Role of secondary mismatch repair (MMR) frameshifts in the evolution of microsatellite instable (MSI) colorectal cancer

Mismatch repair deficient (MMRd) cancers face a delicate balance. Whilst hypermutation fuels adaptive evolution, it also comes at the cost of immunogenic neoantigens and other deleterious mutations. How MMRd cancers navigate the costs versus benefits of hypermutation is unknown. By visualising the clonal architecture of MMRd colorectal cancer in situ I show that the mismatch repair system unfolds in reversible steps to adapt cellular mutability to immune selection. Mechanistically microsatellite instability unmasks two hypermutable homopolymers in the mismatch repair genes MSH6 and MSH3. Spontaneous frameshift mutation and reversion at these homopolymers allows them to act as a molecular switch, regulating expression of MutSα and MutSß respectively. Frameshift switching at these homopolymer sites modulates the rate and spectrum of mutations across the genome. In this manner stochastic mutation bursts combined with stringent immune selection, drive continuous adaptation. This work is supported by a bespoke clonally resolved exome sequencing dataset, validated using two large publicly available genomic datasets and tested in a mathematical model of mutation rate switching. In summary, this work identifies that adaptive mutability associates with increased immune escape and intratumour heterogeneity during mismatch repair deficient cancer evolution. Knowledge of this mechanism of adaptation may inform strategies to target resistance evolution in cancer treatment

Innervation and Muscle Cell Infiltration of Plastic Compressed Collagen Constructs

Abstract not available

Three-dimensional tumour microenvironment reconstruction and tumour-immune interactions' analysis

Tumours arise within complex 3D microenvironments, but the routine 2D analysis of tumours often underestimates the spatial heterogeneity. In this paper, we present a methodology to reconstruct and analyse 3D tumour models from routine clinical samples allowing 3D interactions to be analysed at cellular resolution. Our workflow involves cutting thin serial sections of tumours followed by labelling of cells using markers of interest. Serial sections are then scanned, and digital multiplexed data are created for computational reconstruction. Following spectral unmixing, a registration method of the consecutive images based on a pre-alignment, a parametric and a non-parametric image registration step is applied. For the segmentation of the cells, an ellipsoidal model is proposed and for the 3D reconstruction, a cubic interpolation method is used. The proposed 3D models allow us to identify specific interaction patterns that emerge as tumours develop, adapt and evolve within their host microenvironment. We applied our technique to map tumour-immune interactions of colorectal cancer and preliminary results suggest that 3D models better represent the tumor-immune cells interaction revealing mechanisms within the tumour microenvironment and its heterogeneity

Tertiary lymphoid structures (TLS) identification and density assessment on H&E-stained digital slides of lung cancer

Tertiary lymphoid structures (TLS) are ectopic aggregates of lymphoid cells in inflamed, infected, or tumoral tissues that are easily recognized on an H&E histology slide as discrete entities, distinct from lymphocytes. TLS are associated with improved cancer prognosis but there is no standardised method available to quantify their presence. Previous studies have used immunohistochemistry to determine the presence of specific cells as a marker of the TLS. This has now been proven to be an underestimate of the true number of TLS. Thus, we propose a methodology for the automated identification and quantification of TLS, based on H&E slides. We subsequently determined the mathematical criteria defining a TLS. TLS regions were identified through a deep convolutional neural network and segmentation of lymphocytes was performed through an ellipsoidal model. This methodology had a 92.87% specificity at 95% sensitivity, 88.79% specificity at 98% sensitivity and 84.32% specificity at 99% sensitivity level based on 144 TLS annotated H&E slides implying that the automated approach was able to reproduce the histopathologists’ assessment with great accuracy. We showed that the minimum number of lymphocytes within TLS is 45 and the minimum TLS area is 6,245μm2. Furthermore, we have shown that the density of the lymphocytes is more than 3 times those outside of the TLS. The mean density and standard deviation of lymphocytes within a TLS area are 0.0128/μm2 and 0.0026/μm2 respectively compared to 0.004/μm2 and 0.001/μm2 in non-TLS regions. The proposed methodology shows great potential for automated identification and quantification of the TLS density on digital H&E slides

Modelling outcomes after paediatric brain injury with admission laboratory values: a machine-learning approach.

BACKGROUND: Severe traumatic brain injury (TBI) is a leading cause of mortality in children, but the accurate prediction of outcomes at the point of admission remains very challenging. Admission laboratory results are a promising potential source of prognostic data, but have not been widely explored in paediatric cohorts. Herein, we use machine-learning methods to analyse 14 different serum parameters together and develop a prognostic model to predict 6-month outcomes in children with severe TBI. METHODS: A retrospective review of patients admitted to Cambridge University Hospital's Paediatric Intensive Care Unit between 2009 and 2013 with a TBI. The data for 14 admission serum parameters were recorded. Logistic regression and a support vector machine (SVM) were trained with these data against dichotimised outcomes from the recorded 6-month Glasgow Outcome Scale. RESULTS: Ninety-four patients were identified. Admission levels of lactate, H+, and glucose were identified as being the most informative of 6-month outcomes. Four different models were produced. The SVM using just the three most informative parameters was the best able to predict favourable outcomes at 6 months (sensitivity = 80%, specificity = 99%). CONCLUSIONS: Our results demonstrate the potential for highly accurate outcome prediction after severe paediatric TBI using admission laboratory data

Modelling the inflammatory response of traumatic brain injury using human induced pluripotent stem cell derived microglia

The neuroinflammatory response after traumatic brain injury (TBI) is implicated as a key mediator of secondary injury in both the acute and chronic periods after primary injury. Microglia are the key innate immune cell in the central nervous system, responding to injury with the release of cytokines and chemokines. In this context, we aimed to characterise the downstream cytokine response of human induced pluripotent stem cell (iPSC)-derived microglia when stimulated with five separate cytokines identified following human TBI. iPSC-derived microglia were exposed to IL-1β, IL-4, IL-6, IL-10 and TNF in the concentration ranges identified in clinical TBI studies. The downstream cytokine response was measured against a panel of 37 separate cytokines over a 72-hour time-course. The secretome revealed concentration-, time- and combined concentration and time-dependent downstream responses. TNF appeared to be the strongest inducer of downstream cytokine changes (51), followed by IL-1β (26) and IL-4 (19). IL-10 (11) and IL-6 (10) produced fewer responses. We also compare these responses to our previous studies of iPSC-derived neuronal and astrocyte cultures and the in-vivo human TBI cytokine response. Notably, we found microglial culture to induce both a wider range of downstream cytokine responses and a greater fold change in concentration for those downstream responses, as compared to astrocyte and neuronal cultures. In summary, we present a dataset for human microglial cytokine responses specific to the secretome found in the clinical context of TBI. This reductionist approach complements our previous datasets for astrocyte and neuronal responses and will provide a platform to enable future studies to unravel the complex neuroinflammatory network activated after TBI

Radiological Correlates of Raised Intracranial Pressure in Children: A Review.

Radiological assessment of the head is a routine part of the management of traumatic brain injury. This assessment can help to determine the requirement for invasive intracranial pressure (ICP) monitoring. The radiological correlates of elevated ICP have been widely studied in adults but far fewer specific pediatric studies have been conducted. There is, however, growing evidence that there are important differences in the radiological presentations of elevated ICP between children and adults; a reflection of the anatomical and physiological differences, as well as a difference in the pathophysiology of brain injury in children. Here in, we review the radiological parameters that correspond with increased ICP in children that have been described in the literature. We then describe the future directions of this work and our recommendations in order to develop non-invasive and radiological markers of raised ICP in children

Thresholds for identifying pathological intracranial pressure in paediatric traumatic brain injury.

Intracranial pressure (ICP) monitoring forms an integral part of the management of severe traumatic brain injury (TBI) in children. The prediction of elevated ICP from imaging is important when deciding on whether to implement invasive ICP monitoring for a patient. However, the radiological markers of pathologically elevated ICP have not been specifically validated in paediatric studies. Here in, we describe an objective, non-invasive, quantitative method of stratifying which patients are likely to require invasive monitoring. A retrospective review of patients admitted to Cambridge University Hospital's Paediatric Intensive Care Unit between January 2009 and December 2016 with a TBI requiring invasive neurosurgical monitoring was performed. Radiological biomarkers of TBI (basal cistern volume, ventricular volume, volume of extra-axial haematomas) from CT scans were measured and correlated with epochs of continuous high frequency variables of pressure monitoring around the time of imaging. 38 patients were identified. Basal cistern volume was found to correlate significantly with opening ICP (r = -0.53, p < 0.001). The optimal threshold of basal cistern volume for predicting high ICP ([Formula: see text]20 mmHg) was a relative volume of 0.0055 (sensitivity 79%, specificity 80%). Ventricular volume and extra-axial haematoma volume did not correlate significantly with opening ICP. Our results show that the features of pathologically elevated ICP in children may differ considerably from those validated in adults. The development of quantitative parameters can help to predict which patients would most benefit from invasive neurosurgical monitoring and we present a novel radiological threshold for this.We gratefully acknowledge financial support as follows. Research support: the Medical Research Council (MRC, Grant Nos. G0600986 ID79068 and G1002277 ID98489) and the National Institute for Health Research Biomedical Research Centre (NIHR BRC) Cambridge (Neuroscience Theme; Brain Injury and Repair Theme). Authors’ support: Peter J Hutchinson – NIHR Research Professorship, Academy of Medical Sciences/Health Foundation Senior Surgical Scientist Fellowship, NIHR Global Health Research Group on Neurotrauma, and NIHR Cambridge BRC. Joseph Donnelly is supported by a Woolf Fisher Scholarship. MC- NIHR BRC

Toward a New Approach to Evaluating Significance in Recent-Past Preservation Planning with a Case Study of 1960s Properties in Philadelphia County

In evaluating a stock of recent-past buildings, it is important to stay alert to the ways in which recent-past heritage is more difficult to assess, and what we might be prone to do to make it easier to assess. It is not enough to involve numerous people in the process and to articulate our method of analysis. We as preservation professionals must also consciously strive to avoid cognitive shortcuts. We must set evaluative standards and choose priorities, without simply dismissing a great portion of the built environment as “crap” or accepting self-evidence as a measure of significance. Complexity should not be a cause for despair. We must lead the public in a more self-reflexive view of built heritage, without getting stuck in never-ending philosophizing and debating. The field would benefit from a more systematic, methodical approach to championing pluralism in heritage and recognizing the polysemy in cultural objects, which nonetheless helps to uncover priorities of highest significance. In sum, prior to, and in addition to, preservation advocacy efforts to publicize and popularize buildings of the recent-past, preservation planning efforts must establish better methods for identifying resources and assessing their significance. In light of the issues and caveats just introduced, this study asks: what is an optimal inventory method for a municipal/county-level commission or nonprofit organization to identify priorities for preservation planning for the recent-past