Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Lung Motion Model Validation Experiments, Free-Breathing Tissue Densitometry, and Ventilation Mapping using Fast Helical CT Imaging

The uncertainties due to respiratory motion present significant challenges to accurate characterization of cancerous tissues both in terms of imaging and treatment. Currently available clinical lung imaging techniques are subject to inferior image quality and incorrect motion estimation, with consequences that can systematically impact the downstream treatment delivery and outcome. The main objective of this thesis is the development of the techniques of fast helical computed tomography (CT) imaging and deformable image registration for the radiotherapy applications in accurate breathing motion modeling, lung tissue density modeling and ventilation imaging.Fast helical CT scanning was performed on 64-slice CT scanner using the shortest available gantry rotation time and largest pitch value such that scanning of the thorax region amounts to just two seconds, which is less than typical breathing cycle in humans. The scanning was conducted under free breathing condition. Any portion of the lung anatomy undergoing such scanning protocol would be irradiated for only a quarter second, effectively removing any motion induced image artifacts. The resulting CT data were pristine volumetric images that record the lung tissue position and density in a fraction of the breathing cycle. Following our developed protocol, multiple fast helical CT scans were acquired to sample the tissue positions in different breathing states. To measure the tissue displacement, deformable image registration was performed that registers the non-reference images to the reference one. In modeling breathing motion, external breathing surrogate signal was recorded synchronously with the CT image slices. This allowed for the tissue-specific displacement to be modeled as parametrization of the recorded breathing signal using the 5D lung motion model. To assess the accuracy of the motion model in describing tissue position change, the model was used to simulate the original high-pitch helical CT scan geometries, employed as ground truth data. Image similarity between the simulated and ground truth scans was evaluated. The model validation experiments were conducted in a patient cohort of seventeen patients to assess the model robustness and inter-patient variation. The model error averaged over multiple tracked positions from several breathing cycles was found to be on the order of one millimeter.In modeling the density change under free breathing condition, the determinant of Jacobian matrix from the registration-derived deformation vector field yielded volume change information of the lung tissues. Correlation of the Jacobian values to the corresponding voxel Housfield units (HU) reveals that the density variation for the majority of lung tissues can be very well described by mass conservation relationship. Different tissue types were identified and separately modeled. Large trials of validation experiments were performed. The averaged deviation between the modeled and the reference lung density was 30 HU, which was estimated to be the background CT noise level. In characterizing the lung ventilation function, a novel method was developed to determine the extent of lung tissue volume change. Information on volume change was derived from the deformable image registration of the fast helical CT images in terms of Jacobian values with respect to a reference image. Assuming the multiple volume change measurements are independently and identically distributed, statistical formulation was derived to model ventilation distribution of each lung voxels and empirical minimum and maximum probability distribution of the Jacobian values was computed. Ventilation characteristic was evaluated as the difference of the expectation value from these extremal distributions. The resulting ventilation map was compared with an independently obtained ventilation image derived directly from the lung intensities and good correlation was found using statistical test. In addition, dynamic ventilation characterization was investigated by estimating the voxel-specific ventilation distribution. Ventilation maps were generated at different percentile levels using the tissue volume expansion metrics

A client-server framework for 3D remote visualization of radiotherapy treatment space.

Radiotherapy is safely employed for treating wide variety of cancers. The radiotherapy workflow includes a precise positioning of the patient in the intended treatment position. While trained radiation therapists conduct patient positioning, consultation is occasionally required from other experts, including the radiation oncologist, dosimetrist, or medical physicist. In many circumstances, including rural clinics and developing countries, this expertise is not immediately available, so the patient positioning concerns of the treating therapists may not get addressed. In this paper, we present a framework to enable remotely located experts to virtually collaborate and be present inside the 3D treatment room when necessary. A multi-3D camera framework was used for acquiring the 3D treatment space. A client-server framework enabled the acquired 3D treatment room to be visualized in real-time. The computational tasks that would normally occur on the client side were offloaded to the server side to enable hardware flexibility on the client side. On the server side, a client specific real-time stereo rendering of the 3D treatment room was employed using a scalable multi graphics processing units (GPU) system. The rendered 3D images were then encoded using a GPU-based H.264 encoding for streaming. Results showed that for a stereo image size of 1280 × 960 pixels, experts with high-speed gigabit Ethernet connectivity were able to visualize the treatment space at approximately 81 frames per second. For experts remotely located and using a 100 Mbps network, the treatment space visualization occurred at 8-40 frames per second depending upon the network bandwidth. This work demonstrated the feasibility of remote real-time stereoscopic patient setup visualization, enabling expansion of high quality radiation therapy into challenging environments

A client-server framework for 3D remote visualization of radiotherapy treatment space.

Radiotherapy is safely employed for treating wide variety of cancers. The radiotherapy workflow includes a precise positioning of the patient in the intended treatment position. While trained radiation therapists conduct patient positioning, consultation is occasionally required from other experts, including the radiation oncologist, dosimetrist, or medical physicist. In many circumstances, including rural clinics and developing countries, this expertise is not immediately available, so the patient positioning concerns of the treating therapists may not get addressed. In this paper, we present a framework to enable remotely located experts to virtually collaborate and be present inside the 3D treatment room when necessary. A multi-3D camera framework was used for acquiring the 3D treatment space. A client-server framework enabled the acquired 3D treatment room to be visualized in real-time. The computational tasks that would normally occur on the client side were offloaded to the server side to enable hardware flexibility on the client side. On the server side, a client specific real-time stereo rendering of the 3D treatment room was employed using a scalable multi graphics processing units (GPU) system. The rendered 3D images were then encoded using a GPU-based H.264 encoding for streaming. Results showed that for a stereo image size of 1280 × 960 pixels, experts with high-speed gigabit Ethernet connectivity were able to visualize the treatment space at approximately 81 frames per second. For experts remotely located and using a 100 Mbps network, the treatment space visualization occurred at 8-40 frames per second depending upon the network bandwidth. This work demonstrated the feasibility of remote real-time stereoscopic patient setup visualization, enabling expansion of high quality radiation therapy into challenging environments

A Method for Assessing Ground-Truth Accuracy of the 5DCT Technique

PurposeTo develop a technique that assesses the accuracy of the breathing phase-specific volume image generation process by patient-specific breathing motion model using the original free-breathing computed tomographic (CT) scans as ground truths.MethodsSixteen lung cancer patients underwent a previously published protocol in which 25 free-breathing fast helical CT scans were acquired with a simultaneous breathing surrogate. A patient-specific motion model was constructed based on the tissue displacements determined by a state-of-the-art deformable image registration. The first image was arbitrarily selected as the reference image. The motion model was used, along with the free-breathing phase information of the original 25 image datasets, to generate a set of deformation vector fields that mapped the reference image to the 24 nonreference images. The high-pitch helically acquired original scans served as ground truths because they captured the instantaneous tissue positions during free breathing. Image similarity between the simulated and the original scans was assessed using deformable registration that evaluated the pointwise discordance throughout the lungs.ResultsQualitative comparisons using image overlays showed excellent agreement between the simulated images and the original images. Even large 2-cm diaphragm displacements were very well modeled, as was sliding motion across the lung-chest wall boundary. The mean error across the patient cohort was 1.15 ± 0.37 mm, and the mean 95th percentile error was 2.47 ± 0.78 mm.ConclusionThe proposed ground truth-based technique provided voxel-by-voxel accuracy analysis that could identify organ-specific or tumor-specific motion modeling errors for treatment planning. Despite a large variety of breathing patterns and lung deformations during the free-breathing scanning session, the 5-dimensionl CT technique was able to accurately reproduce the original helical CT scans, suggesting its applicability to a wide range of patients

Peritumoral radiomics features predict distant metastasis in locally advanced NSCLC.

PURPOSE:Radiomics provides quantitative tissue heterogeneity profiling and is an exciting approach to developing imaging biomarkers in the context of precision medicine. Normal-appearing parenchymal tissues surrounding primary tumors can harbor microscopic disease that leads to increased risk of distant metastasis (DM). This study assesses whether computed-tomography (CT) imaging features of such peritumoral tissues can predict DM in locally advanced non-small cell lung cancer (NSCLC). MATERIAL AND METHODS:200 NSCLC patients of histological adenocarcinoma were included in this study. The investigated lung tissues were tumor rim, defined to be 3mm of tumor and parenchymal tissue on either side of the tumor border and the exterior region extended from 3 to 9mm outside of the tumor. Fifteen stable radiomic features were extracted and evaluated from each of these regions on pre-treatment CT images. For comparison, features from expert-delineated tumor contours were similarly prepared. The patient cohort was separated into training and validation datasets for prognostic power evaluation. Both univariable and multivariable analyses were performed for each region using concordance index (CI). RESULTS:Univariable analysis reveals that six out of fifteen tumor rim features were significantly prognostic of DM (p-value < 0.05), as were ten features from the visible tumor, and only one of the exterior features was. Multivariablely, a rim radiomic signature achieved the highest prognostic performance in the independent validation sub-cohort (CI = 0.64, p-value = 2.4×10-5) significantly over a multivariable clinical model (CI = 0.53), a visible tumor radiomics model (CI = 0.59), or an exterior tissue model (CI = 0.55). Furthermore, patient stratification by the combined rim signature and clinical predictor led to a significant improvement on the clinical predictor alone and also outperformed stratification using the combined tumor signature and clinical predictor. CONCLUSIONS:We identified peritumoral rim radiomic features significantly associated with DM. This study demonstrated that peritumoral imaging characteristics may provide additional valuable information over the visible tumor features for patient risk stratification due to cancer metastasis