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Brian Sims,1–3,* Anitra L Farrow,4,* Sparkle D Williams,1,2 Anju Bansal,4 Alexandre Krendelchtchikov,1,2,4 Linlin Gu,5 Qiana L Matthews3,4,6 1Division of Neonatology, Department of Pediatrics, 2Department of Cell, Developmental and Integrative Biology, 3Center for AIDS Research, 4Division of Infectious Diseases, 5Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Alabama at Birmingham, Birmingham, 6Microbiology Program, Department of Biological Sciences, College of Science, Technology, Engineering and Mathematics, Alabama State University, Montgomery, AL, USA *These authors contributed equally to this work Abstract: Exosomes, 30–200 nm nanostructures secreted from donor cells and internalized by recipient cells, can play an important role in the cellular entry of some viruses. These microvesicles are actively secreted into various body fluids, including blood, urine, saliva, cerebrospinal fluid, and breast milk. We successfully isolated exosomes from human breast milk and plasma. The size and concentration of purified exosomes were measured by nanoparticle tracking, while Western blotting confirmed the presence of the exosomal-associated proteins CD9 and CD63, clathrin, and T cell immunoglobulin and mucin proteins (TIMs). Through viral infection assays, we determined that HIV-1 utilizes an exosome-dependent mechanism for entry into human immune cells. The virus contains high amounts of phosphatidylserine (PtdSer) and may bind PtdSer receptors, such as TIMs. This mechanism is supported by our findings that exosomes from multiple sources increased HIV-1 entry into T cells and macrophages, and viral entry was potently blocked with anti-TIM-4 antibodies. Keywords: exosomes, HIV-1, T cell immunoglobulin and mucin proteins, phosphatidylserine, nanoparticle tracking analysis

Brian Sims,1,2,* Linlin Gu,3,* Alexandre Krendelchtchikov,3 Qiana L Matthews3,4 1Division of Neonatology, Department of Pediatrics, 2Department of Cell, Developmental, and Integrative Biology, 3Division of Infectious Diseases, Department of Medicine, 4Center for AIDS Research, University of Alabama at Birmingham, Birmingham, AL, USA *These authors contributed equally to this work Background: Viruses enter host cells through interactions of viral ligands with cellular receptors. Viruses can also enter cells in a receptor-independent fashion. Mechanisms regarding the receptor-independent viral entry into cells have not been fully elucidated. Exosomal trafficking between cells may offer a mechanism by which viruses can enter cells.Methods: To investigate the role of exosomes on cellular viral entry, we employed neural stem cell-derived exosomes and adenovirus type 5 (Ad5) for the proof-of-principle study. Results: Exosomes significantly enhanced Ad5 entry in Coxsackie virus and adenovirus receptor (CAR)-deficient cells, in which Ad5 only had very limited entry. The exosomes were shown to contain T-cell immunoglobulin mucin protein 4 (TIM-4), which binds phosphatidylserine. Treatment with anti-TIM-4 antibody significantly blocked the exosome-mediated Ad5 entry.Conclusion: Neural stem cell-derived exosomes mediated significant cellular entry of Ad5 in a receptor-independent fashion. This mediation may be hampered by an antibody specifically targeting TIM-4 on exosomes. This set of results will benefit further elucidation of virus/exosome pathways, which would contribute to reducing natural viral infection by developing therapeutic agents or vaccines. Keywords: neural stem cell-derived exosomes, adenovirus type 5, TIM-4, viral entry, phospholipids

Abstract Adenoviral vectors have been utilized for a variety of gene therapy applications. Our group has incorporated bioluminescent, fluorographic reporters, and/or suicide genes within the adenovirus genome for analytical and/or therapeutic purposes. These molecules have also been incorporated as capsid components. Recognizing that incorporations at either locale yield potential advantages and disadvantages, our report evaluates the benefits of transgene incorporation versus capsid incorporation. To this end, we have genetically incorporated firefly luciferase within the early region 3 or at minor capsid protein IX and compared vector functionality by means of reporter readout.

Abstract Despite the many potential advantages of Ad vectors for vaccine application, the full utility of current Ad vaccines may be limited by the host anti-vector immune response. Direct incorporation of antigens into the adenovirus capsid offers a new and exciting approach for vaccination strategies; this strategy exploits the inherent antigenicity of the Ad vector. Critical to exploiting Ad in this new context is the placement of antigenic epitopes within the major Ad capsid protein, hexon. In our current study we illustrate that we have the capability to place a range of antigenic epitopes within Ad5 capsid protein hexon hypervariable regions (HVRs) 2 or 5, thus producing viable Ad virions. Our data define the maximal incorporation size at HVR2 or HVR5 as it relates to identical antigenic epitopes. In addition, this data suggests that Ad5 HVR5 is more permissive to a range of insertions. Most importantly, repeated administration of our hexon-modified viruses resulted in a secondary anti-antigen response, whereas minimal secondary effect was present after administration of Ad5 control. Our study describes antigen placement and optimization within the context of the capsid incorporation approach of Ad vaccine employment, thereby broadening this new methodology.

While estimation of prognosis for patients with oligometastatic cancer is important to aid in treatment decision making, at present, prognostication tools are lacking. The purpose of this study was to develop predictive models for polymetastatic progression free survival (PPFS) and overall survival (OS), based on patient outcomes on the SABR-5 clinical trial. In the SABR-5 trial (a multi-center, single arm, phase II clinical trial), 381 patients with 1-5 oligometastases or oligoprogressing lesions were treated with stereotactic ablative radiotherapy (SABR). Prostate cancer was the most common histology (32%), followed by colorectal (16%), breast (11%), lung (9%), and renal (9%). Most patients (91%) were treated for 1-2 metastases. PPFS was defined as time from SABR until polymetastatic failure (≥ 6 metastases, or malignant pleural effusion/malignant ascites) or death. We trained separate Cox models for PPFS and OS. An elastic net penalty was used to select from candidate covariates. The model was internally validated using 10-fold cross validation with three repetitions and evaluated using partial likelihood. The proportional hazards and linearity assumptions were tested by examination of scaled Schoenfold Residuals and Martingale Residuals, respectively. After a median follow-up time of 28.0 months (interquartile range [IQR]19.1 – 39.1), median PPFS was 34.3 months (95% confidence interval [CI] 28.8 – 39.9), and median OS was 50.5 months (95% CI 45.2 – 58.9). The resulting PPFS model contained four covariates: primary tumor type (prostate, colorectal, lung, breast, renal, other), ECOG (1-2 vs 0), oligoprogression (yes vs no), number of organs with metastases (single vs multiple). The cross-validated C-Index was 0.66 [95% CI 0.64 – 0.68]. The OS model had five covariates: age (continuous), primary tumor type (prostate, colorectal, lung, breast, renal, other), ECOG (1-2 vs 0), number of organs with metastases (single vs multiple), presence of brain metastasis (yes vs no) and had a corresponding C-Index of 0.67 [95% CI 0.64 – 0.700]. These nomograms can be a useful guiding tool for clinicians in predicting PPFS and OS in patients treated with SABR in oligometastatic or oligoprogressive setting. These models should be externally validated. [ABSTRACT FROM AUTHOR]

While SABR is known for its overall low toxicity and safety, there remains a research gap regarding its combined use with specific systemic therapies. This study aims to evaluate the toxicity of SABR in combination with various systemic therapies. The hypothesis is that certain systemic therapies would significantly increase the risk of Grade 2+ and Grade 3+ radiation therapy-related toxicities when used concurrently with Stereotactic Ablative Radiotherapy (SABR). A secondary analysis of the SABR-5 trial compared grade 2+ and 3+ toxicities associated with SABR until the last follow-up in patients receiving high-risk or non-high-risk systemic therapy at intervals of 3 months, 2 weeks, 1 week, and concurrently with SABR. High-risk systemic therapy was a priori defined, based on previous literature, as drugs that, when given close to SABR, may increase treatment toxicity. This category encompasses cytotoxic chemotherapy drugs, multi-targeted tyrosine kinase inhibitors, cyclin-dependent kinase 4/6 inhibitors, epidermal growth factor receptor inhibitors, anti-vascular endothelial growth factor agents, and anti-cytotoxic T-lymphocyte-associated protein 4 agents. Among the 381 patients, the actuarial rates of grade 2+ and 3+ toxic effects were as follows: for patients not on systemic therapy 3 months prior to SABR (n = 202), the rates were 17.3% and 3.5%, respectively; for patients on non-high-risk systemic therapy concurrent with SABR (n = 102), the rates were 18.6% and 3.9%, respectively; and for patients on high-risk systemic therapy concurrent with SABR (n = 5), the rates were notably higher at 60% and 40%, respectively. On multivariable analysis, concurrent use of high-risk systemic therapy was associated with a higher risk of grade 2+ (OR = 7.15, P = 0.043) or 3+ toxic effects (OR = 13.9, P = 0.015). Significance was not observed when high-risk drugs were used only within 1 week, 2 weeks, or 3 months of SABR, nor with the use of any non-high-risk drugs. A second adverse factor included increased tumor diameter (per 1 cm increment; G2+ OR = 1.25, P < 0.001; G3+ OR = 1.27, P = 0.015). High-risk drugs have demonstrated a potential of increased SABR-related toxicity, warranting caution in their concurrent use with SABR. In contrast, the combination of non-high-risk drugs with SABR may be safe. Ongoing efforts are essential to identify potential risks and uncertainties associated with this therapeutic combination. [ABSTRACT FROM AUTHOR]

Objectives: We aimed to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) platform for the rapid detection of chikungunya virus (CHIKV) in both patient and mosquito samples from Brazil., Methods: We optimized an RT-LAMP assay and then evaluated the specificity and sensitivity using visual detection. In comparison with the RT-qPCR reference method, we validated the utility of this assay as a molecular diagnostic test in a reference laboratory for arbovirus diagnostics using 100 serum samples collected from suspected CHIKV cases., Results: Our RT-LAMP assay specifically detected CHIKV without cross-reactivity against other arboviruses. The limit of detection of our RT-LAMP was estimated in -1.18 PFU (confidence interval [CI] ranging from -2.08 to 0.45), resulting in a similar analytical sensitivity when directly compared with the reference standard RT-qPCR assay. Then, we demonstrate the ability of our RT-LAMP assay to detect the virus in different human specimens (serum, urine, and saliva), and crude lysate of Aedes aegypti mosquitoes in as little as 20-30 minutes and without a separate RNA isolation step. Lastly, we showed that our RT-LAMP assay could be lyophilized and reactivated by adding water, indicating potential for room-temperature storage. Our RT-LAMP had a clinical sensitivity of 100% (95% CI, 90.97-100.00%), clinical specificity of 96.72% (95% CI, 88.65-99.60%), and overall accuracy of 98.00% (95% CI, 92.96-99.76%)., Discussion: Taken together, these findings indicate that the RT-LAMP assay reported here solves important practical drawbacks to the deployment of molecular diagnostics in the field and can be used to improve testing capacity, particularly in low- and middle-income countries., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: The optimal sequencing of local and systemic therapy for oligometastatic cancer has not been established. This study retrospectively compared progression-free survival (PFS), overall survival (OS), and SABR-related toxicity between upfront versus delay of systemic treatment until progression in patients in the SABR-5 trial., Methods and Materials: The single-arm phase 2 SABR-5 trial accrued patients with up to 5 oligometastases across SABR-5 between November 2016 and July 2020. Patients received SABR to all lesions. Two cohorts were retrospectively identified: those receiving upfront systemic treatment along with SABR and those for whom systemic treatment was delayed until disease progression. Patients treated for oligoprogression were excluded. Propensity score analysis with overlap weighting balanced baseline characteristics of cohorts. Bootstrap sampling and Cox regression models estimated the association of delayed systemic treatment with PFS, OS, and grade ≥2 toxicity., Results: A total of 319 patients with oligometastases underwent treatment on SABR-5, including 121 (38%) and 198 (62%) who received upfront and delayed systemic treatment, respectively. In the weighted sample, prostate cancer was the most common primary tumor histology (48%) followed by colorectal (18%), breast (13%), and lung (4%). Most patients (93%) were treated for 1 to 2 metastases. The median follow-up time was 34 months (IQR, 24-45). Delayed systemic treatment was associated with shorter PFS (hazard ratio [HR], 1.56; 95% CI, 1.15-2.13; P = .005) but similar OS (HR, 0.90; 95% CI, 0.51-1.59; P = .65) compared with upfront systemic treatment. Risk of grade 2 or higher SABR-related toxicity was reduced with delayed systemic treatment (odds ratio, 0.35; 95% CI, 0.15-0.70; P < .001)., Conclusions: Delayed systemic treatment is associated with shorter PFS without reduction in OS and with reduced SABR-related toxicity and may be a favorable option for select patients seeking to avoid initial systemic treatment. Efforts should continue to accrue patients to histology-specific trials examining a delayed systemic treatment approach., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Background: Radiotherapy delivery regimens can vary between a single fraction (SF) and multiple fractions (MF) given daily for up to several weeks depending on the location of the cancer or metastases. With limited evidence comparing fractionation regimens for oligometastases, there is support to explore toxicity levels to nearby organs at risk as a primary outcome while using SF and MF stereotactic ablative radiotherapy (SABR) as well as explore differences in patient-reported quality of life and experience., Methods: This study will randomize 598 patients in a 1:1 ratio between the standard arm (MF SABR) and the experimental arm (SF SABR). This trial is designed as two randomized controlled trials within one patient population for resource efficiency. The primary objective of the first randomization is to determine if SF SABR is non-inferior to MF SABR, with respect to healthcare provider (HCP)-reported grade 3-5 adverse events (AEs) that are related to SABR. Primary endpoint is toxicity while secondary endpoints include lesional control rate (LCR), and progression-free survival (PFS). The second randomization (BC Cancer sites only) will allocate participants to either complete quality of life (QoL) questionnaires only; or QoL questionnaires and a symptom-specific survey with symptom-guided HCP intervention. The primary objective of the second randomization is to determine if radiation-related symptom questionnaire-guided HCP intervention results in improved reported QoL as measured by the EuroQoL-5-dimensions-5levels (EQ-5D-5L) instrument. The primary endpoint is patient-reported QoL and secondary endpoints include: persistence/resolution of symptom reporting, QoL, intervention cost effectiveness, resource utilization, and overall survival., Discussion: This study will compare SF and MF SABR in the treatment of oligometastases and oligoprogression to determine if there is non-inferior toxicity for SF SABR in selected participants with 1-5 oligometastatic lesions. This study will also compare patient-reported QoL between participants who receive radiation-related symptom-guided HCP intervention and those who complete questionnaires alone., Trial Registration: Clinicaltrials.gov identifier: NCT05784428. Date of Registration: 23 March 2023., (© 2024. The Author(s).)

The aim of this retrospective, secondary analysis study was to quantify the dosimetric impact of the lack of interobserver agreement on gross tumor volume (GTV) delineation for canine meningioma. This study used a previously reported population of 13 dogs with GTVs contoured on CT alone and on registered CT-MR by 18 radiation oncologists. The "true" GTV was generated for each dog using a simultaneous truth and performance-level estimation algorithm, and "true" brain was defined as the whole brain minus true GTV. Treatment plans were generated for each dog and observer combination, using criteria applied to the observer's GTV and brain contours. Plans were then categorized as a pass (met all planning criteria for true GTV and true brain) or fail. A mixed-effects linear regression was performed to examine differences in metrics between CT and CT-MR plans and mixed-effects logistic regression was performed to examine differences in percentages of pass/fail between CT and CT-MRI plans. The mean percent coverage of true GTV by prescribed dose was higher for CT-MR plans than for CT plans (mean difference 5.9%; 95% CI, 3.7-8.0; P < 0.001). There was no difference in the mean volume of true brain receiving ≥24 Gy and in maximum true brain dose between CT plans and CT-MR plans (P ≥ 0.198). CT-MR plans were significantly more likely to pass the criteria for true GTV and true brain than CT plans (OR 1.75; 95% CI, 1.02-3.01; P = 0.044). This study demonstrated significant dosimetric impact when GTV contouring was performed on CT alone compared with CT-MR., (© 2023 American College of Veterinary Radiology.)

Background and Purpose: Stereotactic ablative radiotherapy (SABR) for oligometastases may improve survival, however concerns about safety remain. To mitigate risk of toxicity, target coverage was sacrificed to prioritize organs-at-risk (OARs) during SABR planning in the population-based SABR-5 trial. This study evaluated the effect of this practice on dosimetry, local recurrence (LR), and progression-free survival (PFS)., Methods: This single-arm phase II trial included patients with up to 5 oligometastases between November 2016 and July 2020. Theprotocol-specified planning objective was to cover 95 % of the planning target volume (PTV) with 100 % of the prescribed dose, however PTV coverage was reduced as needed to meet OAR constraints. This trade-off was measured using the coverage compromise index (CCI), computed as minimum dose received by the hottest 99 % of the PTV (D99) divided by the prescription dose. Under-coverage was defined as CCI < 0.90. The potential association between CCI and outcomes was evaluated., Results: 549 lesions from 381 patients were assessed. Mean CCI was 0.88 (95 % confidence interval [CI], 0.86-0.89), and 196 (36 %) lesions were under-covered. The highest mean CCI (0.95; 95 %CI, 0.93-0.97) was in non-spine bone lesions (n = 116), while the lowest mean CCI (0.71; 95 % CI, 0.69-0.73) was in spine lesions (n = 104). On multivariable analysis, under-coverage did not predict for worse LR (HR 0.48, p = 0.37) or PFS (HR 1.24, p = 0.38). Largest lesion diameter, colorectal and 'other' (non-prostate, breast, or lung) primary predicted for worse LR. Largest lesion diameter, synchronous tumor treatment, short disease free interval, state of oligoprogression, initiation or change in systemic treatment, and a high PTV Dmax were significantly associated with PFS., Conclusion: PTV under-coverage was not associated with worse LR or PFS in this large, population-based phase II trial. Combined with low toxicity rates, this study supports the practice of prioritizing OAR constraints during oligometastatic SABR planning., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Purpose: A subset of patients with oligometastatic cancer experience early widespread cancer dissemination and do not benefit from metastasis-directed therapy such as SABR. This study aimed to identify factors associated with early polymetastatic relapse (PMR)., Methods and Materials: The SABR-5 trial was a single arm phase 2 study conducted at all 6 regional cancer centers across British Columbia (BC), Canada. SABR for oligometastases was only offered on trial. Patients with up to 5 oligometastatic lesions (total, progressing, or induced) received SABR to all lesions. Patients were 18 years of age or older, Eastern Cooperative Oncology Group 0 to 2 and life expectancy ≥6 months. This secondary analysis evaluated factors associated with early PMR, defined as disease recurrence within 6 months of SABR, which is not amenable to further local treatment. Univariable and multivariable analyses were performed using binary logistic regression. The Kaplan-Meier method and log-rank tests assessed PMR-free survival and differences between risk groups, respectively., Results: Between November 2016 and July 2020, 381 patients underwent treatment on SABR-5. A total of 16% of patients experienced PMR. Worse performance status (Eastern Cooperative Oncology Group 1-2 vs 0; hazard ratio [HR] = 2.01, P = .018), nonprostate/breast histology (HR = 3.64, P <.001), and oligoprogression (HR = 3.84, P <.001) were independent predictors for early PMR. Risk groups were identified with median PMR-free survival ranging from 5 months to not yet reached at the time of analysis. Rates of 3-year overall survival were 0%, 53% (95% confidence interval [CI], 48-58), 77% (95% CI, 73-81), and 93% (95% CI, 90-96) in groups 1 to 4, respectively (P <.001)., Conclusions: Four distinct risk groups for early PMR are identified, which differ significantly in PMR-free survival and overall survival. The group with all 3 risk factors had a median PMR-free survival of 5 months and may not benefit from local ablative therapy alone. This model should be externally validated with data from other prospective trials., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Purpose: Despite increasing utilization of SABR for oligometastatic cancer, prospective outcomes are lacking. The purpose of this study was to determine progression-free survival (PFS), local control (LC), and prognostic factors from the population-based phase 2 SABR-5 trial., Methods and Materials: The SABR-5 trial was a single-arm phase 2 study with the primary endpoint of toxicity, conducted at the 6 regional cancer centers across British Columbia (BC), Canada, during which time SABR for oligometastases was only offered on trial. Patients with up to 5 oligometastases (total or not controlled by prior treatment and including induced oligometastatic disease) underwent SABR to all lesions. Patients were 18 years of age or older, had an Eastern Cooperative Oncology Group score of 0 to 2, and had life expectancy ≥ 6 months. The secondary outcomes of PFS and LC are presented here., Results: Between November 2016 and July 2020, 381 patients underwent SABR on trial. Median follow-up was 27 months (interquartile range, 18-36). Median PFS was 15 months (95% confidence interval [CI], 12-18). LC at 1 and 3 years were 93% (95% CI, 91-95) and 87% (95% CI, 84-90), respectively. On multivariable analysis, increasing tumor diameter (hazard ratio [HR], 1.09; P < .001), declining performance status (HR, 2.13; P < .001), disease-free interval <18 months (HR, 1.52; P = .003), 4 or more metastases at SABR (HR, 1.48; P = .048), initiation or change in systemic treatment (HR, 0.50; P < .001), and oligoprogression (HR, 1.56; P = .008) were significant independent predictors of PFS. Tumor diameter (sub-hazard ratio [SHR], 1.28; P < .001), colorectal histology (SHR, 4.33; P = .002), and "other" histology (SHR, 3.90; P < .001) were associated with worse LC., Conclusions: In this population-based cohort including patients with genuine oligometastatic, oligoprogressive, and induced oligometastatic disease, the median PFS was 15 months and LC at 3 years was 87%. This supports ongoing efforts to randomize patients in phase 3 trials, even outside the original 1 to 5 metachronous oligometastatic paradigm., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Importance: After the publication of the landmark SABR-COMET trial, concerns arose regarding high-grade toxic effects of treatment with stereotactic ablative body radiotherapy (SABR) for oligometastases., Objective: To document toxic effects of treatment with SABR in a large cohort from a population-based, provincial cancer program., Design, Setting, and Participants: From November 2016 to July 2020, 381 patients across all 6 cancer centers in British Columbia were treated in this single-arm, phase 2 trial of treatment with SABR for patients with oligometastatic or oligoprogressive disease. During this period, patients were only eligible to receive treatment with SABR in these settings in trials within British Columbia; therefore, this analysis is population based, with resultant minimal selection bias compared with previously published SABR series., Interventions: Stereotactic ablative body radiotherapy to up to 5 metastases., Main Outcomes and Measures: Rate of grade 2, 3, 4, and 5 toxic effects associated with SABR., Findings: Among 381 participants (122 women [32%]), the mean (SD; range) age was 68 (11.1; 30-97) years, and the median (range) follow-up was 25 (1-54) months. The most common histological findings were prostate cancer (123 [32%]), colorectal cancer (63 [17%]), breast cancer (42 [11%]), and lung cancer (33 [9%]). The number of SABR-treated sites were 1 (263 [69%]), 2 (82 [22%]), and 3 or more (36 [10%]). The most common sites of SABR were lung (188 [34%]), nonspine bone (136 [25%]), spine (85 [16%]), lymph nodes (78 [14%]), liver (29 [5%]), and adrenal (15 [3%]). Rates of grade 2, 3, 4, and 5 toxic effects associated with SABR (based on the highest-grade toxic effect per patient) were 14.2%; (95% CI, 10.7%-17.7%), 4.2% (95% CI, 2.2%-6.2%), 0%, and 0.3% (95% CI, 0%-0.8%), respectively. The cumulative incidence of grade 2 or higher toxic effects associated with SABR at year 2 by Kaplan-Meier analysis was 8%, and for grade 3 or higher, 4%., Conclusions and Relevance: This single-arm, phase 2 clinical trial found that the incidence of grade 3 or higher SABR toxic effects in this population-based study was less than 5%. Furthermore, the rates of grade 2 or higher toxic effects (18.6%) were lower than previously published for SABR-COMET (29%). These results suggest that SABR treatment for oligometastases has acceptable rates of toxic effects and potentially support further enrollment in randomized phase 3 clinical trials., Trial Registration: ClinicalTrials.gov Identifier: NCT02933242.

Purpose: Standard planning target volume (PTV) margins for lung stereotactic ablative radiation therapy (SABR) are 5 mm. High-dose-rate volumetric modulated arc therapy delivered using flattening filter-free (FFF) beams with modern immobilization systems may allow for PTV margin reduction. This study assesses whether PTV margins can be reduced from 5 to 3 mm., Methods: Target intrafractional motions derived from pretreatment and posttreatment cone beam computed tomography (CBCT) scans for 33 patients receiving lung SABR treated with 10XFFF energy and 5-mm PTV margins from 2016 to 2019 were used to calculate the required PTV margin. Deformable registration of the planning CT scan and internal gross tumor volume (IGTV) contour to posttreatment CBCT scans for 36 consecutive patients with 4 fraction schedules was completed to capture volume changes and intrafractional movement. Plans were replanned with 3-mm margins and recalculated on each deformed CT scan to assess deformed IGTV (d-IGTV) coverage and organ-at-risk doses., Results: Margin analysis showed PTV margins may be reduced to 3 mm. The mean d-IGTV coverage (percentage of the d-IGTV receiving ≥100% of the prescription dose [V100%] and the minimum dose covering 99.9% of the d-IGTV volume [D99.9%]) over 4 fractions for each patient was >95% with both margins. With 5-mm PTV margins, all 144 fractions had a d-IGTV V100% of >95% and a D99.9% >95%. With 3-mm PTV margins, the d-IGTV V100% was >95% in 99.3% of fractions (143 of 144) and the D99.9% was >95% in 98.6% of fractions (142 of 144). With 3-mm PTV margins, significant reductions in body V50%, body V80%, the volume of the lung receiving ≥20 Gy, and the mean lung dose and chest wall dose to 0.035 cm 3 and 30 cm 3 were observed (all P < .001). Using theoretical models, the normal tissue complication probability for radiation pneumonitis decreased by a mean of 0.8% (range, 0.1%-2.7%), and the mean 2-year tumor control probability was 96.1% and 95.2% with 5-mm and 3-mm PTV margins, respectively., Conclusion: With modern treatment and immobilization techniques in lung SABR, 3-mm PTV margins maintain acceptable IGTV coverage, modestly reduce toxicity to organs at risk, and maintain a calculated 2-year local control rate of >95%., (© 2021 The Author(s).)

This work combines single cell Raman spectroscopy (RS) with group and basis restricted non-negative matrix factorisation (GBR-NMF) to identify individual biochemical changes associated with radiation exposure in three human cancer cell lines. The cell lines analysed were derived from lung (H460), breast (MCF7) and prostate (LNCaP) tissue and are known to display varying degrees of radio sensitivity due to the inherent properties of each cell type. The GBR-NMF approach involves the deconstruction of Raman spectra into component biochemical bases using a library of Raman spectra of known biochemicals present in the cells. Subsequently, scores are obtained on each of these bases which can be directly correlated with the contribution of each chemical to the overall Raman spectrum. We validated GBR-NMF through the correlation of GBR-NMF-derived glycogen scores with scores that were previously observed using principal component analysis (PCA). Phosphatidylcholine, glucose, arginine and asparagine showed a distinct differential score pattern between radio-resistant and radio-sensitive cell types. In summary, the GBR-NMF approach allows for the monitoring of individual biochemical radiation-response dynamics previously unattainable with more traditional PCA-based approaches.

The global spread of SARS-CoV-2 has shaken our health care and economic systems, prompting re-evaluation of long-held views on how best to deliver care. This is especially the case for our global diagnostic strategy. While current laboratory-based centralized RT-qPCR will continue to serve as a gold standard diagnostic into the foreseeable future, the shortcomings of our dependence on this method have been laid bare. It is now clear that a robust diagnostics pandemic response strategy, like any disaster planning, must include adaptive, diverse and de-centralized solutions. Here we look at how the COVID-19 pandemic, and previous outbreaks, have set the stage for a new innovative phase in diagnostics and a re-thinking of pandemic preparedness.

There is a lack of information regarding interobserver agreement on canine meningioma gross tumor volume (GTV) delineation, and on the impact of MRI on this agreement. The objectives of this retrospective, secondary analysis, observer agreement study were to describe agreement between veterinary radiation oncologists on GTV for canine intracranial meningioma, and to compare interobserver agreement between delineation based on CT alone and delineation based on fused CT-MRI. Eighteen radiation oncologists delineated GTV for 13 dogs with an imaging diagnosis of meningioma on pre- and postcontrast CT, pre- and postcontrast T1-weighted magnetic resonance, and T2-weighted magnetic resonance images. Dice similarity coefficient (DSC), concordance index (CI), and center of volume (COV) were used to quantify interobserver agreement. Multilevel mixed models were used to examine the difference in volume, DSC, CI and COV 3D distance between CT and CT-MR imaging. The mean volume for GTV contours delineated using fused CT-MRI was larger than when CT alone was used for delineation (mean difference CT-MR - CT = 0.89 cm 3 , 95% CI 0.66 to 1.12, P < .001). Interobserver agreement on GTV was improved when MRI was used; the mean DSC and CI were higher, and the mean COV 3D distance was lower, when fused CT-MRI was used than when CT alone was used (P < .001 for all differences). Based on our results, fused CT-MRI is recommended for radiation therapy planning of canine intracranial meningioma., (© 2020 American College of Veterinary Radiology.)

Background: Bone metastases in the lower spine and pelvis are effectively palliated with radiotherapy (RT), though this can come with side effects such as radiation induced nausea and vomiting (RINV). We hypothesize that high rates of RINV occur in part because of the widespread use of inexpensive simple unplanned palliative radiotherapy (SUPR), over more complex and resource intensive 3D conformal RT, such as volumetric modulated arc therapy (VMAT)., Methods: This is a randomized, multi-centre phase III trial of SUPR versus VMAT. We will accrue 250 patients to assess the difference in patient-reported RINV. This study is powered to detect a difference in quality of life between patients treated with VMAT vs. SUPR., Discussion: This trial will determine if VMAT reduces early toxicity compared to SUPR and may provide justification for this more resource-intensive and costly form of RT., Trial Registration: Clinicaltrials.gov identifier: NCT03694015 . Date of registration: October 3, 2018.

Background and Purpose: Planning complex radiotherapy treatments can be inefficient, with large variation in plan quality. In this study we evaluated plan quality and planning efficiency using real-time interactive planning (RTIP) for head and neck (HN) volumetric modulated arc therapy (VMAT)., Materials and Methods: RTIP allows manipulation of dose volume histograms (DVHs) in real-time to assess achievable planning target volume (PTV) coverage and organ at risk (OAR) sparing. For 20 HN patients previously treated with VMAT, RTIP was used to minimize OAR dose while maintaining PTV coverage. RTIP DVHs were used to guide VMAT optimization. Dosimetric differences between RTIP-assisted plans and original clinical plans were assessed. Five blinded radiation oncologists indicated their preference for each PTV, OAR and overall plan. To assess efficiency, ten patients were planned de novo by experienced and novice planners and a RTIP user., Results: The average planning time with RTIP was <20 min, and most plans required only one optimization. All 20 RTIP plans were preferred by a majority of oncologists due to improvements in OAR sparing. The average maximum dose to the spinal cord was reduced by 10.5 Gy (from 49.5 to 39.0 Gy), and the average mean doses for the oral cavity, laryngopharynx, contralateral parotid and submandibular glands were reduced by 3.5 Gy (39.1-35.7 Gy), 6.8 Gy (42.5-35.7 Gy), 1.7 Gy (17.0-15.3 Gy) and 3.3 Gy (22.9-19.5 Gy), respectively., Conclusions: Incorporating RTIP into clinical workflows may increase both planning efficiency and OAR sparing., (© 2019 The Authors.)

Background: Oligometastases refer to a state of disease where cancer has spread beyond the primary site, but is not yet widely metastatic, often defined as 1-3 or 1-5 metastases in number. Stereotactic ablative radiotherapy (SABR) is an emerging radiotherapy technique to treat oligometastases that require further prospective population-based toxicity estimates., Methods: This is a non-randomized phase II trial where all participants will receive experimental SABR treatment to all sites of newly diagnosed or progressing oligometastatic disease. We will accrue 200 patients to assess toxicity associated with this experimental treatment. The study was powered to give a 95% confidence on the risk of late grade 4 toxicity, anticipating a < 5% rate of grade 4 toxicity., Discussion: SABR treatment of oligometastases is occurring off-trial at a high rate, without sufficient evidence of its efficacy or toxicity. This trial will provide necessary toxicity data in a population-based cohort, using standardized doses and organ at risk constraints, while we await data on efficacy from randomized phase III trials., Trial Registration: Registered through clinicaltrials.gov NCT02933242 on October 14, 2016 prospectively before patient accrual.

Altered cellular metabolism is a hallmark of tumor cells and contributes to a host of properties associated with resistance to radiotherapy. Detection of radiation-induced biochemical changes can reveal unique metabolic pathways affecting radiosensitivity that may serve as attractive therapeutic targets. Using clinically relevant doses of radiation, we performed label-free single cell Raman spectroscopy on a series of human cancer cell lines and detected radiation-induced accumulation of intracellular glycogen. The increase in glycogen post-irradiation was highest in lung (H460) and breast (MCF7) tumor cells compared to prostate (LNCaP) tumor cells. In response to radiation, the appearance of this glycogen signature correlated with radiation resistance. Moreover, the buildup of glycogen was linked to the phosphorylation of GSK-3β, a canonical modulator of cell survival following radiation exposure and a key regulator of glycogen metabolism. When MCF7 cells were irradiated in the presence of the anti-diabetic drug metformin, there was a significant decrease in the amount of radiation-induced glycogen. The suppression of glycogen by metformin following radiation was associated with increased radiosensitivity. In contrast to MCF7 cells, metformin had minimal effects on both the level of glycogen in H460 cells following radiation and radiosensitivity. Our data demonstrate a novel approach of spectral monitoring by Raman spectroscopy to assess changes in the levels of intracellular glycogen as a potential marker and resistance mechanism to radiation therapy.

The drive toward personalized radiation therapy (RT) has created significant interest in determining patient-specific tumor and normal tissue responses to radiation. Raman spectroscopy (RS) is a non-invasive and label-free technique that can detect radiation response through assessment of radiation-induced biochemical changes in tumor cells. In the current study, single-cell RS identified specific radiation-induced responses in four human epithelial tumor cell lines: lung (H460), breast (MCF-7, MDA-MB-231), and prostate (LNCaP), following exposure to clinical doses of radiation (2-10 Gy). At low radiation doses (2 Gy), H460 and MCF-7 cell lines showed an increase in glycogen-related spectral features, and the LNCaP cell line showed a membrane phospholipid-related radiation response. In these cell lines, only spectral information from populations receiving 10 Gy or less was required to identify radiation-related features using principal component analysis (PCA). In contrast, the MDA-MB-231 cell line showed a significant increase in protein relative to nucleic acid and lipid spectral features at doses of 6 Gy or higher, and high-dose information (30, 50 Gy) was required for PCA to identify this biological response. The biochemical nature of the radiation-related changes occurring in cells exposed to clinical doses was found to segregate by status of p53 and radiation sensitivity. Furthermore, the utility of RS to identify a biological response in human tumor cells exposed to therapeutic doses of radiation was found to be governed by the extent of the biochemical changes induced by a radiation response and is therefore cell line specific. The results of this study demonstrate the utility and effectiveness of single-cell RS to identify and measure biological responses in tumor cells exposed to standard radiotherapy doses.

This work applies noninvasive single-cell Raman spectroscopy (RS) and principal component analysis (PCA) to analyze and correlate radiation-induced biochemical changes in a panel of human tumour cell lines that vary by tissue of origin, p53 status and intrinsic radiosensitivity. Six human tumour cell lines, derived from prostate (DU145, PC3 and LNCaP), breast (MDA-MB-231 and MCF7) and lung (H460), were irradiated in vitro with single fractions (15, 30 or 50 Gy) of 6 MV photons. Remaining live cells were harvested for RS analysis at 0, 24, 48 and 72 h post-irradiation, along with unirradiated controls. Single-cell Raman spectra were acquired from 20 cells per sample utilizing a 785 nm excitation laser. All spectra (200 per cell line) were individually post-processed using established methods and the total data set for each cell line was analyzed with PCA using standard algorithms. One radiation-induced PCA component was detected for each cell line by identification of statistically significant changes in the PCA score distributions for irradiated samples, as compared to unirradiated samples, in the first 24-72 h post-irradiation. These RS response signatures arise from radiation-induced changes in cellular concentrations of aromatic amino acids, conformational protein structures and certain nucleic acid and lipid functional groups. Correlation analysis between the radiation-induced PCA components separates the cell lines into three distinct RS response categories: R1 (H460 and MCF7), R2 (MDA-MB-231 and PC3) and R3 (DU145 and LNCaP). These RS categories partially segregate according to radiosensitivity, as the R1 and R2 cell lines are radioresistant (SF(2) > 0.6) and the R3 cell lines are radiosensitive (SF(2) < 0.5). The R1 and R2 cell lines further segregate according to p53 gene status, corroborated by cell cycle analysis post-irradiation. Potential radiation-induced biochemical response mechanisms underlying our RS observations are proposed, such as (1) the regulated synthesis and degradation of structured proteins and (2) the expression of anti-apoptosis factors or other survival signals. This study demonstrates the utility of RS for noninvasive radiobiological analysis of tumour cell radiation response, and indicates the potential for future RS studies designed to investigate, monitor or predict radiation response.

This work investigates the capability of Raman spectroscopy (RS) to study the effects of ionizing radiation on single human tumour cells. Prostate tumour cells (cell line DU145) are cultured in vitro and irradiated to doses between 15 and 50 Gy with single fractions of 6 MV photons. Single-cell Raman spectra are acquired from irradiated and unirradiated cultures up to 5 days post-irradiation. Principal component analysis is used to distinguish the uniquely radiation-induced spectral changes from inherent sources of spectral variability arising from cell cycle differences and other known factors. We observe uniquely radiation-induced spectral changes which are correlated with both the irradiated dose and the incubation time post-irradiation. The spectral changes induced by radiation arise from biochemical differences in lipids, nucleic acids, amino acids and conformational protein structures between irradiated and unirradiated cells. To our knowledge, this study is the first use of RS to observe radiation-induced biochemical differences in single cells, and is the first use of vibrational spectroscopy to observe uniquely radiation-induced biochemical differences in single cells independent of concurrent cell-cycle- or cell-death-related processes.