Microenvironmental reorganization in brain tumors following radiotherapy and recurrence revealed by hyperplexed immunofluorescence imaging.

The tumor microenvironment plays a crucial role in determining response to treatment. This involves a series of interconnected changes in the cellular landscape, spatial organization, and extracellular matrix composition. However, assessing these alterations simultaneously is challenging from a spatial perspective, due to the limitations of current high-dimensional imaging techniques and the extent of intratumoral heterogeneity over large lesion areas. In this study, we introduce a spatial proteomic workflow termed Hyperplexed Immunofluorescence Imaging (HIFI) that overcomes these limitations. HIFI allows for the simultaneous analysis of > 45 markers in fragile tissue sections at high magnification, using a cost-effective high-throughput workflow. We integrate HIFI with machine learning feature detection, graph-based network analysis, and cluster-based neighborhood analysis to analyze the microenvironment response to radiation therapy in a preclinical model of glioblastoma, and compare this response to a mouse model of breast-to-brain metastasis. Here we show that glioblastomas undergo extensive spatial reorganization of immune cell populations and structural architecture in response to treatment, while brain metastases show no comparable reorganization. Our integrated spatial analyses reveal highly divergent responses to radiation therapy between brain tumor models, despite equivalent radiotherapy benefit.

Buds of new bone formation within the Femoral Head of Hip Fracture Patients Coincide with Zones of Low Osteocyte Sclerostin.

Romosozumab treatment reduces the rate of hip fractures and increases hip bone density, increasing bone formation by inhibiting sclerostin protein. We studied the normal pattern of bone formation and osteocyte expression in the human proximal femur because it is relevant to both antisclerostin treatment effects and fracture. Having visualized and quantified buds of new bone formation in trabeculae, we hypothesized that they would coincide with areas of (a) higher mechanical stress and (b) low sclerostin expression by osteocytes. In patients with hip fracture, we visualized each bud of active modeling-based formation (forming minimodeling structure [FMiS]) in trabecular cores taken from different parts of the femoral head. Trabecular bone structure was also measured with high-resolution imaging. More buds of new bone formation (by volume) were present in the higher stress superomedial zone (FMiS density, N.FMiS/T.Ar) than lower stress superolateral (p < 0.05), and inferomedial (p < 0.001) regions. There were fewer sclerostin expressing osteocytes close to or within FMiS. FMiS density correlated with greater amount, thickness, number, and connectivity of trabeculae (bone volume BV/TV, r = 0.65, p < 0.0001; bone surface BS/TV, r = 0.47, p < 0.01; trabecular thickness Tb.Th, r = 0.55, p < 0.001; trabecular number Tb.N, r = 0.47, p < 0.01; and connectivity density Conn.D, r = 0.40, p < 0.05) and lower trabecular separation (Tb.Sp, r = -0.56, p < 0.001). These results demonstrate modeling-based bone formation in femoral trabeculae from patients with hip fracture as a potential therapeutic target to enhance bone structure. © 2023 American Society for Bone and Mineral Research (ASBMR).

Imaging and Molecular Annotation of Xenographs and Tumours (IMAXT): High throughput data and analysis infrastructure.

With the aim of producing a 3D representation of tumors, imaging and molecular annotation of xenografts and tumors (IMAXT) uses a large variety of modalities in order to acquire tumor samples and produce a map of every cell in the tumor and its host environment. With the large volume and variety of data produced in the project, we developed automatic data workflows and analysis pipelines. We introduce a research methodology where scientists connect to a cloud environment to perform analysis close to where data are located, instead of bringing data to their local computers. Here, we present the data and analysis infrastructure, discuss the unique computational challenges and describe the analysis chains developed and deployed to generate molecularly annotated tumor models. Registration is achieved by use of a novel technique involving spherical fiducial marks that are visible in all imaging modalities used within IMAXT. The automatic pipelines are highly optimized and allow to obtain processed datasets several times quicker than current solutions narrowing the gap between data acquisition and scientific exploitation.

Topical treatment of actinic keratoses in organ transplant recipients: a feasibility study for SPOT (Squamous cell carcinoma Prevention in Organ transplant recipients using Topical treatments).

BACKGROUND: The risk of cutaneous squamous cell carcinoma (cSCC) is significantly increased in organ transplant recipients (OTRs). Clearance of actinic keratoses (AKs) is generally regarded as a surrogate biomarker for cSCC prevention. OTR-cSCC chemoprevention with topical AK treatments has not been investigated in randomized controlled trials (RCTs), although there is evidence that 5% 5-fluorouracil (5-FU) may be chemoprotective in immunocompetent patients. OBJECTIVES: To assess the feasibility, activity and evaluation outcomes relevant to the design of a future phase III RCT of topical cSCC chemoprevention in OTRs. METHODS: OTRs with 10 or more AKs in predefined areas were randomized 1 : 1 : 1 to topical 5-FU, 5% imiquimod (IMIQ) or sunscreen (sun-protective factor 30+) in a phase II, open-label RCT over 15 months. Feasibility outcomes included proportions of eligible OTRs randomized, completing treatment and willing to be re-treated. AK activity [AK clearance, new AK development, patient-centred outcomes (toxicity, health-related quality of life, HRQoL)] and evaluation methodology (clinical vs. photographic) were assessed. RESULTS: Forty OTRs with 903 AKs were randomized. All feasibility outcomes were met (56% of eligible OTRs were randomized; 89% completed treatment; 81% were willing to be re-treated). AK activity analyses found 5-FU and IMIQ were superior to sunscreen for AK clearance and prevention of new AKs. 5-FU was more effective than IMIQ in AK clearance and prevention in exploratory analyses. Although toxicity was greater with 5-FU, HRQoL outcomes were similar. CONCLUSIONS: Trials of topical AK treatments in OTRs for cSCC chemoprevention are feasible and AK activity results support further investigation of 5-FU-based treatments in future phase III trials. What is already known about this topic? Cutaneous squamous cell carcinoma (cSCC) is significantly more common in immunocompromised individuals including organ transplant recipients (OTRs) compared with immunocompetent populations. cSCC chemoprevention activity of sunscreen and 5-fluorouracil-based (5-FU) actinic keratosis (AK) treatments has been demonstrated in randomized controlled trials (RCTs) in immunocompetent populations but not in OTRs. AKs are cSCC precursors and their clearance and prevention are generally regarded as surrogate endpoint biomarkers for potential cSCC chemoprevention activity. What does this study add? SPOT (SCC Prevention in OTRs using Topical treatments) has confirmed that RCTs of OTR-cSCC chemoprevention with topical AK treatments are feasible. It also suggests that topical 5-FU may be superior to 5% imiquimod and sunscreen in AK clearance and prevention. Together with recent evidence from several RCTs in the general population, these data provide a compelling rationale for further studies of intervention with 5-FU-based topical chemoprevention approaches in OTR-cSCC prevention.

Romosozumab Enhances Vertebral Bone Structure in Women With Low Bone Density.

Romosozumab monoclonal antibody treatment works by binding sclerostin and causing rapid stimulation of bone formation while decreasing bone resorption. The location and local magnitude of vertebral bone accrual by romosozumab and how it compares to teriparatide remains to be investigated. Here we analyzed the data from a study collecting lumbar computed tomography (CT) spine scans at enrollment and 12 months post-treatment with romosozumab (210 mg sc monthly, n = 17), open-label daily teriparatide (20 µg sc, n = 19), or placebo (sc monthly, n = 20). For each of the 56 women, cortical thickness (Ct.Th), endocortical thickness (Ec.Th), cortical bone mineral density (Ct.bone mineral density (BMD)), cancellous BMD (Cn.BMD), and cortical mass surface density (CMSD) were measured across the first lumbar vertebral surface. In addition, color maps of the changes in the lumbar vertebrae structure were statistically analyzed and then visualized on the bone surface. At 12 months, romosozumab improved all parameters significantly over placebo and resulted in a mean vertebral Ct.Th increase of 10.3% versus 4.3% for teriparatide, an Ec.Th increase of 137.6% versus 47.5% for teriparatide, a Ct.BMD increase of 2.1% versus a -0.1% decrease for teriparatide, and a CMSD increase of 12.4% versus 3.8% for teriparatide. For all these measurements, the differences between romosozumab and teriparatide were statistically significant (p < 0.05). There was no significant difference between the romosozumab-associated Cn.BMD gains of 22.2% versus 18.1% for teriparatide, but both were significantly greater compared with the change in the placebo group (-4.6%, p < 0.05). Cortical maps showed the topographical locations of the increase in bone in fracture-prone areas of the vertebral shell, walls, and endplates. This study confirms widespread vertebral bone accrual with romosozumab or teriparatide treatment and provides new insights into how the rapid prevention of vertebral fractures is achieved in women with osteoporosis using these anabolic agents. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

A cross-sectional study on the age-related cortical and trabecular bone changes at the femoral head in elderly female hip fracture patients.

Bone is in a continuous state of remodeling whereby old bone is absorbed and new bone is formed in its place. During this process, new formations reinforce the bone in the direction of the dominant stress trajectories through a functional adaptation. In normal aging, the balance between bone resorption and formation can be shifted. How this affects the functional adaptation remains to be investigated. Furthermore, how or whether the bone continues to change beyond the age of 85 is not yet studied in detail. In this study we examined the age-related changes in the cortical and trabecular bone in old age, and assessed whether we can find evidence of the presence of functional adaptation. We measured cortical and trabecular parameters from micro-computed tomography scans of the femoral head extracted from hip fracture patients between the age of 70 and 93 years. A significant decrease in global trabecular bone mineral density (38.1%) and cortical thickness (13.0%) was seen from the 9th to the 10th decade of life. The degree of anisotropy was maintained globally as well as locally in both high and low stress regions. The local trabecular bone mineral density decreased in both high stress and low stress regions between the 9th and 10th decade of life with similar trends. This suggests that the role of functional adaptation in maintaining the bone structural integrity in old age may be limited. This study highlights the need for a controlled clinical trial examining the cause of the continued bone degradation throughout old age.

An exploratory study into measuring the cortical bone thickness from CT in the presence of metal implants.

PURPOSE: The aim of this study was to develop and evaluate a method for measuring the cortical bone thickness from computed tomography (CT) scans with metallic implants and to assess the benefits of metal artefact removal software. METHODS: A previously validated technique based on the fitting of a cortical model was modified to also model metal structures when required. Cortical thickness measurements were taken over intact bone segments and compared with the corresponding contralateral bone segment. The evaluation dataset includes post-operative CT scans of a unipolar hemi-arthroplasty, a dynamic hip screw fixation, a bipolar hemi-arthroplasty, a fixation with cannulated screws and a total hip arthroplasty. All CT scans were analysed before and after processing with metal artefact removal software. RESULTS: Cortical thickness validity and accuracy were improved through the use of a modified metalwork-optimised model and metal artefact removal software. For the proximal femoral segments of the aforementioned cases, the cortical thickness was measured with a mean absolute error of 0.55, 0.39, 0.46, 0.53 and 0.69 mm. The hemi-pelvis produced thickness errors of 0.51, 0.52, 0.52, 0.47 and 0.67 mm, respectively. CONCLUSIONS: The proposed method was shown to measure cortical bone thickness in the presence of metalwork at a sub-millimetre accuracy. This new technique might be helpful in assessing fracture healing near implants or fixation devices, and improve the evaluation of periprosthetic bone after hip replacement surgery.

The Effects on the Femoral Cortex of a 24 Month Treatment Compared to an 18 Month Treatment with Teriparatide: A Multi-Trial Retrospective Analysis.

BACKGROUND: Teriparatide (TPTD) is an anabolic agent indicated for the treatment of severely osteoporotic patients who are at high risk of fragility fractures. The originally approved duration of TPTD treatment in several regions, including Europe, was 18 months. However, studies of areal bone mineral density (aBMD) showed additional benefit when treatment is continued beyond 18 months, and the drug is currently licenced for 24 months. Improvements in cortical structure at the proximal femur have already been shown in patients given TPTD for 24 months using quantitative computed tomography (QCT). Here, we investigate whether cortical and endocortical trabecular changes differ between an 18- and 24-month treatment. METHODS: Since an 18- versus 24-month TPTD study using QCT has not been conducted, we studied combined QCT data from four previous clinical trials. Combined femoral QCT data from three 18-month TPTD studies ('18-month group') were compared with data from a fourth 24-month trial ('24-month group'). Cortical parameters were measured over the entire proximal femur which allowed for a comparison of the mean changes as well as a visual comparison of the colour maps of changes after 18 and 24 months TPTD. RESULTS: For both the combined 18-month group and the 24-month group, overall cortical thickness and endocortical trabecular density increased, while overall cortical bone mineral density decreased. While the changes in the 24-month group were of greater magnitude compared to the 18-month group, the differences were only significant for the endocortical trabecular density (ECTD), corrected for age, weight, femoral neck T-score, total hip T-score and the baseline mean ECTD. CONCLUSION: Although the combination of data from different clinical trials is not optimal, these data support the concept that the duration of TPTD in the 18-24 month phase is of clinical relevance when considering improvement in hip structure.

Mapping Bone Changes at the Proximal Femoral Cortex of Postmenopausal Women in Response to Alendronate and Teriparatide Alone, Combined or Sequentially.

Combining antiresorptive and anabolic drugs for osteoporosis may be a useful strategy to prevent hip fractures. Previous studies comparing the effects of alendronate (ALN) and teriparatide (TPTD) alone, combined or sequentially using quantitative computed tomography (QCT) in postmenopausal women have not distinguished cortical bone mineral density (CBMD) from cortical thickness (CTh) effects, nor assessed the distribution and extent of more localized changes. In this study a validated bone mapping technique was used to examine the cortical and endocortical trabecular changes in the proximal femur resulting from an 18-month course of ALN or TPTD. Using QCT data from a different clinical trial, the global and localized changes seen following a switch to TPTD after an 18-month ALN treatment or adding TPTD to the ALN treatment were compared. Ct.Th increased (4.8%, p < 0.01) and CBMD decreased (-4.5%, p < 0.01) in the TPTD group compared to no significant change in the ALN group. A large Ct.Th increase could be seen for the switch group (2.8%, p < 0.01) compared to a significantly smaller increase for the add group (1.5%, p < 0.01). CBMD decreased significantly for the switch group (-3.9%, p < 0.01) and was significantly different from no significant change in the add group. Ct.Th increases were shown to be significantly greater for the switch group compared to the add group at the load bearing regions. This study provides new insights into the effects of ALN and TPTD combination therapies on the cortex of the proximal femur and supports the hypothesis of an increased bone remodeling by TPTD being mitigated by ALN.

3D reconstruction of the lumbar vertebrae from anteroposterior and lateral dual-energy X-ray absorptiometry.

Current vertebral fracture prevention measures use Dual-energy X-ray Absorptiometry (DXA) to quantify the density of the vertebrae and subsequently determine the risk of fracture. This modality however only provides information about the projected Bone Mineral Density (BMD) while the shape and spatial distribution of the bone determines the strength of the vertebrae. Quantitative Computed Tomography (QCT) allows for the measurement of the vertebral dimensions and volumetric densities, which have been shown to be able to determine the fracture risk more reliably than DXA. However, for the high cost and high radiation dose, QCT is not used in clinical routine for fracture risk assessment. In this work, we therefore propose a method to reconstruct the 3D shape and density volume of lumbar vertebrae from an anteroposterior (AP) and lateral DXA image used in clinical routine. The method is evaluated for the L2, L3 and L4 vertebra. Of these vertebrae a statistical model of the vertebral shape and density distribution is first constructed from a large dataset of QCT scans. All three models are then simultaneously registered onto both AP and lateral DXA image. The shape and volumetric BMD at several regions of the reconstructed vertebrae is then evaluated with respect to the ground truth QCT volumes. For the L2, L3 and L4 vertebrae respectively the shape was reconstructed with a mean (2RMS) point-to-surface distance of 1.00 (2.64) mm, 0.93(2.52) mm and 1.34(3.72) mm and a strong correlation (r > 0.82) was found between the trabecular volumetric BMD extracted from the reconstructions and from the same subject QCT scans. These results indicate that the proposed method is able to accurately reconstruct the 3D shape and density volume of the lumbar vertebrae from AP and lateral DXA, which can potentially improve the fracture risk estimation accuracy with respect to the currently used DXA derived areal BMD measurements.

A high-resolution atlas and statistical model of the human heart from multislice CT.

Atlases and statistical models play important roles in the personalization and simulation of cardiac physiology. For the study of the heart, however, the construction of comprehensive atlases and spatio-temporal models is faced with a number of challenges, in particular the need to handle large and highly variable image datasets, the multi-region nature of the heart, and the presence of complex as well as small cardiovascular structures. In this paper, we present a detailed atlas and spatio-temporal statistical model of the human heart based on a large population of 3D+time multi-slice computed tomography sequences, and the framework for its construction. It uses spatial normalization based on nonrigid image registration to synthesize a population mean image and establish the spatial relationships between the mean and the subjects in the population. Temporal image registration is then applied to resolve each subject-specific cardiac motion and the resulting transformations are used to warp a surface mesh representation of the atlas to fit the images of the remaining cardiac phases in each subject. Subsequently, we demonstrate the construction of a spatio-temporal statistical model of shape such that the inter-subject and dynamic sources of variation are suitably separated. The framework is applied to a 3D+time data set of 138 subjects. The data is drawn from a variety of pathologies, which benefits its generalization to new subjects and physiological studies. The obtained level of detail and the extendability of the atlas present an advantage over most cardiac models published previously.

Hip fracture discrimination from dual-energy X-ray absorptiometry by statistical model registration.

Although the areal Bone Mineral Density (BMD) measurements from dual-energy X-ray absorptiometry (DXA) are able to discriminate between hip fracture cases and controls, the femoral strength is largely determined by the 3D bone structure. In a previous work a statistical model was presented which parameterizes the 3D shape and BMD distribution of the proximal femur. In this study the parameter values resulting from the registration of the model onto DXA images are evaluated for their hip fracture discrimination ability with respect to regular DXA derived areal BMD measurements. The statistical model was constructed from a large database of QCT scans of females with an average age of 67.8 ± 17.0 years. This model was subsequently registered onto the DXA images of a fracture and control group. The fracture group consisted of 175 female patients with an average age of 66.4 ± 9.9 years who suffered a fracture on the contra lateral femur. The control group consisted of 175 female subjects with an average age of 65.3 ± 10.0 years and no fracture history. The discrimination ability of the resulting model parameter values, as well as the areal BMD measurements extracted from the DXA images were evaluated using a logistic regression analysis. The area under the receiver operating curve (AUC) of the combined model parameters and areal BMD values was 0.840 (95% CI 0.799-0.881), whilst using only the areal BMD values resulted in an AUC of 0.802 (95% CI 0.757-0.848). These results indicate that the discrimination ability of the areal BMD values is improved by supplementing them with the model parameter values, which give a more complete representation of the subject specific shape and internal bone distribution. Thus, the presented method potentially allows for an improved hip fracture risk estimation whilst maintaining DXA as the current standard modality.

Technical note: comparison between single and multiview simulated DXA configurations for reconstructing the 3D shape and bone mineral density distribution of the proximal femur.

PURPOSE: Dual-energy x-ray absorptiometry (DXA) is used in clinical routine to provide a two-dimensional (2D) analysis of the bone mineral density (BMD). 3D reconstruction methods from 2D DXA images could improve the BMD analysis. To find the optimal configuration that should be used in clinical routine, this paper relies on a 3D reconstruction method from DXA images to compare the accuracy that can be obtained from one single-view and from multiview DXA images (two to four projections). METHODS: The 3D reconstruction method uses a statistical model and a nonrigid registration technique to recover in 3D the shape and the BMD distribution of the proximal femur. The accuracy was evaluated in vivo by comparing 3D reconstructions obtained from simulated DXA images of 30 patients (using between one and four DXA views) with quantitative computed tomography reconstructions. RESULTS: This comparison showed that the use of one single DXA provides accurate 3D reconstructions (mean shape accuracy of 1.0 mm and BMD distribution errors of 7.0%). Among the multiview configurations, the use of two views (0° and 45°) was the best compromise, increasing the accuracy of pose (mean accuracy of 0.7°/1.2°/0.9° against 1.0°/3.5°/3.3° for the single view), reducing slightly the BMD errors (5.7%) while maintaining the same shape accuracy. CONCLUSIONS: The use of two views constitutes an interesting configuration when multiview DXA devices are available in clinical routine. However, the use of only one single view remains an accurate solution to recover the shape and the BMD distribution in 3D, with the advantage of a higher potential for clinical translation.

A statistical model of shape and bone mineral density distribution of the proximal femur for fracture risk assessment.

This work presents a statistical model of both the shape and Bone Mineral Density (BMD) distribution of the proximal femur for fracture risk assessment. The shape and density model was built from a dataset of Quantitative Computed Tomography scans of fracture patients and a control group. Principal Component Analysis and Horn's parallel analysis were used to reduce the dimensionality of the shape and density model to the main modes of variation. The input data was then used to analyze the model parameters for the optimal separation between the fracture and control group. Feature selection using the Fisher criterion determined the parameters with the best class separation, which were used in Fisher Linear Discriminant Analysis to find the direction in the parameter space that best separates the fracture and control group. This resulted in a Fisher criterion value of 6.70, while analyzing the Dual-energy X-ray Absorptiometry derived femur neck areal BMD of the same subjects resulted in a Fisher criterion value of 0.98. This indicates that a fracture risk estimation approach based on the presented model might improve upon the current standard clinical practice.

Reconstructing the 3D shape and bone mineral density distribution of the proximal femur from dual-energy X-ray absorptiometry.

The accurate diagnosis of osteoporosis has gained increasing importance due to the aging of our society. Areal bone mineral density (BMD) measured by dual-energy X-ray absorptiometry (DXA) is an established criterion in the diagnosis of osteoporosis. This measure, however, is limited by its two-dimensionality. This work presents a method to reconstruct both the 3D bone shape and 3D BMD distribution of the proximal femur from a single DXA image used in clinical routine. A statistical model of the combined shape and BMD distribution is presented, together with a method for its construction from a set of quantitative computed tomography (QCT) scans. A reconstruction is acquired in an intensity based 3D-2D registration process whereby an instance of the model is found that maximizes the similarity between its projection and the DXA image. Reconstruction experiments were performed on the DXA images of 30 subjects, with a model constructed from a database of QCT scans of 85 subjects. The accuracy was evaluated by comparing the reconstructions with the same subject QCT scans. The method presented here can potentially improve the diagnosis of osteoporosis and fracture risk assessment from the low radiation dose and low cost DXA devices currently used in clinical routine.