Advancing the Role of Proton Therapy for Spine Metastases Through Diagnostic Scan-Based Planning.

Purpose: Many patients with metastatic cancer live years beyond diagnosis, and there remains a need to improve the therapeutic ratio of metastasis-directed radiation for these patients. This study aimed to assess a process for delivering cost-effective palliative proton therapy to the spine using diagnostic scan-based planning (DSBP) and prefabricated treatment delivery devices. Materials and Methods: We designed and characterized a reusable proton aperture system that adjusts to multiple lengths for spine treatment. Next, we retrospectively identified 10 patients scan treated with thoracic proton therapy who also had a diagnostic computed tomography within 4 months of simulation. We contoured a T6-T9 target volume on both the diagnostic scans (DS) and simulation scans (SS). Using the aperture system, we generated proton plans on the DS using a posterior-anterior beam with no custom range compensator to treat T6-T9 to 8 Gy × 1. Plans were transferred to the SS to compare coverage and normal tissue doses, followed by robustness analysis. Finally, we compared normal tissue doses and costs between proton and photon plans. Results were compared using the Wilcoxon signed-rank test. Results: Median D95% on the DS plans was 101% (range, 100%-102%) of the prescription dose. Median Dmax was 107% (range, 105%-108%). When transferred to SS, coverage and hot spots remained acceptable for all cases. Heart and esophagus doses did not vary between the DS and SS proton plans (P >.2). Robustness analysis with 5 mm X/Y/Z shifts showed acceptable coverage (D95% > 98%) for all cases. Compared with the proton plans, the mean heart dose was higher for both anterior-posterior/posterior-anterior and volumetric modulated arc therapy plans (P < .01). Cost for proton DSBP was comparable to more commonly used photon regimens. Conclusion: Proton DSBP is technically feasible and robust, with superior sparing of the heart compared with photons. Eliminating simulation and custom devices increases the value of this approach in carefully selected patients.

Intravitreal injection of a rationally designed AAV capsid library in non-human primate identifies variants with enhanced retinal transduction and neutralizing antibody evasion.

Adeno-associated virus (AAV) continues to be the gold standard vector for therapeutic gene delivery and has proven especially useful for treating ocular disease. Intravitreal injection (IVtI) is a promising delivery route because it increases accessibility of gene therapies to larger patient populations. However, data from clinical and non-human primate (NHP) studies utilizing currently available capsids indicate that anatomical barriers to AAV and pre-existing neutralizing antibodies can restrict gene expression to levels that are "sub-therapeutic" in a substantial proportion of patients. Here, we performed a combination of directed evolution in NHPs of an AAV2-based capsid library with simultaneous mutations across six surface-exposed variable regions and rational design to identify novel capsid variants with improved retinal transduction following IVtI. Following two rounds of screening in NHP, enriched variants were characterized in intravitreally injected mice and NHPs and shown to have increased transduction relative to AAV2. Lead capsid variant, P2-V1, demonstrated an increased ability to evade neutralizing antibodies in human vitreous samples relative to AAV2 and AAV2.7m8. Taken together, this study further contributed to our understanding of the selective pressures associated with retinal transduction via the vitreous and identified promising novel AAV capsid variants for clinical consideration.

Dual-AAV vector-mediated expression of MYO7A improves vestibular function in a mouse model of Usher syndrome 1B.

Usher syndrome is the most common cause of deafness-blindness in the world. Usher syndrome type 1B (USH1B) is associated with mutations in MYO7A. Patients with USH1B experience deafness, blindness, and vestibular dysfunction. In this study, we applied adeno-associated virus (AAV)-mediated gene therapy to the shaker-1 (Myo7a4626SB/4626SB) mouse, a model of USH1B. The shaker-1 mouse has a nonsense mutation in Myo7a, is profoundly deaf throughout life, and has significant vestibular dysfunction. Because of the ∼6.7-kb size of the MYO7A cDNA, a dual-AAV approach was used for gene delivery, which involves splitting human MYO7A cDNA into 5' and 3' halves and cloning them into two separate AAV8(Y733F) vectors. When MYO7A cDNA was delivered to shaker-1 inner ears using the dual-AAV approach, cochlear hair cell survival was improved. However, stereocilium organization and auditory function were not improved. In contrast, in the vestibular system, dual-AAV-mediated MYO7A delivery significantly rescued hair cell stereocilium morphology and improved vestibular function, as reflected in a reduction of circling behavior and improved vestibular sensory-evoked potential (VsEP) thresholds. Our data indicate that dual-AAV-mediated MYO7A expression improves vestibular function in shaker-1 mice and supports further development of this approach for the treatment of disabling dizziness from vestibular dysfunction in USH1B patients.

Optimization of Capillary-Based Western Blotting for MYO7A.

Myosin VIIA (MYO7A)-associated Usher syndrome type 1B (USH1B) is a severe disorder that impacts the auditory, vestibular, and visual systems of affected patients. Due to the large size (~7.5 kb) of the MYO7A coding sequence, we have designed a dual adeno-associated virus (AAV) vector-based approach for the treatment of USH1B-related vision loss. Due to the added complexity of dual-AAV gene therapy, careful attention must be paid to the protein products expressed following vector recombination. In order to improve the sensitivity and quantifiability of our immunoassays, we adapted our traditional western blot protocol for use with the Jess™ Simple Western System. Following several rounds of testing, we optimized our protocol for the detection of MYO7A in two of our most frequently used sample types, mouse eyes, and infected HEK293 cell lysates.

Development of an AAV-CRISPR-Cas9-based treatment for dominant cone-rod dystrophy 6.

Cone-rod dystrophy 6 (CORD6) is caused by gain-of-function mutations in the GUCY2D gene, which encodes retinal guanylate cyclase-1 (RetGC1). There are currently no treatments available for this autosomal dominant disease, which is characterized by severe, early-onset visual impairment. The purpose of our study was to develop an adeno-associated virus (AAV)-CRISPR-Cas9-based approach referred to as "ablate and replace" and evaluate its therapeutic potential in mouse models of CORD6. This two-vector system delivers (1) CRISPR-Cas9 targeted to the early coding sequence of the wild-type and mutant GUCY2D alleles and (2) a CRISPR-Cas9-resistant cDNA copy of GUCY2D ("hardened" GUCY2D). Together, these vectors knock out ("ablate") expression of endogenous RetGC1 in photoreceptors and supplement ("replace") a healthy copy of exogenous GUCY2D. First, we confirmed that ablation of mutant R838S GUCY2D was therapeutic in a transgenic mouse model of CORD6. Next, we established a proof of concept for "ablate and replace" and optimized vector doses in Gucy2e+/-:Gucy2f-/- and Gucy2f-/- mice, respectively. Finally, we confirmed that the "ablate and replace" approach stably preserved retinal structure and function in a novel knockin mouse model of CORD6, the RetGC1 (hR838S, hWT) mouse. Taken together, our results support further development of the "ablate and replace" approach for treatment of CORD6.

Improving retinal vascular endothelial cell tropism through rational rAAV capsid design.

Vascular endothelial cells (VEC) are essential for retinal homeostasis and their dysfunction underlies pathogenesis in diabetic retinopathy (DR) and exudative age-related macular degeneration (AMD). Studies have shown that recombinant adeno-associated virus (rAAV) vectors are effective at delivering new genetic material to neural and glial cells within the retina, but targeting VECs remains challenging. To overcome this limitation, herein we developed rAAV capsid mutant vectors with improved tropism towards retinal VEC. rAAV2/2, 2/2[QuadYF-TV], and rAAV2/9 serotype vectors (n = 9, capsid mutants per serotype) expressing GFP were generated by inserting heptameric peptides (7AA) designed to increase endothelial targeting at positions 588 (2/2 and 2/2[QuadYF-TV] or 589 (2/9) of the virus protein (VP 1-3). The packaging and transduction efficiency of the vectors were assessed in HEK293T and bovine VECs using Fluorescence microscopy and flow cytometry, leading to the identification of one mutant, termed EC5, that showed improved endothelial tropism when inserted into all three capsid serotypes. Intra-ocular and intravenous administration of EC5 mutants in C57Bl/6j mice demonstrated moderately improved transduction of the retinal vasculature, particularly surrounding the optic nerve head, and evidence of sinusoidal endothelial cell transduction in the liver. Most notably, intravenous administration of the rAAV2/2[QuadYF-TV] EC5 mutant led to a dramatic and unexpected increase in cardiac muscle transduction.

ß-secretase 1 overexpression by AAV-mediated gene delivery prevents retina degeneration in a mouse model of age-related macular degeneration.

We reported previously that ß-site amyloid precursor protein cleaving enzyme (BACE1) is strongly expressed in the normal retina and that BACE1-/- mice develop pathological phenotypes associated with age-related macular degeneration (AMD). BACE1 expression is increased within the neural retina and retinal pigment epithelium (RPE) in AMD donor eyes suggesting that increased BACE1 is compensatory. We observed that AAV-mediated BACE1 overexpression in the RPE was maintained up to 6 months after AAV1-BACE1 administration. No significant changes in normal mouse visual function or retinal morphology were observed with low-dose vector while the high-dose vector demonstrated some early pathology which regressed with time. No increase in ß-amyloid was observed. BACE1 overexpression in the RPE of the superoxide dismutase 2 knockdown (SOD2 KD) mouse, which exhibits an AMD-like phenotype, prevented loss of retinal function and retinal pathology, and this was sustained out to 6 months. Furthermore, BACE1 overexpression was able to inhibit oxidative stress, microglial changes, and loss of RPE tight junction integrity (all features of AMD) in SOD2 KD mice. In conclusion, BACE1 plays a key role in retina/RPE homeostasis, and BACE1 overexpression offers a novel therapeutic target in the treatment of AMD.

Preclinical studies in support of phase I/II clinical trials to treat GUCY2D-associated Leber congenital amaurosis.

Mutations in GUCY2D are associated with severe early-onset retinal dystrophy, Leber congenital amaurosis type 1 (LCA1), a leading cause of blindness in children. Despite a high degree of visual disturbance stemming from photoreceptor dysfunction, patients with LCA1 largely retain normal photoreceptor structure, suggesting that they are good candidates for gene replacement therapy. The purpose of this study was to conduct the preclinical and IND-enabling experiments required to support clinical application of AAV5-hGRK1-GUCY2D in patients harboring biallelic recessive mutations in GUCY2D. Preclinical studies were conducted in mice to evaluate the effect of vector manufacturing platforms and transgene species on the therapeutic response. Dose-ranging studies were conducted in cynomolgus monkeys to establish the minimum dose required for efficient photoreceptor transduction. Good laboratory practice (GLP) studies evaluated systemic biodistribution in rats and toxicology in non-human primates (NHPs). These results expanded our knowledge of dose response for an AAV5-vectored transgene under control of the human rhodopsin kinase (hGRK1) promoter in NHPs with respect to photoreceptor transduction and safety and, in combination with the rat biodistribution and mouse efficacy studies, informed the design of a first-in-human clinical study in patients with LCA1.

Adapting to the Adaptive Radiation Workflow: Incorporating Video Sign Out for Improved Safety and Efficiency as Part of Magnetic Resonance Image Guided Adaptive Radiation.

 : Magnetic resonance image guided adaptive radiation therapy (MRgART) represents a significant improvement in our ability to deliver therapeutic radiation. However, for the process of MRgART to be carried out safely and efficiently, the covering radiation oncologist must be aware of all aspects of a patient's case, because they will be required to recontour and replan the patient before each treatment. In this report, we will demonstrate our initial experience with a video sign-out process to convey the detailed level of information required for the covering physician to treat patients safely and effectively with MRgART. We then describe our optimized video sign-out process to allow for other centers to adopt a similar approach.

Night vision restored in days after decades of congenital blindness.

Signaling of vision to the brain starts with the retinal phototransduction cascade which converts visible light from the environment into chemical changes. Vision impairment results when mutations inactivate proteins of the phototransduction cascade. A severe monogenically inherited blindness, Leber congenital amaurosis (LCA), is caused by mutations in the GUCY2D gene, leading to a molecular defect in the production of cyclic GMP, the second messenger of phototransduction. We studied two patients with GUCY2D-LCA who were undergoing gene augmentation therapy. Both patients had large deficits in rod photoreceptor-based night vision before intervention. Within days of therapy, rod vision in both patients changed dramatically; improvements in visual function and functional vision in these hyper-responding patients reached more than 3 log10 units (1000-fold), nearing healthy rod vision. Quick activation of the complex molecular pathways from retinal photoreceptor to visual cortex and behavior is thus possible in patients even after being disabled and dormant for decades.

Operational Performance of a Compact Proton Therapy System: A 5-Year Experience.

Purpose: We present an analysis of various operational metrics for a novel compact proton therapy system, including clinical case mix, subsystems utilization, and quality assurance trends in beam delivery parameters over a period of 5 years. Materials and Methods: Patient-specific data from a total of 850 patients (25,567 fractions) have been collected and analyzed. The patient mix include a variety of simple, intermediate, and complex cases. Beam-specific delivery parameters for a total of 3585 beams were analyzed. In-room imaging system usage for off-line adaptive purpose is reported. We also report key machine performances metrics based on routine quality assurance in addition to uptime. Results: Our analysis shows that system subcomponents including gantry and patient positioning system have maintained a tight mechanical tolerance over the 5-year period. Various beam parameters were all within acceptable tolerances with no clear trends. Utilization frequency histograms of gantry and patient positioning system show that only a small fraction of all available angles was used for patient deliveries with cardinal angels as the most usable. Similarly, beam-specific metrics, such as range, modulation, and air gaps, were clustered unevenly over the available range indicating that this compact system was more than capable to treat the complex variety of tumors of our patient mix. Conclusion: Our data show that this compact system is versatile, robust, and capable of delivering complex treatments like a large full-gantry system. Utilization data show that a fraction of all subcomponents range of angular motion has been used. Compilation of beam-specific metrics, such as range and modulation, show uneven distributions with specific clustering over the entire usable range. Our findings could be used to further optimize the performance and cost-effectiveness of future compact proton systems.

Dosimetric Comparison of Various Spot Placement Techniques in Proton Pencil Beam Scanning.

Purpose: To present quantitative dosimetric evaluations of five proton pencil beam spot placement techniques. Materials and Methods: The spot placement techniques that were investigated include two grid-based (rectilinear grid and hexagonal grid, both commonly available in commercial planning systems) and three boundary-contoured (concentric contours, hybrid, and optimized) techniques. Treatment plans were created for two different target volumes, one spherical and one conical. An optimal set of planning parameters was defined for all treatment plans and the impact of spot placement techniques on the plan quality was evaluated in terms of lateral/distal dose falloff, normal tissue sparing, conformity and homogeneity of dose distributions, as well as total number of spots used. Results: The results of this work highlight that for grid-based spot placement techniques, the dose conformity is dependent on target cross-sectional shape perpendicular to beam direction, which changes for each energy layer. This variable conformity problem is mitigated by using boundary contoured spot placement techniques. However, in the case of concentric contours, the conformity is improved but at the cost of decreased homogeneity inside the target. Hybrid and optimized spot placement techniques, which use contoured spots at the boundary and gridlike interior spot patterns, provide more uniform dose distributions inside the target volume while maintaining the improved dose conformity. The optimized spot placement technique improved target coverage, homogeneity of dose, and minimal number of spots. The dependence of these results on spot size is also presented for both target shapes. Conclusion: This work illustrates that boundary-contoured spot placement techniques offer marked improvement in dosimetry metrics when compared to commercially available grid-based techniques for a range of proton scanned beam spot sizes.

Proposal and Evaluation of a Physician-Free, Real-Time On-Table Adaptive Radiotherapy (PF-ROAR) Workflow for the MRIdian MR-Guided LINAC.

With the implementation of MR-LINACs, real-time adaptive radiotherapy has become a possibility within the clinic. However, the process of adapting a patient's plan is time consuming and often requires input from the entire clinical team, which translates to decreased throughput and limited patient access. In this study, the authors propose and simulate a workflow to address these inefficiencies in staffing and patient throughput. Two physicians, three radiation therapists (RTT), and a research fellow each adapted bladder and bowel contours for 20 fractions from 10 representative patient plans. Contouring ability was compared via calculation of a Dice Similarity Index (DSI). The DSI for bladder and bowel based on each potential physician-therapist pair, as well as an inter-physician comparison, exhibited good overlap amongst all comparisons (p = 0.868). Plan quality was compared through calculation of the conformity index (CI), as well as an evaluation of the plan's dose to a 'gold standard' set of structures. Overall, non-physician plans passed 91.2% of the time. Of the eight non-physician plans that failed their clinical evaluation, six also failed their evaluation against the 'gold standard'. Another two plans that passed their clinical evaluation subsequently failed in their evaluation against the 'gold standard'. Thus, the PF-ROAR process has a success rate of 97.5%, with 78/80 plans correctly adapted to the gold standard or halted at treatment. These findings suggest that a physician-free workflow can be well tolerated provided RTTs continue to develop knowledge of MR anatomy and careful attention is given to understanding the complexity of the plan prior to treatment.

We need to be braver about the generalizability crisis.

We applaud the effort to draw attention to generalizability concerns in twenty-first-century psychological research. Yet we do not feel that a pessimistic perspective is warranted. We outline a continuum of available methodological tools and perspectives, including incremental steps and meta-analytic approaches that can be readily and easily deployed by researchers to advance generalizability claims in a forward-looking manner.

Practical Considerations for Single Isocenter LINAC Radiosurgery of Multiple Brain Metastases.

The purpose of this paper is to summarize treatment guidelines for the performance of single isocenter LINAC radiosurgery of multiple brain metastases developed and used by 3 experienced centers. This article is not meant to provide consensus guidelines. Rather, this is a practical, "how we do it" reference without substantial discussion. To serve as a treatment reference, the great majority of the information is presented in topic-specific tables.

Research productivity of radiation therapy physics faculty in the United States.

PURPOSE: Research productivity metrics are important for decisions regarding hiring, retention, and promotion in academic medicine, and these metrics can vary widely among different disciplines. This article examines productivity metrics for radiation therapy physicists (RTP) in the United States. METHODS AND MATERIALS: Database searches were performed for RTP faculty at US institutions that have RTP residencies accredited by the Commission on Accreditation of Medical Physics Education Programs (CAMPEP). Demographics, academic rank, number of publications, academic career length, Hirsch index (h-index), m-quotient, and history of National Institutes of Health (NIH) funding as a principal investigator (PI) were collected for each RTP. Logistic regression was performed to determine the probability of academic rank as a function of h-index and m-quotient. Statistical tests used included the Wilcoxon ranked sum test and the Pearson χ2 test. RESULTS: A total of 1038 faculty and staff were identified at 78 institutions with CAMPEP-accredited residencies. The average RTP academic career duration is 13.5 years, with 46.7 total publications, h-index of 10.7, and m-quotient of 0.66. Additionally, 10.5% of RTP have a history of NIH funding as a PI. Large disparities were found in academic productivity of doctoral-prepared physicists compared to those with a terminal master's degree. For differences in junior and senior faculty, statistical tests yielded significance in career duration, number of publications, h-index, and m-quotient. Gender disparities were identified in the overall distribution of RTP consistent with the membership of the American Association of Physicists in Medicine. Further gender disparities were found in the number of doctoral-prepared RTP and physicists in senior faculty roles. CONCLUSIONS: This manuscript provides objective benchmark data regarding research productivity of academic RTP. These data may be of interest to faculty preparing for promotion, and also to institutional leadership.

Impact of Radiation Oncology Alternative Payment Model on Community Cancer Centers.

PURPOSE: An episode-based payment model, the Radiation Oncology Alternative Payment Model (RO-APM), is scheduled to go into effect in January 2022. This article investigates the effects of RO-APM on hospital-based and freestanding community centers. METHODS: Historical Medicare data used to generate the RO-APM base rates were reviewed. A sensitivity analysis was performed to show how the RO-APM reimbursements compare with current reimbursements for commonly accepted treatment schedules and with current reimbursements at a large community practice. RESULTS: The RO-APM base rates represent a 2.2% decrease in overall Medicare reimbursement. Freestanding centers have historically billed at higher rates than hospital-based centers, however, and the RO-APM base rates represent a 6% decrease in global reimbursement for freestanding centers. The sensitivity analysis showed that, except for proton therapy, moderately hypofractionated treatment schedules will receive comparable reimbursement under RO-APM. Treatments using higher numbers of fractions of intensity-modulated radiation therapy or protons will see larger decreases in reimbursement. Application of the RO-APM base rates to the 2020 Medicare treatments in our health care network would result in small changes in expected reimbursement, but our sensitivity analysis indicated that Medicare reimbursement reductions could be as large as 23%. CONCLUSION: Compared with historical Medicare reimbursement, RO-APM base rates provide lower reimbursement for many common treatment scenarios, and this will have a larger effect on centers that use complex treatment techniques and longer fractionation schedules or have a large Medicare population.

Safety and improved efficacy signals following gene therapy in childhood blindness caused by GUCY2D mutations.

A first-in-human clinical trial of gene therapy in Leber congenital amaurosis due to mutations in the GUCY2D gene is underway, and early results are summarized. A recombinant adeno-associated virus serotype 5 (rAAV5) vector carrying the human GUCY2D gene was delivered by subretinal injection to one eye in three adult patients with severe visual loss, nystagmus, but preserved retinal structure. Safety and efficacy parameters were monitored for 9 months post-operatively. No systemic toxicity was detected; there were no serious adverse events, and ocular adverse events resolved. P1 and P2 showed statistically significant rod photoreceptor vision improvement by full-field stimulus testing in the treated eye. P1 also showed improvement in pupillary responses. Visual acuity remained stable from baseline in P1 and P2. P3, however, showed a gain of 0.3 logMAR in the treated eye, indicating greater cone-photoreceptor function. The results show safety and both rod- and cone-mediated efficacy of this therapy.