Proton Treatment Suppresses Exosome Production in Head and Neck Squamous Cell Carcinoma.

Proton therapy (PT) is emerging as an effective and less toxic alternative to conventional X-ray-based photon therapy (XRT) for patients with advanced head and neck squamous cell carcinomas (HNSCCs) owing to its clustered dose deposition dosimetric characteristics. For optimal efficacy, cancer therapies, including PT, must elicit a robust anti-tumor response by effector and cytotoxic immune cells in the tumor microenvironment (TME). While tumor-derived exosomes contribute to immune cell suppression in the TME, information on the effects of PT on exosomes and anti-tumor immune responses in HNSCC is not known. In this study, we generated primary HNSCC cells from tumors resected from HNSCC patients, irradiated them with 5 Gy PT or XRT, and isolated exosomes from cell culture supernatants. HNSCC cells exposed to PT produced 75% fewer exosomes than XRT- and non-irradiated HNSCC cells. This effect persisted in proton-irradiated cells for up to five days. Furthermore, we observed that exosomes from proton-irradiated cells were identical in morphology and immunosuppressive effects (suppression of IFN-γ release by peripheral blood mononuclear cells) to those of photon-irradiated cells. Our results suggest that PT limits the suppressive effect of exosomes on cancer immune surveillance by reducing the production of exosomes that can inhibit immune cell function.

Initiating and imaging cavitation from infused echo contrast agents through the EkoSonic catheter.

Ultrasound-enhanced delivery of therapeutic-loaded echogenic liposomes is under development for vascular applications using the EkoSonic Endovascular System. In this study, fibrin-targeted echogenic liposomes loaded with an anti-inflammatory agent were characterized before and after infusion through an EkoSonic catheter. Cavitation activity was nucleated by Definity or fibrin-targeted, drug-loaded echogenic liposomes infused and insonified with EkoSonic catheters. Passive cavitation imaging was used to quantify and map bubble activity in a flow phantom mimicking porcine arterial flow. Cavitation was sustained during 3-min infusions of Definity or echogenic liposomes along the distal 6 cm treatment zone of the catheter. Though the EkoSonic catheter was not designed specifically for cavitation nucleation, infusion of drug-loaded echogenic liposomes can be employed to trigger and sustain bubble activity for enhanced intravascular drug delivery.

GABA(A) receptor activation drives GABARAP-Nix mediated autophagy to radiation-sensitize primary and brain-metastatic lung adenocarcinoma tumors.

In non-small cell lung cancer (NSCLC) treatment, targeted therapies benefit only a subset of NSCLC, while radiotherapy responses are not durable and toxicity limits therapy. We find that a GABA(A) receptor activator, AM-101, impairs viability and clonogenicity of NSCLC primary and brain metastatic cells. Employing an ex vivo 'chip', AM-101 is as efficacious as the chemotherapeutic docetaxel, which is used with radiotherapy for advanced-stage NSCLC. In vivo , AM-101 potentiates radiation, including conferring a survival benefit to mice bearing NSCLC intracranial tumors. GABA(A) receptor activation stimulates a selective-autophagic response via multimerization of GABA(A) Receptor-Associated Protein (GABARAP), stabilization of mitochondrial receptor Nix, and utilization of ubiquitin-binding protein p62. A targeted-peptide disrupting Nix binding to GABARAP inhibits AM-101 cytotoxicity. This supports a model of GABA(A) receptor activation driving a GABARAP-Nix multimerization axis triggering autophagy. In patients receiving radiotherapy, GABA(A) receptor activation may improve tumor control while allowing radiation dose de-intensification to reduce toxicity. Highlights: Activating GABA(A) receptors intrinsic to lung primary and metastatic brain cancer cells triggers a cytotoxic response. GABA(A) receptor activation works as well as chemotherapeutic docetaxel in impairing lung cancer viability ex vivo . GABA(A) receptor activation increases survival of mice bearing lung metastatic brain tumors.A selective-autophagic response is stimulated by GABA(A) receptor activation that includes multimerization of GABARAP and Nix.Employing a new nanomolar affinity peptide that abrogates autophagosome formation inhibits cytotoxicity elicited by GABA(A) receptor activation.

Fluoroscopic 3D Image Generation from Patient-Specific PCA Motion Models Derived from 4D-CBCT Patient Datasets: A Feasibility Study.

A method for generating fluoroscopic (time-varying) volumetric images using patient-specific motion models derived from four-dimensional cone-beam CT (4D-CBCT) images was developed. 4D-CBCT images acquired immediately prior to treatment have the potential to accurately represent patient anatomy and respiration during treatment. Fluoroscopic 3D image estimation is performed in two steps: (1) deriving motion models and (2) optimization. To derive motion models, every phase in a 4D-CBCT set is registered to a reference phase chosen from the same set using deformable image registration (DIR). Principal components analysis (PCA) is used to reduce the dimensionality of the displacement vector fields (DVFs) resulting from DIR into a few vectors representing organ motion found in the DVFs. The PCA motion models are optimized iteratively by comparing a cone-beam CT (CBCT) projection to a simulated projection computed from both the motion model and a reference 4D-CBCT phase, resulting in a sequence of fluoroscopic 3D images. Patient datasets were used to evaluate the method by estimating the tumor location in the generated images compared to manually defined ground truth positions. Experimental results showed that the average tumor mean absolute error (MAE) along the superior-inferior (SI) direction and the 95th percentile in two patient datasets were 2.29 and 5.79 mm for patient 1, and 1.89 and 4.82 mm for patient 2. This study demonstrated the feasibility of deriving 4D-CBCT-based PCA motion models that have the potential to account for the 3D non-rigid patient motion and localize tumors and other patient anatomical structures on the day of treatment.

Coupling surface cameras with on-board fluoroscopy: a feasibility study.

PURPOSE: To investigate the feasibility of using three-dimensional surface imaging cameras as an external surrogate of tumor motion through a temporal synchronization with kV imaging. METHODS: To obtain an "x-ray on" signal from the on-board kV fluoroscopy system (XVI, Elekta), a hardware controller (Gate Controller) was interfaced between the kV fluoroscopy and Gate CT (VisionRT Ltd., London) computers. First, phantom experiments were performed using a programmable respiratory motion platform (sinusoidal motion, period = 3-5 s). The platform included a chest-wall component (A-P amplitude = 1 cm) tracked with the surface camera, while tumorlike objects translated in the superior-inferior direction were tracked using kV fluoroscopy (300 frames, frequency 5.5 fps). Accuracy of tracking the chest-wall platform was assessed, and the latency of the system was characterized by performing linear regression between the peak times obtained from Gate CT and fluoroscopy. Increasing the complexity of experiments, tumor displacement curves from three patients were simulated using synchronous tumor-abdomen data (RTRT). Our approach was further validated by imaging four free-breathing lung cancer patients with simultaneous Gate CT and kV fluoroscopy for approximately 55 s. Consideration was also given to varied sizes and locations of the tracked region of interest on the patient surface. RESULTS: For simple sinusoidal curves, measured amplitude (peak-to-peak) was 1.005 +/- 0.003 cm, 1.013 +/- 0.003 cm, and 1.003 +/- 0.005 cm for periods of 5, 4, and 3.3 s, respectively, demonstrating an excellent agreement with the actual chest platform amplitude of 1.0 cm. Period measurements were within 0.2% of actual using the surface cameras and within 0.9% of actual value using fluoroscopy. For the sinusoidal motion, the system latency was 0.64 +/- 0.02 s. This was further validated for the simulated tumor motion from three patients (latency = 0.65 +/- 0.03 s). Five of the nine patient fractions studied showed the associations between the abdomen and tumor were equivalent or better (Pearson r = 0.93-0.98) than those observed between the diaphragm and tumor (Pearson r = 0.89-0.95). A repeat analysis of five different tracked surfaces on the same patient further demonstrated strong agreement with the diaphragm and tumor, although no improvement in association strength was observed with increased size of region of interest. CONCLUSIONS: The feasibility of using surface imaging cameras to track the patient's abdomen as an external surrogate, while using kV imaging to track internal anatomy in synchrony, has been demonstrated. With further validation through additional patient studies to confirm these findings, gated radiation therapy treatments using surface imaging cameras as the external surrogate can be facilitated.

Motion artifacts occurring at the lung/diaphragm interface using 4D CT attenuation correction of 4D PET scans.

For PET/CT, fast CT acquisition time can lead to errors in attenuation correction, particularly at the lung/diaphragm interface. Gated 4D PET can reduce motion artifacts, though residual artifacts may persist depending on the CT dataset used for attenuation correction. We performed phantom studies to evaluate 4D PET images of targets near a density interface using three different methods for attenuation correction: a single 3D CT (3D CTAC), an averaged 4D CT (CINE CTAC), and a fully phase matched 4D CT (4D CTAC). A phantom was designed with two density regions corresponding to diaphragm and lung. An 8 mL sphere phantom loaded with 18F-FDG was used to represent a lung tumor and background FDG included at an 8:1 ratio. Motion patterns of sin(x) and sin4(x) were used for dynamic studies. Image data was acquired using a GE Discovery DVCT-PET/CT scanner. Attenuation correction methods were compared based on normalized recovery coefficient (NRC), as well as a novel quantity "fixed activity volume" (FAV) introduced in our report. Image metrics were compared to those determined from a 3D PET scan with no motion present (3D STATIC). Values of FAV and NRC showed significant variation over the motion cycle when corrected by 3D CTAC images. 4D CTAC- and CINE CTAC-corrected PET images reduced these motion artifacts. The amount of artifact reduction is greater when the target is surrounded by lower density material and when motion was based on sin4(x). 4D CTAC reduced artifacts more than CINE CTAC for most scenarios. For a target surrounded by water equivalent material, there was no advantage to 4D CTAC over CINE CTAC when using the sin(x) motion pattern. Attenuation correction using both 4D CTAC or CINE CTAC can reduce motion artifacts in regions that include a tissue interface such as the lung/diaphragm border. 4D CTAC is more effective than CINE CTAC at reducing artifacts in some, but not all, scenarios.

Differential transcriptome response to proton versus X-ray radiation reveals novel candidate targets for combinatorial PT therapy in lymphoma.

BACKGROUND AND PURPOSE: Knowledge of biological responses to proton therapy (PT) in comparison to X-ray remains in its infancy. Identification of PT specific molecular signals is an important opportunity for the discovery of biomarkers and synergistic drugs to advance clinical application. Since PT is used for the treatment of lymphoma, we report here transcriptomic responses of lymphoma cell lines to PT vs X-ray and identify potential therapeutic targets. MATERIALS AND METHODS: Two lymphoma cell lines of human (BL41) and murine (J3D) origin were irradiated by X-ray and PT. Differential transcriptome regulation was quantified by RNA sequencing for each radiation type at 12 hours post irradiation. Gene-set enrichment analysis revealed deregulated molecular pathways and putative targets for lymphoma cell sensitization to PT. RESULTS: Transcriptomic gene set enrichment analyses uncovered pathways that contribute to the unfolded protein response (UPR) and mitochondrial transport. Functional validation at multiple time points demonstrated increased UPR activation and decreased protein translation, perhaps due to increased oxidative stress and oxidative protein damage after PT. PPARgamma was identified as a potential regulator of the PT transcriptomic response. Inhibition of PPARgamma by two compounds, T0070907 and SR2595, sensitized lymphoma cells to PT. CONCLUSIONS: Proton vs X-ray radiation leads to the transcriptional regulation of a specific subset of genes in line with diminished protein translation and UPR activation that may be due to oxidative stress. This study demonstrates that different radiation qualities trigger distinct cellular responses in lymphoma cells, and identifies PPARgamma inhibition as a potential strategy for the sensitization of lymphoma to PT.

Melanoma Cell Intrinsic GABAA Receptor Enhancement Potentiates Radiation and Immune Checkpoint Inhibitor Response by Promoting Direct and T Cell-Mediated Antitumor Activity.

PURPOSE: Most patients with metastatic melanoma show variable responses to radiation therapy and do not benefit from immune checkpoint inhibitors. Improved strategies for combination therapy that leverage potential benefits from radiation therapy and immune checkpoint inhibitors are critical. METHODS AND MATERIALS: We analyzed metastatic melanoma tumors in the TCGA cohort for expression of genes coding for subunits of type A γ-aminobutyric acid (GABA) receptor (GABAAR), a chloride ion channel and major inhibitory neurotransmitter receptor. Electrophysiology was used to determine whether melanoma cells possess intrinsic GABAAR activity. Melanoma cell viability studies were conducted to test whether enhancing GABAAR mediated chloride transport using benzodiazepine-impaired viability. A syngeneic melanoma mouse model was used to assay the effect of benzodiazepine on tumor volume and its ability to potentiate radiation therapy or immunotherapy. Treated tumors were analyzed for changes in gene expression by RNA sequencing and presence of tumor-infiltrating lymphocytes by flow cytometry. RESULTS: Genes coding for subunits of GABAARs express functional GABAARs in melanoma cells. By enhancing GABAAR-mediated anion transport, benzodiazepines depolarize melanoma cells and impair their viability. In vivo, benzodiazepine alone reduces tumor growth and potentiates radiation therapy and α-PD-L1 antitumor activity. The combination of benzodiazepine, radiation therapy, and α-PD-L1 results in near complete regression of treated tumors and a potent abscopal effect, mediated by increased infiltration of polyfunctional CD8+ T cells. Treated tumors show expression of cytokine-cytokine receptor interactions and overrepresentation of p53 signaling. CONCLUSIONS: This study identifies an antitumor strategy combining radiation and/or an immune checkpoint inhibitor with modulation of GABAARs in melanoma using benzodiazepine.

Evaluation of the interplay effect when using RapidArc to treat targets moving in the craniocaudal or right-left direction.

PURPOSE: We have investigated the dosimetric errors caused by the interplay between the motions of the LINAC and the tumor during the delivery of a volume modulated arc therapy treatment. This includes the development of an IMRT QA technique, applied here to evaluate RapidArc plans of varying complexity. METHODS: An IMRT QA technique was developed, which involves taking a movie of the delivered dose (0.2 s frames) using a 2D ion chamber array. Each frame of the movie is then moved according to a respiratory trace and the cumulative dose calculated. The advantage of this approach is that the impact of turning the beam on at different points in the respiratory trace, and of different types of motion, can be evaluated using data from a single irradiation. We evaluated this technique by comparing with the results when we actually moved the phantom during irradiation. RapidArc plans were created to treat a 62 cc spherical tumor in a lung phantom (16 plans) and a 454 cc irregular tumor in an actual patient (five plans). The complexity of each field was controlled by adjusting the MU (312-966 MU). Each plan was delivered to a phantom, and a movie of the delivered dose taken using a 2D ion chamber array. Patient motion was modeled by shifting each dose frame according to a respiratory trace, starting the motion at different phases. The expected dose distribution was calculated by blurring the static dose distribution with the target motion. The dose error due to the interplay effect was then calculated by comparing the delivered dose with the expected dose distribution. Peak-to-peak motion of 0.5, 1.0, and 2.0 cm in the craniocaudal and right-left directions, with target periods of 3 and 5 s, were evaluated for each plan (252 different target motion/plan combinations). RESULTS: The daily dose error due to the interplay effect was less than 10% for 98.4% of all pixels in the target for all plans investigated. The percentage of pixels for which the daily dose error could be larger than 5% increased with increasing plan complexity (field MU), but was less than 15% for all plans if the motion was 1 cm or less. For 2 cm motion, the dose error could be larger than 5% for 40% of pixels, but was less than 5% for more than 80% of pixels for MU < 550, and was less than 10% for 99% of all pixels. The interplay effect was smaller for 3 s periods than for 5 s periods. CONCLUSIONS: The interplay between the motions of the LINAC and the target can result in an error in the delivered dose. This effect increases with plan complexity, and with target magnitude and period. It may average out after many fractions.

Use of a realistic breathing lung phantom to evaluate dose delivery errors.

PURPOSE: To compare the effect of respiration-induced motion on delivered dose (the interplay effect) for different treatment techniques under realistic clinical conditions. METHODS: A flexible resin tumor model was created using rapid prototyping techniques based on a computed tomography (CT) image of an actual tumor. Twenty micro-MOSFETs were inserted into the tumor model and the tumor model was inserted into an anthropomorphic breathing phantom. Phantom motion was programed using the motion trajectory of an actual patient. A four-dimensional CT image was obtained and several treatment plans were created using different treatment techniques and planning systems: Conformal (Eclipse), step-and-shoot intensity-modulated radiation therapy (IMRT) (Pinnacle), step-and-shoot IMRT (XiO), dynamic IMRT (Eclipse), complex dynamic IMRT (Eclipse), hybrid IMRT [60% conformal, 40% dynamic IMRT (Eclipse)], volume-modulated are therapy (VMAT) [single-arc (Eclipse)], VMAT [double-arc (Eclipse)], and complex VMAT (Eclipse). The complex plans were created by artificially pushing the optimizer to give complex multileaf collimator sequences. Each IMRT field was irradiated five times and each VMAT field was irradiated ten times, with each irradiation starting at a random point in the respiratory cycle. The effect of fractionation was calculated by randomly summing the measured doses. The maximum deviation for each measurement point per fraction and the probability that 95% of the model tumor had dose deviations less than 2% and 5% were calculated as a function of the number of fractions. Tumor control probabilities for each treatment plan were calculated and compared. RESULTS: After five fractions, measured dose deviations were less than 2% for more than 95% of measurement points within the tumor model for all plans, except the complex dynamic IMRT, step-and-shoot IMRT (XiO), complex VMAT, and single-arc VMAT plans. Reducing the dose rate of the complex IMRT plans from 600 to 200 MU/min reduced the dose deviations to less than 2%. Dose deviations were less than 5% after five fractions for all plans, except the complex single-arc VMAT plan. CONCLUSIONS: Rapid prototyping techniques can be used to create realistic tumor models. For most treatment techniques, the dose deviations averaged out after several fractions. Treatments with unusually complicated multileaf collimator sequences had larger dose deviations. For IMRT treatments, dose deviations can be reduced by reducing the dose rate. For VMAT treatments, using two arcs instead of one is effective for reducing dose deviations.

Use of reduced dose rate when treating moving tumors using dynamic IMRT.

The purpose was to evaluate the effect of dose rate on discrepancies between expected and delivered dose caused by the interplay effect. Fifteen separate dynamic IMRT plans and five hybrid IMRT plans were created for five patients (three IMRT plans and one hybrid IMRT plan per patient). The impact of motion on the delivered dose was evaluated experimentally for each treatment field for different dose rates (200 and 400 MU/min), and for a range of target amplitudes and periods. The maximum dose discrepancy for dynamic IMRT fields was 18.5% and 10.3% for dose rates of 400 and 200 MU/min, respectively. The maximum dose discrepancy was larger than this for hybrid plans, but the results were similar when weighted by the contribution of the IMRT fields. The percentage of fields for which 98% of the target never experienced a 5% or 10% dose discrepancy increased when the dose rate was reduced from 400 MU/min to 200 MU/min. For amplitudes up to 2 cm, reducing the dose rate to 200 MU/min is effective in keeping daily dose discrepancies for each field within 10%.

Quantifying day-to-day variations in 4DCBCT-based PCA motion models.

The aim of this paper is to quantify the day-to-day variations of motion models derived from pre-treatment 4-dimensional cone beam CT (4DCBCT) fractions for lung cancer stereotactic body radiotherapy (SBRT) patients. Motion models are built by (1) applying deformable image registration (DIR) on each 4DCBCT image with respect to a reference image from that day, resulting in a set of displacement vector fields (DVFs), and (2) applying principal component analysis (PCA) on the DVFs to obtain principal components representing a motion model. Variations were quantified by comparing the PCA eigenvectors of the motion model built from the first day of treatment to the corresponding eigenvectors of the other motion models built from each successive day of treatment. Three metrics were used to quantify the variations: root mean squared (RMS) difference in the vectors, directional similarity, and an introduced metric called the Euclidean Model Norm (EMN). EMN quantifies the degree to which a motion model derived from the first fraction can represent the motion models of subsequent fractions. Twenty-one 4DCBCT scans from five SBRT patient treatments were used in this retrospective study. Experimental results demonstrated that the first two eigenvectors of motion models across all fractions have smaller RMS (0.00017), larger directional similarity (0.528), and larger EMN (0.678) than the last three eigenvectors (RMS: 0.00025, directional similarity: 0.041, and EMN: 0.212). The study concluded that, while the motion model eigenvectors varied from fraction to fraction, the first few eigenvectors were shown to be more stable across treatment fractions than others. This supports the notion that a pre-treatment motion model built from the first few PCA eigenvectors may remain valid throughout a treatment course. Future work is necessary to quantify how day-to-day variations in these models will affect motion reconstruction accuracy for specific clinical tasks.

Automatic marker detection and 3D position reconstruction using cine EPID images for SBRT verification.

In previous studies, an electronic portal imaging device (EPID) in cine mode was used for validating respiratory gating and stereotactic body radiation therapy (SBRT) by tracking implanted fiducials. The manual marker tracking methods that were used were time and labor intensive, limiting the utility of the validation. The authors have developed an automatic algorithm to quickly and accurately extract the markers in EPID images and reconstruct their 3D positions. Studies have been performed with gold fiducials placed in solid water and dynamic thorax phantoms. In addition, the authors have examined the cases of five patients being treated under an SBRT protocol for hepatic metastases. For each case, a sequence of images was created by collecting the exit radiation using the EPID. The markers were detected and recognized using an image processing algorithm based on the Laplacian of Gaussian function. To reduce false marker detection, a marker registration technique was applied using image intensity as well as the geometric spatial transformations between the reference marker positions produced from the projection of 3D CT images and the estimated marker positions. An average marker position in 3D was reconstructed by backprojecting, towards the source, the position of each marker on the 2D image plane. From the static phantom study, spatial accuracies of <1 mm were achieved in both 2D and 3D marker locations. From the dynamic phantom study, using only the Laplacian of the Gaussian algorithm, the marker detection success rate was 88.8%. However, adding a marker registration technique which utilizes prior CT information, the detection success rate was increased to 100%. From the SBRT patient study, intrafractional tumor motion (3.1-11.3 mm) in the SI direction was measured using the 2D images. The interfractional patient setup errors (0.1-12.7 mm) in the SI, AP, and LR directions were obtained from the average marker locations reconstructed in 3D and compared to the reference planning CT image. The authors have developed an automatic algorithm to extract marker locations from MV images and have evaluated its performance. The measured intrafractional tumor motion and the interfractional daily patient setup error can be used for off-line retrospective verification of SBRT.

Reconstruction of a high-quality volumetric image and a respiratory motion model from patient CBCT projections.

PURPOSE: To develop and evaluate a method of reconstructing a patient- and treatment day- specific volumetric image and motion model from free-breathing cone-beam projections and respiratory surrogate measurements. This Motion-Compensated Simultaneous Algebraic Reconstruction Technique (MC-SART) generates and uses a motion model derived directly from the cone-beam projections, without requiring prior motion measurements from 4DCT, and can compensate for both inter- and intrabin deformations. The motion model can be used to generate images at arbitrary breathing points, which can be used for estimating volumetric images during treatment delivery. METHODS: The MC-SART was formulated using simultaneous image reconstruction and motion model estimation. For image reconstruction, projections were first binned according to external surrogate measurements. Projections in each bin were used to reconstruct a set of volumetric images using MC-SART. The motion model was estimated based on deformable image registration between the reconstructed bins, and least squares fitting to model parameters. The model was used to compensate for motion in both projection and backprojection operations in the subsequent image reconstruction iterations. These updated images were then used to update the motion model, and the two steps were alternated between. The final output is a volumetric reference image and a motion model that can be used to generate images at any other time point from surrogate measurements. RESULTS: A retrospective patient dataset consisting of eight lung cancer patients was used to evaluate the method. The absolute intensity differences in the lung regions compared to ground truth were 50.8 ± 43.9 HU in peak exhale phases (reference) and 80.8 ± 74.0 in peak inhale phases (generated). The 50th percentile of voxel registration error of all voxels in the lung regions with >5 mm amplitude was 1.3 mm. The MC-SART was also applied to measured patient cone-beam projections acquired with a linac-mounted CBCT system. Results from this patient data demonstrate the feasibility of MC-SART and showed qualitative image quality improvements compared to other state-of-the-art algorithms. CONCLUSION: We have developed a simultaneous image reconstruction and motion model estimation method that uses Cone-beam computed tomography (CBCT) projections and respiratory surrogate measurements to reconstruct a high-quality reference image and motion model of a patient in treatment position. The method provided superior performance in both HU accuracy and positional accuracy compared to other existing methods. The resultant reference image and motion model can be combined with respiratory surrogate measurements to generate volumetric images representing patient anatomy at arbitrary time points.

Low-frequency dynamics of Caldariomyces fumago chloroperoxidase probed by femtosecond coherence spectroscopy.

Ultrafast laser spectroscopy techniques are used to measure the low-frequency vibrational coherence spectra and nitric oxide rebinding kinetics of Caldariomyces fumago chloroperoxidase (CPO). Comparisons of the CPO coherence spectra with those of other heme species are made to gauge the protein-specific nature of the low-frequency spectra. The coherence spectrum of native CPO is dominated by a mode that appears near 32-33 cm(-1) at all excitation wavelengths, with a phase that is consistent with a ground-state Raman-excited vibrational wavepacket. On the basis of a normal coordinate structural decomposition (NSD) analysis, we assign this feature to the thiolate-bound heme doming mode. Spectral resolution of the probe pulse ("detuned" detection) reveals a mode at 349 cm(-1), which has been previously assigned using Raman spectroscopy to the Fe-S stretching mode of native CPO. The ferrous species displays a larger degree of spectral inhomogeneity than the ferric species, as reflected by multiple shoulders in the optical absorption spectra. The inhomogeneities are revealed by changes in the coherence spectra at different excitation wavelengths. The appearance of a mode close to 220 cm(-1) in the coherence spectrum of reduced CPO excited at 440 nm suggests that a subpopulation of five coordinated histidine-ligated hemes is present in the ferrous state at a physiologically relevant pH. A significant increase in the amplitude of the coherence signal is observed for the resonance with the 440 nm subpopulation. Kinetics measurements reveal that nitric oxide binding to ferric and ferrous CPO can be described as a single-exponential process, with rebinding time constants of 29.4 +/- 1 and 9.3 +/- 1 ps, respectively. This is very similar to results previously reported for nitric oxide binding to horseradish peroxidase.

Coherence spectroscopy investigations of the low-frequency vibrations of heme: effects of protein-specific perturbations.

Femtosecond coherence spectroscopy is used to probe the low-frequency (20-200 cm(-1)) vibrational modes of heme proteins in solution. Horseradish peroxidase (HRP), myoglobin (Mb), and Campylobacter jejuni globin (Cgb) are compared and significant differences in the coherence spectra are revealed. It is concluded that hydrogen bonding and ligand charge do not strongly affect the low-frequency coherence spectra and that protein-specific deformations of the heme group lower its symmetry and control the relative spectral intensities. Such deformations potentially provide a means for proteins to tune heme reaction coordinates, so that they can perform a broad array of specific functions. Native HRP displays complex spectral behavior above approximately 50 cm(-1) and very weak activity below approximately 50 cm(-1). Binding of the substrate analog, benzhydroxamic acid, leads to distinct changes in the coherence and Raman spectra of HRP that are consistent with the stabilization of a heme water ligand. The CN derivatives of the three proteins are studied to make comparisons under conditions of uniform heme coordination and spin-state. MbCN is dominated by a doming mode near 40 cm(-1), while HRPCN displays a strong oscillation at higher frequency (96 cm(-1)) that can be correlated with the saddling distortion observed in the X-ray structure. In contrast, CgbCN displays low-frequency coherence spectra that contain strong modes near 30 and 80 cm(-1), probably associated with a combination of heme doming and ruffling. HRPNO displays a strong doming mode near 40 cm(-1) that is activated by photolysis. The damping of the coherent motions is significantly reduced when the heme is shielded from solvent fluctuations by the protein material and reduced still further when T approximately < 50 K, as pure dephasing processes due to the protein-solvent phonon bath are frozen out.

Management of the interplay effect when using dynamic MLC sequences to treat moving targets.

Interplay between organ motion and leaf motion has been shown to generally have a small dosimetric impact for most clinical intensity-modulated radiation therapy treatments. However, it has also been shown that for some MLC sequences there can be large daily variations in the delivered dose, depending on details of patient motion or the number of fractions. This study investigates guidelines for dynamic MLC sequences that will keep daily dose variations due to the interplay between organ motion and leaf motion within 10%. Dose distributions for a range of MLC separations (0.2-5.0 cm) and displacements between adjacent MLCs (0-1.5 cm) were exported from ECLIPSE to purpose-written software, which simulated the dose distribution delivered to a moving target. Target motion parallel and perpendicular to the MLC motion was investigated for a range of amplitudes (0.5-4.0 cm), periods (1.5-10 s), and MLC speeds (0.1-3.0 cm/s) with target motions modeled as sin. Results were confirmed experimentally by measuring the dose delivered to an ion chamber array in a moving phantom for different MLC sequences. The simulation results were used to identify MLC sequences that kept dose variations within 10% compared to the dose delivered with no motion. The maximum allowable MLC speed, when target motion is parallel to the MLC motion, was found to be a simple function of target period and MLC separation. When the target motion is perpendicular to MLC motion, the maximum allowable MLC speed can be described as a function of MLC separation and the displacement of adjacent MLCs. These guidelines were successfully applied to two-dimensional motion, and a simple program was written to import MLC sequence files and evaluate whether the maximum daily dose discrepancy caused by the interplay effect will be larger than 10%. This software was experimentally evaluated, and found to conservatively predict whether a given MLC sequence could give large daily dose discrepancies.

A novel method for estimating SBRT delivered dose with beam's-eye-view images.

Stereotactic body radiation therapy is predicated on a high degree of targeting accuracy. However, inaccurate patient setup as well as intra-fractional motion can hinder the delivery of high doses preferentially to the target. To ensure that the coverage delivered to the patient is as planned, an image-guided verification system has been created to estimate the delivered dose retrospectively. This will not only aid the assessment of treatment techniques, but will also allow for more accurate dose response analysis. Patients with limited hepatic metastases from solid tumors were treated with SBRT. Implanted gold markers were used as target surrogates and a body frame and compression plate provided stereotactic localization and target immobilization, respectively. During treatment, an electronic portal imaging device (EPID), operating in cine mode, collected the exit dose. The sequences of images for each field were processed off-line using in-house software for registration and seed localization. The beam's-eye-view seed positions in the treatment images were compared to those in the DRR's to determine the target shifts in the imaging plane. These target shifts were then imported into the treatment planning software. Each original field was multiplied by the number of images taken during treatment. The calculated shift from each image was then applied to each of the new subfields. Summing all of these subfields together gives the dose distribution that was actually delivered to the patient. The dose-volume histograms for the planned and delivered distributions for four patients' complete treatments are shown. For two of the patients, underdosing due to a setup error or intra-fractional drift was not wholly resolved by subsequent fractions. For one of these patients two alternative corrective strategies have been applied, retrospectively, and the prescribed target coverage recovered for both. The delivered dose can be estimated using the information contained in cine EPID images acquired during irradiation. Calculating the dose actually delivered to the target will allow us to assess treatment procedures as well as more accurately report clinical results.

Evaluation of the combined effects of target size, respiratory motion and background activity on 3D and 4D PET/CT images.

Gated (4D) PET/CT has the potential to greatly improve the accuracy of radiotherapy at treatment sites where internal organ motion is significant. However, the best methodology for applying 4D-PET/CT to target definition is not currently well established. With the goal of better understanding how to best apply 4D information to radiotherapy, initial studies were performed to investigate the effect of target size, respiratory motion and target-to-background activity concentration ratio (TBR) on 3D (ungated) and 4D PET images. Using a PET/CT scanner with 4D or gating capability, a full 3D-PET scan corrected with a 3D attenuation map from 3D-CT scan and a respiratory gated (4D) PET scan corrected with corresponding attenuation maps from 4D-CT were performed by imaging spherical targets (0.5-26.5 mL) filled with (18)F-FDG in a dynamic thorax phantom and NEMA IEC body phantom at different TBRs (infinite, 8 and 4). To simulate respiratory motion, the phantoms were driven sinusoidally in the superior-inferior direction with amplitudes of 0, 1 and 2 cm and a period of 4.5 s. Recovery coefficients were determined on PET images. In addition, gating methods using different numbers of gating bins (1-20 bins) were evaluated with image noise and temporal resolution. For evaluation, volume recovery coefficient, signal-to-noise ratio and contrast-to-noise ratio were calculated as a function of the number of gating bins. Moreover, the optimum thresholds which give accurate moving target volumes were obtained for 3D and 4D images. The partial volume effect and signal loss in the 3D-PET images due to the limited PET resolution and the respiratory motion, respectively were measured. The results show that signal loss depends on both the amplitude and pattern of respiratory motion. However, the 4D-PET successfully recovers most of the loss induced by the respiratory motion. The 5-bin gating method gives the best temporal resolution with acceptable image noise. The results based on the 4D scan protocols can be used to improve the accuracy of determining the gross tumor volume for tumors in the lung and abdomen.

The evaluation of a hybrid biomechanical deformable registration method on a multistage physical phantom with reproducible deformation.

BACKGROUND: Advanced clinical applications, such as dose accumulation and adaptive radiation therapy, require deformable image registration (DIR) algorithms capable of voxel-wise accurate mapping of treatment dose or functional imaging. By utilizing a multistage deformable phantom, the authors investigated scenarios where biomechanical refinement method (BM-DIR) may be better than the pure image intensity based deformable registration (IM-DIR). METHODS: The authors developed a biomechanical-model based DIR refinement method (BM-DIR) to refine the deformable vector field (DVF) from any initial intensity-based DIR (IM-DIR). The BM-DIR method was quantitatively evaluated on a novel phantom capable of ten reproducible gradually-increasing deformation stages using the urethra tube as a surrogate. The internal DIR accuracy was inspected in term of the Dice similarity coefficient (DSC), Hausdorff and mean surface distance as defined in of the urethra structure inside the phantom and compared with that of the initial IM-DIR under various stages of deformation. Voxel-wise deformation vector discrepancy and Jacobian regularity were also inspected to evaluate the output DVFs. In addition to phantom, two pairs of Head&Neck patient MR images with expert-defined landmarks inside parotids were utilized to evaluate the BM-DIR accuracy with target registration error (TRE). RESULTS: The DSC and surface distance measures of the inner urethra tube indicated the BM-DIR method can improve the internal DVF accuracy on masked MR images for the phases of a large degree of deformation. The smoother Jacobian distribution from the BM-DIR suggests more physically-plausible internal deformation. For H&N cancer patients, the BM-DIR improved the TRE from 0.339 cm to 0.210 cm for the landmarks inside parotid on the masked MR images. CONCLUSIONS: We have quantitatively demonstrated on a multi-stage physical phantom and limited patient data that the proposed BM-DIR can improve the accuracy inside solid organs with large deformation where distinctive image features are absent.