Browsing by Author "Oldham, Mark"

Purpose: The purpose of this work is to characterize three novel formulations of a radiochromic material Presage and identify optimal imaging procedures for accurate 3D dosimetry. The dosimetric qualities of interest were studied for each formulation of Presage dosimeter in the context of accurate 3D dosimetry. The formulation of Presage showing the most promise is compared to a clinical 3D quality assurance device to investigate the accuracy of a complex state-of-the-art brain IMRT treatment.

Methods and Materials: Three novel formulations of Presage were studied for their temporal stability, sensitivity, linearity of dose response, and feasibility of absolute dose calibration in large volume dosimeters (1 kg) with small volume cuvettes (4g). Large cylindrical dosimeters with 11 cm diameter and 10 cm height were irradiated with 5 2x2 cm fields on the upper flat surface with 3 distinct dose levels (3, 6 and 9.5 Gy, representing low, medium and high). This irradiation pattern is used to determine the dosimetric characteristics mentioned above and was chosen because of its repeatability and it lends to simple measurements of linearity and sensitivity. Measurements were taken at various time points from 0 hours to 24 hours post-irradiation using the high resolution (6.45 m pixels) Duke Medium-Sized Optical-CT Scanner (DMOS) and reconstructed with a Matlab-based reconstruction GUI created in-house. Analysis of the pertinent dosimetric characteristics was performed in the GUI. A comprehensive end-to-end QA test was performed on the optimal formulation using optimal scan timing determined from the formulation studies described above. A 5-field IMRT plan was created for head treatment. The plan was delivered both to a head phantom containing a Presage insert, and to the Delta4 QA device. Comparison of both delivered distributions together with the Eclipse predicted dose distribution enabled investigation of the accuracy of the delivery, and the consistency of independent measurement devices.

Results: The DEA-1 formulation showed up to 10% variation from 0-2 hours post-irradiation, but showed excellent temporal stability (<2% variation) between 3-7 hours post irradiation, and maintained good stability until 24 hours post-irradiation (up to 3% variation). The DEA-2 also showed up to 10% variation from 0-2 hours post-irradiation. The DEA-2 formulation then showed good stability (up to 2.1% variation) from 3-7 hours, but optical density values dropped by up to 11% after 24 hours. The DX formulation did not maintain stability of optical density for any significant time with values decreasing by ~20% by the 24-hour time point and optical density decreasing at different rates for different dose levels. Linearity of dose response was good for all formulations with an R2 value > 0.99. Gamma analysis with criteria of 3%/2mm was performed on two irradiations of the 5-field pattern on DEA-1 formulation. Voxel passing rates were 96.68% and 97.96%. Comparison of the DEA-1 formulation large dosimeter was done with small volume cuvettes of the same formulation and batch. Sensitivity of the large dosimeter was less than half the sensitivity of the cuvettes. For clinical 3D QA comparison, the DEA-1 formulation was used because it had optimal performance showed the most promise for accurate 3D dosimetry. Line dose profiles showed that Presage compared very well with the Eclipse calculation and had a much better 3D gamma passing rate for 3%/3mm criteria than the Delta4 (>99% vs 75%).

Conclusions: The DEA-1 formulation shows the most promise because of its temporal stability and linearity of dose response. The optimal imaging window for this formulation was determined to be 3-24 hours post-irradiation. The DEA-2 and DX formulation also showed potential for accurate dosimetry. The optimal imaging window for the DEA-2 formulation was determined to be 2-6 hours post-irradiation. The optimal scan time for the DX formulation was determined to be immediately post-irradiation. The amount of accuracy loss depending on the scan time is dependent on the formulation and when the dosimeter is scanned. Line dose profiles and gamma analysis results from the comparison of Presage and Eclipse calculation provide strong validation of the accuracy of the IMRT treatment delivery. Comparison of Presage to the Delta4 show the Delta4 to be somewhat lacking in its ability to calculate 3D dose in the phantom/Presage geometry.

Introduction: RECA (Radiotherapy Enhanced with Cherenkov photo- Activation) is a novel treatment that induces a synergistic therapeutic effect by combining conventional radiation therapy with phototherapy using the anti-cancer and potentially immunogenic drug, psoralen. This work presents a novel method to investigate the therapeutic effect of RECA using rat brain slices and the agarose- based tissue equivalent material. Methods: 4T1 mCherry Firefly Luciferase mouse breast cancer cells are placed on the brain slice after exposed to psoralen solution. Taking fluorescent imaging of the brain slices every day after irradiation, an independent luciferase imaging was taken after the fifth fluorescence imaging. Using different imaging processing and analysis method to identify the cells. Result: Four analyzing method give different result about the fluorescence signal or luminescence signal. The overall trend of the fluorescence signal is rising over day, reaches the lowest point at 48 hours after irradiation. Control group (no radiation and no Cherenkov lights) has the lowest signal compared with other groups. The signal of brain slices with 4T1 cells exposed to psoralen solution is lower than that of brain slices without psoralen exposition. Conclusion: This work shows that rat brain slice can be used to simulate in vivo environment in exploring the therapeutic effect of RECA. Future work should focus on improving the image analyze method to better identify cells and noises.

Purpose: Radiosurgery has become a widely used procedure in the treatment of both solid tumors and secondary metastases in the brain. In cases with multiple brain lesions, isocenters are typically set up for each target, a process which can take hours and become very uncomfortable for the patient. Recently, multifocal treatments with a single isocenter have emerged as a solution. With the high doses delivered to small regions during radiosurgery, the importance of treatment verification is paramount, especially when delivering high doses to regions off isocenter.

Methods: A 5-arc RapidArc radiosurgery plan with a single isocenter and 5 targets was used to treat a dosimeter placed within a RPC-type head and neck phantom. The treatment was delivered five times at varying prescription doses, depending on the sensitivity of the PRESAGE dosimeter used. The delivered dose distribution was measured using an in-house optical-CT system and compared to the Eclipse-planned dose distribution using dose volume histograms and Gamma analysis.

Results: Reasonable dose agreement was measured between the majority of the dosimeters and the Eclipse plan (80-85% pass rate at 5%/3 mm Gamma critera). The failing voxels were located on the periphery of the dosimeter at regions of extremely high or low dose, suggesting a dose dependent stability of the PRESAGE formulation. The formulation with the best temporal stability had a much higher Gamma pass rate of 98% at 3%/2mm criteria.

Conclusions: The potential of accurate delivery of the complex radiosurgery plan was demonstrated with one of the three formulations of PRESAGE. While agreement was worse in the other formulations, the problem seemed to be an easily-fixable stability issue, resulting in improper scaling of doses. Replication of the most stable formulation would provide an excellent tool for verification of radiosurgery treatment delivery and other complex procedures.

As complex radiotherapy techniques become more readily-practiced, comprehensive 3D dosimetry is a growing necessity for advanced quality assurance. However, clinical implementation has been impeded by a wide variety of factors, including the expense of dedicated optical dosimeter readout tools, high operational costs, and the overall difficulty of use. To address these issues, a novel dry-tank optical CT scanner was designed for PRESAGE 3D dosimeter readout, relying on 3D printed components and omitting costly parts from preceding optical scanners. This work details the design, prototyping, and basic commissioning of the Duke Integrated-lens Optical Scanner (DIOS).

The convex scanning geometry was designed in ScanSim, an in-house Monte Carlo optical ray-tracing simulation. ScanSim parameters were used to build a 3D rendering of a convex ‘solid tank’ for optical-CT, which is capable of collimating a point light source into telecentric geometry without significant quantities of refractive-index matched fluid. The model was 3D printed, processed, and converted into a negative mold via rubber casting to produce a transparent polyurethane scanning tank. The DIOS was assembled with the solid tank, a 3W red LED light source, a computer-controlled rotation stage, and a 12-bit CCD camera. Initial optical phantom studies show negligible spatial inaccuracies in 2D projection images and 3D tomographic reconstructions. A PRESAGE 3D dose measurement for a 4-field box treatment plan from Eclipse shows 95% of voxels passing gamma analysis at 3%/3mm criteria. Gamma analysis between tomographic images of the same dosimeter in the DIOS and DLOS systems show 93.1% agreement at 5%/1mm criteria. From this initial study, the DIOS has demonstrated promise as an economically-viable optical-CT scanner. However, further improvements will be necessary to fully develop this system into an accurate and reliable tool for advanced QA.

Pre-clinical animal studies are used as a conventional means of translational research, as a midpoint between in-vitro cell studies and clinical implementation. However, modern small animal radiotherapy platforms are primitive in comparison with conventional linear accelerators. This work also investigates a series of 3D printed tools to expand the treatment capabilities of the X-RAD 225Cx orthovoltage irradiator, and applies them to a feasibility study of hippocampal avoidance in rodent whole-brain radiotherapy.

As an alternative material to lead, a novel 3D-printable tungsten-composite ABS plastic, GMASS, was tested to create precisely-shaped blocks. Film studies show virtually all primary radiation at 225 kVp can be attenuated by GMASS blocks of 0.5cm thickness. A state-of-the-art software, BlockGen, was used to create custom hippocampus-shaped blocks from medical image data, for any possible axial treatment field arrangement. A custom 3D printed bite block was developed to immobilize and position a supine rat for optimal hippocampal conformity. An immobilized rat CT with digitally-inserted blocks was imported into the SmART-Plan Monte-Carlo simulation software to determine the optimal beam arrangement. Protocols with 4 and 7 equally-spaced fields were considered as viable treatment options, featuring improved hippocampal conformity and whole-brain coverage when compared to prior lateral-opposed protocols. Custom rodent-morphic PRESAGE dosimeters were developed to accurately reflect these treatment scenarios, and a 3D dosimetry study was performed to confirm the SmART-Plan simulations. Measured doses indicate significant hippocampal sparing and moderate whole-brain coverage.

Radiation therapy (RT) has been a standard-of-care treatment for many localized cancers for decades. Despite being an effective treatment modality for many clinical presentations, the efficacy of RT against cancer can be limited due to local recurrence, metastatic spread, and radiation resistance from tumor hypoxia. These limitations provide opportunity for innovative approaches to enhance the overall efficacy of RT. This thesis explores the potential novel approach to enhancing RT through the paradigm changing approach of adding a phototherapeutic component initiated simultaneously with RT. X-ray Psoralen Activated Cancer Therapy (X-PACT) is one such approach, where diagnostics-energy kilovoltage (kV) x-ray coupled with energy modulators (phosphors) converts kV photon to ultraviolet (UV) light, which in turn activates psoralen. Radiotherapy Enhanced with Cherenkov photo-Activation (RECA) is another approach, where therapeutic megavoltage (MV) x-ray generates UV light via Cherenkov phenomenon. Both approaches could increase local control in RT, increase treatment effectiveness in hypoxic tumors, and amplify anti-cancer systemic response. The overarching hypothesis that drives this dissertation is that X-PACT and RECA can activate psoralen to enhance cytotoxicity in-vitro and tumor growth control in-vivo compared to RT alone. In line with this hypothesis, this work explores the feasibility of both X-PACT and RECA via in-vitro and in-vivo verification as well as optimization of radiation techniques to maximize the therapeutic benefit of the approach.

X-PACT and RECA in-vitro / in-vivo studies indicate radiotherapy enhancement is plausible with psoralens activated by secondary UV light production from radiation, though further investigation is required to establish feasibility of RECA in-vivo. For X-PACT in-vitro, a substantial reduction in cell viability and increase in apoptosis was observed in various murine cancer cells (4T1, KP-B, and CT2A) when treated with a combination of 50µg/mL phosphor, 10µM psoralen (8-MOP), and 1Gy of 80kVp x-ray (viability < 20%), compared to any of these components alone (viability > 70%). This suggests a synergistic interaction between the components congruent with the X-PACT scheme, where x-ray induces phosphor UV emission, which in turn activates psoralen. The X-PACT in-vivo mice study showed improved survival with X-PACT versus saline control with flank 4T1 tumors (30.7 days for X-PACT vs. 21.6 days for saline) for survival criteria of 1000, 1500, and 2000mm3, respectively. For RECA, in-vitro results seem promising, where reductions in viability of 20% and 9.5% were observed for 4T1 and B16 murine cancer cell lines treated with RECA (radiation + trioxsalen, a potent psoralen derivative) versus radiation alone. A substantial increase in MHC I expression was observed for B16 cells treated with RECA versus those treated with radiation alone. A small RECA in-vivo pilot study using 8-MOP was inconclusive. Further in-vivo trials with a greater number mice per arm of are required to establish the RECA feasibility to enhance radiotherapy.

Feasibility of treatment optimization for both X-PACT and RECA were demonstrated with kV and MV beams respectively, by optimization of optical output per radiation dose delivered. It was found that in both X-PACT and RECA scheme, the energy of the photon radiation beam (i.e. tube voltage and LINAC energy settings) affected optical output the most. With kV beams for X-PACT, accurate beam delivery within the target volume to reduce normal tissue damage typically expected of kV beams was demonstrated with a 3D-printing-based preclinical irradiation scheme, which is expected to help X-PACT translation into the clinics. In addition, for X-PACT, novel MV-responding phosphors were characterized under MV radiation beam, suggesting the possibility of MV-radiation-mediated X-PACT. Immediate future studies should investigate the efficacy of the optimized X-PACT and RECA, as well as MV X-PACT in-vitro and in-vivo. Studies beyond these immediate ones should investigate X-PACT and RECA efficacy against hypoxic and metastatic tumor sites, where radiation can traditionally fail.

Purpose: GRID therapy (Spatially Fractionated RT) has the potential to amplify systemic anti-tumor immune effect. The optimal GRID design, radiation dosage and combination with immunotherapies are not well understood. In this work, we characterized two novel, high-resolution GRIDs of smaller width and spacing had been was previously employed at Duke University. By combining these GRIDs with anti-PD-1 immune checkpoint blockade, we investigated the efficacy of this combination therapy in a preclinical mouse model. This work has two main aims. First, to observe any anti-tumor response from GRID therapy that arises from sparing T cell lymphocytes in the valleys adjacent to high peak doses of radiation facilitating tumor antigen presentation. Second, to investigate the robustness and replicability of a previously published influential work (Markovsky et al., 2019) which demonstrated that hemi-irradiation can produce similar levels of tumor control as conventional radiation therapy [1]. This is one of the first studies we are aware of that combines mini-GRID treatment with immunotherapies. Methods: Prior to in vivo studies, two novel, high-resolution in-house mini-GRIDs were characterized using the Small Animal Radiation Research Platform (SARRP). To perform this characterization, the SARRP (225 kV,13 mAs) irradiated EBT3 film. Using the Epson 11000XL scanner, EBT3 films were scanned prior to and post irradiation. Median filters were applied to avoid artificially suppressing peak and valley dose distributions. A calibration curve was generated using irradiations of a known dosage to determine what dose was delivered to the high and low-dose regions of the GRIDs. From this, peak-to-valley dose ratios as well as output factors were be calculated. Then, two pilot studies were performed using SARRP to deliver RT to C57BL/6J mice with subcutaneous LLC1 (Lewis Lung Carcinoma) flank tumors. The first study tested the therapeutic efficacy of single dose radiation while the second investigated fractionated radiation utilizing the newer, high-resolution GRIDs. In the first study, mice were randomized to four groups: 15 Gy to an open 20 mm x 20 mm field (n=5), 15 Gy to a GRID with 1mm width and spacing (n=5), and 24 Gy to a GRID with 1mm width and spacing (n=5). For the second study, mice were randomized to four groups: an open 20 mm x 20 mm field (n=6), the same field irradiating only half the tumor (n=6) (following Markovsky et al., 2019), a GRID with 1 mm width and spacing (n=6) and a GRID with 254 µm width and spacing (n=7). For both in vivo studies, all mice in this study were treated with 200 μg of anti-PD-1 antibody prior to 15 Gy of RT (single AP field) on days 0, 3, and 6. Anti-PD-1 was then administered weekly until mice reached humane endpoint (>15 mm in any dimension or ulceration). Tumor growth was measured thrice weekly using digital calipers. Results: • Film Characterizations: The peak to valley dose ratios for the 254 µm and 152 µm GRIDS were 19.8 ± 0.7 and 9.37 ± 0.33 respectively. The output factors for these GRIDs were 0.62 ± 0.09 and 0.59 ± 0.03. • First in vivo Study: Tumor quadrupling times (days, ± SD) were: 8.94 ± 1.17 (open field, 15 Gy), 7.75 ± 0.91 (1mm GRID, 15 Gy) and 7.98 ± 1.08 (1mm GRID, 24 Gy). Mean survival times (days, ± SD) were: 16.00 ± 0.00 (open field, 15 Gy), 12.8 ± 1.09 (1mm GRID, 15 Gy and 24 Gy). None of these differences were statistically significant. The width of the valleys for the 254 µm GRID is 544 ± 33.94 µm and for the 152 µm GRID is 548 µm ± 31.57. Assuming a clinically that 100 cells with a diameter of 5µm represent a clinically relevant sample for irradiation, this is a sufficient area for irradiation. • Second in vivo Study: Tumor quadrupling times (days, ± SD) were: 12.8 ± 2.6 (open field), 8.4 ± 2.8 (hemi-irradiation), 9.7 ± 2.4 (1mm GRID), and 6.4 ± 4.4 (0.25 mm GRID). Mean survival times (days, ± SD) were: 14.2 ± 2.1 (open field), 12.2 ± 1.0 (hemi-irradiation), 11.3 ± 1.6 (1mm GRID), and 10.4 ± 2.2 (254 µm GRID). Compared to the open field, time to tumor quadrupling was lower in all groups, significantly so in the hemi-irradiated and 0.25 mm GRID groups (p<0.05). Both the hemi-irradiated and GRID groups showed significantly shorter mean survival times compared to conventional open-field treatment (p<0.05 for 1 mm GRID, p<0.01 for hemi-irradiation and 0.25 mm GRID). Conclusion: Two novel mini-GRIDs were successfully characterized using the SARRP for preclinical work, and sufficiently kept valley doses below 1.5 Gy for infiltrative T cell function [2] with peak doses greater than 15 Gy, thereby enabling tumor antigen presentation. However, neither single dose nor fractionated GRID therapy with anti-PD-1 improved tumor growth delay or survival in a preclinical LLC flank model. In contrast to published data with this model, hemi-irradiation worsened tumor control compared to conventional treatment. Our work, therefore, does support the conclusion drawn in the Markovsky paper that hem-irradiation provides comparable tumor control using hemi-irradiation to conventional treatment [1]. The development of new technologies such as FLASH radiotherapy may present new opportunities for future studies utilizing GRID therapy.

Purpose: To develop and implement novel methods of extreme spatially fractionated radiation therapy (including GRID therapy) and subsequent evaluation in pre-clinical mice trials investigating the potential of novel radiation treatments with potential for promoting anti-cancer immunogenic response.

Methods: Spatially fractionated GRIDs were designed and precision-milled from 3mm thick lead sheets compatible with mounting on a 225 kVp small animal irradiator (X-Rad). Three pencil-beam GRIDs created arrays of 1mm diameter beams, and three “bar” GRIDs created 1x20mm rectangular fields. GRIDs projected 20x20mm fields at isocenter and beamlets were spaced at 1, 1.25, and 1.5mm, respectively. Output factors, peak-to-valley ratios, and dose distributions were determined with Gafchromic film. The bar GRID with 1mm beamlet spacing (50:50 open:closed ratio) was selected for the pre-clinical trial. Soft-tissue sarcoma (p53/MCA) was transplanted into C57BL/6 mice’s flanks. Four treatment arms were compared: unirradiated control (n=18), conventional radiation therapy (n=16), GRID therapy (n=17), and hemi-irradiation (n=17) where one-half of the beam was blocked. All irradiated mice received a single fraction of 15 Gy to irradiated regions. To date, this is the first study to compare GRID treatment against conventional RT at the same dose.

Results: Very high peak-to-valley ratios were achieved (bar GRIDs: 11.9±0.9, 13.6±0.4, 13.8±0.5; pencil-beam GRIDs: 18.7±0.6, 26.3±1.5, 31.0±3.3). Pencil-beam GRIDs spared twice the number of intra-tumor immune cells as bar GRIDs but left more of the tumor untreated (2-3% vs 14-17% area receiving 95% prescription, respectively). Penumbra was halved when GRIDs were 50% closer to treatment isocenter. The GRID selected for mouse trials was capable of sparing approximately 15% of intra-tumor CD8+ and CD4+ T cells. Preliminary results indicate mean times to tumor quintupling were: 12, 13, 14, and 20 days for unirradiated, GRID, hemi-irradiated, and conventional treatment groups, respectively. To date, all tumors have quintupled except for nine in the AP control group.

Conclusions: Peak-to-valley ratios with kV grids were substantially superior to MV grids, which historically achieve ratios between 2.5 and 6.5. In data collected to date, GRID and hemi-irradiation did not significantly delay tumor growth as compared to an unirradiated control (P = 0.122 and P = 0.2437, respectively, P-values from logrank analysis). Differences between GRID and hemi-irradiation were not statistically significant (P = 0.5257). To date, the AP control group has performed significantly better than all other groups (P<0.001). These results do not corroborate the success of hemi-irradiation in Markovsky et al. 2019. GRID treatments may be more effective if a substantially higher dose and/or multiple fractions were employed.

Purpose: RECA (Radiotherapy Enhanced by Cherenkov photo-Activation) is a novel treatment with potential to add an anti-cancer immunogenic component through Cherenkov activation of a photo-chemotherapeutic agent (psoralen). This work investigates RECA in a novel tissue-representative in-vitro model consisting of 4T1 murine cancer cells grown on thin slices of viable rat-brain tissue.Methods: Accurate estimation of viable tumor burden is of foundational importance to this work. A CellProfiler pipeline was created and optimized and validated on realistic simulated data/images where the ground truth of number of colonies and integrated intensity was known. Simulated data sets mimicked key features of real experimental data including colony spatial and size distributions, contaminant and stray light signals, colony overlap, and noise. The optimized CellProfiler pipeline was then applied to the original 4T1 tumor cell images to determine colony growth over five days. Several experiments were conducted prior to the RECA experiment to determine the best protocol. The first tested the optimal concentration of psoralen, loading technique, and type of psoralen by co-incubating 4T1 cells with psoralen for differing times and concentrations and subsequently exposing them to 365nm radiation at variable energy fluences. The plates were tested for 4T1 cell viability using Celltiter-glo and luciferase assay 48-72 hours later depending on confluence of the control plate. Another experiment tested the output of the CellProfiler image analysis for relative growth over time measuring 4T1 mCherry cells plated on rat brain slices at 10k,20k,30k,40k,50k cells per hemisphere. For the RECA experiment, six 12-well plates, each containing 1cm of agarose supporting a 400 µm thick coronal slice of viable rat brain tissue were created. Each plate represented one arm of an experiment incorporating the psoralen derivative 4’-aminomethyl trioxsalen (AMT): MV control, MV+AMT, kV control, kV+AMT, no irradiation control, and AMT alone control. 20,000 4T1 cells expressing both mCherry-flourescent and firefly luciferase-luminescent reporter proteins plated on each rat brain slice hemisphere. For the AMT arms, the cells were co-incubated with 1 µM AMT for 1 hour prior to plating. The MV arms received 4 Gy from a 15 MV linear accelerator beam, and the kV arms received 4 Gy from 160 keV photons. Images were taken of the plates each day for 5 days with a Zeiss Lumar microscope with rhodamine filter for the mCherry protein signal. Results: The CellProfiler pipeline measured integrated intensity of the 10 simulated images that best approximated the images from the experiment with an accuracy of 99.23% ± 0.75%. Further analysis on images with increasing colonies, background, and noise showed the pipeline was accurate on images with variable features. These results gave confidence that the same pipeline could be used on images from this experiment. AMT was found to be a more effective psoralen (0.43 ± 0.22% cell survival after 48hr) relative to 8-MOP (31.3% ± 3.7% cell survival after 72hr). The psoralen cell loading was found to be optimal at 1µM for 1 hr prior to plating. The analysis of the cell titration images showed a significant increase in signal for each increase in cells plated on day one and for all subsequent days except for the 20k cell arm. Additionally, the growth in signal for the plates was consistent between the arms except for the 20k arm due to extra signal on the periphery of the slice likely from displaced cells. Integrated intensity analysis of the 4T1 mCherry cells revealed a significant decrease in tumor proliferation by day 5 between the MV control (5.65±0.78-fold growth) and MV AMT (3.49±-0.52-fold growth) arms. This result is consistent with the hypothesis that psoralen is being activated, causing the decreased proliferation seen in MV AMT arm. The kV control and kV AMT arms had a smaller decrease in proliferation when compared to their MV counterparts (6.73±1.24 and 5.26±0.59-fold growth respectively). The growth observed in the Dark control arm was consistent with the 13.6 ± 1.5 hour doubling time for 4T1 cells. In the MV AMT arm, there were punctuated regions of increased signal in 7/12 wells not corresponding to colonies, making segmentation for this arm challenging. The viability of the brain slice was assessed each day and found to be stable over the 5 days. Conclusions: The technique of testing image analytic software on simulated images proved to be an effective tool to verify the software’s accuracy. A similar technique can be applied to images with new and challenging features. The rat brain slice model gives the opportunity to both generate Cherenkov in real tissue while providing a 3D matrix for the colonies to grow, which is an improvement to the 2D well plate culture for this experiment. This new model adds challenges of proper image analysis with cell autofluorescence as well as cell clumping. The preliminary results are consistent with psoralen activated in RECA treated cells causing decreased proliferation for the MV arm. Further work is needed to confirm and quantify the effect.

Quality assurance (QA) is a critical component of radiation oncology medical physics for both effective treatment and patient safety, particularly as innovations in technology allow movement toward advanced treatment techniques that require increasingly higher accuracy in delivery. Comprehensive 3D dosimetry with PRESAGE® 3D dosimeters read out via optical CT has the potential to detect errors that would be missed by current systems of measurement, and thereby improve the rigor of current QA techniques through providing high-resolution, full 3D verification for a wide range of clinical applications. The broad objective of this dissertation research is to advance and strengthen the standards of QA for radiation therapy, both by driving the development and optimization of PRESAGE® 3D dosimeters for specific clinical and research applications and by applying the technique of high resolution 3D dosimetry toward addressing clinical needs in the current practice of radiation therapy. The specific applications that this dissertation focuses on address several topical concerns: (1) increasing the quality, consistency, and rigor of radiation therapy delivery through comprehensive 3D verification in remote credentialing evaluations, (2) investigating a reusable 3D dosimeter that could potentially facilitate wider implementation of 3D dosimetry through improving cost-effectiveness, and (3) validating deformable image registration (DIR) algorithms prior to clinical implementation in dose deformation and accumulation calculations.

3D Remote Dosimetry: The feasibility of remote high-resolution 3D dosimetry with the PRESAGE®/Optical-CT system was investigated using two nominally identical optical-CT scanners for 3D dosimetry were constructed and placed at the base (Duke University) and remote (IROC Houston) institutions. Two formulations of PRESAGE® (SS1, SS2) were investigated with four unirradiated PRESAGE® dosimeters imaged at the base institution, then shipped to the remote institution for planning and irradiation. After each dosimeter was irradiated with the same treatment plan and subsequently read out by optical CT at the remote institution, the dosimeters were shipped back to the base institution for remote dosimetry readout 3 days post-irradiation. Measured on-site and remote relative 3D dose distributions were registered to the Pinnacle dose calculation, which served as the reference distribution for 3D gamma calculations with passing criteria of 5%/2mm, 3%/3mm, and 3%/2mm with a 10% dose threshold. Gamma passing rates, dose profiles, and dose maps were used to assess and compare the performance of both PRESAGE® formulations for remote dosimetry. Both PRESAGE® formulations under study maintained high linearity of dose response (R2>0.996) over 14 days with response slope consistency within 4.9% (SS1) and 6.6% (SS2). Better agreements between the Pinnacle plan and dosimeter readout were observed in PRESAGE® formulation SS2, which had higher passing rates and consistency between immediate and remote results at all metrics. This formulation also demonstrated a relative dose distribution that remained stable over time. These results provide a foundation for future investigations using remote dosimetry to study the accuracy of advanced radiation treatments.

A Reusable 3D Dosimeter: New Presage-RU formulations made using a lower durometer polyurethane matrix (Shore hardness 30-50A) exhibit a response that optically clears following irradiation and opens up the potential for reirradiation and dosimeter reusability. This would have the practical benefit of improving cost-effectiveness and thereby facilitating the wider implementation of comprehensive, high resolution 3D dosimetry. Three formulations (RU-3050-1.7, RU-3050-1.5, and RU-50-1.5) were assessed with multiple irradiations of both small volume samples and larger volume dosimeters, then characterized and evaluated for dose response sensitivity, optical clearing, dose-rate independence, dosimetric accuracy, and the effects of reirradiation on dose measurement. The primary shortcoming of these dosimeters was the discovery of age-dependent gradients in dose response sensitivity, which varied dose response by as much as 30% and prevented accurate measurement. This is unprecedented in the standard formulations and presumably caused by diffusion of a desensitizing agent into the lower durometer polyurethane. The effect of prior irradiation on the dosimeters would also be a concern as it was seen that the relative amount of dose delivered to any given region of the dosimeter will affect subsequent sensitivity in that area, which would in effect create spatially-dependent variable dose sensitivities throughout the dosimeter based on the distributions of prior irradiations. While a successful reusable dosimeter may not have been realized from this work, these studies nonetheless contributed useful information that will affect future development, including in the area of deformable dosimetry, and provide a framework for future reusable dosimeter testing.

Validating Deformable Image Registration Algorithms: Deformable image registration (DIR) algorithms are used for multi-fraction dose accumulation and treatment response assessment for adaptive radiation therapy, but the accuracy of these methods must be investigated prior to clinical implementation. 12 novel deformable PRESAGE® 3D dosimeter formulations were introduced and characterized for potential use in validating DIR algorithms by providing accurate, ground-truth deformed dose measurement for comparison to DIR-predicted deformed dose distributions. Two commercial clinical DIR software algorithms were evaluated for dose deformation accuracy by comparison against a measured deformed dosimeter dose distribution. This measured distribution was obtained by irradiating a dosimeter under lateral compression, then releasing it from compression so that it could return to its original geometry. The dose distribution within the dosimeter deformed along with the dosimeter volume as it regained to its original shape, thus providing a measurable ground truth deformed dose distribution. Results showed that intensity-based DIR algorithms produce high levels of error and physically unrealistic deformations when deforming a homogeneous structure; this is expected as lack of internal structure is challenging for intensity-based DIR algorithms to deform accurately as they rely on matching fairly closely spaced heterogeneous intensity features. A biomechanical, intensity-independent DIR algorithm demonstrated substantially closer agreement to the measured deformed dose distribution with 3D gamma passing rates (3%/3mm) in the range of 90-91%. These results underscore the necessity and importance of validating DIR algorithms for specific clinical scenarios prior to clinical implementation.

X-PACT as a novel cancer therapy utilizes kilovoltage x-ray beam, phosphor and psoralen to treat solid tumors. Since x-ray beam is not commonly used for radiation therapy treatment purposes, a lack of treatment planning tool and plan based dose calculation is hindering the development of X-PACT. In this study, we try to approach the challenge by creating a Monte Carlo model that is an accurate representation of the actual treatment. The Monte Carlo model will be validated with commissioning measurements and is applicable to clinical data.

FLUKA is used as the Monte Carlo package for our simulations. The Monte Carlo model is created based on the Variantm OBI system. The geometry is created and optimized in Flair. To improve simulation efficiency, we collected the filtered simulated 80 kVp x-ray spectrum and used that as the source file for photon simulation. This way, the x-ray tube is bypassed, and 80 kVp photon can be simulated directly.

The validation process consists of two qualities: the back-scatter factor and the percentage depth dose. The commissioning was done separately from this study [1], and the commissioning data was used to compare with simulation results. The Model shows good overall matching to the commissioning PDD data. At small or large field size, a discrepancy between the simulation PDD data and commissioning PDD data can be observed at the surface, the differences are with 3-4%.

The final step is to apply the model to the Phase I canine trial. The clinical trial includes six dog study cases. In this thesis, Monte Carlo dose calculation based on the CBCT images of each dog study was performed for all six dog studies. The result is a 3D dose matrix for the CBCT images. According to the prescription dose for each dog study, the simulated dose was normalized, and the dose distribution for each dog was generated..

Purpose: To validate commissioning results associated with the Xstrahl Small Animal Radiation Research Platform (SARRP) installed at Duke University in October 2021, verify the accuracy of the Xstrahl Point Dose Calculator (PDC) and MuriPlan dose calculation in simple geometries, and design and characterize a novel in-house kV mini-GRID capability on the SARRP.Methods: After installation at Duke University, Xstrahl SARRP TG-61 output was measured for independent verification using a Farmer ion chamber at reference conditions (33 cm SSD, 2 cm depth, open field, 220 kVp, 13 mA). Half-value layer was measured using the same ion chamber, with copper sheets to vary thickness. The accuracy of the PDC was investigated in simple water and bolus stack phantoms using EBT3 film. A range of field sizes (10x10, 20x20, 30x30, 40x40, 10x20, 20x10, 15x40, 10x40, 30x70 mm2) and depths (1 cm, 2 cm) were spot-checked. MuriPlan simulations of treatment delivery to the bolus phantom and water phantom were compared to results of EBT3 film measurements. Metal Oxide Semiconductor Field Effect Transistor (MOSFET) detectors were also used for independent verification, with detectors being embedded within a tissue-equivalent mouse phantom at 1 cm depth. GRID irradiations were performed with the SARRP, using a 220 kVp beam, 13 mA, and a 40 mm x 40 mm field size. Pencil and bar GRIDs with beamlet spacings of 1 mm and 1.25 mm were characterized by first inserting GRID into a 3D-printed mount and positioning the mount on top of a PLA plastic block, surrounded by distilled water. EBT3 films were infixed to the top level of the PLA block and positioned at isocenter. PDC was utilized to determine irradiation time. The beam was turned on for 102 seconds—sufficient time to administer 6 Gy with a 40 mm x 40 mm field size to the surface film at isocenter with no GRID blocking the beam. EBT3 film results were analyzed to determine the output factors, peak-to-valley ratios, integral dose relative to open field, relative dose maps, as well as to produce dose volume histograms for each GRID. Results were compared to GRID characterizations in Johnson et al [18]. GRID characterizations were used to inform experimental plan for pre-clinical trial evaluating treatment efficacy of GRID therapy with PD-L1 checkpoint inhibition compared to conventional radiation therapy. Results: TG-61 dose rate and half-value layer measured during on-site commissioning showed excellent agreement with Xstrahl factory commissioning results (≈1% difference). The PDC and MuriPlan dose calculation predicted results for field sizes and depths demonstrated acceptable agreement with actual results measured by EBT3 film (.2% to 12%), with exception of several outliers. Using EBT3 film dosimetry for verification (tissue-equivalent bolus medium), MuriPlan simulations were within 2% and 12% difference from the film measured dose for 5/7 field sizes in the bolus phantom and within 3% and 13% for 4/5 field sizes in the water phantom. MOSFET detector measurements using the mouse phantom demonstrated improved agreement with the PDC-predicted dose, with percent errors ranging from .12% to 5.97% (with a single outlier at 18.3%). Measured output factors using the SARRP for the 20 mm x 20 mm pencil GRIDs were .77 ± .03 and .74 ± .02 (1 mm and 1.25 mm beamlet spacing, respectively). For the bar GRIDs, these values were evaluated to be .83 ± .03 and .80 ± .03 (1 mm and 1.25 mm beamlet spacing, respectively). Peak:valley ratios for the 1 mm and 1.25 mm beamlet spacing pencil GRIDs were determined to be 24.5 ± 0.6 and 25.1 ± 1.3, respectively. Peak:valley ratios for the 1 mm and 1.25 mm beamlet spacing bar GRIDs were found to be lower than for pencil GRIDs with equivalent beamlet spacing, being evaluated to be 13.2 ± 1.1 and 18.5 ± 1.2, respectively. Output factors, peak:valley ratios, integral dose relative to open field, and dose volume histograms for the pencil and bar GRIDs using the SARRP largely corroborated the results of Johnson et al in terms of experimental trends (peak:valley ratios being higher for pencil GRIDs and increasing with increasing beamlet spacing, output factors decreasing with increasing beamlet spacing for both GRID types, and decreasing integral dose with increasing beamlet spacing for pencil GRIDs and increasing integral dose with increasing beamlet spacing for bar GRIDs). 4.67% to 30.5% difference was observed for experimentally measured peak:valley ratios relative to the results for the same GRIDs in Johnson et al.. Better agreement was demonstrated in GRID output factor measurements (≈0% to 14%). Integral dose experimental measurements demonstrated exceptional agreement with Johnson et al.., with percent differences ranging from 1% to 2.1%. These measured differences are likely a result of using the SARRP versus the XRAD 225Cx used in Johnson et al, but lend significant credence to reproducibility of results found using the XRAD 225Cx. Conclusions: The PDC and MuriPlan computations provide an effective estimate of the exposure time necessary to deliver dose for corresponding MVC field sizes and depths (within 6% error using the MOSFET for verification). EBT3 film was determined to be an unreliable measure of SARRP dose delivery; MOSFET detectors demonstrated more consistency and effectiveness for treatment planning verification. Xstrahl’s SARRP was able to replicate the kV mini-GRID capabilities of the XRAD 225Cx used in Johnson et al. and can be used for mini-GRID characterizations and preclinical mouse trials.

3D dose verification has important advantages for comprehensive verification of advanced radiation treatments. We have designed, constructed, installed, and commissioned an in-house prototype three dimensional (3D) dose verification system for a joint international collaboration (Duke and Princess Margaret Cancer Centre (PMCC), Toronto) to investigate the comprehensive accuracy of stereotactic body radiotherapy (SBRT) treatments. The potential of this system to achieve sufficient performance (1 mm resolution, 3 % noise, within 3 % bias) for SBRT verification was investigated.

The system, termed PMOS, was designed utilizing a parallel ray geometry instigated by precision telecentric lenses and an 630 nm LED light source. Using PRESAGE® radiochromic dosimeters, a 3D dosimetric comparison with our gold-standard system and treatment planning software (Eclipse®) was performed for a four-field box treatment (8 Gy per fraction), under gamma passing criteria of 3 %/3 mm/10 % dose threshold. Post off-site installation, deviations in the system's dose readout performance was assessed by rescanning the four-field box irradiated dosimeter and using line-profiles to compare on-site and off-site mean and noise levels in four distinct dose regions. As a final step, an end-to-end test of the system was completed at the off-site location, including CT-simulation, irradiation of the dosimeter and a 3D dosimetric comparison of the planned dose (Pinnacle3) to delivered dose for a spinal SBRT treatment(12 Gy per fraction).

The gamma pass rate for the 3D gamma comparison between the PMOS and our gold-standard Duke Large Optical Scanner (DLOS) dose distribution was 99.1% under (3 %, 2mm, 5 % threshold) criteria. Under 3 %, 1mm, 5 % threshold, the gamma comparison between the systems was 95.6 %. Using line profiles, the dose difference between the readout of the PMOS at Duke and PMCC for the same irradiated dosimeter was between 0-1 Gy. At 1mm reconstructed dose voxels, the gamma pass rate was 95. 27% (3 %, 2 mm, 5 % threshold) for the gamma comparison between the measured PMOS and calculate Pinnacle3 dose distribution. The majority of the failing voxels of the gamma map were in the high dose regions of the dose distribution. By rescanning the dosimeter in the DLOS, the PMOS' dose readout was ruled out as the source of the high dose (>14Gy) disagreement. The sensitivity of the PRESAGE® dosimeter and the parameters for the Pinnacle3 dose calculation will be further investigated as possible sources for the error.

This work will describe the end-to-end process and results of designing, installing, and commissioning a state-of-the-art 3D dosimetry system created for verification of advanced radiation treatments including spinal radiosurgery.

As an initial step towards this goal, I worked on a related project to evaluate a prototype of our in-house 3D dosimetry system using Fresnel lens and a `solid tank' made of PRESAGE® like material.

Translational cancer research has been around for many years and has resulted

in many advancements in cancer treatment. Preclinical radiation therapy is an important

tool used in some studies to better understand the biological effects due to radiation.

Current preclinical radiation treatment techniques do not emulate the advanced

techniques used in cancer clinics, such as intensity modulated radiation therapy (IMRT).

In this work we explore the possibility of developing and implementing an IMRT

treatment capability for an orthovoltage micro irradiator used for small animal research.

In order to implement IMRT to the micro irradiator, every step of the radiation

therapy treatment process had to be evaluated, developed, and tested. The first step was

to develop and treatment planning software that can be used for small animal studies.

Using the open source Computational Environment for Radiotherapy Research (CERR)

and adapting it for use with an orthovoltage irradiator, monte carlo dose calculations

could be performed for small animal data sets. CERR does not have the ability to

optimize dose calculations, so a Matlab script was developed and written for inverse

optimization for treatment planning. Treatment plans were designed and optimized for

several small animal cases to evaluate the optimization algorithm. Following successful

simulation development, treatment delivery techniques needed to be developed. 3D

printing was used as a tool to create physical compensators that could be used as an

add-on device to the micro irradiator. With the capability of submillimeter printing

resolution, 3D printing has the capability to handle the high resolution required for very

small structures inside of small animals. Using the simulation data, another Matlab

script was developed to create both compensator and inverse compensator 3D models.

Many materials and techniques were evaluated to determine the best method for

compensator production. Materials were tested for attenuation properties, printing

capabilities, and ease of use until a satisfactory result was achieved.

Once the simulation and delivery techniques were developed to a satisfactory

level, an end to end test was designed to verify the IMRT capability. Using a 2.2 cm

diameter cylindrical Presage® dosimeter as the quality assurance (QA) device/patient, a

treatment plan was created based on the geometry of the Radiologic Physics Center

(RPC) Head and Neck phantom design. The dose tolerances used for the inverse

optimization were the same as the RPC Head and Neck protocol with a stricter tolerance

for the organ at risk (OAR). Compensators were produced for the plan and both 2D and

3D analysis was performed. Radiochromic film was used for 2D dose map analysis.

Gamma analysis was performed using 2D film data with varying criteria for distance to

agreement and dose difference. 3D analysis was done by delivering the treatment plan

to the Presage® dosimeter. Using optical-CT for dose readout of the dosimeter,

qualitative analysis was performed to show the 3D delivered dose data.

The end to end test showed strong evidence that IMRT could be implemented on

the small animal irradiator. The 9 field treatment plan was delivered in under 30

minutes with no mechanical or collisional issues. The 2D dose analysis showed 7 out of 9

treatment fields had a passing rate greater than 90% for a gamma analysis using 10%/0.5

mm tolerances. 3D dose analysis showed promising spatial resolution of the dose

modulation. As a feasibility and an initial testing study for a new treatment technique on

the small animal irradiator, these results showed the capability of the 3D printed

compensators to modulate dose with high spatial precision and moderately accurate

dose delivery.

Purpose: Radiotherapy Enhanced by Cherenkov photo-Activation (RECA) is a novel radiation treatment method that seeks an anti-cancer effect with the introduction of a psoralen compound administered for treatment. The goal of the RECA method is to enhance standard radiation therapy treatments with the addition of psoralen being photo-activated by Cherenkov radiation that is generated during radiotherapy. The purpose of this work is to investigate the effectiveness of RECA on 4T1 mCherry FLuc breast cancer cells seeded on a psoralen-baked-agarose-based rat brain slice.Methods: A previously established CellProfiler pipeline, developed in our lab by Holden et al., was used to assess tumor burden on rat brain slices used for a tissue-equivalent medium for cell culturing. The CellProfiler pipeline was implemented on images of 4T1 breast cancer cells growing over the course of four to five days post-treatment to measure the average intensity of fluorescing cells. Prior to the RECA experiment, multiple preparatory experiments were conducted to refine and optimize experimental techniques. The first preparatory experiment tested the possibility of a plate reader bias effect, i.e., signal from nearby wells contributing to signal of other wells, seen during measurements of cell luminescence within individual wells of a clear-bottom 96-well plate. A CellTiter-Glo endpoint readout was taken 48-hours post-treatment for an endpoint measure to assess the if there was any added signal from nearby wells in the clear-bottom plates. The next experiment tested whether fractionation of dose was feasible and preferrable to single dose treatment by irradiating 4T1 mCherry Fluc cells with 2 Gy and 4 Gy of kV radiation with and without fractionation. An endpoint CellTiter-Glo readout was conducted 72 hours post-treatment to assess cell viability between the treatment plans. Additional preparatory experiments investigated whether psoralen-doped agarose was an effective method for cell loading. A 30 µM AMT-baked agar base was placed in half of the wells in plates with 4T1 mCherry Fluc cells seeded on brain slices on top of the agar. One plate received no treatment and one plate received treatment of 365 nm UVA, and an endpoint Firefly Luciferase reporter assay was conducted 48 hours post-treatment to assess cell viability between the conditions. For the RECA experiment, five 12-well plates, each containing 1 cm of agar with a 400 µm thick coronal slice of rat brain tissue, were given one of five conditions of treatment: no treatment, 4.95 Gy of fractionated kV or MV treatment, or 4.95 Gy of whole kV of MV treatment. Each plate condition consisted of six wells containing AMT-baked agar and six wells containing a standard agar base. After irradiation, images were taken of each of the plates for each day over the course of five days five days with a Zeiss Lumar microscope. The microscope was equipped with a rhodamine filter to analyze the luminescence readings from each well for assessment of cell viability. Results: The preparatory experiments all yielded results that allowed for development of the RECA experiment procedure. Investigation of the plate reader effect showed that background signal from nearby wells was not leaking into well signal readout, with all wells having nearly consistent signal throughout all the wells. Fractionating the dose was found to be preferable because it decreased cell viability less than delivering all dose at once, which floored cell viability. Testing psoralen-doped agar demonstrated that this is an effective delivery method for psoralen to intercalate with cells. The RECA experiment utilizing kV and MV whole dose conditions allowed comparison between irradiations with and without a fractionation scheme. The Firefly Luciferase reporter assay signal for the MV treatment conditions showed less cell viability than the Dark control conditions for both AMT and DMSO. Additionally, the whole dose MV conditions demonstrated a more pronounced decrease in cell viability than the fractionated MV conditions, as expected. The CellProfiler analysis demonstrated the same trends with the whole dose MV AMT condition (8.54 ± 0.99-fold increase) and whole dose MV DMSO condition (11.80 ± 0.70-fold increase) demonstrating less cell viability than the Dark AMT (13.41 ± 0.83-fold increase) and Dark DMSO (14.11 ± 0.62-fold increase). Interestingly, there was not a significant difference in cell viability seen between the fractionated and whole dose conditions. Conclusions: The procedural techniques developed for the analysis of the RECA effect during the preparatory experiments ruled out a plate reader effect and demonstrated that introducing fractionation and psoralen-baked agar is effective. The testing of the fractionation scheme used for kV irradiations proved to be sufficient for decreasing cell viability without killing all the cells. Additionally, the testing of the psoralen-baked agarose slabs proved to be an adequate psoralen delivery method when compared to methods that used cells suspended in psoralen treated media in prior studies. When these changes to the procedure were introduced together during MV irradiations, the RECA effect did not clearly replicate the results demonstrated during kV irradiations in the preparatory experiments. Further investigation is required to confirm and validate the RECA effect generated during radiotherapy.

Introduction: RECA (Radiotherapy Enhanced with Cherenkov photo-Activation) is a novel treatment that induces a synergistic therapeutic effect by combining conventional radiation therapy with phototherapy using the anti-cancer and potentially immunogenic drug, psoralen. Psoralen is photo-activated in-situ by UVA (UltravioletA, 320-400nm) Cherenkov Light (CL), produced in tissue directly by the treatment beam. In this study, we develop methods to image and quantify relative CL production (primarily in the UVA range) from a range of tissue and phantom materials upon photon irradiation. These methods are further applied to identify a tissue-equivalent optical phantom, mimicking CL production in the UVA range, in order to facilitate further RECA experiments.

Methods: The imaging system included a deep-cooled, high-sensitivity CCD camera, equipped with either a visible range lens (sensitive to 400-700nm photons) or a UVA-compatible lens assembly and a band-pass filter (sensitive to 320-400nm photons). CL emission was quantified in bulk tissue samples, solid waters (SW brown and white), and agarose gels in a series of experiments. The samples and imaging equipment were placed in a dark, light-blocking chamber to avoid contamination from other light sources. In addition, the camera was carefully positioned with respect to the LINAC head and was also shielded using lead bricks to minimize radiation noise.The samples were then irradiated with clinical photon beams, while simultaneously being imaged by the camera.

Results: In the visible range, solid water had similar CL emission to that from bulk tissue samples (34% less than the maximum and 44% higher than the minimum UVA emitting tissue). A 25% reduction in radiation noise in the UVA spectrum was achieved using lead block shielding of the camera. In the UVA range at 15MV, white SW emitted 66±5%, 64±5% and 76±3% less UVA than chicken, pork loin and pork belly respectively. Similar under-response was observed at 6MV. Brown SW had 21±8% less UVA emission than white SW at 15MV, and no significant emission at 6MV. Agarose samples (1% by weight) doped with 250ppm India Ink exhibited equivalent UVA CL emission to chicken breast (within 8%).

Conclusion: The results confirm that for the same absorbed dose, SW emits lessUVA light than the tissue samples, indicating that prior in-vitro studies utilizing SW as the CL-generating source may have underestimated the RECA therapeutic effect. Agarose gel doped with 250ppm India Ink is a convenient tissue equivalent phantom for further work.

Purpose: An interest in FLASH radiotherapy has been reawakened due to its noted ability to spare normal tissue, equal tumor control compared to conventional irradiation methods and technological advancement allowing for ultra-high dose rates required for FLASH radiotherapy to be more accessible compared to previous decades. The underlying biological mechanism of the FLASH effect are unknown and developing an in vitro model to study it has proven difficult. This work aims to combine two unique technologies, an organotypic rat brain slice model which models the in-vivo micro-environment in an in vitro setting and a linear accelerator capable of delivering variable FLASH pulses to design experiments which will facility the study of the FLASH effect.Methods: The experiments utilize a 35 MeV electron beam provided by Triangle Universities Nuclear Laboratory’s (TUNL) High Intensity Gamma-ray Source linac (HIGS). The beam can supply electron pulses with a temporal width of 1.2 s or 100 ns and work was performed with Gafchromic EBT3 and EBT-XD film to accurately determine the dose and dose rates of each pulse. Experiments were performed over 5 sessions to establish the use and effectiveness of the HIGS linac and biological rat brain model. A 2D translational stage was developed and targeting procedures were developed to ensure accurate targeting of each well containing an organotypic rat brain slice in a 12 well plate. Each rat brain was shot with a yellow fluorescent protein marker and seeded with 4T1 cancer cells tagged with mCherry and firefly luciferase. An imaging analysis workflow was developed to effectively capture and segment mCherry signal and determine the 4T1 proliferation four to five days after irradiation. These were compared to a final firefly luciferase readout. Each experiment was followed by a conventional irradiation as a control group. Monte Carlo model using TOPAS was created to simulate the HIGS linac dose profiles. Results: The HIGS linac can provide a mean dose rate up to 100 Gy/s and an instantaneous dose rate up to 100 MGy/s. The repeatability of the pulse dose was found to be within 4-5% of the average dose for a given experiment. Targeting was repeatable and dose superposition was confirmed. Well targeting quality assurance procedures of the translational stage allowed for consistent targeting of the pulse to each well. Yellow fluorescent protein bleed through in the mCherry signal was effectively filtered out and mCherry analysis reflects the end readout of firefly Luciferase. A gamma analysis between simulated and measured dose demonstrates a passing rate of 99.4% when using a criteria of 2%/2mm and threshold of 10%. Conclusions: FLASH capable dose rates can be supplied by the HIGS linac and is amongst the highest instantaneous dose rates currently available. The HIGS linac and organotypic rat brain model can be combined to irradiate and measure radiation effects to 4T1 cancer cell growth. There is qualitative data to support the observation of the FLASH effect in the rat brain model and the mCherry and firefly luciferase analysis agreement demonstrates the capabilities of the model to measure radiation effects to cancer cells in the 1-10 Gy range. Future work will be to quantitively measure the neuron health of the brain slices and DNA damage differences between FLASH and conventional irradiation.

Mice are the predominant animal model used in radiation therapy research for investigating radiobiological kinetics and evaluating new therapeutics to achieve a higher therapeutic ratio in the clinic. A novel carcinogen-induced and genetically engineered head and neck squamous cell carcinoma mouse model was developed at Duke to study head and neck cancer, one of the most widely spread cancers in the world. However, platforms that are able to perform precise and reproducible radiation therapy on these mice to mimic human radiation therapy are lacking. To address this issue, a platform based on the X-RAD 225Cx orthovoltage irradiator was developed. 3D printing technique was used to generate imaging phantoms, immobilization devices, and blocks. A simulation was conducted to optimize imaging protocol. Results were verified on the measurement on both the 3D-printed phantom and the actual mouse. Prior to irradiation, mice were placed on the immobilization device in a supine position, and the isocenter was determined by the position of the device since the irradiator does not have a laser localizer system. The performance of the immobilization was obtained by scanning several mice separately at various time points, ranging from several hours post-imaging to two months post-imaging. In order to make up the deficiency that irradiator only have rectangular and circular collimators which cannot provide moderate protection for organs at risk. Blocks with 3% transmission were designed based on the contours of central nervous system by a state-of-art program, BlockGen.

A protocol was developed for immobilization and image acquisition. 60 kVp was found to give the highest contrast of iodine, so it was set as the tube voltage for image acquisition. The deviations of positioning, i.e. the same mouse in separate scanning, are measured as 0.22±0.44 mm in LR axis, 0.15±0.30 mm in PA axis, and -0.24±0.25 mm in IS axis. Blocks with a 1.5 mm margin which can shield brain and spinal cord even in the worst case, were printed for opposed lateral beams; they were verified on fluoroscopy.

The block system was modified to eliminate potential human errors. Comparison on brain and spinal cord among different mice showed the largest deviation in 2.6 mm, however, with manually selection of the middle one, 1.5 mm margin is enough to shield central nervous system. Indicating that a generic block could be used in the experiment that does not require a very accurate treatment. The generic block can significant save time and effort for preclinical radiation treatment experiment. In this study, a platform that is capable of enhancing contrast imaging and allowing precise radiation therapy to be performed on genetically-engineered mice with head and neck cancer has been developed. This paves the way for more accurate head and neck mice model radiation therapy studies. In addition, the platform could be used in other types of preclinical studies.

Purpose: To investigate and characterize a novel 3D dosimetry system consisting of ClearView radiochromic gel dosimeters and a state-of-the-art telecentric optical CT scanner: The Duke Large Field of View Optical-CT Scanner (DLOS). Methods and Materials: ClearView radiochromic dosimeters (Modus QA) are gellan gum based radiochromic dosimeters containing a water-soluble tetrazolium salt which reduces into an insoluble formazan dye (with associated color change) under ionizing radiation. Initial spectrophotometric studies investigated linearity of dose response on small volumes of ClearView in optical cuvettes. Simple, single beam benchmark radiation therapy treatments (central axis photon, lateral photon, and electron deliveries) were delivered to 10 and 15-cm diameter ClearView dosimeters. Additionally, a “stacking pyramid” delivery was developed consisting of overlaid fields of increasing size, delivered down the central axis of each dosimeter, with a 1x1 cm2 small field at the center. The treatments were modeled with a commissioned Eclipse treatment planning system. Dosimeters were scanned with the DLOS, submerged in a refractive index (RI) matching fluid (ca. 10% propylene glycol) both pre- and post-irradiation (within 24h) and 3D reconstructions of the change in linear-optical-attenuation was determined using in-house software and 3D Slicer. Percent depth-dose (PDD), cross plane and in-plane profiles, and relative 3D gamma analysis were performed and compared to the commissioned Eclipse dose, which served as ground truth. All experiments followed a standardized workflow for consistency. Results: Linearity of dose response was confirmed in the cuvette study with excellent agreement (R2 ≥ 0.9986) at two wavelengths (520-and 632 nm) at 3 post-irradiation time points: 21 hours, 6 and 10 days. Dosimeter reconstructions were performed at 1mm³ resolution in full 3D. Relative dose profiles of all irradiations, in both 15-and 10 cm dosimeters, show good agreement in comparison to Eclipse dose calculations, with root mean square errors (RMSE) 0.00107-0.006649, and R2 ≥ 0.9808. Relative 3D gamma analysis was performed at 7%4mm, 5%3mm, 3%3mm, 3%2mm, and 2%2mm for all deliveries on both 10-and 15-cm dosimeters. 15-cm benchmark irradiations passed with ≥ 94% at 2%2mm, ≥ 90% at 3%3mm, and ≥ 90% at 2%2mm, for the central axis, left lateral, and electron deliveries, respectively. 10-cm benchmark irradiations passed with ≥ 93% at 3%2mm, ≥ 91% at 3%3mm, and ≥ 90 at 3%2mm, for the central axis, left lateral, and electron deliveries, respectively. 15-cm stacking field irradiations passed with ≥ 94% at 3%2mm, and 10-cm stacking field irradiations passed with ≥ 96% at 2%2mm. Regions of known artifacts were excluded from gamma analysis (jar base, neck, wall). Some artifacts remain unaccounted for (e.g., ring and cupping artifacts). Conclusion: This work presents the first use of a telecentric optical-CT scanner with ClearView. The system shows substantive promise for a new, comprehensive 3D dosimetry system, and this effort lays the groundwork for further, more specialized applications. Both the benchmark irradiations and stacked field deliveries performed exceptionally well in the various gamma analyses and investigation of the profiles, even in the presence of artifacts that were not completely accounted for in the data analysis. General differences between the 15-cm and 10-cm dosimeters are not made abundantly clear with the small sample size of this work, but both seem viable. There seems to be no difference between photon and electron deliveries, and both are viable with the system given the experiments performed.

Micro-irradiators enable exploration of the efficacy of novel radiation treatment approaches by providing the capability to reproduce realistic treatment delivery in small animal models. An approach of current topical interest is hypofractionated stereotactic body radiation therapy (SBRT), and the study of associated tumor and normal tissue radio-biology. Rodent SBRT is extremely challenging, requiring the precise delivery of radiation beams on the order of several millimeters. At present there are no methods to comprehensively verify these delivery techniques due to the requirements for ultra-high resolution and ability to measure the dose in 3 dimensions (3D).

This work introduces a potential solution to the rodent SBRT verification challenge: radiochromic rodent-morphic 3D dosimeters compatible with ultra-high resolution optical computed tomography (optical-CT) dose read-out. Rodent-morphic dosimeters were produced by 3D-printing molds of rodent anatomy directly from X-ray CT data, and using these molds to create tissue-equivalent phantoms both with and without high-Z spinal inserts for cone-beam CT targeting. Feasibility was evaluated through a series of irradiations, including a 180-degree spinal arc treatment. Dose distributions were measured in high-resolution (0.5mm isotropic voxels) with an in-house built optical-CT system, which determined dose from the change in optical density throughout the dosimeters from pre-and post-irradiation scans. Optical-CT data was calibrated to absolute dose using a calibration curve determined from irradiating small volumes of radiochromic material from the same batch as the rodent-morphic dosimeters to known doses in a 6MV beam (negligible energy response was assumed). Independent verification of absolute dose at a point was made with a novel scintillator comprised of europium and lithium doped yttrium oxide nanocrystals, with a sub-mm active length. Independent verification of the dose distribution was performed using EBT2 radiochromic film positioned in the dosimeters, which had been sliced in half. Contrast-to-noise ratio between high-Z spinal inserts and tissue-equivalent PRESAGE material was found to be ~10, sufficient for bony alignment and isocenter targeting with on-board CBCT image guidance. Absolute dose calculated at isocenter through optical-CT was found to agree with nano-detector measurement within 3%, while relative dose distributions in two orthogonal planes were found to agree with film within 4%. PRESAGE rodent-morphic dosimeters demonstrated much promise in the verification of precise radiation treatment given by the X-Rad 225Cx micro-irradiator.

Practical challenges involved in optical-CT imaging were addressed through the investigation of an in-house Fresnel-based optical-CT system with considerably less refractive index-matching fluid. The "DFOS" (Duke Fresnel-based Optical-CT System) system differed from current optical-CT systems by replacing cumbersome convex telecentric lenses with a lighter and much less expensive Fresnel system. A second major modification was the replacement of the refractive index-matching fluid bath with a solid polyurethane tank. PRESAGE radiochromic dosimeters were irradiated with orthogonal parallel-opposed treatments and a brain IMRT treatment and dose distributions were readout by the DFOS system and compared to both treatment planning software prediction and other in-house optical-CT systems. Gamma index passing rate at the 3%/3mm threshold for the two parallel-opposed and brain IMRT treatments were 89.3%, 92.1%, and 87.5%, respectively. The DFOS system showed promise for 3D dosimetry, but the performance is still substantially inferior at present to the gold-standard systems.