In vitro cellular radiosensitivity in relationship to late normal tissue reactions in breast cancer patients: a multi-endpoint case-control study

Purpose: A minority of patients exhibits severe late normal tissue toxicity after radiotherapy (RT), possibly related to their inherent individual radiation sensitivity. This study aimed to evaluate four different candidate in vitro cellular radiosensitivity assays for prediction of late normal tissue reactions, in a retrospective matched case-control set-up of breast cancer patients. Methods: The study population consists of breast cancer patients expressing severe radiation toxicity (12 cases) and no or minimal reactions (12 controls), with a follow-up for at least 3 years. Late adverse reactions were evaluated by comparing standardized photographs pre-and post-RT resulting in an overall cosmetic score and by clinical examination using the LENT-SOMA scale. Four cellular assays on peripheral blood lymphocytes reported to be associated with normal tissue reactions were performed after in vitro irradiation of patient blood samples to compare case and control radiation responses: radiation-induced CD8+ late apoptosis, residual DNA double-strand breaks, G0 and G2 micronucleus assay. Results: A significant difference was observed for all cellular endpoints when matched cases and controls were compared both pairwise and grouped. However, it is important to point out that most casecontrol pairs showed a substantial overlap in standard deviations, which questions the predictive value of the individual assays. The apoptosis assay performed best, with less apoptosis seen in CD8+ lymphocytes of the cases (average: 14.45%) than in their matched controls (average: 30.64%) for 11 out of 12 patient pairs (p<.01). The number of residual DNA DSB was higher in cases (average: 9.92 foci/cell) compared to their matched control patients (average: 9.17 foci/cell) (p<.01). The average dose response curve of the G0MN assay for cases lies above the average dose response curve of the controls. Finally, a pairwise comparison of the G2MN results showed a higher MN yield for cases (average: 351MN/1000BN) compared to controls (average: 219MN/1000BN) in 9 out of 10 pairs (p<.01). Conclusion: This matched case-control study in breast cancer patients, using different endpoints for in vitro cellular radiosensitivity related to DNA repair and apoptosis, suggests that patients' intrinsic radiosensitivity is involved in the development of late normal tissue reactions after RT. Larger prospective studies are warranted to validate the retrospective findings and to use in vitro cellular assays in the future to predict late normal tissue radiosensitivity and discriminate individuals with marked RT responses.