Over 150 000 Americans are diagnosed with colorectal cancer (CRC) every year, and annually over 50 000 individuals will die from CRC, necessitating improvements in screening, prognostication, disease management, and therapeutic options. Tumor metastasis is the primary factor related to the risk of recurrence and mortality. Yet, screening for nodal and distant metastasis is costly, and invasive and incomplete resection may hamper adequate assessment. Signatures of the tumor-immune microenvironment (TIME) at the primary site can provide valuable insights into the aggressiveness of the tumor and the effectiveness of various treatment options. Spatially resolved transcriptomics technologies offer an unprecedented characterization of TIME through high multiplexing, yet their scope is constrained by cost. Meanwhile, it has long been suspected that histological, cytological, and macroarchitectural tissue characteristics correlate well with molecular information . Thus, a method for predicting transcriptomics data through inference of RNA patterns from whole slide images (WSI) is a key step in studying metastasis at scale. In this work, we collected tissue from 4 stage-III (pT3) matched colorectal cancer patients for spatial transcriptomics profiling. The Visium spatial transcriptomics (ST) assay was used to measure transcript abundance for 17 943 genes at up to 5000 55-micron  spots per patient sampled in a honeycomb pattern, co-registered with hematoxylin and eosin (H&E) stained WSI. The Visium ST assay can measure expression at these spots through tissue permeabilization of mRNAs, which are captured through spatially  barcoded, gene specific oligo probes. WSI subimages were extracted around each co-registered Visium spot and were used to predict the expression at these spots using machine learning models. We prototyped and compared several convolutional, transformer, and graph convolutional neural networks to predict spatial RNA patterns at the Visium spots under the hypothesis that the transformer- and graph-based approaches better capture relevant spatial tissue architecture. We further analyzed the model’s ability to recapitulate spatial autocorrelation statistics using SPARK and SpatialDE. Overall, the results indicate that the transformer- and graph-based approaches were unable to outperform the convolutional neural network architecture, though they exhibited optimal performance for relevant disease-associated genes. Initial findings suggest that different neural networks that operate on different scales are relevant for capturing distinct disease pathways . We add further evidence that deep learning models can accurately predict gene expression in whole slide images and comment on understudied factors which may increase its external applicability . Our preliminary work will motivate further investigation of inference for molecular patterns from whole slide images as metastasis predictors and in other applications. Colorectal cancer (CRC) is the third leading cause of cancer-related death in the United States, and there are disparities in screening and outcomes between age, race, and gender.,Spatially resolved transcriptomics , as enabled through technologies such as 10x Genomics Spatial Transcriptomics  or Nanostring GeoMX Digital Spatial Profiling , is an actively growing area of research that provides rich information about how different areas of tissue interact by analyzing highly multiplexed gene expression at staggering spatial resolution. These technologies can be configured to study the distribution, density, and spread of tumor-infiltrating lymphocytes (TILs) as they may relate to concomitant somatic alterations.Deep learning approaches, which rely on using multi-layer artificial neural networks (ANNs), have proven instrumental for image analyses in the context of digital pathology.,Zeng et al. (2022) and Pang et al. (2021) investigated approaches to integrate broader spatial context into their gene expression prediction models by using vision transformers and demonstrated that it is possible to outperform convolutional architectures that make predictions on individual patches .Here, we compared a convolutional neural network and a graph attention network for inferring spatially co-registered gene expression from WSI. We comment on the role the tissue macroarchitecture may play in RNA inference as elucidated by changing subimage size and the use of contextual models. We also assessed performance on a subset of immune-related genes and how spatial characterization varies across these models/factors and slides. These explorations will motivate future downstream work to characterize factors pertaining to tumor nodal/distant metastasis.1.Data collection: Acquire H&E whole slide images (WSI), and spatially registered Visium assayed spatial transcriptomics slides from 4 stage-pT3 matched colorectal cancer patients at Dartmouth Hitchcock Medical Center, 2 patients without metastasis, 1 with nodal metastasis only, and 1 with both nodal and distant metastasis.2.Preprocess: Preprocess gene expression and WSI subarrays.3.Model development: Configure 2 modeling approaches: convolutional neural network and graph attention neural networks, the latter leveraging larger spatial context. These models will predict both binary (dichotomized expression) and count-based  objectives. We also configured additional approaches  for comparison.4.Capture surrounding tissue context at different scales: Ablation study over patch size to determine whether relevant biological information is encoded outside the Visium collection spot area.5.Leave one-patient-out cross-validation: Evaluation on held-out slides/patients as a measure of external applicability.6.Recover spatial biology inferences: Spatial autocorrelation tests for the capacity of models to draw similar spatial inferences.The primary goal of this work is to predict the gene expression detected by a Visium spot at any given location on the slide. Our method is as follows :1.Data ct-tests for continuous variables and Fisher’s exact tests for categorical variables. The 4 patients were subselected to restrict the tumor site , grade , node status (2 metastasis cases with N-1a), and account for differences in sex . We restricted the cohort to patients without MMR deficiencies as determined through immunohistochemistry (IHC) to control for microsatellite instability status. Tissue blocks were sectioned into 10-micron thick layers, and specific capture areas that contained various distinct macroarchitectural regions  were annotated by the practicing pathologist. A histotechnician carefully extracted/manually cut these capture areas from the tissue, and slides were sent to the Single Cell Genomics Core in the Center for Quantitative Biology for simultaneous H&E staining, imaging, and Visium profiling.The primary dataset utilized in this study was acquired from 4 pathologic T Stage-III (pT3) matched (pTNM system) colorectal cancer patients at Dartmouth Hitchcock Medical Center, determined through a retrospective review of pathology reports from 2016 to 2019 following IRB review and approval. These 3 patients were drawn from a set of 36 patients included in a prior studyVisium spatial transcriptomics profiling: Traditional transcriptome-wide association studies use bulk/dissociated specimens, which disregards spatial information . Prior spatial assays  only allow for targeted analyses (1–10 molecular markers).,untargeted high multiplexing  at high spatial resolution. The 10X Genomics Visium Spatial Transcriptomics (ST) assay elucidates spatial variation for expression of tens of thousands of protein coding genes at up to 5000 (6.5 mm) or 14 000 (11 mm) 55-micron capture areas (1–10 cells) per tissue section.,SpaceRanger package to preprocess the Visium reads into gene count matrices.Spatial gene expression profiles contain information for 17 943 genes at almost 5000 locations per slide , sampled in a honeycomb formation. Each Visium spot covers a circular capture area with a diameter of 130 pixels at 20x magnification. After sequencing, we used the As whole slide images (WSIs) derived from the Visium capture areas (size of capture area– 6.5 × 6.5 mm) span thousands of pixels along each dimension, we subdivided the image into square patches centered on the Visium spot. We associated the gene expression of each patch based on the Visium spot at the center of the patch and ignored expression at other spots contained within the patch.We used the SpatialDE library to select the top 1000 genes based on their mean fraction of spatial variance (FSV) across all slides . We tested the capacity of our models to recover expression for all 1000 genes based on dichotomized expression (binary classification) and the original counts (regression).For binary classification, we classify tiles as having a “high” expression for a gene if its individual expression is greater than the median for that slide, as shown by Levy-Jurgensen et al. For binary tasks, we used a weighted binary cross-entropy loss. The loss was independently taken for positive and negative Visium spots and summed together to account for unbalanced labels.μ, dispersion factor σ, and inflation of zero count π) and is optimized with negative log-likelihood loss. The inferred value is equal to the expected value of the negative binomial distribution (accounting for zero inflation): (1 − π)μ + π(0) = (1 − π)μ.We model the gene expression distribution for regression tasks as a negative binomial distribution with zero inflation. The model predicts the parameters of the distribution  and training graph attention networks (GATs) positively impacted our capacity to predict spatial gene expression. All models featured an output layer, which simultaneously predicted the expression of all selected genes . DetailsWe initialized our model using InceptionV3 weights .(p16) GAT models were labeled by the number of graph attention layers used to make predictions : GAT-1 (1 layer), GAT-2 (2 layers), and GAT-4 (4 layers) (model suffix indicates the number of layers). ViT models were labeled by the size of the patch used to form contextual embeddings: ViT-224 (224-pixel patch sizes) and ViT-384 (384-pixel patch sizes).The Visium spots lie on a hexagonal array, each of which can be treated as a node in a graph, each connected to other Visium spots within 150 pixels. After training the InceptionV3 models from our local patch classification experiments, we extracted patch image embeddings  for each Visium spot from the penultimate layer of the trained Inception-256 model. We test a graph attention network (GAT) to see how iterative message-passing can improve model performance. We also compared these results to that obtained using a Vision Transformer (ViT).To improve the robustness of the model results to new tissue contexts, we transformed patch images by shifting and scaling the pixel intensities by the mean and variance of ImageNet. Then, we applied color jitter and random rotations; between −15 and 15 degrees. Random horizontal and vertical flips also augment patches if they are not used in the GAT. Through a coarse hyperparameter search, learning rates were set to 1e-4, and models were trained for 25 epochs. Batch sizes for the Inception model were set to 32, save for the regression models for patch sizes 512 and 768, where batch sizes were set to 16 and 8, respectively.InceptionV3 embeddings to train the contextual models  that corresponded to the same cross-validation fold. We acknowledge that these statistics could also vary by models, patch size, and slides, so we plotted scatters of test AUCs of each gene to compare these factors in a pairwise manner  to draw additional inferences on suitable modeling approaches for a subset of genes. Identifying a subset of genes that obtained optimal performance for one approach versus another was as important as comparing overall performance. These performance differences could suggest relevant tissue features at different scales. For instance, the GAT could extract features from the larger macroarchitecture, indicating its relevance for a gene that does not predict well from InceptionV3.To compare model and patch-size performances, we performed leave-one-slide-out cross-validation (CV), where 3 of the 4 slides were used for training/validation, and the final slide was used for testing. This scheme was repeated 4 times to report on test performance across all 4 slides in an unbiased manner using macro-averaged (across slides) median (across genes) area under the receiver operating curve (AUROC) and average precision (AP) statistics for the binary outcome and correlation coefficients  to compare true versus predicted counts. Non-parametric bootstrapping was used to assess statistical significance through the calculation of a 95% confidence interval. During cross-validation, we use the same set of Elsevier Pathways database available through the Enrichr package, we performed a pathway analysis of the top 250 genes ranked by AUROC averaged across the CV folds.Due to fundamental limitations in tissue biology, it is unrealistic to expect that every gene can be predicted from tissue histology. We sought to establish which types of genes could be inferred from histology through pathway analysis. Using the InceptionV3 and GAT.InceptionV3, GAT). Mapper, a Topological Data Analysis method, was used to provide topological summaries of the embeddings by reducing the number of points based on overlap and connectivity.InceptionV3 and GAT were colored using the cluster information from the ground truth expression to qualitatively assess topological consistency . This procedure was repeated across the held-out test slides.We also sought to assess how well the predicted gene expression profiles recapitulated relationships/clustering between the Visium spots. This was accomplished through the comparison of Uniform Manifold Approximation and Projection (UMAP) embeddings for the ground truth and predicted expression profiles (on held-out slides) using In addition to tissue clustering, spatial variation is often used as a proxy for explaining the diversity of cellular lineages interacting across the tissue slide. While this factor alone is not an exhaustive assessment of spatial biology, this served as a target for our preliminary assessment . After performing cross-validation, we sought to investigate the ability of our algorithms to recapitulate known spatially variable genes on each of the held-out slides from the cross-validation folds. We used two libraries to determine spatially variable (SV) genes: SpatialDE and SPARK-X.MSigDB Hallmarks gene sets via the Enrichr software.Using SpatialDE, we extracted the Fraction of Spatial Variance (FSV) and P-values for each gene from the ground-truth set. In addition, we used SpatialDE’s Automatic Expression Histology (AEH) to identify 5 groups of genes that were co-expressed spatially from the ground-truth data. Separately, we ran SPARK/SPARK-X on the 1000 genes from each held-out validation slide, reporting projection and adjusted P-value (Bonferroni-corrected) statistics. We separately ran this procedure for the ground-truth expression and predicted expression profiles. As an indication of performance, we expected P-values derived for the projection covariance function for both the inferred and original expression data to correlate well with each other for each slide. This was accomplished using the Fisher’s exact test after dichotomizing spatial autocorrelation statistics into “high autocorrelation”  and “low autocorrelation”  and similar dichotomization for statistics extracted from the inferred expression. Thresholds for dichotomization were chosen to maximize the magnitudes of the Fisher’s exact test statistics. Dichotomized spatial autocorrelation was cross-tabulated across the genes to report odds ratios with P-values. An odds ratio and corresponding confidence interval of more than one  would suggest the ability of the model to recapitulate spatial autocorrelation from the slide. Separately, we sought to assess whether genes that were predicted with high accuracy (AUC) were spatially autocorrelated using a similar methodology . We also compared model accuracies between AEH groups of co-expressed genes identified from SpatialDE using Kruskal-Wallis ANOVA statistical tests. Similar to the AUC comparison, we performed comparisons across models, patch sizes, and slides. It should be noted that these analyses were done on the top 1000 spatially variable genes; dichotomized autocorrelation is for this reference group. For the original and inferred expression, genes which exhibited spatial autocorrelation which differed between patients with and without metastasis were selected for a pathway analysis using the First, we assessed the ability of the Inception model to predict gene expression without considering the surrounding tissue macroarchitecture. Across the whole transcriptome, we noted moderately strong concordance between the predicted and actual expression across the held-out slide folds.Importantly, the model achieved optimal performance by increasing access to the surrounding macroarchitecture by increasing the patch size.After optimizing model architectures, the Inceptionv3 model appeared to outperform the GAT and Transformer approaches for the whole transcriptome assessment. This was true for both classification and regression modeling objectives. The GAT-1 model outperformed the GAT-4 model, which incorporated more of the surrounding tissue context, for the classification task, though GAT-4 outperformed GAT-1 for regression and demonstrated a similar capacity to predict count outcomes as Inception-512 ; Table 1While overall, Inception outperformed GAT, it is important to recognize that there are many genes for which GAT achieves superior performance   between the predicted expression and the ground truth . QualitaWe also compared the capabilities of each modeling approach for their ability to recapitulate slide-level spatial autocorrelation parameters. Results indicate a large significant positive association between predicted and actual spatial autocorrelation for both Inception and GAT approaches. For half of the held-out slides, GAT demonstrated a larger effect estimate than Inception .Table 3SSeparately, results indicate that highly spatially autocorrelated genes, as determined using the actual gene expression, were predicted with higher accuracy using Inception and GAT versus genes which lacked spatial variation . These mUsing the ground truth, Inception, and GAT results, we identified the set of genes, which exhibited different spatial variation for primary sites with  and without metastasis  based on the dichotomized thresholds. Through a pathway analysis , genes that were differently autocorrelated were related to the epithelial to mesenchymal transition (Supplementary Table 3).,Assessment of colon cancer tumor metastasis status and recurrence risk depends on the ability to assess lymph node involvement. For cases where lymph nodes are unable to be completely assessed, leveraging information found in the tumor immune microenvironment (TIME) at the primary resection site presents a viable alternative. Studying TIME can help us understand how immune cells infiltrates and interactions with other cell lineages to form a coordinated anti-tumoral immune response. These interactions can inform us about immune suppression, activation, exhaustion, tumor cell energy metabolism, pluripotency, cytokine secretion/recruitment, immune escape pathways, and collagen/extracellular matrix remodeling. These pathways play a significant role in creating an environment that is permissive for metastasis. Several assays have been developed to study the TIME—perhaps the most notable among them being the Immunoscore assay which assesses T-cell cytotoxicity (CD3/CD8 density) both within and around the tumor as both a metastasis predictor and independent recurrence risk factor.CDH1, KRT18, PABPC1, RAB25, SOX9, EPCAM) while others benefited from considering a broader architectural context . For instance, we noticed that COL6A1 and COL6A2 were consistently predicted better by the GAT model as compared to Inceptionv3.COL6A1 is a crucial component of collagen secretion, extracellular matrix maintenance, and mesenchymal phenotype promotion. Of the other markers listed in this paragraph, an interesting observation is that genes which were better predicted using the Inception approach have been previously implicated with cellular adhesion ,,SOX9),,PABPC1), PECAM1),COL6A1), glycolysis/energy metabolism ,IL1R1, LAPTM5),We add further evidence that deep learning models can accurately predict gene expression in whole slide images, reporting an AUC of 0.8. Furthermore, we demonstrate that increasing the receptive field can improve the performance of certain subsets of genes . Interestingly, although InceptionV3 (AUC=0.79) outperformed other modeling approaches overall , this did not apply to all genes. We noted that certain genes were predicted more effectively at local spatial resolutions  were found in common between both methods. This suggests that each method may capture different molecular aspects of the E2M phenotype.Spatial autocorrelation was recapitulated for these slides from the deep learning model predictions. We noticed that increases in modeling accuracy were associated with spatial gene expression variation. Genes that were differentially autocorrelated between metastatic and non-metastatic tumors corroborated with well-established oncogenic pathways. For instance, both the GAT and Inception models recaptured genes related to the Epithelial to Mesenchymal (E2M) transition with greater statistical power than the ground-truth expression data. For the Inception model, differentially spatially autocorrelated genes included: Nonetheless, these findings supports our overarching modeling approach to characterize spatial heterogeneity. Yet, identified pathways from the spatial autocorrelation analysis once again differed between Inception and GAT. The graph neural networks prioritized the pathways of angiogenesis, myogenesis, glycolysis, and apical junction, which are related to cell migration and energy metabolism as we discussed before. These pathways correspond to the genes that the GAT model could accurately predict. The Inception model selected the pathways of TNF-alpha signaling via NF-kB, TGF-beta signaling, and coagulation, which are mainly involved in inflammation, immune escape mechanisms, and injury responses. These pathways may overlap with or encompass the ones derived from GAT, which had inferior model performance. As some genes were better predicted using the broader spatial architecture, it would make sense to decide which modeling approach to utilize on a gene-by-gene basis and whether the local versus spatial context is preferred. This allows for the selection of optimal modeling approaches in a gene-specific manner to extend the broad applicability of our framework across all disease-relevant genes.Importantly, the techniques featured in this work will prove useful for inferring gene expression on slides from external cohorts. This is necessary due to the prohibitive costs of spatial transcriptomics. If validated properly on a slightly larger set of slides while carefully controlling for potential confounders , these methods may overcome sources of patient-specific batch effects to allow the report of less-biased effect estimates for large-scale cohort studies. Validation efforts on these held-out slides can leverage assessment methods such as Spark-X and SpatialDE to ensure spatial heterogeneity from the inferred expression data is similar to the initial internal validation cohort. To this end, we achieved remarkable performance for the ability to predict spatial heterogeneity, which will potentially help power future studies elucidating spatial transcriptomic predictors of colon metastasis.Our results did not support the hypotheses that, overall, spatial gene expression estimation would benefit from message-passing between patch locations via transformer models and graph convolutional networks. However, these neural networks outperform local patch prediction across a large set of genes, suggesting the relevance of these methods for genes which leverage the spatial context. For GAT, underperforming genes can potentially be explained by the fact that graph convolutional networks tend to smoothen features,There were a few study limitations of note. Our validation scheme featured the use of held-out slides. Generally, as 2 cases had tumor metastasis, whereas the other 2 did not have concurrent metastasis, we believed held-out slides would have similar heterogeneity in expression and morphology as the training/validation slides. Indeed, it is possible that tissue expression/morphology existed outside of this range, differentially impacting the GAT/transformer models designed to capture long-range spatial dependencies. As we had manually selected capture areas, we did not expect slides to have exactly matching/analogous architectural features; thus, the GAT/transformer model could have been unable to generalize as it aims to integrate this higher-level information. There were 4 whole slide images for both training and cross-validation, and while these provided nearly 15 000 patches in the training set when taken individually, they provided limited opportunities for models to learn diverse global contexts that transformer models would have benefitted from. For instance, different molecular alterations  and tissue location  are factors related to patient prognosis and may confound/modify disease associations. Models aiming to infer molecular alterations across a large cohort of CRC patients should be privy to these factors. These challenges could have been ameliorated by pretraining the GAT/Transformer on a variety of colorectal cancer tissue contexts, which does not explicitly require spatial omics data. Furthermore, the assessment of tumor metastasis using the ground truth and inferred spatial expression information in this study uses differences in spatial autocorrelation between patients with and without metastasis to determine the presence of metastasis. There are many other parameters related to tumor metastasis which warrant further consideration—for instance, how different cell-types may co-localize within distinct architectures , differential expression of cell-type specific markers at the invasive margin , and associating tumors which have metastasized with risk of recurrence/mortality. Integration of single cell RNAseq information can help facilitate these nuanced assessments by mapping single cell expression profiles to the Visium spots. Assessing different parameters of metastatic pathogenesis  will be the subject of future work which will be enhanced through inferring expression across large study cohorts.We also acknowledge the impact of evolving workflows for spatial resolution of transcriptomics information. Many workflows rely on the prediction of tissue features from fresh or fresh frozen tissue. We utilized formalin-fixed paraffin-embedded (FFPE) tissue slides. Only recently have deparaffinization and assay workflows been developed for FFPE. As many of these specimen processing workflows are still under development, manual staining and imaging could have both introduced batch effects and impacted tissue quality. For instance, while a plethora of data preprocessing workflows offer capabilities to combat batch effects through normalization, a robust comparison of preprocessing workflows was outside of the study scope. Our group is keen to adopt future iterations of the FFPE workflows. We expect that protocol updates for the FFPE workflow and data collection will provide major improvements in specimen processing, data quality, and resolution, improving prediction models. Enlarging the capture area and evaluating complementary molecular assays  will improve the resolution and scope of our findings.Tumor metastasis is heavily tied to poor prognostic outcomes and risk of recurrence. Assessment of key niches at the primary site  may reveal histomorphological or biological factors relating to nodal involvement or distant metastasis. In this work, we investigated the potential to infer spatially resolved transcriptomics from the tissue histology of colorectal cancer patients through several sophisticated neural network approaches. Our findings indicate that neural networks can be effectively employed in this capacity and that selection of a neural network model could be informed by its relevance to a molecular pathway resolved from histological features at different scales. Findings reaffirm the role of the epithelial-to-mesenchymal transition as an important metastasis-related process. In the future, with additional algorithmic fine-tuning, data curation, and standardization, there are opportunities to generalize these findings to perform large-scale spatial molecular assessments.None to disclose.


Dermoid cysts are benign developmental anomalies that can occur anywhere along the neuroaxis or embryonic lines of fusion. While intracranial dermoid cysts at the midline frequently have an associated nasal or subcutaneous sinus tract, it is quite rare to encounter an intracranial dermoid cyst off the midline with a lateral sinus tract. Standard practice for the treatment of dermoid cysts is surgical resection to minimize the risks of meningitis, abscess, mass effect, neurologic deficit, and/or death. A 3-year-old male with a history of DiGeorge syndrome presented with right orbital cellulitis and a right-sided dermal pit. Computed Tomography (CT) imaging demonstrated a dermal sinus tract with an associated lytic bone lesion within the right sphenoid wing and posterolateral orbital wall with intracranial extension. The patient was taken to the operating room in conjunction with plastic surgery for resection of the dermal sinus tract and intraosseous dermoid. This case presents a rare occurrence of a non-midline, frontotemporal dermal sinus tract associated with a dermoid cyst with intracranial extension presenting with pre- and post-septal orbital cellulitis. Important considerations include preservation of the frontal branch of the facial nerve, preservation of orbital structure and volume, complete surgical resection to prevent infectious complications including meningitis, and a multidisciplinary surgical approach with plastic surgery, ophthalmology, and/or otolaryngology.  Dermoid cysts are benign developmental anomalies originating embryologically from the entrapment of ectodermal elements consisting of a stratified squamous epithelium lining containing keratin and hair . IntracrHistoryThe patient presented as a 3-year-old male with a past medical history including DiGeorge syndrome, single cardiac ventricle anatomy following aortic reconstruction, Glenn, and Damus-Kaye-Stansel procedures, heart block with pacemaker, T-cell lymphopenia, and gastrostomy tube dependence, in addition to developmental delay and intellectual disability. He presented to the emergency department with a fever and 24 hours of progressively worsening swelling and erythema of the right eye in addition to mild proptosis concerning for orbital cellulitis. Of note, pupils were equal in size and reactive to light and the patient was without obvious visual field deficit. Interestingly, the parents reported a history of a skin dimple over the right pterion that has been present since birth that intermittently extruded hair and purulent material Figure .The patient was febrile on presentation (38.3°C) with associated leukocytosis (14.65 thousand). Other pertinent laboratory data included C-reactive protein (CRP) of 5.0 mg/dL , erythrocyte sedimentation rate (ESR) of 18 mm/h , and neutrophil count 10.66 thousand . Blood cultures obtained in the emergency department demonstrated no growth, though antibiotics were started prior to the acquisition of blood cultures. The patient was also started on intravenous steroids in addition to intravenous vancomycin and ceftriaxone for broad-spectrum antibiotic coverage for presumed orbital cellulitis. Preoperative evaluationGiven his history of cardiac pacemaker placement, computed tomography (CT) imaging of the head and orbits with and without contrast was obtained as he was not a candidate for magnetic resonance imaging (MRI). The obtained images demonstrated an intraorbital, extraconal rim-enhancing fluid collection along the superolateral wall of the right orbit measuring approximately 1.4 x 1.4 x 0.5cm immediately adjacent to a lytic bone lesion within the right sphenoid wing/posterolateral orbital wall with intracranial epidural extension anterior to the right temporal pole. This was associated with a circuitous cutaneous tract extending through the right temporal soft tissue Figure .Considering the complex presentation, a multidisciplinary team was assembled to review the case and develop a surgical plan including neurosurgery, plastic surgery, ophthalmology, infectious disease, otolaryngology, and critical care medicine. Given the concern for an infected intracranial/intraorbital dermoid cyst and orbital abscess, the decision was made to proceed with a right craniotomy for resection of the dermal sinus tract and sphenoid mass. Operative treatmentThe patient was taken to the operating room in conjunction with pediatric plastic and reconstructive surgery. The frontal branch of the facial nerve was percutaneously mapped based on surface landmarks and found its course in close proximity to the deeper portion of the tract. Considering this, preoperative discussion with parents included the risk of transient neuropraxia versus permanent frontal branch palsy. An incision was made circumferentially around the sinus tract and blunt dissection with limited cautery was used to dissect along the tract while minimizing the risk of injury to the facial nerve. Dissection was carried through the subcutaneous tissue and temporalis muscle down to the periosteum. At this point, a coronal incision was made Figure  and the Postoperative coursePorphyromonas endodontalis. Infectious disease initiated a six-week course of intravenous ceftriaxone, daptomycin, and oral metronidazole. He recovered appropriately and was discharged home on postoperative day seven in stable condition. The patient was monitored in the intensive care unit. Post-operative CT imaging demonstrated resection of the mass and anatomic cranial and orbital reconstruction. The patient did have transient neuropraxia of the right frontalis muscle following the operation. Intraoperative cultures revealed gram-positive cocci on gram stain though no growth was observed. Subsequent broad-range PCR demonstrated positivity for Pathologic analysisPathologic analysis of the tract and mass revealed “portion of skin and underlying tissue with a squamous epithelium lined tract” and “ruptured and inflamed structure with focal presence of squamous epithelium and keratinous debris with associated foreign body type giant cell reaction” Figure . Dermoid cysts are benign inclusion cysts that arise from ectodermal tissue and may be associated with a dermal sinus tract . Dermal The presentation of dermoid cysts varies from the discovery of a benign cutaneous pit with extrusion of keratinous debris to local or systemic infection. These masses can also rupture and, depending on the location, lead to chemical meningitis secondary to the inflammatory nature of fat and keratin. Patients may also exhibit cerebrospinal fluid leakage from the sinus tract, and there have been reports of cranial nerve palsy caused by sequelae of dermoid cysts and associated sinus tracts . As the The presence of dermoid cysts can be confirmed with skull radiographs, CT, and MRI. Dermoid cysts are typically osteolytic with well-defined, sclerotic margins on skull radiographs. CT imaging demonstrates hypodense lesions since the cysts typically contain fat. On MRI, dermoid cysts are hypointense on T1-weighted imaging and T2-weighted imaging and can exhibit marked diffusion restriction. These lesions typically do not contrast enhance. Dermoid cysts may also be associated with developmental anomalies such as Klippel-Feil anomaly, encephaloceles, coccygeal sinus, and venous sinus anomalies . Given tThe treatment of choice for dermoid cysts is surgical resection . A multiAnother important consideration during the resection of frontotemporal dermoid cysts is the preservation of the frontal branch of the facial nerve. Before making an incision, the frontal branch can be mapped using cutaneous landmarks or using compound muscle action potentials . FurtherStaphylococcus aureus [Actinomyces, Peptoniphilus, Propionibacterium species [Escherichia coli [Porphyromonas species, in addition to Finegoldia, Campylobacter, and Bacteroides [Porphyromonas in our patient was of a different species, however. Infectious complications include posterior fossa abscess, ventriculitis, meningitis, osteomyelitis, orbital cellulitis, and epidural abscess [Pathogens associated with dermal sinus tract infections have been documented in prior case reports. These include s aureus , Actinom species , and Eschia coli . There hteroides . The gen abscess  as seen In this report, we document a rare occurrence of a non-midline, frontotemporal dermal sinus tract associated with a dermoid cyst with intracranial extension presenting with pre- and post-septal orbital cellulitis. These lesions commonly go unnoticed until severe symptoms arise as was the case in this patient. Prompt recognition and surgical resection of these masses are the sole means of treatment, thereby minimizing the risk of potentially life-threatening intracranial and orbital infections.


Currently, many neurological disorders lack effective treatment options due to biological barriers that effectively separate the central nervous system (CNS) from the periphery. CNS homeostasis is maintained by a highly selective exchange of molecules, with tightly controlled ligand-specific transport systems at the blood–brain barrier (BBB) playing a key role. Exploiting or modifying these endogenous transport systems could provide a valuable tool for targeting insufficient drug delivery into the CNS or pathological changes in the microvasculature. However, little is known about how BBB transcytosis is continuously regulated to respond to temporal or chronic changes in the environment. The aim of this mini-review is to draw attention to the sensitivity of the BBB to circulating molecules derived from peripheral tissues, which may indicate a fundamental endocrine-operating regulatory system of receptor-mediated transcytosis at the BBB. We present our thoughts in the context of the recent observation that low-density lipoprotein receptor-related protein 1 (LRP1)-mediated clearance of brain amyloid-β (Aβ) across the BBB is negatively regulated by peripheral proprotein convertase subtilisin/kexin type 9 (PCSK9). We hope that our conclusions will inspire future investigations of the BBB as dynamic communication interface between the CNS and periphery, whose peripheral regulatory mechanisms could be easily exploited for therapeutic purposes. Many disorders of the central nervous system (CNS) are lacking effective therapeutic treatment options due to the insufficiency of conventional drugs to enter the brain following peripheral administration . DespiteThe majority of the extravascular CNS is separated from the providing vasculature by the blood brain barrier (BBB) and the blood–cerebrospinal fluid barrier (BCSFB) . The BBBSince the extremely dense brain microcapillary system represents the largest blood–brain interface, regulating the immediate microenvironment of cerebral cells throughout the brain, and possesses the structural prerequisites for controlled but efficient transport, crossing the BBB is the primary, and in certain circumstances the exclusive route of entry for peripheral substances . HoweverDue to the absence of paracellular or transcellular channels at the brain microvasculature, the required transfer of larger and/or polar solutes through the semi-permeable BBB is physiologically performed by ligand-specific endogenous transport systems such as carrier- or receptor-mediated transcytosis ,28. The Exploiting endogenous transcellular transport systems for drug delivery into the brain would ideally allow for extensive drug distribution within the CNS by utilizing the widely branched brain microcapillary system without interfering with the structure and function of the BBB ,36. IndiHowever, a well-known procedure to achieve efficient BBB transcytosis is the conjugation of short peptides or monoclonal antibodies mimicking ligand epitopes (peptidomimetics) to the nanocarrier surface, which are recognized by the respective endogenous receptors (“Trojan horse” strategy) . Since eTranscytosis across an intact brain endothelial microvasculature is strictly downregulated . DespiteMembers of the LDL receptor family, including the eponym LDL receptor and the prominent low-density lipoprotein receptor-related protein 1 (LRP1), are essential cell surface receptors participating in diverse physiological processes . ReceptoLRP1 is expressed in certain cell types, such as hepatocytes, fibroblasts, macrophages, smooth muscle cells, neurons, and astrocytes, making it present in most tissues of the body ,89. In cDue to the structural prerequisites, members of the LDL receptor family are inherently endocytic receptors, which bind to specific ligands from the extracellular space and translocate in complex with the ligand from the plasma membrane into an intracellular vesicle. However, when it comes to extremely thin (~200 nm) and polarized endothelial cells of the brain microvasculature ,8,37, thThe receptor surface levels of members of the LDL receptor family can be regulated at different cellular levels in response to physiological or pathophysiological conditions. A central physiological feedback loop is transcriptional regulation due to cellular levels of a ligand. The cellular depletion of cholesterol initiates the two-step proteolysis of the transcription factor sterol-regulatory element binding protein (SREBP)-2 in the Golgi apparatus, which allows its translocation from the endoplasmic reticulum (ER) membrane into the nucleus . While nIn addition to transcriptional regulation, cell surface activity of transmembrane receptors is limited by proteolytic shedding. Membrane-anchored metalloproteases, including ADAM10, ADAM17, and BACE1 are capable of cleaving adjacent LRP1 within the extracellular chain, releasing a large fragment into the extracellular space ,103. In 2+-independent autocatalysis of its prodomain early in the ER, which is required for its secretion into the extracellular space . T. T132]. The use of the receptor-binding epitopes of ApoB and E allows endocytosis by members of the LDL receptor family and subsequent transcellular transport from the luminal to the abluminal membrane of brain endothelial cells. Subsequently, Apo-like structures are exocytosed into the CNS and internalized by proximal neurons or astrocytes . StudiesOur results demonstrate that receptor-mediated Aβ clearance across the BBB by LRP1 can be significantly enhanced due to peripheral PCSK9 inhibition. Besides the use of high-affinity monoclonal antibodies at substantial concentrations in therapeutic contexts to silence circulating PCSK9, PCSK9 receptor affinity is physiologically reduced by furin cleavage or the binding to Apo-containing lipoproteins as demonstrated for LDL particles . In addiIlluminating the complex crosstalk between peripheral tissues, which strongly influence the plasma composition, and the CNS at the BBB as a dynamic endocrine interface, might reveal potential approaches to increase the efficiency of drug penetration via endogenous pathways.PCSK9 is a potent post-transcriptional regulator of members of the LDL receptor family. Circulating, liver-derived PCSK9 reduces cell surface receptor concentration within the vascular system, including the brain microvascular endothelium, whereas inhibition of peripheral PCSK9 has the opposite effect. Therapeutic FDA-approved inhibition of circulating PCSK9 by peripheral application of monoclonal antibodies to raise cell surface LDL receptor levels is a more recently developed but well-established treatment for patients with high plasma LDL cholesterol . PCSK9 iThe mere knowledge that receptors at the BBB might be sensitive to, and their number regulated by, vascular circulating molecules could be beneficial in several ways. Transient upregulation could improve the translocation of peripherally administered therapeutic agents, such as nanoparticles, into the brain. In contrast, receptor downregulation could decrease off-target brain penetration and subsequent side effects of highly concentrated circulating drugs . As shown for enhanced LRP1-mediated Aβ clearance due to peripheral PCSK9 inhibition, altering the cell surface receptor concentration itself could be a therapeutic option by increasing or decreasing receptor-mediated transcytosis to or from the CNS. This would avoid the need to introduce a therapeutic agent into the CNS, and potential cross-reactions. BBB permeability changes dynamically due to aging or pathophysiological conditions, such as AD, which could be addressed by exploiting regulatory mechanisms as a preventive measure or therapeutic option after disease onset. Due to the relative simplicity of access and monitoring, endocrine regulatory pathways as described for circulating PCSK9 would be predestined. Thus, a deeper understanding of how BBB transport systems are organized and regulated might be crucial to efficiently exploit the BBB transcytosis machinery for drug delivery to the brain, and to modify the compromised BBB during pathophysiological states as a therapeutic option.


Bladder cancer poses a significant public health burden, with high recurrence and progression rates in patients with non-muscle-invasive bladder cancer (NMIBC). Current treatment options include bladder-sparing therapies (BST) and radical cystectomy, both with associated risks and benefits. However, evidence supporting optimal management decisions for patients with recurrent high-grade NMIBC remains limited, leading to uncertainty for patients and clinicians. The CISTO  Study aims to address this critical knowledge gap by comparing outcomes between patients undergoing BST and radical cystectomy.The CISTO Study is a pragmatic, prospective observational cohort trial across 36 academic and community urology practices in the US. The study will enroll 572 patients with a diagnosis of recurrent high-grade NMIBC who select management with either BST or radical cystectomy. The primary outcome is health-related quality of life (QOL) at 12 months as measured with the EORTC-QLQ-C30. Secondary outcomes include bladder cancer-specific QOL, progression-free survival, cancer-specific survival, and financial toxicity. The study will also assess patient preferences for treatment outcomes. Statistical analyses will employ targeted maximum likelihood estimation (TMLE) to address treatment selection bias and confounding by indication.The CISTO Study is powered to detect clinically important differences in QOL and cancer-specific survival between the two treatment approaches. By including a diverse patient population, the study also aims to assess outcomes across the following patient characteristics: age, gender, race, burden of comorbid health conditions, cancer severity, caregiver status, social determinants of health, and rurality. Treatment outcomes may also vary by patient preferences, health literacy, and baseline QOL. The CISTO Study will fill a crucial evidence gap in the management of recurrent high-grade NMIBC, providing evidence-based guidance for patients and clinicians in choosing between BST and radical cystectomy. The CISTO study will provide an evidence-based approach to identifying the right treatment for the right patient at the right time in the challenging clinical setting of recurrent high-grade NMIBC.ClinicalTrials.gov, NCT03933826. Registered on May 1, 2019.The online version contains supplementary material available at 10.1186/s12885-023-11605-8. Most bladder cancer patients (74%) present with non-muscle-invasive bladder cancer (NMIBC), where the cancer is limited to the lining or support layer of the bladder [Bladder cancer is the sixth most common cancer in the United States and affects all genders. bladder , 3 high atients 7% present bladder . Most hi bladder , 6. Howe bladder , 3. Sele bladder . Thus, tWe conducted an Agency for Healthcare Research and Quality (AHRQ)-funded evidence review of the comparative effectiveness of numerous intravesical treatments and identified a paucity of evidence for the effectiveness of management strategies for patients with recurrent high-grade NMIBC . Thus, cTo address this clinical and patient-centered gap, we designed the Comparison of Intravesical Therapy and Surgery as Treatment Options (CISTO) Study in partnership with patients and other stakeholders engaged in NMIBC care, including clinicians and policymakers, and received funding from PCORI to support the CISTO Study as a 36-site pragmatic trial  , 15. TheThe primary objective of the CISTO Study is to compare patient-reported and patient-centered clinical outcomes between patients undergoing radical cystectomy and those receiving BST for recurrent high-grade NMIBC. We hypothesize that: 1) patients undergoing radical cystectomy will have worse health-related QOL at 12 months compared with patients who choose BST; 2) 12-month disease-free survival and metastasis-free survival will be better among radical cystectomy patients than among patients who choose BST; and 3) treatment with BST will result in better QOL within important subgroups, such as those aged 75 years or older, women, and patients with multiple comorbid health conditions. A secondary objective of the study is to characterize the heterogeneity of treatments received and corresponding patient preferences.The CISTO Study is a pragmatic, prospective observational cohort study of patients with recurrent high-grade NMIBC who have selected management with BST or radical cystectomy. Since radical cystectomy is selected less frequently than BST, the study design includes a cap of two enrollments into the BST arm for every one enrollment into the cystectomy arm at each site. This block recruitment strategy ensures adequate enrollment into the BST arm and limits unbalanced accrual by sites. A prospective observational cohort study design was chosen for the CISTO Study in response to critical input from the BCAN Patient Survey Network, as only 11% of respondents reported being willing to consent to randomization for a study of BST versus radical cystectomy .The CISTO Study is informed by a conceptual model of the relationship between BST and radical cystectomy and patient-centered outcomes Fig. . As suchThe CISTO Study protocol was developed through multistakeholder involvement as described previously,  applyingThe study setting is academic and community urology practices in the United States. A total of 36 clinical sites were selected based on geography, patient volume, patient demographics, research infrastructure, and previous experience with clinical trials.The CISTO Study will include 572 patients with a diagnosis of recurrent high-grade NMIBC who chose either cystectomy or BST as their treatment option. Participants must have attempted or received induction BCG at some point and have received their last treatment for NMIBC within the previous 12 months  and those receiving BST  for recurrent high-grade NMIBC.The primary outcome is patient-reported physical QOL measured with the European Organisation for Research and Treatment of Cancer Core Quality of Life (EORTC QLQ-C30) physical functioning domain at 12 months  and serious adverse events (SAEs) and unanticipated problems to the applicable Institutional Review Boards (IRBs) or other monitoring bodies. SAEs are defined as 1) death during the study period; 2) a life-threatening event related to the treatment or significant disability/incapacity related to the treatment; 3) inpatient hospitalization (other than for cystectomy); and 4) prolonged hospitalization following cystectomy (14 days or more). The number of adverse events and related unexpected SAEs will be summarized by arm, by grade, and by the National Cancer Institute’s Common Terminology Criteria for Adverse Events (CTCAE) system organ class. In addition, for each toxicity, the proportion of affected participants overall and by arm will be summarized by the maximum CTCAE grade experienced.P-values from Exact Conditional Tests, such as Fisher's exact test and its multi-degree of freedom extensions, will be used to compare categorical data.We will use descriptive statistics to characterize the treatments received, PROs, and clinical outcomes. Prior to statistical analysis, we will first compare continuous demographic and clinical characteristics by treatment group with Wilcoxon rank-sum tests to protect against violations of normality assumptions. potential outcomes: two predicted outcomes for each individual patient participant, under the hypothetical assumption that they had been treated with either radical cystectomy or intravesical therapy. Next, as with a propensity score approach, we will estimate the probability of treatment with radical cystectomy using either logistic regression, machine learning  or super learning approaches. Finally, we will use the probabilities obtained in the second step to update the initial estimate of each patient participant’s pair of potential outcomes and the TMLE estimate is interpreted as the causal difference in QOL outcomes if all patients had been treated with chemoradiation versus having been treated with radical cystectomy [To address treatment selection bias and potential confounding by indication in the comparison of BST vs. radical cystectomy, we will utilize targeted maximum likelihood estimation (TMLE) as the primary analytic approach for causal effect estimation using observational data –34. Unlistectomy . As a sePatient data will be analyzed according to an intention-to-treat (ITT) framework for the primary analysis, where the treatment arm is decided upon at enrollment regardless of whether or not that treatment was initiated. Longitudinal trajectories of the effect of treatment on patient QOL over time will be modeled and characterized using standard linear mixed effects models  or generalized estimating equations . Because each patient has a pair of potential outcomes under each treatment, we will include covariates in the TMLE and longitudinal data models  . Power aRecurrent high-grade NMIBC is a public health burden, with limited evidence to inform the complex decision confronted by patients and clinicians as they consider BST and radical cystectomy. This complex decision-making engages patients and their caregivers, who may be impacted by the urinary, sexual, and bowel dysfunctions that can occur with NMIBC treatment. Optimal decision-making would include evidence-based selection of treatment that optimizes cancer-specific and QOL outcomes consistent with the patient’s values and preferences. The CISTO Study aims to fill the current knowledge gaps regarding the comparative effectiveness and harms of management options for recurrent high-grade NMIBC to inform these critically important decisions. By including a diverse patient population, the study also aims to assess outcomes across the following patient characteristics: age, gender, race, burden of comorbid health conditions, cancer severity, caregiver status, social determinants of health, and rurality. Treatment outcomes may also vary by patient preferences, health literacy, and baseline QOL. The results of the CISTO Study are anticipated to help identify which patients for whom radical cystectomy represents the best option, and similarly which patients are best managed with BST.The patient-centered and pragmatic CISTO Study has numerous strengths. The primary question this study will address was prioritized by patients, the design of the study was conducted in partnership with patients and clinicians, and the primary and secondary outcomes were selected based on patient and clinician input. The broad inclusion criteria and pragmatic design of the CISTO Study will improve the generalizability of the study results. The experiences of patients will be assessed with validated PROs, and all statistical analyses will apply rigorous methods and sensitivity analyses. Since treatment selection bias is an inherent part of any observational cohort study, we will utilize TMLE as our primary analytic strategy. TMLE is an innovative, state-of-the-art analytic method for causal effect estimation using observational data. Unlike general propensity score-based or G-computation approaches, TMLE is doubly robust to misspecification arising from an omitted confounding variable in the models for the exposure (treatment) or the outcome measure –34. To sAlthough the CISTO Study will address important evidence gaps in the management of recurrent high-grade NMIBC, there are limitations to the study design. Most notably, participants in the CISTO Study are not randomized to a treatment arm but instead select their treatment. However, similar randomized controlled trials comparing radical surgery with more conservative management strategies for genitourinary malignancies suffered from substantial accrual challenges , 39. BRADespite these limitations, the CISTO Study is the only ongoing clinical trial evaluating radical cystectomy as a comparator in NMIBC. A recent Clinical Trial Planning Meeting of the National Cancer Institute’s Genitourinary Cancers Steering Committee Bladder Cancer Task Force entitled “Defining the Next Generation of Clinical Trials with Combination Therapies in Non-Muscle Invasive Bladder Cancer” reviewed concepts for next generation trials in NMIBC, none of which included radical cystectomy as a comparator intervention . Yet gui1 Future work will focus on the dissemination and implementation of findings through risk prediction models that integrate PRO and clinical outcomes data to permit the identification of which patients benefit from radical cystectomy and which patients benefit from BST. The CISTO study will provide an evidence-based approach to identifying the right treatment for the right patient at the right time in the challenging clinical setting of recurrent high-grade NMIBC.The CISTO Study will advance clinical care by generating new and actionable evidence to support the decision-making of patients with recurrent high-grade NMIBC considering BST or radical cystectomy, including PRO and clinical outcomes. The results from this study will have direct patient care implications as bladder cancer is among the most common cancers in the United States.Additional file 1: Supplemental Figure 1. CISTO Study sites. A map of the 36 sites participating in the CISTO Study. Supplemental Table 1. Candidate baseline [baseline -43.


We present a case of a 51-year-old female who presented for evaluation of a large scalp mass found to have a different quartet of vascular malformations- a persistent scalp arteriovenous malformation (sAVM) with sinus pericranii, an inoperable intracranial SM-V brain arteriovenous malformation (bAVM), and a Cognard I dural arteriovenous fistula (dAVF). This is the first reported instance with four distinct vascular pathologies. We review the etiologies of multiple vascular abnormalities in the cerebral circulation that could contribute to this patient's findings and review strategies for treatment. We conducted a retrospective review of the clinical and angiographic records for a single adult female patient, including a management approach and an in-depth literature review. Given the high baseline vascularity of these complex lesions, surgery was not considered the initial therapy. We focused primarily on the sAVM with a staged embolization involving both transarterial and transvenous approaches. Transarterial coil embolizes 5 feeding artery branches of the right external carotid artery, followed by transvenous coil embolization into the common venous pouch accessed through the transosseous sinus pericranii via the SSS, dramatically reduced the size and filling of the large sAVM and eliminated a significant source of hypertensive venous outflow. Serial endovascular treatments of her sAVM led to a significant reduction in size and pulsatility, and the pain from tenderness to palpation was concurrently decreased. Despite multiple treatments, serial angiographic evaluations of her scalp lesion showed continued new development of collaterals. Ultimately the patient elected to forego further treatment for her sAVM.To our knowledge, there has not been another report of a single adult patient with a quartet of vascular malformations in the literature. Treatment paradigms for sAVMs are limited to case reports and small series; however, we purport that the most successful therapeutic approaches are multimodal and likely should incorporate surgical resection when feasible. We emphasize the caution required for patients with multiple other underlying intracranial vascular malformations. The altered intracranial flow dynamics can drastically hinder the success of a unimodal approach involving endovascular therapy alone. Due to their rarity, scalp arteriovenous malformations (sAVM) escape a robust classification scheme ,2. TheseClinical presentationA 51-year-old female presented to our institution to evaluate a large pulsating scalp mass on the patient's forehead, which was found to be a scalp AVM. Workup also revealed the large sAVM was associated with sinus pericranii, a large Spetzler-Martin (SM) grade V brain AVM (bAVM), as well as a Cognard grade I dural arteriovenous fistula (dAVF). Her scalp mass and brain AVM were discovered in childhood after an initial bleeding episode from her forehead. She was recommended for conservative management of both lesions. When she presented, she had noticed progressive enlargement of her right-sided frontal scalp AVM. Approximately five years before her presentation to our institution, she had a right frontotemporal intracranial hemorrhage prompting reimaging and subtotal endovascular embolization of the complicated bAVM nidus. She underwent multiple endovascular treatments over one year, which unfortunately culminated in an iatrogenic stoke producing left-sided hemiparesis and homonymous hemianopsia, which improved significantly with intensive rehabilitation. Notably, serial angiograms revealed a marked progression of the sAVM.The patient had no other significant past medical history and was a non-smoker. On examination, she had mild dysarthria and slurred speech with left-sided homonymous hemianopsia, relative left-sided hemiparesis graded 4/5, with mild residual paresthesias and numbness in the left hand. She was ambulatory independently without the need for assistive devices. A large purple pulsatile lesion was noted in the region of her right forehead. The overlying skin was noted to be taut and tender to palpation with an overlying scab secondary to the multiple times it had bled and healed in recent history.Angiographic resultsDiagnostic digital subtraction angiography (DSA) via a transfemoral approach was performed under general anesthesia. The right external carotid artery showed hypertrophy of the superficial temporal artery, middle meningeal artery, and various branches supplying the scalp. The scalp AVM was identified, receiving input primarily from the right superficial temporal artery and possessing a large venous varix measuring approximately 8 cm x 6cm x 4 cm in greatest dimensions with drainage into superficial facial veins as well as intracranial drainage into the superior sagittal sinus (SSS) via a transosseous channel indicating the presence of a sinus pericranii Figure .Injection of the right internal carotid artery revealed the SM-V bAVM within the right frontotemporal lobe again figure . ArteriaManagement of the scalp AVMGiven the nature of her neurological morbidity and complex quartet of vascular malformations, treatment was focused primarily on the sAVM with a staged embolization involving both transarterial and transvenous approaches. A right-sided transfemoral artery embolization successfully deposited coils into five feeding branches of the right external carotid artery supplying the sAVM, namely from distal branches of the superficial temporal artery Figure . Next, aSerial endovascular treatments of her sAVM led to a significant reduction in size and pulsatility. Moreover, the pain from the lesions regarding tenderness to palpation was concurrently decreased. We ultimately discussed surgical resection along with the aid of facial plastic surgery. Despite multiple treatments, serial angiographic evaluations of her scalp lesion showed persistent new development of collaterals. Ultimately the patient elected to forego further treatment for her sAVM.To our knowledge, there has yet to be another report of a single patient with a quartet of vascular malformations within the literature. Scalp AVMs, on their own, are rare extracranial homologs of their more commonly encountered intracranial counterparts. Thus far, congenital, traumatic, iatrogenic, and physiological etiologies  are described, although predominantly within the pediatric and adolescent population -4,7-13. Genetic syndromes predisposing individuals to multiple vascular malformations include ataxia telangiectasia, von Hippel-Lindau syndrome, and hereditary hemorrhagic telangiectasia ,8,14,15.A network of neoangiogenic factors and pathways critical for vessel remodeling have progressively been uncovered by studying human cerebrovascular tissue. A non-exhaustive list, including procollagen-lysin, 2-oxoglutarate 5-dioxygenase 2 (PLOD), KRASG12V, Ras/MAPK, Wnt, BMP/TGF- β, and Sry-box 2 (SOX2), likely contribute to de novo vascular malformations in the carotid circulation ,11,14-17This invariably generates an ischemic state that promotes discordant angiogenesis of the existing vasculature to form de novo shunts that target nearby dural sinuses. Chronicity of both lesions can result in an enlargement of the scalp AVM to a point. Furthermore, if venous hypertension persists, it may stimulate the formation of another malformation, such as the dAVF noted in our patient. It represents another high-capacitance, low-resistance outlet, particularly when considering the sinus pericranii . A quart Scalp AVMs have historically received varied treatments based on patient and lesion characteristics. Chowdury et al. review their institutional experience with a series of eight patients with sAVM. After triaging these lesions as either high-flow or low-flow via multimodal imaging, all patients underwent surgical resection with an average follow-up of 3.6 years. With an age range from 4 - 72 years, and male predominance, the majority experienced lesional enlargement in the third decade of life, and 3/8 had previously experienced recurrent hemorrhage. With most of their patients having high-flow lesions, they state that surgical treatment offers the most definitive hemostatic and cosmetic result due to the ability to manage multiple arterial feeders supplying the nidus and better control of the tissue for wound healing . Other gGiven the high baseline vascularity of this complex sAVM with sinus pericranii in tandem with the bAVM and dAVF, surgery was not considered the initial therapy. Coil embolization alone was insufficient to prevent previously angiographically occult feeders from arising. Transvenous-trans-sinus pericranii coil embolization successfully reduced the flow through the sinus pericrania and, subsequently, the sAVM nidus by eliminating a significant source of hypertensive venous outflow. Although our patient refused further intervention, we still recommend prioritizing definitive treatment options whenever possible, such as surgical resection or sclerotherapy as an adjuvant to endovascular therapy, especially in the case of a large sAVM in tandem with other vascular malformations. An isolated endovascular approach may limit patient morbidity secondary to a long treatment course.We provide the first report of an adult patient with four distinct vascular malformations involving three separate compartments - a persistent scalp AVM with sinus pericranii, an inoperable intracranial SM-V bAVM, and a Cognard I dAVF. Treatment paradigms for sAVMs are limited to case reports and small series; however, we purport that the most successful therapeutic approaches are multimodal and likely should incorporate surgical resection when feasible. We emphasize the caution required for patients with multiple other underlying intracranial vascular malformations. We believe the altered intracranial flow dynamics can drastically hinder the success of a unimodal approach involving endovascular therapy alone.


The gastrointestinal tract is densely innervated by the peripheral nervous system and populated by the immune system. These two systems critically coordinate the sensations of and adaptations to dietary, microbial, and damaging stimuli from the external and internal microenvironment during tissue homeostasis and inflammation. The brain receives and integrates ascending sensory signals from the gut and transduces descending signals back to the gut via autonomic neurons. Neurons regulate intestinal immune responses through the action of local axon reflexes or through neuronal circuits via the gut-brain axis. This neuroimmune crosstalk is critical for gut homeostatic maintenance and disease resolution. In this review, we discuss the roles of distinct types of gut-innervating neurons in the modulation of intestinal mucosal immunity. We will focus on the molecular mechanisms governing how different immune cells respond to neural signals in host defense and inflammation. We also discuss the therapeutic potential of strategies targeting neuroimmune crosstalk for intestinal diseases. C. elegans, to vertebrates, including fish and mammals [While the immune and nervous systems have traditionally been studied separately, it is increasingly clear that these two complex systems are intricately linked at the functional level. Neuronal crosstalk with the immune system is an old concept. Two thousand years ago, the Roman physician Aulus Cornelius Celsus defined the four cardinal signs of inflammation as pain, redness, swelling, and heat; the first sign was driven by the sensory nervous system, and the latter three were linked to vascular and immune functions . Neurona mammals . The pas mammals . The focThe primary function of the gastrointestinal tract is food processing and digestion. As such, it is densely innervated by peripheral neurons, including sensory and autonomic neurons, that coordinate the detection of nutrients, gut motility, and the secretion of enzymes necessary for proper digestive functions. The GI tract is also populated by a myriad of immunocytes, including both innate and adaptive immune cells, which critically mediate gut tissue homeostasis and host defense against enteric pathogens . FurtherThe neuroanatomy of the gut is composed of both sensory and autonomic neurons that reside within and outside the organ Fig. . Gut-extThe nervous system and immune system have evolved a common language to communicate with each other at every step of their response to environmental insults from inception to resolution . NeuronsHere, we focus on mechanisms underlying the neuronal regulation of inflammation and immunity in the gut. We discuss how neurons and neurotransmitters are involved in immune responses during tissue repair and host defense. Advances and challenges coexist in this fascinating field. We highlight the most recent studies that take advantage of new tools to overcome the limitations, and we argue that targeting neurons is a promising strategy for the treatment of diseases in the gut. We are unable to comprehensively cover this topic, as it is a fast-moving field. For further reading on the gut neuro-immune axis and how the microbiome regulates these interactions, we recommend these excellent reviews –18.Gut-innervating sensory neurons arise from the DRG and the nodose/jugular vagal ganglia (VG), transducing signals from the gut to the CNS to mediate the perception of various luminal stimuli, including mechanical stretch, nutrients, microbial cues and immune mediators Fig. . SensoryAn important aspect of sensory neurons is their ability to mediate local neurogenic inflammation . In thisSalmonella infection, as mice lacking its receptor neurokinin-1 receptor (NK-1R) had enhanced host protection [Nociceptor neurons communicate with immune cells such as mast cells in the gut to regulate pain and inflammation. Mast cell expansion is a hallmark of irritable bowel syndrome, which is usually accompanied by visceral pain. Mast cells are located close to sensory nerves in the gut mucosa, and mediators derived from mast cells, such as histamine, PGE2 and tryptase, are able to enhance the excitation of DRG neurons, leading to visceral pain , 22. Oneotection . Whetherotection . These votection . This ilRecent studies have shown that DRG neurons play critical roles in gut barrier protection, microbial homeostasis, and protection against colitis , 28. GobSalmonella typhimurium in mice [S. typhimurium penetrates the gut barrier through microfold (M) cells, specialized epithelial cells in the small intestine ileum Peyer’s patch (PP) follicle-associated epithelia (FAE) [S. typhimurium infection by releasing CGRP, which reduces M cell numbers to limit S. typhimurium infection. Nociceptors also regulate the level of the gut commensal microbe segmented filamentous bacteria (SFB), which colonizes the ileum villi and PP FAE to promote host defense against infection [Citrobacter rodentium and enterotoxigenic Escherichia coli [Nociceptor neurons are also critical for host defense against enteric infections. TRPV1+Nav1.8 + DRG neurons protect against infections caused by the gram-negative bacterial pathogen  in mice . S. typhia (FAE) . Nocicepnfection . TRPV1+ hia coli .3R expressed on neurons to modulate afferent mechanosensory functions [3R signaling blocks toxin-induced nausea-like behaviors [Sensory neuron activity is also dictated by multiple other cell types in the gut. Enteroendocrine cells (EECs) are specialized gut epithelial cells that form synaptic-like structures with enteric and sensory neurons. In response to microbial products, chemical irritants and dietary stimuli, EECs secrete the neurotransmitter serotonin, which binds to the receptor 5HTunctions . EECs alehaviors .Therefore, sensory neurons play pleiotropic roles in gut barrier protection under both homeostasis and host defense by signaling to immune and epithelial cells Fig. . RemainiAutonomic neurons that regulate involuntary physiologic processes in the gut, including digestion and motility, also play a critical role in controlling gut immunity. Autonomic neurons that innervate the gastrointestinal system include enteric neurons, sympathetic neurons, and parasympathetic neurons Fig. . While sEnteric neurons are capable of sensing various gut luminal stimuli and crosstalk between immune cells and epithelial cells in both healthy and disease states via the production of neurotransmitters, neuropeptides, and cytokines Fig. . EntericS. typhimurium infection [Enteric neurons also express cytokines that can regulate both immune and epithelial barrier function as a part of host defense. IL-6, a typical inflammatory cytokine produced by immune cells such as macrophages, is also expressed in enteric neurons . Entericnfection . It has N. brasiliensis helminth infection, leading to improved host protection and worm clearance [N. brasiliensis can trigger NMU production from enteric neuron organoids in a MyD88-dependent manner in vitro, suggesting that these neurons are also responding in a transcriptionally plastic manner to type 2 immune triggers [Enteric neurons also play a major role in coordinating the function of innate lymphoid cells (ILCs) in the gut. ILCs are early responding innate lymphocytes that coordinate downstream adaptive immunity. Enteric neurons are in close proximity to ILCs, laying a cellular basis for these neurons to regulate ILC functions , 40. ILClearance . Both thtriggers . ILC2s ttriggers . Given tCitrobacter rodentium infection [+ ILC3s, along with enhanced susceptibility to DSS-induced colitis [Citrobacter rodentium infection [Citrobacter rodentium or Trichuris muris infection, respectively [Enteric neurons also play a key role in regulating the function of type 3 ILCs (ILC3s) in the gut lamina propria. ILC3s express high levels of VIPR2, a receptor for the neuropeptide VIP. A subset of enteric neurons  express high levels of VIP, and they have been found to regulate ILC3 function at homeostasis and during host defense , 43. In nfection . A contrnfection . Interesectively . These sSympathetic neurons are efferent neurons of the gut-brain axis and the major branch of the stress signaling response. Their main effector neurotransmitters are catecholamines, which bind to adrenergic receptors on target cells. Sympathetic neuron activity is modulated by luminal microbial cues in the gut. Loss of the microbiome leads to increased expression of cFos, a marker of neuronal activation, in sympathetic neurons, suggesting that the endogenous flora plays a regulatory role in suppressing sympathetic neuron activation . AccompaSalmonella typhimurium (SpiB mutant) infection activates sympathetic neurons, resulting in NE release in the myenteric plexus; this signals to MMs to polarize their transcriptional profile in vivo in a β2AR-dependent manner [S. typhimurium infection also leads to the loss of intrinsic enteric-associated neurons (iEAN), resulting in decreased gut motility [S. typhimurium Spib infection-induced neuronal loss, while sympathetic neuron activation protects the gut from pathogen-induced iEAN damage [Salmonella, Yersinia, and helminths [In addition to regulating gut motility and immune cell migration, sympathetic neurons modulate intestinal immune cell plasticity. Tyrosine hydroxylase (TH+) sympathetic neurons have close proximity to muscularis macrophages (MMs) in the intestinal muscularis layer. MMs display different morphology and cell dynamics compared with lamina propria macrophages (LpMs), with the former having a tissue protective and wound healing “M2-like” macrophage signature and the latter having an “M1-like” macrophage signature . MMs spet manner . S. typhmotility . The symmotility . β2AR abN damage . This syelminths . In thiselminths .+ and IL-13+ ILC2s and protects the host from gastrointestinal helminth Nippostrongylus brasiliensis (N. brasiliensis) infection, while β2AR treatment inhibits ILC2-mediated type 2 immune responses and compromises the hosts protection against helminths [Sympathetic neurons also signal to ILC2s in the small intestine, where these cells are closely located in the villi and submucosa. Similar to macrophages, ILC2s also highly express the receptor β2AR . β2AR sielminths .These studies together show that sympathetic neurons and local stress signaling through catecholamines can powerfully regulate gut homeostasis and immunity against pathogens Fig. . Allowin+ T cells are recruited into the colon during C. rodentium infection, and T-cell-specific ChAT deficiency renders the host more susceptible to C. rodentium infection, accompanied by increased expression of TNFα, IL1β, and IL6, indicating that T-cell-derived Ach mediates antimicrobial gut defenses [Parasympathetic neurons reside in the brainstem and innervate peripheral organs via the vagus nerve. The vagus nerve may directly innervate the gut or signal through intermediary ganglia that in turn innervate the gut. The main neurotransmitter produced by these neurons is acetylcholine (ACh). It is increasingly clear that vagal efferent parasympathetic neurons have major immunomodulatory effects Fig. . The “chdefenses . Vagal pdefenses . Intestidefenses .Fig. 5PaN. brasiliensis and S. typhimurium intestinal infection. M3R-deficient mice are more susceptible to both infections and display compromised T-cell activation and cytokine release during infections, suggesting a T-cell-dependent role for Ach in protection against pathogens [Vagal-derived acetylcholine could also regulate T-cell function during adaptive immunity responses to helminth and enteric pathogen infections. The M3 muscarinic Ach receptor (M3R) has been found to mediate host defense against athogens . Ach andathogens .Ach also plays a major role in regulating gut epithelial barrier protection. Ach is the most well-known neurotransmitter that regulates goblet cell mucus secretion . Goblet Of note, one of the challenges in studying how vagal parasympathetic neurons regulate gut immunity is the lack of more specific tools to target only these neurons in the brainstem, as these motor efferents are molecularly diverse. It is also difficult to study because cholinergic neurons reside in both the brainstem and the enteric nervous system. Future work will require investigating the role of distinct vagal efferent subsets in regulating gut immunity.A major question in this field is how the brain senses inflammation and crosstalks with the gut from a top-down perspective. Recent work suggests that there could be specific brain regions that respond to colitis and that specific neural circuits provide feedback to the gut to regulate inflammation. Using cFOS-TRAP-based techniques, a recent study showed that intestinal inflammation induced by DSS treatment leads to neuron activation in the thalamus, paraventricular hypothalamic (PVH) nuclei, central amygdala (CeA), anterior cingulate cortex (ACC), supplementary somatosensory cortex, and insular cortex . ChemogeAnother way the brain affects gut immunity is via neuroendocrine signals, in particular, the hypothalamus pituitary adrenal gland (HPA) axis, which can be activated under conditions of stress and inflammation . In thisNeurological disorders, including autism spectrum disorder (ASD), have been shown to be accompanied by GI dysfunction , suggestTherefore, future work is needed to determine how brain activity and signals can be transduced to the GI tract to affect immunity. It also remains to be determined whether GI dysfunction is the result or cause of neurological disorders.Neuroimmunology is a fast-growing field. Recent breakthroughs in how different neuronal subsets regulate immune responses in the gut deepen our understanding of intestinal immunity under physiological and pathological conditions. In addition to passively accepting and responding to harmful insults, the immune system signals to the nervous system to initiate defensive responses. Meanwhile, under the anticipation and perception of potential threats, the nervous system actively modulates the immune response in the gut. Coordination between the nervous system and immune system allows the host to properly deal with complex stimuli and an ever-changing environment. The cellular neural basis of how distinct neuron types regulate gut immunity and the molecular mechanism of how different neurotransmitters modulate immune cells are major questions that need to be characterized in the future.An important consideration is that neurons and immune cells integrate signals from a complex tissue microenvironment. Therefore, analysis of neuroimmune signaling will require analysis of other cell types. Enteric glial cells, which support enteric neuron development, exhibit immunoregulatory functions by signaling to both immune cells and epithelial cells in the gut –70. EndoThe gut microbiota is also a critical arm that regulates both neuronal and immune activation in the gut-brain axis. The used of gnotobiotic mice with bacterial genetic manipulation remains the most powerful approach in uncoupling microbiota from the gut-brain axis. Multi-omic studies combining the transcriptome, proteome, metagenome and microbiome will improve our understanding of how neurons shape the intestinal ecosystem .Recent technical advancements have promoted the identification and manipulation of neurons in regulating gut immunity. Optogenetics and chemogenetics have allowed for the manipulation of neuronal activity in a temporally and spatially controlled manner , 73, 74,The immunoregulatory role of the brain in gut immunity is an open area of exploration. This includes mapping how emotional and cognitive brain regions could signal to the body to induce intestinal dysfunction. The communication between the brain and gut is bidirectionally mediated by neural, endocrine, immune, and humoral links . One recThese findings also have therapeutic implications for targeting neuroimmune interactions for inflammatory disease treatment. Since the discovery that electrical vagal efferent neuron stimulation attenuates endotoxin-induced inflammation in vivo , bioelecThe advances and breakthroughs in neuroimmune research in the gut during the last several years have demonstrated the potential of therapeutic strategies that manipulate neuroimmune interactions for clinical disease treatment. There are still challenges that need to be addressed before we can understand this interaction. The neuronal connections and neurotransmitters that mediate neuroimmune crosstalk need to be further characterized, and the role of brain networks in the regulation of peripheral immunity remains elusive. These are the fundamental questions in this realm that we expect to witness more breakthroughs in relation to in the coming years.


Mirounga leonina) breeding at Marion Island in the Southern Ocean (1989–2019), and to identify the factors that correlate with phenological shifts within and between individuals. We found strong support for covariation in the timing of breeding arrival dates and the timing of the preceding moult. Breeding arrival dates were more repeatable at the individual level, as compared with the population level, even after accounting for individual traits  associated with phenological variability. Mother–daughter similarities in breeding phenology were also evident, indicating that additive genetic effects may contribute to between-individual variation in breeding phenology. Over 30 years, elephant seal phenology did not change towards earlier or later dates, and we found no correlation between annual fluctuations in phenology and indices of environmental variation. Our results show how maternal genetic (or non-genetic) effects, individual traits and linkages between cyclical life-history events can drive within- and between-individual variation in reproductive phenology.Population-level shifts in reproductive phenology in response to environmental change are common, but whether individual-level responses are modified by demographic and genetic factors remains less well understood. We used mixed models to quantify how reproductive timing varied across 1772 female southern elephant seals ( For example, long-term warming trends have advanced spring events such as flowering, migration and breeding in many plants and animals . AlteredPopulation-level phenological change in life-history events (e.g. birth or egg-laying dates) arises from individual responses to changing environmental conditions, and/or via changes in population structure that may then shape trait distributions . We can Although animals might exhibit phenotypic plasticity (responsiveness to changing environments) at the within-individual level, there often remain consistent between-individual differences in average phenology, even after environmental and demographic factors are accounted for. One explanation for consistent between-individual differences is that phenotypic flexibility within individuals is constrained by linkages between cyclical life-history events . Carry-over effects between different phenological events may therefore limit phenotypic flexibility within individuals ,14. AnotMirounga leonina; hereafter elephant seals) are long-lived Southern Ocean marine mammals that breed and moult on land. Female elephant seals maintain highly synchronized seasonal reproduction, probabaly through photoperiodic cueing but also through variables in addition to daylength  )  . We calcp = 0.001, h2 = 0.40) (R2 = 0.05). Permutation tests showed that the predicted slope between arrival dates of random pairs of animals was mostly non-significant and centred around zero , and that the observed parent–offspring regression slope coefficient was higher than expected by chance  .Parent–offspring regression analysis indicated that the arrival dates of daughters were significantly correlated to their mothers’ arrival dates  . Daughte. 4Our 30-year study of female elephant seal breeding phenology demonstrated five important points. It (1) highlighted a strong relationship between the timing of different parts of the annual cycle (moult and breeding phenology), (2) showed associations between breeding arrival dates and individual traits , (3) revealed mother–daughter similarities in breeding phenology, and (4) indicated high repeatabilities  of arrival date within females. We did not find evidence for (5) any strong relationship between breeding season arrival dates and measures of environmental variation .Leptonychotes weddellii) [Halichoerus grypus) [Mirounga angustirostris) [Arrival dates were consistently early among young first-time breeding females, aligning with the findings of previous studies on elephant seals . In contddellii) , grey se grypus)  and nortrostris) . In thesElephant seal females time their pre-breeding migrations so that they return to the breeding colony a few days before birth ,29. BreePhotoperiod is a major determinant of the reproductive cycles of seals, but other environmental cues and physiological condition would also determine when implantation occurs and embryonic growth commences, leading to annual variability in the timing of births . The reaParent–offspring regression showed that a significant proportion of the total variance in arrival dates was explained by additive genetic effects. Our permutation analysis, which drew dyads at random from the population, showed that the probability of obtaining a heritability estimate as high or higher than ours by chance was negligible. Establishing parent–offspring relationships in the field is not always easy, and our heritability analysis was therefore conducted on a subset of mother–pup pairs (paternity could not be established). Nevertheless, the significantly positive linear slope of the weaning date covariate in our main analysis supports the mother–daughter regression results. Pup weaning dates are related to the arrival date of a mother: mothers that arrive early in the breeding season have pups with earlier weaning dates, and these pups tend towards earlier breeding arrival dates as adults, plausibly because of a heritable component. Parent–offspring regression sometimes overestimates heritability of traits compared with analyses based on more complex pedigrees . This maPhoca vitulina [Our results describe, for the first time to our knowledge, how maternal genetic (or non-genetic) effects, individual traits and linkages between cyclical life-history events can drive within- and between-individual variation in reproductive phenology in elephant seals. By contrast, breeding arrival dates did not show a relationship with measures of environmental variation. Irrespective of the level of environmental variability, secondary consumers like elephant seals often show lower levels of phenological variability compared with primary producers and consumers . Limitedvitulina  and greyvitulina ). These vitulina . Marked vitulina ) or longvitulina ,66). Elevitulina ,29. Unlivitulina ), but fe (a)We found no correlation between annual fluctuations in phenology and indices of environmental variation, but this does not mean that environmental controls of breeding phenology are absent. For example, the timing of breeding may be affected by unmeasured environmental conditions, or at a finer scale than we could detect. Indeed, for wide-ranging marine predators with extensive distributions, such as elephant seals, it is often challenging to obtain environmental covariates that accurately reflect changes in marine food webs and thus the availability of essentail resources .Our inferences were based on AIC model selection, which selects models based on their ability to reproduce the in-sample data (the data used to estimate the parameters of the model). The identified relationships therefore do not necessarily extend to causality . Cause-aWe acknowledge that improved data or analysis methods can potentially increase confidence in our results. Alternative observation methods (e.g. passive integrated transponders or geolocation tags) or increased survey frequencies would allow a more accurate estimation of breeding arrival times in our population, but these methods are difficult to implement under current field conditions. Analytical approaches such as state–space models make it possible to model the true state of the system  as a latent process, with the observed data conditional on this process and an observation error. Bayesian state–space models could potentially be used to incorporate an observation error distribution in one direction. Furthermore, site occupancy models ,71 and n. 5Our analysis of long-term individual-based data revealed that additive genetic effects, early-life traits, age and breeding experience, and the timing of other parts of the annual cycle influence the reproductive phenology of elephant seals. While many studies consider phenological shifts in animal life-history events in relation to changes in current environmental conditions, fewer consider how transgenerational effects, early-life events, demography and carry-over effects between cyclical life-history events may contribute to shape individual and population phenology distributions. Yet, population-level studies that do not account for these effects may reach misleading conclusions about environmental drivers of phenological change . Much of


Breast cancer is the most common type of cancer diagnosed among women worldwide. It significantly contributes to cancer-related mortality in females. Early-stage breast cancers have a high cure rate. However, distant metastasis of breast cancer due to haematological and lymphatic spread often leads to a poor prognosis. Gastric and duodenal metastasises are rarely observed in invasive lobular carcinoma of the breast. Early diagnosis is challenging due to the non-specific symptoms, the limited specificity of radiological investigations and the difficulty in obtaining adequate tissue biopsy. Herein, we report the clinical, radiological, and macroscopic features of a 72-year-old female with rare gastric metastasis of breast cancer and likely concurrent duodenal metastasis. Breast cancer is the second most common cancer worldwide and the most common cause of cancer-related mortality among females . MetastaA 72-year-old female was diagnosed with 3 weeks of vomiting associated with dull right-sided abdominal pain and 5 kg weight loss in four weeks. The patient had a history of stage IIIc (pT2N3) multifocal left pleomorphic lobular carcinoma diagnosed two years prior to admission with axilla lymph node metastasis. She underwent a left mastectomy and axillary lymph node dissection, followed by adjuvant radiotherapy to the left chest wall, axilla, the supraclavicular fossa and the internal mammary chain (42.4Gy/16 fractions). The patient declined chemotherapy due to her frailty (ECOG score = 2–3) and high-risk comorbidities.An abdominal computed tomography (CT) performed at this admission demonstrated soft tissue thickening around the second part of duodenum (D2), suggestive of a possible malignant stricture . Magnetiin situ hybridisation was not performed to confirm HER2 copy number secondary to diagnostic rather than treatment intent of the biopsy. The duodenal biopsy contained no abnormalities.A fluorodeoxyglucose positron emission tomography (FDG-PET) scan did not demonstrate definite evidence of local recurrence. There was no increased uptake in the region surrounding D2, although FDG-avidity was observed in a right crural lymph node. Gastroscopy identified a nodule in the gastric antrum with an unusual vascular surface pattern . The firIt was hypothesized that the duodenal obstruction was caused by metastases from the recurrence of invasive lobular carcinoma of the breast. However, a definitive histopathological diagnosis was not possible as both radiological and surgical approaches to biopsying the paraduodenal lesion were deemed too high-risk. Treatment options were subsequently discussed at a multidisciplinary team meeting and systemic, endoscopic, laparoscopic and open surgical options were considered. The patient was too frail for palliative chemotherapy. The benefits of endoscopic stenting were outweighed by the risks of stent migration, erosion and obstruction. The patient and her family declined any surgical intervention. Palliative radiotherapy was the most appropriate treatment given the circumstance.Diagnosis of gastrointestinal metastasis of breast cancer can be challenging. Patients usually present with non-specific symptoms including epigastric pain, nausea, vomiting, dyspepsia and dysphagia . The simThe radiological distinction between primary gastric cancer and breast cancer with gastric metastasis is difficult. Abdominal CT and barium study findings are usually non-specific. PET CT is reported to have a low sensitivity in diagnosing the origin of metastasis. However, it could be useful in assessing treatment response . EndoscoBiopsy and subsequent immunohistochemical analysis remain the most accurate method in differentiating between metastatic breast cancer and primary gastric cancer. However, the initial gastric biopsies could be reported as normal in 46–50% of cases as invasion of malignant cells are limited to the submucosa and the seromuscular layers and can be missed with superficial sampling (biopsy sampling encompassing the lamina propria only). Therefore, deep and extensive biopsies are recommended . The limIn conclusion, we reported a rare case of gastric metastasis of invasive lobular carcinoma of the breast. The rarity of breast cancer with gastric metastasis, the non-specific symptoms associated with the gastric metastasis, the low specificity of the radiological imaging and the difficulty in obtaining adequate tissue biopsies are challenges hinder early diagnosis. As a result, the possibility of gastric metastasis should not be overlooked in patients with a history of ILC of the breast presenting with a suspicious gastric or duodenal lesion.