Removal of scatter radiation in paediatric cardiac catheterisation: a randomised controlled clinical trial.

OBJECTIVE: This study sought to determine if DNA integrity was compromised by ionising radiation from paediatric cardiac catheterisations and if dose optimisation techniques allowed DNA integrity to be maintained. MATERIALS AND METHODS: Children were imaged using either: (i) an anti-scatter grid (current departmental protocol), (ii) no anti-scatter grid or, (iii) no anti-scatter grid and a 15 cm air-gap between the child and the x-ray detector. Dose area product and image quality were assessed, lifetime attributable cancer risk estimates were calculated and DNA double-strand breakages quantified using the γH2AX assay. RESULTS: Consent was obtained from 70 parents/guardians/children. Image quality was sufficient for each procedure performed. Removal of the anti-scatter grid resulted in dose reductions of 20% (no anti-scatter grid) and 30% (15 cm air-gap), DNA double-strand break reductions of 30% (no anti-scatter grid) and 20% (15 cm air-gap) and a reduction of radiation-induced cancer mortality risk of up to 45%. CONCLUSION: Radiation doses received during paediatric cardiac catheterisation procedures resulted in a significant increase in DNA damage while maintaining acceptable image quality and diagnostic efficacy. It is feasible to remove the anti-scatter grid resulting in a reduction in DNA damage to the patient. The γH2AX assay may be used for assessment of dose optimisation strategies in children.

Prostate cancer treated with brachytherapy; an exploratory study of dose-dependent biomarkers and quality of life.

BACKGROUND: Low-dose-rate permanent prostate brachytherapy (PPB) is an attractive treatment option for patients with localised prostate cancer with excellent outcomes. As standard CT-based post-implant dosimetry often correlates poorly with late treatment-related toxicity, this exploratory (proof of concept) study was conducted to investigate correlations between radiation - induced DNA damage biomarker levels, and acute and late bowel, urinary, and sexual toxicity. METHODS: Twelve patients treated with 125I PPB monotherapy (145Gy) for prostate cancer were included in this prospective study. Post-implant CT based dosimetry assessed the minimum dose encompassing 90% (D90%) of the whole prostate volume (global), sub-regions of the prostate (12 sectors) and the near maximum doses (D0.1cc, D2cc) for the rectum and bladder. Six blood samples were collected from each patient; pre-treatment, 1 h (h), 4 h, 24 h post-implant, at 4 weeks (w) and at 3 months (m). DNA double strand breaks were investigated by staining the blood samples with immunofluorescence antibodies to γH2AX and 53BP1 proteins (γH2AX/53BP1). Patient self-scored quality of life from the Expanded Prostate Cancer Index Composite (EPIC) were obtained at baseline, 1 m, 3 m, 6 m, 9 m, 1 year (y), 2y and 3y post-treatment. Spearman's correlation coefficients were used to evaluate correlations between temporal changes in γH2AX/53BP1, dose and toxicity. RESULTS: The minimum follow up was 2 years. Population mean prostate D90% was 144.6 ± 12.1 Gy and rectal near maximum dose D0.1cc = 153.0 ± 30.8 Gy and D2cc = 62.7 ± 12.1 Gy and for the bladder D0.1cc = 123.1 ± 27.0 Gy and D2cc = 70.9 ± 11.9 Gy. Changes in EPIC scores from baseline showed high positive correlation between acute toxicity and late toxicity for both urinary and bowel symptoms. Increased production of γH2AX/53BP1 at 24 h relative to baseline positively correlated with late bowel symptoms. Overall, no correlations were observed between dose metrics (prostate global or sector doses) and γH2AX/53BP1 foci counts. CONCLUSIONS: Our results show that a prompt increase in γH2AX/53BP1foci at 24 h post-implant relative to baseline may be a useful measure to assess elevated risk of late RT - related toxicities for PPB patients. A subsequent investigation recruiting a larger cohort of patients is warranted to verify our findings.

Assessment of DNA double-strand breaks induced by intravascular iodinated contrast media following in vitro irradiation and in vivo, during paediatric cardiac catheterization.

Paediatric cardiac catheterizations may result in the administration of substantial amounts of iodinated contrast media and ionizing radiation. The aim of this work was to investigate the effect of iodinated contrast media in combination with in vitro and in vivo X-ray radiation on lymphocyte DNA. Six concentrations of iodine (15, 17.5, 30, 35, 45, and 52.5 mg of iodine per mL blood) represented volumes of iodinated contrast media used in the clinical setting. Blood obtained from healthy volunteers was mixed with iodinated contrast media and exposed to radiation doses commonly used in paediatric cardiac catheterizations (0 mGy, 70 mGy, 140 mGy, 250 mGy and 450 mGy). Control samples contained no iodine. For in vivo experimentation, pre and post blood samples were collected from children undergoing cardiac catheterization, receiving iodine concentrations of up to 51 mg of iodine per mL blood and radiation doses of up to 400 mGy. Fluorescence microscopy was performed to assess γH2AX-foci induction, which corresponded to the number of DNA double-strand breaks. The presence of iodine in vitro resulted in significant increases of DNA double-strand breaks beyond that induced by radiation for ≥ 17.5 mg/mL iodine to blood. The in vivo effects of contrast media on children undergoing cardiac catheterization resulted in a 19% increase in DNA double-strand breaks in children receiving an average concentration of 19 mg/mL iodine to blood. A larger investigation is required to provide further information of the potential benefit of lowering the amount of iodinated contrast media received during X-ray radiation investigations.

Alpha particles induce pan-nuclear phosphorylation of H2AX in primary human lymphocytes mediated through ATM.

The use of high linear energy transfer radiations in the form of carbon ions in heavy ion beam lines or alpha particles in new radionuclide treatments has increased substantially over the past decade and will continue to do so due to the favourable dose distributions they can offer versus conventional therapies. Previously it has been shown that exposure to heavy ions induces pan-nuclear phosphorylation of several DNA repair proteins such as H2AX and ATM in vitro. Here we describe similar effects of alpha particles on ex vivo irradiated primary human peripheral blood lymphocytes. Following alpha particle irradiation pan-nuclear phosphorylation of H2AX and ATM, but not DNA-PK and 53BP1, was observed throughout the nucleus. Inhibition of ATM, but not DNA-PK, resulted in the loss of pan-nuclear phosphorylation of H2AX in alpha particle irradiated lymphocytes. Pan-nuclear gamma-H2AX signal was rapidly lost over 24h at a much greater rate than foci loss. Surprisingly, pan-nuclear gamma-H2AX intensity was not dependent on the number of alpha particle induced double strand breaks, rather the number of alpha particles which had traversed the cell nucleus. This distinct fluence dependent damage signature of particle radiation is important in both the fields of radioprotection and clinical oncology in determining radionuclide biological dosimetry and may be indicative of patient response to new radionuclide cancer therapies.

DNA double-strand break repair and induction of apoptosis in ex vivo irradiated blood lymphocytes in relation to late normal tissue reactions following breast radiotherapy.

This study aimed to test whether induction of apoptosis following ex vivo X-irradiation of unstimulated blood lymphocytes correlated with clinical radiosensitivity and DNA double-strand break (DSB) repair in breast radiotherapy patients and healthy volunteers. Using small molecule inhibitors, the relationship between DSB repair and radiation-induced apoptosis was examined. Sixteen breast cancer patients with minimal (controls, n = 8) or extremely marked late radiation-induced change (cases, n = 8) and eight healthy volunteers were selected. DSBs were quantified by γH2AX/53BP1 immunofluorescence, and apoptosis was measured using a fluorogenic inhibitor of caspases assay. Mean γH2AX/53BP1 focus levels 24 h after exposure to 4 Gy were higher in cases (12.7 foci per cell) than in controls (10.3 foci per cell, p = 0.002). In contrast, the mean apoptotic fraction 48 h after 8 Gy was comparable, 37.2 % in cases and 34.7 % in controls (p = 0.442). Residual focus and apoptosis levels were not correlated within individuals (Spearman's R = -0.0059, p = 0.785). However, cells treated with DNA-PK inhibitor Nu7441 had higher focus and apoptosis levels 48 h after 1 Gy compared to mock-treated cells, suggesting that apoptosis induction following irradiation is modulated by DSB repair. This effect required functional ATM since cells treated simultaneously with Nu7441 and the ATM inhibitor Ku55933 were resistant to apoptosis despite high levels of residual foci. One clinical case displayed an impaired DNA-PK-dependent end-joining cellular phenotype. In summary, clinical radiosensitivity may be associated with impaired DSB repair in some patients. Although pharmaceutical inhibition of ATM and DNA-PK affected apoptosis induction and DSB repair, no association was observed between apoptosis and residual focus levels in patients and volunteers.

Combined analysis of gamma-H2AX/53BP1 foci and caspase activation in lymphocyte subsets detects recent and more remote radiation exposures.

Analysis of gamma-H2AX foci in blood lymphocytes is a promising approach for rapid dose estimation to support patient triage after a radiation accident but has one major drawback: the rapid decline of foci levels post-exposure cause major uncertainties in situations where the exact timing between exposure and blood sampling is unknown. To address this issue, radiation-induced apoptosis (RIA) in lymphocytes was investigated using fluorogenic inhibitors of caspases (FLICA) as an independent biomarker for radiation exposure, which may complement the gamma-H2AX assay. Ex vivo X-irradiated peripheral blood lymphocytes from 17 volunteers showed dose- and time-dependent increases in radiation-induced apoptosis over the first 3 days after exposure, albeit with considerable interindividual variation. Comparison with gamma-H2AX and 53BP1 foci counts suggested an inverse correlation between numbers of residual foci and radiation-induced apoptosis in lymphocytes at 24 h postirradiation (P = 0.007). In T-helper (CD4), T-cytotoxic (CD8) and B-cells (CD19), some significant differences in radiation induced DSBs or apoptosis were observed, however no correlation between foci and apoptosis in lymphocyte subsets was observed at 24 h postirradiation. While gamma-H2AX and 53BP1 foci were rapidly induced and then repaired after exposure, radiation-induced apoptosis did not become apparent until 24 h after exposure. Data from six volunteers with different ex vivo doses and post-exposure times were used to test the capability of the combined assay. Results show that simultaneous analysis of gamma-H2AX and radiation-induced apoptosis may provide a rapid and more accurate triage tool in situations where the delay between exposure and blood sampling is unknown compared to gamma-H2AX alone. This combined approach may improve the accuracy of dose estimations in cases where blood sampling is performed days after the radiation exposure.

Gamma-H2AX-based dose estimation for whole and partial body radiation exposure.

Most human exposures to ionising radiation are partial body exposures. However, to date only limited tools are available for rapid and accurate estimation of the dose distribution and the extent of the body spared from the exposure. These parameters are of great importance for emergency triage and clinical management of exposed individuals. Here, measurements of γ-H2AX immunofluorescence by microscopy and flow cytometry were compared as rapid biodosimetric tools for whole and partial body exposures. Ex vivo uniformly X-irradiated blood lymphocytes from one donor were used to generate a universal biexponential calibration function for γ-H2AX foci/intensity yields per unit dose for time points up to 96 hours post exposure. Foci--but not intensity--levels remained significantly above background for 96 hours for doses of 0.5 Gy or more. Foci-based dose estimates for ex vivo X-irradiated blood samples from 13 volunteers were in excellent agreement with the actual dose delivered to the targeted samples. Flow cytometric dose estimates for X-irradiated blood samples from 8 volunteers were in excellent agreement with the actual dose delivered at 1 hour post exposure but less so at 24 hours post exposure. In partial body exposures, simulated by mixing ex vivo irradiated and unirradiated lymphocytes, foci/intensity distributions were significantly over-dispersed compared to uniformly irradiated lymphocytes. For both methods and in all cases the estimated fraction of irradiated lymphocytes and dose to that fraction, calculated using the zero contaminated Poisson test and γ-H2AX calibration function, were in good agreement with the actual mixing ratios and doses delivered to the samples. In conclusion, γ-H2AX analysis of irradiated lymphocytes enables rapid and accurate assessment of whole body doses while dispersion analysis of foci or intensity distributions helps determine partial body doses and the irradiated fraction size in cases of partial body exposures.

gamma-H2AX as protein biomarker for radiation exposure.

For large scale exposures of the human population to ionising radiation, there is a need for cost-effective high throughput assessment of radiation exposure levels from biological samples to allow triage decisions to be made. Here we discuss the usefulness of the DNA damage marker gamma-H2AX for this purpose. Foci of gamma-H2AX form in response to radiation-induced DNA doublestrand breaks and can be quantified by immunofluorescence microscopy or flow cytometry. Several studies have analysed this marker in patients' blood samples to determine radiation exposures during various diagnostic or therapeutic radiation treatments. Such planned exposures involve only a moderate number of samples which can be obtained at a prearranged time following exposure. In contrast, application of this method as a triage tool in large scale radiological emergencies demands high throughput sample processing and analysis. The rapid kinetics of gamma-H2AX induction and loss presents a major challenge to its successful application as a triage tool. These and other as yet unresolved questions are discussed.