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Background Advanced hepatocellular carcinoma (HCC) is commonly diagnosed using non-invasive radiological criteria (NIRC) defined by the European Association for the Study of the Liver or the American Association for the Study of Liver Diseases. In 2017, The National Institute for Clinical Excellence mandated histological confirmation of disease to authorise the use of sorafenib in the UK. Methods This was a prospective multicentre audit in which patients suitable for sorafenib were identified at multidisciplinary meetings. The primary analysis cohort (PAC) was defined by the presence of Child-Pugh class A liver disease and performance status 0–2. Clinical, radiological and histological data were reported locally and collected on a standardised case report form. Results Eleven centres reported 418 cases, of which 361 comprised the PAC. Overall, 76% had chronic liver disease and 66% were cirrhotic. The diagnostic imaging was computed tomography in 71%, magnetic resonance imaging in 27% and 2% had both. Pre-existing histology was available in 45 patients and 270 underwent a new biopsy, which confirmed HCC in 93.4%. Alternative histological diagnoses included cholangiocarcinoma (CC) and combined HCC-CC. In cirrhotic patients, NIRC criteria had a sensitivity of 65.4% and a positive predictive value of 91.4% to detect HCC. Two patients (0.7%) experienced mild post-biopsy bleeding. Conclusion The diagnostic biopsy is safe and feasible for most patients eligible for systemic therapy

Background: Neuroendocrine tumours (NET) overexpress somatostatin receptors (SSTR) that can be targeted for therapy. Somatostatin receptor expression is routinely measured by molecular imaging but the resolution is insufficient to define heterogeneity. We hypothesised that SSTR expression could be measured on circulating tumour cells (CTCs) and used to investigate heterogeneity of expression and track changes during therapy. Methods: MCF-7 cells were transfected with SSTR2 or 5 and spiked into donor blood for analysis by CellSearch. Optimum anti-SSTR antibody concentration and exposure time were determined, and flow cytometry was used to evaluate assay sensitivity. For clinical evaluation, blood was analysed by CellSearch, and SSTR2/5 immunohistochemistry was performed on matched tissue samples. Results: Flow cytometry confirmed CellSearch was sensitive and that detection of SSTR was unaffected by the presence of somatostatin analogue up to a concentration of 100 ng ml −l . Thirty-one NET patients were recruited: grade; G1 (29%), G2 (45%), G3 (13%), primary site; midgut (58%), pancreatic (39%). Overall, 87% had SSTR-positive tumours according to somatostatin receptor scintigraphy or 68-Ga-DOTATE PET/CT. Circulating tumour cells were detected in 21 out of 31 patients (68%), of which 33% had evidence of heterogeneous expression of either SSTR2 ( n =5) or SSTR5 ( n =2). Conclusions: Somatostatin receptors 2 and 5 are detectable on CTCs from NET patients and may be a useful biomarker for evaluating SSTR-targeted therapies and this is being prospectively evaluated in the Phase IV CALMNET trial (NCT02075606).

Background Bone metastases are associated with a worse outcome in patients with neuroendocrine tumours (NETs). Tumour overexpression of C-X-C chemokine receptor 4 (CXCR4) appears predictive of skeletal involvement. We investigated the role of circulating tumour cells (CTCs) and CXCR4 expression on CTCs as potential predictors of skeleton invasion. Methods Blood from patients with metastatic bronchial, midgut or pancreatic NET (pNET) was analysed by CellSearch. CXCR4 immunohistochemistry was performed on matched formalin-fixed paraffin-embedded (FFPE) samples. Results Two hundred and fifty-four patients were recruited with 121 midgut and 119 pNETs, of which 51 and 36% had detectable CTCs, respectively. Bone metastases were reported in 30% of midgut and 23% of pNET patients and were significantly associated with CTC presence ( p  = 0.003 and p  < 0.0001). In a subgroup of 40 patients, 85% patients with CTCs had CTCs positive for CXCR4 expression. The proportion of CXCR4-positive CTCs in patients with bone metastases was 56% compared to 35% in those without ( p  = 0.18) it. Staining for CXCR4 on matched FFPE tissue showed a trend towards a correlation with CXCR4 expression on CTCs ( p  = 0.08). Conclusions CTC presence is associated with bone metastases in NETs. CXCR4 may be involved in CTC osteotropism and present a therapeutic target to reduce skeletal morbidity.

Identification of the molecular alterations that drive cancer is critical for precision oncology. Profiling of a single tissue biopsy is insufficient to interrogate the full spectrum of molecular heterogeneity that exists within a patient's tumour, and is not without risk to the patient. The analysis of CTCs as part of a liquid biopsy circumvents this issue and allows single-cell analysis as well as longitudinal monitoring over time and in response to therapy. The aim of this thesis is to perform the first molecular characterisation of CTCs derived from NEN patients with a view to evaluating therapeutic targets and characterising tumour heterogeneity and evolution at the single-cell level. Firstly, I developed an assay to enable detection of the therapeutic targets SSTR2 and SSTR5 on individual NEN CTCs. Applied to a cohort of 31 metastatic NEN patients, I identified an SSTR+ subpopulation in 33% of patients and demonstrated significant intra- and inter-patient heterogeneity of SSTR expression. Next, I evaluated the size-based Parsortix platform for CTC enrichment against the gold standard EpCAM-dependent CellSearch in a pilot study of NEN patients, demonstrating that a higher number of CTCs could be isolated in a greater proportion of NEN patients using this technique. Furthermore, the presence of CTCs with low and absent EpCAM expression was observed for the first time in NEN alongside significant intra-patient heterogeneity in EpCAM expression. In order to fully dissect the heterogeneity observed in this early work, I developed DEPArraybased workflows to allow the single-cell evaluation of CTC copy number profiles using next-generation sequencing. The developed methodologies were subsequently tested in a representative cohort of NEN patients. By performing comprehensive copy number profiling of 125 single CTCs, I was able to identify recurrent and therapeutically relevant rearrangements, such as the amplification of CDK4/6, MET and BRAF and loss of BRCA2. Unsupervised hierarchical clustering demonstrated CTCs with distinct clonal lineages and significant heterogeneity was seen in CNV profiles between and within patient samples. In conclusion, this thesis describes successful workflows for the genomic analysis of CTCs at the single-cell level and is a step towards the implementation of precision oncology in neuroendocrine patients. This analysis has identified CTC heterogeneity at the single-cell level with implications for the identification of therapeutic targets, mechanisms of resistance to therapy, tracking of evolutionary change and biomarker refinement in NEN.

There has been little change to the standard treatment for osteosarcoma (OS) over the last 25 years and there is an unmet need to identify new biomarkers and novel therapeutic approaches if outcomes are to improve. Furthermore, there is limited evidence on the impact of OS treatment on patient-reported outcomes (PROs). ICONIC (Improving Outcomes through Collaboration in Osteosarcoma; NCT04132895) is a prospective observational cohort study recruiting newly diagnosed OS patients across the United Kingdom (UK) with matched longitudinal collection of clinical, biological, and PRO data. During Stage 1, which assessed the feasibility of recruitment and data collection, 102 patients were recruited at 22 sites with representation from patient groups frequently excluded in OS studies, including patients over 50 years and those with less common primary sites. The feasibility of collecting clinical and biological samples, in addition to PRO data, has been established and there is ongoing analysis of these data as part of Stage 2. ICONIC will provide a unique, prospective cohort of newly diagnosed OS patients representative of the UK patient population, with fully annotated clinical outcomes linked to molecularly characterised biospecimens, allowing for comprehensive analyses to better understand biology and develop new biomarkers and novel therapeutic approaches.

Hepatocellular carcinoma (HCC) is characterized as a hypervascular tumor which derives the majority of its blood supply from the hepatic artery while the liver parenchyma is supplied by the portal vein. This chapter reviews the evidence base for treatment interventions and highlights areas for future development. Therapeutic options include those that are potentially curative such as liver transplantation, surgical resection, or ablation, and those that are considered non‐curative or palliative and include transarterial embolization or transarterial chemoembolization, transarterial radioembolization, and systemic therapy. There are a range of ablative techniques available to treat liver tumors including percutaneous ethanol injection and radiofrequency ablation for which there are extensive data available, and newer techniques such as microwave ablation, cryoablation, and irreversible electroporation. Targeted therapies aimed at specific molecular aberrations have the potential to improve outcomes in advanced HCC as part of a \"personalized medicine\" approach.

In this chapter, we will cover the oncological management of hepatocellular carcinoma, cholangiocarcinoma, and colorectal carcinoma with liver metastases, focusing mainly on palliative therapies. Colorectal cancer and hepatocellular carcinoma are the third and sixth most commonly diagnosed cancers worldwide, respectively. Cholangiocarcinoma remains a rare malignancy although the incidence is increasing for unknown reasons. Previously, these three cancer types had an extremely poor prognosis, but some improvements in progression‐free and overall survival have been seen with advances in treatment options.

Globally, pesticides improve crop yields but at great environmental cost, and their overuse has caused resistance. This incurs large financial and production losses but, despite this, very diversified farm management that might delay or prevent resistance is uncommon in intensive farming. We asked farmers to design more diversified cropping strategies aimed at controlling herbicide resistance, and estimated resulting weed densities, profits, and yields compared to prevailing practice. Where resistance is low, it is financially viable to diversify pre-emptively; however, once resistance is high, there are financial and production disincentives to adopting diverse rotations. It is therefore as important to manage resistance before it becomes widespread as it is to control it once present. The diverse rotations targeting high resistance used increased herbicide application frequency and volume, contributing to these rotations’ lack of financial viability, and raising concerns about glyphosate resistance. Governments should encourage adoption of diverse rotations in areas without resistance. Where resistance is present, governments may wish to incentivise crop diversification despite the drop in wheat production as it is likely to bring environmental co-benefits. Our research suggests we need long-term, proactive, food security planning and more integrated policy-making across farming, environment, and health arenas.

Genetic determinants of stroke, the leading neurological cause of death and disability, are poorly understood and have seldom been explored in the general population. Our aim was to identify additional loci for stroke by doing a meta-analysis of genome-wide association studies. For the discovery sample, we did a genome-wide analysis of common genetic variants associated with incident stroke risk in 18 population-based cohorts comprising 84 961 participants, of whom 4348 had stroke. Stroke diagnosis was ascertained and validated by the study investigators. Mean age at stroke ranged from 45·8 years to 76·4 years, and data collection in the studies took place between 1948 and 2013. We did validation analyses for variants yielding a significant association (at p<5 × 10−6) with all-stroke, ischaemic stroke, cardioembolic ischaemic stroke, or non-cardioembolic ischaemic stroke in the largest available cross-sectional studies (70 804 participants, of whom 19 816 had stroke). Summary-level results of discovery and follow-up stages were combined using inverse-variance weighted fixed-effects meta-analysis, and in-silico lookups were done in stroke subtypes. For genome-wide significant findings (at p<5 × 10−8), we explored associations with additional cerebrovascular phenotypes and did functional experiments using conditional (inducible) deletion of the probable causal gene in mice. We also studied the expression of orthologs of this probable causal gene and its effects on cerebral vasculature in zebrafish mutants. We replicated seven of eight known loci associated with risk for ischaemic stroke, and identified a novel locus at chromosome 6p25 (rs12204590, near FOXF2) associated with risk of all-stroke (odds ratio [OR] 1·08, 95% CI 1·05–1·12, p=1·48 × 10−8; minor allele frequency 21%). The rs12204590 stroke risk allele was also associated with increased MRI-defined burden of white matter hyperintensity—a marker of cerebral small vessel disease—in stroke-free adults (n=21 079; p=0·0025). Consistently, young patients (aged 2–32 years) with segmental deletions of FOXF2 showed an extensive burden of white matter hyperintensity. Deletion of Foxf2 in adult mice resulted in cerebral infarction, reactive gliosis, and microhaemorrhage. The orthologs of FOXF2 in zebrafish (foxf2b and foxf2a) are expressed in brain pericytes and mutant foxf2b−/− cerebral vessels show decreased smooth muscle cell and pericyte coverage. We identified common variants near FOXF2 that are associated with increased stroke susceptibility. Epidemiological and experimental data suggest that FOXF2 mediates this association, potentially via differentiation defects of cerebral vascular mural cells. Further expression studies in appropriate human tissues, and further functional experiments with long follow-up periods are needed to fully understand the underlying mechanisms. NIH, NINDS, NHMRC, CIHR, European national research institutions, Fondation Leducq.

The genetic and molecular basis of flagellar motility has been investigated for several decades, with innovative research strategies propelling advances at a steady pace. Furthermore, as the phenomenon is examined in diverse bacteria, new taxon-specific regulatory and structural features are being elucidated. Motility is also a straightforward bacterial phenotype that can allow undergraduate researchers to explore the palette of molecular genetic tools available to microbiologists. This study, driven primarily by undergraduate researchers, evaluated hundreds of flagellar motility mutants in the Gram-negative plant-associated bacterium Agrobacterium fabrum . The nearly saturating screen implicates a total of 37 genes in flagellar biosynthesis, including genes of previously unknown function.