Biodosimetric analysis of head and neck cancer patients undergoing radiotherapy by dicentric chromosome aberration assay.

BACKGROUND: Biodosimetry is the quantification of absorbed radiation dose using biological material obtained from an exposed individual. Radiation can cause different types of chromosomal aberrations, including stable aberrations like translocations and unstable ones like micronuclei, dicentric chromosomes (DC), acentric, and ring forms. Dicentric chromosome assay has become the "gold standard" for cytogenetic biodosimetry due to its reproducibility, specificity (low baseline rates), and sensitivity to low doses. Using existing calibration curves and models obtained from in vitro irradiation of blood, the yield of DCs can be used to estimate the average whole-body absorbed dose. PURPOSE: To evaluate and compare the in vivo dose-response relation of DC aberration formation in peripheral blood lymphocytes of head and neck cancer (HNC) patients undergoing radiotherapy (RT) alone, cisplatin-based chemoradiation (CCRT), accelerated fractionation RT (AFRT), and CCRT with gefitinib (GCRT). METHODOLOGY: This prospective observational and analytical study was conducted from 2018 to 2021 in the Department of Radiation Oncology and Genetic Lab of tertiary care, teaching hospital after approval from the Institutional Ethics Committee. Biodosimetric analysis was done weekly in patients undergoing RT (n = 20) versus CCRT (n = 20), CCRT (n = 12) versus AFRT (n = 12), and CCRT (n = 6) versus GCRT (n = 6). The yield of DCs was measured in blood samples taken before starting treatment, that is, day 0 and during RT on days 6, 11, and 16 in RT alone versus CCRT; on days 7 and 13 in CCRT versus AFRT; and days 6 and 11 in CCRT versus GCRT from a blood sample drawn 1-2 h after RT. Phytohemagglutinin-stimulated lymphocytes were cultured using heparinized blood in RPMI-1640 medium supplemented with fetal bovine serum. Cells were arrested at metaphase using demecolcine, harvested by centrifugation, mounted, and stained with Giemsa. Cytogenetic analysis was performed by analyzing at least 100 metaphases with well-spread chromosomes. DC aberrations and acentric fragments were identified and recorded. To standardize the findings as per the customized field for every patient, the mean DC yield per cm2 of the irradiated area was calculated and compared. RESULTS: The mean yield of DC/cm2 in the CCRT group was greater than the RT alone group by 16.33%, 28.57%, and 18.68% on days 6, 11, and 16 of treatment, respectively. This difference between the two groups at day 6 (P = 0.001), day 11 (P < 0.001), and day 16 (P < 0.001) was found to be statistically significant. The mean yield of DC/cm2 in the CCRT group was greater than the AFRT group by 7.9% and 18.3% on days 7 and 13 of treatment, respectively. This difference at day 7 (P < 0.001) and day 13 (P < 0.001) was found to be statistically significant. The mean yield of DC/cm2 in the CCRT group was greater than the GCRT group by 22.7% and 21.8% on days 6 and 11 of treatment, respectively. The difference at day 6 (P = 0.01) was statistically significant but, on day 11 (P = 0.065) this difference was found insignificant. CONCLUSION: There is a dose-dependent increase in the yield of DCs in lymphocytes of HNC patients undergoing RT with subsequent fractions. Cisplatin-based chemoradiation is the superior method of treatment intensification radio-biologically proven by higher DC yield.

Radiation exposure lymphocyte damage assessed by γ-H2AX level using flow cytometry.

DNA double-strand breaks (DSBs) are considered the most relevant lesions to the DNA damage of ionizing radiation (IR), and γ-H2AX foci in peripheral blood lymphocytes are regarded as an adequate marker for DSB quantitative studies. This study aimed to investigate IR-induced DNA damage in mice through γ-H2AX fluorescence analyses by flow cytometry (FCM). The levels of γ-H2AX in CD4/CD8/B220-positive lymphocytes were quantified by FCM through mean fluorescence intensity (MFI) values. Peripheral venous blood samples were collected for evaluation, and all the control groups were restrained from irradiation. For external irradiation experiments, the dose-dependency of MFI values and temporal alternations were assessed both in vitro and in vivo. External radiation exposure damage was positively correlated with the absorbed radiation dose, and the lymphocyte recovered from damage within 3 days. I-131 sodium iodide solution (74 MBq) was injected into the mice intraperitoneally for internal irradiation experiments. Gamma counting and γH2AX foci analyses were performed at 1 h and 24 h by the group. The blood-to-blood S values (Sblood←blood) were applied for the blood-absorbed dose estimation. Internal low-dose-irradiation-induced damage was proved to recover within 24 h. The FCM method was found to be an effective way of quantitatively assessing IR-induced DNA damage.

Peripheral blood lymphocytes differ in DNA damage response after exposure to X-rays with different physical properties.

Introduction: In radiology, low X-ray energies (<140 keV) are used to obtain an optimal image while in radiotherapy, higher X-ray energies (MeV) are used to eradicate tumor tissue. In radiation research, both these X-ray energies being used to extrapolate in vitro research to clinical practice. However, the energy deposition of X-rays depends on their energy spectrum, which might lead to changes in biological response. Therefore, this study compared the DNA damage response (DDR) in peripheral blood lymphocytes (PBLs) exposed to X-rays with varying beam quality, mean photon energy (MPE) and dose rate.Methods: The DDR was evaluated in peripheral blood lymphocytes (PBLs) by the É£-H2AX foci assay, the cytokinesis-block micronucleus assay and an SYTOX-based cell death assay, combined with specific cell death inhibitors. Cell cultures were irradiated with a 220 kV X-ray research cabinet (SARRP, X-Strahl) or a 6 MV X-ray linear accelerator (Elekta Synergy). Three main physical parameters were investigated: beam quality (V), MPE (eV) and dose rate (Gy/min). Additional copper (Cu) filtration caused variation in the MPE (78 keV, 94 keV, 118 keV) at SARRP; dose rates were varied by adjusting tube current for 220 kV X-rays (0.33-3 Gy/min) or water-phantom depth in the 6 MV set-up (3-6 Gy/min).Results: The induction of chromosomal damage and initial (30 min) DNA double-stranded breaks (DSBs) were significantly higher for 220 kV X-rays compared to 6 MV X-rays, while cell death induction was similar. Specific cell death inhibitors for apoptosis, necroptosis and ferroptosis were not capable of blocking cell death after irradiation using low or high-energy X-rays. Additional Cu filtration increased the MPE, which significantly decreased the amount of chromosomal damage and DSBs. Within the tested ranges no specific effects of dose rate variation were observed.Conclusion: The DDR in PBLs is influenced by the beam quality and MPE. This study reinforces the need for consideration and inclusion of all physical parameters in radiation-related studies.

Low-dose radiation from CT examination induces DNA double-strand breaks and detectable changes of DNA methylation in peripheral blood cells.

BACKGROUND: Radiation burden from CT examinations increases rapidly with the increased clinical use frequency. Previous studies have disclosed the association between radiation exposure and increased double-strand breaks (DSBs) and changes in DNA methylation. However, whether the induced DSBs by CT examination recover within 24h and whether a CT examination induces detectable gene-specific methylation changes are still unclear. The aim of the present study was to analyze γ-H2AX in the peripheral blood lymphocyte (PBL) of healthy adults before and after CT examination and to discover the differentially methylated positions (DMPs) along with an analysis of DNA methylation changes caused by CT examination. MATERIALS AND METHODS: Peripheral blood samples of 4 ml were drawn from 20 healthy volunteers at three time points: before CT examination, after CT examination 1h, and after CT examination 24h. γ-H2AX immunofluorescence and Illumina Infinium Human Methylation EPIC BeadChip (850k BeadChip) were used respectively for the test of DSBs and the epigenome-wide DNA methylation analysis. Linear mixed-effect (LME) models were used to evaluate the impacts of doses represented by different parameters and foci on genome-wide DNA methylation. RESULTS: The number of γ-H2AX foci per cell at 1h showed linear dose-responses for the radiation doses represented by CT index volume (CTDIvol), dose length product (DLP), and blood absorbed dose, respectively. Residual γ-H2AX foci was observed after CT examination at 24h (p < .001). DMPs and γ-H2AX foci changes could be found within 1h. One CpG site related to PAX5 was significantly changed by using most of the parameters in LME models and did not recover till 24h. CONCLUSIONS: Residual γ-H2AX foci exist after CT examination at 24h. The DNA methylation changes induced by CT examination may not recover within 24h. The DNA methylation had been changed as early as at 1h. The PAX5-related CpG site may be a potential biomarker of low-dose radiation. CLINICAL RELEVANCE: The biological effects and the cancer risks of CT examination are still unclear. The present study is an effort to document the CT scan-induced events in 24h in vivo. The CT scanning area should be strictly limited, and non-essential repeated operations shouldn't be performed within 24h.

Biodosimetry Based on Gamma-H2AX Quantification in Human Peripheral Blood Lymphocytes after Partial-body Irradiation.

ABSTRACT: Quantification of gamma-H2AX foci can estimate exposure to ionizing radiation. Most nuclear and radiation accidents are partial-body irradiation, and the doses estimated using the total-body irradiation dose estimation formula are often lower than the actual dose. To evaluate the dose-response relation of gamma-H2AX foci in human peripheral blood lymphocytes after partial-body irradiation and establish a simple and high throughput model to estimate partial-body irradiation dose, we collected human peripheral blood and irradiated with 0-, 0.5-, 1-, 2-, 3-, 4-, 5-, 6-, and 8-Gy gamma rays to simulate total-body irradiation in vitro. Gamma-H2AX foci were quantitated by flow cytometry at 1 h after irradiation, and a dose-response curve was established for total-body irradiation dose estimation. Then, a partial-body irradiation dose-response calibration curve was established by adding calibration coefficients based on the Dolphin method. To reflect the data distribution of all doses more realistically, the partial-body irradiation dose-response calibration curve was divided into two sections. In addition, partial-body irradiation was simulated in vitro, and the PBI data were substituted into curves to verify the accuracy of the two partial-body irradiation calibration curves. Results showed that the dose estimation variations were all less than 30% except the 25% partial-body irradiation group at 1 Gy, and the partial-body irradiation calibration dose-response curves were YF 1 = - 3.444 x 2 + 18.532 x + 3.109, R 2 = 0.92 (YF ≤ 27.95); YF 2 = - 2.704 x 2 + 37.97 x - 56.45, R 2 = 0.86 (YF > 27.95). Results also suggested that the partial-body irradiation dose-response calibration curve based on the gamma-H2AX foci quantification in human peripheral blood lymphocytes is a simple and high throughput model to assess partial-body irradiation dose.

Effects of genistein supplemented before or after irradiation on DNA injury in human lymphocytes in vitro.

Background: Ionizing radiation (IR) carry adequate energy to ionize or remove electrons from an atom. Particles interact with water to produce reactive oxygen species (ROS). Genistein (GEN) is a naturally occurring phytoestrogen and the basic isoflavonoid in soybeans and soybean-enriched products and is believed to have the strongest antioxidant activity. Objective: The study aimed at the investigation if application of GEN at different time prior or past irradiation may ameliorate or reduce injury of DNA in human lymphocytes. Material and Methods: The isolated lymphocytes were exposed to X-irradiation (0.5; 1 Gy). GEN (1 µM/ml; 10 µM/ ml) was appended to attempts at various times prior or past irradiation (1 h prior, immediately prior, immediately past, 1 h past). We joined each X-rays dose with each GEN dose. After 1h of incubation DNA damages were examined using Comet assay. Results: Combination of 1 µM/ml of GEN given 1 h before irradiation with low or high dose markedly decreased induced by irradiation DNA injury. Higher dose of GEN applied immediately before or after irradiation markedly extended the frequency of DNA injury generated by irradiation. The result of application 1 µM/ml GEN 1 h after irradiation was not significantly different compared to control. The effect of 1 Gy + 10 µM/ml GEN was not significantly lower compared to each agent alone. Conclusions: Only a very low concentration of GEN applied before irradiation, may be considered as a potential radiomitigator/radioprotector. High doses of GEN work as a radiosentitizer and may potent the effects of radiotherapy.

[Analysis of whole blood cells and genetic influencing factors in medical radiation workers].

Objective: To explore the influencing factors of whole blood cells and genetics of medical radiation workers, and provide technical support for improving occupational health management and strengthening radiation protection. Methods: In January 2022, a total of 4180 medical radiation workers who underwent occupational health examination in Gansu Provincial Center for Disease Control and Prevention from January 2020 to December 2021 were collected as the research objects, and the results of demographic characteristics, whole blood cells, chromosome aberrations, lymphocyte micronucleus and other results were collected. The whole blood cells and genetic abnormalities of different demographic characteristics of medical radiation workers were compared. And the influencing factors of whole blood cells and genetic abnormalities were analyzed by multivariate logistic regression. Results: The rates of hemoglobin (HGB), chromosome aberration and lymphocyte micronucleus abnormality were the highest in the nuclear medicine group, and the rate of white blood cell (WBC) abnormality in the radiotherapy group was higher than those in other occupational groups, the differences were statistically significant (P<0.05). The abnormal rates of WBC, HGB and lymphocyte micronucleus in female radiation workers were significantly higher than those in male radiation workers (P<0.001). The abnormal rates of HGB and lymphocyte micronucleus were statistically different among different working years and different age radiation workers (P<0.001). And the abnormal rate of platelet (PLT) was statistically different among different working years radiation workers (P<0.05). The abnormal rate of HGB in radiation workers of different hospital levels was statistically different (P<0.001). Logistic regression analysis showed that the risk of abnormal WBC and HGB in females radiation workers were 3.048 times and 13.122 times of those in males, respectively (P<0.001). The abnormal risks of WBC in the 6-20 working years group and >20 working years group were 1.517 times and 1.874 times of that in the ≤5 working years group, respectively (P<0.05). The abnormal risk of PLT in the >20 working years group was 2.643 times of that in ≤5 working years group (P<0.05). The abnormal risk of WBC in radiotherapy group and intervention group were 2.407 times and 1.341 times of that in general radiotherapy group, respectively (P<0.05) . Conclusion: Ionizing radiation has different effects on the whole blood cells and genetic indexes of workers in the nuclear medicine, interventional group and radiotherapy group. The occupational health protection of female radiation workers should be paid attention to.

Evaluation of the relationship between γ-H2AX biomarker levels and dose received after radiation exposure in abdominal-pelvic and chest CT scans.

Background: As one of the most informative diagnostic radiation instruments, computed tomography (CT) has seen considerable improvement since its implementation in the 1970s; however, the possibility of low-dose radiation risk after CT procedures is still challenging and little is known about the biological effects of CT exposure on patients. As a result, this research aimed to look at the biological and cytogenetic effects of low-dose abdominal-pelvic and chest CT scans on adults, focusing on the number of γ-H2AX foci formation. Materials and Methods: Blood tests were taken before and 10 min after CT exams on patients aged 25-55 who were undergoing abdominal-pelvic and chest CT exams with very low-ionizing radiation exposure (TLD doses of 15.67-63.45 mGy). Blood lymphocytes that had been isolated, fixed, and stained were dyed with γ-H2AX antibodies. Finally, the percentage of phosphorylation of histone H2AX as an indicator of double-strand breaks was determined using a cytometry technique. Results: Our findings showed that after CT examination, the mean value of γ-H2AX foci in patients increased (P < 0.0001). A statistically significant correlation between dose radiation and the number of γ-H2AX foci was also found (P = 0.047, r = 0.4731). The current study also found a pattern of elevated γ-H2AX foci in patients over 40 years of age relative to younger patients. Conclusion: A Significant activation of γ-H2AX foci was found in lymphocytes of peripheral blood samples of patients after CT compared to before CT scan. This increase in γ-H2AX foci levels in blood cells may be a useful quantitative biomarker of low-level radiation exposure in humans.

[Radiation-induced lymphopenia: Lymphocytes as a new organ at risk]. / Lymphopénie radio-induite : les lymphocytes comme nouvel organe à risque.

Taking the immune system into account in the fight against tumors has upset the cancer treatment paradigm in the 21st century. Combination treatment strategies associating radiotherapy with immunotherapy are being increasingly implemented in clinical practice. In this context, lymphocytes, whether lymphocytes infiltrating the tumour, circulating blood lymphocytes or lymphocytes residing within the lymph nodes, are key players in cellular and humoral anti-tumor immunity. The significant radiosensitivity of lymphocytes was demonstrated in the early 1990s. Along with the cells of the digestive mucosa, lymphocytes are thus among the most radiosensitive cell types in the body. Compared to the old practices of external radiotherapy, current intensity modulated treatments have allowed a considerable improvement in acute and late toxicity, at the cost of a significant increase in the volume irradiated at low doses. This is not without consequence on the incidence of radiation-induced lymphopenia, with prognostic implications for many tumor types. Thus, in order not to hinder the action of antitumor immunity and the efficacy of immunotherapy, it is essential to consider lymphocytes as a new organ at risk in its own right. In this development, based on current data from the literature, we will begin by justifying the necessary prevention of radiation-induced lymphopenia, before providing the tools currently known to apprehend lymphocytes as a new multicompartments. Finally, we will broaden the perspective by outlining ways to develop research in this area.

Chemotherapy and cryopreservation affects DNA repair foci in lymphocytes of breast cancer patients.

PURPOSE: Although breast cancer (BC) patients benefit from radiotherapy (RT), some radiosensitive (RS) patients suffer from side effects caused by ionizing radiation in healthy tissues. It is thought that RS is underlaid by a deficiency in the repair of DNA double-strand breaks (DSB). DNA repair proteins such as p53-binding protein 1 (53BP1) and phosphorylated histone H2AX (γH2AX), form DNA repair foci at the DSB locations and thus serve as DSB biomarkers. Peripheral blood lymphocytes (PBL) are commonly believed to be an appropriate cell system for RS assessment using DNA repair foci. The amount of DSB may also be influenced by chemotherapy (CHT), which is often chosen as the first treatment modality before RT. As it is not always possible to analyze blood samples immediately after collection, there is a need for cryopreservation of PBL in liquid nitrogen. However, cryopreservation may potentially affect the number of DNA repair foci. In this work, we studied the effect of cryopreservation and CHT on the amount of DNA repair foci in PBL of BC patients undergoing radiotherapy. MATERIALS AND METHODS: The effect of cryopreservation was studied by immunofluorescence analysis of 53BP1 and γH2AX proteins at different time intervals after in vitro irradiation. The effect of chemotherapy was analyzed by fluorescent labelling of 53BP1 and γH2AX proteins in PBL collected before, during, and after RT. RESULTS: Higher number of primary 53BP1/γH2AX foci was observed in frozen cells indicating that cryopreservation affects the formation of DNA repair foci in PBL of BC patients. In CHT-treated patients, a higher number of foci were found before RT, but no differences were observed during and after the RT. CONCLUSIONS: Cryopreservation is the method of choice for analyzing DNA repair residual foci, but only similarly treated and preserved cells should be used for comparison of primary foci. CHT induces DNA repair foci in PBL of BC patients, but this effect disappears during radiotherapy.

Comet Assay Profiling of FLASH-Induced Damage: Mechanistic Insights into the Effects of FLASH Irradiation.

Numerous studies have demonstrated the normal tissue-sparing effects of ultra-high dose rate 'FLASH' irradiation in vivo, with an associated reduction in damage burden being reported in vitro. Towards this, two key radiochemical mechanisms have been proposed: radical-radical recombination (RRR) and transient oxygen depletion (TOD), with both being proposed to lead to reduced levels of induced damage. Previously, we reported that FLASH induces lower levels of DNA strand break damage in whole-blood peripheral blood lymphocytes (WB-PBL) ex vivo, but our study failed to distinguish the mechanism(s) involved. A potential outcome of RRR is the formation of crosslink damage (particularly, if any organic radicals recombine), whilst a possible outcome of TOD is a more anoxic profile of induced damage resulting from FLASH. Therefore, the aim of the current study was to profile FLASH-induced damage via the Comet assay, assessing any DNA crosslink formation as a putative marker of RRR and/or anoxic DNA damage formation as an indicative marker of TOD, to determine the extent to which either mechanism contributes to the "FLASH effect". Following FLASH irradiation, we see no evidence of any crosslink formation; however, FLASH irradiation induces a more anoxic profile of induced damage, supporting the TOD mechanism. Furthermore, treatment of WB-PBLs pre-irradiation with BSO abrogates the reduced strand break damage burden mediated by FLASH exposures. In summary, we do not see any experimental evidence to support the RRR mechanism contributing to the reduced damage burden induced by FLASH. However, the observation of a greater anoxic profile of damage following FLASH irradiation, together with the BSO abrogation of the reduced strand break damage burden mediated by FLASH, lends further support to TOD being a driver of the reduced damage burden plus a change in the damage profile mediated by FLASH.

Evaluation of γ-H2AX foci distribution among different peripheral blood mononucleated cell subtypes.

INTRODUCTION: The detection of γ-H2AX foci in peripheral blood mononucleated cells (PBMCs) has been incorporated as an early assay for biological dosimetry. However, overdispersion in the γ-H2AX foci distribution is generally reported. In a previous study from our group, it was suggested that overdispersion could be caused by the fact that when evaluating PBMCs, different cell subtypes are analyzed, and that these could differ in their radiosensitivity. This would cause a mixture of different frequencies that would result in the overdispersion observed. OBJECTIVES: The objective of this study was to evaluate both the possible differences in the radiosensitivities of the different cell subtypes present in the PBMCs and to evaluate the distribution of γ-H2AX foci in each cell subtype. MATERIALS AND METHODS: Peripheral blood samples from three healthy donors were obtained and total PBMCs, and CD3+, CD4+, CD8+, CD19+, and CD56+ cells were separated. Cells were irradiated with 1 and 2 Gy and incubated at 37 °C for 1, 2, 4, and 24 h. Sham-irradiated cells were also analyzed. γ-H2AX foci were detected after immunofluorescence staining and analyzed automatically using a Metafer Scanning System. For each condition, 250 nuclei were considered. RESULTS: When the results from each donor were compared, no observable significant differences between donors were observed. When the different cell subtypes were compared, CD8+ cells showed the highest mean of γ-H2AX foci in all post-irradiation time points. The cell type that showed the lowest γ-H2AX foci frequency was CD56+. The frequencies observed in CD4+ and CD19+ cells fluctuated between CD8+ and CD56+ without any clear pattern. For all cell types evaluated, and at all post-irradiation times, overdispersion in γ-H2AX foci distribution was significant. Independent of the cell type evaluated the value of the variance was four times greater than that of the mean. CONCLUSION: Although different PBMC subsets studied showed different radiation sensitivity, these differences did not explain the overdispersion observed in the γ-H2AX foci distribution after exposure to IR.

Chromosomal radiosensitivity of human breast carcinoma cells and blood lymphocytes following photon and proton exposures.

Breast carcinomas (BC) are among the most frequent cancers in women. Studies on radiosensitivity and ionizing radiation response of BC cells are scarce and mainly focused on intrinsic molecular mechanisms but do not include clinically relevant features as chromosomal rearrangements important for radiotherapy. The main purpose of this study was to compare the ionizing radiation response and efficiency of repair mechanisms of human breast carcinoma cells (Cal 51) and peripheral blood lymphocytes (PBL) for different doses and radiation qualities (60Co γ-rays, 150 MeV and spread-out Bragg peak (SOBP) proton beams). The radiation response functions obtained using the conventional metaphase assay and premature chromosome condensation (PCC) technique enabled us to determine the number of chromosomal breaks at different time after irradiation. Both cytogenetic assays used confirmed the higher biological radiosensitivity for proton beams in tumor cells compared to PBL, corresponding to higher values of the linear LQ parameter α. additionally, the ratio of the LQ parameters ß/α describing efficiency of the repair mechanisms, obtained for chromosome aberrations, showed higher numbers for PBL than for Cal 51 for all exposures. Similar results were observed for the ratio of PCC breaks determined directly after irradiation to that obtained 12 h later. This parameter (t0/t12) showed faster decrease of the repair efficiency with increasing LET value for Cal 51 cells. This finding supports the use of the proton therapy for breast cancer patients.

Predicting Severity of Radiation Induced Lymphopenia in Individual Proton Therapy Patients for Varying Dose Rate and Fractionation Using Dynamic 4-Dimensional Blood Flow Simulations.

PURPOSE: Radiation-induced lymphopenia has gained attention recently as the result of its correlation with survival in a range of indications, particularly when combining radiation therapy (RT) with immunotherapy. The purpose of this study is to use a dynamic blood circulation model combined with observed lymphocyte depletion in patients to derive the in vivo radiosensitivity of circulating lymphocytes and study the effect of RT delivery parameters. METHODS AND MATERIALS: We assembled a cohort of 17 patients with hepatocellular carcinoma treated with proton RT alone in 15 fractions (fx) using conventional dose rates (beam-on time [BOT], 120 seconds) for whom weekly absolute lymphocyte counts (ALCs) during RT and follow-up were available. We used HEDOS, a time-dependent, whole-body, blood flow computational framework, in combination with explicit liver blood flow modeling, to calculate the dose volume histograms for circulating lymphocytes for changing BOTs (1 second-300 seconds) and fractionations (5 fx, 15 fx). From this, we used the linear cell survival model and an exponential model to determine patient-specific lymphocyte radiation sensitivity, α, and recovery, σ, respectively. RESULTS: The in vivo-derived patient-specific α had a median of 0.65 Gy-1 (range, 0.30-1.38). Decreasing BOT to 1 second led to an increased average end-of-treatment ALC of 27.5%, increasing to 60.3% when combined with the 5-fx regimen. Decreasing to 5 fx at the conventional dose rate led to an increase of 17.0% on average. The benefit of both increasing dose rate and reducing the number of fractions was patient specificࣧpatients with highly sensitive lymphocytes benefited most from decreasing BOT, whereas patients with slow lymphocyte recovery benefited most from the shorter fractionation regimen. CONCLUSIONS: We observed that increasing dose rate at the same fractionation reduced ALC depletion more significantly than reducing the number of fractions. High-dose-rates led to an increased sparing of lymphocytes when shortening the fractionation regimen, particularly for patients with radiosensitive lymphocytes at elevated risk.

Dose-Dependent Transmissibility of Chromosome Aberrations in Human Lymphocytes at First Mitosis. II. Biological Effectiveness of Heavy Charged Particles Versus Gamma Rays.

Chromosome aberrations (CAs) are large scale structural rearrangements to the genome that have been used as a proxy endpoint of mutagenic and carcinogenic potential. And yet, many types of CAs are incapable of causing either of these effects simply because they are lethal. Using 24-color multi-fluor combinatorial painting (mFISH), we examined CAs in normal human lymphocytes exposed to graded doses of 1 GeV/nucleon accelerated 56Fe ions and 662 keV 137Cs gamma rays. As expected, the high-linear energy transfer (LET) heavy ions were considerably more potent per unit dose at producing total yields of CAs compared to low-LET gamma rays. As also anticipated, the frequency distribution of aberrations per cell exposed to 56Fe ions was significantly overdispersed compared to the Poisson distribution, containing excess numbers of cells devoid of aberrations. We used the zero-inflated negative binomial (ZINB) distribution to model these data. Based on objective cytogenetic criteria that are subject to caveats we discuss, each cell was individually evaluated in terms of likely survival (i.e., its ability to transmit to daughter cell progeny). For 56Fe ion irradiations, the frequency of surviving cells harboring complex aberrations represented a significant portion of aberration-bearing cells, while for gamma irradiation no survivable cells containing complex aberrations were observed. When the dose responses for the two radiation types were compared, and the analysis was limited to surviving cells that contained aberrations, we were surprised to find the high-LET 56Fe ions only marginally more potent than the low-LET gamma rays for doses less than 1 Gy. In fact, based on dose-response modeling, they were predicted to be less effective than gamma rays at somewhat higher doses. The major implication of these findings is that measures of relative biological effectiveness that fail to account for coincident lethality will tend to overstate the impact of transmissible chromosomal damage from high-LET particle exposure.

Effects of Flash Radiotherapy on Blood Lymphocytes in Humans and Small Laboratory Animals.

A mathematical model, which describes the level of surviving lymphocytes in the blood after ultra-high (FLASH) and lower dose rates of partial-body irradiation, is developed. The model is represented by simple analytic formulae that involve a few parameters, namely, physiologic parameters (characteristics of the blood flow through the blood circulatory system and its irradiated part), a biophysical parameter (a characteristic of the blood lymphocytes radiosensitivity), and the physical parameters (characteristics of irradiation). The model predicts that the level of surviving blood lymphocytes increases as the dose rate increases and approaches the limiting level of (1 - vR), where vR is the fraction of the blood volume in the irradiated part of the blood circulatory system. The model also predicts that the level of surviving blood lymphocytes after the same exposure is higher for lower vR. It is found that FLASH irradiation in humans with doses of 10 to 40 Gy and with exposure times significantly less (<1 s) than the blood circulation time (∼60 s) leads to the maximal blood lymphocyte sparing. Simple formula, which determines effective dose rates for optimal blood lymphocyte sparing, is derived in the framework of the developed model. For the dose range specified above, the obtained modeling prediction of the range of effective dose rates for optimal blood lymphocyte sparing in humans (namely, N ≥40 Gy/s) coincides with the dose rate range in FLASH radiation therapy. It is revealed that the respective effective dose rates for mice are higher than those for humans (for the same dose range) due to the shorter blood circulation time in mice than in humans. Proceeding from the findings obtained in this paper, a hypothesis elucidating the mechanisms of the abscopal effect of FLASH radiation therapy (namely, an antitumor response on metastases located outside of irradiated part of a body) is proposed.

Long-Term Immunological Consequences of Radiation Exposure in a Diverse Cohort of Rhesus Macaques.

PURPOSE: The aim of this study was to develop an improved understanding of the delayed immunologic effects of acute total body irradiation (TBI) using a diverse cohort of nonhuman primates as a model for an irradiated human population. METHODS AND MATERIALS: Immune recovery was evaluated in 221 rhesus macaques either left unirradiated (n = 36) or previously irradiated (n = 185) at 1.1 to 8.5 Gy TBI (median, 6.5 Gy) when aged 2.1 to 15.5 years (median, 4.2 years). Blood was drawn annually for up to 5 years total between 0.5 and 14.3 years after exposure. Blood was analyzed by complete blood count, immunophenotyping of monocytes, dendritic cells (DC) and lymphocytes by flow cytometry, and signal joint T-cell receptor exclusion circle quantification in isolated peripheral blood CD4 and CD8 T cells. Animals were categorized by age, irradiation status, and time since irradiation. Sex-adjusted means of immune metrics were evaluated by generalized estimating equation models to identify cell populations altered by TBI. RESULTS: Overall, the differences between irradiated and nonirradiated animals were subtle and largely restricted to younger animals and select cell populations. Subsets of monocytes, DC, T cells, and B cells showed significant interaction effects between radiation dose and age after adjustment for sex. Irradiation at a young age caused transient increases in the percentage of peripheral blood myeloid DC and dose-dependent changes in monocyte balance for at least 5 years after TBI. TBI also led to a sustained decrease in the percentage of circulating memory B cells. Young irradiated animals exhibited statistically significant and prolonged disruption of the naïve/effector memory/central memory CD4 and CD8 T-cell equilibrium and exhibited a dose-dependent increase in thymopoiesis for 2 to 3 years after exposure. CONCLUSIONS: This study indicates TBI subtly but significantly alters the circulating proportions of cellular mediators of adaptive immune memory for several years after irradiation, especially in macaques under 5 years of age and those receiving a high dose of radiation.

Validation of a High-Throughput Dicentric Chromosome Assay Using Complex Radiation Exposures.

Validation of biodosimetry assays is routinely performed using primarily orthovoltage irradiators at a conventional dose rate of approximately 1 Gy/min. However, incidental/ accidental exposures caused by nuclear weapons can be more complex. The aim of this work was to simulate the DNA damage effects mimicking those caused by the detonation of a several kilotons improvised nuclear device (IND). For this, we modeled complex exposures to: 1. a mixed (photons + IND-neutrons) field and 2. different dose rates that may come from the blast, nuclear fallout, or ground deposition of radionuclides (ground shine). Additionally, we assessed whether myeloid cytokines affect the precision of radiation dose estimation by modulating the frequency of dicentric chromosomes. To mimic different exposure scenarios, several irradiation systems were used. In a mixed field study, human blood samples were exposed to a photon field enriched with neutrons (ranging from 10% to 37%) from a source that mimics Hiroshima's A-bomb's energy spectrum (0.2-9 MeV). Using statistical analysis, we assessed whether photons and neutrons act in an additive or synergistic way to form dicentrics. For the dose rates study, human blood was exposed to photons or electrons at dose rates ranging from low (where the dose was spread over 32 h) to extremely high (where the dose was delivered in a fraction of a microsecond). Potential effects of cytokine treatment on biodosimetry dose predictions were analyzed in irradiated blood subjected to Neupogen or Neulasta for 24 or 48 h at the concentration recommended to forestall manifestation of an acute radiation syndrome in bomb survivors. All measurements were performed using a robotic station, the Rapid Automated Biodosimetry Tool II, programmed to culture lymphocytes and score dicentrics in multiwell plates (the RABiT-II DCA). In agreement with classical concepts of radiation biology, the RABiT-II DCA calibration curves suggested that the frequency of dicentrics depends on the type of radiation and is modulated by changes in the dose rate. The resulting dose-response curves suggested an intermediate dicentric yields and additive effects of photons and IND-neutrons in the mixed field. At ultra-high dose rate (600 Gy/s), affected lymphocytes exhibited significantly fewer dicentrics (P < 0.004, t test). In contrast, we did not find the dose-response modification effects of radiomitigators on the yields of dicentrics (Bonferroni corrected P > 0.006, ANOVA test). This result suggests no bias in the dose predictions should be expected after emergency cytokine treatment initiated up to 48 h prior to blood collection for dicentric analysis.

Mitotic index maximization with no effect on radiation-induced dicentric chromosome frequency.

PURPOSE: The dicentric chromosome (Dic) assay, which is the gold standard for biological dose assessment in radiation emergency medicine, requires an analysis of at least 500 lymphocyte metaphases or 100 Dic aberrations. Therefore, peripheral blood culture conditions able to obtain a high frequency of metaphases for efficient dose evaluation should be optimized. However, the type of blood cultures [i.e. whole blood (WB) or isolated peripheral blood mononuclear cell (PBMC)-culture] and blood volume differ between biodosimetry laboratories. The purpose of this study is to investigate the blood volume at which a high mitotic index (MI) is obtained in peripheral WB-culture and isolated PBMC-culture, and to examine the possible effect of blood volume on radiation-induced Dic frequency. MATERIALS AND METHODS: Peripheral blood was collected from three healthy donors with their informed consent. The complete and differential blood counts were performed using an automated hematology analyzer. After blood count, peripheral blood was irradiated with 0 or 2 Gy X-ray. Blood was cultured with phytohemagglutinin (180 µg/ml) and demecolcine　(0.05 µg/ml) for 48 h. The MI and Dic frequency were analyzed in 5, 10, 15, 20, 25, and 30% WB-cultures and 0.6, 1.2, 1.8, 2.4, 3.0, 3.6, and 4.2 ml WB-equivalent PBMC-cultures. RESULTS: In WB-culture, MI showed the highest value (∼22%) in 5-15% WB-culture and then gradually decreased to ∼9% with 30% WB-culture. MI peaked at 36 and 31% in 1.8 and 2.4 ml-WB equivalent volumes for PMBC-cultures, respectively. MI progressively decreased as the amount of PBMCs increased. Although individual differences were observed in the MI values among the three subjects, all the subjects showed the same tendency and higher MI was seen in PBMC than WB-cultures. However, these factors had no significant impact on the yield of Dics. In all culture conditions, the estimated dose calculated based on the Dic frequency was equivalent to the absorbed dose of ex vivo X-ray-irradiated blood. CONCLUSION: While MI was affected by the blood culture type and the volume of cultured blood, Dic yield did not differ significantly between these conditions. These results could be used by relevant laboratories to optimize MI in certain circumstances.

The Intensity of Blood Lymphocyte Apoptosis in Chronically Exposed Individuals with Obligate Forms of Precancerous Conditions.

We studied the apoptotic death of peripheral blood lymphocytes in individuals chronically exposed to radiation with a history of obligate forms of precancerous conditions. The study included 242 subjects chronically exposed to low-intensity radiation in the range of low and medium doses (mean dose of irradiation of the red bone marrow 555±39 mGy). Of these, 121 subjects had obligate forms of precancerous conditions. In irradiated individuals with obligate forms of precancers, a statistically significant increase in the frequency of apoptosis of peripheral blood lymphocytes at an early and late stages was revealed. Correlation analysis revealed no dependence of the apoptotic death of peripheral blood lymphocytes on the dose of red bone marrow irradiation.