The TLR2/TLR6 ligand FSL-1 mitigates radiation-induced hematopoietic injury in mice and nonhuman primates.

Thrombocytopenia, hemorrhage, anemia, and infection are life-threatening issues following accidental or intentional radiation exposure. Since few therapeutics are available, safe and efficacious small molecules to mitigate radiation-induced injury need to be developed. Our previous study showed the synthetic TLR2/TLR6 ligand fibroblast stimulating lipopeptide (FSL-1) prolonged survival and provided MyD88-dependent mitigation of hematopoietic acute radiation syndrome (H-ARS) in mice. Although mice and humans differ in TLR number, expression, and function, nonhuman primate (NHP) TLRs are like those of humans; therefore, studying both animal models is critical for drug development. The objectives of this study were to determine the efficacy of FSL-1 on hematopoietic recovery in small and large animal models subjected to sublethal total body irradiation and investigate its mechanism of action. In mice, we demonstrate a lack of adverse effects, an easy route of delivery (subcutaneous) and efficacy in promoting hematopoietic progenitor cell proliferation by FSL-1. NHP given radiation, followed a day later with a single subcutaneous administration of FSL-1, displayed no adversity but showed elevated hematopoietic cells. Our analyses revealed that FSL-1 promoted red blood cell development and induced soluble effectors following radiation exposure. Cytologic analysis of bone marrow aspirates revealed a striking enhancement of mononuclear progenitor cells in FSL-1-treated NHP. Combining the efficacy of FSL-1 in promoting hematopoietic cell recovery with the lack of adverse effects induced by a single administration supports the application of FSL-1 as a viable countermeasure against H-ARS.

Exogenous Growth Hormone Exacerbates Post-Irradiation Atherosclerosis in Susceptible Epicardial Coronary Arteries.

Cardiac exposure to ionizing radiation can damage both the microvasculature and coronary arteries, as well as increase the long-term risk of heart disease, myocardial fibrosis, and conduction abnormalities. Therapeutic agents capable of promoting recovery from radiation injury to the heart are limited. Growth hormone is linked to improved cardiac function following injury. Here, we leveraged a cynomolgus macaque model to determine the long-term outcomes of recombinant human growth hormone (rhGH) therapy on the heart following low-dose ionizing radiation. Macaques were exposed to 2 Gy radiation, treated with rhGH for one month, and assessed after 2 years. Overall, plasma lipid profile, cardiac function, and coronary artery disease were similar between rhGH and placebo treated animals. However, a subgroup of rhGH-treated animals exhibited more extensive atherosclerotic plaques in the coronary arteries. Together, these findings indicate that transient human growth hormone therapy subsequent to a single low dose of ionizing radiation involving the heart does not result in long-term changes to plasma cholesterol but may promote exacerbated coronary artery disease in a subset of individuals.

Outcomes of radiation-induced meningiomas treated with stereotactic radiosurgery.

PURPOSE: Data on the efficacy and safety of stereotactic radiosurgery (SRS) for treatment of radiation-induced meningiomas (RIMs) are limited. METHODS: A single institution database of Cobalt-60 SRS cases from 08/1999 to 10/2020 was reviewed. Radiation-induced meningiomas were identified using Cahan's criteria. Endpoints included overall survival (OS), progression free survival (PFS), local control (LC), treatment failure, and treatment toxicity. Univariate and multivariate analyses were performed using cox proportional hazard models. RESULTS: A total of 29 patients with 86 RIM lesions were identified. Median follow-up after SRS was 59 months. The median dose prescribed to the 50% isodose line was 14 Gy (range 12-20 Gy). The actuarial 5-yr OS and PFS were 96% and 68%, respectively. Patients treated for recurrent RIMs had a significantly lower PFS (45% vs 94% at 3 yr, p < 0.005) than patients treated in the upfront setting. Patients with presumed or WHO grade I RIMs had a significantly greater PFS (3-year PFS 96% vs 20%) than patients with WHO grade II RIMs (p < 0.005). On a per-lesion basis, local control (LC) at 1-, 3-, and 5-yrs was 82%, 76%, 74%, respectively. On multivariate analysis, female gender was associated with improved LC (p < 0.001), while marginal doses > 14 Gy were associated with worse local control (p < 0.001). Grade I-III toxicity following treatment was 9.0%. CONCLUSIONS: Stereotactic radiosurgery is a safe and effective treatment option for radiographic RIMs, WHO grade I RIMs, or lesions treated in the upfront setting. WHO grade II lesions and recurrent lesions are at increased risk for disease progression.

Initial brain metastasis velocity: does the rate at which cancers first seed the brain affect outcomes?

PURPOSE/OBJECTIVE(S): Brain metastasis velocity (BMV) is a metric that describes the rate of development of new brain metastases (BM) after initial stereotactic radiosurgery (SRS). A limitation in the application of BMV is it cannot be applied until time of first BM failure after SRS. We developed initial BM velocity (iBMV), a new metric that accounts for the number of BM at first SRS and the time since initial cancer diagnosis. MATERIALS/METHODS: We reviewed patients with BM treated at our institution with upfront SRS without WBRT. iBMV was calculated as the number of BM at initial SRS divided by time (years) from initial cancer diagnosis to first SRS. We performed a linear regression to correlate BMV as a continuous variable and with low, intermediate, and high BMV risk groups. Kaplan-Meier estimation of OS was calculated from time of first SRS to death. iBMV was not calculated for patients who presented with BM at initial cancer diagnosis. RESULTS: 994 patients were treated with upfront SRS without WBRT between 2000 and 2017. Median OS was 8.5 mos. 595 (60%) patients developed BM after cancer diagnosis and median time to first SRS from time of initial diagnosis was 2.2 years. Median iBMV was 0.79 BM/year. iBMV correlated with BMV (ß = 1.57 p = 0.021) and independently predicted for mortality [Cox proportional hazard ratio (HR) 1.11, p = 0.036] after accounting for histology, number of initial brain metastases (HR 1.03, p = 0.32), time from cancer diagnosis to SRS (HR 0.98, p = 0.157) in a multivariate model. CONCLUSION: iBMV correlates with BMV and OS. With further validation, iBMV could serve as a metric to risk stratify patients for WBRT or SRS at time of first BM presentation.

Clinical and economic outcomes of patients with brain metastases based on symptoms: an argument for routine brain screening of those treated with upfront radiosurgery.

BACKGROUND: Insurers have started to deny reimbursement for routine brain surveillance with magnetic resonance imaging (MRI) after stereotactic radiosurgery (SRS) for brain metastases in favor of symptom-prompted imaging. The authors investigated the clinical and economic impact of symptomatic versus asymptomatic metastases and related these findings to the use of routine brain surveillance. METHODS: Between January 2000 and December 2010, 442 patients underwent upfront SRS for brain metastases. In total, 127 asymptomatic patients and 315 symptomatic patients were included. Medical records were used to determine the presenting symptoms, distant and local brain failure, retreatment, and need for hospital and rehabilitative care. Cost-of-care estimates were based on Medicare payment rates as of January 2013. RESULTS: Symptomatic patients had an increased hazard for all-cause mortality (hazard ratio, 1.448) and were more likely to experience neurologic death (42% vs 20%; P < .0001). Relative to asymptomatic patients, symptomatic patients required more craniotomies (43% vs 5%; P < .0001), had more prolonged hospitalization (2 vs 0 days; P < .0001), were more likely to have Radiation Therapy Oncology Group grade 3 and 4 post-treatment symptoms (24% vs 5%; P < .0001), and required $11,957 more on average to manage per patient. Accounting for all-cause mortality rates and the probability of diagnosis at each follow-up period, the authors estimated that insurers would save an average $1326 per patient by covering routine surveillance MRI after SRS to detect asymptomatic metastases. CONCLUSIONS: Patients who presented with symptomatic brain metastases had worse clinical outcomes and cost more to manage than asymptomatic patients. The current findings argue that routine brain surveillance after radiosurgery has clinical benefits and reduces the cost of care.

Local control and toxicity outcomes in brainstem metastases treated with single fraction radiosurgery: is there a volume threshold for toxicity?

Gamma Knife Radiosurgery (GKRS) has been reported in the treatment of brainstem metastases while dose volume toxicity thresholds remain mostly undefined. A retrospective review of 52 brainstem metastases in 44 patients treated with GKRS was completed. A median dose of 18 Gy (range 10-22 Gy) was prescribed to the tumor margin (median 50 % isodose). 25 patients had undergone previous whole brain radiation therapy. Toxicity was graded by the LENT-SOMA scale. Mean and median follow-up was 10 and 6 months. Only 3 of the 44 patients are living. Multiple brain metastases were treated in 75 % of patients. Median size of lesions was 0.134 cc, (range 0.013-6.600 cc). Overall survival rate at 1 year was 32 % (95 % CI 51.0-20.1 %) with a median survival time of 6 months (95 % CI 5.0-16.5). Local control rate at 6 months and 1 year was 88 % (95 % CI 70-95 %) and 74 % (95 % CI 52-87 %). Cause of death was neurologic in 17 patients, non-neurologic in 20 patients, and unknown in four. Four patients experienced treatment related toxicities. Univariate analysis of tumor volume revealed that volume greater than 1.0 cc predicted for toxicity. A strategy of using lower marginal doses with GKRS to brain stem metastases appears to lead to a lower local control rate than seen with lesions treated within the standard dose range in other locations. Tumor size greater than 1.0 cc predicted for treatment-related toxicity.

Single-institution retrospective series of gamma knife radiosurgery in the treatment of multiple sclerosis-related trigeminal neuralgia: factors that predict efficacy.

BACKGROUND: Gamma knife radiosurgery (GKRS) has been reported as a treatment option for multiple sclerosis (MS)-related trigeminal neuralgia. OBJECTIVE: To report the outcomes of a single-institution retrospective series of MS-related trigeminal neuralgia. METHODS: Between 2002 and 2010, 35 patients with MS-related trigeminal neuralgia were treated with GKRS. The median maximum dose was 90 Gy. Data were analyzed to determine the response to GKRS and factors that may predict for efficacy. RESULTS: Of the 35 patients, 88% experienced a Barrow Neurological Institute (BNI) pain score of I-III at 3 months after GKRS. Kaplan-Meier estimates of 1-, 2- and 5-year freedom from BNI IV-V pain relapse were 57, 57 and 52%, respectively. Numbness was experienced by 39% of patients after GKRS, though no patients reported bothersome numbness. Several differences were noted between how the MS-related variant responded to GKRS and what has previously been reported for idiopathic trigeminal neuralgia. These include the observations that development of post-GKRS numbness did not predict for treatment response (p = 0.62) and that dorsal root entry zone dose did not predict for freedom from pain relapse (odds ratio 1.01, p = 0.1). Active smoking predicted for freedom from pain relapse (odds ratio 67.4, p = 0.04). CONCLUSION: GKRS is a viable noninvasive treatment option for MS-related trigeminal neuralgia.

Understanding radiation-generated electronic traps in radiation dosimeters based on organic field-effect transistors.

Organic dosimeters offer unique advantages over traditional technologies, and they can be used to expand the capabilities of current radiation detection systems. In-depth knowledge of the mechanisms underlying the interaction between radiation and organic materials is essential for their widespread adoption. Here, we identified and quantitatively characterized the electronic traps generated during the operation of radiation dosimeters based on organic field-effect transistors. Spectral analysis of the trap density of states, along with optical and structural studies, revealed the origin of trap states as local structural disorder within the crystalline films. Our results provide new insights into the radiation-induced defects in organic dosimeters, and pave the way for the development of more efficient and reliable radiation detection devices.

Seven Challenges in Radiology Practice: From Declining Reimbursement to Inadequate Labor Force: Summary of the 2023 ACR Intersociety Meeting.

This white paper discusses the seven most important challenges facing radiology today: declining reimbursement, corporatization and consolidation, inadequate labor force, imaging appropriateness, burnout, turf wars with nonphysicians, and workflow efficiency. Participants in the Intersociety Summer Conference-2023 focused their effort on identifying potential solutions given how critical these topics are to the sustainability of the profession.

Patterns of recurrence after stereotactic radiosurgery for treatment of meningiomas.

OBJECT: The purpose of this study was to evaluate patterns of failure after stereotactic radiosurgery (SRS) for meningiomas and factors that may influence these outcomes. METHODS: Based on a retrospective chart review, 279 patients were treated with SRS for meningiomas between January 1999 and March 2011 at Wake Forest Baptist Health. Disease progression was determined using serial imaging, with a minimum follow-up of 6 months (median 34.2 months). RESULTS: The median margin dose was 12.0 Gy (range 8.8-20 Gy). Local control rates for WHO Grade I tumors were 96.6%, 84.4%, and 75.7% at 1, 3, and 5 years, respectively. WHO Grade II and III tumors had local control rates of 72.3%, 57.7%, and 52.9% at 1, 3, and 5 years, respectively. Tumors without pathological grading had local control rates of 98.7%, 97.6%, and 94.2% at 1, 3, and 5 years, respectively. Of the local recurrences, 63.1% were classified as marginal (within 2 cm of treatment field). The 1-, 3-, and 5-year rates of distant failure were 6.5%, 10.3%, and 16.6%, respectively, for Grade I tumors and 11.4%, 17.2%, and 22.4%, respectively, for Grade II/III tumors. Tumors without pathological grading had distant failure rates of 0.7%, 3.2%, and 6.5% at 1, 3, and 5 years, respectively. Wilcoxon analysis revealed that multifocal disease (p < 0.001) and high-grade histology (WHO Grade II or III; p < 0.001) were significant predictors of local recurrence. Additionally, male sex was a significant predictor of distant recurrence (p = 0.04). Multivariate analysis also showed that doses greater than or equal to 12 Gy were associated with improved local control (p = 0.015). CONCLUSIONS: In this patient series, 12 Gy was the minimum sufficient margin dose for the treatment of meningiomas. Male sex is a risk factor for distant failure, whereas high-grade histology and multifocal disease are risk factors for local failure.

Quantitative Assessment and Comparative Analysis of Longitudinal Lung CT Scans of Chest-Irradiated Nonhuman Primates.

Computed tomography (CT) imaging has been used to diagnose radiation-induced lung injury for decades. However, histogram-based quantitative tools have rarely been applied to assess lung abnormality due to radiation-induced lung injury (RILI). Here, we used first-order summary statistics to derive and assess threshold measures extracted from whole lung histograms of CT radiodensity in rhesus macaques. For the present study, CT scans of animals exposed to 10 Gy of whole thorax irradiation were utilized from a previous study spanning 2-9 months postirradiation. These animals were grouped into survivors and non-survivors based on their clinical and experimental endpoints. We quantified the change in lung attenuation after irradiation relative to baseline using three density parameters; average lung density (ALD), percent change in hyper-dense lung volume (PCHV), hyperdense volume as a percent of total volume (PCHV/TV) at 2-month intervals and compared each parameter between the two irradiated groups (non-survivors and survivors). We also correlated our results with histological findings. All the three indices (ALD, PCHV, PCHV/TV) obtained from density histograms showed a significant increase in lung injury in non-survivors relative to survivors, with PCHV relatively more sensitive to detect early RILI changes. We observed a significant positive correlation between histologic pneumonitis scores and each of the three CT measurements, indicating that CT density is useful as a surrogate for histologic disease severity in RILI. CT-based three density parameters, ALD, PCHV, PCHV/TV, may serve as surrogates for likely histopathology patterns in future studies of RILI disease progression.

Summary of the 2022 ACR Intersociety Meeting.

The ACR Intersociety Committee meeting of 2022 (ISC-2022) was convened around the theme of "Recovering From The Great Resignation, Moral Injury and Other Stressors: Rebuilding Radiology for a Robust Future." Representatives from 29 radiology organizations, including all radiology subspecialties, radiation oncology, and medical physics, as well as academic and private practice radiologists, met for 3 days in early August in Park City, Utah, to search for solutions to the most pressing problems facing the specialty of radiology in 2022. Of these, the mismatch between the clinical workload and the available radiologist workforce was foremost-as many other identifiable problems flowed downstream from this, including high job turnover, lack of time for teaching and research, radiologist burnout, and moral injury.

Breast cancer subtype affects patterns of failure of brain metastases after treatment with stereotactic radiosurgery.

We investigate the variance in patterns of failure after Gamma Knife™ radiosurgery (GKRS) for patients with brain metastases based on the subtype of the primary breast cancer. Between 2000 and 2010, 154 breast cancer patients were treated with GKRS for brain metastases. Tumor subtypes were approximated based on hormone receptor (HR) and HER2 status of the primary cancer: Luminal A/B (HR+/HER2(-)); HER2 (HER2+/HR(-)); Luminal HER2 (HR+/HER2+), Basal (HR(-)/HER2(-)), and then based on HER2 status alone. The median follow-up period was 54 months. Kaplan-Meier method was used to estimate survival times. Multivariable analysis was performed using Cox regression models. Median number of lesions treated was two (range 1-15) with a median dose of 20 Gy (range 9-24 Gy). Median overall survival (OS) was 7, 9, 11 and 22 months for Basal, Luminal A/B, HER2, and Luminal HER2, respectively (p = 0.001), and was 17 and 8 months for HER2+ and HER(-) patients, respectively (p < 0.001). Breast cancer subtype did not predict time to local failure (p = 0.554), but did predict distant brain failure rate (76, 47, 47, 36 % at 1 year for Basal, Luminal A/B, HER2, and Luminal HER2 respectively, p < 0.001). An increased proportion of HER2+ patients experienced neurologic death (46 vs 31 %, p = 0.066). Multivariate analysis revealed that HER2+ patients (p = 0.007) independently predicted for improved survival. Women with basal subtype have high rates of distant brain failure and worsened survival. Our data suggest that differences in biologic behavior of brain metastasis occur across breast cancer subtypes.

Gamma knife radiosurgery in the treatment of tumor-related facial pain.

BACKGROUND: Intracranial neoplasms can cause pain similar to trigeminal neuralgia. Literature regarding radiosurgery for this is limited. We present a retrospective review of patients with tumor-related facial pain from benign lesions treated with gamma knife radiosurgery (GKRS) at Wake Forest University. OBJECTIVES: The primary objectives were to determine long-term pain relief and predictive factors for pain alleviation. METHODS: We reviewed 515 patients treated with GKRS for benign meningioma, vestibular schwannoma or trigeminal schwannoma between August 1999 and August 2010. Twenty-one eligible patients had tumor-related facial pain prior to GKRS. The median marginal tumor dose was 12 Gy. Long-term pain relief data were obtained by chart review and telephone interview. RESULTS: The median follow-up for symptom evaluation was 3.8 years. Seventeen of 21 patients (81%) experienced a Barrow Neurological Institute (BNI) score of I-III at 6 months following GKRS. Kaplan-Meier estimates of freedom from BNI IV-V relapse were 66% at 1 year and 53% at 2 years. No pain relapses occurred after 2 years. CONCLUSION: GKRS of benign lesions is a noninvasive option for patients with tumor-related facial pain. Pain relief is modest, with the majority of pain relapses occurring within 2 years and approximately one half of patients maintaining relief beyond 2 years.

Pseudo Pelger-Huët anomalies as potential biomarkers for acute exposure radiation dose in rhesus macaques (Macaca mulatta).

PURPOSE: The potential for malicious use of radiation, or radiation accidents could potentially lead to acute, high radiation doses to the public. Following acute accidental exposure to high doses of radiation, medical intervention is pivotal to the survivability of the patient, and the sooner the appropriate measures are taken the better the odds for survival. Early estimates of acute accidental radiation doses can be determined via biomarkers such as dicentric chromosome analysis or scenario reconstruction using computer software. However, both take valuable time and can be expensive. Increased frequencies of abnormal neutrophils in peripheral blood, referred to as pseudo Pelger-Huët anomalies (PPHAs), have been shown to be potential biomarkers of radiation exposure in several scenarios, including the 1958 Y-12 criticality accident and the radium dial painters. PPHAs are potentially a faster and cheaper quantitative biomarker for radiation exposure, and here they were evaluated in acutely exposed rhesus macaques. METHODS AND MATERIALS: Peripheral blood smears from acutely exposed rhesus macaques were evaluated for the percentage of neutrophils that displayed the PPHA morphology using light microscopy. Irradiated animals received 0 to 8.5 Gy total body radiation using one of two strategies: (1) linear accelerator-produced 6 MV photons delivered at 80 cGy/minute; or (2) Cobalt 60-produced gamma irradiation delivered at 60 cGy/min. Zero dose animals were used to determine a baseline percentage of PPHAs, and blood smears taken periodically throughout the lifetime of exposed animals post-irradiation were used to determine the persistence and biokinetics of PPHAs. RESULTS: The baseline prevalence of the PPHA in rhesus macaques was determined to be 0.58 ± 0.46%. The dose-response curve with doses ranging from 0 Gy to 8.5 Gy (LD90/30) displayed a strong positive correlation between PPHA percentage and acute radiation dose (R2 of 0.88 p = 3.62 × 10-22). Statistically significant differences were found when animals were separated into dose cohorts of 0, 4, 6.4-6.5, and 8-8.5 Gy. The biokinetics model utilized only 4 Gy exposures and blood smears taken periodically over 3.1 years post-irradiation. PPHA morphology increases quickly following irradiation and appears stable over 3.1 years post-irradiation. CONCLUSION: PPHA morphology was confirmed to be present in rhesus macaques, a dose-response relationship was constructed, and it is stable over 3 years post-irradiation. This study demonstrates that PPHA analysis can be a fast and cheap method of biodosimetry. Future studies will work to determine the accuracy of dose determination and lower limits of detection.

Staged gamma knife radiosurgery for large cerebral arteriovenous malformations.

BACKGROUND: We present our experience over a 10-year period of staged radiosurgery for large arteriovenous malformations (AVMs) including patient outcomes and methods. METHODS: From July 2000 to December 2010, 80 patients with AVMs were treated with gamma knife radiosurgery (GKS) at our institution; of these patients, 5 were treated for large AVMs with staged GKS (volumes >20 cm(3)). The mean interval between treatments was 10 months (range 7-16). The mean dose for the margin used was 18.0 Gy (range 16-20). The mean volume treated was 37.2 cm(3) (range 22-50). The mean total follow-up was 76.5 months (range 42-120). RESULTS: Two patients had complete obliteration of the AVM nidus. One patient had 95% obliteration (31 months after radiosurgery), one had 90% obliteration (38 months after radiosurgery), and one had less than 50% obliteration at 53 months with a 16-month interval between staged treatments. CONCLUSIONS: Staged radiosurgery is an effective and safe method for the treatment of large AVMs. We report achieving higher marginal doses with staging in planned intervals of 6-9 months between staged treatments. It appears that the longer the wait between treatments, the less likely it is that complete obliteration will be achieved.

Clinicopathologic and Transcriptomic Analysis of Radiation-Induced Lung Injury in Nonhuman Primates.

PURPOSE: Radiation-induced lung injury (RILI) is a progressive condition with an early phase (radiation pneumonitis) and a late phase (lung fibrosis). RILI may occur after partial-body ionizing radiation exposures or internal radioisotope exposure, with wide individual variability in timing and extent of lung injury. This study aimed to provide new insights into the pathogenesis and progression of RILI in the nonhuman primate (NHP) rhesus macaque model. METHODS AND MATERIALS: We used an integrative approach to understand RILI and its evolution at clinical and molecular levels in 17 NHPs exposed to 10 Gy of whole-thorax irradiation in comparison with 3 sham-irradiated control NHPs. Clinically, we monitored respiratory rates, computed tomography (CT) scans, plasma cytokine levels, and bronchoalveolar lavage (BAL) over 8 months and lung samples collected at necropsy for molecular and histopathologic analyses using RNA sequencing and immunohistochemistry. RESULTS: Elevated respiratory rates, greater CT density, and more severe pneumonitis with increased macrophage content were associated with early mortality. Radiation-induced lung fibrosis included polarization of macrophages toward the M2-like phenotype, TGF-ß signaling, expression of CDKN1A/p21 in epithelial cells, and expression of α-SMA in lung stroma. RNA sequencing analysis of lung tissue revealed SERPINA3, ATP12A, GJB2, CLDN10, TOX3, and LPA as top dysregulated transcripts in irradiated animals. In addition to transcriptomic data, we observed increased protein expression of SERPINA3, TGF-ß1, CCL2, and CCL11 in BAL and plasma samples. CONCLUSIONS: Our combined clinical, imaging, histologic, and transcriptomic analysis provides new insights into the early and late phases of RILI and highlights possible biomarkers and potential therapeutic targets of RILI. Activation of TGF-ß and macrophage polarization appear to be key mechanisms involved in RILI.

Long-Term Recovery of the Adaptive Immune System in Rhesus Macaques After Total Body Irradiation.

PURPOSE: Ionizing radiation causes acute damage to hematopoietic and immune cells, but the long-term immunologic consequences of irradiation are poorly understood. We therefore performed a prospective study of the delayed immune effects of radiation using a rhesus macaque model. METHODS AND MATERIALS: Ten macaques received 4 Gy high-energy x-ray total body irradiation (TBI) and 6 control animals received sham irradiation. TBI caused transient lymphopenia that resolved over several weeks. Once white blood cell counts recovered, flow cytometry was used to immunophenotype the circulating adaptive immune cell populations 4, 9, and 21 months after TBI. Data were fit using a mixed-effects model to determine age-dependent, radiation-dependent, and interacting effects. T cell receptor (TCR) sequencing and quantification of TCR Excision Circles were used to determine relative contributions of thymopoiesis and peripheral expansion to T cell repopulation. Two years after TBI, the cohort was vaccinated with a 23-valent pneumococcal polysaccharide vaccine and a tetravalent influenza hemagglutinin vaccine. RESULTS: Aging, but not TBI, led to significant changes in the frequencies of dendritic cells, CD4 and CD8 T cells, and B cells. However, irradiated animals exhibited increased frequencies of central memory T cells and decreased frequencies of naïve T cells. These consequences of irradiation were time-dependent and more prolonged in the CD8 T cell population. Irradiation led to transient increases in CD8+ T cell TCR Excision Circles and had no significant effect on TCR sequence entropy, indicating T cell recovery was partially mediated by thymopoiesis. Animals that were irradiated and then vaccinated showed normal immunoglobulin G binding and influenza neutralization titers in response to the 4 protein antigens but weaker immunoglobulin G binding titers to 10 of the 23 polysaccharide antigens. CONCLUSIONS: These findings indicate that TBI causes subtle but long-lasting immune defects that are evident years after recovery from lymphopenia.

CytoRADx: A High-Throughput, Standardized Biodosimetry Diagnostic System Based on the Cytokinesis-Block Micronucleus Assay.

In a large-scale catastrophe, such as a nuclear detonation in a major city, it will be crucial to accurately diagnose large numbers of people to direct scarce medical resources to those in greatest need. Currently no FDA-cleared tests are available to diagnose radiation exposures, which can lead to complex, life-threatening injuries. To address this gap, we have achieved substantial advancements in radiation biodosimetry through refinement and adaptation of the cytokinesis-block micronucleus (CBMN) assay as a high throughput, quantitative diagnostic test. The classical CBMN approach, which quantifies micronuclei (MN) resulting from DNA damage, suffers from considerable time and expert labor requirements, in addition to a lack of universal methodology across laboratories. We have developed the CytoRADx™ System to address these drawbacks by implementing a standardized reagent kit, optimized assay protocol, fully automated microscopy and image analysis, and integrated dose prediction. These enhancements allow the CytoRADx System to obtain high-throughput, standardized results without specialized labor or laboratory-specific calibration curves. The CytoRADx System has been optimized for use with both humans and non-human primates (NHP) to quantify radiation dose-dependent formation of micronuclei in lymphocytes, observed using whole blood samples. Cell nuclei and resulting MN are fluorescently stained and preserved on durable microscope slides using materials provided in the kit. Up to 1,000 slides per day are subsequently scanned using the commercially based RADxScan™ Imager with customized software, which automatically quantifies the cellular features and calculates the radiation dose. Using less than 1 mL of blood, irradiated ex vivo, our system has demonstrated accurate and precise measurement of exposures from 0 to 8 Gy (90% of results within 1 Gy of delivered dose). These results were obtained from 636 human samples (24 distinct donors) and 445 NHP samples (30 distinct subjects). The system demonstrated comparable results during in vivo studies, including an investigation of 43 NHPs receiving single-dose total-body irradiation. System performance is repeatable across laboratories, operators, and instruments. Results are also statistically similar across diverse populations, considering various demographics, common medications, medical conditions, and acute injuries associated with radiological disasters. Dose calculations are stable over time as well, providing reproducible results for at least 28 days postirradiation, and for blood specimens collected and stored at room temperature for at least 72 h. The CytoRADx System provides significant advancements in the field of biodosimetry that will enable accurate diagnoses across diverse populations in large-scale emergency scenarios. In addition, our technological enhancements to the well-established CBMN assay provide a pathway for future diagnostic applications, such as toxicology and oncology.