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.

progression of the DNA-repair process. Here, we analyzed whether different modes of 23 irradiation influence the formation of DSBs in the PBLs of patients with cancer, and whether 24 accompanying chemotherapy influences the DSB-appearance.

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We obtained 86 blood samples before and 0.1, 0.5 and 24 h after irradiation from patients 26 with head and neck, or rectal cancers receiving radiotherapy (RT) or radio-chemotherapy 27 (RCT). 53BP1-foci were quantified by IFM. In addition, TEM was used to quantify gold-28 labelled pKu70-dimers and 53BP1-clusters within euchromatin and heterochromatin of PBLs.

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dose intensity per fraction, number of irradiation fields, or isodose volume. However, more 33 53BP1-foci were detected in PBLs of patients treated with accompanying chemotherapy.

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To perform TEM-analysis, blood samples were collected directly before and 0.1, 0.5 and 24 h 126 after the first RT-fraction for immediate processing.

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For ex-vivo-experiments, blood from healthy donors was obtained, PBLs isolated,

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Briefly, blood samples in heparin tubes were diluted with 6 ml RPMI and incubated at 37°C.

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PBLs were isolated using a kit (PAA Laboratories; Cölbe, Germany). Blood samples were 138 layered on Percol 400 and centrifuged at 1200 g for 20 minutes. 5 ml PBS was added to the 139 resulting interphase and centrifuged at 300 g for 10 minutes. The separation yielded ~80% and kept for 24 h at -20°C followed by ultraviolet light exposure until resin was polymerized.

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Ultrathin 70 nm slices were sectioned off the samples using a Microtome Ultracut UCT identified pKu70-dimers (two 10 nm gold particles) and 53BP1 bead-clusters (6 nm) visually 176 at 48000-86000x magnification and counted these in 50 randomly chosen nuclear sections.

Statistical analysis 178
A one sided Mann-Whitney Test was performed using the statistical software OriginPro 179 (version 8.5, OriginLab Corporation, Northampton, USA) to evaluate potential differences 180 between data groups. The criterion for statistical significance was p ≤ 0.05.

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The dispersion index test was used to determine the deviation of foci per cell-distribution at 182 the 0.5 h data point from Poisson statistics to demonstrate that -in the setting of partial body 183 irradiation to the head and neck or pelvic region -only a proportion of PBLs was exposed to 184 irradiation [28,29]. The test was performed with the software Dose Estimate, version 3.0

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The study population consisted of nine individuals with head and neck or rectal cancers (5 188 patients received RCT and 4 RT without chemotherapy).

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To compare the appearance of 53BP1-foci among samples from different treatment types,

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we looked at the PTV-size, radiation duration and exposed blood volume from delineated 210 blood vessels (>1 cm diameters) encompassed by the 50%-isodose line. Table 2 shows the 211 53BP1-foci distribution analysis results measured by IFM 0.5 h after the first RT-fraction.

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The control showed a low number of foci in patients with head and neck cancer (0.37 ± 0.02 234 53BP1-foci/cell) and in those with rectal cancer (0.33 ± 0.08 53BP1-foci/cell  (Fig 2A).

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The 53BP1-foci-levels in PBLs of patients with head and neck cancer tended to outnumber 247 those of patients with rectal cancer; however, the differences were not significant. Moreover,

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we analyzed the number of 53BP1-foci according to whether patients received 249 accompanying chemotherapy (Fig 2B)

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Additionally, 53BP1-clusters, consisting of 5 to 12 gold beads and without pKu70 304 colocalization, were observed 8 h and 24 h after irradiation, potentially marking chromatin 305 changes in areas where DNA-damage was present.

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To expand our knowledge on DNA-damage, we investigated PBLs of patients with head and 313 neck cancer after RT and RCT using TEM, before and 0.1, 0.5 and 24 h after the first fraction 314 by quantifying pKu70-dimers and 53BP1-clusters in euchromatin and heterochromatin of 50 315 nuclear sections per sample. Figs 4A and 4B show representative TEM-micrographs.

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Visualization of 10-nm (pKu70-dimers) and 6-nm-gold beads (53BP1-cluster) was improved 317 by overlaying with red and green circles respectively. The RCT-patient (Figs 4C-F) showed a 318 higher level of repair proteins than the RT-patient in both chromatin domains (Fig 4C) 0.5 h 319 after the first fraction.

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In this study, we questioned whether the DNA damage repair in PBLs during radiotherapy for

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Following homogeneous PBL irradiation, IFM showed a linear 53BP1 dose correlation up to 365 1 Gy ( Fig 1C) and TEM showed similar correlation up to 4 Gy ( Fig 3C). With increasing doses (> 1 Gy), the fluorescent signal of individual adjacent foci overlapped, making 367 quantification in IFM difficult or almost impossible (Fig 1A). This limitation does not occur with 368 quantitative TEM as gold beads were either present and quantifiable or absent. However,

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when LR-gold-resin embedded PBL sections are investigated and quantified using TEM it is  RCT, independent of the collective.

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By using the higher resolution of TEM in combination with the immunogold-labeling of pKu70 418 and 53BP1 within the intact nuclear cell ultrastructure, we were able to detect a higher 419 number of pKu70-dimers in the euchromatin and in the heterochromatin of PBLs in RCT-420 patients than in those of patients after a single RT 24 h post-IR (Fig 4). In addition, we 421 visualized pKu70-dimers individually as well as collectively (2x or 3x pKu70-dimers) which grant number 02NUK035A (grant coordinator Claudia E. Rübe). The funders had no role in 450 study design, data collection and analysis, decision to publish, or preparation of the 451 manuscript.

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The above-mentioned authors state that there are no actual or potential conflicts of interest 454 to disclose.

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Data Availability

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All relevant data are within the paper and its supporting information files.  antibody, bound to a secondary antibody coupled to a 10 nm colloidal gold particle.