Broad transcriptomic dysregulation occurs across the cerebral cortex in ASD.

Neuropsychiatric disorders classically lack defining brain pathologies, but recent work has demonstrated dysregulation at the molecular level, characterized by transcriptomic and epigenetic alterations1-3. In autism spectrum disorder (ASD), this molecular pathology involves the upregulation of microglial, astrocyte and neural-immune genes, the downregulation of synaptic genes, and attenuation of gene-expression gradients in cortex1,2,4-6. However, whether these changes are limited to cortical association regions or are more widespread remains unknown. To address this issue, we performed RNA-sequencing analysis of 725 brain samples spanning 11 cortical areas from 112 post-mortem samples from individuals with ASD and neurotypical controls. We find widespread transcriptomic changes across the cortex in ASD, exhibiting an anterior-to-posterior gradient, with the greatest differences in primary visual cortex, coincident with an attenuation of the typical transcriptomic differences between cortical regions. Single-nucleus RNA-sequencing and methylation profiling demonstrate that this robust molecular signature reflects changes in cell-type-specific gene expression, particularly affecting excitatory neurons and glia. Both rare and common ASD-associated genetic variation converge within a downregulated co-expression module involving synaptic signalling, and common variation alone is enriched within a module of upregulated protein chaperone genes. These results highlight widespread molecular changes across the cerebral cortex in ASD, extending beyond association cortex to broadly involve primary sensory regions.

Mathematical modeling links Wnt signaling to emergent patterns of metabolism in colon cancer.

Cell-intrinsic metabolic reprogramming is a hallmark of cancer that provides anabolic support to cell proliferation. How reprogramming influences tumor heterogeneity or drug sensitivities is not well understood. Here, we report a self-organizing spatial pattern of glycolysis in xenograft colon tumors where pyruvate dehydrogenase kinase (PDK1), a negative regulator of oxidative phosphorylation, is highly active in clusters of cells arranged in a spotted array. To understand this pattern, we developed a reaction-diffusion model that incorporates Wnt signaling, a pathway known to upregulate PDK1 and Warburg metabolism. Partial interference with Wnt alters the size and intensity of the spotted pattern in tumors and in the model. The model predicts that Wnt inhibition should trigger an increase in proteins that enhance the range of Wnt ligand diffusion. Not only was this prediction validated in xenograft tumors but similar patterns also emerge in radiochemotherapy-treated colorectal cancer. The model also predicts that inhibitors that target glycolysis or Wnt signaling in combination should synergize and be more effective than each treatment individually. We validated this prediction in 3D colon tumor spheroids.

Enhancer-targeted CRISPR-Activation Rescues Haploinsufficient Autism Susceptibility Genes.

Autism Spectrum Disorder (ASD) is a highly heritable condition with diverse clinical presentations. Approximately 20% of ASD's genetic susceptibility is imparted by de novo mutations of major effect, most of which cause haploinsufficiency. We mapped enhancers of two high confidence autism genes - CHD8 and SCN2A and used CRISPR-based gene activation (CRISPR-A) in hPSC-derived excitatory neurons and cerebral forebrain organoids to correct the effects of haploinsufficiency, taking advantage of the presence of a wildtype allele of each gene and endogenous gene regulation. We found that CRISPR-A induced a sustained increase in CHD8 and SCN2A expression in treated neurons and organoids, with rescue of gene expression levels and mutation-associated phenotypes, including gene expression and physiology. These data support gene activation via targeting enhancers of haploinsufficient genes, as a therapeutic intervention in ASD and other neurodevelopmental disorders.

Challenges and opportunities for precision medicine in neurodevelopmental disorders.

Neurodevelopmental Disorders (NDDs) encompass a broad spectrum of disorders, linked because of their origins in brain developmental processes, including diverse conditions across the age span, including autism spectrum disorders (ASD) and schizophrenia (SCZ). Clinical treatment of these disorders has traditionally focused on symptom management, as the severity of developmental disruption varies widely and the precise molecular mechanisms, timing, and progression of these disorders is usually not known. Several hundred genes have been identified as major risk factors for ASD and SCZ, which creates new potential therapeutic avenues, and there is strong evidence that these genes converge upon key molecular pathways, pointing to opportunities for precision medicine. In this review, we focus on forms of ASD and SCZ with known genetic etiologies and discuss advances in research technologies that enable a more systemic understanding of disease progression. We highlight recent advances in targeted clinical treatment and discuss ongoing preclinical efforts as well as new initiatives aimed at developing scalable platforms for NDD precision medicine.

Disruption of ß-Catenin-Dependent Wnt Signaling in Colon Cancer Cells Remodels the Microenvironment to Promote Tumor Invasion.

The recent classification of colon cancer into molecular subtypes revealed that patients with the poorest prognosis harbor tumors with the lowest levels of Wnt signaling. This is contrary to the general understanding that overactive Wnt signaling promotes tumor progression from early initiation stages through to the later stages including invasion and metastasis. Here, we directly test this assumption by reducing the activity of ß-catenin-dependent Wnt signaling in colon cancer cell lines at either an upstream or downstream step in the pathway. We determine that Wnt-reduced cancer cells exhibit a more aggressive disease phenotype, including increased mobility in vitro and disruptive invasion into mucosa and smooth muscle in an orthotopic mouse model. RNA sequencing reveals that interference with Wnt signaling leads to an upregulation of gene programs that favor cell migration and invasion and a downregulation of inflammation signatures in the tumor microenvironment. We identify a set of upregulated genes common among the Wnt perturbations that are predictive of poor patient outcomes in early-invasive colon cancer. Our findings suggest that while targeting Wnt signaling may reduce tumor burden, an inadvertent side effect is the emergence of invasive cancer. IMPLICATIONS: Decreased Wnt signaling in colon tumors leads to a more aggressive disease phenotype due to an upregulation of gene programs favoring cell migration in the tumor and downregulation of inflammation programs in the tumor microenvironment; these impacts must be carefully considered in developing Wnt-targeting therapies.

Proton radiography and fluoroscopy of lung tumors: a Monte Carlo study using patient-specific 4DCT phantoms.

PURPOSE: Monte Carlo methods are used to simulate and optimize a time-resolved proton range telescope (TRRT) in localization of intrafractional and interfractional motions of lung tumor and in quantification of proton range variations. METHODS: The Monte Carlo N-Particle eXtended (MCNPX) code with a particle tracking feature was employed to evaluate the TRRT performance, especially in visualizing and quantifying proton range variations during respiration. Protons of 230 MeV were tracked one by one as they pass through position detectors, patient 4DCT phantom, and finally scintillator detectors that measured residual ranges. The energy response of the scintillator telescope was investigated. Mass density and elemental composition of tissues were defined for 4DCT data. RESULTS: Proton water equivalent length (WEL) was deduced by a reconstruction algorithm that incorporates linear proton track and lateral spatial discrimination to improve the image quality. 4DCT data for three patients were used to visualize and measure tumor motion and WEL variations. The tumor trajectories extracted from the WEL map were found to be within 1 mm agreement with direct 4DCT measurement. Quantitative WEL variation studies showed that the proton radiograph is a good representation of WEL changes from entrance to distal of the target. CONCLUSIONS: MCNPX simulation results showed that TRRT can accurately track the motion of the tumor and detect the WEL variations. Image quality was optimized by choosing proton energy, testing parameters of image reconstruction algorithm, and comparing to ground truth 4DCT. The future study will demonstrate the feasibility of using the time resolved proton radiography as an imaging tool for proton treatments of lung tumors.

An in vitro vascularized micro-tumor model of human colorectal cancer recapitulates in vivo responses to standard-of-care therapy.

Around 95% of anti-cancer drugs that show promise during preclinical study fail to gain FDA-approval for clinical use. This failure of the preclinical pipeline highlights the need for improved, physiologically-relevant in vitro models that can better serve as reliable drug-screening and disease modeling tools. The vascularized micro-tumor (VMT) is a novel three-dimensional model system (tumor-on-a-chip) that recapitulates the complex human tumor microenvironment, including perfused vasculature, within a transparent microfluidic device, allowing real-time study of drug responses and tumor-stromal interactions. Here we have validated this microphysiological system (MPS) platform for the study of colorectal cancer (CRC), the second leading cause of cancer-related deaths, by showing that gene expression, tumor heterogeneity, and treatment responses in the VMT more closely model CRC tumor clinicopathology than current standard drug screening modalities, including 2-dimensional monolayer culture and 3-dimensional spheroids.

Variations in tumor size and position due to irregular breathing in 4D-CT: a simulation study.

PURPOSE: To estimate the position and volume errors in 4D-CT caused by irregular breathing. METHODS: A virtual 4D-CT scanner was designed to reproduce axial mode scans with retrospective resorting. This virtual scanner creates an artificial spherical tumor based on the specifications of the user, and recreates images that might be produced by a 4D-CT scanner using a patient breathing waveform. 155 respiratory waveforms of patients were used to test the variability of 4D-CT scans. Each breathing waveform was normalized and scaled to 1, 2, and 3 cm peak-to-peak motion, and artificial tumors with 2 and 4 cm radius were simulated for each scaled waveform. The center of mass and volume of resorted 4D-CT images were calculated and compared to the expected values of center of mass and volume for the artificial tumor. Intrasubject variability was investigated by running the virtual scanner over different subintervals of each patient's breathing waveform. RESULTS: The average error in the center of mass location of an artificial tumor was less than 2 mm standard deviation for 2 cm motion. The corresponding average error in volume was less than 4%. In the worst-case scenarios, a center of mass error of 1.0 cm standard deviation and volume errors of 30%-60% at inhale were found. Systematic errors were observed in a subset of patients due to irregular breathing, and these errors were more pronounced when the tumor volume is smaller. CONCLUSIONS: Irregular breathing during 4D-CT simulation causes systematic errors in volume and center of mass measurements. These errors are small but depend on the tumor size, motion amplitude, and degree of breathing irregularity.

Evaluation and commissioning of a surface based system for respiratory sensing in 4D CT.

The purpose of this study is to assess the temporal and reconstruction accuracy of a surface imaging system, the GateCT under ideal conditions, and compare the device with a commonly used respiratory surrogate: the Varian RPM. A clinical CT scanner, run in cine mode, was used with two optical devices, GateCT and RPM, to detect respiratory motion. A radiation detector, GM-10, triggers the X-ray on/off to GateCT system, while the RPM is directly synchronized with the CT scanner through an electronic connection. Two phantoms were imaged: the first phantom translated on a rigid plate along the anterior-posterior (AP) direction, and was used to assess the temporal synchronization of each optical system with the CT scanner. The second phantom, consisting of five spheres translating 3 cm peak-to-peak in the superior-inferior direction, was used to assess the quality of rebinned images created by GateCT and RPM. Calibration assessment showed a nearly perfect synchronization with the scanner for both the RPM and GateCT systems, thus demonstrating the good performance of the radiation detector. Results for the volume rebinning test showed discrepancies in volumes for the 3D reconstruction (compared to ground truth) of up to 36% for GateCT and up to 40% for RPM. No statistical difference was proven between the two systems in volume sorting. Errors are mainly due to phase detection inaccuracies and to the large motion of the phantom. This feasibility study assessed the consistency of two optical systems in synchronizing the respiratory signal with the image acquisition. A new patient protocol based on both RPM and GateCT will be soon started.

Accuracy in breast shape alignment with 3D surface fitting algorithms.

Surface imaging is in use in radiotherapy clinical practice for patient setup optimization and monitoring. Breast alignment is accomplished by searching for a tentative spatial correspondence between the reference and daily surface shape models. In this study, the authors quantify whole breast shape alignment by relying on texture features digitized on 3D surface models. Texture feature localization was validated through repeated measurements in a silicone breast phantom, mounted on a high precision mechanical stage. Clinical investigations on breast shape alignment included 133 fractions in 18 patients treated with accelerated partial breast irradiation. The breast shape was detected with a 3D video based surface imaging system so that breathing was compensated. An in-house algorithm for breast alignment, based on surface fitting constrained by nipple matching (constrained surface fitting), was applied. Results were compared with a commercial software where no constraints are utilized (unconstrained surface fitting). Texture feature localization was validated within 2 mm in each anatomical direction. Clinical data show that unconstrained surface fitting achieves adequate accuracy in most cases, though nipple mismatch is considerably higher than residual surface distances (3.9 mm vs 0.6 mm on average). Outliers beyond 1 cm can be experienced as the result of a degenerate surface fit, where unconstrained surface fitting is not sufficient to establish spatial correspondence. In the constrained surface fitting algorithm, average surface mismatch within 1 mm was obtained when nipple position was forced to match in the [1.5; 5] mm range. In conclusion, optimal results can be obtained by trading off the desired overall surface congruence vs matching of selected landmarks (constraint). Constrained surface fitting is put forward to represent an improvement in setup accuracy for those applications where whole breast positional reproducibility is an issue.

Exploration of the potential of liquid scintillators for real-time 3D dosimetry of intensity modulated proton beams.

In this study, the authors investigated the feasibility of using a 3D liquid scintillator (LS) detector system for the verification and characterization of proton beams in real time for intensity and energy-modulated proton therapy. A plastic tank filled with liquid scintillator was irradiated with pristine proton Bragg peaks. Scintillation light produced during the irradiation was measured with a CCD camera. Acquisition rates of 20 and 10 frames per second (fps) were used to image consecutive frame sequences. These measurements were then compared to ion chamber measurements and Monte Carlo simulations. The light distribution measured from the images acquired at rates of 20 and 10 fps have standard deviations of 1.1% and 0.7%, respectively, in the plateau region of the Bragg curve. Differences were seen between the raw LS signal and the ion chamber due to the quenching effects of the LS and due to the optical properties of the imaging system. The authors showed that this effect can be accounted for and corrected by Monte Carlo simulations. The liquid scintillator detector system has a good potential for performing fast proton beam verification and characterization.

Quantification and visualization of charged particle range variations.

PURPOSE: Range variations during respiration affect the penetration of charged particle beams and can result in beam overshoot or undershoot to the target. We have developed analysis tools to quantify the water equivalent pathlength (WEL) variations resulting from respiration (WEL analyzer, Aqualyzer), as well as a data explorer to view WEL variations interactively. METHODS AND MATERIALS: The metrics to characterize and quantify penetration of a charged particle beam during respiration were calculated semiautomatically. The analysis involved the generation of images that (1) encode the radiologic pathlength across a beam's eye view image during the respiratory phase, (2) display the variation of the radiologic pathlength relative to a reference respiratory phase, (3) display isopenetration as a function of breathing, and (4) show range fluctuation images for a compensating bolus when applied to four-dimensional computed tomography. Additional quantities relevant to the analysis of charged particle beams in a breathing patient that are calculated include the beam overshoot volume and beam overshoot distance. These quantities are calculated as a function of time, gantry angle, and position. RESULTS: The software was applied to test cases to illustrate its utility in the analysis of range variations of charged particle beams in the treatment of lung tumors. CONCLUSION: WEL analysis is useful in the rapid assessment of range variations in the treatment of lung tumors and in determining the optimal gantry angle and respiratory gating window. The extension of encoding range fluctuations to a beam's eye view display is helpful in designing plans that are more robust in the presence of motion.

Maximum-intensity volumes for fast contouring of lung tumors including respiratory motion in 4DCT planning.

PURPOSE: To assess the accuracy of maximum-intensity volumes (MIV) for fast contouring of lung tumors including respiratory motion. METHODS AND MATERIALS: Four-dimensional computed tomography (4DCT) data of 10 patients were acquired. Maximum-intensity volumes were constructed by assigning the maximum Hounsfield unit in all CT volumes per geometric voxel to a new, synthetic volume. Gross tumor volumes (GTVs) were contoured on all CT volumes, and their union was constructed. The GTV with all its respiratory motion was contoured on the MIV as well. Union GTVs and GTVs including motion were compared visually. Furthermore, planning target volumes (PTVs) were constructed for the union of GTVs and the GTV on MIV. These PTVs were compared by centroid position, volume, geometric extent, and surface distance. RESULTS: Visual comparison of GTVs demonstrated failure of the MIV technique for 5 of 10 patients. For adequate GTV(MIV)s, differences between PTVs were <1.0 mm in centroid position, 5% in volume, +/-5 mm in geometric extent, and +/-0.5 +/- 2.0 mm in surface distance. These values represent the uncertainties for successful MIV contouring. CONCLUSION: Maximum-intensity volumes are a good first estimate for target volume definition including respiratory motion. However, it seems mandatory to validate each individual MIV by overlaying it on a movie loop displaying the 4DCT data and editing it for possible inadequate coverage of GTVs on additional 4DCT motion states.

Quantitative assessment of range fluctuations in charged particle lung irradiation.

PURPOSE: Water equivalent path length (WEL) variations due to respiration can change the range of a charged particle beam and result in beam overshoot to critical organs or beam undershoot to tumor. We have studied range fluctuations by analyzing four-dimensional computed tomography data and quantitatively assessing potential beam overshoot. METHODS AND MATERIALS: The maximal intensity volume is calculated by combining the gross tumor volume contours at each respiratory phase in the four-dimensional computed tomography study. The first target volume calculates the maximal intensity volume for the entire respiratory cycle (internal target volume [ITV]-radiotherapy [RT]), and the second target volume is the maximal intensity volume corresponding to gated RT (gated-RT, approximately 30% phase window around exhalation). A compensator at each respiratory phase is calculated. Two "composite" compensators for ITV-RT and gated-RT are then designed by selecting the minimal compensator depth at the respective respiratory phase. These compensators are then applied to the four-dimensional computed tomography data to estimate beam penetration. Analysis metrics include range fluctuation and overshoot volume, both as a function of gantry angle. We compared WEL fluctuations observed in treating the ITV-RT versus gated-RT in 11 lung patients. RESULTS: The WEL fluctuations were <21.8 mm-WEL and 9.5 mm-WEL for ITV-RT and gated-RT, respectively for all patients. Gated-RT reduced the beam overshoot volume by approximately a factor of four compared with ITV-RT. Such range fluctuations can affect the efficacy of treatment and result in an excessive dose to a distal critical organ. CONCLUSION: Time varying range fluctuation analysis provides information useful for determining appropriate patient-specific treatment parameters in charged particle RT. This analysis can also be useful for optimizing planning and delivery.

Comparison of target registration errors for multiple image-guided techniques in accelerated partial breast irradiation.

PURPOSE: External beam accelerated partial breast irradiation requires accurate localization of the target volume for each treatment fraction. Using the concept of target registration error (TRE), the performance of several methods of target localization was compared. METHODS AND MATERIALS: Twelve patients who underwent external beam accelerated partial breast irradiation were included in this study. TRE was quantified for four methods of image guidance: standard laser-based setup, kilovoltage imaging of the chest wall, kilovoltage imaging of surgically implanted clips, and three-dimensional surface imaging of the breast. The use of a reference surface created from a free-breathing computed tomography scan and a reference surface directly captured with three-dimensional video imaging were compared. The effects of respiratory motion were also considered, and gating was used for 8 of 12 patients. RESULTS: The median value of the TRE for the laser, chest wall, and clip alignment was 7.1 mm (n=94), 5.4 mm (n=81), and 2.4 mm (n=93), respectively. The median TRE for gated surface imaging based on the first fraction reference surface was 3.2 mm (n=49), and the TRE for gated surface imaging using the computed tomography-based reference surface was 4.9 mm (n=56). The TRE for nongated surface imaging using the first fraction reference surface was 6.2 mm (n=25). CONCLUSIONS: The TRE of surface imaging using a reference surface directly captured with three-dimensional video and the TRE for clip-based setup were within 1 mm. Gated capture is important for surface imaging to reduce the effects of respiratory motion in accelerated partial breast irradiation.

Effects of intrafractional motion on water equivalent pathlength in respiratory-gated heavy charged particle beam radiotherapy.

PURPOSE: To analyze the water equivalent pathlength (WEL) fluctuations resulting from cardiac motion and display these variations on a beam's-eye-view image; the analysis provides insight into the accuracy of lung tumor irradiation with heavy charged particle beams. MATERIALS AND METHODS: Volumetric cine computed tomography (CT) images were obtained on 7 lung cancer patients under free-breathing conditions with a 256-multislice CT scanner. Cardiac phase was determined by selecting systole and diastole. A WEL difference image (DeltaWEL) was calculated by subtracting the WEL image at end-systole from that at end-diastole at respiratory exhalation phase. Two calculation regions were defined: Region 1 was limited to the volume defined by planes bounding the heart; Region 2 included the entire body thickness for a given beam's-eye-view angle. RESULTS: The DeltaWEL values observed in Region 1 showed fluctuations at the periphery of the heart that varied from 20.4 (SD, 5.2) mm WEL to -15.6 (3.2) mm WEL. The areas over which these range perturbation values were observed were 36.8 (32.4) mm(2) and 6.0 (2.8) mm(2) for positive and negative WEL, respectively. The WEL fluctuations in Region 2 increased by approximately 3-4 mm WEL, whereas negative WEL fluctuations changed by approximately -4 to -5 mm WEL, compared with WEL for Region 1; areas over 20 mm WEL changes in Region 2 increased by 9 mm(2) for positive DeltaWEL and 2 mm(2) for negative DeltaWEL. CONCLUSIONS: Cine CT with a 256-multislice CT scanner captures both volumetric cardiac and respiratory motion with a temporal resolution sufficient to estimate range fluctuations by these motions. This information can be used to assess the range perturbations that charged particle beams may experience in irradiation of lung or esophageal tumors adjacent to the heart.

Four-dimensional imaging and treatment planning of moving targets.

Four-dimensional CT acquisition is commercially available, and provides important information on the shape and trajectory of the tumor and normal tissues. The primary advantage of four-dimensional imaging over light breathing helical scans is the reduction of motion artifacts during scanning that can significantly alter tumor appearance. Segmentation, image registration, visualization are new challenges associated with four-dimensional data sets because of the overwhelming increase in the number of images. Four-dimensional dose calculations, while currently laborious, provide insights into dose perturbations due to organ motion. Imaging before treatment (image guidance) improves accuracy of radiation delivery, and recording transmission images can provide a means of verifying gated delivery.

Inhibition of nuclear Wnt signalling: challenges of an elusive target for cancer therapy.

The highly conserved Wnt signalling pathway plays an important role in embryonic development and disease pathogenesis, most notably cancer. The 'canonical' or ß-catenin-dependent Wnt signal initiates at the cell plasma membrane with the binding of Wnt proteins to Frizzled:LRP5/LRP6 receptor complexes and is mediated by the translocation of the transcription co-activator protein, ß-catenin, into the nucleus. ß-Catenin then forms a complex with T-cell factor (TCF)/lymphoid enhancer binding factor (LEF) transcription factors to regulate multiple gene programmes. These programmes play roles in cell proliferation, migration, vasculogenesis, survival and metabolism. Mutations in Wnt signalling pathway components lead to constitutively active Wnt signalling that drives aberrant expression of these programmes and development of cancer. It has been a longstanding and challenging goal to develop therapies that can interfere with the TCF/LEF-ß-catenin transcriptional complex. This review will focus on the (i) structural considerations for targeting the TCF/LEF-ß-catenin and co-regulatory complexes in the nucleus, (ii) current molecules that directly target TCF/LEF-ß-catenin activity and (iii) ideas for targeting newly discovered components of the TCF/LEF-ß-catenin complex and/or downstream gene programmes regulated by these complexes. LINKED ARTICLES: This article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc.

Image interpolation in 4D CT using a BSpline deformable registration model.

PURPOSE: To develop a method for deriving the phase-binned four-dimensional computed tomography (4D CT) image sets through interpolation of the images acquired at some known phases. METHODS AND MATERIALS: Four-dimensional computed tomography data sets for 3 patients were acquired. For each patient, the correlation between inhale and exhale phases was studied and quantified using a BSpline deformable model. Images at an arbitrary phase were deduced by an interpolation of the deformation coefficients. The accuracy of the proposed scheme was assessed by comparing marker trajectories and by checkerboard/difference display of the interpolated and acquired images. RESULTS: The images at intermediate phases could be derived by an interpolation of the deformation field. An analysis of marker movements indicated that 3 mm accuracy is achievable by the interpolation. The subtraction of image analysis indicated a similar level of success. The proposed technique was useful also for automatically mapping the organ contours in a known phase to other phases, and for designing patient-specific margins in the presence of respiratory motion. Finally, the technique led to a 90% reduction in the acquired data, because in the BSpline model, a lattice of only a few thousand values is sufficient to describe a CT data set of 25 million pixels. CONCLUSIONS: Organ deformation can be well modeled by using a BSpline model. The proposed technique may offer useful means for radiation dose reduction, binning artifacts removal, and disk storage improvement in 4D imaging.