and non-diploid tumors (gain on chromosome 17). Differences were also noted between tumors progressing to distant metastases within the period of follow-up and those which do not (gains on 11q13 and 12q24; loss on 12q). Significant univariate correlations (p 0.05) with the metastasis-free survival of patients were found for lymph node status, the cytometrical determined DNA ploidy (diploid/non-diploid) and anisokaryosis, and for DNA gains on 11q13, 12q24, 17, and 18p. An unexpected inverse correlation was found between clinical outcome and gains on 11q13 and 12q24. In multivariate analysis independent prognostic value, in addition to lymph node status, was found for chromosomal gains on 11q13, 12q24, 17 and 18p. Amplification on 20q, which did not correlate with metastasis-free survival in a univariate analysis, showed weak prognostic significance in combination with the nodal status. The prognostic value of chromosomal alterations -some of them by inverse correlation -suggests an interaction and/or compensation of the involved amplified genes and their effects on the occurrence of distant metastases in breast cancer patients

Natural Cubic Spline Regression Modeling Followed by Dynamic Network Reconstruction for the Identification of Radiation-Sensitivity Gene Association Networks from Time-Course Transcriptome Data.

Gene expression time-course experiments allow to study the dynamics of transcriptomic changes in cells exposed to different stimuli. However, most approaches for the reconstruction of gene association networks (GANs) do not propose prior-selection approaches tailored to time-course transcriptome data. Here, we present a workflow for the identification of GANs from time-course data using prior selection of genes differentially expressed over time identified by natural cubic spline regression modeling (NCSRM). The workflow comprises three major steps: 1) the identification of differentially expressed genes from time-course expression data by employing NCSRM, 2) the use of regularized dynamic partial correlation as implemented in GeneNet to infer GANs from differentially expressed genes and 3) the identification and functional characterization of the key nodes in the reconstructed networks. The approach was applied on a time-resolved transcriptome data set of radiation-perturbed cell culture models of non-tumor cells with normal and increased radiation sensitivity. NCSRM detected significantly more genes than another commonly used method for time-course transcriptome analysis (BETR). While most genes detected with BETR were also detected with NCSRM the false-detection rate of NCSRM was low (3%). The GANs reconstructed from genes detected with NCSRM showed a better overlap with the interactome network Reactome compared to GANs derived from BETR detected genes. After exposure to 1 Gy the normal sensitive cells showed only sparse response compared to cells with increased sensitivity, which exhibited a strong response mainly of genes related to the senescence pathway. After exposure to 10 Gy the response of the normal sensitive cells was mainly associated with senescence and that of cells with increased sensitivity with apoptosis. We discuss these results in a clinical context and underline the impact of senescence-associated pathways in acute radiation response of normal cells. The workflow of this novel approach is implemented in the open-source Bioconductor R-package splineTimeR