Codon usage influences fitness through RNA toxicity.

Many organisms are subject to selective pressure that gives rise to unequal usage of synonymous codons, known as codon bias. To experimentally dissect the mechanisms of selection on synonymous sites, we expressed several hundred synonymous variants of the GFP gene in Escherichia coli, and used quantitative growth and viability assays to estimate bacterial fitness. Unexpectedly, we found many synonymous variants whose expression was toxic to E. coli Unlike previously studied effects of synonymous mutations, the effect that we discovered is independent of translation, but it depends on the production of toxic mRNA molecules. We identified RNA sequence determinants of toxicity and evolved suppressor strains that can tolerate the expression of toxic GFP variants. Genome sequencing of these suppressor strains revealed a cluster of promoter mutations that prevented toxicity by reducing mRNA levels. We conclude that translation-independent RNA toxicity is a previously unrecognized obstacle in bacterial gene expression.

Membrane and synaptic defects leading to neurodegeneration in Adar mutant Drosophila are rescued by increased autophagy.

BACKGROUND: In fly brains, the Drosophila Adar (adenosine deaminase acting on RNA) enzyme edits hundreds of transcripts to generate edited isoforms of encoded proteins. Nearly all editing events are absent or less efficient in larvae but increase at metamorphosis; the larger number and higher levels of editing suggest editing is most required when the brain is most complex. This idea is consistent with the fact that Adar mutations affect the adult brain most dramatically. However, it is unknown whether Drosophila Adar RNA editing events mediate some coherent physiological effect. To address this question, we performed a genetic screen for suppressors of Adar mutant defects. Adar5G1 null mutant flies are partially viable, severely locomotion defective, aberrantly accumulate axonal neurotransmitter pre-synaptic vesicles and associated proteins, and develop an age-dependent vacuolar brain neurodegeneration. RESULTS: A genetic screen revealed suppression of all Adar5G1 mutant phenotypes tested by reduced dosage of the Tor gene, which encodes a pro-growth kinase that increases translation and reduces autophagy in well-fed conditions. Suppression of Adar5G1 phenotypes by reduced Tor is due to increased autophagy; overexpression of Atg5, which increases canonical autophagy initiation, reduces aberrant accumulation of synaptic vesicle proteins and suppresses all Adar mutant phenotypes tested. Endosomal microautophagy (eMI) is another Tor-inhibited autophagy pathway involved in synaptic homeostasis in Drosophila. Increased expression of the key eMI protein Hsc70-4 also reduces aberrant accumulation of synaptic vesicle proteins and suppresses all Adar5G1 mutant phenotypes tested. CONCLUSIONS: These findings link Drosophila Adar mutant synaptic and neurotransmission defects to more general cellular defects in autophagy; presumably, edited isoforms of CNS proteins are required for optimum synaptic response capabilities in the brain during the behaviorally complex adult life stage.

Pin1 and WWP2 regulate GluR2 Q/R site RNA editing by ADAR2 with opposing effects.

ADAR2 catalyses the deamination of adenosine to inosine at the GluR2 Q/R site in the pre-mRNA encoding the critical subunit of AMPA receptors. Among ADAR2 substrates this is the vital one as editing at this position is indispensable for normal brain function. However, the regulation of ADAR2 post-translationally remains to be elucidated. We demonstrate that the phosphorylation-dependent prolyl-isomerase Pin1 interacts with ADAR2 and is a positive regulator required for the nuclear localization and stability of ADAR2. Pin1(-/-) mouse embryonic fibroblasts show mislocalization of ADAR2 in the cytoplasm and reduced editing at the GluR2 Q/R and R/G sites. The E3 ubiquitin ligase WWP2 plays a negative role by binding to ADAR2 and catalysing its ubiquitination and subsequent degradation. Therefore, ADAR2 protein levels and catalytic activity are coordinately regulated in a positive manner by Pin1 and negatively by WWP2 and this may have downstream effects on the function of GluR2. Pin1 and WWP2 also regulate the large subunit of RNA Pol II, so these proteins may also coordinately regulate other key cellular proteins.

Editing independent effects of ADARs on the miRNA/siRNA pathways.

Adenosine deaminases acting on RNA (ADARs) are best known for altering the coding sequences of mRNA through RNA editing, as in the GluR-B Q/R site. ADARs have also been shown to affect RNA interference (RNAi) and microRNA processing by deamination of specific adenosines to inosine. Here, we show that ADAR proteins can affect RNA processing independently of their enzymatic activity. We show that ADAR2 can modulate the processing of mir-376a2 independently of catalytic RNA editing activity. In addition, in a Drosophila assay for RNAi deaminase-inactive ADAR1 inhibits RNAi through the siRNA pathway. These results imply that ADAR1 and ADAR2 have biological functions as RNA-binding proteins that extend beyond editing per se and that even genomically encoded ADARs that are catalytically inactive may have such functions.

Functional conservation in human and Drosophila of Metazoan ADAR2 involved in RNA editing: loss of ADAR1 in insects.

Flies with mutations in the single Drosophila Adar gene encoding an RNA editing enzyme involved in editing 4% of all transcripts have severe locomotion defects and develop age-dependent neurodegeneration. Vertebrates have two ADAR-editing enzymes that are catalytically active; ADAR1 and ADAR2. We show that human ADAR2 rescues Drosophila Adar mutant phenotypes. Neither the short nuclear ADAR1p110 isoform nor the longer interferon-inducible cytoplasmic ADAR1p150 isoform rescue walking defects efficiently, nor do they correctly edit specific sites in Drosophila transcripts. Surprisingly, human ADAR1p110 does suppress age-dependent neurodegeneration in Drosophila Adar mutants whereas ADAR1p150 does not. The single Drosophila Adar gene was previously assumed to represent an evolutionary ancestor of the multiple vertebrate ADARs. The strong functional similarity of human ADAR2 and Drosophila Adar suggests rather that these are true orthologs. By a combination of direct cloning and searching new invertebrate genome sequences we show that distinct ADAR1 and ADAR2 genes were present very early in the Metazoan lineage, both occurring before the split between the Bilateria and Cnidarians. The ADAR1 gene has been lost several times, including during the evolution of insects and crustacea. These data complement our rescue results, supporting the idea that ADAR1 and ADAR2 have evolved highly conserved, distinct functions.

Stereotactic body radiation therapy for nonresectable tumors of the pancreas.

BACKGROUND: Stereotactic body radiation therapy (SBRT) has emerged as a potential treatment option for local tumor control of primary malignancies of the pancreas. We report on our experience with SBRT in patients with pancreatic adenocarcinoma who were found not to be candidates for surgical resection. METHODS: The prospective database of the first 20 consecutive patients receiving SBRT for unresectable pancreatic adenocarcinomas and a neuroendocrine tumor under an IRB approved protocol was reviewed. Prior to SBRT, cylindrical solid gold fiducial markers were placed within or around the tumor endoscopically (n = 13), surgically (n = 4), or percutaneously under computerized tomography (CT)-guidance (n = 3) to allow for tracking of tumor during therapy. Mean radiation dose was 25 Gray (Gy) (range 22-30 Gy) delivered over 1-3 fractions. Chemotherapy was given to 68% of patients in various schedules/timing. RESULTS: Patients had a mean gross tumor volume of 57.2 cm(3) (range 10.1-118 cm(3)) before SBRT. The mean total gross tumor volume reduction at 3 and 6 mo after SBRT were 21% and 38%, respectively (P < 0.05). Median follow-up was 14.57 mo (range 5-23 mo). The overall rate of freedom from local progression at 6 and 12 mo were 88% and 65%. The probability of overall survival at 6 and 12 mo were 89% and 56%. No patient had a complication related to fiducial markers placement regardless of modality. The rate of radiation-induced adverse events was: grade 1-2 (11%) and grade 3 (16%). There were no grade 4/5 adverse events seen. CONCLUSION: Our preliminary results showed SBRT as a safe and likely effective local treatment modality for pancreatic primary malignancy with acceptable rate of adverse events.

Quantifying risk using FMEA: An alternate approach to AAPM TG-100 for scoring failures and evaluating clinical workflow.

PURPOSE: Renovation of the brachytherapy program at a leading cancer center utilized methods of the AAPM TG-100 report to objectively evaluate current clinical brachytherapy workflows and develop techniques for minimizing the risk of failures, increasing efficiency, and consequently providing opportunities for improved treatment quality. The TG-100 report guides evaluation of clinical workflows with recommendations for identifying potential failure modes (FM) and scoring them from the perspective of their occurrence frequency O, failure severity S, and inability to detect them D. The current study assessed the impact of differing methods to determine the risk priority number (RPN) beyond simple multiplication. METHODS AND MATERIALS: The clinical workflow for a complex brachytherapy procedure was evaluated by a team of 15 staff members, who identified discrete FM using alternate scoring scales than those presented in the TG-100 report. These scales were expanded over all clinically relevant possibilities with care to emphasize mitigation of natural bias for scoring near the median range as well as to enhance the overall scoring-system sensitivity. Based on staff member perceptions, a more realistic measure of risk was determined using weighted functions of their scores. RESULTS: This new method expanded the range of RPN possibilities by a factor of 86, improving evaluation and recognition of safe and efficient clinical workflows. Mean RPN values for each FM decreased by 44% when changing from the old to the new clinical workflow, as evaluated using the TG-100 method. This decreased by 66% when evaluated with the new method. As a measure of the total risk associated with an entire clinical workflow, the integral of RPN values increased by 15% and decreased by 31% with the TG-100 and new methods, respectively. CONCLUSIONS: This appears to be the first application of an alternate approach to the TG-100 method for evaluating the risk of clinical workflows. It exemplifies the risk analysis techniques necessary to rapidly evaluate simple clinical workflows appropriately.

Novel and programmatic improvements to the workflow associated with the AccuBoost breast brachytherapy procedure.

PURPOSE: While the noninvasive breast brachytherapy (NIBB) treatment procedure, known as AccuBoost, for breast cancer patients is well established, the treatment quality can be improved by the efficiency of the workflow delivery. A formalized approach evaluated the current workflow through failure modes and effects analysis and generated insight for developing new procedural workflow techniques to improve the clinical treatment process. METHODS AND MATERIALS: AccuBoost treatments were observed for several months while gathering details on the multidisciplinary workflow. A list of possible failure modes for each procedure step was generated and organized by timing within the treatment process. A team of medical professionals highlighted procedural steps that unnecessarily increased treatment time, as well as introduced quality deficiencies involving applicator setup, treatment planning, and quality control checks preceding brachytherapy delivery. Procedural improvements and their impact on the clinical workflow are discussed. RESULTS: The revised clinical workflow included the following key procedural enhancements. Prepatient arrival: Improvement of prearrival preparation requires advance completion of dose calculation documentation with patient-specific setup data. Patient arrival pretreatment: Physicists carry out dwell time calculations and check the plan, while the therapist concurrently performs several checks of the ensuing hardware configuration. TREATMENT: An electronic method to export the associated HDR brachytherapy paperwork to the electronic medical record system with electronic signatures and captured approvals was generated. Posttreatment: The therapist confirms the applicators were appropriately positioned, and treatment was delivered as expected. CONCLUSIONS: The procedural improvements reduced the overall treatment time, improved consistency across users, and eased performance of this special procedure for all participants.

Stereotactic body radiosurgery for pelvic relapse of gynecologic malignancies.

Clinical management of pelvic relapses from gynecologic malignancies remains challenging. Bulky pelvic relapses often lead to symptomatic cancer-related complications and poor clinical outcomes. Options may be limited by prior surgical, chemotherapeutic, and radiation treatment. Stereotactic body radiosurgery is a novel treatment modality which allows high radiation dose delivery in a non-coplanar fashion with sub-millimeter precision utilizing a linear accelerator mounted on a robotic arm. This study details our clinical experience with stereotactic body radiosurgery for treatment of patients with pelvic relapses of gynecologic malignancies after prior pelvic radiation.

Cyberknife radiosurgery for squamous cell carcinoma of vulva after prior pelvic radiation therapy.

Limited options exist for patients experiencing a local recurrence of vulvar malignancies after surgery and pelvic radiation. These recurrences often are associated with cancer-related skin desquamation and poor clinical outcomes. A new radiotherapeutic treatment modality for the previously irradiated patient is cyberknife radiosurgery, which uses a linear accelerator mounted on an industrial robotic arm to allow non-coplanar radiation therapy delivery with sub-millimeter precision. This study describes the first reported use of cyberknife radiosurgery for the treatment of recurrent vulvar cancer in three women.

Method for estimating skeletal spongiosa volume and active marrow mass in the adult male and adult female.

UNLABELLED: Active bone marrow is one of the more radiosensitive tissues in the human body and, hence, it is important to predict and possibly avoid myelotoxicity in radionuclide therapies. The MIRD schema currently used to calculate marrow dose generally requires knowledge of the patient's total skeletal active marrow mass -- a value that, at present, cannot be directly measured. Conceptually, the active marrow mass in a given skeletal region may be obtained given knowledge of the trabecular spongiosa volume (SV) of the bone site. A recent study has established a multiple regression model to easily calculate total skeletal SV (or TSSV) based on simple skeletal measurements obtained from a pelvic CT scan or radiograph. This model, based on data from only 20 cadavers, did not account for sex differences in TSSV. This study thus extends this work toward sex-specific models. METHODS: Twenty male and 20 female cadavers were subjected to whole-body CT. Bone sites containing active bone marrow were manually segmented to obtain SV at each site. In addition to age and height, 14 CT-based skeletal measurements were recorded for each cadaver. Multiple linear regression techniques were used to determine the best subset of measurements that allowed an accurate prediction of TSSV. RESULTS: A pooled model (R(2) = 0.76) and a sex-specific model (R(2) = 0.79) are provided. A leave-one-out analysis reveals that these models predict total SV with less than 10% error for 50%-70% of subjects, and with less than 20% error for 70%-90% of subjects. Tables were constructed that provide the percent distribution of SV in active-marrow containing bone sites for both males and females. CONCLUSION: This study provides models that can be used to simply, yet accurately, predict total SV in individuals within the clinical setting. The models require only 2 or 3 skeletal measurements that can be easily measured on a pelvic CT scan. Even though this study does not conclusively determine which model is best at predicting TSSV, the sex-specific model is most consistent at providing reasonable estimates of TSSV. This study also explains how the predictive TSSV model can be used to estimate patient-specific active bone marrow mass under the assumption of reference values of marrow volume fraction and bone marrow cellularity by skeletal site.

Linear regression model for predicting patient-specific total skeletal spongiosa volume for use in molecular radiotherapy dosimetry.

UNLABELLED: The toxicity of red bone marrow is widely considered to be a key factor in restricting the activity administered in molecular radiotherapy to suboptimal levels. The assessment of marrow toxicity requires an assessment of the dose absorbed by red bone marrow which, in many cases, requires knowledge of the total red bone marrow mass in a given patient. Previous studies demonstrated, however, that a close surrogate-spongiosa volume (combined tissues of trabecular bone and marrow)-can be used to accurately scale reference patient red marrow dose estimates and that these dose estimates are predictive of marrow toxicity. Consequently, a predictive model of the total skeletal spongiosa volume (TSSV) would be a clinically useful tool for improving patient specificity in skeletal dosimetry. METHODS: In this study, 10 male and 10 female cadavers were subjected to whole-body CT scans. Manual image segmentation was used to estimate the TSSV in all 13 active marrow-containing skeletal sites within the adult skeleton. The age, total body height, and 14 CT-based skeletal measurements were obtained for each cadaver. Multiple regression was used with the dependent variables to develop a model to predict the TSSV. RESULTS: Os coxae height and width were the 2 skeletal measurements that proved to be the most important parameters for prediction of the TSSV. The multiple R(2) value for the statistical model with these 2 parameters was 0.87. The analysis revealed that these 2 parameters predicted the estimated the TSSV to within approximately +/-10% for 15 of the 20 cadavers and to within approximately +/-20% for all 20 cadavers in this study. CONCLUSION: Although the utility of spongiosa volume in estimating patient-specific active marrow mass has been shown, estimation of the TSSV in active marrow-containing skeletal sites via patient-specific image segmentation is not a simple endeavor. However, the alternate approach demonstrated in this study is fairly simple to implement in a clinical setting, as the 2 input measurements (os coxae height and width) can be made with either pelvic CT scanning or skeletal radiography.

CT volumetry of the skeletal tissues.

Computed tomography (CT) is an important and widely used modality in the diagnosis and treatment of various cancers. In the field of molecular radiotherapy, the use of spongiosa volume (combined tissues of the bone marrow and bone trabeculae) has been suggested as a means to improve the patient-specificity of bone marrow dose estimates. The noninvasive estimation of an organ volume comes with some degree of error or variation from the true organ volume. The present study explores the ability to obtain estimates of spongiosa volume or its surrogate via manual image segmentation. The variation among different segmentation raters was explored and found not to be statistically significant (p value >0.05). Accuracy was assessed by having several raters manually segment a polyvinyl chloride (PVC) pipe with known volumes. Segmentation of the outer region of the PVC pipe resulted in mean percent errors as great as 15% while segmentation of the pipe's inner region resulted in mean percent errors within approximately 5%. Differences between volumes estimated with the high-resolution CT data set (typical of ex vivo skeletal scans) and the low-resolution CT data set (typical of in vivo skeletal scans) were also explored using both patient CT images and a PVC pipe phantom. While a statistically significant difference (p value <0.002) between the high-resolution and low-resolution data sets was observed with excised femoral heads obtained following total hip arthroplasty, the mean difference between high-resolution and low-resolution data sets was found to be only 1.24 and 2.18 cm3 for spongiosa and cortical bone, respectively. With respect to differences observed with the PVC pipe, the variation between the high-resolution and low-resolution mean percent errors was a high as approximately 20% for the outer region volume estimates and only as high as approximately 6% for the inner region volume estimates. The findings from this study suggest that manual segmentation is a reasonably accurate and reliable means for the in vivo estimation of spongiosa volume. This work also provides a foundation for future studies where spongiosa volumes are estimated by various raters in more comprehensive CT data

Correlations of total pelvic spongiosa volume with both anthropometric parameters and computed tomography-based skeletal size measurements.

Patient-specific dosimetry within the field of molecular radiotherapy continues to pose a challenge owing to the difficulty in predicting marrow toxicity. This study examined the correlation between total pelvic spongiosa volume (TPSV) and independent variables, which include both readily measured or calculated anthropometric parameters (AP), and image-based skeletal measurements requiring computed tomography (CT) images or skeletal radiographs. Fourteen (14) patients (5 male and 9 female) undergoing total hip arthroplasty (THA) were subjected to modified pelvic CT scans. These scans were utilized to estimate TPSV, which was comprised of the volumes of spongiosa within the L5 vertebra, os coxae, sacrum, and both proximal femurs. The APs investigated included total body height (TBH), total body mass (TBM), body mass index (BMI), body surface area (BSA), maximum effective mass (MEM), lean body mass (LBM), and fat-free mass (FFM). Skeletal measurements were also obtained from the CT images of the pelvic region. Correlation coefficients (r) were obtained for TPSV and each set of APs as well as each set of skeletal measurements. Total body height (r = 0. 80) and os coxae height (r = 0.83) had the highest correlation coefficients of all the APS and skeletal measurements, respectively. FFM (r = 0.50), LBM (r = 0.42), TBM (r = 0.11), and BSA (r = 0.11) did not correlate well with TPSV, which accounts for approximately 45% of total spongiosa seen throughout the skeleton at sites associated with active bone marrow. Skeletal height measurements appear to have a much higher correlation with TPSV than either their corresponding skeletal width measurements or parameters that are a function of an individual's TBM.

Micro-processing events in mRNAs identified by DHPLC analysis.

Post-transcriptional processes such as alternative splicing and RNA editing have a huge impact on the diversity of the proteome. Detecting alternatively spliced transcripts is difficult when they are rare. In addition, edited transcripts often differ from the genomic sequence by only a few nucleotides. Denaturing high performance liquid chromatography (DHPLC) is routinely used for single nucleotide polymorphism detection and we used this method to detect alternatively spliced or edited transcripts. As the sites of RNA editing appear to be conserved within gene families, we investigated whether editing sites are conserved in the murine homologue of the Drosophila cacophony transcript encoding the alpha1 subunit of a voltage-gated calcium channel that is edited at 10 independent positions. Although DHPLC analysis detects RNA editing at as low as 3% in control transcripts, no evidence of RNA editing was found in the analysed murine transcript. However an alternative exon was identified at the 3' end of the mouse Cacna1alpha transcript and an alternative micro-exon encoding only two amino acids (NP) was found in the extracellular loop before the IVS4 helix in the same transcript. In the homologous Drosophila transcript a micro-exon also encoding two amino acids was found at the same position before the IVS4 helix.

Investigation of Nonuniform Dose Voxel Geometry in Monte Carlo Calculations.

The purpose of this work is to investigate the efficacy of using multi-resolution nonuniform dose voxel geometry in Monte Carlo (MC) simulations. An in-house MC code based on the dose planning method MC code was developed in C++ to accommodate the nonuniform dose voxel geometry package since general purpose MC codes use their own coupled geometry packages. We devised the package in a manner that the entire calculation volume was first divided into a coarse mesh and then the coarse mesh was subdivided into nonuniform voxels with variable voxel sizes based on density difference. We name this approach as multi-resolution subdivision (MRS). It generates larger voxels in small density gradient regions and smaller voxels in large density gradient regions. To take into account the large dose gradients due to the beam penumbra, the nonuniform voxels can be further split using ray tracing starting from the beam edges. The accuracy of the implementation of the algorithm was verified by comparing with the data published by Rogers and Mohan. The discrepancy was found to be 1% to 2%, with a maximum of 3% at the interfaces. Two clinical cases were used to investigate the efficacy of nonuniform voxel geometry in the MC code. Applying our MRS approach, we started with the initial voxel size of 5 × 5 × 3 mm(3), which was further divided into smaller voxels. The smallest voxel size was 1.25 × 1.25 × 3 mm(3). We found that the simulation time per history for the nonuniform voxels is about 30% to 40% faster than the uniform fine voxels (1.25 × 1.25 × 3 mm(3)) while maintaining similar accuracy.

Establishing a process of irradiating small animal brain using a CyberKnife and a microCT scanner.

PURPOSE: Establish and validate a process of accurately irradiating small animals using the CyberKnife G4 System (version 8.5) with treatment plans designed to irradiate a hemisphere of a mouse brain based on microCT scanner images. METHODS: These experiments consisted of four parts: (1) building a mouse phantom for intensity modulated radiotherapy (IMRT) quality assurance (QA), (2) proving usability of a microCT for treatment planning, (3) fabricating a small animal positioning system for use with the CyberKnife's image guided radiotherapy (IGRT) system, and (4)in vivo verification of targeting accuracy. A set of solid water mouse phantoms was designed and fabricated, with radiochromic films (RCF) positioned in selected planes to measure delivered doses. After down-sampling for treatment planning compatibility, a CT image set of a phantom was imported into the CyberKnife treatment planning system--MultiPlan (ver. 3.5.2). A 0.5 cm diameter sphere was contoured within the phantom to represent a hemispherical section of a mouse brain. A nude mouse was scanned in an alpha cradle using a microCT scanner (cone-beam, 157 × 149 pixels slices, 0.2 mm longitudinal slice thickness). Based on the results of our positional accuracy study, a planning treatment volume (PTV) was created. A stereotactic body mold of the mouse was "printed" using a 3D printer laying UV curable acrylic plastic. Printer instructions were based on exported contours of the mouse's skin. Positional reproducibility in the mold was checked by measuring ten CT scans. To verify accurate dose delivery in vivo, six mice were irradiated in the mold with a 4 mm target contour and a 2 mm PTV margin to 3 Gy and sacrificed within 20 min to avoid DNA repair. The brain was sliced and stained for analysis. RESULTS: For the IMRT QA using a set of phantoms, the planned dose (6 Gy to the calculation point) was compared to the delivered dose measured via film and analyzed using Gamma analysis (3% and 3 mm). A passing rate of 99% was measured in areas of above 40% of the prescription dose. The final inverse treatment plan was comprised of 43 beams ranging from 5 to 12.5 mm in diameter (2.5 mm size increments are available up to 15 mm in diameter collimation). Using the Xsight Spine Tracking module, the CyberKnife system could not reliably identify and track the tiny mouse spine; however, the CyberKnife system could identify and track the fiducial markers on the 3D mold.In vivo positional accuracy analysis using the 3D mold generated a mean error of 1.41 mm ± 0.73 mm when fiducial markers were used for position tracking. Analysis of the dissected brain confirmed the ability to target the correct brain volume. CONCLUSIONS: With the use of a stereotactic body mold with fiducial markers, microCT imaging, and resolution down-sampling, the CyberKnife system can successfully perform small-animal radiotherapy studies.

The RNA-editing enzyme ADAR1 controls innate immune responses to RNA.

The ADAR RNA-editing enzymes deaminate adenosine bases to inosines in cellular RNAs. Aberrant interferon expression occurs in patients in whom ADAR1 mutations cause Aicardi-Goutières syndrome (AGS) or dystonia arising from striatal neurodegeneration. Adar1 mutant mouse embryos show aberrant interferon induction and die by embryonic day E12.5. We demonstrate that Adar1 embryonic lethality is rescued to live birth in Adar1; Mavs double mutants in which the antiviral interferon induction response to cytoplasmic double-stranded RNA (dsRNA) is prevented. Aberrant immune responses in Adar1 mutant mouse embryo fibroblasts are dramatically reduced by restoring the expression of editing-active cytoplasmic ADARs. We propose that inosine in cellular RNA inhibits antiviral inflammatory and interferon responses by altering RLR interactions. Transfecting dsRNA oligonucleotides containing inosine-uracil base pairs into Adar1 mutant mouse embryo fibroblasts reduces the aberrant innate immune response. ADAR1 mutations causing AGS affect the activity of the interferon-inducible cytoplasmic isoform more severely than the nuclear isoform.

Stereotactic radiosurgery for gynecologic cancer.

Stereotactic body radiotherapy (SBRT) distinguishes itself by necessitating more rigid patient immobilization, accounting for respiratory motion, intricate treatment planning, on-board imaging, and reduced number of ablative radiation doses to cancer targets usually refractory to chemotherapy and conventional radiation. Steep SBRT radiation dose drop-off permits narrow 'pencil beam' treatment fields to be used for ablative radiation treatment condensed into 1 to 3 treatments. Treating physicians must appreciate that SBRT comes at a bigger danger of normal tissue injury and chance of geographic tumor miss. Both must be tackled by immobilization of cancer targets and by high-precision treatment delivery. Cancer target immobilization has been achieved through use of indexed customized Styrofoam casts, evacuated bean bags, or body-fix molds with patient-independent abdominal compression.(1-3) Intrafraction motion of cancer targets due to breathing now can be reduced by patient-responsive breath hold techniques,(4) patient mouthpiece active breathing coordination,(5) respiration-correlated computed tomography,(6) or image-guided tracking of fiducials implanted within and around a moving tumor.(7-9) The Cyberknife system (Accuray [Sunnyvale, CA]) utilizes a radiation linear accelerator mounted on a industrial robotic arm that accurately follows patient respiratory motion by a camera-tracked set of light-emitting diodes (LED) impregnated on a vest fitted to a patient.(10) Substantial reductions in radiation therapy margins can be achieved by motion tracking, ultimately rendering a smaller planning target volumes that are irradiated with submillimeter accuracy.(11-13) Cancer targets treated by SBRT are irradiated by converging, tightly collimated beams. Resultant radiation dose to cancer target volume histograms have a more pronounced radiation "shoulder" indicating high percentage target coverage and a small high-dose radiation "tail." Thus, increased target conformality comes at the expense of decreased dose uniformity in the SBRT cancer target. This may have implications for both subsequent tumor control in the SBRT target and normal tissue tolerance of organs at-risk. Due to the sharp dose falloff in SBRT, the possibility of occult disease escaping ablative radiation dose occurs when cancer targets are not fully recognized and inadequate SBRT dose margins are applied. Clinical target volume (CTV) expansion by 0.5 cm, resulting in a larger planning target volume (PTV), is associated with increased target control without undue normal tissue injury.(7,8) Further reduction in the probability of geographic miss may be achieved by incorporation of 2-[(18)F]fluoro-2-deoxy-D-glucose ((18)F-FDG) positron emission tomography (PET).(8) Use of (18)F-FDG PET/CT in SBRT treatment planning is only the beginning of attempts to discover new imaging target molecular signatures for gynecologic cancers.

Phase II Clinical Trial of Robotic Stereotactic Body Radiosurgery for Metastatic Gynecologic Malignancies.

BACKGROUND: Recurrent gynecologic cancers are often difficult to manage without significant morbidity. We conducted a phase II study to assess the safety and the efficacy of ablative robotic stereotactic body radiosurgery (SBRT) in women with metastatic gynecologic cancers. METHODS: A total of 50 patients with recurrent gynecologic cancer who had single or multiple (≤4) metastases underwent robotic-armed Cyberknife SBRT (24Gy/3 daily doses). Toxicities were graded prospectively by common toxicity criteria for adverse events (version 4.0). SBRT target responses were recorded following RECIST criteria (version 1.0). Rates of clinical benefit for SBRT and non-radiosurgical disease relapse were calculated. Disease-free and overall survivals were estimated by the Kaplan-Meier method and the Cox proportional hazards model was used to control for prognostic variables. FINDINGS: SBRT was safely delivered, with 49 (98%) of 50 patients completing three prescribed fractions. The most frequent grade 2 or higher adverse events attributed to SBRT included fatigue (16%), nausea (8%), and diarrhea (4%). One (2%) grade four hyperbilirubinemia occurred. SBRT target response was 96% (48 of 50 patients). A 6-month clinical benefit was recorded in 34 [68% (95% CI, 53.2, 80.1)] patients. No SBRT targeted disease progressed. Non-radiosurgical disease relapse occurred in 31 (62%) patients. Median disease-free survival was 7.8 months (95% CI, 4.0, 11.6). Median overall survival was 20.2 months (95% CI, 10.9, 29.5). INTERPRETATION: SBRT safely controlled metastatic gynecologic cancer targets. Given an observed high rate of non-radiosurgical disease relapse, a phase I trial assessing co-administration of SBRT and cytotoxic chemotherapy is underway. FUNDING: Case Comprehensive Cancer Center.