Subgroup Analysis of Functional Associations Between In Vitro Radiation Response of Fibroblasts and Late Toxicity After Breast Radiation Therapy

Purpose/Objective(s)
Late toxicity after tumor radiotherapy (RT) is considered to be determined by variations in multiple, low-penetrance genes each associated with moderate hazard ratios. Here we test the hypothesis that functional assays can distinguish subgroups of patients characterized by different mechanisms associated with patients' risk of late reaction after radiotherapy.

Materials/Methods
Early-passage fibroblast cultures were established with informed consent from selected breast cancer patients with minimal (RT-resistant, n = 15) or marked breast changes (RT-sensitive, n = 19) after breast conserving therapy and were irradiated with 6MV X-rays in vitro. Residual DNA double-strand breaks (DSBs) were scored by 53BP1 foci 24h after irradiation with 4 Gy. Molecular markers p53 and Ki-67 were detected by immunofluorescence microscopy and the plating efficiency (PE) was determined by the colony formation assay. The non-parametric Wilcoxon/Mann-Whitney test, Spearman's rank correlation test, and a linear model, were used to test for statistical significance.

Results
RT-sensitive patients showed significantly increased residual 53BP1 foci (2.48 ± 0.22 vs 1.86 ± 0.11 per cell; P = 0.007) and an increased basal p53-positive fraction of cells without irradiation (7.1 ± 1.5 vs 2.5 ± 1.0 pct.; P = 0.02). In addition, the subgroup of RT-sensitive patients with severe reaction relative to known risk factors (n = 10) showed vigorous early upregulation of p53 2h after irradiation (P = 0.005) in contrast to RT-sensitive patients with more moderate reaction (n = 9) and RT-resistant patients. RT sensitivity showed no significant correlation with proliferation marker Ki-67. However, in the patient subgroup with low fibroblast proliferation activity (Ki-67 index), RT sensitivity correlated with basal levels of p53 (P < 0.001) and residual 53BP1 (P = 0.013) whereas in the patient subgroup with higher Ki-67 index, severity correlated with early upregulation at 2h (P < 0.001) after irradiation. Multivariate analysis suggested that these potentially predictive parameters were largely independent.

Conclusion
These results establish an association between the radiation response of fibroblasts and late reaction of the breast after RT and provide novel evidence for the existence of patient subgroups. We suggest that defects in the p53 stress response or DSB repair pathways may contribute to late reaction in RT-sensitive patients, possibly via increased misrepair and genomic instability. Functional studies may synergize with “omics” approaches and help unravel the mechanisms of normal-tissue reaction after RT.