Concomitant chemotherapy increases radiotherapy-mediated DNA-damage in peripheral blood lymphocytes

53BP1-foci detection in peripheral blood lymphocytes (PBLs) by immunofluorescence microscopy (IFM) is a sensitive and quantifiable DNA-double-strand-break (DSB) marker. In addition, high-resolution transmission-electron-microscopy (TEM) with immunogold-labeling of 53BP1 and DSB-bound phosphorylated Ku70 (pKu70) can be used to determine the progression of the DNA-repair process. Here, we analyzed whether different modes of irradiation influence the formation of DSBs in the PBLs of patients with cancer, and whether accompanying chemotherapy influences the DSB-appearance. We obtained 86 blood samples before and 0.1, 0.5 and 24 h after irradiation from patients with head and neck, or rectal cancers receiving radiotherapy (RT) or radio-chemotherapy (RCT). 53BP1-foci were quantified by IFM. In addition, TEM was used to quantify gold-labelled pKu70-dimers and 53BP1-clusters within euchromatin and heterochromatin of PBLs. During radiotherapy, persisting 53BP1-foci accumulated in PBLs with increasing numbers of administered RT-fractions. This 53BP1-foci accumulation was not influenced by irradiation technique applied (3D-conformal radiotherapy versus intensity-modulated radiotherapy), dose intensity per fraction, number of irradiation fields, or isodose volume. However, more 53BP1-foci were detected in PBLs of patients treated with accompanying chemotherapy. TEM analyses showed that DSBs, indicated by pKu70, were present for longer periods in PBLs of RCT-patients than in PBLs of RT-only-patients. Moreover, not every residual 53BP1-focus was equivalent to a remaining DSB, since pKu70 was not present at every damage site. Persistent 53BP1-clusters, visualized by TEM, without colocalizing pKu70 likely indicate chromatin alterations after repair completion, or possibly, defective repair. Therefore, IFM-53BP1-foci analyses alone are not adequate to determine individual repair capacity after irradiation of PBLs, as a DSB may be indicated by a 53BP1-focus but not every 53BP1-focus represents a DSB. The level of DNA-damage during RT is influenced by the presence of accompanying chemotherapy.