Assessment of individual radiosensitivity in human lymphocytes of cancer patients and its correlation with adverse side effects to radiation therapy

Background and purpose: Individual radiosensitivity is an inherent characteristic, associated with an increased reaction to ionizing radiation on the human body. Biological endpoints such as clonogenic survival, chromosome aberration formation and repair capacity of radiation-induced damage have been applied to evaluate individual radiosensitivity in vitro. 5%-7% of cancer patients develop adverse side effects to radiation therapy in normal tissues within the treatment field, which are referred as 'clinical radiation reactions' and include acute effects, late effects and cancer induction. It has been hypothesized that the occurrence and severity of these reactions are mainly influenced by genetic susceptibility to radiation. Additionally, the nature of the genetic disorders associated with hypersensitivity to radiotherapy suggests that DNA repair mechanisms are involved. Consequently, the characterization of DNA repair in lymphocytes through cytokinesis blocked micronucleus (MN) and alkaline single-cell micro gel electrophoresis (comet) assays could be suitable approaches to evaluate individual radiosensitivity in vitro. The MN assay is an established cytogenetic technique to evaluate intrinsic cell radiosensitivity in tumor cells and lymphocytes; comet assay is a sensitive and rapid method for measuring DNA damage and repair in individual cells. The aims of this study were: 1) To assess the in vitro radiosensitivity of peripheral blood lymphocytes from two groups of cancer patients (retrospectively and prospectively studied), using MN and comet assays, in comparison with the observed clinical response; and 2) To test the predictive potential of both techniques. Materials and methods: 38 cancer patients receiving radiation therapy were enrolled in this study. The tumor sites were: head and neck (n 25) and cervix (n = 13). Nineteen patients were evaluated about 6-18 month after radiotherapy (retrospective group) and 19 patients were evaluated prior, mid-way an of a treatment (prospective group). In the retrospective group, blood samples were irradiated in vitro with 0 (control) or 2 Gy and evaluated using MN test. Cytogenetic data were analyzed comparing expected MN frequencies (calibration curve from healthy donors) with values observed after in vitro irradiation. One over reactor was also evaluated trough comet assay. DNA damage and repair capacity were quantified by the tail moment. Lymphocytes of healthy individuals were used as reference sample. Cytogenetic data from the prospective group were analyzed using a mathematical model to evaluate the attenuation of the cytogenetic effect as a function of the time between a single exposure and blood sampling, estimating a cytogenetic recovery factor k. Results: In the retrospective evaluation, lymphocytes of 3 from 4 patients that had developed late reactions were significantly more radiosensitive than lymphocytes from the rest of the patients and normal donors. The individual cytogenetic response suggests a correlation with the maximum grade of late reaction (osteonecrosis, fibrosis and trismus). The patient additionally evaluated by comet assay showed reduced DNA repair capacity. In the prospective evaluation, factor k correlated with the individual radiosensitivity. Patients with low recovery from the cytogenetic effect (k tending to zero) developed late toxicity (fibrosis and actinic rectitis). Conclusions: MN and comet tests could be suitable predictive assays to evaluate individual radiosensitivity in vitro, contributing with the detection of patients that develop late adverse reactions. However, further studies are required to confirm the validity of these tests.