Eur J Nucl Med Mol Imaging (2012) 39:1712–1719 DOI 10.1007/s00259-012-2201-1

ORIGINAL ARTICLE

Induction and repair of DNA double-strand breaks in blood lymphocytes of patients undergoing 18F-FDG PET/CT examinations Matthias S. May & Michael Brand & Wolfgang Wuest & Katharina Anders & Torsten Kuwert & Olaf Prante & Daniela Schmidt & Simone Maschauer & Richard C. Semelka & Michael Uder & Michael A. Kuefner Received: 29 February 2012 / Accepted: 16 July 2012 / Published online: 2 August 2012 # Springer-Verlag 2012

Abstract Purpose The purpose of this study was to evaluate DNA double-strand breaks (DSBs) in blood lymphocytes of patients undergoing positron emission tomography (PET)/ CT using γ-H2AX immunofluorescence microscopy and to differentiate between 18F-fluorodeoxyglucose (FDG) and CT-induced DNA lesions. Methods This study was approved by the local Ethics Committee and complies with Health Insurance Portability and Accountability Act (HIPAA) requirements. After written informed consent was obtained, 33 patients underwent whole-body 18F-FDG PET/CT (3 MBq/kg body weight, 170/100 reference mAs at 120 kV). The FDG PET and CT portions were performed as an initial CT immediately followed by the PET. Blood samples were obtained before, at various time points following 18F-FDG application and up to 24 h after the CT scan. Distinct foci representing DSBs

were quantified in isolated lymphocytes using fluorescence microscopy after staining against the phosphorylated histone variant γ-H2AX. Results The DSB values at the various time points were significantly different (p<0.001). The median baseline level was 0.08/cell (range 0.06–0.12/cell). Peaks of radiationinduced DSBs were found 30 min after 18F-FDG administration (median excess foci 0.11/cell, range 0.06–0.27/cell) and 5 min after CT (median excess foci 0.17/cell, range 0.05–0.54/cell). A significant correlation between CTinduced DSBs and dose length product was obtained (ρ0 0.898, p<0.001). After 24 h DSB values were still slightly but significantly elevated (median foci 0.11/cell, range 0.10–0.14/cell, p00.003) compared to pre-exposure levels. Conclusion PET/CT-induced DSBs can be monitored using γ-H2AX immunofluorescence microscopy. Peak values may be obtained 30 min after 18F-FDG injection and 5 min after CT. The radionuclide contributes considerably to the total DSB induction in this setting.

M. S. May : M. Brand : W. Wuest : K. Anders : M. Uder : M. A. Kuefner Department of Radiology, University Hospital Erlangen, Erlangen, Germany

Keywords DNA double-strand breaks . Positron emission tomography . Computed tomography . Immunofluorescence microscopy . Gamma-H2AX . Radiation exposure

T. Kuwert : O. Prante : D. Schmidt : S. Maschauer Department of Nuclear Medicine, University Hospital Erlangen, Erlangen, Germany

Introduction

R. C. Semelka Department of Radiology, University of North Carolina, Chapel Hill, NC, USA M. S. May (*) Department of Radiology, University Hospital Erlangen, Maximiliansplatz 1, 91054 Erlangen, Germany e-mail: [email protected]

The combination of positron emission tomography (PET) and computed tomography (CT) allows for functional and topographical imaging with high accuracy by fusion of the two separately acquired data sets [1, 2]. Use of PET/CT has increased due to its roles in the diagnosis of malignant disease, benign disease evaluation, tumour staging, imageguided therapy planning and treatment monitoring, and this

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has been accompanied by rising concern because of its rather high radiation exposure [3]. This is especially true for repetitive examinations in benign diseases and younger patients [4]. Examinations that include a diagnostic CT scan might account for effective doses ten times higher than natural background radiation per year [5, 6]. Established dose calculations are based on physiological and biokinetic models delineating the detailed distribution of the radionuclides [7, 8]. However, due to the complex interaction of various compartments and tissues these estimates remain assumptions with undetermined inaccuracies [9]. Recently, an immunofluorescence technique has been established to assess radiation-induced DNA double-strand breaks (DSBs) as biomarkers of exposure in cells, based on the phosphorylation of the histone variant H2AX [10]. This method has been shown to be reliable and sensitive for monitoring in vivo induction and repair of DSBs in blood lymphocytes after CT and angiography. All prior studies have described either in vitro irradiation or irradiation of defined body regions [11, 12]. The DSB levels obtained are dependent on both local radiation exposure and the exposed blood volume [13]. Whole-body exposure and thus irradiation of almost all resting and circulating lymphocytes has not been previously reported. Furthermore, the biological effect of radionuclides at diagnostic dosages has not yet been evaluated. 18F-Fluorodeoxyglucose (FDG), frequently used in PET/CT, is trapped within the cytoplasm after uptake by glucose transporters and subsequent phosphorylation by hexokinase. The radioactive decay of 18F occurs through the emission of a positron (β+, Emax 0 635 keV) followed by annihilation resulting in γ-radiation (511 MeV). The impact on the biological radiation damage by this intracellular radiation source has not yet been investigated in vivo. Therefore, the aim of this study was to analyse kinetics of DSB formation and repair after combined 18F-FDG PET/CT examinations and to differentiate between the radiation damage induced by the radionuclide and the CT scan.

Materials and methods Patients This prospective, single-centre study complied with the Declaration of Helsinki and the Health Insurance Portability and Accountability Act (HIPAA). Written informed consent was obtained in accordance with local Ethics Committee approval. Of 165 consecutive patients with clinical indication for whole-body 18F-FDG PET/CT examination, 33 patients were enrolled and 132 patients were excluded. Exclusion criteria were radiation or chemotherapy within the last 6 months, X-ray examination within the last 3 days

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and history of lymphoma or leukaemia (in order to avoid including patients with preexistent lymphocyte damage or intrinsic deficits that may affect DSB repair). 18

F-FDG PET/CT examinations

Patient preparation was performed according to standard operational procedures. After acquisition of blood glucose concentration, body weight-adapted 18 F-FDG activity (3 MBq/kg) was injected through an antecubital vein. Blood glucose concentration >7.77 mmol/l was a contraindication for 18F-FDG administration. The combined PET/CT examination was performed using a 64-slice scanner (Siemens Biograph True Point 64, Siemens Healthcare, Forchheim, Germany). CT was performed at 120 kV either using a standard protocol (SP) with 170 reference mAs in 27 subjects or a low-dose (LD) protocol with 100 reference mAs in 6 subjects. In all patients, anatomy-based tube current modulation (Care Dose 4D™) was used. The scan range included head, neck, chest, abdomen, pelvis and proximal upper legs. In eight individuals, an additional examination of both legs was appended 31–34 min (median 32 min) after the first CT scan. Twenty-three patients received 0.5 g/kg iodinated contrast agent intravenously (iopromide, Ultravist 370®, Bayer-Schering Healthcare, Berlin, Germany) for the diagnostic CT scan. The mean delay between FDG administration and PET acquisition was 80±22 min. The resulting tube current time product, CT index (CTDIvol) and dose length product (DLP) were recorded as provided by the scanner’s patient protocols. Standardized uptake values (SUV) were measured in the liver and in the blood pool (left ventricle, right ventricle, aorta) and the biodistribution of 18F-FDG was evaluated by a nuclear medicine specialist with 16 years of experience. Determination of in vivo DSBs and time-activity curves Patients’ blood samples were taken from a contralateral antecubital vein prior to 18F-FDG administration to obtain DSB baseline levels, 30 min after 18F-FDG application, and before, 5 min, 30 min and 24 h after whole-body CT using ethylenediaminetetraacetic acid (EDTA)-containing vials (see Fig. 1). In those eight patients undergoing wholebody as well as additional leg scan, blood samples were also obtained before and 5 min after the additional CT scan. For determination of individual in vivo time courses of 18FFDG-related DSB induction and repair, additional serial blood draws were obtained from eight patients within short intervals following 18F-FDG administration (in patient 23: at 10, 30, 45, 94 min; patient 24: at 13, 30, 49, 65, 82 min; patient 25: at 4, 28, 45, 65 min; patient 26: at 1, 16, 31, 50, 68 min; patient 27: at 15, 28, 30, 31, 33, 35, 45, 69 min; patient 28: at 15, 25, 27, 30, 32, 35, 45, 68 min; patient 29:

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Eur J Nucl Med Mol Imaging (2012) 39:1712–1719 Blood samples to obtain baseline DSB levels prior to 18F-FDG administration (PREFDG, n=33)

Blood samples 30 minutes after 18

F-FDG application (FDG30, n=11)

Blood samples prior to CT (PRECT1, n=33)

Blood samples 5 and 30 minutes following CT (CT1, n=33)

Blood samples immediately before and 5 min following additional leg scan (PRECT2, CT2, n=8)

γ-H2AX immunofluorescence microscopy Isolated leucocytes were stained as described in detail in prior studies [10]. Briefly, after fixation and permeabilization each sample was washed, labelled using specific γH2AX [Anti-H2A.X-Phosphorylated (Ser139), BioLegend, Uithoorn, The Netherlands] and fluorescent secondary antibodies (Alexa Fluor 488-conjugated goat anti-mouse secondary antibody, Invitrogen, Paisley, UK), washed again and covered with a mounting medium. γ-H2AX foci were quantified using a DM6000 B fluorescence microscope (Leica, Wetzlar, Germany) equipped with a ×63 magnification objective. In each sample, clearly visually detectable foci were counted by a medical technical assistant with 6 years of daily experience in γ-H2AX foci counting until 40 γ-H2AX foci were detected. The foci numbers were expressed in relation to the number of enumerated cells. The foci size and intensity were not assessed. Each focus represents one DSB [16]. Numbers of 18F-FDG-induced DSBs were calculated by subtracting DSB levels obtained before (PREFDG) from those obtained 30 min after 18FFDG (FDG30) application, and CT-induced DSBs were assessed by subtracting foci numbers of corresponding pre-CT (PRECT1/2) from post-CT (CT1/2) samples and were called excess foci. Statistical analyses

Blood samples 24 hours following whole body CT (H24, n=11)

Fig. 1 Flow chart depicting time points of blood withdrawal

at 25, 27, 30, 33, 36, 73 min; patient 33: at 2, 7, 13, 20, 27, 30, 33, 40, 50, 60, 95 min). All samples were stored at 4 °C during transport to the laboratory. In order to prevent DSB foci loss by DNA repair 1 nM calyculin A was added to each vial, which is a potent inhibitor of the dephosphorylation of γ-H2AX [14]. Lymphocytes were isolated by using gradient centrifugation, as described in detail previously [10]. Cells were spotted onto microscope slides for 10 min at room temperature followed by fixation in 100 % methanol (20 min, −20 °C) and permeabilization in 100 % acetone (1 min, −20 °C). To determine comparative in vivo radioactivity time curves, additional blood samples were withdrawn from two patients (1 male, 1 female) at different time points after 18F-FDG administration (1–120 min) and radioactivity measurements were performed using a gamma counter (1470 Wallac Wizard Gamma Counter, PerkinElmer, Waltham, MA, USA). For estimation of the initial radioactivity concentration, the injected radioactivity was divided by the calculated total blood volume [15].

In order to assess the dependence of in vivo foci levels on the duration after exposure one-way analysis of variance was performed. Paired Wilcoxon tests were performed to compare pre- and post-exposure values. The Pearson product–moment correlation coefficient was calculated for CT-induced DSBs and DLP. A p value<0.05 was considered to be statistically significant. Statistical analyses were performed using the software package PASW Statistics 18.0 (SPSS Inc., Chicago, IL, USA).

Results Patients The median age of the 33 patients (22 male, 11 female) was 57 years (range 29–81 years) and the median body mass index 25 kg/m2 (range 17–40 kg/m2). Clinical indications for whole-body PET/CT were the following: evaluation of malignant disease (21 patients; lung cancer n011, ENT malignancies n03, carcinoma of unknown primary n03, melanoma n02, breast cancer n01, osteosarcoma n01) and benign disease (12 patients; inflammation workup n0 9, vasculitis n02, Ormond’s disease n01).

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Exposure parameters Body weight-adapted FDG activity dose administration (median 223 MBq, range 138–354 MBq) and individual blood volume calculations resulted in a median initial blood activity concentration of 50 kBq/ml (range 36–56 kBq/ml). SUVs ranged from 1.7 to 3.8 in the liver (median 2.7) and averaged SUVs in the blood pool ranged from 1.3 to 2.6 (median 2.0). Systemic biodistribution of 18F-FDG was considered normal in all of the patients. Anatomy-based tube current modulation resulted in a median tube current time product of 107 mAs (range 80–178 mAs) in the SP (170 reference mAs) and 61 mAs (range 45–63 mAs) in the LD protocol (100 reference mAs) subgroup. Median CTDIvol and DLP were 7.3 mGy (range 5.4–17.4 mGy) and 885 mGy×cm (range 545– 1,751 mGy×cm) in SP and 4.1 mGy (range 3.1–4.3 mGy) and 430 mGy×cm (range 311–494 mGy×cm) in LD protocol, respectively. In vivo formation and repair of PET/CT-induced DSBs Data from the eight individuals, in whom multiple in vivo time plots of DSB induction and repair were determined, all showed similar kinetics. Following 18F-FDG administration increasing foci levels were observed with the maximum number of DNA damages found at 30 min; thereafter the amount of DNA lesions declined. Five minutes after the CT scan a second peak was observed followed by a rapid decrease of γ-H2AX foci. In all of the patients foci values 30 min after CT were lower compared to levels obtained 5 min after CT. An illustration of this is shown in two patients in Figs. 2 (patient 33) and 3 (patient 25). The corresponding time curve of the in vivo radioactivity

Fig. 2 In vivo DSBs. Illustrative in vivo time course of DSB (foci per cell) induction and repair in patient 33 undergoing combined PET/CT (18F-FDG 211 MBq, DLP 841 mGy×cm). The radionuclide was injected at 0 min (18F-FDG), whole-body CT was performed after 95 min (CT1) using the SP (black arrows). Data points represent the medians and error bars indicate the ranges of three independent measurements

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concentrations of 18F-FDG in the blood of patient 25 who underwent gamma well counter readings is plotted on the second y-axis in Fig. 3. The in vivo foci levels of all individuals are shown in Fig. 4a. The median pre-exposure foci level was 0.08/cell (range 0.06–0.12/cell). The median foci level obtained 30 min after 18F-FDG application was 0.20/cell (range 0.16–0.36/cell). Median levels before and 5 min after CT were 0.15/cell (range 0.11–0.26/cell) and 0.33/cell (range 0.17–0.66/cell), respectively. Median levels 30 min after CT were 0.24/cell (range 0.12–0.53/cell). Before and after additional leg scans median foci levels were 0.22/cell (range 0.12–0.41/cell) and 0.29/cell (range 0.16–0.45/cell), respectively (Fig. 4b). This corresponds to a median of 0.11 DSBs per cell (range 0.06–0.27) induced by 18 F-FDG, 0.17 DSBs per cell (range 0.04–0.54) induced by whole-body CT and 0.04 DSBs per cell (range 0.02–0.08) induced by additional leg scan. The LD protocol led to a reduction of CT-induced DSBs of more than 50 % compared to the SP (Fig. 5). The median DSB level 24 h after PET/CT (0.11/cell, range 0.10– 0.14/cell) was slightly, but significantly, elevated compared to the pre-exposure baseline values (p00.003, Fig. 4b). One-way analysis of variance yielded statistically significant differences between the foci levels at the different points PREFDG, FDG30, PRECT1, CT1, PRECT2, CT2 and H24 (p<0.001). According to Wilcoxon analysis, the DSB increases following 18F-FDG administration (FDG),

Fig. 3 In vivo DSBs and 18F-FDG radioactivity concentration. Illustrative in vivo time course of DSB (foci per cell, solid line) induction and repair in patient 25 undergoing combined PET/CT (18F-FDG 354 MBq, DLP 1,399 mGy×cm) and 18F-FDG radioactivity as measured in blood samples (kBq/ml, dashed line). The radionuclide was injected at 0 min (18F-FDG), whole-body CT (CT1) was performed using the SP at 70 and additional leg scan (CT2) at 100 min (black arrows). Data points represent the medians and error bars indicate the ranges of three independent measurements of γ-H2AX foci

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Fig. 5 In vivo radiation-induced DSBs. Amount of excess foci representing DSBs induced by 18F-FDG (FDG), full dose diagnostic CT using the SP (CT1-SP), LD CT (CT1-LD) and additional leg scan (CT2). Boxes show interquartile ranges, the middle horizontal line represents the median and the error bars indicate the range of the non-outlying data points

Fig. 4 Individual and median in vivo DSBs. DNA DSBs (foci per cell) at different points before (PREFDG), 30 min after 18F-FDG administration (FDG30), before (PRECT1), 5 min after whole-body CT (CT1), before (PRECT2) and 5 min after additional leg scan (CT2), and 24 h after PET/CT (H24). a Individual data: each bar represents one patient. b Summarized data: boxes show interquartile ranges, the middle horizontal line represents the median and the error bars indicate the range of the non-outlying data points

and 58 % using the LD CT protocol. This agrees with recently published mathematical dose calculations [4]. In contrast to experience with short-term external X-ray irradiation, continuous combined intra- and extracellular ß-

whole-body CT (CT1) and additional leg scan (CT2) were statistically significant (p<0.001 each). A significant linear correlation between CT-induced DSB levels and DLP was obtained (ρ00.898 and p<0.001, Fig. 6).

Discussion γ-H2AX immunofluorescence microscopy represents a reliable technique for determination of radiation-induced DNA DSBs as biomarkers of exposure in distinct cells [10]. Using this approach we were able to detect a statistically significant DSB increase in blood lymphocytes both by exposure to the radionuclide and X-rays. 18F-FDG-induced DSBs contributed 35 % to the total DNA damage induced by the combined PET/CT examination using the SP

Fig. 6 Correlation between CT-induced DSBs and DLP. Number of excess foci representing CT-induced DSBs obtained for all patients 5 min after SP and LD whole-body CT in relation to DLP. The line represents linear fit to all data points from all patients. Pearson ρ was 0.898 (p<0.001)

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and γ-irradiation, as occurs with incorporated radiopharmaceuticals, has not been well investigated. The only in vivo data, from a recent study, provided a peak of γ-H2AX foci in blood lymphocytes of patients undergoing radioiodine therapy 2 h after administration of therapeutic 131I doses [17]. Andrievski and Wilkins obtained peak values at 1.5 h after 137Cs irradiation with doses up to 10 Gy in an in vitro study design and concluded that the relatively quick lifetime kinetics of γ-H2AX foci limit its suitability for biodosimetry [18]. In our study, DSB induction and repair following 18FFDG exposure was triphasic. The first phase occurred within 5 min after tracer injection, which represented a fast increase in foci. We interpret this rapidly occurring peak of γ-H2AX foci as reflecting DNA damage in circulating blood lymphocytes due to high, primarily in plasma, radioactivity concentrations. In the second phase, the increase of foci progresses moderately until peak values are obtained around 30 min. Two different mechanisms might account for this: firstly, the fast elimination of 18F-FDG from plasma to the extracellular space and to the cytoplasm of tissue cells coupled with radioactive decay results in reduced in vivo blood radioactivity concentrations (Fig. 3), leading to a slower DSB induction. The second process is that DNA repair mechanisms are ongoing, which serves to dampen the rate of increased foci production. Concurrent decrease of DSB induction in association with development of DNA repair likely accounts for a lower increase of γ-H2AX foci in this second phase (Fig. 2). In the third phase, the γ-H2AX foci levels decrease by almost two thirds, until CT acquisition is initiated. We explain this third phase as reflecting DSB repair outbalancing lesser DSB induction, with further decay of cytoplasmic 18F-FDG. The relation of concurrent DSB induction and repair is well known. In a recent study comparable DSB kinetics were reported in patients undergoing angiographic procedures where radiation dose is applied in fractions over a longer period. Peak values were found at 15 min after the end of the exposure [13]. Our current study showed in vivo DSB peak values around 30 min, suggesting that blood withdrawals should be performed 30 min after 18F-FDG application if assessment is to be made of the amount of 18 F-FDG-induced DSBs as markers of exposure. We observed persistent, albeit only slightly, elevated levels of foci after 24 h, which is different than the findings reported by Löbrich et al. describing CT-induced DSBs which were nearly completely repaired within 24 h [10]. Our opinion is that, in contrast to CT or angiography, in which patients are exposed to X-rays for a shorter period, remnants of the radionuclide might lead to further DSB induction for several hours after intravenous 18F-FDG injection. Therefore, excess foci detected 24 h after PET/CT are likely due to prolonged DSB induction from persistent

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radionuclide and incomplete repair. In addition bystander effects which are biological responses in cells not traversed by radiation tracks could be responsible for DSB induction beyond the longevity of the radionuclides (based upon the half-life of 18F-FDG, after 20 h no significant radioactivity should remain) [19, 20]. It is also possible that DSB frequency could be affected in patients with altered biodistribution of the radionuclide. However, in comparison with previously published data, in this collective SUVs were in a normal range and the systemic biodistribution of 18F-FDG in our patients was normal [21]. In this present study, we have described for the first time DSB induction at nearly whole-body X-ray exposure, with virtually complete irradiation of all blood lymphocytes. This is in contrast to previous studies where only a part of the total blood volume had been exposed and therefore a substantial dilution of irradiated by non-irradiated cells had occurred [22, 23]. The effect of this whole-body radiation is that despite motion throughout the body the circulating blood lymphocytes would experience similar radiationinduced injury as stationary cells, such as breast cells, and therefore we would anticipate that the DSBs should be comparable between them. Hence findings in lymphocytes would be more reflective of all cell types. Some limitations of this study merit consideration. Despite the high sensitivity of γ-H2AX immunofluorescence microscopy there are some open issues concerning this method. Interindividual variations and a recent exposure to genotoxic agents may affect the background levels. Therefore, we determined background levels in every patient and subtracted them from post-exposure levels. H2AX phosphorylation can occur in the absence of DSBs in cells undergoing replication, but this does not apply for lymphocytes used in our study. None of the software packages offered for automated foci scoring is properly validated yet. Rapid γ-H2AX formation kinetics and foci loss present a major challenge. Additionally the effect of protracted radiation exposure is not sufficiently evaluated yet [24]. However, it was our purpose to contribute to the understanding of the kinetics after protracted irradiation. Foci size and intensity may be influenced by various factors (e.g. radiation dose, radiation quality). Unfortunately we did not have a validated scoring system available and therefore were not able to assess foci characteristics, which would have probably provided further interesting information. 18F-FDG activities and CT scan protocols differ between countries and even centres in the same country. Therefore, our findings may not be generalizable throughout the world. Only 11 of 33 patients had blood taken at 30 min after 18F-FDG application. Serial blood withdrawals after FDG administration to determine individual time curves were performed in only eight patients, and the intervals between the respective blood samples were different in these

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individuals. Because this study was performed in human subjects and clinical workflow, various factors between the individuals did not allow us to perform exact timing in each case. Detailed time curves of DSB induction were only elucidated after FDG administration and not after CT. Indepth evaluation of the time course of effects of CT was not within the scope of the present evaluation, as prior studies have looked specifically at CT [10, 23]. Since peak values were described between 5 and 30 min following CT in these studies, we chose both time points for determination of CTinduced DSBs in the current study. In all of our patients foci values after 30 min were lower compared to the early measurements after 5 min. It is important to note that, using the γ-H2AX immunofluorescence technique for biological dose estimation, creation of calibration curves deriving from individual in vitro irradiations is mandatory. Correlation of in vivo data with the absorbed dose expressed by calibration curves can be used for calculation of the radiation dose to the blood [11, 23]. In our study we unfortunately did not perform such in vitro experiments and therefore we were not able to estimate the biological radiation dose. However, our purpose was to assess the kinetics of formation and repair of γ-H2AX foci which we used as biomarkers of exposure. The evaluation of radiation-induced DNA DSBs was limited to blood lymphocytes, as under clinical conditions a solid tissue specimen cannot be obtained easily and without additional risk for the patient. However, a previous study in mice has shown that DSB induction in various tissues does not differ significantly from that in blood lymphocytes [25]. Thus lymphocytes serve as a good in vivo indicator for radiation-induced biological damage. As blood cells are in continuous circulation, DSBs that are measured in lymphocytes in the setting of regional radiation exposure may underestimate the exposure of stationary tissues. In our present study nearly the entire patient was exposed, both to the radionuclide and to X-rays; thus radiation damage in lymphocytes would closely reflect the experience in stationary tissues. Radiation dose distribution is strongly influenced by biokinetics and biodistribution with 18F-FDG and by anatomy with CT. Thus radiation dose and resulting DSB induction might be considerably elevated in tissues with a higher exposure to 18F-FDG (such as urinary bladder) and in superficial organs with CT (such as female breast). Lastly, the correlation between DSBs and carcinogenesis is contentious. Evaluation of γ-H2AX foci can be used for the monitoring of DSB induction and repair, but this may not correlate directly with cancer risk estimations, even if increased DSB turnover might statistically be associated with an increased rate of DNA misrepair [26]. In conclusion, γ-H2AX immunofluorescence microscopy may be used for monitoring radiation-induced in vivo

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DNA DSBs by 18F-FDG and CT separately in patients undergoing combined PET/CT. Peak values were obtained 30 min after 18F-FDG injection and 5 min after CT. We observed that the total amount of 18F-FDG-induced DSBs is comparable to CT and ranged from one to two thirds of the total DNA damage, depending on the CT protocol employed. Acknowledgments We thank Christina Engert, Tobias Löwe, Matthias Sommer, Bernhard Schmidt and Wolfram Nitsch for excellent technical assistance and Luitpold Distel for extraordinary collaboration in the lab. We also thank Ulrich Kahl, Ulrich Gärtner, Gerson Schütze, Kilian Bose, Rainer Linke and Roland Wondra for their support in patient examination and acquisition of blood samples. Conflicts of interest None.

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