RENEB Inter-Laboratory comparison 2017: limits and pitfalls of ILCs.

PURPOSE: In case of a mass-casualty radiological event, there would be a need for networking to overcome surge limitations and to quickly obtain homogeneous results (reported aberration frequencies or estimated doses) among biodosimetry laboratories. These results must be consistent within such network. Inter-laboratory comparisons (ILCs) are widely accepted to achieve this homogeneity. At the European level, a great effort has been made to harmonize biological dosimetry laboratories, notably during the MULTIBIODOSE and RENEB projects. In order to continue the harmonization efforts, the RENEB consortium launched this intercomparison which is larger than the RENEB network, as it involves 38 laboratories from 21 countries. In this ILC all steps of the process were monitored, from blood shipment to dose estimation. This exercise also aimed to evaluate the statistical tools used to compare laboratory performance. MATERIALS AND METHODS: Blood samples were irradiated at three different doses, 1.8, 0.4 and 0 Gy (samples A, C and B) with 4-MV X-rays at 0.5 Gy min-1, and sent to the participant laboratories. Each laboratory was requested to blindly analyze 500 cells per sample and to report the observed frequency of dicentric chromosomes per metaphase and the corresponding estimated dose. RESULTS: This ILC demonstrates that blood samples can be successfully distributed among laboratories worldwide to perform biological dosimetry in case of a mass casualty event. Having achieved a substantial harmonization in multiple areas among the RENEB laboratories issues were identified with the available statistical tools, which are not capable to advantageously exploit the richness of results of a large ILCs. Even though Z- and U-tests are accepted methods for biodosimetry ILCs, setting the number of analyzed metaphases to 500 and establishing a tests' common threshold for all studied doses is inappropriate for evaluating laboratory performance. Another problem highlighted by this ILC is the issue of the dose-effect curve diversity. It clearly appears that, despite the initial advantage of including the scoring specificities of each laboratory, the lack of defined criteria for assessing the robustness of each laboratory's curve is a disadvantage for the 'one curve per laboratory' model. CONCLUSIONS: Based on our study, it seems relevant to develop tools better adapted to the collection and processing of results produced by the participant laboratories. We are confident that, after an initial harmonization phase reached by the RENEB laboratories, a new step toward a better optimization of the laboratory networks in biological dosimetry and associated ILC is on the way.

Interphase Cytogenetic Analysis of G0 Lymphocytes Exposed to α-Particles, C-Ions, and Protons Reveals their Enhanced Effectiveness for Localized Chromosome Shattering-A Critical Risk for Chromothripsis.

For precision cancer radiotherapy, high linear energy transfer (LET) particle irradiation offers a substantial advantage over photon-based irradiation. In contrast to the sparse deposition of low-density energy by χ- or γ-rays, particle irradiation causes focal DNA damage through high-density energy deposition along the particle tracks. This is characterized by the formation of multiple damage sites, comprising localized clustered patterns of DNA single- and double-strand breaks as well as base damage. These clustered DNA lesions are key determinants of the enhanced relative biological effectiveness (RBE) of energetic nuclei. However, the search for a fingerprint of particle exposure remains open, while the mechanisms underlying the induction of chromothripsis-like chromosomal rearrangements by high-LET radiation (resembling chromothripsis in tumors) await to be elucidated. In this work, we investigate the transformation of clustered DNA lesions into chromosome fragmentation, as indicated by the induction and post-irradiation repair of chromosomal damage under the dynamics of premature chromosome condensation in G0 human lymphocytes. Specifically, this study provides, for the first time, experimental evidence that particle irradiation induces localized shattering of targeted chromosome domains. Yields of chromosome fragments and shattered domains are compared with those generated by γ-rays; and the RBE values obtained are up to 28.6 for α-particles (92 keV/µm), 10.5 for C-ions (295 keV/µm), and 4.9 for protons (28.5 keV/µm). Furthermore, we test the hypothesis that particle radiation-induced persistent clustered DNA lesions and chromatin decompaction at damage sites evolve into localized chromosome shattering by subsequent chromatin condensation in a single catastrophic event-posing a critical risk for random rejoining, chromothripsis, and carcinogenesis. Consistent with this hypothesis, our results highlight the potential use of shattered chromosome domains as a fingerprint of high-LET exposure, while conforming to the new model we propose for the mechanistic origin of chromothripsis-like rearrangements.

Individual Radiosensitivity in Oncological Patients: Linking Adverse Normal Tissue Reactions and Genetic Features.

Introduction: Adverse effects of radiotherapy (RT) significantly affect patient's quality of life (QOL). The possibility to identify patient-related factors that are associated with individual radiosensitivity would optimize adjuvant RT treatment, limiting the severity of normal tissue reactions, and improving patient's QOL. In this study, we analyzed the relationships between genetic features and toxicity grading manifested by RT patients looking for possible biomarkers of individual radiosensitivity. Methods: Early radiation toxicity was evaluated on 143 oncological patients according to the Common Terminology Criteria for Adverse Events (CTCAE). An individual radiosensitivity (IRS) index defining four classes of radiosensitivity (highly radiosensitive, radiosensitive, normal, and radioresistant) was determined by a G2-chromosomal assay on ex vivo irradiated, patient-derived blood samples. The expression level of 15 radioresponsive genes has been measured by quantitative real-time PCR at 24 h after the first RT fraction, in blood samples of a subset of 57 patients, representing the four IRS classes. Results: By applying univariate and multivariate statistical analyses, we found that fatigue was significantly associated with IRS index. Interestingly, associations were detected between clinical radiation toxicity and gene expression (ATM, CDKN1A, FDXR, SESN1, XPC, ZMAT3, and BCL2/BAX ratio) and between IRS index and gene expression (BBC3, FDXR, GADD45A, and BCL2/BAX). Conclusions: In this prospective cohort study we found that associations exist between normal tissue reactions and genetic features in RT-treated patients. Overall, our findings can contribute to the identification of biological markers to predict RT toxicity in normal tissues.

Investigation of the influence of calibration practices on cytogenetic laboratory performance for dose estimation.

PURPOSE: In the frame of the QA program of RENEB, an inter-laboratory comparison (ILC) of calibration sources used in biological dosimetry was achieved to investigate the influence of calibration practices and protocols on the results of the dose estimation performance as a first step to harmonization and standardization of dosimetry and irradiation practices in the European biological dosimetry network. MATERIALS AND METHODS: Delivered doses by irradiation facilities used by RENEB partners were determined with EPR/alanine dosimetry system. Dosimeters were irradiated in the same conditions as blood samples. A short survey was also performed to collect the information needed for the data analysis and evaluate the diversity of practices. RESULTS: For most of partners the deviation of delivered dose from the targeted dose remains below 10%. Deviations larger than 10% were observed for five facilities out of 21. Origins of the largest discrepancies were identified. Correction actions were evaluated as satisfactory. The re-evaluation of some ILC results for the fluorescence in situ hybridization (FISH) and premature chromosome condensation (PCC) assays has been performed leading to an improvement of the overall performances. CONCLUSIONS: This work has shown the importance of dosimetry in radiobiology studies and the needs of harmonization, standardization in irradiation and dosimetry practices and educational training for biologists using ionizing radiation.

Dose assessment intercomparisons within the RENEB network using G0-lymphocyte prematurely condensed chromosomes (PCC assay).

PURPOSE: Dose assessment intercomparisons within the RENEB network were performed for triage biodosimetry analyzing G0-lymphocyte PCC for harmonization, standardization and optimization of the PCC assay. MATERIALS AND METHODS: Comparative analysis among different partners for dose assessment included shipment of PCC-slides and captured images to construct dose-response curves for up to 6 Gy γ-rays. Accident simulation exercises were performed to assess the suitability of the PCC assay by detecting speed of analysis and minimum number of cells required for categorization of potentially exposed individuals. RESULTS: Calibration data based on Giemsa-stained fragments in excess of 46 PCC were obtained by different partners using galleries of PCC images for each dose-point. Mean values derived from all scores yielded a linear dose-response with approximately 4 excess-fragments/cell/Gy. To unify scoring criteria, exercises were carried out using coded PCC-slides and/or coded irradiated blood samples. Analysis of samples received 24 h post-exposure was successfully performed using Giemsa staining (1 excess-fragment/cell/Gy) or centromere/telomere FISH-staining for dicentrics. CONCLUSIONS: Dose assessments by RENEB partners using appropriate calibration curves were mostly in good agreement. The PCC assay is quick and reliable for whole- or partial-body triage biodosimetry by scoring excess-fragments or dicentrics in G0-lymphocytes. Particularly, analysis of Giemsa-stained excess PCC-fragments is simple, inexpensive and its automation could increase throughput and scoring objectivity of the PCC assay.