Dried blood spots (DBS) are drops of capillary blood collected onto filter paper from a finger prick. They have many advantages compared with traditional phlebotomy and enable patients to take samples at home. A DBS collection device was developed and incorporated into a CE marked DBS collection kit. This was successfully used in an international direct access vitamin D DBS service. A random access DBS CRP method was established for use with the DBS collection device and a new microsampling device called the Mitra. The quality of DBS received and the impact of lancet type was assessed and the effect of blood spot characteristics on CRP and vitamin D concentration was examined. The vitamin D service uptake and the population using it was analysed. The vitamin D concentration and status of users was compared to serum samples received in the laboratory from the local GP population. Significant differences between the populations were seen, with DBS users showing higher levels of vitamin D. In addition, the response to vitamin D testing for both populations was analysed. A higher rate of high to toxic vitamin D levels was seen in the blood spot population and the reasons for this were explored.

In human, cancer immunotherapeutic strategies using blocking antibodies anti-CTLA-4 and anti-PD-1 have achieved significant clinical responses. However, this has been associated with significant off target toxic autoimmune side effects that have restricted dose escalation efficacy of these therapies. Previous work in our lab generated a FoxP3K0 mouse model without FoxP3+ dependent CD4 driven autoimmunity when the TNFRSF receptors OX40 and CD30 signals are abrogated simultaneously. Thus suggests a novel approach which can be applied to abrogate off target CD4 driven side effects in cancer immunotherapy, without compromising CD8 anti-tumour immune responses. Here we showed that the CD30K0 0X40KO FoxP3K0 (tKO) mice are resistant to tumour progression in a mouse model of melanoma where anti-tumour immunity is CDS dependent. This was mimicked in CD30K0 0X40K0 (dKO) mice and WT mice treated respectively with anti-CTLA-4, anti-PD-1, or anti-CTLA-4, anti-PD-1 and anti-OX40L and anti-CD30L blocking mAbs. Interestingly, the CD4 driven autoimmunity has been abrogated using the fourfold combination: anti-(CTLA-4, PD-1. OX40L and CD30L), while an excellent CD8 anti-tumour response is preserved in C57BI/6 mice harbouring melanoma tumours. I have also investigated the effects of overexpression of IFNy in our mouse model of melanoma, using sanroque mice that show IFNy overexpression phenotype. I have shown that tumours do grow more slowly in sanroque mice. A striking finding in sanroque mice was that PD1 expression on tumour infiltrating CD4 and CD8 T cells was virtually completely abrogated, indicating that this might explain the delayed tumour growth. I found that agonistic OX40 mAbs in WT mice also downregulated PD-l expression on tumour infiltrating CD4 and CD8 T cells.

Adenovirus early region proteins, ElA, E1B-55K, E4orf3 and E4orf6 regulate host cell processes to facilitate viral replication. E4orf3 suppress host cell anti-viral activities through association with host cell proteins in E4orf3 nuclear-track structures, whilst E1B-55K, E4orf3 and E4orf6 are all recruited to viral replication centres during infection to promote viral DNA replication and inhibit host cell antiviral activities. Immunoprecipitation coupled to mass spectrometry identified Toplla as an Ad12 E4orf3-binding protein that localized with E4orf3 in adenovirus-infected cells. It was determined that Toplla expression was induced during infection, and that Toplla was required for the adenovirusdependent stabilization of p53. It was also established that, despite their ability to cooperate functionally, Toplla and p53 do not associate physically during infection. Immunoprecipitation coupled to mass spectrometry was also used to identify host cell proteins recruited to viral replication centres during adenovirus infection. The RP A-1 binding protein, Smarcall, and the FACT complex histone chaperone protein, SSRPl were identified as host cell proteins recruited to viral replication centres during infection. Following recruitment to viral replication centres Smarcall was found to be degraded in an E1B-55K and E4orf6 dependent manner, whilst SSRPl was found to be stable during infection and was not targeted for degradation.

Gastric cancer (GC), the fourth most common malignancy worldwide, has poor prognosis and treatment innovation is needed. The aims of this project were to investigate immune targets and treatment strategies for GC. I identified new T-cell epitopes in three Epstein-Barr virus (EBV) tumor antigens, LMP1, LMP2 and BARF1, expressed in the 10% of GC cases positive for EBV. T-cell clones showed that a BARF1-specific CD4 T-cell epitope restricted by HLA-DR51, an allele common in the population, was presented by an EBV-positive epithelial cancer cell line. Analysing blood and fresh tumor from newly diagnosed GC patients, I detected T-cell responses to MAGEA1, MAGEA4 and NY-ESO-1 tumour antigens in blood but not tumor. Compared to healthy donors, patients had: higher frequencies of LAG3 or CTLA4 positive CD8 T-cells, TIM-3 or CTLA4 CD4+ T-cells, T-regs, NKT-cells and gamma-delta T-cells in blood and tissue. Patients also had high granulocytic MDSC frequencies in PBMC. The CD4:CD8 ratio was low in some patients' blood, potentially indicating immunosenesence, but was always higher in tumor tissue. I successfully generated tumour infiltrating lymphocytes (TILs) from nine patients' tumors. These comprised high T-cells and NK-cells and low T-reg and MDSC. LAG-3 was increased, but PD1, was decreased on TIL T-cells. Using 3-dimensional organoids established from two patients, I showed that TIL NK-cells, but not TIL T-cells, recognized autologous tumor organoids. My results are the first proof of principle that TILs can readily be generated from gastric tumors, can target tumors cells and therefore be used to treat gastric cancer.

The investigation of human imprinting disorders has provided important insights into the role of genomic imprinting in normal health and development. Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth disorder associated with abnormal function of 11p15.5 imprinted genes that’s result, most commonly, from the epimutation (loss of maternal allele methylation) at the imprinting centre KCNQ1OT1:TSS-DMR (BWS_IC2). In contrast, Silver-Russell syndrome (SRS) is characterised by pre- and postnatal growth retardation and, most commonly, epimutations (loss of paternal allele methylation) at H19/IGF2:IG-DMR. Using Infinium 450K methylation array, I performed methylation profiling at 46 imprinted differentially methylated regions in 90 BWS and 21 SRS patients. I report epimutations at other imprinting centres outside of chromosome 11p15.5 in 40% of BWS_IC2 but not in SRS_IC1. The investigation of the potential underlying causes of this multilocus methylation disturbances (MLID) epigenotype in BWS_IC2 individuals indicated that several factors might contribute to the BWS phenotype and MLID epigenotype. Although not an universal finding, the use of assisted reproductive technology was significantly associated with MLID in my cohort of BWS_IC2 patients. Furthermore, using whole-exome sequencing strategy, I describe new potential candidate genes for trans-acting factors regulating methylation at imprinting DMRs.

Over the last two decades genetic advances have provided novel insights into the molecular basis of familial and sporadic cancers and provided the basis for the development of novel therapeutic approaches. For example, the identification of the gene for von Hippel Lindau disease provided seminal insights into its role in most clear cell renal carcinomas (RCC) and led to new treatments for RCC. In this thesis I investigated three related genetic aspects of neoplasia. Firstly, I analyzed the results of genetic testing for inherited phaeochromocytoma and investigated how clinical features could be used to stratify patients and improve the cost effectiveness of genetic testing. Secondly, I sought to identify novel causes of inherited neoplasia. Through exome sequencing of familial RCC kindreds, \(CDKN2B\) was identified as a novel familial RCC gene. The role of \(CDKN2B\) mutations in neoplasia was evaluated in familial and sporadic RCC and phaeochromocytoma. \(In\) \(vitro\) assays confirmed that germline \(CDKN2B\) mutations associated with inherited RCC caused an abrogation of tumour suppressor function. Finally, I explored how a gene-based strategy might be used to identify novel therapeutic strategies, Thus, using a siRNA library screen, in RCC cells with inactivated \(VHL\), potential candidate targets (e.g. \({PLK1/STK}\)-\(10\)) were identified for selectively decreasing the viability of RCC cells with inactivated \(VHL\).

The aim of this study was to characterise the genetic and epigenetic profiles of rare forms of sporadic renal cancers (RCC) and identify differential patterns of DNA methylation or somatic mutations that may permit distinction between different subtypes of RCC and could facilitate disease prognosis or identify molecular pathways that could be targeted therapeutically. Illumina Infinium HumanMethylation 450K BeadChip permitted the comparison of the epigenome of the malignant chromophobe RCC and the benign renal oncocytoma. This study identified several genes to be differentially hypermethylated in chromophobe RCC, and renal oncocytoma showing that although both visually and pathologically similar, both tumours have a distinct methylation pattern. Whole exome sequencing (WES) of renal oncocytoma samples identified somatic mutations in eighteen genes involved in a variety of cellular functions. Sanger sequencing was then used to confirm the mutations identified, followed by further screening by Sanger in a cohort of additional renal oncocytoma samples to identify if the somatic mutations are recurrent. Modern high throughput and quantitative techniques have permitted further characterisation of these rare renal cancers and have enabled unique insights into their molecular genetics, findings that may hopefully be of clinical benefit in the future.

Chronic lymphocytic leukaemia (CLL) is characterised by marked disease heterogeneity and is currently incurable. This thesis presents work undertaken to discover novel biological and therapeutic insights through the investigation of spontaneous CLL regression, the evaluation of ATR inhibition as a therapeutic strategy, and the assessment of the impact of post-treatment minimal residual disease (MRD) in CLL. Firstly, spontaneous regressed CLL tumours were found to express somatically mutated IGHV genes, display unresponsiveness to IgM and IgD BCR stimulation and exhibit a phenotype of short telomeres with low CD49d expression, suggesting a model in which the CLL clone undergoes an initial period of proliferation which subsequently subside into a state of anergy and low proliferation. Secondly, ATR inhibition was found to be a promising therapeutic target for CLL tumours with TP53 or ATM defects. Treatment with ATR inhibitor induced synthetic lethality and selective cytotoxicity to these tumours in vitro and in vivo, and sensitised them to chemotherapy and ibrutinib. Finally, MRD negativity was found to predict for 10-year survival in CLL independent of the type and line of treatment, as well as known prognostic factors including adverse cytogenetics, supporting its use as a prognostic marker and potential therapeutic goal in CLL.

Hypermethylation of CpG islands is one of the many processes that a developing cancer cell may use for the inactivation of tumour suppressor genes. The Sav/Hippo/Warts pathway was originally identified in Drosophila and shown to be responsible for controlling both growth and apoptosis, implying this is a tumour suppressor pathway. This pathway is both evolutionarily and functionally conserved in mammals. Work presented here shows that apart from FAT1 and YAP other pathway members are not epigenetically silenced in common epithelial or haematological cancers. FAT1 and YAP were frequently methylated in childhood acute lymphoblastic leukaemia (ALL) but unmethylated in epithelial cancers. Childhood ALL is a blood cancer with peak prevalence between the ages of 3-5 years. The epigenetics of this cancer were examined with three separate approaches; the first, a candidate gene approach, second a NotI restriction enzyme based array examining the methylation of genes residing on chromosome 3, and thirdly the methylated-CpG island recovery assay (MIRA) combined with CpG island arrays examining methylation on a genome-wide scale. These approaches identified a large number of novel genes which were frequently methylated in ALL. Many of the identified genes were new methylation targets and were shown to be likely targets for methylation in both common epithelial and haematological cancers. A series of these genes was seen to be specifically methylated in different leukaemia sub types, and to cluster T-ALL and B-ALL samples into high and low methylation clusters. When examined in chronic lymphoblastic leukaemia (CLL) methylation of two of the above genes was associated with disease progression and methylation of another gene was associated with response to clinical treatment.

Iron is central to the aetiology of gastrointestinal disease. Specifically, the toxic effects of excess, unabsorbed "luminal" iron ingested from the diet has been shown to be important in the development of inflammatory bowel disease and intestinal cancer. A platform for therapeutic intervention is likely to involve chelation of this luminal pool of iron. As such, a range of dietary iron chelators have been tested for their iron binding capacity. Natural biopolymers extracted from seaweed (alginates) and a variety of natural polyphenolic compounds were stratified in terms of their iron binding potential. One alginate, Manucol LD, was unique in its iron binding and demonstrated luminal iron chelation properties. With respect to the polyphenols, only one of the tested compounds (quercetin) displayed iron chelation activity in vitro and was able to suppress cellular concentrations of reactive oxygen species acting as an antioxidant. As such, it has been demonstrated that a unique alginate, Manucol LD, is an excellent candidate for sequestering luminal iron present in the gastrointestinal tract. These results underpin the rationale in utilising these types of natural and safe bio-polymers for the prevention and treatment of gastrointestinal disease.