Cancer treatment by adoptive immune cell therapy (AIT) is a form of immunotherapy that relies on the in vitro activation and/or expansion of immune cells. In this approach, immune cells, particularly CD8+ T lymphocytes, can potentially be harvested from a tumor-bearing patient, then activated and/or expanded in vitro in the presence of cytokines and other growth factors, and then transferred back into the same patient to induce tumor regression. AIT allows the in vitro generation and activation of T-lymphocytes away from the immunosuppressive tumor microenvironment, thereby providing optimum conditions for potent anti-tumor activity. The overall objective of this study is to: a) develop multi-modality (optical- and radionuclide-based) molecular imaging approaches to study the overall kinetics of labeled adoptively transferred T- lymphocytes in vivo, b) to non-invasively image and assess in-vivo, targeting and retention of adoptively transferred labeled T-lymphocytes at the tumor site. T-lymphocytes obtained from draining lymph nodes of 4T1 (murine breast cancer cell) sensitized BALB/C mice were activated in vitro with Bryostatin/ Ionomycin for 18 hours, and were grown in either Interleukin-2 (IL-2) or combination of Interleukin-7 and Interleukin-15 (IL-7/IL-15) for 13 days, (cells grown in IL-2 called IL2 cells, and cells grown in IL7/15 called IL7/15 cells). In order to validate the methodology and to offer future clinical translation, both direct and indirect cell labeling methods were expanded and employed. The first method was based on direct in vitro cell labeling by lipophilic near-infrared (NIR) fluorescent probe, 1,1- dioctadecyltetramethyl indotricarbocyanine iodide, (DiR), followed by intravenous (i.v.) injection into BALB/C mice for multi-spectral fluorescence imaging (MSFI). The second method was based on indirect labeling of T- lymphocytes through transduction of a reporter gene (cell cytoplasm labeling Herpes Simplex Virus type 1- thymidine kinase (HSV-1 tk). The product of this reporter gene is an enzyme (HSV-1TK) which phosphorylates a radio labeled substrate 2-fluoro-2-deoxy-1 β- D- arabinofuranosyl-5-iodouracil ([124I]-FIAU) for Positron Emission tomography (PET) imaging. ATP based cell viability assay, flow cytometry and interferon-γ (IFN-γ) ELISA were used to investigate if there are any changes in cell viability, proliferation and function respectively, before and after direct and indirect labeling. The results showed that cell viability, proliferation, and function of labeled 4T1 specific T-lymphocytes were not affected by labeling for direct labeling methods at DiR concentration of 320µg/ml. For the indirect labeling method, the viability and proliferation results showed that cell viability decreases as multiplicity of infectious (MOI) increases. In particular, at MOI of 10 almost all cells die 3 days post transduction. At MOI of 5, cells viability was ≤ 30% and at MOI of 2 was ≤ 60%. Cell viability was 80% at MOI of 1. The results of optical imaging were as follows: when the recipient mice with established 4T1 tumors were injected with DiR labeled 4T1 specific T-lymphocytes, the 4T1 specific T-lymphocytes (IL2 cells) infused into tumor-bearing mice showed high tumor retention, which peaked 3 or 6 days post infusion depending on the tumor size and persisted at the tumor site for 3 weeks. In contrast, IL7/15 cells showed lower signal at the tumor site and this peaked on day 8. On the other case when 4T1 tumor cells were implanted 1-week post-infusion of labeled T-lymphocytes. IL2 T-lymphocytes moved out of lymphoid compartments to the site of subsequent 4T1 inoculation within two hours and peaked on day 3 and the signal persisted for 2 more weeks. In contrast with infusion of IL7/15 cells, the signal was barely detected and did not show a similar trafficking pattern as with IL2 cells. The results of the indirect labeling method, PET reporter gene (PRG) system (HSV-1tk / [124I ] FIAU ) showed that both IL2 and IL7/15 cells were successfully transduced as verified ex vivo by real time PCR and western blot. T Cells transduction efficiency was assessed from cell uptake study in comparison to stable transduced Jurkat cells which have transduction efficiency of 100 %. Both IL2 and IL7/15 cells showed lower transduction efficiency (≤ 30%) compared to Jurkat cells. Consequently, PET imaging did not show a detectable signal of transduced T cells in vivo. Biodistribution study was carried out on day 3 post [124I]-FIAU injections. Results were consistent with the optical imaging results, except for IL7/15 cells. Transduced and untransduced IL2 and IL7/15 cells were labeled with DiR and injected ( i.v.) into Balb / C mice and then imaged by both imaging modalities (MSFI and PET) at the same time. MSFI images of transduced IL2 cell showed detectable signal starting from 2 hours, peaked at 72 hours and persisted up to 2 weeks, while IL7/15 cells were detectable at the tumor site starting at 24 hours, peaked at 72 hours and persisted up to 2 weeks. By the end of this study animals were dissected and tissue activities were counted using gamma counting and expressed as % Injected dose/gram of tissue (%ID/gm). Transduced IL2 and IL7/15 cells showed higher %ID/gm than other organs at lungs, liver, spleen, tumor, lymph nodes and bone/bone marrow. IL7/15 cells compared to IL2 cells showed higher %ID/gm at same organs. Neither IL2 nor IL7/15 untransduced DiR labeled cells showed any activity at tumor site, and their activities at other organs was very low compared to transduced cells. To investigate whether labeled T-lymphocytes will localize at tumor metastases or not, and to study the difference in their migration patterns to the tumor site versus tumor metastases, 4T1 tumor cells were successfully transduced with HSV-1tk as confirmed by RT-PCR , western blot and cell uptake study. Transduced 4T1 cells were implanted in the right flank or in the mammary fat pad of the mouse. Serial PET imaging was carried out in the third and fourth week post tumor implantation to know when the tumor will metastasizes. PET imaging showed only signal at the tumor site and no metastasis were detected.

Interstitial and intracavitary brachytherapy plays an essential role in management of several malignancies. However, the achievable accuracy of brachytherapy treatment for prostate and cervical cancer is limited due to the lack of intraoperative planning and adaptive replanning. A major problem in implementing TRUS-based intraoperative planning is an inability of TRUS to accurately localize individual seed poses (positions and orientations) relative to the prostate volume during or after the implantation. For the locally advanced cervical cancer patient, manual drawing of the source positions on orthogonal films can not localize the full 3D intracavitary brachytherapy (ICB) applicator geometry. A new iterative forward projection matching (IFPM) algorithm can explicitly localize each individual seed/applicator by iteratively matching computed projections of the post-implant patient with the measured projections. This thesis describes adaptation and implementation of a novel IFPM algorithm that addresses hitherto unsolved problems in localization of brachytherapy seeds and applicators. The prototype implementation of 3-parameter point-seed IFPM algorithm was experimentally validated using a set of a few cone-beam CT (CBCT) projections of both the phantom and post-implant patient’s datasets. Geometric uncertainty due to gantry angle inaccuracy was incorporated. After this, IFPM algorithm was extended to 5-parameter elongated line-seed model which automatically reconstructs individual seed orientation as well as position. The accuracy of this algorithm was tested using both the synthetic-measured projections of clinically-realistic Model-6711 125I seed arrangements and measured projections of an in-house precision-machined prostate implant phantom that allows the orientations and locations of up to 100 seeds to be set to known values. The seed reconstruction error for simulation was less than 0.6 mm/3o. For the physical phantom experiments, IFPM absolute accuracy for position, polar angle, and azimuthal angel were (0.78 ± 0.57) mm, (5.8 ± 4.8)o, and (6.8 ± 4.0)o, respectively. It avoids the need to match corresponding seeds in each projection and accommodates incomplete data, overlapping seed clusters, and highly-migrated seeds. IFPM was further generalized from 5-parameter to 6-parameter model which was needed to reconstruct 3D pose of arbitrary-shape applicators. The voxelized 3D model of the applicator was obtained from external complex combinatorial geometric modeling. It is then integrated into the forward projection matching method for computing the 2D projections of the 3D ICB applicators, iteratively. The applicator reconstruction error for simulation was about 0.5 mm/2o. The residual 2D registration error (positional difference) between computed and actual measured applicator images was less than 1 mm for the intrauterine tandem and about 1.5 mm for the bilateral colpostats in each detector plane. By localizing the applicator’s internal structure and the sources, the effect of intra and inter-applicator attenuation can be included in the resultant dose distribution and CBCT metal streaking artifact mitigation. The localization accuracy of better than 1 mm and 6o has the potential to support more accurate Monte Carlo-based or 2D TG-43 dose calculations in clinical practice. It is hoped the clinical implementation of IFPM approach to localize elongated line-seed/applicator for intraoperative brachytherapy planning may have a positive impact on the treatment of prostate and cervical cancers.

Blast wave induced traumatic brain injury (bTBI) is a modality of injury that has come into prominence at the current time due to the large number of military and civilian personnel who have experienced the localized shock wave produced by explosive devices. The shock wave will travel concentrically outward from the explosive center, being absorbed and transmitted thru soft objects, such as tissue, and reflecting off stationary obstructions. Transmission and absorption in tissues can result in a number of physiological measureable injuries, the most common of which being what is frequently called “blast lung”. Blast lung involves the spalling effect at air-tissue interfaces. Another documented effect involves the asynchronous motion of tissue, particularly in the cranium, as the shock wave passes by. This predominately manifests itself in what is believed to be diffuse axonal injury and initiation of secondary injury mechanism. This study is designed to explore the relationship between shock waves and bTBI. A blast device was constructed for generating a free field shock wave through the high pressure rupture of a polycarbonate membrane. Air pressure in a small chamber is increased to a value several orders of magnitude greater than ambient air pressure and is held in place with the polycarbonate member. At the rupture of this membrane a shock wave is created. Measurements of this blast event, carried out with a piezoelectric pressure transducer, have shown that this shock wave is reproducible for the different membrane materials tested and is symmetrical with respect to the central axis of the high pressure chamber and exit nozzle. Having characterized the shock wave properties in the blast field, a location was chosen at which maximum shock wave pressure could be applied to the cranium for inducing bTBI. Experiments involving blast wave exposure were performed on two separate groups of animals in an attempt at establishing injury. One group was placed at a fixed distance directly below the blast nozzle, thereby experiencing both the shock wave and the associated air blast from the residual air in the chamber, and one placed at a defined distance off-axis to avoid the air blast, yet receiving two sequential blast exposures. All animal studies were approved by the VCU Institutional Animal Care and Use Committee. The degree of injury was then assessed with the use of magnetic resonance imaging (MRI) and spectroscopy (MRS). Image Data was acquired on a 2.4 Tesla magnet for assessing changes in either the total percent water concentration or the apparent diffusion coefficients (ADC) of selected regions of interest in the brain of rats. Localized proton spectroscopic data was acquired from a voxel placed centrally in the brain. The baseline values of these parameters were established before the induction of bTBI. After the blast exposure, the animals were followed up with MRI and MRS at defined intervals over a period of one week. The first group of animals received blast exposure directly underneath the blast device nozzle and the MR data does suggest changes in some of the measureable parameters from baseline following blast exposure. This blast wave data though is confounded with additional and undesirable characteristics of the blast wave. The second group of animals that received a pure shock wave blast exposure revealed no remarkable changes in the MR data pre- to post- blast exposure. The percent water concentration, ADC and spectroscopic parameters were for statistical purposes identical before and after the blast. The resolution of this negative result will require reconsideration of the free field blast exposure concept.

Statistical iterative reconstruction (SIR) algorithms for x-ray computed tomography (CT) have the potential to reconstruct images with less noise and systematic error than the conventional filtered backprojection (FBP) algorithm. More accurate reconstruction algorithms are important for reducing imaging dose and for a wide range of quantitative CT applications. The work presented herein investigates some potential advantages of one such statistically motivated algorithm called Alternating Minimization (AM). A simulation study is used to compare the tradeoff between noise and resolution in images reconstructed with the AM and FBP algorithms. The AM algorithm is employed with an edge-preserving penalty function, which is shown to result in images with contrast-dependent resolution. The AM algorithm always reconstructed images with less image noise than the FBP algorithm. Compared to previous studies in the literature, this is the first work to clearly illustrate that the reported noise advantage when using edge-preserving penalty functions can be highly dependent on the contrast of the object used for quantifying resolution. A polyenergetic version of the AM algorithm, which incorporates knowledge of the scanner’s x-ray spectrum, is then commissioned from data acquired on a commercially available CT scanner. Homogeneous cylinders are used to assess the absolute accuracy of the polyenergetic AM algorithm and to compare systematic errors to conventional FBP reconstruction. Methods to estimate the x-ray spectrum, model the bowtie filter and measure scattered radiation are outlined which support AM reconstruction to within 0.5% of the expected ground truth. The polyenergetic AM algorithm reconstructs the cylinders with less systematic error than FBP, in terms of better image uniformity and less object-size dependence. Finally, the accuracy of a post-processing dual-energy CT (pDECT) method to non-invasively measure a material’s photon cross-section information is investigated. Data is acquired on a commercial scanner for materials of known composition. Since the pDECT method has been shown to be highly sensitive to reconstructed image errors, both FBP and polyenergetic AM reconstruction are employed. Linear attenuation coefficients are estimated with residual errors of around 1% for energies of 30 keV to 1 MeV with errors rising to 3%-6% at lower energies down to 10 keV. In the ideal phantom geometry used here, the main advantage of AM reconstruction is less random cross-section uncertainty due to the improved noise performance.

Accelerated tumor cell repopulation during radiation therapy is one of the leading causes for low survival rates of head-and-neck cancer patients. The therapeutic effectiveness of radiotherapy could be improved by selectively targeting proliferating tumor subvolumes with higher doses of radiation. Positron emission tomography (PET) imaging with 3´-deoxy-3´-[18F]-fluorothymidine (FLT) has shown great potential as a non-invasive approach to characterizing the proliferation status of tumors. This thesis focuses on histopathological validation of FLT PET imaging specifically for image-guidance applications in biologically adaptive radiotherapy. The lack of experimental data supporting the use of FLT PET imaging for radiotherapy guidance is addressed by developing a novel methodology for histopathological validation of PET imaging. Using this new approach, the spatial concordance between the intratumoral pattern of FLT uptake and the spatial distribution of cell proliferation is demonstrated in animal tumors. First, a two-dimensional analysis is conducted comparing the microscopic FLT uptake as imaged with autoradiography and the distribution of active cell proliferation markers imaged with immunofluorescent microscopy. It was observed that when tumors present a pattern of cell proliferation that is highly dispersed throughout the tumor, even high-resolution imaging modalities such as autoradiography could not accurately determine the extent and spatial distribution of proliferative tumor subvolumes. While microscopic spatial coincidence between high FLT uptake regions and actively proliferative subvolumes was demonstrated in tumors with highly compartmentalized/aggregated features of cell proliferation, there were no conclusive results across the entire set of utilized tumor specimens. This emphasized the need for addressing the limited resolution of FLT PET when imaging microscopic patterns of cell proliferation. This issue was emphasized in the second part of the thesis where the spatial concordance between volumes segmented on FLT simulated FLT PET images and the three dimensional spatial distribution of cell proliferation markers was analyzed.

Dose verification is a critical component of adaptive radiotherapy, as it provides a measurement of treatment delivery success. Based on the measured outcome, the plan may be adapted to account for differences between the planned dose and the delivered dose. Although placement of an EPID behind the patient during treatment allows for exit dosimetry which may be used to reconstruct the delivered patient dose via backprojection of the fluence, there have not been any studies examining the basic assumption of backprojection-based dose verification: that deviations between the expected and delivered exit fluences are totally caused by errors in the delivered fluence, and not caused by patient geometry changes. In this dissertation, the validity of this assumption is tested. Exit fluence deviations caused by machine fluence delivery errors are measured as well as those caused by interfractional changes in the patient anatomy. Dose reconstruction errors resulting from the backprojection assumption are assessed. Correlations are examined between exit fluence deviations and patient dose reconstruction deviations. Based on these correlations, a decision tree is proposed detailing when caution should be taken in performing dose reconstruction to achieve delivery verification. Finally, a semi-automated dose verification tool is constructed for both clinical and research purposes.

The objective of this dissertation is to develop treatment planning techniques that have the potential to improve radiation therapy of time-dependent (4D) anatomy. Specifically, this study examines dose estimation, dose evaluation, and decision making in the context of optimizing lung cancer radiation therapy. Two methods of dose estimation are compared in patients with locally advanced and early stage lung cancer: dose computed on a single image (3D-dose) and deformably registered, accumulated dose (or 4D-dose). The results indicate that differences between 3D- and 4D- dose are not significant in organs at risk (OARs), however, 4D-dose to a moving lung cancer target can deviate from 3D-dose. These differences imply that optimization of the 4D-dose through multiple-anatomy optimization (MAO) can improve radiation therapy in 4D-anatomy. MAO incorporates time-dependent target and OAR geometry while enabling a simple, clinically realizable delivery. MAO has the potential to enhance the therapeutic ratio in terms of target coverage and OAR sparing in 4D-anatomy. In dose evaluation within 4D-anatomy; dose-to-mass is a more intuitive and precise metric in estimating the effects of radiation in tissues. Assuming physical density is proportional to functional tissue density, dose-to-mass has a 1-1 correspondence with radiation damage. Dose-to-mass optimization boosts dose in massive regions of lung cancer targets and can reduce integral dose to lung by preferentially treating through regions of low-density lung tissue. Finally, multi-criteria optimization (MCO) is implemented in order to clarify decision making during plan design for lung cancer treatment. An MCO basis set establishes a patient-specific decision space which reveals trade-offs in OAR-dose at a fixed, constrained target dose. By interpolating the MCO basis set and evaluating the plan on 4D-anatomy, patient- and organ- specific conservatism in plan design can be expressed in real time. Through improved methods of dose estimation, dose evaluation, and decision making, this dissertation will positively impact radiation therapy of time-dependent anatomy.

The development of hybrid PET/MRI imaging systems needs to be paralleled with the development of a hybrid intrinsic PET/MRI probes. The aim of this work was to develop and validate a novel radio-superparamagnetic nanoparticle (r-SPNP) for hybrid PET/MRI imaging. This was achieved with the synthesis of superparamagnetic iron oxide nanoparticles (SPIONs) that intrinsically incorporated 59Fe and manganese iron oxide nanoparticles (MIONs) that intrinsically incorporated 52Mn. Both [59Fe]-SPIONs and [52Mn]-MIONs were produced through thermal decomposition synthesis. The physiochemical characteristics of the r-SPNPs were assessed with TEM, DLS, and zeta-potential measurements, as well as in imaging phantom studies. The [59Fe]-SPIONs were evaluated in vivo with biodistribution and MR imaging studies. The biodistrubution studies of [59Fe]-SPIONs showed uptake in the liver. This corresponded with major MR signal contrast measured in the liver. 52Mn was produced on natural chromium through the 52Cr(p,n)52Mn reaction. The manganese radionuclides were separated from the target material through a liquid-liquid extraction. The αVβ3 integrin binding of [52Mn]-MION-cRGDs was evaluated with αVβ3 integrin solid phase assays, and the expression of αVβ3 integrin in U87MG xenograft tumors was characterized with fluorescence flow cytometry. [52Mn]-MION-cRGDs were used for in vivo PET and MR imaging of U87MG xenograft tumor bearing mice. PET data showed increased [52Mn]-MION-cRGD uptake compared with untargeted [52Mn]-MIONs. ROI analysis of PET and MRI data showed that MR contrasted corresponded with PET signal. Future work will utilize [52Mn]-MION-cRGDs in other tumor models and with hybrid PET/MRI imaging systems.

Platinum based chemotherapy is amongst the mainstream DNA-damaging agents used in clinical cancer therapy today. Agents such as cisplatin, carboplatin are clinically prescribed for the treatment of solid tumors either as single agents, in combination, or as part of multi-modality treatment strategy. Despite the potent anti-tumor activity of these drugs, overall effectiveness is still hampered by inadequate delivery and retention of drug in tumor and unwanted normal tissue toxicity, induced by non-selective accumulation of drug in normal cells and tissues. Utilizing molecular imaging and nanoparticle technologies, this thesis aims to contribute to better understanding of how to improve the profile of platinum based therapy. By developing a novel fluorinated derivative of carboplatin, incorporating a Flourine-18 (18F) moiety as an inherent part of the molecule, quantitative measures of drug concentration in tumors and normal tissues can be directly determined in vivo and within the intact individual environment. A potential impact of this knowledge will be helpful in predicting the overall response of individual patients to the treatment. Specifically, the aim of this project, therefore, is the development of a fluorinated carboplatin drug derivative with an inherent positron emission tomography (PET) imaging capability, so that the accumulation of the drug in the tumor and normal organs can be studied during the course of therapy . A secondary objective of this research is to develop a proof of concept for simultaneous imaging of a PET radiolabeled drug with a SPECT radiolabeled liposomal formulation, enabling thereby bi-modal imaging of drug and delivery vehicle in vivo. The approach is challenging because it involves development in PET radiochemistry, PET and SPECT imaging, drug liposomal encapsulation, and a dual-modal imaging of radiolabeled drug and radiolabeled vehicle. The principal development is the synthesis of fluorinated carboplatin 19F-FCP using 2-(5-fluoro-pentyl)-2-methyl malonic acid as the labeling agent to coordinate with the cisplatin aqua complex. It was then used to treat various cell lines and compared with cisplatin and carboplatin at different concentrations ranging from 0.001 µM to 100 µM for 72 hrs and 96 hrs. IC50 values calculated from cell viability indicated that 19F-FCP is a more potent drug than Carboplatin. Manual radiosynthesis and characterization of [18F]-FCP was performed using [18F]-2-(5-fluoro-pentyl)-2-methyl malonic acid with coordination with cisplatin aqua complex. Automated radiosynthesis of [18F]-FCP was optimized using the manual synthetic procedures and using them as macros for the radiosynthesizer. [18F]-FCP was evaluated in vivo with detailed biodistribution studies and PET imaging in normal and KB 3-1 and KB 8-5 tumor xenograft bearing nude mice. The biodistribution studies and PET imaging of [18F]-FCP showed major uptake in kidneys which attributes to the renal clearance of radiotracer. In vivo plasma and urine stability demonstrated intact [18F]-FCP. [111In]-Labeled Liposomes was synthesized and physiochemical properties were assessed with DLS. [111In]-Labeled Liposome was evaluated in vivo with detailed pharmacokinetic studies and SPECT imaging. The biodistribution and ROI analysis from SPECT imaging showed the spleen and liver uptake of [111In]-Labeled Liposome and subsequent clearance of activity with time. [18F]-FCP encapsulated [111In]-Labeled Liposome was developed and physiochemical properties were characterized with DLS. [18F]-FCP encapsulated [111In]-Labeled Liposome was used for in vivo dual tracer PET and SPECT imaging from the same nanoconstruct in KB 3-1 (sensitive) and COLO 205 (resistant) tumor xenograft bearing nude mice. PET imaging of [18F]-FCP in KB 3-1 (sensitive) and COLO 205 (resistant) tumor xenograft bearing nude mice was performed. Naked [18F]-FCP and [18F]-FCP encapsulated [111In]-Labeled Liposome showed different pharmacokinetic profiles. PET imaging of [18F]-FCP showed major uptake in kidneys and bladder. However, [18F]-FCP encapsulated [111In]-Labeled Liposome showed major uptake in RES in both PET and SPECT images. ROI analysis of SPECT image enabled by 111In corresponded with PET image enabled by 18F demonstrating the feasibility of dual tracer imaging from the single nanoconstruct. Future work involves the intensive in vitro characterization of [18F]-FCP encapsulated [111In]-Labeled Liposome and detailed in vivo evaluation of [18F]-FCP encapsulated [111In]-Labeled Liposome in various tumor models.

In radiation therapy, interfraction organ motion introduces a level of geometric uncertainty into the planning process. Plans, which are typically based upon a single instance of anatomy, must be robust against daily anatomical variations. For this problem, a model of the magnitude, direction, and likelihood of deformation is useful. In this thesis, principal component analysis (PCA) is used to statistically model the 3D organ motion for 19 prostate cancer patients, each with 8-13 fractional computed tomography (CT) images. Deformable image registration and the resultant displacement vector fields (DVFs) are used to quantify the interfraction systematic and random motion. By applying the PCA technique to the random DVFs, principal modes of random tissue deformation were determined for each patient, and a method for sampling synthetic random DVFs was developed. The PCA model was then extended to describe the principal modes of systematic and random organ motion for the population of patients. A leave-one-out study tested both the systematic and random motion model’s ability to represent PCA training set DVFs. The random and systematic DVF PCA models allowed the reconstruction of these data with absolute mean errors between 0.5-0.9 mm and 1-2 mm, respectively. To the best of the author’s knowledge, this study is the first successful effort to build a fully 3D statistical PCA model of systematic tissue deformation in a population of patients. By sampling synthetic systematic and random errors, organ occupancy maps were created for bony and prostate-centroid patient setup processes. By thresholding these maps, PCA-based planning target volume (PTV) was created and tested against conventional margin recipes (van Herk for bony alignment and 5 mm fixed [3 mm posterior] margin for centroid alignment) in a virtual clinical trial for low-risk prostate cancer. Deformably accumulated delivered dose served as a surrogate for clinical outcome. For the bony landmark setup subtrial, the PCA PTV significantly (p30, D20, and D5 to bladder and D50 to rectum, while increasing rectal D20 and D5. For the centroid-aligned setup, the PCA PTV significantly reduced all bladder DVH metrics and trended to lower rectal toxicity metrics. All PTVs covered the prostate with the prescription dose.