Radiation therapy is an effective cancer treatment, although damage to healthy tissues is common. Here we analyzed cell-free, methylated DNA released from dying cells into the circulation to evaluate radiation-induced cellular damage in different tissues. To map the circulating DNA fragments to human and mouse tissues, we established sequencing-based, cell-type-specific reference DNA methylation atlases. We found that cell-type-specific DNA blocks were mostly hypomethylated and located within signature genes of cellular identity. Cell-free DNA fragments were captured from serum samples by hybridization to CpG-rich DNA panels and mapped to the DNA methylation atlases. In a mouse model, thoracic radiation-induced tissue damage was reflected by dose-dependent increases in lung endothelial and cardiomyocyte methylated DNA in serum. The analysis of serum samples from patients with breast cancer undergoing radiation treatment revealed distinct dose-dependent and tissue-specific epithelial and endothelial responses to radiation across multiple organs. Strikingly, patients treated for right-sided breast cancers also showed increased hepatocyte and liver endothelial DNA in the circulation, indicating the impact on liver tissues. Thus, changes in cell-free methylated DNA can uncover cell-type-specific effects of radiation and provide a readout of the biologically effective radiation dose received by healthy tissues.

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Background Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications across clinical research, including monitoring radiation exposure. A key limitation to their implementation is minimal standardization in EV isolation and analytical methods. Further, most urinary EV isolation protocols necessitate large volumes of sample. This study aimed to compare and optimize isolation and analytical methods for EVs from small volumes of urine. Methods 3 EV isolation methods were compared: ultracentrifugation, magnetic bead-based, and size-exclusion chromatography from 0.5 mL or 1 mL of rat and human urine. EV yield and mass spectrometry signals (Q-ToF and Triple Quad) were evaluated from each method. Metabolomic profiling was performed on EVs isolated from the urine of rats exposed to ionizing radiation 1-, 14-, 30- or 90-days post-exposure, and human urine from patients receiving thoracic radiotherapy for the treatment of lung cancer pre- and post-treatment. Results Size-exclusion chromatography is the preferred method for EV isolation from 0.5 mL of urine. Mass spectrometry-based metabolomic analyses of EV cargo identified biochemical changes induced by radiation, including altered nucleotide, folate, and lipid metabolism. We have provided standard operating procedures for implementation of these methods in other laboratories. Conclusions We demonstrate that EVs can be isolated from small volumes of urine and analytically investigated for their biochemical contents to detect radiation induced metabolomic changes. These findings lay a groundwork for future development of methods to monitor response to radiotherapy and can be extended to an array of molecular phenotyping studies aimed at characterizing EV cargo.

Introduction: Review the early experience with a single-room gantry mounted active scanning proton therapy system. Material and Methods: All patients treated with proton beam radiotherapy (PBT) were enrolled in an institutional review board-approved patient registry. Proton beam radiotherapy was delivered with a 250 MeV gantry mounted synchrocyclotron in a single-room integrated facility within the pre-existing cancer center. Demographic data, cancer diagnoses, treatment technique, and geographic patterns were obtained for all patients. Treatment plans were evaluated for mixed modality therapy. Insurance approval data was collected for all patients treated with PBT. Results: A total of 132 patients were treated with PBT between March 2018 and June 2019. The most common oncologic subsites treated included the central nervous system (22%), gastrointestinal tract (20%), and genitourinary tract (20%). The most common histologies treated included prostate adenocarcinoma (19%), non-small cell lung cancer (10%), primary CNS gliomas (8%), and esophageal cancer (8%). Rationale for PBT treatment included limitation of dose to adjacent critical organs at risk (67%), reirradiation (19%), and patient comorbidities (11%). Patients received at least one x-ray fraction delivered as prescribed (36%) or less commonly due to unplanned machine downtime (34%). Concurrent systemic therapy was administered to 57 patients (43%). Twenty-six patients (20%) were initially denied insurance coverage and required peer-to-peers (65%), written appeals (12%), secondary insurance approval (12%), and comparison x-ray to proton plans (8%) for subsequent approval. Proton beam radiotherapy approval required a median of 17 days from insurance submission. Discussion: Incorporation of PBT into our existing cancer center allowed for multidisciplinary oncologic treatment of a diverse population of patients. Insurance coverage for PBT presents as a significant hurdle and improvements are needed to provide more timely access to necessary oncologic care.

Introduction Review the early experience with a single-room gantry mounted active scanning proton therapy system implemented in the modern era. Materials and Methods All patients treated with proton beam radiotherapy (PBT) were enrolled in an institutional review board-approved patient registry. Proton beam radiotherapy was delivered with a 250 MeV gantry mounted synchrocyclotron in a single-room integrated facility within the pre-existing cancer center. Demographic data, cancer diagnoses, treatment technique, and geographic patterns were obtained for all patients. Treatment plans were evaluated for mixed modality therapy. Insurance approval data was collected for all patients treated with PBT. Results A total of 132 patients were treated with PBT between March 2018 and June 2019. The most common oncologic subsites treated included the central nervous system (22%), gastrointestinal tract (20%), and genitourinary tract (20%). The most common histologies treated included prostate adenocarcinoma (19%), non-small cell lung cancer (10%), primary CNS gliomas (8%), and esophageal cancer (8%). Rationale for PBT treatment included limitation of dose to adjacent critical organs at risk (67%), reirradiation (19%), and patient comorbidities (11%). Patients received at least one x-ray fraction delivered as prescribed (36%) or less commonly due to unplanned machine downtime (34%). Concurrent systemic therapy was administered to 57 patients (43%). Twenty-six patients (20%) were initially denied insurance coverage and required peer-to-peers (65%), written appeals (12%), secondary insurance approval (12%), and comparison x-ray to proton plans (8%) for subsequent approval. Proton beam radiotherapy approval required a median of 17 days from insurance submission. Conclusion Incorporation of PBT into our existing cancer center allowed for multidisciplinary oncologic treatment of a diverse population of patients. Insurance coverage for PBT presents as a significant hurdle and improvements are needed to provide more timely access to necessary oncologic care.