A digital pathology tool for quantification of color features in histologic specimens.

In preclinical research, histological analysis of tissue samples is often limited to qualitative or semiquantitative scoring assessments. The reliability of this analysis can be impaired by the subjectivity of these approaches, even when read by experienced pathologists. Furthermore, the laborious nature of manual image assessments often leads to the analysis being restricted to a relatively small number of images that may not accurately represent the whole sample. Thus, there is a clear need for automated image analysis tools that can provide robust and rapid quantification of histologic samples from paraffin-embedded or cryopreserved tissues. To address this need, we have developed a color image analysis algorithm (DigiPath) to quantify distinct color features in histologic sections. We demonstrate the utility of this tool across multiple types of tissue samples and pathologic features, and compare results from our program to other quantitative approaches such as color thresholding and hand tracing. We believe this tool will enable more thorough and reliable characterization of histological samples to facilitate better rigor and reproducibility in tissue-based analyses

Targeted Nucleic Acid Delivery to the Endothelium

The human endothelium extends throughout nearly all tissues in the body, and has an estimated total surface area of up to seven thousand square meters [1]. This expansive monolayer of endothelial cells (ECs) serves as the interface between materials circulating in the bloodstream and the internal tissues of the body. Even in its quiescent state, the endothelium is actively signaling and reacting in order to support the basic functions of the vascular system – transporting oxygen, nutrients, and waste. A closer look reveals that the cells that make up this endothelial lining are diverse in their phenotypes and functions, dependent on the organ or tissue in which they reside. When activated under inflammatory conditions or in other disease states, endothelial cells further differentiate themselves through expression of surface antigens. Being a large, easily accessible surface that interfaces with almost all other tissues in the body, and consisting of distinctly identifiable subcategories as well as some universal characteristics, the endothelium is an attractive therapeutic target. Of particular interest in this dissertation is applying gene therapies to treat or prevent inflammation.We describe a polymeric delivery system for nucleic acids that can be modulated by exchanging polymer end-groups and conjugating cell surface targeting molecules to the delivery vehicle. We show that conjugating EC targeting antibodies to the surface of a cationic poly(amine-co-ester) (PACE) nucleic acid delivery vehicle enhances its transfection efficiency in cultured human ECs. This can enable delivery of gene therapies to ECs, either in vitro as cellular components of an engineered vascular graft, ex vivo in donated human organs for transplantation, or for targeting the endothelium in vivo. In the work presented here we apply the EC-targeted polymeric delivery vehicle to deliver siRNA against IL-15, a cytokine involved the activation of T cells during acute inflammation. Our work with transplant-declined human organs motivated the development of a new digital pathology tool for color-based quantification of histologic specimens, which we have applied to quantify vascular assembly in engineered grafts as well as vascular pathologies in human and animal tissue samples. We demonstrate the benefits of an automated program for color-based detection of pathological features in histologic specimens, in particular in a setting in which large numbers of images are generated. We propose further investigations into the antibody-targeted PACE polyplex delivery platform including a broader exploration of targeting in different cell types in vitro and in vivo. The work described in this dissertation aims to advance the therapeutic potential of targeted nanocarriers for treating pathologies in the endothelium

An Assessment of Universal Tumour Associated Antigens in Primary Liver Neoplasms

Rates of primary liver cancer are increasing in the Western world, a fact yet to be reflected in research with most currently undertaken in Asia. Whilst here in the UK, during a diagnostic and therapeutic workup within the NHS, surgically resected tumours are routinely kept in storage for up to thirty years. Meaning there is a huge, currently unused, resource that could potentially be included in clinical research. The regional specialist centre in the Southwest of England has been performing liver resections since 2005, no basic-science research has been performed using these archived specimens. In this research I have assessed the presence of genetic alterations, attempted to quantify the transcriptome, and measured protein expression in patients with liver cancer using a digital pathology platform. Telomerase and Survivin, the two targets of these endeavours, have previously been shown to be expressed in numerous cancer types, earning the title ‘universal tumour associated antigen.’ The techniques used in this research project are, mostly, already used in healthcare diagnostics, meaning there is potential for vastly increasing the power of these results should the study be increased. Genetic alterations in the promoter sequences were amplified and sequenced using DNA from archived samples. Attempts to quantify the transcribed component which have been locked away in tiny exosomes (which lack degradation enzymes) were made using a quantitative polymerase chain reaction. Protein expression was detected in tumours and background liver tissues using immunohistochemistry and quantified using digital pathology techniques on whole-slide images. Haematological protein levels were assessed using the enzyme linked immunosorbent assay. All of these characteristics were then compared with clinical measures such as tumour size, grade, stage, vascular invasion, overall survival, and diseases associated with liver carcinogenesis. Lessons learned from my work, particularly the techniques used on the source material, could be used in any NHS department without the need for significant financial investment required for a formal research facility. Access to these precious resources allows a more accurate representation of these antigens in the local clinical cohort. Below I have provided evidence that Telomerase promoter mutations are an HCC-specific alteration, and are present in tumours with vascular invasion. There is also early evidence that these mutations may correlate with a reduced overall survival. The Survivin promoter has been found to be a germline characteristic, whilst Survivin protein expression has been found to correlate with numerous adverse clinical features including tumour stage, grade, vascular invasion, perineural invasion and overall survival. These results are very encouraging and could possibly even be used as a risk-stratification tool during future routine clinical liver tumour workup, as an aid to identify patients at a higher risk of adverse clinical outcomes

Impact of Community Factors on the Donor Quality Score in Liver Transplantation

An increasing prevalence of metabolic syndrome and obesity has been linked to the rise in transplant indication for cryptogenic cirrhosis and nonalcoholic fatty liver disease (NAFLD), creating a growing challenge to public health. NAFLD liver transplant (LT) candidates are listed with low priority, and their waiting mortality is high. The impact of community/geographic factors on donor risk models is unknown. The purpose of this study was to develop a parsimonious donor risk-adjusted model tailored to NAFLD recipients by assessing the impact of donor, recipient, transplant, and external factors on graft survival. The theoretical framework was the social ecological model. Secondary data were collected from 3,165 consecutive recipients from the Scientific Registry of Transplant Recipients and Community Health Scores, a proxy of community health disparities derived from the Robert Wood Johnson Foundation\u27s community health rankings. Data were examined using univariate and multivariate analyses. The donor risk-adjusted model was developed using donor-only factors and supplemented with recipient and transplant factors, classifying donors as low, medium, and high risk. NAFLD residents in high-risk counties had increased likelihood of liver graft failure. Findings may be used to allocate high-risk donors to a subset of NAFLD with excellent outcomes, increasing the donor pool and decreasing mortality on the wait list

Liver Tumors

This book is oriented towards clinicians and scientists in the field of the management of patients with liver tumors. As many unresolved problems regarding primary and metastatic liver cancer still await investigation, I hope this book can serve as a tiny step on a long way that we need to run on the battlefield of liver tumors

Investigating the role of MR and PLVAP in hepatic leukocyte recruitment during chronic liver inflammation

Chronic liver diseases (CLDs) are characterised by inflammation and fibrosis which are driven by aberrant leukocyte recruitment. This process is mediated by specialised discontinuous endothelia, known as hepatic sinusoidal endothelial cells (HSEC), which act as the liver gatekeepers. During chronic inflammation, HSEC undergo substantial phenotypic changes, including upregulation of adhesion molecules, further driving leukocyte recruitment and exacerbating inflammation. The composition of the hepatic immune microenvironment determines the outcome of liver injury and understanding the mechanisms which regulate this process is critical to identify novel therapeutic targets. Mannose receptor (MR) and plasmalemma vesicle-associated protein (PLVAP) have previously been implicated in immune cell trafficking but their contribution to hepatic leukocyte recruitment remains undefined. This study aimed to characterise the expression of MR and PLVAP in normal and diseased human liver, understand their regulation in the hepatic sinusoids, and investigate their potential role in hepatic leukocyte recruitment. Immunohistochemistry and gene expression studies demonstrated that MR and PLVAP are differentially expressed in liver endothelium, with MR being downregulated and PLVAP being upregulated, in CLD and hepatocellular carcinoma. MR was resistant to regulation in primary human HSEC and did not seem to be involved in lymphocyte recruitment. In contrast, PLVAP was upregulated by several soluble factors, most notably by the senescence-associated secretory phenotype (SASP). The SASP, comprised of several cytokines and chemokines, is known to facilitate senescence surveillance by stimulating leukocyte recruitment. Additional experiments using patient tissue samples uncovered a previously unreported link between hepatic senescence, immune cell infiltration, and PLVAP expression in CLD. Furthermore, in vitro flow adhesion assays showed that PLVAP plays a selective functional role in monocyte paracellular transmigration, whilst having no impact on lymphocyte recruitment. These findings suggest that senescent cell-endothelial crosstalk drives PLVAP expression and shapes the immune landscape in chronic liver inflammation

Impact of Particle Sizes on MRI Signal Relaxation in Phantoms for Assessment of Hepatic Steatosis and Iron Overload

Magnetic Resonance Imaging (MRI) is emerging as a powerful tool to non-invasively evaluate diffuse liver diseases such as hepatic steatosis and iron overload. To test new MRI techniques, phantom studies are utilized in place of patients but often do not consider the microscopic interactions of particles suspended in the media which may cause a notable difference in the signal. Hence, this study investigates the impact of differing particle sizes on the magnetic resonance signal using phantoms. To accomplish this, steatosis phantoms were created using two different mixing methods to control droplet size and while combination iron-fat phantoms featuring iron particles of differing diameters were used to emulate hepatic iron overload. Signal behavior from both sets of phantoms were resolved using linear calibrations to determine values from two known biomarkers, fat fraction and R2*, for steatosis and iron overload, respectively. Overall, evidence showing that particle size impacts the signal to a significant degree remains inconclusive, but fitting model performance in biomarker quantification varied. This study demonstrates different sequencing and post-processing assessments are critical for the analysis of sensitive biomarkers such as R2* and FF

The use of an evidence based approach to guide optimal surgical management of colorectal liver metastases

The aim of this thesis was to validate the optimal surgical management of colorectal liver metastases (CLM) by using an evidence based approach. A meta-analysis comparing combined and sequential resection for synchronous CLM demonstrated that combined resection is associated with reduced hospital stay and with comparable perioperative mortality and morbidity, operative blood loss, and survival rates as sequential resection. Nevertheless, combined resection was associated with lower metastatic disease severity compared to sequential resection. A meta-analysis assessed liver resection for CLM in the presence of hepatic lymph node involvement and demonstrated that survival rates are lower in node positive disease patients compared to node negative disease patients, irrespective of whether the positive disease nodes were detected by routine or selective lymphadenectomy, or whether nodal involvement was microscopic or macroscopic. A network meta-analysis comparing different treatment strategies aiming to decrease operative blood loss found no difference in mortality, length of hospital stay or ITU stay between the treatment strategies. The use of radiofrequency dissecting sealer resulted in more serious adverse events compared to the clamp-crush method in the absence of vascular occlusion and fibrin sealant. Simple methods, such as clamp-crush method, gave overall equivalent outcomes to methods which require special equipment. Not reporting the period of follow-up was investigated as a potential source of study bias. Overall analysis did not identify a significant difference in mortality and disease recurrence, but sensitivity analysis of more recent reviews and larger reviews showed that the trials reporting the period of follow-up had a significantly lower hazard ratio for disease recurrence compared to trials not reporting the period of follow-up. A network meta-analysis comparing interventions aiming to decrease ischaemia-reperfusion injury during liver resection, demonstrated that ischaemic preconditioning resulted in fewer serious adverse events, lower operative blood loss, fewer transfusion proportions, and shorter operative time

Modelling the hepatitis C virus disease burden among injecting drug users in Scotland

A forward projection model was used to estimate the numbers of, both current and former, IDUs who acquired HCV infection and progressed to mild, moderate and severe HCV disease in Glasgow and Scotland between 1960 and 2030. The model was developed initially for Glasgow because more epidemiological information exists for this region, than elsewhere in Scotland, to calibrate model outcomes with local data relating to HCV and its consequence. Insights gained from the model fitting process in Glasgow were then used to extend the model to the rest of Scotland. First, the incidence and cessation of injecting drug use in Glasgow during 1960-2000 were derived through the use of a modified Delphi approach. Instead of the usual iterative process to refine experts’ estimates, the elicitation of IDU incidence and cessation provided an opportunity to combine these data and examine coherence with capture-recapture IDU prevalence estimates. Coherent estimates indicated that incidence (median: 28 to 49) and cessation (1 to 24%) remained low and stable during 1960-1975, rose steeply between 1975-1985 (incidence from 49 to 1,335; cessation from 2% to 6%), and by 2000 there had been a decline in incidence (1,195) but a further rise in cessation (15%). Secondly, stochastic simulation was used to model the transmission of HCV among current IDUs in Glasgow, according to their injecting risk behaviours, and estimate the past incidence of HCV infection. The model that considered higher infectivity during acute viraemia following infection produced seroprevalences (median: 62-72%) and incidences (18-30 per 100 susceptible injector-years) consistent with observed data during the 1990s. The annual number of new HCV infections among current IDUs in Glasgow was estimated to be low during 1960-1976 (median: 10-60), rise steeply during 1960-1976 (median: 10-60), rise steeply during the early 1980s to peak in 1985 (1,120), stabilise during 1991-1997 (510-610) and rise again during 1998-2000 (710-780). Scenario analyses indicated that potentially as many as 4,500 HCV infections (10th and 90th percentiles: 2,400-7,700) had been prevented in Glasgow during 1988-2000 as a result of harm-reduction measures. Scenario analyses also permitted the gauging of changes in risk behaviours required to effect appreciable reductions in the incidence of HCV infection. Incidence can be successfully reduced if IDUs who, unavoidably, share needles/syringes confine their borrowing to one person; with this strategy alone, an estimated 5,300 HCV infections (10th and 90th percentiles: 4,100-6,700) could have been averted in Glasgow during 1988-2000. Such insights will inform those responsible for developing new ways to prevent HCV transmission among IDU populations. Thirdly, linkage of laboratory data on diagnosed HCV antibody positive persons in Scotland to clinical data from hospital and death records provided a unique national epidemiological dataset to estimate the number who had progressed to severe HCV disease